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UBC Theses and Dissertations

Variability in an enzyme-linked, immunosorbent assay (ELISA) for Erwinia carotovora subsp. atroseptica Caron, Michel 1982

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VARIABILITY IN AN ENZYME-LINKED, IMMUNOSORBENT ASSAY (ELISA) FOR ERWINIA CAROTOVORA SUBSP. ATROSEPTICA by MICHEL CARON B . S c . A . , U n i v e r s i t e ' L a v a l , 1976 M . S c , U n i v e r s i t e L a v a l , 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of P l a n t Sc ience) We accept t h i s t h e s i s as conforming t o the r e q u i r e d s tandard THE UNIVERSITY OF BRITISH COLUMBIA May 1982 Q M i c h e l Caron, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of PLANT SCIENCE  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date /ffifj Jf /fU DE-6 (3/81) i i ABSTRACT Factors a f f e c t i n g a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Erwinia carotovora subsp. atroseptica were investigated. Optimum reaction conditions for detecting known c e l l numbers of Eca were found to be 2.0ug/ml of coating y - g l o b u l i n and 1:4-00 enzyme-y-globu-l i n conjugate d i l u t i o n . These conditions were determined using antiserum produced against glutaraldehyde-fixed, whole b a c t e r i a l c e l l s of s t r a i n E82 of Eca (serogroup I ) , and polystyrene m i c r o t i t r a t i o n plates (Dynatech substrate p l a t e s ) . In s p i t e of these optimized conditions, v a r i a b i l i t y was observed between sets of data obtained under i d e n t i c a l experimental conditions. In order to minimize or eliminate t h i s v a r i a b i l i t y , d i f f e r e n t parameters were investigated. The washing procedure was standardized by the use of a con-t r o l l e d pressure-washing system employing d i s t i l l e d water, and two 15-sec washes at 34.4-8 kPa (5 p s i ) , with 180° r o t a t i o n of the plate between each wash. Tween-20 was eliminated from the washing s o l u t i o n , since i t i n t e r f e r r e d with the s e n s i t i v i t y of the assay. This e f f e c t could not be r e l a t e d to the age of the Tween-20 employed. Well to well v a r i a b i l i t y was observed with the polystyrene m i c r o t i t r a t i o n plates employed but i t was not exclusive to the outside rows. The pattern of d i s t r i b u t i o n of the "odd" wells within a plate changed, and the number of "odd" wells decreased with time. The maximum v a r i a t i o n from the mean also decreased with time. Addition to d i f f e r e n t wells of an extra 5% of coating y-globulin, sample, and enzyme -y-globulin conjugate i n d i v i d u a l l y or i n d i f f e r e n t combinations, f a i l e d to reproduce the v a r i a b i l i t y observed thereby eliminating pipetting errors as a source of v a r i a b i l i t y . The ^05 values were influenced by the buffer solutions employed for sample and conjugate d i l u t i o n . Any given buffer had a greater e f f e c t when used for i i i conjugate d i l u t i o n . The complete buffer of phosphate buffered s a l i n e (PBS)+ 0.05% Tween-20 +2.0% polyvinylpyrrolidone+0.2% egg albumin commonly used i n vi r u s work, was found to be s u i t a b l e for the Eca system although i t s e f f i -ciency i n the presence of plant material containing bacteria remains to be evaluated. This ELISA for Eca employing optimized coating and conjugate, a stan-dardized washing procedure and a complete buffer for samples and conjugate d i l u t i o n , r o u t i n e l y detected 105 to 106 c e l l s / m l of only serogroup I of Eca when pure cultures of both homologous and heterologous s t r a i n s were tested. At concentrations >107 c e l l s / m l , s t r a i n s from serogroups XVIII, XX, and XXII of subsp. atroseptica and a few s t r a i n s from serogroups I I , I I I , IV, and V of subsp. carotovora also reacted. Even at high b a c t e r i a l concentration (10° cells/ml) no cross reactions were observed with Pseudomonas marginalis and Corynebacterium sepedonicum. Heat treatment of c e l l suspensions of serogroup I at 60 C for 3-6 min enhanced A^ os values but the l e v e l of s e n s i t i v i t y was not reduced below 105 c e l l s / m l . Cross reactions with s t r a i n s of subsp. caro- tovora serogroups I II and V, observed at 10 c e l l s / m l , were reduced but not eliminated by t h i s heat treatment. Both h e a t - l a b i l e and heat-stable water-soluble antigens were detected by t h i s ELISA for Eca. The media upon which c e l l s were grown also affected the A l + 0 5 values but t h i s e f f e c t was not propor-t i o n a l to the amount of growth observed. Based on these r e s u l t s i t was concluded that u n t i l well to well v a r i a -b i l i t y i s eliminated, and s e n s i t i v i t y increased, there w i l l be l i t t l e incen-t i v e to use the double sandwich ELISA technique with plant sap where a reduc-t i o n i n s e n s i t i v i t y i s l i k e l y . At t h i s point ELISA seems to have l i t t l e p o t e n t i a l i n routine surveys for detecting latent blackleg i n f e c t i o n s i n c e r t i f i e d seed potatoes. i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES ix ACKNOWLEDGEMENTS x i INTRODUCTION 1 CHAPTER I. EFFECT OF THE PLATE WASHING PROCEDURE ON THE DETECTION OF ERWINIA CAROTOVORA SUBSP. ATROSEPTICA (VAN HALL) DYE BY THE ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) 8 INTRODUCTION 8 MATERIALS AND METHODS 11 Ba c t e r i a l Cultures, Antiserum Production, and y-globulin P u r i f i c a t i o n 11 Conjugation of Al k a l i n e Phosphatase with y - g l o b u l i n 12 ELISA 12 Determination of the Optimun Coating y - g l o b u l i n Concentration and Enzyme-y -globulin Conjugate D i l u t i o n ... 15 Kin e t i c s of the Reaction 16 Washing Procedure Standardization 16 S t a t i s t i c a l Analysis 18 RESULTS 19 Determination of the Optimum Coating y - g l o b u l i n Concentration and Enzyme-y -globulin Conjugate D i l u t i o n ... 19 Ki n e t i c s of the Reaction 21 Washing Procedure Standardization 26 DISCUSSION 35 LITERATURE CITED 40 V CHAPTER II. FACTORS CONTRIBUTING TO THE VARIABILITY OF THE ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR ERWINIA CAROTOVORA SUBSP. ATROSEPTICA (VAN HALL) DYE 43 INTRODUCTION 43 MATERIALS AND METHODS 46 B a c t e r i a l Cultures, Antiserum Production, y - g l o b u l i n P u r i f i c a t i o n , and Al k a l i n e Phosphatase-y-globulin Conjugation 46 Basic ELISA Procedure 46 Plate Uniformity 47 Ef f e c t of Pipetting Errors 47 E f f e c t of Dif f e r e n t Buffers on ELISA 48 S t a t i s t i c a l Analysis 49 RESULTS 50 Plate Uniformity 50 Ef f e c t of Pip e t t i n g Errors 52 E f f e c t of Dif f e r e n t Buffers on ELISA 55 DISCUSSION 61 LITERATURE CITED 66 CHAPTER III. SPECIFICITY AND SENSITIVITY OF THE ERWINIA CAROTOVORA SUBSP. ATROSEPTICA (VAN HALL) DYE (SEROGROUP I) ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) 69 INTRODUCTION 69 MATERIALS AND METHODS 72 B a c t e r i a l Cultures, Antiserum Production, y-globulin P u r i f i c a t i o n , and Alka l i n e Phosphatase-y - g l o b u l i n Conjugation 72 Basic ELISA Procedure 72 E f f e c t s of Culture Conditions on ELISA 73 Ef f e c t of Heat Treatment of B a c t e r i a l C e l l s on ELISA 73 Ef f e c t of Washing B a c t e r i a l C e l l s on the ELISA 74 v i S p e c i f i c i t y of ELISA to Dif f e r e n t Strains of Erwinia carotovora 75 S t a t i s t i c a l Analysis 76 RESULTS 77 DISCUSSION 90 LITERATURE CITED 94 DISCUSSION 97 SUMMARY 100 SUPPLEMENTARY LITERATURE CITED 102 v i i LIST OF TABLES CHAPTER I . Page Table 1. The ef f e c t of coating y - g l o b u l i n concentration and enzyme-y-glo b u l i n conjugate d i l u t i o n on mean A+Q5 of controls (buffer only) treatments i n an ELISA for Erwinia carotovora subsp. atroseptica 22 Table 2. The e f f e c t of coating concentration and enzyme-y-globulin conjugate-dilution on the absorbance r a t i o s (AR) obtained by ELISA for known concentrations of Erwinia carotovora subsp. atroseptica 23 Table 3. The ef f e c t of washing system and washing s o l u t i o n on absor-bance (A+os ) and absorbance r a t i o (AR) obtained with known c e l l concentrations i n an ELISA optimized for detection of Erwinia carotovora subsp. atroseptica 27 Table 4. E f f e c t of washing s o l u t i o n and forced a i r drying on absor-bance (A+05 ) and absorbance r a t i o (AR) obtained with known c e l l concentrations i n an ELISA optimized for detection of Erwinia carotovora subsp. atroseptica 29 Table 5. E f f e c t of d i f f e r e n t washing solutions applied by co n t r o l l e d pressure washing system on the absorbance (A+05 ) a n d absorbance r a t i o s (AR) obtained with known c e l l concentrations i n an ELISA optimized for detection of Erwinia carotovora subsp. atroseptica 31 Table 6. The e f f e c t of number, duration and pressure of d i s t i l l e d water washes applied by a controlled-pressure washing system on the absorbance (A+os ) and absorbance r a t i o s (AR) obtained with known c e l l concentrations i n an ELISA optimized for detection of Erwinia carotovora subsp. atroseptica 32 Table 7. E f f e c t of washing method on absorbance (A + 05 ) and absorbance r a t i o s (AR) obtained with known antigen concentrations i n ELISA optimized for the detection of dandelion v i r u s S and Erwinia  carotovora subsp. atroseptica 34 CHAPTER I I . Table 1. E f f e c t of pip e t t i n g error on mean \ Q 5 values obtained with 1( ce l l s / m l i n an ELISA optimized for Erwinia carotovora subsp. atroseptica 56 Table 2. E f f e c t of 10 p i of additi o n a l coating, sample and/or conjugate on the mean A+05 values obtained with 106 c e l l s / m l i n an ELISA optimized for Erwinia carotovora subsp. atroseptica 57 v i i i Page Table 3. Comparison of the e f f e c t of d i f f e r e n t sample and/or conjugate buffers on absorbance (A+05 ) obtained with 10 6cells/ml i n an ELISA optimized for detection of Erwinia carotovora subsp. atroseptica 59 Table 4. Ef f e c t of buffers used for sample and conjugate d i l u t i o n on absorbance (A+Q5 ) and absorbance r a t i o (AR) obtained with known c e l l concentrations i n an ELISA optimized for Erwinia carotovora subsp. atroseptica 60 CHAPTER III. Table 1. Ef f e c t of culture media on absorbance (A+os ) and absorbance r a t i o (AR) obtained with known concentrations of 48-h c e l l s of st r a i n s E82 and E193 i n an ELISA optimized for the detection of Erwinia carotovora subsp. atroseptica 78 Table 2. E f f e c t of heat treatment on the absorbance (A+os ) and the absorbance r a t i o s (AR) obtained with known c e l l concentrations i n an ELISA optimized for Erwinia carotovora subsp. atroseptica 79 Table 3. Detection by ELISA of Erwinia carotovora subsp. at r o s e p t i c a antigens i n d i s t i l l e d water c e l l wash f l u i d s and washed c e l l suspensions of subsp. atroseptica ( s t r a i n E193) and subsp. carotovora ( s t r a i n E95) 84 Table 4. E f f e c t of a 15 min 80 C heat treatment on detection by ELISA of Erwinia carotovora subsp. atroseptica antigens i n d i s t i l l e d water c e l l wash f l u i d s and washed suspensions of ( s t r a i n E193) 85 Table 5. E f f e c t of a 90 min heat treatment at 121 C on detection by ELISA of Erwinia carotovora subsp. atroseptica antigens i n undiluted d i s t i l l e d water c e l l wash f l u i d s and washed c e l l suspensions (108 cells/ml) of f l u i d s s t r a i n E193 86 Table 6. Absorbance (A+os)? absorbance r a t i o (AR) and q u a l i t a t i v e v i s u a l estimate of color development for 108 c e l l s / m l of s t r a i n s representing 24 serogroups of Erwinia carotovora and other b a c t e r i a l pathogens i n an ELISA optimized for the detection of Erwinia carotovora subsp. atroseptica serogroup I 88 Table 7. E f f e c t of a 5 min 60 C heat treatment on the detection by ELISA of known concentrations of selected s t r a i n s of Erwinia carotovora 89 ix LIST OF FIGURES CHAPTER I . Page Figure 1. Description of the controlled-pressure washing system. The co n t r o l l e d pressure washing system consisted of a time clock (2) linked to both the pump (3), and the solenoid valve (6) c o n t r o l l i n g l i q u i d movement. The pressure was adjusted with the pressure regulator (4), and monitored on the pressure gauge (5). The pump was linked to a reser v o i r (1) containing the appropriate washing s o l u t i o n . The " r i n s i n g box" (7) was composed of a p l e x i g l a s s frame, supporting an inverted m i c r o t i t r a t i o n plate, each well of which had been replaced by a 1 cc tuberculin syringe cover provided with a 45" angle needle hole at t h e i r t i p (designed by Dr. R. Stace-Smith, Vancouver Research Station, Agriculture Canada). (8) M i c r o t i t r a t i o n plate to be washed, (a) 2.0 cm g l a s s - f i b e r r e i n f o r c e d tygon tubing (b) 1.5 cm copper pipe. (c) 1.0 cm tygon tubing). Figure 2. E f f e c t of coating y -globulin concentration and enzyme-y -gl o b u l i n conjugate d i l u t i o n (1:1001 I , 1:200GS33 , 1:400 nTTTffl , 1:800E223 ) on absorbance (A+os ) in an ELISA for Erwinia carotovora subsp. atroseptica at known c e l l concentrations. Each value i s the mean of three r e p l i c a t e s . Figure 3. E f f e c t of eating y - g l o b u l i n concentration and enzyme-y -gl o b u l i n conjugate d i l u t i o n (1:100[ZZ] , 1 : 2 0 0 ^ ^ , 1:400 r r r n T r n , 1:800 ES33) on mean absorbance (\ 0 5 ) with 105 c e l l s / ml i n an ELISA for Erwinia carotovora subsp. atroseptica. Each value i s the mean of three r e p l i c a t e s . Corrected values = uncorrected - c o n t r o l . 14 20 24 Figure 4. E f f e c t of reaction time, b a c t e r i a l s t r a i n and c e l l concentra-t i o n on absorbance ( A + o s ) i n an ELISA for Erwinia carotovora subsp. a t r o s e p t i c a . Each point i s the mean A+05 °^ s ^ x r e p l i c a t e s . 25 Figure 5. E f f e c t of washing system and washing so l u t i o n (DWI I , PBS V////A , PBS+TffUnil ) on absorbance ( A + o s ) obtained with known c e l l concentrations i n an ELISA optimized for the detection of Erwinia carotovora subsp. atroseptica. Each value repre-sents the mean of six r e p l i c a t e s . Corrected values = uncor-rected - c o n t r o l . 28 CHAPTER I I . Figure 1. V a r i a b i l i t y i n A»os values obtained at three reaction times i n an ELISA optimized for detection of Erwinia carotovora subsp. a t r o s e p t i c a . A l l wells received 10 c e l l s / m l (0 > mean plus 10% of mean, • < mean minus 10% of the mean). 51 X Figure 2. Well to well v a r i a b i l i t y i n A+os values obtained at d i f f e r e n t reaction times when a l l wells received 106 c e l l s / m l i n an ELISA optimized for Erwinia carotovora subsp. atroseptica (0 > mean + indicated % of mean; • < mean - indicated % of mean). 53 Figure 3. Maximum percent v a r i a t i o n from the mean A + o s values obtained at 5-min i n t e r v a l s for known b a c t e r i a l concentrations of s t r a i n s E82 and E193 i n a ELISA optimized for detection of Erwinia carotovora subsp. atroseptica . 54 CHAPTER I I I . Figure 1. E f f e c t of heat treatment bance (A+os ) obtained i n Erwinia carotovora subsp. (temperature and duration) on absor-an ELISA with known concentrations of atro s e p t i c a . 81 Figure 2. Eff e c t of heat treatment (60 C) duration on absorbance ( A + o s ) i n an ELISA with known concentrations of Erwinia carotovora subsp. atroseptica . 82 x i ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. R. 3. Copeman, th e s i s supervisor for h i s assistance and guidance throughout the course of t h i s study. Special appreciation i s also extended to Dr. S. H. DeBoer, Agriculture Canada Research Station, Vancouver, for his c r i t i c a l review of the th e s i s p r i o r to submission. I wish to thank Dr. V. C. Runeckles (Chairman), Dr. N. S. Wright ( A g r i -c u l t u r e Canada Research Station, Vancouver), and Dr. 3. Gordon (Department of Microbiology, U.B.C.) for t h e i r presence on my th e s i s committee. Sincere thanks also to Lois Johns, former technician at the Agriculture Canada Research Station, Vancouver, for her tec h n i c a l help i n the p u r i f i c a t i o n of the y - g l o b u l i n , and to Dr. R. Stace-Smith of the same i n s t i t u t i o n for the use of equipment. Appreciation i s also extended to Dr. B. Todd (Manitoba Department of Agriculture) for h i s help i n the preparation of the s t a t i s t i c a l a n a l y s i s . Sincere appreciation i s also expressed to a l l my fri e n d s for t h e i r encouragement and support without which t h i s work would have ended prematurely. I wish to thank the National Research Council of Canada for a scholar-ship which permitted t h i s study. - 1 -INTRODUCTION Plant pathogenic bacteria, unlike fungi, cannot be i d e n t i f i e d on the basis of morphology. As a consequence, r e l i a b l e and s p e c i f i c detection and i d e n t i f i c a t i o n techniques are required. Because many b a c t e r i a l pathogens exi s t i n low numbers i n a latent state u n t i l triggered into a c t i v i t y by favor-able environmental conditions, these detection techniques must also be very s e n s i t i v e i f they are to be of any p r a c t i c a l use. A n t i b i o t i c - r e s i s t a n t s t r a i n s and the lack of e f f e c t i v e substitutes r e g i s t e r e d for use have resulted in generally poor chemical c o n t r o l . This in turn has resulted in c e r t i f i c a -t i o n becoming an important component i n the strategy for b a c t e r i a l disease c o n t r o l . However, the existence of latent i n f e c t i o n s makes c e r t i f i c a t i o n on the basis of f i e l d symptoms d i f f i c u l t to j u s t i f y . In the absence of r e l i a b l e , s p e c i f i c and s e n s i t i v e techniques there i s no a l t e r n a t i v e . U n t i l recently, such was the s i t u a t i o n with Erwinia carotovora. Erwinia carotovora i s now thought to overwinter primarily in latent i n f e c t i o n s i n the l e n t i c e l s of potato tubers. The environmental conditions during storage and the growing season, determine when and i f the tuber or seed piece w i l l s t a r t to r o t . Thus, storage r o t , seed piece decay, f i e l d blackleg or tuber rot at harvest may or may not occur. Even though the seed piece breaks down and contaminates the daughter tuber and the rhizosphere, the tops may not show any v i s i b l e symptoms. Thus, techniques to detect these latent i n f e c t i o n s which could be used to predict storage a b i l i t y or for seed potato c e r t i f i c a t i o n purposes are surely needed. Fortunately i n the l a s t few years, several d i f f e r e n t techniques have been developed so that the detection of Erwinia carotovora i s further advanced than with most other plant pathogenic b a c t e r i a . - 2 -The use of s e l e c t i v e and enrichment media i s one means by which phytopathogenic bacteria can be detected. These media, designed to select f or growth of a c e r t a i n organism, can not always prevent the growth of unwanted organisms, and are generally of low s e n s i t i v i t y . For example, c r y s t a l v i o l e t pectate (CVP) (Cuppels and Kelman 1974) allows a minimum detection of 106 -107 c e l l s / g dry wt s o i l of Erwinia carotovora, but permits growth of Pseudo- monas spp. and other bacteria. I f used i n combination with Meneley and S t a n g h e l l i n i ' s enrichment medium, the l e v e l of s e n s i t i v i t y can be increased by 100 to 1,000-fold (Meneley and S t a n g h e l l i n i 1976). The long standing c a p a b i l i t y of c l i n i c a l medical microbiology to provide rapid and accurate s e r o l o g i c a l i d e n t i f i c a t i o n of bacteria, would suggest a s i m i l a r p o t e n t i a l e x i s t s for plant pathogenic b a c t e r i a . Only recently have d i f f e r e n t techniques based on t h i s p r i n c i p l e been f u l l y evaluated. The agglu-t i n a t i o n t e s t has been employed to i d e n t i f y or d i f f e r e n t i a t e numerous phyto-pathogenic bacteria including Erwinia atroseptica (Graham 1963), F_. aroideae (Okabe and Goto 1955), F_. carotovora (Goto and Okabe 1957), and F_. amylovora ( L e l l i o t t 1968). The general experience with t h i s technique i s that high c e l l concentrations are required. While no figures appear to be a v a i l a b l e for Erwinia carotovora, Slack et _al. (1979) have reported that Corynebacterium  sepedonicum could be detected only at a l e v e l of 2 x 107 c e l l s / m l by t h i s technique. In the same study, the immunodiffusion t e s t permitted a s i m i l a r l e v e l of s e n s i t i v i t y . Vruggink and Maas. Geesteranus (1975) detected 107 c e l l s / m l of Erwinia carotovora subsp. atroseptica (Van Hall) Dye (Eca) by immunodiffusion. However, the value of the immunodiffusion technique i s i n determining r e l a t i o n s h i p s (DeBoer et a l . 1979), rather than i n detection. The latex agglutination t e s t represents another p o s s i b i l i t y that has not been evaluated for use with Erwinia carotovora, probably because of the early - 3 -a p p l i c a t i o n of immunofluoroscence (IF) to detection of Eca by A l l a n and Kelman (1977). Using d i r e c t IF they were able to detect 10s - 106 c e l l s / m l . The i n d i r e c t technique (Slack ^ t a l . 1979) has been reported to be even more se n s i t i v e (101 - 102 cells/ml) with Corynebacterium sepedonicum. DeBoer (1980) has s u c c e s s f u l l y used the d i r e c t technique to detect cross reaction between Erwinia carotovora serogroups, on the basis of t h e i r f l a g e l l a r antigen. However, the IF technique i s not always easy to perform (Bar-Ooseph et£ al^. 1979), i s time consuming, and depends upon subjective assessment ( V o l l e r et a l . 1974, 1976, 1977). The enzyme-linked immunosorbent assay (ELISA) which has found widespread a p p l i c a t i o n i n medicine ( b a c t e r i a l , mycotic, v i r a l and p a r a s i t i c diseases), and veterinary science ( v i r a l , mycoplasmal, and p a r a s i t i c diseases) (Voller et a l . 1979), i s a s e r o l o g i c a l technique that has become av a i l a b l e to plant pathology only recently with the adaptation of the technique by Clark and Adams (1977) for plant viruses. It probably o f f e r s the greatest p o t e n t i a l for pathogen detection i n plant pathology (Clark 1981). The basic ELISA te s t depends on two p r i n c i p l e s . The f i r s t , i s that an antigen or antibody can be attached to a s o l i d phase support yet r e t a i n immunological a c t i v i t y . The second, i s that either antigen or antibody can be linked to an enzyme, and the complex retains both immunological and enzymatic a c t i v i t y (Voller et a l . 1976, 1977). Di f f e r e n t types of assays employing these basic p r i n c i p l e s have been described. The competitive method, the double antibody sandwich method, and the modified double antibody sandwich can a l l be used for detection and measurement of antigen. The i n h i b i t i o n ELISA for antigen detection i s e s p e c i a l l y suited for use with small molecular weight substances. The - 4 -i n d i r e c t method can be used for detection and measurement of antibodies and has found widespread app l i c a t i o n e s p e c i a l l y with v i r a l diseases (Voller et a l . 1979). The double antibody sandwich ELISA method i s the most commonly employed with plant viruses, bacteria, and spiro plasma. It i s c a r r i e d out as follows: the s p e c i f i c antibody i s adsorbed to a s o l i d phase, and the excess i s removed by washing. The te s t solution containing the antigen i s added, and the excess i s washed away aft e r an appropriate incubation period. The enzyme la b e l l e d s p e c i f i c antibody i s added, and the excess removed by washing. The enzyme substrate i s added and the amount of substrate hydrolysis detectable as color development i s proportional to the amount of antigen present (Voller et a l . 1979). Generally the technique has been recognized for i t s accuracy, r e l i a b i l i t y , r e p r o d u c i b i l i t y , s p e c i f i c i t y , s e n s i t i v i t y , speed, economy, and quantitative p o t e n t i a l (Bar-Ooseph et a l . 1979; Birch et j i l . 1979; Brodeur et a l . 1978; Bruins et a l . 1978; Bullock and Walls 1977; C a r l i e r et a l . 1979; Carlsson et a l . 1972, 1975, 1976; Chia and Spence 1979; Clark and Adams 1977; Engvall et al. 1971; Engvall and Perlmann 1971, 1972; Gugerli and Gehriger 1980; Ito et a l . 1980; Kishinevsky and Bar-3oseph 1978; L i s t e r and Rochow 1979; Marco and Cohen 1979; Nachmias et a l . 1979; Russell et a l . 1976; Tresh et a l . 1977; V o l l e r et a l . 1974, 1976, 1977, 1979; Vruggink 1978). When compared to the other s e r o l o g i c a l techniques currently employed with d i f f e r e n t antigen systems, ELISA proved to have many advantages. Flegg and Clark (1979) reported that ELISA was more s e n s i t i v e than the tube p r e c i p i -t i n test when employed for Apple Ch l o r o t i c Leafspot Virus. S i m i l a r l y , ELISA was better than the complement f i x a t i o n t e s t for detection of cytomegalovirus IgG antibody (Chia and Spence 1979). It was more s e n s i t i v e than agglutination - 5 -and immunodiffusion for detection of Rhizobium (Kishinevsky and Bar-Joseph 1978), and a l o t more s e n s i t i v e (10 3 - 105 x) than double d i f f u s i o n for the detection of Phoma t r a c h e i p h i l a responsible for Mai Secco disease i n lemon (Nachmias et a l . 1979). V o l l e r et a l . (1974), in t h e i r work with malaria, found a p o s i t i v e c o r r e l a t i o n between ELISA and immunofluorescence, but also pointed out that the l a t t e r was more time consuming. S i m i l a r l y , Bar-Joseph et a l . (1979) reported that ELISA was easier to use than immunofluorescence with Ci t r u s T r i s t e z a V i r u s . ELISA was also found to have a comparable s e n s i t i v i t y to the Radio Immuno Assay (RIA) (Engvall et a l . 1971; Engvall and Perlmann 1972; V o l l e r et a l . 1976, 1977; Yolken et al. 1977), while overcoming the s u b j e c t i v i t y , expense, r i s k s , and r e s t r i c t e d use associated with RIA (Voller et a l . 1976, 1977, 1979). Furthermore, the p o s s i b i l i t i e s of the ELISA tech-nique for large scale sampling (Bar-Joseph et a l . 1979; C a r l i e r et a l . 1979; Chia and Spence 1979; Clark and Adams 1977; Clark et a l 1978; Flegg and Clark 1979; Kishinevsky and Bar-Joseph 1978; Marco and Cohen 1980; Stevens and Tsiantos 1979; Tamada and Harrison 1980; Tresh et a l . 1977), make i t an a t t r a c t i v e technique for c e r t i f i c a t i o n programs, where several v i r u s and b a c t e r i a l diseases could eventually be assayed at once. In much of the preliminary work with p l a n t - i n f e c t i n g bacteria, the tech-nique has not shown the expected high l e v e l of s e n s i t i v i t y predicted from the success obtained i n v i r u s detection (Tresh et a U 1977; Clark and Adams 1977; Bar-Joseph et a l . 1979). Kishinevsky and Bar-Joseph (1978) reported a l e v e l of detection of only 1 & - 108 c e l l s / ml for Rhizobium under normal condi-t i o n s . Weaver and Guthrie (1978) considered 106 - 107 c e l l s / m l a high l e v e l of s e n s i t i v i t y for Pseudomonas phaseolicola. Stevens and Tsiantos (1979) used ELISA to detect whole Corynebacterium michiganense c e l l s , both in culture - 6 -suspensions of known concentrations and tomato plant extracts, and reported a l e v e l of detection of 103 c e l l s / m l from pure culture suspensions. However, t h i s conclusion i s not supported by t h e i r data, and a l e v e l of 105 - 106 c e l l s / m l seems more r e a l i s t i c . C l a f l i n and Uyemoto (1978) reported detecting Corynebacterium sepedonicum i n infected stems and tubers of potato, and i n culture suspensions. Unfortunately, no l e v e l of s e n s i t i v i t y was reported. Cross reactions have also been a frequent problem i n these preliminary reports. Berger et a l . (1979) demonstrated weak cross reactions in ELISA with heterologous antisera for Rhizobium s t r a i n s . Weaver and Guthrie (1978) reported cross reactions between Pseudomonas phaseolicola and other unspecified b a c t e r i a . S i m i l a r l y , Vruggink (1978) reported cross reactions between Xanthomonas pe l a r g o n i i and Aplanobacter populi. The only instance where ELISA has been used with some success with phytopathogenic bacteria has been with Eca (Vruggink 1978; Cother and Vruggink 1980). However, while cautioning about the need for determining the l i m i t s and s p e c i f i c i t y of the assay, Vruggink did not provide experimental evidence that many of the factors a f f e c t i n g the assay with other organisms had been investigated for the Eca system. The detection of a r t i f i c i a l l y - c r e a t e d latent i n f e c t i o n s of Eca i n potato tubers (Cother and Vruggink 1980) indicated the p o t e n t i a l usefulness of the ELISA technique. The ELISA technique i f i t i s to be widely used for bacteria must over-come the lack of s e n s i t i v i t y and s p e c i f i c i t y observed i n the preliminary studies. These studies have lar g e l y employed reaction conditions used for plant virus assays. Whether these f a i l u r e s are due to the fact that b a c t e r i a l and v i r a l systems have d i f f e r e n t optimal reaction conditions needs to be determined. I d e n t i f i c a t i o n of the sources of v a r i a b i l i t y observed in other - 7 -systems (Bullock and Walls 1979; Carlsson et at. 1972, 1975; Engvall et a l . 1971, and L i s t e r and Rochow 1979) and means for t h e i r removal are e s s e n t i a l to any attempt to increase s e n s i t i v i t y i n b a c t e r i a l systems. Of the phytopatho-l o g i c a l b a c t e r i a l systems studied to date only that involving Eca seems to o f f e r much promise. In addition, considerably more i s known about the serolo-g i c a l r e l a t i o n s h i p s of t h i s organism (cross reactions between serogroups, presence of f l a g e l l a r and somatic antigens) and there i s an immediate p r a c t i -c a l a p p l i c a t i o n for an ELISA for t h i s organism. Thus, Eca was selected as the model ELISA system for study with the following objectives: 1. to devise a standardized working system and determine the optimum working conditions; 2. to determine the optimum buffers for sample and congugate prepara-t i o n ; 3. to evaluate the m i c r o t i t r a t i o n plates, p i p e t t i n g errors and washing technique as sources of v a r i a b i l i t y ; 4. to determine the e f f e c t on s e n s i t i v i t y of heat treatment and washing of b a c t e r i a l c e l l s ; 5. to determine whether the immunodiffusion serogroup s p e c i f i c i t y was retained i n the ELISA. - 8 -CHAPTER I. EFFECT OF THE PLATE WASHING PROCEDURE ON THE DETECTION OF ERWINIA  CAROTOVORA SUBSP. ATROSEPTICA (VAN HALL) DYE BY THE ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) INTRODUCTION The enzyme-linked immunosorbent assay (ELISA) has found widespread application in medicine ( b a c t e r i a l , mycotic, v i r a l and p a r a s i t i c diseases), veterinary science ( v i r a l , mycoplasmal and p a r a s i t i c diseases), and more recently a g r i c u l t u r e ( v i r a l d i s e a s e s ) ( V o l l e r et _al. 1979). However, as Lehtonen and Viljanen (1980) have pointed out i t has been widely used without adequate standardization. To r e a l i z e the f u l l p o t e n t i a l of ELISA, optimal conditions must be p r e c i s e l y determined for each system (Clark 1981; V o l l e r et a l . 1977). The need for standardization of reagents, operational procedures and methods for the analysis and presentation of r e s u l t s , as well as the need for a better appreciation of the p r i n c i p l e s involved becomes correspondingly greater as the technique becomes more widely used in plant pathology (Clark 1981). The operational procedure for each system requiring standardization should include determination of a suitable coating-conjugate combination, choice of buffer for preparation of sample and conjugation d i l u t i o n s , time and temperature of incubation, enzyme system, reaction time for substrate and a convenient method of data analysis. The procedure employed to wash the m i c r o t i t r a t i o n plates between steps has been described as the key to the successful use of t h i s method (Birch et a l . 1979; V o l l e r et al_. 1977). A survey of the l i t e r a t u r e indicated that not only has a variety of washing solutions been employed, e.g. tap water (Bidwell - 9 -et aL. 1977; Henrikson 1979), d i s t i l l e d water (Bruins et _al. 1978), and phos-phate buffered s a l i n e containing 0.05% Tween-20 (PBS+T) (Berger et al. 1979; Birch et a l . 1979; Brodeur et al. 1978; Carlsson ^ t j i l . 1976; Clark and Adams 1977; Engvall and Perlmann 1971; Flegg and Clark 1979; Holmgren and Svenner-holm 1973; V o l l e r jet _al. 1979), but that the number of washes, and the washing time varied g r e a t l y . PBS+T i s the most widely employed washing s o l u t i o n , presumably because the addition of Tween-20 was reported to increase s e n s i t i -v i t y by Bullock and Walls (1977) working with Toxoplasma gon d i i . Bruins et a l . (1978) have reported that i t s i n c l u s i o n i n the washing sol u t i o n a c t u a l l y decreased s e n s i t i v i t y probably because the detergent action removed from the polystyrene walls the lipopolysaccharide of the rough mutant of Salmonella  minnesota being studied. The washing procedure i t s e l f has r a r e l y been described. Excepting the few instances where eit h e r an automatic washer (Denmark and Chessum 1978), or a homemade washing device (Henriksen 1979) was employed, the use of a wash bot t l e must be assumed. Although ELISA has been employed with several genera of plant pathogenic b a c t e r i a including Corynebacterium michiganense (E. F. Sm) Jensen (Stevens and Tsiantos 1979), Corynebacterium sepedonicum (Spieck. & Kotth.) Skapt. & Burkh. ( C l a f f i n and Uyemoto 1978), Pseudomonas phaseolicola (Burkh.) Dowson (Weaver and Guthrie 1978) and Xanthomonas pe l a r g o n i i (N.A. Brown) Starr & Burkh. (Vruggink 1978), no data on the determination of optimal conditions were presented for these systems. Vruggink (1978) and Cother and Vruggink (1980), working with Erwinia carotovora subsp. atroseptica (van Hall) Dye (Eca) cautioned about the need for determining the l i m i t s and s p e c i f i c i t y of the assay, but did not provide experimental evidence that many of the factors a f f e c t i n g ELISA in other systems had been investigated in the Erwinia system. - 10 -However, t h e i r work did indicate that the technique had p o t e n t i a l with t h i s organism. The objectives of t h i s work were to determine optimal reaction condi-tions for the detection of F_. carotovora subsp. atroseptica (van Hall) Dye by ELISA and to determine the e f f e c t s of d i f f e r e n t washing procedures, and wash-ing solutions on a standardized model system. - 11 -MATERIALS AND METHODS Ba c t e r i a l Cultures, Antiserum Production, and y -globulin P u r i f i c a t i o n Potato s t r a i n s (E82, E193) of Eca conforming to serogroup I (DeBoer et a l . 1979) were employed throughout t h i s study. Stock cultures were maintained on Difco Nutrient Agar (NA) slants at 4 C. Unless otherwise noted, b a c t e r i a l c e l l suspensions used i n the assays were prepared from 48-hour cultures grown on NA at 27 C. Antiserum against glutaraldehyde-fixed, whole c e l l s of s t r a i n E82 was prepared i n rabbit by the procedure of A l l e n and Kelman (1977). To avoid excessive loss of y -globulin by adsorption to glass surfaces, a l l glassware employed i n the p u r i f i c a t i o n procedure was s i l i c o n i z e d with a 5% solution of dichlorodimethyl s i l a n e ( (CH3)2SiCl 2)(Matheson, Coleman and B e l l Manufacturing Chemists)) i n chloroform. The y - g l o b u l i n p u r i f i c a t i o n procedure of Clark and Adams (1977) was followed, with some modifications. One ml of crude antiserum was d i l u t e d i n 9.0 ml of s t e r i l e d i s t i l l e d water, and the Y - g l o b u l i n p r e c i p i t a t e d by dropwise addition of 10 ml of saturated ammonium s u l f a t e . After s t i r r i n g for 60 min at room temperature, the r e s u l t i n g suspen-sion was centrifuged at 2500 g for 5 min at 4 C (IEC Refrigerated Centrifuge, Model B-20, Rotor 870). The p e l l e t was resuspended i n 1.0 ml of half strength phosphate buffered s a l i n e (PBS), 0.005 M, pH 7.4. The d i l u t i o n , p r e c i p i t a t i o n and c e n t r i f u g a t i o n steps were repeated and the p e l l e t obtained resuspended i n 1.0 ml of half-strength PBS, and dialyzed at 4 C against three changes (two, 1-hour periods and overnight) of 500 ml of half-strength PBS. The dialyzate was passed through a DEAE-22 Sephadex column (3.0 - 5.0 x .9 cm) p r e e q u i l i b -rated by passing 10x, 5x, 1x, and half-strength PBS, pH 7.4 through a batch of DEAE-22 u n t i l pH and conductivity matched half-strength PBS. The y -globulin - 12 -was washed through with half-strength PBS and the ef f l u e n t monitored at 280 nm with an ISCO Model UA-4 Absorbance Monitor. The f i r s t major peak was c o l l e c t e d and the combined f r a c t i o n s were adjusted to A+ 0 5 =1.4 (approxi-mately 1 mg of y-globulin/ml) using a G i l f o r d Spectrophotometer 250. The Y- g l o b u l i n was divided into 0.5 ml aliquots in s i l i c o n i z e d 15 x 45 mm, screw-cap glass specimen bottles,and stored at -18 C. Conjugation of Al k a l i n e Phosphatase with y - g l o b u l i n . The conjugation procedure of Clark and Adams (1977) was followed. S i l i c o n i z e d glassware was used in a l l experiments. A 0.5 ml aliquot of alka-l i n e phosphatase (Phosphatase Alkaline No P-4502, Sigma Chemical Co.) was centrifuged at 2500 g for 5 min at 4 C (IEC Rotor 870). The p e l l e t was resus-pended i n 1.0 ml of p u r i f i e d y - g l o b u l i n and dialyzed at 4 C against three changes (two, 1-hour periods, and overnight) of 0.01 M, pH 7.4 PBS (500 ml). Glutaraldehyde was added d i r e c t l y to the suspension to give a f i n a l concentra-ti o n of 0.06%, and the mixture incubated 4 h in the dark at room temperature. The excess glutaraldehyde was removed by d i a l y s i s as previously described. The a l k a l i n e phosphatase-y-globulin conjugate was stored i n s i l i c o n i z e d v i a l s at 4 C and approximately 0.0001% of sodium azide was added as a preservative. ELISA The ELISA procedure described by Clark and Adams (1977) was employed with modifications as noted f o r i n d i v i d u a l experiments. F l a t bottom polysty-rene m i c r o t i t r a t i o n plates were employed ( M i c r o t i t e r ^ Immulon Substrate Plates, Dynatech Laboratories Inc., Alexandria, V i r g i n i a ) . P r i o r to coating with y - g l o b u l i n a l l plates required for a given experiment were numbered, and scanned at 405 nm on a Ti t e r t e k Multiskan plate reader (Flow Laboratories, - 13 -Model 310C) . Column one of p l a t e one was employed as a b lank to c a l i b r a t e the i n s t r u m e n t . Only p l a t e s w i th uni form A+os va lues were used . The c o a t i n g Y - g l o b u l i n , prepared i n carbonate b u f f e r , pH 9 .6 (0.15% NaC03 + 0.29% NaHCC^ + 0.026% NaN3 ) , was added (0 .2 ml) to each w e l l . The p l a t e s were p laced i n d i v i -d u a l l y i n p l a s t i c bags and incubated at 37 C f o r 4 h. A p l a t e washing dev ice (F igure 1) which permi t ted c o n t r o l of the d u r a -t i o n and pressure of the wash s o l u t i o n was used to s t a n d a r d i z e the washing c o n d i t i o n s . P l a t e s to be washed were emptied of c o n t e n t s , shaken v i g o r o u s l y t o remove d r o p l e t s t rapped i n the w e l l s , and p laced i n v e r t e d on the " r i n s i n g box" . F o l l o w i n g a 15 -sec wash wi th d i s t i l l e d water (DW) at 34.48 kPa (5 p s i ) the p l a t e s were removed, shaken t o remove d r o p l e t s , r o t a t e d 180 degrees and s i m i l a r l y washed a second t i m e . P l a t e s were removed, shaken and p laced on a s i m i l a r box connected t o compressed a i r (L inde M e d i c a l A i r , B r e a t h i n g Grade, CGA Type 1, Grade F ) . A f t e r a 5 -sec d r y i n g pe r iod at 172.38 kPa (25 p s i ) , the p l a t e s were removed, r o t a t e d 180 degrees , and the t reatment r e p e a t e d . B a c t e r i a l c e l l s grown as p r e v i o u s l y d e s c r i b e d , were suspended i n a f r e s h s o l u t i o n of phosphate b u f f e r e d s a l i n e c o n t a i n i n g 0.05% Tween-20 ( p o l y -oxyethy lene (20) s o r b i t a n monolaurate) ( F i s h e r S c i e n t i f i c ) , 2.0% p o l y v i n y l -p y r r o l i d o n e (PVP) (M.W. approx. 4 4 , 0 0 0 , BDH C h e m i c a l s ) , and 0.2% egg albumin (EA) (Egg albumine (ovalbumine) Grade I I I , Sigma, No -5378) , h e r e a f t e r r e f e r r e d t o as PSB+T+PVP+EA. The b a c t e r i a l suspension was ad jus ted to an absorbance at 540 nm ( A 5 i + 0 ) of 0 . 1 , which corresponded to 108 c e l l s / m l . A t e n - f o l d d i l u t i o n s e r i e s was prepared i n the same sample b u f f e r . B u f f e r a lone wi th no b a c t e r i a l c e l l s added, served as the c o n t r o l . A l i q u o t s (0 .2 ml) of the a p p r o p r i a t e b a c t e r i a l c o n c e n t r a t i o n were added to the w e l l s . The p l a t e s were p laced i n d i v i d u a l l y i n p l a s t i c bags, incubated at 4 C overn ight (16 hours) and were - 14 -F i g u r e 1 - D e s c r i p t i o n of the c o n t r o l l e d - p r e s s u r e washing system. The c o n -t r o l l e d p ressure washing system c o n s i s t e d of a t ime c l o c k (2) l i n k e d to both the pump (3), and the s o l e n o i d va l ve (6) c o n t r o l l i n g l i q u i d movement. The pressure was ad jus ted w i th the pressure r e g u -l a t o r ( 4 ) , and monitored on the pressure gauge ( 5 ) . The pump was l i n k e d t o a r e s e r v o i r (1) c o n t a i n i n g the a p p r o p r i a t e washing s o l u -t i o n . The " r i n s i n g box" (7) was composed of a p l e x i g l a s s frame, s u p p o r t i n g an i n v e r t e d m i c r o t i t r a t i o n p l a t e , each w e l l of which had been r e p l a c e d by a 1 cc t u b e r c u l i n s y r i n g e cover prov ided wi th a 45° angle needle ho le at t h e i r t i p (designed by Dr . R. S t a c e - S m i t h , Vancouver Research S t a t i o n , A g r i c u l t u r e Canada) . (8) M i c r o t i t r a -t i o n p l a t e t o be washed, (a) 2.0 cm g l a s s - f i b e r r e i n f o r c e d tygon tub ing (b) 1.5 cm copper p i p e . (c) 1.0 cm tygon t u b i n g ) . - 15 -washed as previously described. The a l k a l i n e phosphatase-y-globulin conjugate was also prepared in a fresh solution of PBS+T+PVP+EA and 0.2 ml added to each well. The plates were placed i n d i v i d u a l l y i n p l a s t i c bags, and incubated at 37 C for 4 h p r i o r to washing as previously described. Aliquots of 0.2 ml of a l k a l i n e phosphatase substrate (p-nitrophenyl phosphate disodium, c r y s t a l l i n e , Sigma, Sigma 104) at 0.6 mg/ml in a 10% diethanolamine s o l u t i o n , pH 9.8, were added to the plates, with the use of an eight channel Multi Channel Pipetter ( T l t e r t e k ) , c a l i b r a t e d to de l i v e r 0.2 ml. The reactions were allowed to proceed for 30 min at room temperature. A q u a l i t a t i v e v i s u a l estimate of the degree of yellow color development was made, and the plates were read spectrophotometrically at 405 nm with a Tl t e r t e k Multiskan plate reader c a l i b r a t e d against the f i r s t column wells at the s t a r t of the reaction. A+05 sample An absorbance r a t i o ( ) greater than 2.0 was considered a p o s i t i v e A+os c o n t r o l r e s u l t . Determination of the Optimum Coating y - g l o b u l i n Concentration and Enzyme-y - globulin Conjugate D i l u t i o n Coating y - g l o b u l i n concentrations of 4.0 ug, 2.0 yg, 1.0 ug, and 0.5 \i g/ml were prepared; a treatment with coating buffer alone served as the c o n t r o l . A l k a l i n e phosphatase-y-globulin conjugate d i l u t i o n s of 1:100, 1:200, 1:400, and 1:800 were prepared; conjugate buffer alone served as the con t r o l . A ten-fold d i l u t i o n series of bacteria ranging from 10 to 10 ce l l s / m l was employed; buffer alone with no b a c t e r i a l c e l l s added served as the co n t r o l . Two m i c r o t i t r a t i o n plates were required to contain a l l possible coating-- 16 -sample-conjugate combinations. Three r e p l i c a t e s were employed. On each plate, wells of columns 1, 12, and Row A from column 1 to 6 received only the buffers, and the substrate. K i n e t i c s of the Reaction B a c t e r i a l d i l u t i o n s e r i e s ranging from 103 to 108 c e l l s / m l of s t r a i n E193 and E82 were prepared. Each d i l u t i o n of each s t r a i n was r e p l i c a t e d six times on one m i c r o t i t r a t i o n plate (84 wells), and compared to the sample buffer c o n t r o l . The reaction was assessed immediately a f t e r addition of the substrate and every 5 min thereafter. Washing Procedure Standardization In a l l experiments with the washing procedure, a coating y - g l o b u l i n concentration of 2.0 pg/ml, conjugate d i l u t i o n of 1:400, b a c t e r i a l concentra-tions of 103 to 107 c e l l s / m l , and a buffer only c o n t r o l were employed. Each b a c t e r i a l concentration including the control represented a treatment and was r e p l i c a t e d 10 times/plate. The outside rows of each plate contained only the buffer control and the substrate. The ELISA procedure was performed as pre-viously described, except that the washing procedure was modified. The wash-ing solutions evaluated were phosphate buffered s a l i n e (PBS), PBS containing 0.05% Tween-20 (PBS+T), and d i s t i l l e d water (DW). In i n i t i a l experiments the use of a wash bot t l e was compared with the controlled-pressure washing system. One m i c r o t i t r a t i o n plate was employed/buffer/system. One wash bot t l e was used for each buffer and the m i c r o t i t r a t i o n plates were washed three times for 1 min at each washing step. A compressed a i r drying period was not employed with t h i s conventional washing technique, but plates were shaken to remove droplets a f t e r each washing step. With the controlled-pressure wash-- 17 -ing system, the washing solutions were kept i n i n d i v i d u a l containers, and the system flushed with the washing solution p r i o r to use to remove the previous washing s o l u t i o n . To f a c i l i t a t e the procedure, the buffers were employed i n the following order: DW, PBS, and PBS+T. To eliminate any trace of Tween-20 the system was flushed with DW for 2 to 3 min at the completion of each PBS+T wash. The plates were washed twice for 10 sec at 34.48 kPa (5 p s i ) , and dried with compressed a i r twice for 5 sec at 172.38 kPa (25 p s i ) . The e f f e c t of a i r drying the plates with compressed a i r , was evaluated i n only the c o n t r o l l e d pressure washing system, employing two washes of 10 sec at 34.48 kPa (5 p s i ) . At each wash step, one serie s of plates were only shaken, while a second s e r i e s were shaken and dried with compressed a i r previously described. A comparison was also made between d i f f e r e n t l o t s of Tween-20 (Fisher S c i e n t i f i c ) used for the preparation of PBS+T. The f i r s t two Tween-20 l o t s (T1, T2) were approximately one year old, T1 representing the l o t previously used; the t h i r d l o t (T3) was recently purchased and had 6 months l e f t before the expiration date. The plates were washed twice for 10 sec at 5 p s i , and dried twice for 5 sec at 172.38 kPa (25 psi) at each wash step. Up to t h i s point, a l l experiments involving the c o n t r o l l e d pressure washing system, were conducted with an a r b i t r a r y choice of conditions i n the duration, number of washes, and pressure. To optimize the washing procedure, an experiment was designed with the following v a r i a b l e s : one, two, or three washes at each step, for periods of 5, 10, 15, or 20 sec, and a pressure of 34.48 kPa (5 psi) or 68.95 kPa (10 p s i ) . Only DW was employed, and two drying periods of 5 sec at 172.38 kPa (25 psi) applied at each wash step. - 18 -To determine the e f f e c t of washing on a system employing only a protein antigen, s i m i l a r experiments were conducted with a p u r i f i e d preparation of a s t r a i n of Dandelion Virus S (DVS) obtained from L. Johns (Canada Department of Agricu l t u r e , Vancouver Research S t a t i o n ) . Previous work (L. Johns personal  communication) had established that 1.0 yg/ml of coating y-globulin and a conjugate d i l u t i o n of 1:800 were optimal. A t e n - f o l d d i l u t i o n s e r i e s was pre-pared from a stock DVS preparation containing 140 ng/ml. The conventional washing procedure u t i l i z i n g a wash bot t l e with PBS+T, (three washes of 1 min at each wash step without drying) was compared with the standardized washing conditions of two washes of 15 sec at 34.48 kPa (5 psi) with DW, followed by two drying periods of 5 sec at 172.38 kPa (25 p s i ) , at each step. One micro-t i t r a t i o n plate was employed for each system. Wells of columns 1, 12, Row A from column 1 to 5, and Row H from column 8 to 12 of each plate, contained only the buffer control and the substrate. S t a t i s t i c a l Analysis In a l l experiments, a l l treatments were completely randomized. In some experiments, Tukey's multiple range te s t was performed on \ Q 5 values uncor-rected for t h e i r controls with a 5.0% s i g n i f i c a n c e l e v e l for the F-value. - 19 -RESULTS Determination of the Optimum Coating y - g l o b u l i n Concentration and Enzyme-y - gl o b u l i n Conjugate D i l u t i o n At b a c t e r i a l c e l l concentrations of 105 - 107 c e l l s / m l , color develop-ment (A+05) in t h i s ELISA for Eca decreased as the d i l u t i o n of enzyme-y-globu-l i n conjugate was increased (Fig. 2). At 107 and 106 b a c t e r i a l c e l l s / m l , the mean values obtained for each conjugate d i l u t i o n within each coating concen-t r a t i o n were s i g n i f i c a n t l y d i f f e r e n t from one another. However with 105 c e l l s / m l , t h i s pattern was not maintained. At t h i s concentration and at 4.0 ug/ml a l l conjugate d i l u t i o n s were s i g n i f i c a n t l y d i f f e r e n t from each other except 1:200 and 1:400. At 2.0 ug/ml 1:100 and 1:200, and 1:200 and 1:400 did not d i f f e r , but 1:100 was s i g n i f i c a n t l y d i f f e r e n t from 1:400. The highest d i l u t i o n (1:800) was s i g n i f i c a n t l y lower than a l l of the others. Only the dif f e r e n c e s between 1:100 and 1:400, 1:100 and 1:800, and 1:200 and 1:800 were s i g n i f i c a n t l y d i f f e r e n t at 1.0 ug/ml. At 0.5 ug/ml only 1:100 and 1:800 d i f f e r e d s i g n i f i c a n t l y . Color development was also a function of b a c t e r i a l concentration and coating y - g l o b u l i n concentration. Most of the mean A t 0 5 values among the coating concentrations within the same conjugate d i l u t i o n at 10 5, 106 and 107 c e l l s / m l were s i g n i f i c a n t l y d i f f e r e n t . Exceptions were at 107 c e l l s / m l with 1.0 and 2.0 ug/ml coating y - g l o b u l i n with both 1:100 and 1:200 conjugate d i l u t i o n s and with 2.0 and 4.0 ug/ml coating y - g l o b u l i n and 1:800 conjugate d i l u t i o n . Further exceptions were at 105 c e l l s / m l at 1:400 and 1:800 conjugate d i l u t i o n s were there was not a s i g n i f i c a n t difference between the 2.0 ug/ml - 20 -o U l U Z < o t/t OS] controls ~ COATING (pg/ml ) 2.0 4.0 2JOH 1.0H 0.5H 10 bacteria/ml 0.5 1.0 COATING (jig/ml ) 0.5 1.0 2.0 10 bacteria^ml 1 1.0 2.0 \-2JD -1.5 -1.0 •0.5 • F i g . 2. E f f e c t of coating y - g l o b u l i n concentration and enzyme-y-globulin conjugate d i l u t i o n (1:100 1 I . 1:200 T 1:400 InTirm , 1:800 ^ ^ ) on absorbance (A+ 0 5 ) i n a n ELISA for Erwinia carotovora subsp. atro- septica at known c e l l concentrations. Each value i s the mean oT three r e p l i c a t e s . - 21 -and 4.0yg/ml coatings, and at 1:800 where the differ e n c e between the 1.0 and 2.0ug/ml coatings was also not s i g n i f i c a n t . The mean control values (A+05) also decreased with a decrease i n coating concentration and an increase i n conjugate d i l u t i o n ( F i g . 2 and Table 1). Because the background was a function of the coating-conjugate combination, the high coating and conjugate combinations r a p i d l y gave v i s u a l l y p o s i t i v e backgrounds. Those combinations which developed p o s i t i v e background readings within 30 min were not further considered. When the data i n F i g . 1 were examined i n terms of absorbance r a t i o s (AR) (Table 2), not a l l coating-conju-gate combinations having p o s i t i v e values (AR > 2.0) at high b a c t e r i a l c e l l concentrations also had AR > 2.0 at a l l concentrations approaching the l i m i t s of s e n s i t i v i t y (10 5 cells/ml) of the technique. Only one of nine combinations giving a p o s i t i v e v i s u a l estimate at 105 c e l l s / m l also had a p o s i t i v e absor-bance r a t i o . By contrast, four of the seven v i s u a l l y negative combinations had p o s i t i v e r a t i o s . Moreover, at 1& c e l l s / m l none of the three combinations rated v i s u a l l y p o s i t i v e had p o s i t i v e absorbance r a t i o s , while three of the t h i r t e e n v i s u a l l y negative combinations did. This apparent discrepancy i s related to the fact that each coating-conjugate combination had d i f f e r e n t c o n t r o l values (Table 1). The e f f e c t of t h i s v a r i a b l e background was to greatly reduce apparently large differences i n the uncorrected A+os v a l u e s among d i f f e r e n t combinations (Fig. 3). As a consequence, the absorbance r a t i o s generally increased with increasing conjugate d i l u t i o n s and decreasing coating concentrations which had lower backgrounds. K i n e t i c s of the Reaction At a l l b a c t e r i a l concentrations the mean A+05 values increased with an increased incubation time (Fig. 4). With both b a c t e r i a l s t r a i n s , the A+gs - 22 -Table 1. The e f f e c t of coating y - g l o b u l i n concentration and enzyme-y-globulin conjugate d i l u t i o n on mean r\os of controls (buffer only) treatments in an ELISA for Erwinia carotovora subsp. atroseptica Coating Mean \Q5 at conjugate d i l u t i o n concentration (ug/ml) 1 :100 1:200 1:400 1:800 4.0 0.308 a* 0.221 b 0.132 de 0.106 efg 2.0 0.203 be 0.160 cd 0.114 def 0.078 fgh 1 .0 0.116 def 0.067 ghi 0.023 i j 0.043 h i j 0.5 0.024 i j 0.035 i j 0.014 j 0.018 i j *Means (of three r e p l i c a t e s ) followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P=0.05) according to Tukey's multiple range t e s t . * * V i s u a l l y negative (A+QS <. 0.140 not detectable v i s u a l l y ) Table 2 . The e f f e c t of c o a t i n g c o n c e n t r a t i o n and enzyme-y -g Iobu l in c o n j u g a t e - d i l u t i o n on the absorbance r a t i o s (AR) obta ined by ELISA f o r known c o n c e n t r a t i o n s of E rw in ia caro tovora subsp. a t r o s e p t i c a Absorbance r a t i o s f o r b a c t e r i a l c o n c e n t r a t i o n s ( c e l l s / m l ) 107 106 105 id* C o a t i n g Conjugate d i l u t i o n Conjugate d i l u t i o n Conjugate d i l u t i o n Conjugate d i l u t i o n c o n c e n t r a t i o n <ug/ml) 1:100 1:200 1:400 1:800 1:100 1:200 1:400 1:800 1:100 1:200 1:400 1:800 1:100 1:200 1:400 1:800 4 . 0 6 . 5 8 . 9 14.6 14.4 3 .2 3 . 8 4 .9 4 . 9 1.3 1.2 1.6 1.5 1.1 1.1 1.3 1.0 2 . 0 9 . 6 12.0 15.2 19.4 4 . 3 4 . 5 5.1 6 . 5 1.2 1.3 1.5 1.4 0 . 9 1.0 0 .7 0 .8 1.0 16.7 28.6 72.7 30 .0 6 . 6 9 . 9 22.3 10.2 1.4 2.2 4 .6 1.8 1.2 1.1 2.4 0 . 9 0 . 5 52 .3 29 .3 59.1 35.8 14.7 8 . 7 18.9 11.8 3.1 1.7 3.1 2 .0 2 .0 0 . 9 2.1 0 . 9 *Each AR based on t h e mean A+Q5 of t h r e e rep l i c a f e s * * V i s u a l l y n e g a t i v e ( A ^ g _^ 0 .140 were not d e t e c t a b l e v i s u a l l y ) - 2k -corrected values o.H j f l k i n k E c m o u z < o 0.4 0.3-0.2-0.1-control values o> 0.4-0.3-o> 0.1-1 uncorrected values 1.0 2.0 4.0 COATING (ug/ml) F i g . 3 . E f f e c t of c o a t i n g y - g l o b u l i n c o n c e n t r a t i o n and e n z y m e - y - g l o b u l i n conjugate d i l u t i o n (1:1001 I , 1:200 ^ 223 , 1:4-00 011100 * 1:800 EE3) on mean absorbance (A+05 ) w i th 105 c e l l s / m l i n an ELISA f o r E r w i n i a c a r o t o v o r a subsp. a t r o s e p t i c a . Each va lue i s the mean of th ree r e p l i c a t e s . Cor rec ted va lues = u n c o r r e c t e d - c o n t r o l . - 25 -Strain E193 c i TIMES (minutes) UJ U Z < g Strain E 82 TIMES (minutes) F i g . 4. E f f e c t of reaction time, b a c t e r i a l s t r a i n and c e l l concentration on absorbance (A+os ) in an ELISA for Erwinia carotovora subsp. atrosep- t i c a . Each point i s the mean A+os of six r e p l i c a t e s . - 26 -values were proportional to the b a c t e r i a l concentrations at 106 , 107 and 10s c e l l s / m l . Mean A+os values corresponding to 103 , /\& and 105 c e l l s / m l were equivalent to the co n t r o l . Washing Procedure Standardization Use of a controlled-pressure washing system compared to washing by wash bo t t l e (Table 3) resulted i n higher mean A+os values at a l l b a c t e r i a l concen-t r a t i o n s regardless of the washing s o l u t i o n . However, none of the \ Q 5 values obtained with 10s or fewer c e l l s / m l i n either system, with any of the washing solutions, was s i g n i f i c a n t l y d i f f e r e n t from t h e i r corresponding controls. Although the differences between washing solutions i n the controls were not s i g n i f i c a n t l y d i f f e r e n t , PBS-T gave the highest background A+05 i n both, wash-ing systems and the lowest corrected values (Fig. 5). Increases of 28.5%, 28.2% and 19.0% i n the corrected \ 0 5 values were obtained by using the stan-dardized DW r i n s e for c e l l concentrations of 10 , 10 and 10 re s p e c t i v e l y . S i m i l a r l y , increases of 48.5%, 66.6% and 114.5% were obtained with the stan-dardized PBS r i n s e . By contrast there was either no change (10 7 and 105 cells/ml) or a s l i g h t decrease (10 6 cells/ml) when PBS+T was used. Drying the plates i n a stream of a i r a f t e r each washing had no e f f e c t on the mean A+os values obtained (Table 4). Regardless of whether the plates were dried or not, a f i l m was observed on the bottom of the wells whenever PBS+T was employed as the washing s o l u t i o n . The mean A+Q5 values for the PBS+T controls were s i g n i f i c a n t l y higher than the DW or PBS controls. Although the absolute A+os values i n t h i s experiment were higher than compar-able treatments i n the previous experiments (Table 3), s i m i l a r trends were observed. DW and PBS alone gave s i g n i f i c a n t l y higher mean \ 0 5 values than PBS+T at 106 and 107 c e l l s / m l . In t h i s experiment at 105 c e l l s / m l the A+os Table 3. The ef f e c t of washing system and washing solution on absorbance (A+05) a n c* absorbance r a t i o (AR) obtained with known c e l l concentrations i n an ELISA optimized for detection of Erwinia carotovora subsp atroseptica Absorbance (A+05) a n c ' absorbance r a t i o (AR) for b a c t e r i a l concentrations (cell/ml) Washing system Washing solu t i o n 107 106 * 105 10** 103 0 AR A+05 AR \05 AR AR \05 AR \05 Wash DW 0.847c** 60.5 0.177 ef 12.6 0.035 ghi 2.5 0.016 i 1.1 0.014 i 1.0 0.014 i B o t t l e PBS 0.835 c 64.2 0.160 f 12.3 0.027 h i 2.1 0.012 i 0.9 0.012 i 0.9 0.013 i PBS+T 0.845 c 14.1 0.209 def 3.5 0.070 ghi 1.2 0.063 ghi 1.1 0.056 ghi 0.9 0.058 ghi Controlled DW 1.092 b 52.0 0.230. de 11.0 0.046 ghi 2.2 0.023 h i 1.1 0.023 h i 1.1 0.021 h i Pressure PBS 1.237 a 61.9 0.265 d 13.3 0.050 ghi 2.5 0.027 h i 1.4 0.024 h i 1.2 0.020 h i PBS+T 0.856 c 12.4 0.227 de 3.3 0.091 g 1.3 0.077 gh 1.1 0.068 ghi 1.0 0.069 ghi *Limit of v i s u a l estimation *Means (of six r e p l i c a t e s ) followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P=0.05) according to Tukey's multiple range t e s t . - 28 -1.H i.(H 0.5H nn corrected values 1.5-1.0-as-E c u-l O z < co Of O 0.5H 2J0-1.5-1.0-as-[ Mk \ mm r^jm i i mm i mm io7 io6 io5BACTERIA/ML 10 7 10° 10 5 control values asJ ITU i mM JUL. uncorrected values 20-15-l.(H 0.54 mM i nJUl WASH BOTTLE SYSTEM REGULATED PRESSURE WASHING SYSTEM i g . 5. E f f e c t of washing system and washing sol u t i o n (DW I 1 , P B S ^ i ^ , PBS+T llilLLUj ) on absorbance (A+05) obtained with known c e l l concentrations i n an ELISA optimized for the detection of Erwinia  carotovora subsp. atroseptica . Each value represents the mean of r e p l i c a t e s . Corrected values = uncorected-control. Table 4. E f f e c t of washing solution and forced a i r drying on absorbance (\o5^ a n d absorbance r a t i o (AR) obtained with known c e l l concentrations i n an ELISA optimized for detection of Erwinia carotovora subsp. atroseptica Absorbance (\ 0 5 ) and absorbance r a t i o (AR) for b a c t e r i a l concentrations (cell/ml) Washing solution 107 106 105 103 0 Drying AR AR AR AR AR No DW PBS 1.375 1.562 b* a 62.5 104.1 0.463 0.566 f e 21.4 37.7 0.094 h i j 4.3 0.092 hijk 6.1 0.029 ijklm 0.027 jklm 1.3 1.8 0.022 0.017 lm m 1.0 1.1 0.022 lm 0.015 m PBS+T 1.091 c 10.4 0.392 g 3.7 0.152 h 1.4 0.101 h 1.0 0.099 h 0.9 0.105 h * Yes DW PBS 1.540 1.509 a a 140.0 100.6 0.598 0.579 e e 54.4 38.6 0.095 h i j 0.096 hi 8.6 6.4 0.019 m 0.024 klm 1.7 1.6 0.008 0.018 m m 0.7 1.2 0.011 m 0.015 m PBS+T 0.914 d 9.7 0.340 g 3.6 0.129 h 1.4 0.089 h i j k l 0.9 0.089 h i j k l 0.9 0.094 h i j *Means (of six r e p l i c a t e s ) followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P=0.05) according to Tukey's multiple range t e s t . **Limit of v i s u a l estimation. - 30 -values for DW and PBS but not PBS+T were s i g n i f i c a n t l y greater than t h e i r cor-responding c o n t r o l s . The l i m i t of detection both v i s u a l l y and by absorbance r a t i o s was 10 5 c e l l s / m l for DW and PBS but 1 0 6 c e l l s / m l for PBS+T. Three d i f f e r e n t l o t s of Tween-20 employed i n the PBS+T washing s o l u t i o n , gave higher mean background values (A+os) than DW and PBS (Table 5 ) . But only Lo t - 1 at b a c t e r i a l concentrations of 10 6 and 10 7 c e l l s / m l resulted i n s i g n i f i -cantly higher mean A+05 values than DW or PBS. Absorbance r a t i o s obtained at b a c t e r i a l concentrations of 10 6 and 10 7 c e l l s / m l were greater with DW and PBS than any of the Tween -20 l o t s employed r e f l e c t i n g the e f f e c t s of the higher background A+05 values obtained with Tween -20. The l i m i t of s e n s i t i v i t y was 10 6 b a c t e r i a l c e l l s / m l for a l l washing solutions, both v i s u a l l y and by absor-bance r a t i o s . The duration, pressure and number of rinses at each washing step were a l l found to influence mean A+0s values (Table 6 ) . At any pressure with a 5 or 10 sec duration, increasing the number of washes decreased the background values i n the c o n t r o l . With combinations involving longer times, the f i r s t wash apparently removed everything that could be removed. S i m i l a r l y at a b a c t e r i a l concentration of 10p c e l l s / m l and the combinations of 15 s e c - 6 8 . 9 5 kPa (10 p s i ) - 2 or 3 washes, 20 sec-3 4 . 4 8 kPa (5 p s i ) - 3 washes and 20 s e c - 6 8 . 9 5 kPa (10 p s i ) - 1 , 2 or 3 washes, the increased washing resulted i n A+05 values decreased to a l e v e l (<0 .05) considered too low to be r e l i a b l y used, compared to the remaining combinations where strong c o l o r a t i o n s t i l l developed at 1 0 5 b a c t e r i a l c e l l s / m l with very l i t t l e background i n the co n t r o l s . Two 15 sec washes at 3 4 . 4 8 kPa were selected as the optimum washing conditions because the background was minimized but the sample mean A+05 values were high at 10 5 , 10 6 and 10 7 c e l l s / m l . The l i m i t of v i s u a l estimation was 10 6 b a c t e r i a l Table 5. E f f e c t of d i f f e r e n t washing solutions applied by controlled pressure washing system on the absorbance (\o5 ) a n c ' absorbance r a t i o s (AR) obtained with known c e l l concentrations i n an ELISA optimized for detection of Erwinia carotovora subsp. atroseptica Absorbance (A+os ) a n c ' absorbance r a t i o s (AR) for b a c t e r i a l concentrations (cells/ml) 107 106 * 105 1(f 103 0 Washing Solution +^05 AR +^05 AR A+05 AR AR AR \05 DW 1.687 b** 29.6 0.464 d 8.1 0.108 ef 1.9 0.059 f 1.0 0.055 f 1.0 0.057 f PBS 1.707 b 31.0 0.443 d 8.1 0.098 ef 1.8 0.053 f 1.0 0.058 f 1.1 0.055 f PBS+T Lot 1 1.805 a 23.4 0.529 c 6.9 0.128 e 1.7 0.089 ef 1.2 0.080 ef 1.0 0.077 ef PBS+T Lot 2 1.651 b 23.9 0.462 d 6.7 0.103 ef 1.5 0.073 ef 1.1 0.067 ef 1.0 0.069 ef PBT+T Lot 3 1.708 b 21.1 0.465 d 5.7 0.115 ef 1.4 0.086 ef 1.1 0.078 ef 1.0 0.081 ef *Limit of v i s u a l estimation. **Means (of six r e p l i c a t e s ) followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P=0.05) according to Tukey's multiple range t e s t . Table 6. The e f f e c t of number, duration and pressure of d i s t i l l e d water washes applied by a controlled-pressure washing system on the absorbance (A+os ) a n d absorbance r a t i o s (AR) obtained with known c e l l concentrations i n an ELISA optimized for detection of Erwinia carotovora subsp. atroseptica Absorbance (A+os ) and absorbance r a t i o (AR) for b a c t e r i a l concentrations (cells/ml) 107 10 6* 10s id* 103 0 Time Pressure No* • * -~" ' sec kPa Washes A+ 0 5 A R \ o 5 A R \o5 A R ^ 0 5 A R ^ 0 5 A R \ u 5 5 34.48 1 1.131** 37.7 0.427 2 1.045 41.8 0.367 3 0.766 40.3 0.230 5 68.95 1 0.927 21.1 0.318 2 1.117 34.9 0.400 3 0.778 37.0 0.238 10 34.48 1 1.057 22.5 0.399 2 0.942 37.7 0.322 3 1.014 46.1 0.363 10 68.95 1 0.821 23.5 0.243 2 1.112 41.2 0.388 3 0.748 34.0 0.219 15 34.48 1 1.662 92.3 0.579 2 1.599 319.8 0.457 3 1.520 190.0 0.449 15 68.95 1 1.535 511.7 0.491 2 1.523 380.8 0.413 3 1.314 328.5 0.339 20 34.48 1 1.692 105.8 0.509 2 1.699 242.7 0.521 3 1.493 OO 0.409 20 68.95 1 1.573 CO 0.450 2 1.257 OO 0.305 3 1.344 OO 0.357 14.2 0.078 2.6 0.034 1.1 0.039 1.3 0.030 14.7 0.066 2.6 0.021 0.8 0.019 0.8 0.025 12.1 0.050 2.6 0.020 1.1 0.017 0.9 0.019 7.2 0.078 1.8 0.044 1.0 0.037 0.8 0.044 12.5 0.072 2.3 0.026 0.8 0.032 1.0 0.032 11.3 0.052 2.5 0.020 1.0 0.018 0.9 0.021 8.5 0.088 1.9 0.048 1.0 0.036 0.8 0.047 12.9 0.070 2.8 0.031 1.2 0.023 0.9 0.025 16.5 0.064 2.9 0.019 0.9 0.021 1.0 0.022 6.9 0.071 2.0 0.043 1.2 0.037 1.1 0.035 14.4 0.064 2.4 0.032 1.2 0.028 1.0 0.027 10.0 0.049 2.2 0.015 0.7 0.015 0.7 0.022 32.2 0.071 3.9 0.018 1.0 0.022 1.2 0.018 91.4 0.059 11.8 0.006 1.2 0.018 3.6 0.005 56.1 0.057 7.1 0.005 0.6 0.010 1.3 0.008 163.7 0.054 18.0 0.007 2.3 0.009 3.0 0.003 103.3 0.026 6.5 0.004 1.0 0.002 0.5 0.004 84.8- 0.026 6.5 0.001 0.3 0.002 0.5 0.004 31.8 0.074 4.6 0.020 1.3 0.023 1.4 0.016 74.4 0.054 7.7 0.005 0.7 0.009 1.3 0.007 CO 0.021 OO 0.000 0.0 0.000 0.0 .0.000 CO 0.021 OO 0.000 0.0 0.000 0.0 0.000 OO 0.000 0.0 0.000 0.0 0.000 0.0 0.000 CO 0.005 OO 0.000 0.0 0.000 0.0 0.000 *Limit of v i s u a l estimation **Mean of six r e p l i c a t e s - 33 -c e l l s / m l for a l l combinations. Absorbance r a t i o s were greater than 2.0 for a l l combinations at 105 b a c t e r i a l c e l l s / m l with the exception of 5 sec-68.95 kPa (10 psi)-1 wash, 10 sec-34.48 kPa (5 psi)-1 wash. The controlled-pressure DW washing system when applied to a protein antigen system (the Dandelion Virus S assay) also reduced the background com-pared to the conventional PBS-T wash bottle r i n s e (Table 7). In t h i s system also, the marked contrast between the uncorrected mean A+os values and the absorbance r a t i o s for the two washing systems c l e a r l y i l l u s t r a t e d the impor-tance of thorough washing. This more thorough washing res u l t e d i n a 10-fold decrease i n the l i m i t of v i s u a l estimation i n the virus system (Table 11), but a s i m i l a r increase i n s e n s i t i v i t y when absorbance r a t i o s were determined. The b a c t e r i a l system, included for comparison, had 105 b a c t e r i a l c e l l s / m l as the v i s u a l l i m i t of s e n s i t i v i t y for both washing systems. Based on absorbance r a t i o s the l i m i t of s e n s i t i v i t y was 106 b a c t e r i a l c e l l s / m l with the conven-t i o n a l system, and 105 b a c t e r i a l c e l l s / m l with the controlled-pressure DW washing system. Table 7. E f f e c t of washing method on absorbance ( A 4 0 5 ) and absorbance r a t i o s (AR) obtained with known antigen concentrations i n ELISA optimized for the detection of dandelion virus S and Erwinia  carotovora subsp atroseptica Washing System Virus d i l u t i o n from stock (140 ug/ml) 10" 10" 10" 1 0 - 1 0 - Control B a c t e r i a l concentration (cells/ml) 107 106 105 ^0k 103 Wash b o t t l e 05 1.408* 0.478 0.218 0.194** 0.168 0.105 1.396 0.456 0.200** 0.168 0.152 0.145 AR 13.4 4.6 2.1 1.8** 1.6 9.6 3.1 1.4** 1.2 1.0 Standardized, c o n t r o l l e d -pressure A+05 0.920 0.308 0.104** 0.067 0.045 0.016 0.970 0.284 0.094** 0.057 0.057 0.042 AR 57.5 19.3 6.5** 4.2 2.8 23.0 6.8 2.2** 1.4 1.4 *Mean of six r e p l i c a t e s **Limit of v i s u a l estimate - 35 -DISCUSSION The enzyme-linked immunosorbent assay (ELISA) i s usually evaluated on the basis of one of two c r i t e r i a : v i s u a l estimates or absorbance r a t i o s >2.0 calculated from spectrophotometry determinations of color development ( L i s t e r and Rochow 1979, V o l l e r et a l . 1979, Yolken et a l . 1977). The former method which i s simply q u a l i t a t i v e , i s gradually being replaced as automated plate reading devices become more a v a i l a b l e . Both c r i t e r i a were employed simultane-ously in t h i s study which enabled a d i r e c t comparison and contrasting of the r e s u l t s obtained by each method. A l l r e s u l t s were based on determinations made aft e r 30 min which was selected as a reasonable processing time based on previous reports that timing errors could be brought about by shorter times (Bidwell et j a l . 1977). Prolonging incubation beyond that time to increase the s e n s i t i v i t y as suggested by Engvall and Perlmann (1972), resulted in increased color development in the controls as well as samples which could make v i s u a l estimation d i f f i c u l t i f not impossible. Both assessment methods gave s i m i l a r r e s u l t s when high concentrations of the t e s t antigen were employed. However, at the l i m i t s of s e n s i t i v i t y (10 5 cells/ml) of the technique d i f f e r e n t r e s u l t s were obtained. At high coating concentrations and low conjugate d i l u t i o n s (high conjugate concentrations), v i s u a l l y p o s i t i v e r e s u l t s were recorded at 105 c e l l s / m l . Because the back-ground i s a function of coating-conjugate concentrations, the measured back-grounds were higher at these combinations which reduced the p o s s i b i l i t y of absorbance r a t i o s being > 2.0. At the other extreme, low coating concentra-t i o n s and d i l u t e conjugates, absorbance r a t i o s > 2.0 were recorded for some samples which were not v i s u a l l y p o s i t i v e (Table 2). Because of the very low - 36 -backgrounds obtained at these combinations, any color development or small differences between wells were maximized and probably contributed to the v a r i -a b i l i t y observed, and the f a l s e p o s i t i v e s (AR > 2.0) recorded at c e l l concen-t r a t i o n s considered below the l i m i t s of detection. Because both c r i t e r i a were employed i n the s e l e c t i o n of the optimum coating and conjugate d i l u t i o n s in the Erwinia carotovora subsp. at r o s e p t i c a system, any combinations which did not give a v i s i b l e color development i n 30 min at the l i m i t of s e n s i t i v t y (10 5 cells/ml) were discarded. The combina-ti o n s of 2.0 ug/ml, and 4.0 ug/ml with conjugate d i l u t i o n s of both 1:100 and 1:200, for which v i s u a l l y p o s i t i v e controls were obtained within 30 min were also discarded (Table 3). The 4.0 ug/ml-1:800 combination was eliminated because of the high background associated with t h i s high coating concentra-t i o n . Because there was no s i g n i f i c a n t d i f f e r e n c e between 2.0 and 4.0 ug/ml at 1:400, the lower l e v e l was favored for conservation of antiserum as well as high background considerations. S i m i l a r l y , because there was no s i g n i f i c a n t difference between 1:100 and 1:200 at 1.0 ug/ml, the greater d i l u t i o n was favored. Thus, the choice of an optimum coating-conjugate combination was narrowed down to 2.0 ug/ml-1:400, and 1.0 ug/ml-1:200. While the l a t t e r was the only combination to give both a v i s u a l p o s i t i v e , and an AR > 2.0 at a bac-t e r i a l concentration of 105 c e l l s / m l , the 2.0 ug/ml-1:400 combination was selected as a compromise to economize on the conjugated antiserum. As Herrmann and C o l l i n s (1976), and L i s t e r and Rochow (1979) have pointed out, conservation of reagents i s an important consideration when l i t t l e i s gained by the use of higher concentrations. The only previous work involving detection of Erwinia carotovora subsp. atroseptica by ELISA (Cother and Vruggink 1980, Vruggink 1978) employed a - 37 -combination of 1.0 ug/ml of coating and a conjugate d i l u t i o n of 1:400, with no mention of the time allowed for the reaction to proceed. Furthermore, because of differences i n antiserum preparation ( l i v e c e l l s versus glutaraldehyde-fixed c e l l s ) , and s l i g h t l y d i f f e r e n t glutaraldehyde concentrations in the conjugation procedure, which could be c r i t i c a l according to Korpraditskul et  a l . (1979), the systems cannot be d i r e c t l y compared. Based on these r e s u l t s , the widespread use of Tween-20 as a component of the washing sol u t i o n for ELISA i s not j u s t i f i e d . I t s i n c l u s i o n resulted i n higher background A+os values, and s i g n i f i c a n t l y lower sample A+05 values at high b a c t e r i a l concentrations (10 6 and 107 cells/ml) compared to d i s t i l l e d water or PBS. The consequence was a loss i n s e n s i t i v i t y due to the high back-ground which determines absorbance r a t i o s (Table 4, 10-fold loss in s e n s i t i -v i t y ) . This r e s u l t supports Bruins et _al. (1978) who also reported that the use of Tween-20 in the washing solution i n t e r f e r e d with the detection of a lipopolysaccharide antigen. However, i t does not confirm Bullock and Walls (1977) report that the presence of Tween-20 e f f e c t i v e l y eliminated background reactions and increased s e n s i t i v i t y i n assays for Toxoplasma gondii antigen. The f i l m observed on the bottom of a l l plates washed with Tween-20, could not be r e l a t e d to the q u a l i t y (age) of the material employed. This f i l m provides an explanation for the occurrence of increased background A+ 0 5 values without any increase i n v i s u a l l y detected color development. Because the A+QS values were determined d i r e c t l y through the wells, t h i s f i l m could increase back-ground A+05 values without a f f e c t i n g the rate of v i s i b l y detectable color i n the wells. The reduction in sample A+os values i n spite of the f i l m can,also be explained. It i s possible that the detergent action of Tween-20 may be removing some of the c e l l s at high b a c t e r i a l concentrations so that the f i n a l - 38 -antigen concentration resulted i n a reduced A+os value which resulted i n a net reduction i n s p i t e of the f i l m on the plates. Further work w i l l be required to determine whether antigens of d i f f e r e n t s i z e s and chemical composition are equally prone to t h i s proposed mechanism. However in t h i s work, both protein and LPS antigens responded s i m i l a r l y to the use of Tween-20 i n the washing solu t i o n (Table 7). Because no s i g n i f i c a n t difference was obtained between d i s t i l l e d water and PBS, and because the former proved much easier to use with the c o n t r o l l e d pressure washing system, d i s t i l l e d water was adopted as the washing solu-t i o n . The use of tap water as suggested by Bidwell et a l . (1977) and Henrikson (1979) was not considered because of the v a r i a b i l i t y i n tap water from one lo c a t i o n to another. The importance of thorough and uniform washing (Birch et^ _al. 1979, Vo l l e r et al^. 1979) was confirmed in t h i s work. The fact that increases of 19.0% to 38.5% i n sample A+QS values corrected for background were obtained by the standardized method employing d i s t i l l e d water i l l u s t r a t e s the importance of a thorough washing. However, as shown by successive washes, sample as well as background can be removed i f optimum conditions are not determined. These r e s u l t s support the conclusion of Lehtonen and Viljanen (1980) that the number of washes i s c r i t i c a l in determining the f i n a l surface concentration on the wells. Because d i f f e r e n t combinations of pressure and time resulted i n d i f f e r e n t degrees of background and/or antigen removal (Table 6), uniform and reproducible washing should be employed. A combination involving two washes rather than one longer wash was chosen so that the plate could be rotated 180u on the washer to minimize error. A 15-sec wash at 34.48 kPa (5 psi) i n the system employed, - 39 -corresponded to about 1600 ml. Henriksen (1979) employed a somewhat si m i l a r washing device without pressure c o n t r o l . He reported reproducible r e s u l t s with 10 to 15 sec washings where approximately 1000 ml/5 sec were poured d i r e c t l y on the spreader p l a t e . When the same treatments were run under apparently s i m i l a r conditions in successive experiments, the A+05 varied considerably. While the trends in the data were consistent, the l i m i t of s e n s i t i v i t y for detection of Erwinia caro- tovora subsp. atroseptica varied between 105 and 106 c e l l s / m l , i n spite of optimized coating-conjugate conditions, and reproducible washing conditions. L i s t e r and Rochow (1979) have noted s i m i l a r v a r i a b i l i t y in an ELISA for Barley Yellow Dwarf Virus. V o l l e r ^ t _al_. (1979) have reported that small departures from precise optimal conditions of pH, time, temperature or reagent concentra-t i o n s can i n t e r f e r e with the r e p r o d u c i b i l i t y of the r e s u l t s . Thus a thorough study of other factors influencing the A+os values i n t h i s system should be undertaken to i d e n t i f y a d d i t i o n a l sources of v a r i a b i l i t y that would i n t e r f e r e with the detection of Erwinia carotovora subsp. at r o s e p t i c a . - 40 -LITERATURE CITED ALLAN, E. and A. KELMAN. 1977. Immunofluorescent s t a i n procedures f or detection and i d e n t i f i c a t i o n of Erwinia carotovora var. atroseptica. Phytopathology 67:1305-1312. BERGER, 3.A., S.N. MAY, L.R. BERGER and B.B. B0HL00L. 1979. Colorimetric enzyme-linked immunosorbent assay for the i d e n t i f i c a t i o n of s t r a i n s of Rhizobium in culture and i n the nodules of l e n t i l s . Appl. and Environm. Mi c r o b i o l . 37:642-646. BIDWELL, D.E., A.A. BUCK, H.O. DIESFEL, B. ENDERS, 3. HAWORTH, G. HULDT, N.H. KENT, C. KIRSTEN, P. MATTERN, E.3. RUITENBERG and A. VOLLER. 1977. Le t i t r a g e avec immunoadsorbant l i e a une enzyme (ELISA). B u l l . WHO. 55:557-568. BIRCH, C.3., N.I. LEHMANN, A.3. HAWKER, 3.A. MARSHALL and I.D. GUST. 1979. Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and i n d i r e c t immunofluorescence for detection of human ro t a v i r u s antigen i n faeces. 3. C l i n . Path. 32:700-705. BR0DEUR, B.R., F.E. ASHT0N and B.B. DIENA. 1978. Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae s p e c i f i c antibodies. Can. 3. M i c r o b i o l . 24:1300-1305. BRUINS, S.C., I. INGWER, M.L. ZECKEL and A.C. WHITE. 1978. Parameters a f f e c t i n g the enzyme linked immunosorbent assay of immunoglobulin G antibody to a rough mutant of Salmonella minnesota. Inf. Imm. 21:721-728. BULLOCK, S.L. and K.W. WALLS. 1977. Evaluation of some of the parameters of the enzyme-linked immunospecific assay. 3. Infect. Dis. 136 (Supplement):S279-S285. CARLSS0N, H.E., B. HURVELL and A.A. LINDBERG. 1976. Enzyme-linked immunosorbent assay (ELISA) for t i t r a t i o n of antibodies against B r u c e l l a  abortus and Ye r s i n i a e n t e r o c o l i t i c a . Acta Path. M i c r o b i o l . Scand. Sect. C. 84:168-176. CLAFLIN, L.E. and 3. K. UYEM0T0. 1978. Serodiagnosis of Corynebacterium  sepedomicum by enzyme-linked immunosorbent assay. Phytopathology News 12(9): 156 (Abstract). CLARK, M.F. 1981. Immunosorbent assays in plant pathology. Ann. Rev. Phytopathol. 19: 83-106. CLARK, M.F. and A.N. ADAMS. 1977. C h a r a c t e r i s t i c s of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. 3. Gen. V i r o l . 34:475-483. C0THER, E.3. and H. VRUGGINK. 1980. Detection of v i a b l e and nop viable c e l l s of Erwinia carotovora var. atroseptica i n inoculated tubers of var. B i n t j e with enzyme-linked immunosorbent assay (ELISA). Potato Res. 23:133-135. - 41 -DE BOER, S.H., R.J. COPEMAN and H. VRUGGINK. 1979. Serogroups of Erwinia ' carotovora potato s t r a i n s determined with d i f f u s i b l e somatic antigens. Phytopathology 69:316-319. DENMARK, J.R. and B.S. CHESSUM. 1978. Standardization of enzyme-linked immunosorbent assay (ELISA) and the detection of Toxoplasma antibody. Med. Lab. Sc. 35:227-232. ENGVALL, E. and P. PERLMANN. 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871-874. ENGVALL, E. and P. PERLMANN. 1972. Enzyme-linked immunosorbent assay, ELISA. I l l - Quantitation of s p e c i f i c antibodies i n enzyme-labeled anti-immunoglobulin i n antigen coated tubes. J. Immunol. 109:129-135. FLEGG, C.L. and M.F. CLARK. 1979. The detection of apple c h l o r o t i c leafspot virus by a modified procedure of enzyme-linked immunosorbent assay (ELISA). Ann. appl. B i o l . 91:61-65. HENRIKSEN, Sv. Aa. 1979. A simple washing unit for micro-ELISA. Acta Vet. Scand. 20:598-600. HERRMANN, J.E. and M.F. COLLINS. 1976. Quantitation of immunoglobulin adsorption to p l a s t i c s . J. Immunol. Methods. 10:363-366. HOLMGREN, J. and A.M. SVENNERH0LM. 1973. Enzyme-linked immunsorbent assays for cholera serology. Inf. Immunity. 7:759-763. JOHNS, L.J. 1979. P u r i f i c a t i o n and properties of a new c a r l a v i r u s from dandelion. M.Sc. Thesis. University of B.C., Vancouver, B.C. 66 pp. KORPRADITSKUL, P., R. CASPER, and D.E. LESEMANN. 1979. Evaluation of short reaction times and some c h a r a c t e r i s t i c s of the enzyme-conjugation i n enzyme-linked immunosorbent assay (ELISA). Phytopath. Z. 96:281-285. LEHT0NEN, O.P. and M.K. VILJANEN. 1980. Antigen attachment i n ELISA. J. Immunol. Methods. 34:61-70. LISTER, R.M. and W.F. R0CH0W. 1979. Detection of barley yellow dwarf v i r u s by enzyme-linked immunosorbent assay. Phytopathology. 69:649-654. STEVENS, W.A. and J. TSIANT0S. 1979. The use of enzyme-linked immunosorbent assay (ELISA) for the detection of Corynebacterium michiganense i n tomatoes. Microbios Letters 10:29-32. VOLLER, A., D.E. BIDWELL and A. BARTLETT. 1977. The enzyme-linked immunosor-bent assay (ELISA). Published by Dynatech Laboratories, 900 Slaters Lane, Alexandria, V i r g i n i a 22314. 48 pp. VOLLER, A., D.E. BIDWELL and A. BARTLETT. 1979. The enzyme-linked immuosor-bent assay (ELISA). Published by Dynatech Laboratories, 900 Slaters Lane, Alexandria, V i r g i n a 22314. 128 pp. - 42 -VRUGGINK, H. 1978. Enzyme-linked immunosorbent assay (ELISA) i n the sero-diagnosis of plant pathogenic ba c t e r i a . Proc. 4th Int. Conf. Plant. Path. Bact. Angers, p. 307-310. WEAVER, W.M. and 3.W. Guthrie. 1978. Enzyme-linked immunospecific assay app l i c a t i o n to the detection of seed borne b a c t e r i a . Phytopathology News 12:156-157 (Abstract). YOLKEN, R.H., H.B. GREENBERG, M.H. MERSON, R.B. SACK and A.Z. KAPIKIAN. 1977. Enzyme-linked immunosorbent assay for detection of Escherichia c o l i h e a t - l a b i l e enterotoxin. 3. C l i n . Microbiol. 6:439-444. - 43 -CHAPTER I I . FACTORS CONTRIBUTING TO THE VARIABILITY OF THE ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR ERWINIA CAROTOVORA SUBSP. ATRO-SEPTICA (VAN HALL) DYE. INTRODUCTION The enzyme-linked immunosorbent assay (ELISA), i s often referred to as an accurate, r e l i a b l e , and reproducible technique (Bar-Joseph et _al. 1979; Birch et at. 1979; Brodeur et a l . 1978; Bullock and Walls 1977; Carlsson et a l . 1975; Engvall and Perlmann 1971; Gugerli and Gehriger 1980; Kishinevsky and Bar-Joseph 1978; L i s t e r and Rochow 1979; Marco and Cohen 1979; V o l l e r et a l . 1976, 1977). These q u a l i t i e s have resulted i n the several authors (Clark and Adams; 1977; Engvall et j i l . 1971; Vruggink 1978) pointing out the quantitative p o t e n t i a l of the technique. However, t h i s p o t e n t i a l has never been f u l l y r e a l i z e d due to the v a r i a t i o n observed between r e p l i c a t e s within the same test (Bullock and Walls 1977; Carlsson et al. 1972, 1975; Engvall et a l . 1971; L i s t e r and Rochow 1979). Although t h i s v a r i a b i l i t y has been att r i b u t e d p r i m a r i l y to v a r i a t i o n s within the m i c r o t i t r a t i o n plates (Clark and Adams 1977; Denmark and Chessum 1978; McMurray and Blanchflower 1979), other factors may be involved. V o l l e r et _al. (1979) have pointed out that even small departures from the optimal conditions determined for a given system, can i n t e r f e r e with r e p r o d u c i b i l i t y of the r e s u l t s . The l i m i t e d studies with plant pathogenic bacteria, including Erwinia  carotovora subsp. atroseptica (van Hall) Dye (Cother and Vruggink 1980; Vruggink 1978); Corynebacterium sepedonicum (Spieck. & Kotth.) Skapt. & Burkh. - 44 -( C l a f l i n and Uyemoto 1978) Corynebacterium michiganense (E.F. Sm) Oensen (Stevens and Tsiantos 1979); Pseudomonas phaseolicola (Burkh.) Dawson (Weaver and Guthrie 1978); Xanthomonas pelargonii (N.A. Brown) Starr & Burkh. (Vruggink 1978) and Rhizobium spp. (Berger et a l . 1979; Kishinevsky and Bar-Ooseph 1978) have d i r e c t l y applied techniques developed for use with plant viruses to the b a c t e r i a l systems. The extreme s e n s i t i v i t y characterizing the v i r u s assays has not generally been observed with the b a c t e r i a . Whether reaction conditions optimal for plant virus detection are also s u i t a b l e for b a c t e r i a l detection remains to be determined. The large number of d i f f e r e n t reaction conditions reported i n the more thoroughly-researched systems sug-gests that comparative studies would be i n order even here. The reaction condition upon which there seems to be the least agreement i n the ELISA l i t e r a t u r e i s the choice of buffers for antigen samples and enzyme-conjugates. Phosphate buffered s a l i n e plus 0.05% Tween-20 (PBS+T) has been used for sample preparation involving antibodies (Bullock and Walls 1979; Carlsson et a l . 1972, 1975, 1976; Engvall and Perlmann 1972; Russell et a l . 1976) viruses (Crook and Payne 1980) and b a c t e r i a l enterotoxin (Yolken et a l . 1977) . PBS+T plus 2.0% polyvinylpyrrolodine (PBS+T+PVP) has been employed for sample preparation i n studies involving plant viruses (Bar-3oseph et _al. 1979; L i s t e r and Rochow 1979; Marco and Cohen 1979; Tresh et al. 1977) and phyto-pathogenic b a c t e r i a (Vruggink 1978). PBS+T+PVP plus 0.2% egg albumin (PBS+T+-PVP+EA) has been used with plant viruses (Gugerli and Gehringer 1980) and phytopathogenic b a c t e r i a (Stevens and Tsiantos 1979). Many of these same buffers have also been employed with the conjugate preparations. Phosphate buffered s a l i n e (PBS) (Birch et a l . 1979) and PBS+T have been widely used (Bullock and Walls 1977; Crook and Payne 1980; Russell et a l . 1976; V o l l e r et - 45 -a l . 1976), PBS+T+PVP (Nachmias et a l . 1979) and PBS+T+EA (Vruggink 1978) have also been used. The objectives of t h i s work were to determine the r e l a t i v e importance of well to well v a r i a t i o n i n m i c r o t i t r a t i o n plates, of d i f f e r e n t sample and conjugate buffers and of pipetting errors as sources of v a r i a b i l i t y in an ELISA for the detection of Erwinia carotovora subsp. atroseptica. - 46 -MATERIALS AND METHODS Ba c t e r i a l Cultures, Antiserum Production, y -globulin P u r i f i c a t i o n , and Alka- l i n e Phosphatase-^ -glo b u l i n Conjugation Potato s t r a i n s (E82, E193) of Erwinia carotovora subsp. atroseptica conforming to Serogroup I (DeBoer et ail. 1979) were employed throughout t h i s study. Stock cultures were maintained on Difco Nutrient Agar (NA) slants at 4 C. Unless otherwise noted, c e l l suspension from cultures grown for 48 h on NA at 27 C were employed i n a l l t e s t s . Antiserum against glutaraldehyde-fixed whole c e l l s of s t r a i n E82 was prepared i n rabbit by the procedure of Al l a n and Kelman (1977). Crude a n t i -serum was p a r t i a l l y p u r i f i e d by saturated ammonium s u l f a t e p r e c i p i t a t i o n and passage through a DEAE-22 Sephadex column p r i o r to conjugation with a l k a l i n e phosphatase (Chapter 1). Basic ELISA Procedure The basic procedure employed was i d e n t i c a l to that previously described (Chapter 1) unless indicated otherwise. The optimum coating y-globulin concentration of 2.0ug/ml and conjugation d i l u t i o n of 1:400 were employed i n a l l experiments. Plates were washed with d i s t i l l e d water for 15 sec at 34.48 kPa (5 p s i ) , rotated 180° on the washing devise and washed a second time. Plates were dried by two 5-sec applications of compressed a i r (medical grade) at 172.38 kPa (25 p s i ) . The enzyme substrate reaction was allowed to proceed for 30 minutes at room temperature. A q u a l i t a t i v e estimate of color development was made and plates were read spectrophotometrically at 405 nm with a T i t e r t e k Multiskan plate reader c a l i b r a t e d at the time of substrate addition. An absorbance - 47 -sample r a t i o ( ) >^  2 was considered a p o s i t i v e r e s u l t . A+Q5 cont r o l Plate Uniformity To determine the well to well v a r i a t i o n in the m i c r o t i t r a t i o n plates, a l l 96 wells in each of six plates received the same treatment. A suspension of 105 c e l l s / m l of Erwinia carotovora subsp. atroseptica s t r a i n E193 was employed. Samples were d i s t r i b u t e d in the wells using a 8 channel Multi Channel P i p e t t e r . The plates were read spectrophotometrically after 30, 45 and 60 min. B a c t e r i a l d i l u t i o n series ranging from 103 to 108 c e l l s / m l of s t r a i n s E193 and E82 were prepared. Each d i l u t i o n of each s t r a i n was r e p l i c a t e d six times on one m i c r o t i t r a t i o n plate, (84 wells) and compared to the sample buffer c o n t r o l . The reaction was assessed immediately a f t e r addition of the substrate and every 5 minutes thereafter. E f f e c t of Pipetting Errors To determine whether pi p e t t i n g errors due to t i p replacement affected ELISA, the standard assay was employed but coating, sample and conjugate were added 10 w e l l s / t i p , 5 w e l l s / t i p or 1 w e l l / t i p . One m i c r o t i t r a t i o n plate was employed. Columns 1 and 12 received only the buffers, and the substrate. A b a c t e r i a l suspension of 106 c e l l s / m l of s t r a i n E193 was employed. Each seri e s of wells was r e p l i c a t e d f i v e times. To determine the e f f e c t of p i p e t t i n g errors at d i f f e r e n t stages of the assay, an a d d i t i o n a l 10 u1 of coating, sample or conjugate was added i n d i v i -- 48 -dually to wells which had already received normal coating, sample or conju-gate. One m i c r o t i t r a t i o n plate was employed. Columns 1 and 12 received only the buffers, and the substrate. A b a c t e r i a l suspension of 105 c e l l s / m l ( s t r a i n E193) was used. Each combination coating, sample and conjugate was re p l i c a t e d 10 times. E f f e c t of Dif f e r e n t Buffers on ELISA To determine the e f f e c t of the sample buffer on ELISA, the standard ELISA procedure was followed, except that b a c t e r i a l suspensions of 105 c e l l s / ml ( s t r a i n E193) were made up in d i f f e r e n t buffers. A b a c t e r i a l suspension adjusted to A540 =0.1 (10 8 cells/ml) was prepared i n s t e r i l i z e d d i s t i l l e d water, and d i l u t e d to 105 c e l l s / m l i n each of the following: d i s t i l l e d water (DW); phosphate buffered s a l i n e (PBS); PBS + 0.05% Tween-20 (polyoxyethylene (20) sorbitan monolaurate, Fisher S c i e n t i f i c ) (PBS + T); PBS + 2.0% p o l y v i n y l -pyrrolidone (M.W. approx. 44000, BDH Chemicals) (PBS+PVP); PBS + 0.2% egg albumin (Egg albumin (ovalbumin) Grade I I I , Sigma, No-5378) (PBS+EA); PBS+T+ PVP; PBS+T+EA; PBS+PVP+EA; and PBS+T+PVP+EA. A l l treatments were run on one plat e . Columns 1 and 12 and Row A from 1 to 5, and Row H from 8 to 12, received only DW and the substrate. Each treatment was r e p l i c a t e d eight times. S i m i l a r l y , to determine the e f f e c t of the conjugate buffer on ELISA, the standard procedure was followed, except that conjugate d i l u t i o n s of 1:400 were prepared i n the d i f f e r e n t buffers described above. The buffers employed, and the plate design were described above. A b a c t e r i a l suspension of 106 c e l l s / m l ( s t r a i n E193) i n PBS+T+PVP+EA was employed. A comparison of i n d i v i d u a l buffers used for both sample and conjugate preparation, was undertaken using a modified standard procedure. In - 49 -preliminary experiments only b a c t e r i a l suspensions of 10b c e l l s / m l ( s t r a i n E193) and conjugate d i l u t i o n s of 1:400 were made up in the buffer series l i s t e d above. An i n d i v i d u a l treatment consisted of the same buffer being used for both sample and conjugate preparations. In l a t e r experiments a b a c t e r i a l concentration s e r i e s ranging from 103 to 107 c e l l s / m l were employed with the appropriate buffer only c o n t r o l . Only 54 wells from each of six m i c r o t i t r a -t i o n plates were employed. The outside wells, and wells from Row B (1 - 4) and G (9-12) received only DW and the substrate. Each treatment was r e p l i -cated six times, once per plate. S t a t i s t i c a l Analysis In each experiment, a l l treatments or combinations of treatments on one plate, were completely randomized. In some experiments, Tukey's multiple range t e s t was performed on the A+ 0 5 values uncorrected for t h e i r controls with a 5.0% s i g n i f i c a n c e l e v e l for the F-value. - 50 -RESULTS Plate Uniformity When a l l wells i n six plates were treated i d e n t i c a l l y , the \ Q 5 values within plates and between plates d i f f e r e d considerably. To determine whether there was any pattern to t h i s v a r i a t i o n an acceptable range around the average value equal to ± 10% of the mean was a r b i t r a r i l y established for each plate ( F i g . 1). Some plates showed more uniformity (plates three and six) than others (plates four and five) a f t e r a 30 min reaction time. Values f a l l i n g below the acceptable range were concentrated i n the middle part of plates one and two, were located more on the l e f t side of plates three and four, and were generally dispersed on plates f i v e and s i x . Values above the acceptable range were concentrated i n the bottom t h i r d of plates four and f i v e , the top t h i r d of plates one and s i x , and more generally d i s t r i b u t e d on plates two and three. Values both above and below the range were observed i n the outside rows, and were more numerous in some plates (plates four and fi v e ) than others (plates one and s i x ) . Both types of values were frequently found i n the same column or row. The number of wells with values greater or smaller than the 10% l i m i t of acceptable v a r i a b i l i t y decreased as the reaction time increased. The number of wells outside the acceptable range on plate one went from 31 a f t e r 30 min, to 20 after 45 min, to 11 after 60 min. Similar decreases in the number of wells were observed with the other plates. As reaction time increased, the d i s t r i b u t i o n of these wells outside the acceptable range changed. Some wells previously within the ±10% range of acceptable v a r i a b i l -i t y were now outside, where others previously outside were now within. For - 51 -TIMES (minutes) 30 5 5 3 0 5 3 • PP • rj 3 • • - U U L C X U f i B U • • J U K J K J U L J M I I | 3 J 45 0 e i • c> 3 • S ft ft • • • i a 5 60 5 0 J ft 3 3 ft • ft ft ft 4 Si 11 ppi P T T P — wn 1*1: 3 3 0 ft ft •LU •LU •SU ft 3 a MMJ * 3 • 3 3 • 3 3 ft 3 J | J J 3 3 3 J • • C 0 • • mu J 1 3 • • * Jl 3 3 M 1 J 1 mm mm L M 3 3 A u C l i 5 D * 3 _ P * # 5 3 3 3 0 3 • 3 3 3 3 3 ft • 3 3 * 3 ft 3 3 3 14 • • 0 1 c 3 S C 3 C c; • 1 m • • • 3 a 0 U c 1 . J I I 1 1 ft 3 5 3 • ? > 1 ft J S M > » M " 3 3 « H J 1 3 m 3 j o 3 l j ft * D 3 3 3 3 J ; J j A B C ? 0 e 5 0 0 3 ft ft • 3 i • • ft • • • f 8 ft i d c • 3 0 ft ft am 3 3 C 3 ft am ft I au 3 urn 3 3 r 3 ft 3 3 ft 1 ft 5 3 3 3 » ft 3 ft * ft ft 3 ft 3 0 Figure 1. V a r i a b i l i t y in \ 0 5 values obtained at three reaction times in an ELISA optimized for detection of Erwinia carotovora subsp. atrosep-t i c a . A l l wells received 106 c e l l s / m l (0 > mean plus 10% of mean, • < mean minus 10% of the mean). - 52 -example on plate one ( F i g . 1) wells D-5, H-6 and G-11 were within the accep-table range af t e r 30 min but were outside i t after 45 min. Conversely wells A-1, E-3, C-4, A-5, C-5, D-6, A-7, E-7, F-8, F-9, F-10, H-11, B-12 and C-12 f e l l outside the range at 30 min but within i t aft e r 45 min. After 60 min a l l of the above wells plus several others (F-3, C-5, E-5, C-6, E-8, E-9 and H-9) were within the range. If the acceptable range was increased from ± 5% to ± 10% of the mean or to ± 15% of the mean, the number of wells f a l l i n g outside the range decreased ( F i g . 2). The combination of the largest range and the longest incubation period gave a "uniform" plate in one instance but not the other. In another experiment the maximum percent v a r i a t i o n from the mean was determined for A+os values read at 5 min i n t e r v a l s . Regardless of s t r a i n or b a c t e r i a l c e l l concentration, the maximum percent v a r i a t i o n decreased with time as the A+QJ mean value increased ( F i g . 3). After about 20 min at most b a c t e r i a l c e l l concentrations, the maximum percent v a r i a t i o n tended to " l e v e l -o f f " . An examination of the raw data revealed that a d i f f e r e n t r e p l i c a t e was usually responsible for the maximum v a r i a t i o n at successive readings, suggest-ing that well to well v a r i a b i l i t y was again the causal f a c t o r . The lowest percent maximum v a r i a t i o n was associated with treatments giving the highest A+0 5 mean values and vice versa. The maximum percent v a r i a t i o n observed af t e r 30 min at the l i m i t of detection (10 5 cells/ml) f o r t h i s experiment was 16.0% for both s t r a i n s . E f f e c t of Pip e t t i n g Errors Retention of l i q u i d in the pipette t i p was often observed when several wells were f i l l e d with the same t i p . This retention appeared to add to the - 53 -30 PLATE 1 TIMES (minutes) 45 Q1 35 21 S21SJBSBB SSBBBBU S l B J J J B J •JBSSiBBTJ J J B J B L 1 J J M S M B L J B BBBSlB'arti 33 3a 30 10% \ 5 3 3 3 3 5 3 b C 1 i • • 3 D 3 j E F B B L J L H J J U J I J B B J B B I X J B U B J J ' J B B G l 1 I 4 | LI J r> 0 • 1 B B B J J J B U J B B J B B J J B J B B B B B B 11 111 N 1 U H I 3 3 3 1 3 J 1 1 1 1 1 o < 3 15%' 3 1 | • B B J J J J U J • B • _ | z o D C < > PLATE 2 5% I F lafflUJUBISBBiSi j a a a a B B B K B M J •MMBBBiBlKIKSJSU B J J B J J B B 5 3 °c •••1KB 1 1 3 ' f iSBI 3 , i . • j B s i « i a 3 i J J B J J • ft » ii • J B B B J ft » BBBBJ 1 -:R I •««]«* L-1 B B B J J J B B B B » B _ J K a B W * ] J B M H S B B S S J f f l S B M M S l B B J B B B B J B J J J B U J B H J S B J B S J B B B J J B _ 3 22 51 F ft C D B E E B J S J J B L J U L U J U U B J U J B U U B B J B B B J L ^ S B • I B 3 a 3 J 3 3 • 1 B J J • • J •BJ ! J MM ! • 3 • 3 0 B 3 3 3 » 3 J 1 J • J 3 J J 15% 3 3 3 D 3 3 V • » • • 1 • ft 3 J a • A • • j j • j • j • • • a B B 3 • J 3 J 3 J J J B F i g . 2. Well to well v a r i a b i l i t y i n \ 0 5 values obtained at d i f f e r e n t reaction times when a l l wells received 106 c e l l s / m l in an ELISA optimized for Erwinia carotovora subsp. atroseptica (0 > mean + indicated % of mean; • < mean - indicated % of mean). - 54 -c F i g . 3. Maximum percent v a r i a t i o n from the mean A+05 values obtained at 5-min i n t e r v a l s for known b a c t e r i a l concentrations of s t r a i n s E82 and E193 in an ELISA optimized for detection of Erwinia carotovora subsp. atr o s e p t i c a . - 55 -v a r i a b i l i t y only when 10 wells were f i l l e d from the same t i p (Table 1). The well to well v a r i a t i o n previously noted would account for the v a r i a b i l i t y between wells f i l l e d with separate t i p s and between wells f i l l e d i n groups of f i v e with the same t i p . This i n t e r p r e t a t i o n was supported by the fact that i experiments where deliberate p i p e t t i n g errors of 5% (V/V) were made at a l l possible points i n the assay there were no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r -ences obtained between the A+05 values (Table 2). Ef f e c t of Di f f e r e n t Buffers on ELISA Preliminary studies (Table 3) on the e f f e c t on A+05 of d i f f e r e n t buffer employed for sample preparation or conjugate d i l u t i o n alone or for both simul taneously, c l e a r l y showed that there were d i f f e r e n c e s . Not only was there a buffer requirement for sample and conjugate preparation but there were also s i g n i f i c a n t differences between the buffers employed. D i s t i l l e d water as a sample diluent resulted i n s i g n i f i c a n t l y lower mean A+os values than a l l of the other buffers. Inclusion of egg albumin i n both PBS and PBS+T as the sample diluent resulted i n higher, but not s i g n i f i c a n t l y higher, mean A+05 values. By contrast, i n c l u s i o n of PVP reduced mean \ 0 5 values and in one case the reduction was s i g n i f i c a n t . The standard buffer (PBS+T+PVP+EA) included for comparison had the fourth highest mean A+os value but was not s i g n i f i c a n t l y d i f f e r e n t from the f i r s t three. D i s t i l l e d water also gave the lowest A+05 "lean value when employed as the conjugate diluent, but was not s i g n i f i c a n t l y d i f f e r e n t from PBS+T, and PBS+T+PVP. By contrast, PBS and PBS+ PVP as conjugate diluents gave mean A+05 values which were s i g n i f i c a n t l y higher than a l l other buffers including the standard PBS+T+PVP+EA. Again d is t i l l e d water had the lowest mean A+05 value when i t was used to prepare both the sample and the conjugate. However, i t was not s i g n i f i c a n t l y d i f f e r e n t - 56 -Table 1. Ef f e c t of pipetting error on mean \ 0 5 values obtained with 106 c e l l s / m l i n an ELISA optimized for Erwinia carotovora subsp. atro- septica Wells Mean Highest % v a r i a t i o n from mean % v a r i a t i o n of the set f i l l e d / t i p A+os observed within a set from mean of a l l sets 10 0.382 14.1 8.2 10 0.376 15.7 6.5 10 0.367 7.4 4.0 10 0.307 25.7 13.0 10 0.332 25.6 5.9 5 0.343 8.5 0.3 5 0.358 10.9 4.7 5 0.312 6.4 8.8 5 0.345 7.0 0.9 5 0.353 8.5 3.2 ! 0.360 - 3.7 1 0.347 0.0 1 0.310 10.7 1 0.379 9.2 1 0.340 2.0 - 57 -Table 2. Ef f e c t of 10 u l of additional coating, sample and/or conjugate on the mean A+os values obtained with 10 c e l l s / m l i n an ELISA optimized for Erwinia carotovora subsp. atroseptica Coating Sample Conjugate \o5*** -* - - 0.352 a +** - - 0.361 a + - 0.358 a + 0.346 a + + - 0.366 a + + 0.363 a + - + 0.368 a + + + 0.365 a * - normal treatment ** + received an addit i o n a l 10 u1 *** Means (of 10 r e p l i c a t e s ) followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P=0.05) according to Tukey's multiple range test - 58 -from several other buffers. The r e l a t i v e differences between the mean \Q5 values when the same buffer was used for both sample and conjugate preparation were very s i m i l a r to those obtained when the buffers were used only for the conjugate preparation. When the experiment involving common buffers for sample and conjugate preparation was repeated at several b a c t e r i a l concentrations with proper controls (Table 4), the use of PBS, and PBS+PVP resulted i n extremely high mean A+os values for the controls as well as the samples after 30 min. As a consequence, the absorbance r a t i o s (AR) for both treatments were < 2.0 i n spite of the extremely high mean A+05 values, emphasizing the importance of the background i n t h i s assay. S i m i l a r l y , d i s t i l l e d water had a high back-ground r e l a t i v e to the mean A+os values obtained at the d i f f e r e n t b a c t e r i a l concentrations which prevented i t s use. At the l i m i t s of detection (10 5 cells/ml) only buffers containing egg albumin were v i s u a l l y p o s i t i v e after 30 min, and a l l had AR > 2.0 with the exception of PBS+EA (AR = 1.9). Although the mean A+05 values d i f f e r e d , the differences were not s i g n i f i c a n t , so that none represented an improvement upon the complete buffer PBS+T+PVP+EA. Both buffer solutions containing Tween-20 but not egg albumin had AR > 2.0 but were not v i s u a l l y p o s i t i v e at 105 c e l l s / m l . Because the c r i t e r i a for a p o s i t i v e r e s u l t was an AR > 2.0 and a p o s i t i v e v i s u a l reading after 30 min, the l i m i t of detection was considered to be 106 c e l l s / m l for these treatments. - 59 -Table 3. Comparison of the e f f e c t of d i f f e r e n t sample and/or conjugate buffers on absorbance (A+05) obtained with 106 c e l l s / m l i n an ELISA optimized for detection of Erwinia carotovora subsp. atroseptica Diluent used for preparation of Both sample & Sample only Conjugate only conjugate Diluent A+os Rank A+05 Rank A^os Rank DW* 0 .277 f ** 9 0 .110 e 9 0 .217 d 6 PBS 0 .372 bcde 5 0 .775 a 1 1 .012 a 1 PBS+PVP 0 .345 de 7 0 .709 a 2 0 .788 b 2 PBS+EA 0 .399 abc 3 0 .291 be 4 0 .241 cd 4 PBS+PVP+EA 0 .335 e 8 0 .244 cd 5 0 .198 d 9 PBS+T 0 .418 ab 2 0 .182 de 8 0 .199 d 8 PBS+T+PVP 0 .357 cde 6 0 .185 de 7 0 .202 d 7 PBS+T+EA 0 .429 a 1 0 .203 d 6 0 .226 cd 5 PBS+T+PVP+EA*** 0 .392 abed 4 0 .361 b 3 0 .390 c 3 *DW = d i s t i l l e d water; PBS = phosphate buffered s a l i n e ; PVP = p o l y v i n y l -pyrrolidone; EA = egg albumin; T = Tween-20 **Means (of 8 r e p l i c a t e s ) sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P=0.05) according to Tukey's multiple range t e s t . ***Buffer employed i n standard procedure. Table 4 . E f f e c t of b u f f e r s used f o r sample and conjugate d i l u t i o n on absorbance (^5) a n d absorbance r a t i o (AR) ob ta ined w i th known c e l l c o n c e n t r a t i o n s in an ELISA o p t i m i z e d f o r E rw in ia carotovora subsp. a t r o s e p t i c a B a c t e r i a l c o n c e n t r a t i o n ( c e l l s / m l ) 10 7 10 6 10 5 IO4 10 3 0 AR ^05 AR ^ 0 5 AR ^05 AR ^05 AR ^05 DW* 0.212 k** 1.3 0 .138 1 0 . 9 0 .122 1 0 .8 0.137 1 0 .9 0 .182 k 1.1 0 .161 k l * * * PBS 1.604 be 1.5 1.269 efgh 1.2 1.011 i 0 .9 0 .965 h i 0 .9 0 .945 i 0 . 9 1.085 h i PBS+PVP 1.951 1.9 1.475 cdef 1.5 1.250 g 1.2 1.326 defgh 1.3 1.122 ghi 1.1 1.008 f g h i PBS+EA 1.435 cdef 21.4 0 .506 j 7 .6 0 .125 1 1.9 0.061 0 . 9 0 .055 0 .8 0.067 1 PBS+PVP+EA 1.524 bede 40.1 0 . 5 0 6 j 14.7 0.177 kl 4 .7 0 .065 1.7 0 .063 1.7 0.038 1 PBS+T 1.468 cdef 50.6 0 .414 j k 14.3 0.077 1 2.7 0.038 1.3 0 .024 « 0.8 0.029 1 PBS+T+PVP 1.483 cdef 4 4 . 9 0.421 j k 12.8 0 .085 1 2.6 0 .043 1.3 0 . 0 3 0 0 . 9 • 0 .033 1 PBS+T+EA 1.763 ab 55.1 0 .637 j 19.9 0.121 I 3 .8 0 .052 1.6 0.029 0 . 9 0 .032 1 PBS+T+PVP+EA 1.576 bed 38 .4 0 . 4 8 2 j 11.8 0 .092 1 2 .2 0.051 1 1.2 0 .029 1 0.7 0.041 1 *DW = d i s t i l l e d water ; PBS = phosphate bu f fe red s a l i n e ; PVP = p o l y v i n y l p y r r o l i d o n e ; EA = egg a lbumin ; T = Tween-20. **Means (of s i x r e p l i c a t e s ) s h a r i n g t h e same l e t t e r a r e not s i g n i f i c a n t l y d i f f e r e n t (P=0.05) accord ing t o Tukey's m u l t i p l e range t e s t . ***L imi t of v i s u a l d e t e c t i o n . - 61 -DISCUSSION The well to well v a r i a b i l i t y associated with the m i c r o t i t r a t i o n plates was found to be the major source of v a r i a t i o n in the detection of Erwinia  carotovora subsp. atroseptica by ELISA, confirming previous observations of Clark and Adams (1977), Denmark and Chessum (1978), and McMurray and Blanch-flower (1979). The degree of v a r i a b i l i t y was shown to be a function of each i n d i v i d u a l plate (Figure 1). Outside rows did not give mainly higher values as reported by Denmark and Chessum (1978). Moreover, values outside the a r b i t r a r i l y set acceptable l i m i t s of v a r i a b i l i t y were found not only in the outside rows, but anywhere on the plates. This v a r i a b i l i t y was also a func-ti o n of reaction time because the pattern was constantly changing on a plate. In addition, these changes occurred i n both d i r e c t i o n s . When increasingly larger acceptable l e v e l s of v a r i a b i l i t y were applied, a decrease in the number of wells outside the acceptable l i m i t s was obtained for each plate. Increasing the reaction time at any one l e v e l of v a r i a b i l i t y also reduced the number of wells outside the l i m i t s . At the normal reaction time of 30 min, even with the ±15% l i m i t of v a r i a b i l i t y from the mean, some wells s t i l l remained outside. The use of even longer reaction times did reduce the number of wells outside the ±15% l i m i t s , but the controls became p o s i t i v e v i s u a l l y a f t e r 30 min, which i s important in l a b o r a t o r i e s where only a v i s u a l estimate i s possible. The maximum percent v a r i a t i o n from the mean varied with time and to a lesser extent with the concentration of b a c t e r i a l c e l l s employed. Greater percent v a r i a t i o n was associated with low b a c t e r i a l c e l l concentrations and vice versa. Such a r e s u l t i s consistent with the f a c t that a small mean value i s used for comparison i n the f i r s t instance and large one i n the second. The - 62 -decrease with time was expected because the means were increasing as the reaction proceeded and color development i n t e n s i f i e d . What i s not r e a d i l y explainable i s why the maximum percent v a r i a t i o n appeared to decrease less r a p i d l y or l e v e l - o f f after about 20 min. The source of the v a r i a t i o n i s not representable by a constant A+ 05 value which decreases in r e l a t i o n to an increasing mean. Instead i t appears to be a c e r t a i n proportion of the ever changing A+QS values associated with each d i f f e r e n t b a c t e r i a l c e l l concentra-t i o n . I f increased maximum v a r i a b i l i t y i s somehow associated with an a n t i -serum excess which i s i n e v i t a b l e at the l i m i t s of s e n s i t i v i t y , the cause of the v a r i a b i l i t y must be i d e n t i f i e d and eliminated by means that remain to be determined i f the p o t e n t i a l of the ELISA i s to be f u l l y r e a l i z e d . At a b a c t e r i a l concentration of 106 c e l l s / m l , the maximum percentage v a r i a b i l i t y observed for both b a c t e r i a l s t r a i n s a f t e r a 30-min reaction time was 16%. Other workers have reported v a r i a b i l i t y in the order of 17% for four readings (Bullock and Walls 1977), 10% for duplicates (Carlsson et a l . 1972), less than 10% using duplicate samples over a li m i t e d antigen range (Clark 1981), 10 to 20% (Carlsson et _ a l . : 1975), and plus or minus 9% from the mean of t r i p l i c a t e s after correction was made for the background (Engvall et a l . 1971). Thus the v a r i a b i l i t y reported i n these experiments i s t y p i c a l and not peculiar to the Erwinia carotovora subsp. atroseptica system. While conceding that the outside rows were indeed va r i a b l e , avoiding t h e i r use in an experiment design seems unwarranted, because si m i l a r v a r i a b i l -i t y i s also common in other sections of the plate. Replication can reduce the e f f e c t s of v a r i a b i l i t y . If more than one plate i s necessary for a t e s t , i t i s e s s e n t i a l that each plate have i t s own co n t r o l , and that the number of r e p l i -cates be evenly d i s t r i b u t e d between the plates to minimize both within and - 63 -between plate v a r i a b i l i t y . I f the appropriate design precautions are taken, the number of plates to be used, should not be considered a l i m i t a t i o n where pre c i s i o n i s needed. Investigation of p i p e t t i n g errors as a probable source of v a r i a b i l i t y showed that i t i s rather d i f f i c u l t to d i s s o c i a t e the v a r i a b i l i t y r e s u l t i n g from the repeated use of one t i p , from the well to well v a r i a b i l i t y of the m i c r o t i t r a t i o n plate. The p r o b a b i l i t y of an "odd" well being included i n a 10-well s e r i e s i s greater than that i n a 5-well s e r i e s . Furthermore, the v a r i a b i l i t y obtained when only one well i s f i l l e d per t i p could be a t t r i b u t e d to either a well or t i p e f f e c t . The f a i l u r e to reproduce the observed v a r i a -b i l i t y by d e l i b e r a t e p i p e t t i n g errors equal to 5%, supports the i n t e r p r e t a t i o n that well v a r i a t i o n not p i p e t t i n g errors are responsible. The benefits of sample preparation i n a s u i t a b l e buffer are obvious because of the differences which were demonstrated i n t h i s work. High A+os values were obtained i n the Erwinia carotovora subsp. atroseptica ELISA only when PBS + 0.05% Tween-20 was employed for sample preparation, presumably because the Tween-20 prevented nonspecific adsorption (Bullock and Walls: 1977, Clark and Adams: 1977, Engvall and Perlmann: 1972, Yolken et a l . : 1977). Inclusion of egg albumin which also prevents nonspecific retention of antibodies (Gugerli and Gehriger: 1980), further increased t h i s e f f e c t although not s i g n i f i c a n t l y . The complete buffer frequently used (PBS+T+PVP+ EA) gave a lower mean A+05 value which would appear to be due to the PVP component. Clark and Adams (1977) also observed that PVP decresed s e n s i t i v -i t y . PVP probably could be omitted i n further studies involving cultured c e l l s because i t s r o l e i s to remove phenolic compounds present i n plant sap which may a f f e c t antigen and antibody s t a b i l i t y . - 64 -The need for buffering capacity and reagents preventing nonspecific absorption i n the diluent for pure cultures was shown by the lower mean A405 values obtained when c e l l suspensions were made up i n d i s t i l l e d water. Whether s i m i l a r reductions i n A+os values would be observed i f samples were prepared from infected plants remains to be determined. Cother and Vruggink (1980) and Vruggink (1978) have reported detecting F_. carotovora subsp. atro- septica i n potatoes by adding a peel extract d i r e c t l y to the wells. Unfortun-ately no comparisons were made with buffered suspensions of pure c u l t u r e s . Thus, the e f f e c t of plant sap on the detection of t h i s bacterium by ELISA remains to be determined. If the same ef f e c t occurs i n t h i s system as has been observed i n plant v i r u s assays (Clark and Adams 1977; Flegg and Clark 1979), a reduction i n s e n s i t i v i t y can be expected. When d i f f e r e n t buffers were used only i n the preparation of the conju-gate d i l u t i o n s or for the preparation of both the samples and conjugates, the same r e l a t i v e d ifferences between the d i f f e r e n t buffers were obtained. This suggests that the buffer has a greater e f f e c t on ELISA when i t i s the diluent for the conjugate rather than for the sample. This also suggests that the requirements for binding of b a c t e r i a l c e l l s to coating y -globulin may be d i f f e r e n t from these for the binding of enzyme-y -g l o b u l i n to the b a c t e r i a l c e l l s . The use of buffers not containing either Tween-20 or egg albumin re s u l t e d i n high backgrounds which were presumably due to nonspecific adsorption. The i n c l u s i o n of egg albumin i n the sample and conjugate buffer i s important i n the E. carotovora subsp. atroseptica ELISA, as only those buffers containing i t were v i s u a l l y p o s i t i v e after 30 min and had absorbance r a t i o s > 2.0 (with one borderline exception). Based on these r e s u l t s with b a c t e r i a l c e l l s grown in culture, there seems to be no compelling reason to - 65 -adopt a d i f f e r e n t buffer system than that currently used i n plant virus ELISA systems. A s i m i l a r study should be done with b a c t e r i a l c e l l s i n plant sap, to determine whether the complete buffer (PBS+T+PVP+EA) i s also optimal when plant t i s s u e extracts are involved. However, the p o s s i b i l i t y e x i s t s that the high well to well v a r i a t i o n observed i n t h i s study masked buffer e f f e c t s . I f the new, improved m i c r o t i t r a t i o n plates which became a v a i l a b l e after t h i s work was completed are r e a l l y l e s s variable as reported (Clark 1981), a reevaluation of the e f f e c t of buffers and pipetting errors would be in order. - 66 -LITERATURE CITED ALLAN, E and A. KELMAN. 1977. Immunofluorescent s t a i n procedures for detection and i d e n t i f i c a t i o n of Erwinia carotovora var. atroseptica. Phytopathology 67:1305-1312. BAR-JOSEPH, M., S.M. GARNSEY, D. GONSALVES, M. M0SC0VITZ, D.E. PURCIFULL, M.F. CLARK and G. LOEBENSTEIN. 1979. The use of enzyme-linked immunosorbent assay for detection of c i t r u s t r i s t e z a v i r u s . Phytopathology 69:190-194. BERGER, J.A., S.N. MAY, L.R. BERGER and B.B. B0HL00L. 1979. Colorimetric enzyme-linked immunosorbent assay for the i d e n t i f i c a t i o n of s t r a i n s of Rhizobium i n culture and i n the nodules of l e n t i l s . Appl. and Environ. Mi c r o b i o l . 37:642-646. BIRCH, C.J., N.I. LEHMANN, A.J. HAWKER, J.A. MARSHALL and I.D. GUST. 1979. Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and i n d i r e c t immunofluorescence for detection of human rot a v i r u s antigen i n faeces. J. C l i n . Path. 32:700-705. BR0DEUR, B.R., F.E. ASHT0N and B.B. DIENA. 1978. Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae s p e c i f i c antibodies. Can. J. Micr o b i o l . 24:1300-1305. BULLOCK, S.L. and K.W. WALLS. 1977. Evaluation of some of the parameters of the enzyme linked immunospecific assay. J. Infec. Dis. 136 (Supplement):S279-S285. CARLSSON, H.E., B. HURVELL and A.A. LINDBERG. 1976. Enzyme-linked immunosor-bent assay (ELISA) for t i t r a t i o n of antibodies against Brucella abortus and Y e r s i n i a e n t e r o c o l i t i c a . Acta Path. Micr o b i o l . Scand. Sect. C. 84:168-176. CARLSSON, H.E., A.A. LINDBERG and S. HAMMARSTR0M. 1972. T i t r a t i o n of antibodies to Salmonella 0 antigen by enzyme-linked immunosorbent assay. Inf. Imm. 6:703-708. CARLSSON, H.E., A.A. LINDBERG, S. HAMMARSTR0M and A. LJUNGGREN. 1975. Quantitation of Salmonella 0-antibodies in human sera by enzyme-linked immunosorbent assay (ELISA). Int. Archs. Allergy Appl. Immun. 48:485-494. CLAFLIN, L.E. and J.K. UYEM0T0. 1978. Serodiagnosis of Corynebacterium  sepedonicum by enzyme-linked immunosorbent assay. Phytopathol. News 12:156. (Abstr.) CLARK, M.F. 1981. Immunosorbent assays i n plant pathology. Ann. Rev. Phytopathol. 19:83-106. CLARK, M.F. and A.N. ADAMS. 1977. C h a r a c t e r i s t i c s of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. V i r o l . 34:475-483. - 67 -COTHER, E.J. and H. VRUGGINK. 1980. Detection of viable and non viable c e l l s of Erwinia carotovora var. atroseptica in inoculated tubers of var. Bi n t j e with enzyme-linked immunosorbent assay (ELISA). Potato Res. 23:133-135. CROOK, N.E. and C C . PAYNE. 1980. Comparison of three methods of ELISA for Baculoviruses. 3. Gen. V i r o l . 46:29-37. DE BOER, S.H., R.J. COPEMAN and H. VRUGGINK. 1979. Serogroups of Erwinia  carotovora potato s t r a i n s determined with d i f f u s i b l e somatic antigens. Phytopathology 69:316-319. DENMARK, J.R. and B.S. CHESSUM. 1978. Standardization of enzyme-linked immunosorbent assay (ELISA) and the detection of Toxoplasma antibody. Med. Lab. Sc. 35:227-232. ENGVALL, E., K. J0NSS0N and P. PERLMANN. 1971. Enzyme-linked immunosorbent assay. II-Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim. Biophys. Acta. 251:427-434. ENGVALL, E. and P. PERLMANN. 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochem. 8:871-874. ENGVALL, E. and P. PERLMANN. 1972. Enzyme-linked immunosorbent assay, ELISA. I l l - Quantitation of s p e c i f i c antibodies in enzyme-labeled anti-immunoglobulin in antigen coated tubes. J. Immunol. 109:129-135. FLEGG, C L . and M.F. CLARK. 1979. The detection of apple c h l o r o t i c leafspot v i r u s by a modified procedure of enzyme-linked immunosorbent assay (ELISA). Ann. Appl. B i o l . 91:61-65. GUGERLI, P. and W. GEHRIGER. 1980. Enzyme-linked immunosorbent assay (ELISA) for the detection of potato l e a f r o l l v i r u s and potato v i r u s Y in potato tubers after a r t i f i c i a l break of dormancy. Potato Res. 23:353-359. KISHINEVSKY, B. and M. BAR-30SEPH. 1978. Rhizobium s t r a i n i d e n t i f i c a t i o n i n Arachis hypogaea nodules by enzyme-linked immunosorbent assay (ELISA). Can. 3. M i c r o b i o l . 24:1537-1543. LISTER, R.M. and W.F. R0CH0W. 1979. Detection of barley yellow dwarf v i r u s by enzyme-linked immunosorbent assay. Phytopathology 69:649-654. MARCO, S. and S. COHEN. 1979. Rapid detection and t i t e r evaluation of viruses in pepper by enzyme-linked immunosorbent assay. Phytopathology 69:1259-1262. MCMURRAY, C H . and W.J. BLANCHFLOWER. 1979. Multi-channel, probe colorimeter for use with the Micro-ELISA t e s t , which makes use of disposable flat-bottom microhemagglutination pl a t e s . C l i n . Chem. 25:570-576. NACHMIAS, A., M. BAR-JOSEPH, Z. S0LEL and I. BARASH. 1979. Diagnosis of Mai Secco disease in lemon by enzyme-linked immunosorbent assay. Phytopathology 69:559-561. - 68 -RUSSELL, H., R.R. FACKLAM and L.R. EDWARDS. 1976. Enzyme-linked immunosor-bent assay for streptococcal M protein antibodies. 3. C l i n . M i c r o b i o l . 3:501-505. STEVENS, W.A. and 3. TSIANTOS. 1979. The use of enzyme-linked immunosorbent assay (ELISA) for the detection of Corynebacterium michiganense in tomatoes. Microbios. L e t t . 10:29-32. TRESH, O.M., A.N. ADAMS, D.O. BARBARA and M.F. CLARK. 1977. The detection of three viruses of hop (Humulus lupulus) by enzyme-linked immunosorbent assay (ELISA). Ann. appl. B i o l . 87:57-65. VOLLER, A., D. BIDWELL and A. BARTLETT. 1976. Microplate enzyme immunoassays for the immunodiagnosis of virus i n f e c t i o n s , pp. 506-512. Jji N. Rose and H. Friedman, (ed). Manual of C l i n i c a l Immunology. American Soc. for Mic r o b i o l . VOLLER, A., D.E. BIDWELL and A. BARTLETT. 1977. The enzyme-linked immuno-sorbent assay. Dynatech Laboratories, Alexandria, V i r i g i n i a . 48 pp. VOLLER, A., D.E. BIDWELL and A. BARTLETT. 1979. The enzyme-linked immunosor-bent assay (ELISA). Dynatech Laboratories, Alexandria, V i r g i n i a . 48 pp. VRUGGINK, H. 1978. Enzyme-linked immunosorbent assay (ELISA) in the sero-diagnosis of plant pathogenic bacteria. Proc. 4th Int. Conf. Plant Path. Bact. Angers, pp. 307-310. WEAVER, W.M. and J.W. GUTHRIE. 1978. Enzyme-linked immunospecific assay ap p l i c a t i o n to the detection of seed borne ba c t e r i a . Phytopathol. News 12:156-157. (Abstr.) Y0LKEN, R.H., H.B. GREENBERG, M.H. MERS0N, R.B. SACK and A.Z. KAPIKIAN. 1977. Enzyme-linked immunosorbent assay for detection of Escherichia c o l i h e a t - l a b i l e enterotoxin. 0. C l i n . M i c r o b i o l . 6:439-444. - 69 -CHAPTER I I I . SPECIFICITY AND SENSITIVITY OF THE ERWINIA CAROTOVORA SUBSP. ATROSEPTICA (VAN HALL) DYE (SEROGROUP I) ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA). INTRODUCTION The enzyme-linked immunosorbent assay (ELISA) has been employed exten-s i v e l y in c l i n i c a l pathology to detect the presence of a wide range of organisms including protozoa, b a c t e r i a and viruses ( V o l l e r et al_. 1977). In plant pathology, the technique was adapted for virus detection (Clark and Adams 1977), and has been widely employed f o r a v a r i e t y of viruses (Bar-Joseph et a l . 1979; Flegg and Clark 1979; Gugerli 1979; Gugerli and Gehriger 1980; L i s t e r and Rochow 1979; Marco and Cohen 1979; Tamada and Harrison 1980; Tresh et a l . 1977). The detection of b a c t e r i a l c e l l s by ELISA has only r e l a t i v e l y recently been t r i e d . The medical l i t e r a t u r e contains numerous examples where b a c t e r i a l pathogens are being detected by ELISA, but detection i s based on detecting the antibodies in human sera rather than the bacteria per se (Brodeur et aL. 1978; Russel et _al. 1976; Carlsson j^t j i l . 1975, 1976; Holmgren and Svennerholm 1973; Ito et ^1. 1980). Thus, the adaptation of the technique for detection of b a c t e r i a l c e l l s has involved plant-pathogenic b a c t e r i a . Berger et al_. (1979) employed an i n d i r e c t ELISA to detect whole Rhizo- bium c e l l s from cultures and pooled nodule samples. They reported that c e l l s produced in broth culture which were washed to remove most of the e x t r a c e l l u -l a r polysaccharide slime, reacted q u a l i t a t i v e l y in the same manner as unwashed c e l l s from plant nodules where no slime was produced. They suggested that the - 70 -the e x t r a c e l l u l a r slime when present might i n t e r f e r e with the reaction. They also reported that weak cross reactions were obtained with heterologous a n t i -sera. Both Berger et ^1. (1979) and Kishinevsky and Bar-Joseph (1978), reported that heat treatment of the Rhizobium c e l l s improved the reaction i n ELISA. Furthermore, Kishinevsky and Bar-Joseph (1978) pointed out that the A+os v a l u e s the unheated c e l l suspensions were lower so that s t r a i n s were r e a d i l y detected only at high c e l l s concentrations (10 6 - 108 c e l l s / m l ) , compared to 101* - 105 c e l l s / m l for heated c e l l suspensions. They also reported that the s e r o l o g i c a l s p e c i f i c i t y of the Rhizobium s t r a i n s obtained with ELISA generally followed that observed i n the agglutination and immuno-d i f f u s i o n t e s t s . In the preliminary work with plant pathogenic bacteria, the technique has not shown much promise to t h i s point, e s p e c i a l l y with regard to the l e v e l of s e n s i t i v i t y . Weaver and Guthrie (1978) considered 10 - 10 c e l l s / m l a high l e v e l of s e n s i t i v i t y for Pseudomonas phaseolicola (Burkh.) Dawson, and reported cross reactions with other unspecified species. S i m i l a r l y Vruggink (1978) reported cross reactions between Xanthomonas pe l a r g o n i i (N.A. Brown) Starr & Burkh. (_X. campestris pv. pelargonii) and Aplanobacter populi with ELISA. Stevens and Tsiantos (1979) used ELISA to detect whole c e l l s of Corynebacterium michiganense (E.F. Sm.) Jensen both i n suspensions of known concentration and i n tomato plant extracts. They reported that bacteria from culture could be detected at 103 c e l l s / m l . However, t h e i r data do not support t h i s conclusion i f absorbance r a t i o s > 2.0 are used as the c r i t e r i o n for a p o s i t i v e t e s t . A l e v e l of 105 - 10s c e l l s / m l seems more appropriate from t h e i r data. C l a f l i n and Uyemoto (1978) detected whole c e l l s of Corynebacter- ium sepedonicum (Spieck. & Kotth.) Skapt. & Burkh. from infected stems and - 71 -tubers of potato and in suspension of c e l l s grown on a r t i f i c i a l media. Unfortunately no l e v e l of s e n s i t i v i t y was reported. No cross reactions were reported with species of Corynebacterium, Erwinia, Xanthomonas, Pseudomonas or Agrobacterium. The absence of a follow-up paper substantiating t h i s prelimin-ary work casts some doubt on the r e l i a b i l i t y of t h i s report. The only instance where ELISA has been used with some success i s with Erwinia carotovora subsp. atroseptica. Vruggink (1978), using the general procedure employed for plant virus detection, was able to detect t h i s bacter-ium in plant material with a y - g l o b u l i n coating concentration of 1 yg/ml and a conjugate d i l u t i o n of 1:400. He cautioned against the use of the technique f o r a new pathogen without determining s p e c i f i c i t y and reported that some F_. carotovora subsp. carotovora s t r a i n s reacted with E_. carotovora subsp. atro- septica i n ELISA. This caution i s even more relevant because DeBoer (1980) has recently shown that s e r o l o g i c a l r e l a t i o n s h i p s e x i s t among f l a g e l l a r and somatic antigens of several Erwinia carotovora serogroups. Cother and Vruggink (1980) have shown that both l i v e and dead c e l l s of Erwinia carotovora subsp. atroseptica reacted i n ELISA. They also noted that the lower l i m i t of detection of Erwinia carotovora subsp. atroseptica by ELISA was of lO 4 c e l l s / m l , but did not include any data supporting t h e i r conclusion. The ELISA technique as used to date for detecting whole b a c t e r i a l c e l l s has not generally shown the high degree of s e n s i t i v i t y and s p e c i f i c i t y asso-ciated with i t s use for plant viruses. Because of t h i s f a c t , the objectives of t h i s work were to determine the e f f e c t of culture medium, washing b a c t e r i a l c e l l s and heat treatment on the s e n s i t i v i t y and s p e c i f i c i t y of detection of Erwinia carotovora subsp. atroseptica by ELISA. A further objective was to determine whether the serogroups, distinguished by immunodiffusion, were detectable by ELISA. - 72 -MATERIALS AND METHODS Ba c t e r i a l Cultures, Antiserum Production, y -globulin P u r i f i c a t i o n , and  Alk a l i n e Phosphatase-y - g l o b u l i n Conjugation Previously i s o l a t e d potato s t r a i n s of Erwinia carotovora subsp. caro- tovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca) were employed throughout t h i s study. Unless indicated otherwise, b a c t e r i a l suspensions were prepared from 48-h cultures grown at 27 C on Nutrient Agar. Antiserum against glutaraldehyde-fixed whole c e l l s of s t r a i n E82 was prepared i n rabbit by the procedure of Allan and Kelman (1977). Crude a n t i -serum was p a r t i a l l y p u r i f i e d by saturated ammonium s u l f a t e p r e c i p i t a t i o n and passage through a DEAE-22 Sephadex column, p r i o r to conjugation with a l k a l i n e phosphatase (Chapter 1). Basic ELISA Procedure The basic procedure employed was i d e n t i c a l to that previously described (Chapter 1) unless indicated otherwise. The optimum coating y - g l o b u l i n con-centration of 2.0 ug/ml and conjugation d i l u t i o n of 1:400 were employed i n a l l experiments. Plates were washed with d i s t i l l e d water for 15 sec at 34.48 kPa (5 p s i ) , rotated 180° on the washing device and washed a second time. Plates were dried by two 5 second applications of medical a i r at 172.38 kPa (25 p s i ) . The enzyme substrate reaction was allowed to proceed for 30 minutes at room temperature. A q u a l i t a t i v e estimate of color development was made and plates were read spectrophotometrically at 405 nm with a T i t e r t e k Multiskan plate reader c a l i b r a t e d at the time of substrate addition. An absorbance A+os s a m p l e r a t i o ( ) >^  2.0 was considered a p o s i t i v e r e s u l t . A+05 control - 73 -E f f e c t s of Culture Conditions on ELISA To determine the e f f e c t of medium, and age of culture on ELISA, s t r a i n E82 and E193 of Erwinia carotovora subsp. atroseptica (Eca) conforming to serogroup I (DeBoer et a l . , 1979), were grown for 48 h at 27 C on f i v e d i f f e r -ent media: Difco Nutrient Agar (NA), Nutrient Sucrose Agar (NSA), (NA + 5% sucrose), Casamino Peptone Glucose agar (CPG) (Allan and Kelman 1977), King's Medium B (KMB) (King et a l . 1954) and Difco Potato Dextrose Agar (PDA). To ensure uniform growth, c e l l s of 48-h cultures on NA were used to inoculate the plat e s . Ten-fold d i l u t i o n s e r i e s i n phosphate buffered s a l i n e plus 0.05% Tween-20 (polyoxyethylene (20) sorbitan monolaurate, Fisher S c i e n t i f i c ) plus 2.0% polyvinylpyrrolidone (M.W. approx. 44000, BDH Chemicals) plus 0.2% egg albumin (Egg albumin (ovalbumin) Grade I I I , Sigma, No-5378) (PBS+T+PVP+EA) were prepared from 1& to 107 c e l l s / m l , with a control for each d i l u t i o n s e r i e s ( s t r a i n ) . Each treatment was r e p l i c a t e d six times, one r e p l i c a t e / p late. On each plate, the outside rows, and row B from 2 to 6, and row G from 7 to 11 received only the buffers and substrate. E f f e c t of Heat Treatment of B a c t e r i a l C e l l s on ELISA To determine the e f f e c t of heat treatment of the b a c t e r i a l c e l l s on ELISA, s t r a i n E331 (Serogroup I) of Eca was employed. A t e n - f o l d d i l u t i o n s e r i e s (103 to 107 cells/ml) was prepared and a sample buffer only control was included. Each c e l l concentration was further divided into 1.0 ml ali q u o t s . Four aliquots of each c e l l concentration were placed i n each of f i v e water baths adjusted to 21 C (room T), 40 C, 60 C, 80 C, and 100 C. One tube of each c e l l concentration at each temperature was removed af t e r 3, 6, 9, and 12 min heating. The samples were allowed to cool at room temperature. Each treatment combination of temperature and time was r e p l i c a t e d s i x times, one - 74 -r e p l i c a t e / b l o c k , where a b lock c o n s i s t e d of two p l a t e s . Twelve p l a t e s were employed, i n which the o u t s i d e rows r e c e i v e d the b u f f e r s and s u b s t r a t e o n l y . In a subsequent exper iment , temperatures of 21 C (room T) , 30 C, 40 C, 50 C, 60 C, 70 C, and 80 C were a p p l i e d fo r 3 and 6 min . A d i l u t i o n s e r i e s (10 3 t o 106 c e l l s / m l ) of s t r a i n E193 was s i m i l a r l y p repared , and d i v i d e d i n t o 2 .0 ml a l i q u o t s . A b u f f e r only c o n t r o l was s i m i l a r l y t r e a t e d . Each t reatment combinat ion of temperature , and t ime was r e p l i c a t e d s i x t i m e s , one r e p l i c a t e / p l a t e . On each p l a t e , columns 1 and 12, and row A from 2 to 6 , and row H from 7 to 11 r e c e i v e d only the b u f f e r s and s u b s t r a t e . E f f e c t o f Washing B a c t e r i a l C e l l s on the ELISA To determine the e f f e c t of washing b a c t e r i a l c e l l s on ELISA, c e l l s of s t r a i n E95 (Serogroup I I I ) o f Ecc and s t r a i n E193 (Serogroup I) of Eca , grown fo r 48 h at 27 C on NA were suspended i n PBS+T+PVP+EA. A sample of each was ad jus ted to /^i+o = 0.1 (10 8 c e l l s / m l ) , to serve as the normal , unwashed suspension (normal ) . The remain ing suspensions ( « 1 0 1 0 c e l l s / m l ) were mixed on a Vortex mixer f o r 30 s e c , and c e n t r i f u g e d at 2 .5 x 1& g f o r 10 min (Rotor No. 870; IEC R e f r i g e r a t e d C e n t r i f u g e Model B - 2 0 ) . The supernatants ( c e l l wash f l u i d s 1) were f i l t e r e d s t e r i l i z e d (0.22u) ( M i l l i p o r e C o r p o r a t i o n , Bedford , M a s s c h u s e t t s ) . The p e l l e t s were resuspended i n 3 . 0 ml of the sample b u f f e r and a p o r t i o n of each ad jus ted t o A51+ 0 = 0 . 1 to g i v e a once-washed c e l l s u s p e n s i o n . Th is washing s tep was s i m i l a r l y repeated on the remainder of each suspension t o g i v e c e l l wash f l u i d s 2 . A p o r t i o n of each tw ice -washed , p e l l e t was ad jus ted to /^i+g = 0 .1 to g i ve twice-washed c e l l s u s p e n s i o n s . A d i l u t i o n s e r i e s r e p r e s e n t i n g c o n c e n t r a t i o n s of 103 to 10 s c e l l s / m l was t e s t e d f o r each c e l l s u s p e n s i o n . A t e n - f o l d d i l u t i o n s e r i e s of each supernatant ( u n d i l u t e d t o 1 0 " 5 ) was s i m i l a r l y t e s t e d . Sample b u f f e r only c o n t r o l s were i n c l u d e d fo r - 75 -each d i l u t i o n s e r i e s . Each treatment was r e p l i c a t e d s i x times, once per pla t e . Columns 1 and 12 and row A from 2 to 6 and row H from 7 to 11 received only the buffers and substrate. To determine whether heat l a b i l e factors were responsible for the observed reactions, the same b a c t e r i a l c e l l washing procedure was repeated with s t r a i n E193. S e r i a l d i l u t i o n s of the supernatants (0 to 10-1* ) and of the resuspended washed c e l l s (103 to 107 cells/ml) were divided into two sets. One set was heated i n a water bath at 80 C for 15 minutes then allowed to cool back to room temperature. The control set was s i m i l a r l y treated but at 21 C. Each d i l u t i o n of the heat-treated supernatants and the washed c e l l s were i n d i v i d u a l l y tested i n the ELISA. Each treatment was r e p l i c a t e d s i x times, one r e p l i c a t e / p l a t e . The outside rows of each plate received the buffers and substrate only. In a subsequent experiment, s i m i l a r l y prepared supernatants and washed c e l l suspensions ( A ^ g - 0.1) of s t r a i n E193 were employed undiluted. A sample buffer control was included for each supernatant and c e l l suspension. A portion of each supernatant or c e l l suspension (3.0 ml) was autoclaved at 121 C for 90 minutes. Untreated samples were maintained at room temperature. Each treatment was tested i n d i v i d u a l l y with f i v e r e p l i -cates/plate. Columns 1 and 12 received only the buffers and substrate. S p e c i f i c i t y of ELISA to Dif f e r e n t Strains of Erwinia carotovora Representative b a c t e r i a l s t r a i n s of a l l 18 Erwinia carotovora serogroups determined by DeBoer et a l . (1979), and 6 new serogroups (S.H. DeBoer personal  communication) were employed. One s t r a i n of each of Pseudomonas marginalis (Brown) Stevens (PM6 - kindly supplied by Dr. A. Kelman, University of Wiscon-sin) and Corynebacterium sepedonicum were added. A l l s t r a i n s were grown for 48 hours at 27 C on NA, with the exception of C. sepedonicum which was grown - 76 -on NM medium (Katznelson and Sutton 1956) for 4 days at 27 C. B a c t e r i a l concentrations of 10s c e l l s / m l were employed, to maximize the detection of a reaction. A l l wells except those i n column 1 which received only the buffers and substrate, on two plates were employed. Each s t r a i n , and control buffer sample were r e p l i c a t e d three times on each plate. To determine the e f f e c t of heat treatment on s p e c i f i c i t y , varying concentration of only those s t r a i n s found to react i n the previous experiment were heat treated. A non reacting s t r a i n (S62) was included as a c o n t r o l . The s t r a i n s of Eca employed were: E193 (Serogroup I ) , E19 (Serogroup XVIII), E368 (Serogroup XX), and E555 (Serogroup XXII); while the Ecc s t r a i n s were: S21 (Serogroup I I ) , E95 (Serogroup I I I ) , E14 (Serogroup IV), S26 (Serogroup V), and S62 (Serogroup VI I I ) . A te n - f o l d d i l u t i o n s e r i e s (10 4 to 107 c e l l s / ml) and a buffer only co n t r o l were prepared for each s t r a i n . Aliquots (2.0 ml) of each d i l u t i o n s e r i e s were placed in water baths at 21 C (room T) and at 60 C for 5 min. After cooling at room temperature, each dilution-temperature combination was considered a treatment for t e s t i n g by ELISA. Each treatment was r e p l i c a t e d s i x times, 1 r e p l i c a t e per plate. On each plate, columns 1 and 12 received only the buffers and substrate. S t a t i s t i c a l Analysis In each experiment, a l l treatments or combination of treatments on one plate, were always completely randomized. In some experiments Tukey's multi-ple range t e s t was performed on the A+os values uncorrected for t h e i r controls, with a 5.0% s i g n i f i c a n c e l e v e l for the F-value. - 77 -RESULTS The culture medium upon which the bacteria were grown affected the A+05 mean values obtained when known concentrations of Eca were tested by ELISA (Table 1). With s t r a i n E193, c e l l s grown on NSA had a s i g n i f i c a n t l y higher A+os mean value at both 106 and 107 c e l l s / m l than c e l l s produced on a l l other media. By contrast, 10 c e l l s / m l of s t r a i n E82 grown on NA gave the highest A+05 m e a n value which was s i g n i f i c a n t l y d i f f e r e n t only from that of PDA, and KMB. However, at 10 c e l l s / m l the highest mean A+05 value was obtained with c e l l s grown on NSA instead of NA, although the difference between them was not s i g n i f i c a n t . Only mean A+os values obtained with c e l l s grown on NSA, and CPG were s t i l l s i g n i f i c a n t l y d i f f e r e n t from those on KMB. At lower b a c t e r i a l concentrations, with both s t r a i n s , there were no s i g n i f i c a n t differences between the mean A+05 values obtained with c e l l s grown on any of the media, although c e l l s grown on NSA consistently had the highest A+05 value with both s t r a i n s . P o s i t i v e v i s u a l estimates and AR > 2.0 were obtained with 105 c e l l s / m l of both s t r a i n s grown i n a l l of the media. At 1& c e l l s / m l , the v i s u a l estimates obtained with s t r a i n E193 were more va r i a b l e , and only c e l l s grown on NSA resul t e d i n an AR > 2.0. With the homologous s t r a i n E82, 1ft cel l s / m l grown on NA, NSA, and CPG resulted i n both a p o s i t i v e v i s u a l estimate and AR > 2.0. The mean A+05 values for s t r a i n E82 compared to those of s t r a i n E193 at 107 c e l l s / m l , were s i g n i f i c a n t l y greater when the c e l l s were grown on NA, CPG, and PDA but not on NSA or KMB. AT 106 c e l l s / m l , only c e l l s grown on CPG gave s i g n i f i c a n t l y higher mean values with s t r a i n E82. At let and 105 c e l l s / m l the mean A+05 values for both s t r a i n s grown on a l l media were not s t a t i s t i c a l l y d i f f e r e n t . Table 1. E f f e c t of culture media on absorbance (A+os ) and absorbance r a t i o (AR) obtained with known con-centrations of 48-h c e l l s of s t r a i n s E82 and E193 in an ELISA optimized for the detection of Erwinia carotovora subsp. atroseptica B a c t e r i a l concentrations (cells/ml) 107 106 105 10* Str a i n Medium +^05 AR +^05 AR +^05 AR +^05 AR E193 NA 1.025 c 22.3** 0.391 i j k l 8.5 0.142 mno 3.1 0.090 0 1.9 *** NSA 1.255 b 27.3 0.717 ef 15.6 0.234 klmno 5.1 0.096 0 2.1 CPG 0.975 cd 21.2 0.322 jklm 7.0 0.105 0 2.3 0.071 0 1.5 PDA 1.053 c 22.9 0.437 h i j 9.5 0.133 no 2.9 0.082 0 1.8 KMB 0.832 de 18.1 0.316 jklmn 6.8 0.110 0 2.4 0.073 0 1.6 E82 NA 1.545 a 33.6 0.524 ghi 11.4 0.179 mno 3.9 0.096 0 2.1 NSA 1.375 ab 29.9 0.694 efg 15.1 0.211 lmno 4.6 0.110 0 2.4 CPG 1.370 ab 29.8 0.598 fgh 13.0 0.197 mno 4.3 0.092 0 2.0 *** PDA 1.288 b 28.0 0.492 h i j 10.7 0.138 no 3.0 0.069 0 1.5 KMB 0.961 cd 20.9 0.395 i j k 8.6 0.133 no 2.9 0.067 0 1.4 *Means (of s i x r e p l i c a t e s ) sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P=0.05) according to Tukey's multiple range t e s t . **Absorbance r a t i o s based on control (mean of six re p l i c a t e s ) of A+os = 0.046. ***Limit of v i s u a l detection. Table 2. E f f e c t of heat treatment on the absorbance (A+05 ) and the absorbance r a t i o s (AR) obtained with known c e l l concentrations i n an ELISA optimized for Erwinia carotovora subsp. atroseptica B a c t e r i a l concentrations (cells/ml) Temp. (C) Time (min) 10' ^ 05 AR 106 05 AR 105 05 AR 10* 05 AR 103 05 AR 05 21 40 60 80 100 3 6 9 12 3 6 9 12 3 6 9 12 3 6 9 12 3 6 9 12 1.729** 1.458 1.462 1.511 1.620 1.593 1.511 1.527 2.274 2.441 1.804 1.801 1.740 1.947 1.761 1.933 1.642 2.057 2.089 2.034 41.2 34.6 36.6 40.8 37.7 40.8 43.2 25.9 58.3 56.8 44.0 47.4 47.0 52.6 51.-8 53.7 45.6 66.4 58.0 63.6 0.561 0.482 0.457 0.448 0.564 0.543 0.700 0.685 1.078 1.255 1.109 1.223 1.233 1.117 1.160 1.294 1.120 1.201 1.161 1.269 13.4 11.8 11.4 12.1 13.1 13.9 20.0 11.6 27.6 29.2 27.0 32.2 33.3 30.2 34.1 35.9 31.1 38.7 32.3 39.7 0.097 0.103 0.096 0.103 0.113 0.112 0.110 0.126 0.262 0.269 0.262 0.252 0.278 0.237 0.266 0.275 0.272 0.257 0.264 0.227 2.3 0.045 1.1 0.044 1.0 0.042 2.5 0.045 1.1 0.062 1.5 0.041 2.4 0.047 1.2 0.041 1.0 0.040 2.8 0.047 1.3 0.039 1.1 0.037 1.6 0.039 1.0 0.039 0.9 0.043 2.9 0.047 1.2 0.042 1.1 0.039 3.1 0.053 1.5 0.052 1.5 0.035 2.1 0.043 0.7 0.073 1.2 0.059 6.7 0.070 1.8 0.041 1.1 0.039 6.3 0.058 1.3 0.042 1.0 0.043 6.4 0.058 1.4 0.041 1.0 0.041 6.6 0.061 1.6 0.072 1.9 0.038 7.5 0.059 1.6 0.038 1.0 0.037 6.4 0.057 1.5 0.040 1.1 0.037 7.8 0.058 1.7 0.040 1.2 0.034 7.6 0.060 1.7 0.041 1.1 0.036 7.6 0.054 1.5 0.037 1.0 0.036 8.3 0.051 1.6 0.037 1.2 0.031 7.3 0.057 1.6 0.039 1.1 0.036 7.1 0.061 1.9 0.033 1.0 0.032 *Limit of v i s u a l detection. **Mean of s i x r e p l i c a t e s - 80 -The amount of growth of 48 h cultures also varied with the medium, and the s t r a i n . With both s t r a i n s the growth on KMB > NSA > NA > CPG > PDA. On KMB both s t r a i n s produced large mucoid colonies while on NSA lesser amounts of these materials were produced. On NA, CPG and PDA s t r a i n E82 gave small, w e l l - i s o l a t e d , i n d i v i d u a l colonies. S t r a i n 193 produced s i m i l a r colonies on CPG and PDA, but gave s l i g h t l y larger colonies on NA, with very l i t t l e mucoid materials. Thus, i t appears that the type of growth may be important i n t h i s assay. B a c t e r i a l suspensions heated to a temperature of 60 C or greater p r i o r to t e s t i n g by ELISA, had greatly increased mean \ u 5 values compared to simi-l a r unheated suspensions (Table 2). However, heating did not a l t e r the buffer c o n t r o l s . A l l combinations of temperature and time gave v i s u a l l y detectable reactions at 105 c e l l s / m l and had AR > 2.0 (Table 2). The absorbance r a t i o s of heat-treated c e l l s were approximately three times those of the correspond-ing untreated samples at the l i m i t of detection. Subsequent experiments with an expanded temperature range around the c r i t i c a l temperature ( F i g . 1) con-firmed the i n i t i a l r e s u l t that 60 C was the c r i t i c a l minimum temperature required for an increase in A+05 values. Prolonging the heat treatment at the c r i t i c a l temperature beyond the minimum period tested (3 min) did not further enhance the increase in mean A+os observed after the minimum treatment (Table 2 and F i g . 1 and 2). Washing the c e l l s of s t r a i n E193 with d i s t i l l e d water p r i o r to t e s t i n g resulted i n a decrease i n the mean A+os values and absorbance r a t i o s of the once-washed c e l l s compared to the unwashed suspension (Table 3). The high absorbance r a t i o s observed with even 100-fold d i l u t i o n s of the wash f l u i d suggested that at least one of the antigens being detected was soluble. A - 81 -F i g . 1. E f f e c t of heat treatment (temperature and duration) on absorbance (A+os) obtained in an ELISA with known concentrations of Erwinia  carotovora subsp. atroseptica . - 82 -BACTERIA/ML TIMES (minutes) F i g . 2. Ef f e c t of heat treatment (60 C) duration on absorbance (A+ 0 5) i n an ELISA with known concentrations of Erwinia carotovora subsp. atr o s e p t i c a. - 83 -second washing did not r e s u l t i n a further decrease i n absorbance r a t i o for the twice-washed b a c t e r i a l c e l l s but the undiluted second wash f l u i d had an absorbance r a t i o s i m i l a r to that of the f i r s t wash f l u i d . Presumably the concentration of the soluble antigen in t h i s second wash f l u i d was less because the a b i l i t y to detect i t was l o s t at a lower d i l u t i o n . By contrast washing c e l l s of s t r a i n 95 (Ecc) did not r e s u l t i n increased absorbance r a t i o s being associated with the wash f l u i d s . One washing did reduce the absorbance r a t i o s observed for the washed c e l l s but a second washing caused no further reduction. With both s t r a i n s there was good agreement between treatments judged p o s i t i v e on the basis of absorbance r a t i o s greater than 2 compared with v i s u a l estimates. Heating the wash f l u i d s and c e l l suspensions for 15 min at 80 C p r i o r to t e s t i n g resulted i n decreased absorbance r a t i o s for the c e l l wash f l u i d s but increased r a t i o s for the washed c e l l suspensions (Table 4). The l i m i t s of detection for the o r i g i n a l suspension increased from 106 to 10* c e l l s / m l on the basis of absorbance r a t i o s and from 107 to 106 c e l l s / m l v i s u a l l y . A ten-f o l d increase i n s e n s i t i v i t y was observed both v i s u a l l y and spectrophotometri-c a l l y when the once-washed c e l l suspensions were s i m i l a r l y heated. Even greater increases were observed with the twice-washed c e l l suspensions. The absorbance r a t i o s of the wash f l u i d s were decreased by heating but the l i m i t of detection by absorbance r a t i o s remained unchanged with the exception of the second c e l l wash f l u i d at a d i l u t i o n of 10~* (AR < 2.0). By contrast heat-ing caused a 10 to 100-fold decrease i n the v i s u a l l y determined l i m i t of s e n s i t i v i t y . The presence of h e a t - l a b i l e antigens in both the wash f l u i d s and the washed b a c t e r i a l suspensions was indicated by the decreased mean A+os values for a l l samples receiving the 90 min autoclave treatment (Table 5). - 84 -Table 3. Detection by ELISA of Erwinia carotovora subsp. atroseptica antigens i n d i s t i l l e d water c e l l wash f l u i d s and washed c e l l suspensions of subsp. atroseptica ( s t r a i n E193) and subsp. carotovora ( s t r a i n E95) Stra i n Treatment Absorbance r a t i o (AR) at d i l u t i o n factor undiluted 10 AR -1 AR 1 0 - 2 1 0 - 3 AR AR 10 AR 1 0 - 5 AR E193 normal c e l l * suspension f i r s t wash f l u i d once-washed c e l l * suspension second wash f l u i d twice-washed* c e l l suspension E95 normal c e l l * suspension f i r s t wash f l u i d once-washed c e l l * suspension second wash f l u i d twice-washed* c e l l suspension 38.5** 39.7 23.9 37.7 21.3 5.7 27.4 6.8 1.5 2.1 1.6 2.3 1.8 0.9 1.3 38.5 30.4 8.2 10.0 3.0 23.7 5.3 9.7 2.3 2.2 1.3 1.0 0.8 1.3 1.6 1.0 0.9 1.4 1.1 1.1 1.5 1.2 0.8 1.2 1.2 1.6 1.2 1.2 1.0 0.9 1.1 0.8 1.1 0.9 1.2 1.0 1.2 0.9 . 0.9 1.1 1.6 0.9 0.9 1.2 0.9 *Eguivalent b a c t e r i a l concentrations i n c e l l s / m l : undiluted - 10s , 10"1 = 107 ; 10"2 = 106 ; 10-3 = 105 ; 10"^ = ; 10"5 = 103 . **Based on average co n t r o l value of six r e p l i c a t e s A405 = 0.040. ***Limit of v i s u a l detection. - 85 -Table 4. E f f e c t of a 15 min 80 C heat treatment on detection by ELISA of Erwinia carotovora subsp. atroseptica antigens i n d i s t i l l e d water c e l l wash f l u i d s and washed suspensions of ( s t r a i n E193) Absorbance r a t i o (AR) at d i l u t i o n factor Temp undiluted 10"1 10"2 10~3 10"1* Treatment C AR AR AR AR AR *** Normal c e l l 21 7.5* 3.3 1.0 1.1 0.2 suspension** 80 12.9 8.3 3.3 2.2 1.6 F i r s t wash 21 46.0 20.9 11.9 6.8 3.7 f l u i d 80 45.2 9.1 4.8 2.9 3.1 Once-washed c e l l * * 21 4.9 2.0 1.4 1.0 1.0 suspension 80 6.7 8.7 2.8 0.8 1.1 Second wash 21 41.1 21.4 7.6 5.9 3.6 f l u i d 80 32.8 8.3 5.9 4.6 1.7 Twice-washed** 21 3.4 1.7 1.2 0.9 1.1 c e l l suspension 80 7.3 5.6 2.5 1.7 1.3 *Based on average c o n t r o l value (25 r e p l i c a t e s ) of A+os = at 21 C and A+05 = ° - 0 1 8 at 80 C. **Equivalent b a c t e r i a l concentrations i n c e l l s / m l : undiluted = 10 ; 10" = 106 ; 10"2 = 1-5 ; 10-3 = 104 and 10~k = 105 . ***Limit of v i s u a l detection. - 86 -Table 5. E f f e c t of a 90 min heat treatment at 121 C on detection by ELISA of Erwinia carotovora subsp. atroseptica antigens i n undiluted d i s t i l l e d water c e l l wash f l u i d s and washed c e l l suspensions (10s cells/ml) of f l u i d s s t r a i n E193 A+os a f t e r treatment at 20 C 121 C Treatment 109 0 108 0 Normal c e l l suspension 1.159* 0.019 0.763 0.020 F i r s t wash f l u i d 1.321 0.051 0.879 0.036 Once-washed c e l l suspension 1.065 0.019 0.671 0.020 Second wash f l u i d 1.309 0.067 0.791 0.044 Twice-washed c e l l suspension 0.967 0.019 0.762 0.020 *Mean of f i v e r e p l i c a t e s . - 87 -When representative s t r a i n s of the 24 Erwinia carotovora serogroups were tested, predictably the serogroup I st r a i n s gave the highest mean A+Q5 values (Table 6). However at the c e l l concentration used (10 8 cells/ml) s t r a i n s of the three other subsp. atroseptica serogroups (XVIII, XX, XXII) plus three subsp. carotovora serogroups ( I I I , IV, V) were also detected by the test system. Although the mean A+os values obtained were only 10% of those obtained with the homologous s t r a i n s , the absorbance r a t i o s were greater than 2.0 and the wells containing these serogroups were v i s u a l l y detectable. With the exception of the serogroup II s t r a i n which was v i s u a l l y v ariable but spectrophotometrically negative, the remaining serogroups were s i m i l a r to the buffer c ontrols. Two other species often associated with natural Erwinia i n f e c t i o n s were also comparable to the con t r o l s . A 5 min heat treatment at 60 C generally increased the absorbance r a t i o s for the subsp. atroseptica sero-groups but decreased those of the subsp. carotovora serogroups at most c e l l concentrations. In sp i t e of t h i s s e l e c t i v e enhancement, the l i m i t of detec-t i o n for the homologous s t r a i n s remained at 105 c e l l s / m l . The subsp. caroto- vora s t r a i n s , although s t i l l detectable by absorbance r a t i o s at 10 c e l l s / m l , were variable or negative v i s u a l l y after heat treatment. The l i m i t of detec-t i o n of s t r a i n s E368 and E555 corresponding to serogroups XX and XXII were unaffected by heating. Strain E17 (serogroup XVIII) was exceptional among subsp. atroseptica s t r a i n s i n that heating decreased the A +os reading at 106 c e l l s / m l . As a consequence there was a 10-fold loss i n s e n s i t i v i t y as deter-mined by absorbance r a t i o s . - 88 -Table 6. Absorbance (A+os)* absorbance r a t i o (AR) and q u a l i t a t i v e v i s u a l e s t i mate of color development for 108 c e l l s / m l of s t r a i n s representing 2 serogroups of Erwinia carotovora and other b a c t e r i a l pathogens i n an ELISA optimized for the detection of Erwinia carotovora subsp. atro- septica serogroup I Absorbance V i s u a l Absorbance Serogroup Str a i n Organism estimate AR I E68 atroseptica 1.515* + 26.6 E82 atroseptica 1.588 + 27.9 E193 atroseptica 2.164 + 38.0 E331 atroseptica 1.608 + 28.2 II S21 carotovora 0.092 - 1.6 III E95 carotovora 0.154 + 2.7 IV E14 carotovora 0.145 + 2.5 V S26 carotovora 0.145 + 2.5 VI S189 carotovora 0.064 - 1.1 VII S68 carotovora 0.053 - 0.9 VIII S62 carotovora 0.047 - 0.8 IX E84 carotovora 0.054 - 0.9 X S61 carotovora 0.059 - 1.0 XI E385 carotovora 0.049 - 0.9 XII S67 carotovora 0.053 - 0.9 XIII S59 carotovora 0.051 - 0.9 XIV S65 carotovora 0.049 - 0.9 XV S23 carotovora 0.050 - 0.9 XVI E m carotovora 0.051 - 0.9 E315 carotovora 0.055 - 1.0 XVII E6 carotovora 0.055 - 1.0 XVIII E17 atroseptica 0.287 + 5.0 XIX E103 carotovora 0.046 - 0.8 XX E368 atroseptica 0.201 + 3.5 XXI E295 carotovora 0.052 - 0.9 XXII E555 atroseptica 0.143 + 2.5 XXIII S207 carotovora 0.060 - 1.1 XXIV E254 carotovora 0.057 - 1.0 PM6 Ps. marginalis 0.050 - 0.9 CS-3 C. sepedonicum 0.045 - 0.8 Control 0.057 - * *Mean of six r e p l i c a t e s . Table 7. E f f e c t of a 5 min 60 C heat treatment on the detection by ELISA of known concentrations of selected s t r a i n s of Erwinia carotovora Absorbance (A+os) a n d absorbance r a t i o (AR) at b a c t e r i a l concen-t r a t i o n (cells/ml) Temp 107 1 0 6 1 0 5 10** Serogroup S t r a i n Subsp. C A+os A R ^405 A R \o5 A R \o5 A R I E193 atroseptica 21 1.708* 30.5 0.647 14.1 0.150 2.6 0.073 1.6 60 1.822 38.0 1 .152 22.2 0.313 6.1 0.095 1.9 II S21 carotovora 21 0.072 1.3 0.070 1.5 0.060 1.1 0.059 1.3 60 0.081 1.7 0.080 1.5 0.066 1.3 0.057 1.1 I I I E95 carotovora 21 0.184 3.3 0.078 1.7 0.056 1.0 0.054 1.2 60 • 0.109 2.3 0.065 1.3 0.068 1.3 0.050 1.0 IV E14 carotovora 21 0.163 2.9 0.088 1.9 0.060 1.1 0.053 1.2 60 0.132 2.9 0.078 1.5 0.053 1.0 0.059 1.2 V S26 carotovora 21 0.131 2.3 0.073 1.6 0.047 0.8 0.045 1.0 60 0.097 2.0 0.059 1.1 0.051 1.0 0.062 1.2 VIII S62 carotovora 21 0.055 1.0 0.049 1.1 0.054 0.9 0.049 1.1 60 0.049 1.0 0.057 1.1 0.053 1.0 0.045 0.9 XVIII E17 atroseptica 21 0.235 4.2 0.101 2.2 0.065 1.1 0.052 1.1 60 0.245 5.1 0.100 1.9 0.059 1.2 0.054 1.1 XX E368 atroseptica 21 0.158 2.8 0.064 1.4 0.057 1.0 0.055 1.2 60 0.199 4.1 0.084 1.6 0.053 1.0 0.051 1.0 XXII E55 atroseptica 21 0.161 2.9 0.079 1.7 0.053 0.9 0.059 1.3 60 0.169 3.5 0.078 1.5 0.058 1.1 0.056 1.1 Control 21 60 0.056 0.048 0.046 0.052 0.057 0.051 0.046 0.051 *Mean of six r e p l i c a t e s **Limit of v i s u a l detection - 90 -DISCUSSION The detection of bacteria by ELISA, unlike viruses and y-globulins, i s complicated by the presence of several antigenic s i t e s on bacteria, unless the antiserum i s produced against a p a r t i c u l a r antigen. In t h i s study, antiserum to glutaraldehyde-fixed whole c e l l s was employed so that the s p e c i f i c i t y i n ELISA could be d i r e c t l y compared with that i n the double d i f f u s i o n system previously described for Erwinia carotovora (DeBoer et^ _al. 1979). Both water soluble antigen(s) as well as antigen(s) bound to the c e l l are apparently responsible for the reaction i n ELISA. The fact that a small component of both the soluble and i n s o l u b l e f r a c t i o n s was heat l a b i l e (Table 5) supports the conclusion that the primary antigen involved was LPS but that secondary l a b i l e protein antigens were also present. The a v a i l a b i l i t y of these antigens appears to be at least p a r t i a l l y influenced by growing conditions. Equal numbers of c e l l s grown on d i f f e r e n t media were detected to d i f f e r e n t degrees by ELISA. The c e l l s produced on KmB which supported very large mucoid colonies, reacted poorly compared to those grown on media supporting small colonies. Whether t h i s was due to physical masking of the r e a c t i v e s i t e s by the polysaccharides or to s l i g h t changes i n c e l l envelope chemistry was not determined. The fact that washed c e l l s were detected l e s s well by ELISA than unwashed c e l l s , tends to discount the f i r s t p o s s i b i l i t y . However, the enhanced detection after the c e l l s were heated for periods as short as 3 min, suggests that a heat l a b i l e protein may be par-t i a l l y blocking the r e a c t i v e s i t e s . Kishinevsky and Bar-Ooseph (1978) and Berger et ^1_. (1979) have previously reported obtaining increased A+os when Rhizobium c e l l s were heated p r i o r to assay. The l a t t e r authors speculated that the heating uncovers more rea c t i v e s i t e s . - 91 -This ELISA system employing antiserum against glutaraldehyde fixed whole c e l l s of Eca s t r a i n E82 was quite s p e c i f i c . No cross reactions were observed with P_. marginalis, and _C. sepedonicum even at high (10 s cells/ml) concentra-t i o n s . However, at high c e l l concentrations cross reactions were observed with a few s t r a i n s of Ecc belonging to serogroups I I , I I I , IV and V. Simi-l a r l y , s t r a i n s f a l l i n g into the other 3 serogroups which are biochemically subsp. atroseptica also reacted. Vruggink (1978) also reported cross reac-tions with some s t r a i n s although the serogroups were unknown. Such a r e s u l t i s not su r p r i s i n g because DeBoer (1980) has reported that serogroups I, I I I , V and XVIII share a common h e a t - l a b i l e f l a g e l l a r antigen, and that the c e l l wall antigens of serogroups I, II and XVIII are rel a t e d . Heating the c e l l s p r i or to assay did reduce but not completely eliminate the reactions of s t r a i n s i n serogroups III and V at 10 c e l l s / m l , as both AR > 2.0 and p o s i t i v e v i s u a l estimates were recorded. St r a i n E17 belonging to serogroup XVIII was excep-t i o n a l among the subsp. atroseptica s t r a i n s i n that heat treatment at 10 ce l l s / m l resulted i n a 10-fold loss of s e n s i t i v i t y . The p o s s i b i l i t y e x i s t s that as the l i m i t s of s e n s i t i v i t y are approached, removal of the common l a b i l e f l a g e l l a r antigen may have been s u f f i c i e n t to r e s u l t i n a negative reaction. The fact that the somatic antigens of serogroups I and XVIII are rela t e d explains why higher absorbance r a t i o s were obtained than with the subsp. carotovora s t r a i n s except those belonging to serogroup I I . The l a t t e r also shares a common somatic antigen with serogroup I and might have been expected to have a higher value. Why the s t r a i n of t h i s l a t e r serogroup did not react more strongly after heat treatment i s unknown. Heat treatment markedly increased the absorbance r a t i o of the homologous antigen while only s l i g h t l y increasing those of the remaining two subsp. atroseptica serogroups. Thus i n the range of 106 and 105 c e l l s / m l , th i s ELISA was serogroup s p e c i f i c . Heat - 92 -treatment of the samples increased the absorbance r a t i o s but did not change the pattern of s p e c i f i c i t y . At higher c e l l concentrations the p o s s i b i l i t y of cross reactions with serogroup III which i s most commonly found on potato tubers (DeBoer et_ jal. 1979), and the other subsp. atroseptica serogroups (XVIII, XX and XXII) c e r t a i n l y e x i s t s . Depending upon the purpose of the detection work, being able to detect these important serogroups might be d e s i r a b l e . The p o t e n t i a l r e l a t i o n s h i p between serogroup IV and I detected as a cross reaction i n t h i s study was not detected by DeBoer (1980). However, t h i s observation i s not su r p r i s i n g because due to the s e n s i t i v i t y of the ELISA system, cross reactions and r e l a t i o n s h i p s not detectable by other means have been previously detected by ELISA (Carlsson et j i l . 1976). Even af t e r standardized washing and s e l e c t i v e enhancement by heat t r e a t -ment, the maximum l i m i t of s e n s i t i v i t y i n t h i s ELISA for Erwinia carotovora subsp. atroseptica (serogroup I) i s 105 c e l l s / m l based on both AR > 2.0 and p o s i t i v e v i s u a l color development. This compares favorably with the reported l e v e l of s e n s i t i v i t y of 106 - 107 c e l l s / m l for Pseudomonas phaseolicola (Weaver and Guthrie 1978). Stevens and Tsiantos (1979) claimed 103 c e l l s / m l as the l i m i t of detection for Corynebacterium michiganense i n buffer suspen-sions. However, based on t h e i r Figure 2, a more l i k e l y l i m i t of detection i s 105 - 106 c e l l s / m l . Because the curve was e s s e n t i a l l y at the same l e v e l from 103 to 105 c e l l s / m l , i t i s d i f f i c u l t to understand how they can claim sensi-t i v i t y at the lower l e v e l . Cother and Vruggink (1980) working with Erwinia  carotovora subsp. atroseptica reported, as unpublished data, that the l i m i t of detection in t h e i r system was 10* c e l l s / m l , but no data have been published to confirm t h i s f i g u r e . The l e v e l of 105 c e l l s / m l i s at or above the l i m i t of s e n s i t i v i t y of immunofluorescence reported by Slack jjt j i l _ . (10 to 10 2 c e l l s / - 93 -ml) 1979) and A l l a n and Kelman (105 cells/ml) (1977). These data confirm Vruggink's (1978) report that both IFAS and ELISA gave comparable r e s u l t s with Eca. The optimized ELISA technique as used i n t h i s study compared to immuno-fluorescence for detection of Eca o f f e r s only a reduction in operator fatigue, and an objective means of assessment but no greater s e n s i t i v i t y nor a more rapid t e s t . The fact that the primary antigen detected i n t h i s system i s water soluble provides the opportunity for tests based on c e l l suspensions or c e l l e x tracts. The high absorbance r a t i o s associated with c e l l wash f l u i d s i n Tables 3 and 4 require cautious i n t e r p r e t a t i o n . In these experiments suspen-sions containing very high concentrations of c e l l s (« 10 1 0 ) were washed to maximize the presence of soluble antigens. Thus, although the b a c t e r i a l suspensions that were used were standardized to 108 c e l l s / m l , the washing f l u i d s used did not correspond to the washing f l u i d s from a 10s c e l l s / m l suspension but rather to the o r i g i n a l more concentrated suspension. It remains to be determined whether s i m i l a r data would be obtained from the wash f l u i d s of lower c e l l concentrations, and whether a t e s t based on just the soluble antigen i s possible. The ELISA for Eca was found to have serogroup s p e c i f i c i t y at the medium to low concentrations (10 5 - 106 cells/ml) at which i t would be most l i k e l y used. The obstacle to i t s widespread use i s s e n s i t i v i t y . In s p i t e of employing optimized conditions of coating and conjugate concentrations, washing and s e l e c t i v e enhancement by heat, v a r i a b i l i t y i n the plates precludes lowering the l i m i t of s e n s i t i v i t y beyond 105 c e l l s / m l . U n t i l or unless t h i s v a r i a b i l i t y with Erwinia carotovora subsp. atroseptica can be eliminated or reduced, ELISA seems to have l i t t l e p o t e n t i a l i n routine surveys for detecting latent blackleg i n f e c t i o n i n c e r t i f i e d seed potatoes. - 94 -LITERATURE CITED ALLAN, E. and A. KELMAN. 1977. Immunofluorescent st a i n procedures for detec-t i o n and i d e n t i f i c a t i o n of Erwinia carotovora var. at r o s e p t i c a. Phytopathology 67:1305-1312. BAR-JOSEPH, M., S.M. GARNSEY, D. GONSALVES, M. M0SC0VITZ, D.E. PURCIFULL, M.F. CLARK and G. LOEBENSTEIN. 1979. The use of enzyme-linked immunosorbent assay for detection of c i t r u s t r i s t e z a v i r u s . Phytopathology 69:190-194. BERGER, 3.A., S.N. MAY, L.R. BERGER and B.B. B0HL00L. 1979. Colorimetric enzyme-linked immunosorbent assay for the i d e n t i f i c a t i o n of s t r a i n s of Rhizobium i n culture and i n the nodules of l e n t i l s . Appl. Environm. Mi c r o b i o l . 37:642-646. BRODEUR, B.R., F.E. ASHTON and B.B. DIENA. 1978. Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae s p e c i f i c antibodies. Can. 3. M i c r o b i o l . 24:1300-1305. CARLSSON, H.E., B. HURVELL and A.A. LINDBERG. 1976. enzyme-linked immunosor-bent assay (ELISA) for t i t r a t i o n of antibodies against Brucella abortus and Yer s i n i a e n t e r o c o l i t i c a . Acta. Path. M i c r o b i o l . Scand. Sect. C, 84:168-176. CARLLS0N, H.E., A.A. LINDBERG, S. HAMMARSTR0M and A. LOUNGGREN. 1975. Quan-t i t a t i o n of Salmonella 0-antibodies i n human sera by enzyme-linked immunosorbent assay (ELISA). Int. Archs. Allergy Appl. Immun. 48:485-494. CLAFLIN, L.E., and O.K. UYEM0T0. 1978. Serodiagnosis of Corynebacterium  sepedonicum by enzyme-linked immunosorbent assay. Phytopathology News 12:156 (Abstr.). CLARK, M.F. and A.N. ADAMS. 1977. C h a r a c t e r i s t i c s of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. 3. Gen. V i r o l . 34:475-483. COTHER, E.3. and H. VRUGGINK. 1980. Detection of viable and non viable c e l l s of Erwinia carotovora var. atroseptica in inoculated tubers of var Bintje with enzyme-linked immunosorbent assay (ELISA). Pot. Res. 23:133-135. DE BOER, S.H., R.3. C0PEMAN and H. VRUGGINK. 1979. Serogroups of Erwinia  carotovora potato s t r a i n s determined with d i f f u s i b l e somatic antigens. Phytopathology 69:316-319. DE BOER, S.H. 1980. S e r o l o g i c a l r e l a t i o n s h i p s among f l a g e l l a of Erwinia  carotovora var. atroseptica and some E. carotovora var. carotovora serogroups. Can. 3. M i c r o b i o l . 26:567-571 FLEGG, C L . and M.F. CLARK. 1979. The detection of apple c h l o r o t i c leafspot virus by a modified procedure of enzyme-finked immunosorbent assay (ELISA). Ann. appl. B i o l . 91:61-65. - 95 -GUGERLI, P. 1979. Potato Virus A and Potato L e a f r o l l Virus: P u r i f i c a t i o n , antiserum production and se r o l o g i c a l detection i n potato and te s t plants by enzyme-linked immunosorbent assay (ELISA). Phytopath. Z. 96:97-107. GUGERLI, P. and W. GEHRIGER. 1980. Enzyme-linked immunosorbent assay (ELISA) for the detection of potato l e a f r o l l v irus and potato v i r u s Y in potato tubers after a r t i f i c i a l break of dormancy. Pot. Res. 23:353-359. HOLMGREN, 3. and A.M. SVENNERHOLM. 1973. Enzyme-linked immunosorbent assays for cholera. Serology Infection and Immunity 1(5):759-763. ITO, 3.1. 3r., A.C. WUNDERLICH, 3. LYONS, C.E. DAVIS, D.C. GUINEY and A.I. BRAUDE. 1980. Role of magnesium i n the enzyme-linked immunosorbent assay for lipopolysaccharides of rough Escherichia c o l i s t r a i n 35 and Neiss e r i a  gonorrhoeae. 3. Inf. Dis. 142:532-537. KATZNELSON, H. and R.M.D. SUTTON. 1956. Laboratory detection of Corynebac-terium sepedonicum, causa agent of b a c t e r i a l ring rot of potatoes. Can. 3. Bot. 34:48-53. KING, E.O., M.K. WARD and D.E. RANEY. 1954. Two simple media for the demon-st r a t i o n of pyocyanin and f l u o r e s c e i n . 3. Lab. C l i n . Med. 44:301-307. KISHINEVSKY, B. and M. BAR-30SEPH. 1978. Rhizobium s t r a i n i d e n t i f i c a t i o n i n Archis hypogaea nodules by enzyme-linked immunosorbent assay (ELISA). Can. 3. M i c r o b i o l . 24:1537-1543. LISTER, R.M. and W.F. R0CH0W. 1979. Detection of barley yellow dwarf v i r u s by enzyme-linked immunosorbent assay. Phytopathology 69:649-654. MARCO, S. and S. COHEN. 1979. Rapid detection and t i t e r evaluation of viruses in pepper by enzyme-linked immunosorbent assay. Phytopathology 69:1259-1262. RUSSELL, H., R.R. FACKLAM and L.R. EDWARDS. 1976. Enzyme-linked immunosor-bent assay for streptococcal M protein antibodies. 3. C l i n . M i c r o b i o l . 3:501-505. SLACK, S.A., A. KELMAN and L.B. PERRY. 1979. Comparison of three serodiag-nostic assays for detection of Corynebacterium sepedonicum. Phytopathology 69:186-189. STEVENS, W.A. and 3. TSIANTOS. 1979. The use of enzyme-linked immunosorbent assay (ELISA) for the detection of Corynebacterium michiganense i n tomatoes. Microbio. L e t t e r s IU:29-32. TAMADA, T. and B.D. HARRISON. 1980. Application of enzyme-linked immunosor-bent assay to the detection of potato l e a f r o l l v i r u s i n potato tubers. Ann. appl. B i o l . 96:67-78. TRESH, 3.M., A.N. ADAMS, D.3. BARBARA and M.F. CLARK. 1977. The detection of three viruses of hop (Humulus lupulus) by enzyme-linked immunosorbent assay (ELISA). Ann. appl. B i o l . 87:57-65. - 96 -VOLLER, A., D.E. BIDWELL and A. BARTLETT. 1977. The enzyme-linked immunosor-bent assay (ELISA). Dynatech Laboratories, Alexandria, V i r g i n i a . 48 pp. VRUGGINK, H. 1978. Enzyme-linked immunosorbent assay (ELISA) in the sero-diagnosis of plant pathogenic bacteria. Proceed. 4th Int. Conf. Plant Path. Bact. Angers pp. 307-310. WEAVER, W.M. and 3.W. GUTHRIE. 1978. Enzyme-linked immunospecific assay app l i c a t i o n to the detection of seed borne b a c t e r i a . Phytopathol. News 12:156-157. (Abstr.) - 97 -DISCUSSION This enzyme-linked immunosorbent assay (ELISA) model system, employing Erwinia carotovora subsp. atroseptica, was based on c r i t e r i a seldom used simultaneously. The requirement for both absorbance r a t i o s >^  2.0 and a posi-t i v e v i s u a l estimate, following a 30-min reaction time, imposed more stringent requirements for a p o s i t i v e t e s t than are normally used. By requiring both c r i t e r i a , only strong and rapid reactions which had v i s i b l y negative controls were considered. At high b a c t e r i a l concentrations (10 6 - 107 cells/ml) there was consistency of the r e s u l t s with both c r i t e r i a , while at lower concentra-tions (10* - 105 cells/ml) some differences could be observed, and the percent v a r i a b i l i t y remained high. These discrepancies at low b a c t e r i a l concentra-t i o n s were att r i b u t e d mainly to the background. This variable background influenced the AR >_ 2.0 and biased the conclusions about detection at the l i m i t of s e n s i t i v i t y . The use of a standardized washing procedure removed some of the v a r i a -b i l i t y by removing some of the background. Regardless of the wash solution employed, the use of a c o n t r o l l e d pressure device was always much better than a wash b o t t l e . The addition of Tween-20 i n the washing sol u t i o n should be avoided unless a plate reader with dual wavelength i s a v a i l a b l e because i t leaves a f i l m at the bottom of plates, which i n t e r f e r e d with the readings. While the presence of Tween-20 did not increase the v i s u a l background, lower l i m i t s of s e n s i t i v i t y were obtained both v i s u a l l y and on an absorbance r a t i o >_ 2.0 basis, which might be the r e s u l t of the detergent e f f e c t of Tween-20. The use of d i s t i l l e d water was as good as phosphate buffered s a l i n e , and more p r a c t i c a l with the c o n t r o l l e d pressure system adopted. It was also important that the s e l e c t i o n of the coating y -globulin concentration and the enzyme-y-- 98 -g l o b u l i n conjugate d i l u t i o n combination be done under standardized washing conditions, because excessive washing of the various components, resulted in a severe decrease of s e n s i t i v i t y . The well to well v a r i a b i l i t y observed within a plate, as well as the plate to plate v a r i a b i l i t y were primarily responsible for the low l i m i t of s e n s i t i v i t y of the technique. These sources of v a r i a b i l i t y were maintained despite an optimized coating-conjugate combination, and a standardized washing procedure. This type of v a r i a b i l i t y caused problems e s p e c i a l l y at low coating concentration and high conjugate d i l u t i o n s , where background v a r i a t i o n s made i t hard to determine what was p o s i t i v e . Moreover, t h i s v a r i a b i l i t y within or between plates might possibly mask other p o t e n t i a l sources of v a r i a b i l i t y . Among these, the buffers used for the samples or the conjugate d i l u t i o n s showed differences but were not s i g n i f i c a n t l y d i f f e r e n t from the one employed in v i r u s work. Whether the requirements for in v i t r o work employing only pure cu l t u r e s , and those for in vivo work involving plant sap might be d i f f e r e n t , remains to be determined. The d i f f e r e n t r e s u l t s obtained between buffers, and the fa c t that the main e f f e c t was re l a t e d to the conjugate, suggested that the requirements for binding the b a c t e r i a l c e l l s to the coating y-globulin could be d i f f e r e n t from those for binding of the enzyme-y-globulin conjugate to the b a c t e r i a l c e l l s . None of the v a r i a b i l i t y observed could be related to pipet-t i n g e r r o r s . It i s possible that improved plates with le s s inherent v a r i a b i l -i t y might permit uncovering sources of v a r i a t i o n a c t u a l l y masked in t h i s work. At t h i s point, the ELISA technique as employed for Erwinia carotovora subsp. atroseptica does not o f f e r r e a l advantages over immunofluorescence with regard to both s p e c i f i c i t y and s e n s i t i v i t y . Although heat treatment of the c e l l s showed a s e l e c t i v e enhancement, the s e n s i t i v i t y of the technique - 99 -could not be lowered below 105 c e l l s / m l . Furthermore, the serogroup s p e c i f i -c i t y at lower b a c t e r i a l concentrations (10 6 cells/ml) was comparable with or without heat treatment of the c e l l s , to that shown by immunodiffusion. The data obtained by heat treatment, c e l l washing, and the observed s p e c i f i c i t y support the idea that the lipopolysaccharide (LPS) i s probably the major antigen in t h i s t e s t . Since the a v a i l a b i l i t y of the antigen was a function of the growth medium, i t w i l l be i n t e r e s t i n g to know how c e l l s from n a t u r a l l y - i n f e c t e d plants would compare. Because LPS i s p a r t l y soluble i n water, further use of t h i s fact should be made i n future t r i a l s . The work reported here, obviously s u f f e r s the disadvantage that only one antiserum was used throughout. However, encouraging and valuable information was obtained. Whether t h i s i s representative, and whether antisera to other antigens might be better remain to be investigated. U n t i l v a r i a b i l i t y i s eliminated and s e n s i t i v i t y increased, there w i l l be l i t t l e incentive to use the double sandwich ELISA technique with plant sap where reduction in s e n s i t i v t y i s l i k e l y . At t h i s point ELISA seems to have l i t t l e p o t e n t i a l i n routine surveys for detecting latent blackleg i n f e c t i o n in c e r t i f i e d seed potatoes. - 100 -SUMMARY 1. The combination of 2.0 ug/ml coating y - g l o b u l i n and 1:400 enzyme-y-globu-l i n conjugate d i l u t i o n were determined as optimum for the Erwinia caroto- vora subsp. atroseptica system with t h i s p a r t i c u l a r antiserum and type of m i c r o t i t r a t i o n plates. 2. The washing procedure was a variable a f f e c t i n g detection which had to be standardized. A c o n t r o l l e d pressure-washing system employing d i s t i l l e d water, and two 15-sec washes at 34.48 kPa (5 p s i ) , with 180= r o t a t i o n of the plate between each wash was adopted. 3. The well to well v a r i a b i l i t y i n the m i c r o t i t r a t i o n plates was not exclu-sive to the outside rows and was shown to decrease with time of reacti o n . 4. The buffer solutions employed for d i l u t i o n of samples and conjugate i n f l u -enced the r \ Q 5 values. The conjugate buffer had a greater e f f e c t than the sample buffer. 5. The complete buffer used in virus work (PBS + 0.05% Tween-20 + 2.0% PVP + 0.2% egg albumin) was a s u i t a b l e choice for both sample and conjugate preparation. 6. The l i m i t of detection of E. carotovora subsp. atroseptica was 105 - 106 c e l l s / m l . 7. P i p e t t i n g errors of 5% i n coating, sample and/or conjugate did not repro-duce the observed v a r i a b i l i t y . 8. Heat treatment of the c e l l s at 60 C for 3 - 6 min enhanced A+os values but the l e v e l of s e n s i t i v i t y following a heat treatment was 105 c e l l s / m l . 9. The medium upon which c e l l s were grown affected the A+os values but was not proportional to the amount of growth observed. - 101 -10. Washing b a c t e r i a l c e l l s i n d i s t i l l e d water resulted i n the detection by ELISA of soluble antigens i n both the wash f l u i d s and the resuspended c e l l suspensions. 11. Heat-labile and heat-stable antigens were present in both the wash f l u i d s and the resuspended c e l l s . 12. S p e c i f i c i t y of the JE. carotovora subsp. atroseptica serogroup I ELISA was maintained at c e l l concentrations of les s than 105 c e l l s / m l . 13. S e r o l o g i c a l cross reactions among E_. carotovora serogroups at high c e l l concentrations (> 10 cells/ml) were confirmed by ELISA. - 102 -SUPPLEMENTARY LITERATURE CITED CARLIER, Y., D. BOUT, and A. CAPRON. 1979. Automation of enzyme-linked immunosorbent assay (ELISA). J. Immunol. Methods. 31:237-246. CHIA, W.K. and L. SPENCE. 1979. Quantitative determination of cytomegalo-vi r u s IgG antibody by enzyme-linked immunosorbent assay (ELISA). Can. J . Mic r o b i o l . 25:1082-1086. CLARK, M.F., C L . FLEGG, M. BAR-JOSEPH, and S. ROTTEM. 1978. The detection of Spiroplasma c i t r i by enzyme-linked immunosorbent assay (ELISA). Phytopath. Z. 92:322-337. CUPPELS, D. and A. KELMAN. 1974. Evaluation of s e l e c t i v e media for i s o l a t i o n of s o f t - r o t b acteria from s o i l and plant t i s s u e . Phytopathology 64:468-475. GOTO, M. and N. OKABE. 1957. Studies on s t r a i n s of Erwinia carotovora (Jones) Holland. IV. Antigenic v a r i a t i o n s . B u l l . Fac. Agric. Shizuoka Univ. 7:11-20. GRAHAM, D.C 1963. S e r o l o g i c a l diagnosis of potato blackleg and tuber soft r o t . Plant Pathol. 12:142-144. LELLIOTT, R.A. 1968. The diagnosis of f i r e b l i g h t Erwinia amylovora and some diseases caused by Pseudomonas syringae. Report of the Intern. Conf. on F i r e b l i g h t . Eur. Mediterr. Plant Prot. Organ. EPPO Publ. 45:27-34. MENELEY, J.C. and M.E. STANGHELLINI. 1976. I s o l a t i o n of s o f t - r o t Erwinia spp. from a g r i c u l t u r a l s o i l s using an enrichment technique. Phytopathology 66:367-370. OKABE, N. and M. GOTO. 1955. B a c t e r i a l plant diseases i n Japan. I I . Studies on s o f t r ot due to Erwinia aroideae (Townsend) Holland, with s p e c i a l reference to the antigenic structures of f l a g e l l a . B u l l . Fac. Agric. Shizuoka Univ. 5:72-86. VOLLER, A, D. BIDWELL, G. HULDT and E. ENGVALL. 1974. A microplate method of enzyme-linked immunosorbent assay and i t s a p p l i c a t i o n to malaria. B u l l . W.H.0. 51:209-211. VRUGGINK, H. and H.P. MAAS GEESTERANUS. 1975. S e r o l o g i c a l recognition of Erwinia carotovora var. atroseptica, the causal organism of potato blackleg. Potato Res. 18:546-555. 

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