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The role of toxic shock syndrome toxin-1 in the pathogenesis of toxic shock syndrome Rosten, Patricia Melanie 1986

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THE ROLE OF TOXIC SHOCK SYNDROME TOXIN-1 IN THE PATHOGENESIS OF TOXIC SHOCK SYNDROME By PATRICIA MELANIE ROSTEN B.Sc. The University of British Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December.1986 ®Patricia Melanie Rosten, 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of /Vr'cpt) h''o ^°<J^j The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date / X / 7 _ l^^l 3E-6 (3/81) ABSTRACT Toxic shock syndrome toxLn-1 (TSST-1), an exoprotein produced by some strains of Staphylococcus aureus, is implicated in the pathogenesis cf menstrual TSS. However, its role in nonmenstrual TSS is less certain. In order to study the pathogenetic role of TSST-1 in TSS, three approaches were taken: a) to develop an ELISA for detection of TSST-1 in biologic fluids in order to verify TSST-1 production in vivo in TSS patients, b) to quantitate TSST-1 specific antibodies in the serum of TSS patients and controls to detennine whether such antibodies are protective, and c) to attempt to identify other staphylococcal products which may be implicated in some forms of TSS. A sensitive and specific noncompetitive enzyme-linked immunosorbent assay (ELISA) capable of detecting TSST-1 at concentrations from 0.5 to 16 ng/ml was developed. This assay did not detect other staphylococcal enterotoxLns including A, B, C^, 0.^ C3/ D and E. Possible interference by protein A was readily eUniinated by pretreatment of test samples with 10% nonimmune rabbit serum. The assay was adapted for rapid screening of TSST-1 production by S. aureus isolates in culture supernatants in vitro, and for the detection of TSST-1 in vaginal washings and urine of TSS patients and healthy controls in vivo. All 35 S. aureus isolates confirmed to be TSST-1 positive by Ouchterlony immunodiffusion, and 59 of 60 isolates confirmed to be TSST-1 negative, gave concordant results by ELISA. Interestingly, toxigenic S. aureus strains isolated from TSS patients quantitatively produced significantly more toxin in vitro compared to toxigenic control strains (p<0.05, Mann-Whitney rank sum test). TSST-1 could be detected by ELISA in 3 of 4 vaginal washings collected within 3 days of lx»spita]ization from 3 women with acute menstrual TSS, compared to 0 of 17 washings from 9 TSS women collected greater than 3 i i days after hospitalizatiori (p=0.003, Fisher's exact test) and 1 of 15 washings from 14 healthy control women (p=0.016). TSST-1 was not detected in the urine of 4 acute TSS patients, 2 convalescent TSS patients or in 3 control urine tested. A sensitive and reproducible ELISA was also developed for the quantitation of TSST-1 specific IgG in serum. Anti-TSST-1 was assessed in acute and convalescent sera from 16 nonmenstrual (9 female, 7 male) and 14 menstrual TSS patients, and from 87 healthy women and 66 healthy men as controls. Quantitative levels of anti-TSST-1 in the study groups were calculated as the percent of standard activity (POSA) relative to a medium titre reference serum standard. ELISA titers in acute sera from menstrual TSS (26.2 + 5.2, mean POSA + S.E.M.), but not nonmenstrual TSS women (71.8 + 18.6), were significantly lower than in healthy controls (78.9 + 7.3) (p<0.01, Mam-Whitney test). Titers from menstrual TSS patients remained low (25.2 + 10.7) even during late convalescence (mean duration 20 months after illness onset), compared to healthy female controls (p<0.05). Acute titers in males with TSS (37.0 + 15.6) were also significantly lower than those in control men (114.6 + 11.0) (p<0.05). An inverse relationship of recovery of toxigenic S. aureus and anti-TSST-1 titers in acute sera of TSS patients was observed. Ir±erest±ngly, antibody titers in control men were significantLy higher than in control women (p<0.001). No age-dependent effects or interactive effects of age and sex on ELISA titers were observed. To enable immunoblot analyses, TSST-1 was produced and partially purified using column chromatography techniques. Percent recovery of TSST-1 from culture supernatant through to the final procedure was approximately 15.5%. The relative purity of TSST-1 (TSST-l/total protein, w/w) was increased from 0.21% in culture supernatants to 94.4% in the final product. Ouchterlony i i i immunoprecdpitation against reference rabbit antitoxin demonstrated identity with reference TSST-1 as well as with TSST-1 prepared in other laboratories. Physical characterization demonstrated a molecular weight of 24 kd and a pi of 7.0. Using pooled normal human serum as a first antibody probe, several bands in addition to the 24 kd TSST-1 band were visualized by immunoblot against our partially purified toxin as well as similar preparations obtained from other investigators. To determine whether any of the additional bands might be implicated in TSS, acute and convalescent sera from TSS patients were used to probe for immunoreactive bands in our partially purified TSST-1 as well as a commercially obtained preparation. Seroconversion was demonstrated to the 24 kd TSST-1 protein in 7 of 10 TSS patients from whom toxigenic S. aureus was isolated, m addition, seroconversion was noted to a 49 kd band in 4 patients, to a 21 kd band in 3 patients, to a 28 kd band in 1 patient and to a 32 kd band in 2 patients. In conclusion: 1) the ability to measure TSST-1 in biologic fluids lends stronger support for the role of TSST-1 in menstrual TSS patients; 2) the serologic data support the etiologic role of TSST-1 in menstrual TSS and in nonmenstrual TSS patients from whom toxigenic S. aureus could be cultured, but not for nonmenstrual TSS women from whom toxigenic S. aureus was not isolated; 3) iinmunoblotting results with acute and convalescent sera from TSS and control patients, not only add further support to the role of TSST-1 in patients from whom toxigenic S. aureus could be isolated, but also indicate that there may be several other staphylococcal products implicated in TSS, particularly in whom antibody to TSST-1 pre-existed in acute sera. The nonresponsLveness or lack of seroconversion to TSST-1 in some patients could suggest either: a) TSST-1 was not the etiologic agent for such patients; b) TSST-1 was the etiologic agent, but the exposure was ^sufficient for an i v immune response (airular to tetanus), or; c) some immunologic defect may be present. Future studies are required to clarify these possibilities. v Table of Contents Page ABSTRACT i i List of Tables ix List of Figures x List of Abbreviations xii Acknowledgements xiiL L INTRODUCTION AND OBJECTIVES 1 JL LITERATURE REVIEW 3 A. History and Epidemiology 3 B. Characteristics of TSS-associated Staphylococcus aureus 5 C. Isolation and purification of TSST-1 6 D. Biologic effects of TSST-1 10 E. Serologic studies 11 F. Other possible agents involved in TSS 14 UL MATERIALS AND METHODS 16 A. TSST-1 DETECTION BY EKESA 16 1. Ammonium sulphate precipitation of reference rabbit anti-TSST-1 16 2. Staphylococcal enterotoxins 16 3. Conjugation of rabbit antL-TSST-1 to alkaline phosphatase 17 4. EKESA procedure for detection of TSST-1 17 5. TSST-1 detection in S. aureus culture supernatants 18 6. TSST-1 detection in vaginal washings of TSS patients and healthy controls 19 7. TSST-1 detection in urine 20 8. TSST-1 detection in serum 20 v i B. SEROLOGIC STUDIES BY ELISA 20 1. ELISA for TSST-1 specific IgG 20 2. Calculation of relative ELISA titers 21 3. Study populations 22 4. Detection of TSST-1 specific IgG and IgA in vaginal washings 22 C. IMMUNOBLOTTTNG ANALYSES 23 1. Purification of TSST-1 23 a. Preparation of media for TSST-1 production 23 b. Bacterial strain, growth and TSST-1 production 23 c. Ion exchange with SP-Sephadex C-25 24 d. Gel filtration with Sephadex G-75 Superfine 25 e. Protein and endotoxin measurement 25 f. SDS-PAGE analysis of products 26 g. Isoelectric focusing of partially purified TSST-1 26 2. Immunoblot analysis with human serum 27 a. Immunoblot procedure 27 b. TSS patient sera used for probing 27 3. Immunoblot analysis with rabbit serum 28 D. STATISTICAL METHODS 28 IV. RESULTS 29 A. TSST-1 DETECTION BY ELBA 29 1. Standardization of ELTSA procedure for TSST-1 detection 29 2. Reproducibility of the EKESA for TSST-1 detection 29 3. Specificity of the EKESA for TSST-1 detection 31 4. Elimination of protein A effect by normal rabbit serum 35 5. Detection of TSST-1 in culture supernatants of S. aureus isolates 35 vi i 6. Detection of TSST-1 in vaginal washings of TSS patients and healthy women 40 7. Detection of TSST-1 in urine 43 8. Standardization of ELISA for detection of TSST-1 in human serum 43 B. SEROLOGIC STUDIES 43 1. Standardization of ELISA for antL-TSST-1 detection in serum 43 2. Detection of TSST-1 specific igG in serum of TSS patients and healthy controls 46 3. Standardization for detection of TSST-1 specific IgG and igA in vaginal washings 49 C. IMMUNOBLOT ANALYSES 52 1. Purification of TSST-1 52 2. Comparison of four TSST-1 preparations by immunoblots probed with normal human serum 57 3. Comparison of immunoblots of TSST-1 preparations probed with acute and convalescent sera from 12 TSS patients 66 V. DISCUSSION 73 VL CONCLUSIONS 85 VUL REFERENCES 87 v i i i List of Tables Table Title Page 1 Case defmition of toxic shock syndrome. 4 2 Comparison of methods for TSST-1 purification. 8 3 Detection of TSST-1 in vaginal washings of menstrual TSS patients and healthy control women. 41 4 Quantitation of TSST-1 in vaginal washings of TSS and control women. 42 5 Comparison of ELISA rites (POSA) in acute sera of TSS patients with and without associated toxigenic S. aureus. 50 6 Relative purity of TSST-1 at each level of the purification procedure. 56 7 Immunoreactivity of acute and convalescent sera from TSS patients to partially purified TSST-1 in immunohlots. 72 List of Figures Figure Title Page 1 Reproducibility and specificity of ELISA for TSST-1 detection. 30 2 Immunoblot of reference TSST-1 with rabbit antiserum. 32 3 Effect of added protein A and normal rabbit serum on detection of TSST-1 in BHI broth. 36 4 Effect of 10 % normal rabbit serum on elimination of interference by protein A during TSST-1 detection in culture supernatants by ELISA. 37 5 Comparison of quantitative TSST-1 production by toxigenic S. aureus vaginal isolates from TSS patients and controls. 39 6 Neutralization of TSST-1 specific IgG in serum following pretreatment with TSST-1. 44 7 Standard curve for predicting TSST-1 specific IgG titers (POSA). 45 8 TSST-1 specific relative ELISA titers in acute, early convalescent and late convalescent sera of TSS patients and controls. 47 9 Demonstration of dose-response relationship in the detection of TSST-1 specific IgG and IgA in vaginal washings. 51 10 Ion exchange chromatography of TSST-1 from culture supernatant on SP-Sephadex C-25. 53 11 Rechromatography of TSST-1 on SP-Sephadex C-25. 54 12 Gel filtration of TSST-1 through Sephadex G-75 Superfine. 55 13 SDS-PAGE analysis of partially purified TSST-1 at each stage of the purification procedure. 58 14 Demonstration of immunologic identity of TSST-1 preparations. 60 15 immunoblot analysis of four TSST-1 preparations, probed with pooled normal human serum. 61 x l ist of Figures (cont) Figure Title Page 16 immunoblot analysis of partially purified TSST-1 preparations, probed with acute and convalescent patient sera. 68 xi LIST OF ABBREVIATIONS BHI Brain Heart Infusion ELISA Enzyme-linked immunosorbent assay IL-1 Interleukin-1 IRS Immune rabbit serum kd kilodalton LD^Q Lethal dose for 50% of experimental animals LPS Lipopolysaccharide NRS Normal rabbit serum PBS Phosphate-buffered saline PEC Pyrogenic exotoxin C pi Isoelectric point POSA Percent of standard activity SDS-PAGE Sodium dodecyl sulphate (SDS) - polyacrylamide gel electrophoresis SEF Staphylococcal enterotoxin F TSS Toxic shock syndrome TSST-1 Toxic shock syndrome toxLn-1 TBS Tris-buffered saline xi i ACKNOWLEDGEMENTS I gratefully acknowledge the guidance, support and encouragement of Dr. Anthony W. Chow throughout the course of this study. I also wish to thank Karen H. Barrlett for her greatly appreciated technical advice and patience. This thesis is dedicated to Pete. xi i i L INTRODUCTION AND OBJECTIVES Toxic shock syndrome (TSS), first described by Todd et al in 1978, occurs primarily in women, is associated with menstruation and tampon use and is believed to be mediated by TSS toxin-1 (TSST-1). This toxin, identified as a 22-24 kd protein with an isoelectric point (pD of approximately 7.2, is produced by 85-100% of vaginal Staphylococcus aureus isolates associated with menstrual TSS (8,21,66). m contrast, only 16-23% of isolates from healthy control women or non-TSS cases of S. aureus infection produce TSST-1 (54). More recently, cases of TSS that are not associated with menstruation are increasingly recognized in both female and male patients (28,55,56). Nonmenstrual TSS is usually associated with focal S. aureus wound or soft tissue infections, and also occurs more commonly in women (16,55). m contrast to menstrual TSS vaginal isolates, only 62-76% of S. aureus isolates from nonmenstrual TSS are positive for TSST-1 (28,35,54). S. aureus isolates from nonmenstrual and menstrual TSS cases also differ phenotypically in other characteristics (19), suggesting different origins or pathogenesis in these two forms of TSS. For these reasons, the etiology of TSST-1 in nonmenstrual TSS is uncertain. To further clarify the role of TSST-1 in the pathogenesis of TSS, three main objectives of study were pursued. First, a simple, sensitive and specific enzyme-linked immunosorbent assay (ELISA) method was developed, capable of detecting TSST-1 at concentrations of 0.5 to 16 ng/ml in culture supernatants and biologic fluids. The ability to detect TSST-1 in biologic fluids is of utmost importance in order to implicate TSST-1 as the cause of the clinical manifestations of toxic shock syndrome. Second, in order to study the serologic response to TSST-1, a sensitive and reproducible ELISA method for the quantitation of serum IgG specific for TSST-1 was developed. Working with the 1 hypothesis that TSS patients may be susceptible because they have sub-protective levels of TSST-1 specific antibody at onset, antibody responses to TSST-1 were determined in acute and convalescent sera of 16 nonmenstrual (9 female and 7 male) and 14 menstrual TSS patients, as well as in random sera of 87 healthy women and 66 normal male blood donors. A third objective was to seek other factors which may be implicated in those TSS patients whose illness could not be readily attributed to TSST-1. immunoblottLng techniques were employed for this purpose, using partially purified TSST-1 preparations and probed with acute and convalescent sera from TSS patients. With these tools, and a collection of patient specimens with cQinical information, an in-depth study of a group of TSS patients could be accomplished with the hope that the role of TSST-1 in toxic shock syndrome would be better defined and understood. 2 IL Literature Review A. History and Epideiniolagy Todd et al first described the occurrence of toxic shock syndrome in 1978 in children as an illness that was associated with Staphylococcus aureus and toxin mediated (81). The syndrome is now recognized to occcur mainly in young mensrxuating women, although many cases of TSS occurring in nonmenstruating women as well as in men and children are frequently reported (19,56). The case definition of TSS as described by the Center For Disease Control (Atlanta, Georgia) is generally adopted (Table 1) (14). Typical menstrual TSS is clearly associated with menstruation; nonmenstrual TSS in women is not associated with menstruation and the age of onset is generally older compared to menstrual TSS. Menstrual TSS is associated with vaginal infection or colonization by S. aureus, in contrast to nonmenstrual TSS in women and TSS in men, in which the syndrome is usually associated with focal S. aureus wound or soft tissue infections (14). Nonmenstrual TSS also occurs more frequently in women than it does in men (55). The Center For Disease Control (CDC) reported that by April 9, 1982, 96% of 1660 patients with confirmed TSS were women and 92% of these were menstruating at the time of onset of their illness (14). The mean age of patients with menstrual TSS was reported to be 21 years and there was a very low incidence of illness in non-caucasLan women. In addition, a definite geographical pattern in North America with respect to incidence of the syndrome was observed (15). Recurrence in menstrually related TSS has been reported to occur at a rate of at least once in 34% of untreated individuals (27). Discontinuation of tampon use and the use of anti-staphylococcal therapy have been shown to greatly reduce the rate of recurrence (27). Pathologic studies have 3 Table 1. Case D e f i n i t i o n of Toxic Shock Syndrome. 1. Temperature _>• 3°-9°C 2. Rash 3. Desquamation 4. Hypotension ( s y s t o l i c blood pressure <90 mm Hg, syncope, or o r t h o s t a t i c decrease in s y s t o l i c blood pressure.>15 mm Hg). 5. C l i n i c a l or laboratory abnormali t ies of >3 organ systems: gast roi ntes t i na1 hepat i c muscular rena 1 card iovascu lar neurolog i c mucous membrane ( v a g i n i t i s , p h a r y n g i t i s , c o n j u n c t i v i t i s ) 6. Reasonable evidence for absence of other e t i o l o g i e s . * De f in i t e TSS ( a l l c r i t e r i a present) Probable TSS ( a l l but one c r i t e r i o n present) Modif ied from: Follow-up on tox ic shock syndrome. MMWR 1980; 29:441-445. 4 demonstrated that the S. aureus strains associated with TSS are relatively non-invasive (4). i t was also demonstrated that in surgical cases of TSS, the toxin producing strains were isolated from generally uninflamed wounds. ]£ has also been shown that antL^teichoic acid antibodies are absent in the convalescent sera from TSS patients, supporting the non-invasive characteristic of these strains (40). Death among TSS patients has mainly been due to refractory cardiac arrhythmias, irreversible respiratory failure, and hemorrhaging due to coagulation defects (49). The mechanism of action of the toxin has not been clarified, but the toxin is thought to be responsible directly for vasodilation and capillary leakage as well as mucous membrane and skin manifestations. It is also suspected that the toxin can cause irreversible neuromuscular system changes (60). B. Characteristics of TSS associated S. aureus There has generally been a very high incidence of TSST-1 producing S. aureus strains isolated from the vagina or cervix of menstrual TSS patients (85 to 100%) (8,21,66). In contrast is the lower incidence of TSST-1 production (62 to 76%) among S. aureus isolated from nonvaginal sources. Other phenotypic differences are present between menstrual and nonmenstrual TSS isolates (19). Such differences include decreased 8-hemolysis of sheep blood in agar by genital TSS isolates compared to non-genital TSS isolates, suggesting that there may be differences in pathogenesis between menstrual and nonmenstrual TSS. Susceptibility to mercury and resistance to cadmium, arsenate and penicillin are also common features of TSS-associated S. aureus strains. Among non-TSS S. aureus, this same pattern is seen in only 42% of strains (3). Lysogeny also seems to be a common feature of TSS-associated S. aureus. Schutzer et al (73) demonstrated lysogeny by a common bacteriophage in 11 of 12 TSS S. 5 aureus isolates compared with the presence cf temperate phage in only 1 of 18 control S. aureus strains. It is hypothesized that the TSS-associated strains may have a specific receptor for this particular group of phage (73). Phage typing has demonstrated that the majority of TSS-associated S. aureus strains belong in group predominantly types 29 and 52 (1). C. Isolation and purification of TSST-1 Two exoproteins were simultaneously isolated from TSS-associated 5. aureus strains by two groups of researchers in 1981. The two proteins had similar molecular weights and isoelectric points and were believed to be the etiologic agent responsible for TSS. Pyrogenic exotoxin C (PEC) was isolated by Schlievert et al (72). Characterization of the toxin demonstrated an isoelectric point of 7.2 and a molecular weight of 22,000 daltons. Schlievert also reported that 100% of the TSS-associated strains of S. aureus produced PEC. Staphylococcal enterotoxLn F (SEF) was isolated by Bergdoll et al (8) also in 1981 and bore many sinularities to PEC. Bergdoll reported that 93.8% of S. aureus strains from TSS patients produced this marker protein with a molecular weight of 20,000 daltons and isoelectric point of 6.8, while only 4.6% of S. aureus from other clinical sources produced this toxin. A pivotal study by Bonventre et al (13) demonstrated that these two proteins were identical, and this protein was subsequently designated toxic shock syndrome toxin-1, or TSST-1 (TSS Symposium, June 1984, Madison, Wisconsin). Schlievert et al (69) studied the effect of different physical and chemical factors on growth and TSST-1 production, attempting to reproduce the conditions which might occur in the vagina during menses and determined conditions for maximal toxin production in vitro. It was found that while growth was enhanced slightly at 37 °C compared to 30 °C, TSST-1 production increased ten-fold. Vigorous aeration also allowed a 32-fold increase in toxin 6" production and a 2-fold increase in growth compared with anaerobic conditions. Conditions at pH 7 and 8 allowed maximal growth and toxin production while pH 5 or 9 permitted little. There was also an effect due to glucose; glucose levels higher than 0.3% caused inhibition of both growth and toxin production while levels lower than this were permisLve. In addition, it was demonstrated that complex media such as brain heart infusion (BHD broth allowed the greatest TSST-1 production in liquid culture. Several methods for growing S. aureus for TSST-1 production have been used. Membrane-over-agar techniques, growth in semi-^olid agar and sac culture methods allow production of small quantities of much more concentrated TSST-1. The broth culture method is generally preferred if large quantities are to be produced and is less labour intensive. Several schemes for the purification of TSST-1 have been used and have been summarized in Table 2 with respect to the S. aureus strain, TSST-1 characteristics (molecular weight, pD and purification scheme. Of the isolates used for TSST-1 production, the majority are either Bergdoll's FRI 1169 high toxin producing strain or Schlievert's 587 and MN8 strains as these have been the most thoroughly characterized. Methods used fall into the categories of either column chromatographic techniques, of which there are many variations, or Schlievert's method which utilizes an initial ethanol precipitation followed by preparative isoelectric focusing. The main consideration during a purification procedure is to prevent unnecessary exposure of the protein to harsh chemicals or conditions which may result in loss of immunogemcity or molecular configuration. For this reason, ethanol precipitation and isoelectric focusing are suspect and chromatographic techniques, which separate by size and charge without subjecting the protein molecules to denaturing chemicals, are much 7 T a b l e 2. C o m p a r i s o n o f methods o f TSST-1 p u r i f i c a t i o n A u t h o r ( r e f . ) P u r i f i c a t i o n P r o c e d u r e . a u r e u s s t r a i n M o l e c u l a r w e i g h t (kd) I s o e l e c t r i c p o i n t ( p l ) S c h l i e v e r t (72) B a r b o u r (3) Bergdol1 (7) Notermans m Re i s e r (57) B o n v e n t r e (13) I g a r a s h i (35) Pa r s o n n e t (51) Reeves (54) Reeves (53a) EtOH p r e c i p . I EF EtOH p r e c i p . I EF Ion exchange Gel f i l t r a t i o n Ion exchange G e l f i l t r a t i o n Ion exchange G e l p e r m e a t i o n a) Ion exchange b) EtOH p r e c i p . I EF Ion exchange Chromato foeus Gel f i l t r a t i o n EtOH p r e c i p . I EF EtOH p r e c i p . C h r o m a t o f o e u s 587, MN8 TSS s t r a i n FRI 1169 FRI 1183 FRI 1169 FRI 1169 587 FRI 1169/587 587 TSS s t r a i n s A f f i n i t y p u r i f . TSS s t r a i n s 22 22 20 23 24 20-24 20-24 24 23.1 21 .4-22 .1 21.4-22.1 7-2 7-0 6.8 7.2 7.0 6.8-6.9 6.8-6.9 7.0 7.2 7.1-7.2 7.1-7.2 EtOH p r e c i p . : e t h a n o l p r e c i p i t a t i o n IEF : p r e p a r a t i v e i s o e l e c t r i c f o c u s i n g Ion exchange : s e v e r a l v a r i a t i o n s o f t e c h n i q u e s used C h r o m a t o f o c u s : c h r o m a t o f o c u s i n g -v A l l a u r e u s s t r a i n s a r e T S S - a s s o c i a t e d i s o l a t e s . FRI 1169 and 13 83 a r e f rom the Food R e s e a r c h I n s t i t u t e , U n i v e r s i t y o f W i s c o n s i n , M a d i s o n , S t r a i n s 587 and MN8 a r e f rom P . M . S c h l i e v e r t , U n i v e r s i t y o f M i n n e s o t a , M i n n e a p o l i s . 8 production and a 2-fold increase in growth compared with anaerobic conditions. Conditions at pH 7 and 8 allowed maximal growth and toxin production while pH 5 or 9 permitted little. There was also an effect due to glucose; glucose levels higher than 0.3% caused inhibition of both growth and toxin production while levels lower than this were permisive. In addition, it was demonstrated that complex media such as brain heart infusion (BHD broth allowed the greatest TSST-1 production in liquid culture. Several methods for growing S. aureus for TSST-1 production have been used. Membrane-over-agar techniques, growth in semi-solid agar and sac culture methods allow production of small quantities of much more concentrated TSST-1. The broth culture method is generally preferred if large quantities are to be produced and is less labour intensive. Several schemes for the purification of TSST-1 have been used and have been summarized in Table 2 with respect to the S. aureus strain, TSST-1 characteristics (molecular weight, pD and purification scheme. Of the isolates used for TSST-1 production, the majority are either Bergdoll's FRI 1169 high toxin producing strain or Schlievert's 587 and MN8 strains as these have been the most thoroughly characterized. Methods used fall into the categories of either column chromatographic techniques, of which there are many variations, or Schlievert's method which utilizes an initial ethanol precipitation followed by preparative isoelectric focusing. The main consideration during a purification procedure is to prevent unnecessary exposure of the protein to harsh chemicals or conditions which may result in loss of immunogenicity or molecular configuration. For this reason, ethanol precipitation and isoelectric focusing are suspect and chromatographic techniques, which separate by size and charge without subjecting the protein molecules to demturing chemicals, are much preferred. 9 The effect of magnesium ions on TSST-1 production has also been studied. Mills et al (46) reported in 1985 that in vitro production of TSST-1 was enhanced by using medium free of Mg for S. aureus growth. The role of tampons in TSS was also linked to the ability of some tampon fibres to chelate available Mg present in vaginal secretions, thus creating more permissive conditions for S. aureus growth and TSST-1 production in the vagina. However, Mills and co-workers' findings were contradicted by findings (68) that production of TSST-1 is not affected by high levels of Mg . Schlievert et al (68) found that magnesium concentrations as high as 300 ug/ml did not alter cell numbers or toxin concentrations compared to lower levels. Further work by Mills et al (44,45) demonstrated that production of extracellular proteins, indnding TSST-1, is increased when S. aureus is grown in Mg deficient medium. It is evident from these apparently different findings that the role of j | Mg in TSS requires further research. D. Biologic effects of TSST-1 Since Schlievert reported in 1981 that TSST-1 could enhance lethal endotoxic shock in rabbits by a factor of 50,000 (66), possible synergistic actions of TSST-1 and endotoxin have been suggested (66,72). Schlievert has suggested that TSST-1 may act to suppress the host immune response, thereby allowing the growth of opportunistic bacteria which provide endotoxin. The possible involvement of endotoxin also arises from the finding that there is frequent co-isolation of E. coli and toxigenic S. aureus from the vaginas of menstrual TSS patients (22). Of interest is the finding by Sugiyama et al (79) in 1983 that mortality in mice and rabbits administered a highly purified staphylococcal enterotoxin B preparation was significantly increased if animals were pretreated with endotoxin, compared to animals not pretreated with endotoxin. The enterotoxin itself had no apparent effect on the animals. 10 Biological activity was gauged by measuring pyrogenicity and the capacity to enhance lethal endotoxic shock, and myocardial damage in rabbits. Schlievert reported that, while the LD 5Q of endotoxin alone in rabbits was approximately 500 ugAg, death could result from as little as 5 [xq/kg of endotoxin when just 0.1 ug/kg of TSST-1 was co-adndnistered. Fever was also produced when as little as 0.1 ugA<3 of exotoxin alone was given. Nonspecific lymphocyte mitogenicity due to the toxin was also demonstrated at doses as low as 0.01 ng/ml. Maximal suppression of the IgM antibody response by murine splenocytes was observed at TSST-1 doses of 1.0 and 0.1 ng/ml. A key study performed by Kushnaryov et al demonstrated the ability of TSST-1 to bind cultured epithelial cells and be rapidly internalized (37). It has previously been observed that S. aureus strains involved in TSS were relatively noninvasive and, although colonization of the vaginal epithelium by S. aureus occurs in menstrual TSS, these organisms themselves do not adhere to the epithelial surface to an extent greater than do control strains (40). Kushnaryov's study involved incubation of human epithelial cells with iodinated TSST-1 in order to measure the rate of binding and uptake. It was concluded that TSST-1 binding to epithelial cells occurs through high-affinity specific receptors, although such receptors have not yet been isolated or characterized. In addition, Kushnaryov suggests that following receptor mediated endocytosis, internalized TSST-1 may be transported to other epithelial cells via some special transport vesicles. At this time, there is still no suggested possible mechanism for the cytopathic action of TSST-1 against epithelial cells to explain the clinical manifestations of illness. E. Serologic Studies Several studies have been reported in which the prevalence of TSST-1 antibodies in the serum of TSS patients and control populations have been 11 assessed. Vergeront et al (83) reported in 1983 that the absence of antibody to TSST-1 was a good indicator of susceptibility to TSS. He also showed that the prevalence of TSST-1 antibodies in serum varies with age. At age 20, i t was estimated that approximately 87% of the population has a high titer of TSST-1 specific antibody. However, Vergeront's definition of what titer constitutes a positive and negative titer and the rationale behind the definition is somewhat obscure, especially when statistical analyses are performed partially with a 1:5 dilution as a negative cutoff and then at other times using a 1:100 dilution. A study by Notermans et al (48) confirmed Vergeront's findings of the high prevalence of TSST-1 antibodies in the healthy population (91%), indicating widespread exposure to the toxin. In addition, it was observed that TSS patients had lower levels of serum antitoxin than controls, and that increase in antibody levels subsequent to illness was rarely seen. Notermans' method of antibody quantitation was very rudimentary, using non-standardized absorbance values obtained at a single serum dilution to compare different sera. Absorbance values are not a sensitive means of qnantitating antibody levels, as absorbance values are not linearly proportional to antibody titers. Another study reported by Ritz et al (58) in 1984 demonstrated that serum antibody levels were higher among individuals with nasal carriage of TSST-1 producing S. aureus compared to non^arriers. The main observation made was that individuals carrying TSST-1 producing S. aureus nasally had, as a group, significantly higher serum anti-TSST-1 titers than did those individuals who carried non-toxLgenic strains or no S. aureus. However, Ritz's study included only male subjects. In addition, there may be error in assuming that nasal exposure to TSST-1 and the resultant immune response can be directly correlated to vaginal exposure to TSST-1 which presumably occurs in menstrual TSS. Also, antibody titers were expressed as relative OD values which, as discussed earlier, are not linearly proportional to 1 2 antibody titers. In a follow-up study to that of Vergeront, Stolz et al (78) described the development of serum antibody to TSST-1 in TSS patients. In acute sera, 91% of menstrual TSS patients had negative titers. Nonmenstrual TSS patients had a significantly higher age of onset of illness than did menstrual TSS patients and a smaller proportion of nonmenstrual patients had negative antibody titers «1:5) (64% versus 91%). However, Stolz et al failed to report the proportions of male and female nonmenstrual TSS patients that were studied; thus any sex-specific differences within the nonmenstrual TSS group would be obscured by treating all as one group. It was also suggested in this study that the reason for higher antibody titers in nonmenstrual patients might be due to exposure to large doses of TSST-1, in spite of the fact that many nonmenstrual TSS isolates do not produce TSST-1. Seroconversion was noted in a significantly greater number of menstrual TSS patients less than 17 yrs of age compared to older patients. Seroconversion was not studied in nonmenstrual TSS patients. It is generally felt that low or absent antibody to TSST-1 is a marker for susceptibility to TSS (48,58,78). Davis (26) has demonstrated that immunization of rabbits with TSST-1 confers protection from death as a result cf infection with TSST-1 producing S. aureus isolated from patients with TSS. Furthermore, Melish et al (Abstr. ASM Ann Meeting, Las Vegas, Nevada, 1985) has demonstrated the protective nature of anti-TSST-1 antibodies in rabbits in which immune rabbit antisera, administered up to 24 h after challenge with lethal doses of TSST-1, prevented death. Reasons for failure to seroconvert against TSST-1 among TSS patients are unknown. Possibilities include the hypothesis that numerous exposures may be necessary to induce anti-TSST-1 production (75). It has also been suggested that the vaginal route of exposure in menstrual TSS patients may not be as effective 13 in eliciting an immune response compared to other routes of exposure (63). A protective level of antibodies to TSST-1 has not yet been clearly established. Judging from all of the serologic studies above, the association of TSST-1 in menstrual TSS seems to be quite strong. Lacking from the literature is cQarification of the role and association of TSST-1 in nonmenstrual TSS. F. Other possible agents involved in TSS There is increasing evidence that TSST-1 may not be responsible for some cases of nonmenstrual TSS, although its role in menstrual TSS is relatively undisputed. It has also been reported that Staphylococcus species other than S. aureus have the ability to produce TSST-1 (25). Garbe at al (31), studied S. aureus strains from patients with nonmenstrual TSS. It was found that isolates from nonmenstrual TSS patients were less likely to produce TSST-1 compared to isolates associated with menstrual TSS. The case fatality rate was higher among nonmenstrual TSS patients associated with TSST-1 negative S. aureus isolates. When rabbits were inoculated with TSST-1 negative S. aureus, fatality was observed and the cQinLcal illness was the same as that observed in rabbits challenged with TSST-1 positive strains. It was suggested that other toxin (s) may be involved in nonmenstrual TSS. Of interest is a recent report by Scott et al (74) which describes the presence of a 30 kd protein produced by a strain of TSST-1 negative S. aureus isolated from a patient with nonmenstrual TSS. This protein cross-reacts immunologically with TSST-1 by Western blot and the isolate causes TSS-like illness in rabbits. Scott and co-workers have proposed that this protein be designated TSST-2, although they had not attempted to isolate or purify the protein. The possibility that this 30 kd protein may have been a contaminant cf the TSST-1 preparation used to immunize the rabbits for antiserum production was not addressed, even though the toxin preparation used 14 to demonstrate presence of 24 and 30 kd immunoreactive bands by Western blot was the same preparation used for antisera production in the rabbit. It has also been suggested that TSST-1 may induce the production of monocyte products, such as interleukin-1 GL—1) and cachectin (50). CachectLn, or tumour necrosis factor, is an interesting cytokine described more recently by Beutler et al (10). These investigators demonstrated that passive immunization of mice against this protein can prevent endotoxin-induced death (11). It is possible that many of the cQinical mariifestetions of TSS may be mediated by host-produced substances such as IL-1 and cachectin which may be induced by TSST-1 or some other microbial factor(s). 15 UL MATERIALS AND METHODS A. TSST-1 DETECTION BY ELISA 1. Ammonium sulphate precipitation of reference rabbit anti-TSST-1 Reference rabbit antibody to TSST-1 (1:4 diluted stock) was provided by Merlin Bergdoll (University of Wisconsin, Madison). Additional rabbit anti-TSST-1 was obtained from actively immunized New Zealand white rabbits following graded intravenous challenge with purified TSST-1 according to the dose schedule of Bergdoll et al (8). Immunoglobulin fractions of all sera to be used were prepared by precipitation with saturated ammonium sulphate according to standard procedure (84). Briefly, 0.5 ml of saturated ammonium sulphate, pH 7.8, was added drop wise to 1.0 ml of serum and then mixed for 2 h at room temperature. The precipitated protein was centrifuged at 1400 x g for 30 minutes at room temperature, the supernatant discarded and the pellet redissolved in 1 ml of sterile saline. This procedure was repeated twice, and the final pellet was dissolved in sterile phosphate buffered saline (PBS), pH 8.0, and dialyzed (12-14 kd molecular weight exclusion) against PBS, pH 8.0, at 4°C for 48 h with four exchanges of buffer. Protein determinations were performed according to the method of Lowry et al (42). 2. Staphylococcal enterotoxins Reference TSST-1 was provided by Merlin Bergdoll (University of Wisconsin, Madison). In addition, purified staphylococcal enterotoxins A, B, C^, Q^, Cy TSST-1 and crude preparations of SED and SEF, as well as their respective rabbit antisera, were obtained from commercial sources (Toxin Technology Inc., Madison, Wise.). 16 3. Conjugation of rabbit antL-TSST-1 to alkaline phosphatase Ammonium sulphate precipitated anti-TSST-1 was conjugated to alkaline phosphatase accorrling to the method of Voller et al (84). One mg of precipitated anti-TSST-1 and 2.5 mg of alkaline phosphatase enzyme (Cat. no. P-0405, Sigma Diagnostics, St, Louis, Mo.) were mixed, glutaraldehyde (Cat.No. G-5882, Sigma Diagnostics) was added to a final concentration of 0.2% and the mixture was stirred for 2 h at room temperature to allow conjugation. The conjugate was then dialyzed (12-14 kd molecular weight exclusion) against PBS, pH 7.0, for 24 h with two changes of buffer to facilitate removal of residual glutaraldehyde. Further dialysis was performed against 0.05 M TrLs-chloride buffer, pH 8.0, for 24 h with two changes of buffer. The conjugate was then diluted to 2 ml with sterile 0.05 M Tris buffer pH 8.0, containing 1% bovine serum albumin (BSA) and 0.02% sodium azide, and stored at 4°C in the dark. Little reduction in activity of the conjugate was seen for up to 8 months storage at 4°C. 4. ELISA procedure for detection of TSST-1 A noncompetitive, antibody enzyme-conjugated ELISA procedure used for TSST-1 detection was developed. Ammonium sulphate precipitated reference rabbit antL-TSST-1, 100 ul per well, as well as nonimmune control sera, diluted in 0.05 M carbonate buffer, pH 9.6, were passively absorbed to the inner wells of polystyrene microtiter plates CEmmunolon I1*, Dynatech Laboratories, Alexandria, Va.) by incubation at 20°C for 18 h. Unbound antibody was removed by three 2 min washes with PBS containing 0.05% Tween 20, pH 7.4 (PBS-T). Purified TSST-1 reference standard (serially diluted from 16.0 to 0.5 ng/ml in PBS-T), test samples pretreated with normal rabbit serum (10% final concentration, v/v) to etiminate possible sources of protein A, diluted in PBS-T if necessary, and additional reagent controls were pipetted in duplicate in 100 17 ul volumes to their respective wells. This was followed by incubation at 37 °C for 2 h. The plates were then washed as before to remove unbound toxin. The conjugate was diluted in PBS-T and 100 ul was added to each well, followed by another 2 h incubation at 37 °C. The plates were then washed to remove unbound conjugate. The chromogenic enzyme substrate p-rdtrophenyl phosphate, at a concentration of 1 mg/ml in 10 % diethanolamine buffer (BDH, Poole, England), pH 9.8, was added in 100 ul volumes to each well followed by a final incubation at 37°C for 90 min to allow the enzyme reaction to proceed. The intensity of the yellow colour produced was read at 405 nm in a Titertek Mulriscan spectrophotometer (Flow Laboratories, McLean, Va.). Linear regression analyses of the standard reference toxin concentrations were calculated by plotting absorbance (405 nm) versus log 2 (TSST-1 concentration). TSST-1 concentrations in test samples were predicted from the reference toxin regression equations derived during each assay procedure. The specificity of the ELISA for TSST-1 was examined both by inclusion of nonimmune rabbit serum lacking anti-TSST-1, by use of immune rabbit serum pretreated with graded concentrations of purified TSST-1, and by exanunation of possible cross-reactivity with other staphylococcal enterotoxins, including enterotoxins A, B, C-^ , Cy C3' D E ( T o x m Technology Inc., Madison, Wis.) in concentrations of 1000, 100 and 10 ng/ml. 5. TSST-1 detection in S. aureus culture supematants The S. aureus isolates tested for in vitro TSST-1 production were vaginal isolates from TSS patients or healthy control women. Culture supematants were prepared from subculture of S. aureus grown in brain heart infusion (BHD broth (Difco Laboratories, Detroit, Mich.) with shaking at 37°C for 18-20 h in ambient atmosphere. Cells were removed by centrifugation (30,000 x g for 15 min) and culture supernatant filter-sterili zed and frozen at -70°C prior to 18 testing. Each culture supernatant was tested for TSST-1 both by Ouchterlony immunodiffusion and by ELISA with and without overnight preincubation at 4°C with normal rabbit serum (10% v/v). Pretreated test samples were tested by ELISA at neat, 1:10 and 1:100 dilutions in PBS-T. Detection of TSST-1 by Ouchterlony immunodiffusion was examined in 1% Noble agar (Difco Laboratories) with reference rabbit antisera (1:36 dilution) in the center well and culture supernatants, concentrated according to the method of Schlievert et al (72), in the outer wells. 6. TSST-1 detection in vaginal washings of TSS patients and healthy controls Vaginal washings were collected from 9 patients who fulfilled the case definition for menstrual TSS according to the Center for Disease Control, and were studied in Vancouver between August 1980 and February 1985 (22). Washings were also collected from 14 healthy control women from the University of British Columbia Student Health Service. Informed consent was obtained from all subjects. Vaginal washings were obtained with 10 ml sterile pyrogen free saline from the posterior vaginal fornix using a plastic pipette under direct visualization. The washings were immediately centrifuged (90 x g for 10 min at 4°C) and the supernatant filter-sterilized and stored at -70°C. Quantitation of TSST-1 was performed using the above ELISA method with several additional controls. Vaginal washings to be tested for TSST-1 were diluted 1:2, 1:4 and 1:8 in PBS-T and examined in duplicate in 100 ul volumes. Vaginal washings giving positive results were retested with and without 10% normal rabbit serum pretreatment. Pooled vaginal washings devoid of TSST-1 from healthy control women (C-0) served as a negative control and pooled washings with 50 ng/ml TSST-1 added served as a positive control (C-50). These were diluted 1:2, 1:4 and 1:8 in PBS^T and tested in 100 ul volumes in duplicate. A third control for specificity consisted cf reference rabbit 19 antL-TSST-1 added to the C-50 control; this was also diluted and tested in the same manner. 7. TSST-1 detection in urine of TSS patients and healthy controls Urine specimens were collected from 5 female TSS patients studied in Vancouver between August 1980 and February 1985 and stored at -70°C prior to testing. Urine obtained from 12 healthy individuals were pooled and served as a negative control. As a positive control, reference TSST-1 was added to the normal pooled urine and a standard curve was generated with the same concentrations of TSST-1 (0.5 to 16 ng/ml) used to generate a standard curve in PBS-T. Urine specimens to be tested were not pretreated in any way and were tested at neat, 1:2 and 1:4 dilutions in 100 ul volumes in duplicate. 8. Standardization of ELISA for detection of TSST-1 in serum Normal rabbit serum was spiked with reference TSST-1 and tested in parallel with TSST-1 in PBS-T at concentrations of 0.5 to 16 ng/nu, in addition, serum from several healthy human individuals was spiked with TSST-1 at concentrations of 1.0 and 10 ng/ml and tested neat in 100 ul volumes in duplicate. B. SEROLOGIC STUDIES BY ELISA  1. ELISA for TSST-1 specific IgG The internal 60 wells of a Linbro polystyrene ndcrxitLter plate (Flow Laboratories, McLean, Va.) were filled with 100 ul of either a solution of TSST-1 (50 ug/ml, stock SEF 50, lot FT-11, supplied by M.S. Bergdoll, University of Wisconsin) in a carbonate-bicarbonate coating buffer at pH 9.6, or coating buffer alone. The plate was incubated for 18 h at room temperature in a humid chamber. The wells were then washed repeatedly with phosphate buffered saline (PBS, pH 7.2) containing 0.05% Tween 20 (Sigma, St. Louis, 20 Missouri),(PBS-T). Sera to be tested along with a reference serum standard containing a medium level of antibody to TSST-1 (medium standard reference serum) were diluted 1:1000 in PBS-T containing 1% bovine serum albumin (PBS-^-BSA) and added in 100 ul volumes to wells previously treated with TSST-1 in coating buffer, and wells treated with coating buffer alone. The plate was incubated at 37 °C for 2 h, after which time the wells were washed with PBS-T as before. Each of the 60 wells was then filled with 100 ul of a 1:1000 dilution of alkaline phosphatase-conjugated goat anti-human immunoglobulin G (heavy chain specific, Sigma # A-3150) in PBS-T. The plate was again incubated at 37°C for 2 h, and washed with PBS-T. The chromogenic substrate p-nitrophenyl phosphate (Sigma #104-105) in a 1 mg/ml solution in 10% diethanolamine buffer (BDH Chemicals, Poole, England), pH 9.8, was added in 100 ul volumes to all wells, and the plate was incubated at 37°C for 30 niin. Colour reactions developed were measured at a wave length of 405 nm in a Titertek Mulriscan spectrophotometer (Flow Laboratories). Wells containing only the enzyme substrate were used to blank the spectrophotometer. The net optical density (OD) of each serum was calculated by subtracting the mean OD of the two wells treated with coating buffer alone from the two wells treated with TSST-1. The relative OD of each serum was calculated by dividing its net OD by the net OD of the reference standard serum and multiplying the ratio by 1000. With this procedure, values for sera run in different experiments could be more readily compared. 2. Calculation of relative ET.TRA titers To calculate the relative ELISA titer, a standard curve of the percent of standard activity (POSA) as modified from Feigner (30) was used. The curve was constructed from the relative ODs derived from serial two-fold dilutions of the medium standard reference serum beginning with 1:1000. Linear regression 21 analysis was performed on these data by plotting the dog POSA) (Y-axis) versus the Qog relative OD) (x-axis). 3. Study populations in serologic studies All menstrual and nonmenstrual TSS patients fulfilled the case definition for TSS according to the Center for Disease Control and were studied in Vancouver between 1980 and 1985 (22). All 14 menstrual TSS patients experienced onset of illness during menses. Of the 16 nonmenstrual TSS patients, 7 were male and 9 were female (3 menopausal, 2 postpartum, 1 neonatal and the remaining 3 with onset of illness at least 7 days prior to or after onset of menses). The control populations consisted of 87 volunteer women (mean age + S.D., 28.8 + 10.6 yr; including 53 women recruited from the University of British Columbia Student Health Service, and 34 consecutive female blood donors attending the Vancouver Red Cross Clinic during 1985) and 66 consecutive male blood donors (mean age + S.D., 34.9 + 10.3 yr). All sera were stored at -70°C until testing. 4. Detection of TSST-1 specific IgG and IgA in vaginal washings Linbro iaLcrctiter plates (Flow Laboratories) were coated with 100 ul/well of 100 ng/ml solution of reference TSST-1 in carbonate buffer, pH 9.6, and incubated at room temperature overnight. To generate a standard curve, pooled vaginal washings, previously concentrated 20 times by lyophilization, were serially diluted in PBS-T, and 100 ul volumes of each dilution was added in duplicate to coated wells. Plates were incubated 2 h at 37°C then washed with PBS-T. Samples to be tested were diluted 1:100 and 1:1000 and tested in duplicate. Goat anti-human IgG conjugated to alkaline phosphatase (Sigma #A-3150) or goat anti-human IgA (heavy chain specific, Sigma #A-3400) diluted 1:500 was added in 100 ul volumes to each well, followed by another incubation at 37°C. Enzyme substrate p-nitrophenyl 22 phosphate (Sigma, #104-105), 1 mg/ml in 10% diethanclamine buffer, pH 9.8, 100 ul/well, was added to all wells, and plates were incubated for 60 min at 37 °C. Colour reactions were measured in a Titertek Mulfiscan spectrophotometer (Flow Laboratories). C. IMMUNOBLOT ANALYSES  1. Purification of TSST-1 a. Preparation of media for TSST-1 production The dialyzable portion of BHI broth media (Difco) was used to grow S. aureus MN8 for TSST-1 production. A 10 times normal strength solution of BHI broth medium was prepared in pyrogen free distilled water and dialyzed, 12-14 kd molecular weight exclusion, against 10 volumes of pyrogen free distilled water for 72 h at 4°C. The dialyzable portion of the medium (dBHD was sealed in pyrogen-free glass bottles and sterilized by autoclaving in preparation for use. Non-dialyzed BHI broth was also prepared in order to determine possible alterations in TSST-1 production, total protein content or lipopolysacchari.de (LPS) cx)ntemination resulting from these modifications. b. Bacterial strain, growth and TSST-1 production Staphylococcus aureus strain MN8 was obtained from P. Schlievert (University of Minnesota). This organism was a vaginal isolate from a woman who developed typical menstrual TSS. This organism has been shown to be a high producer of TSST-1. The strain was maintained on brain heart infusion (BHD agar (Difco) containing 5% sheep blood at 4°C, and was regularly transferred to fresh media to maintain purity and viability. A 20 ml seed culture of strain MN8 was used for inoculation of large culture flasks. All glassware used was rendered pyrogen-free by baking at 180°C for 2 h and sterilized by autoclaving. Any plasticware used was rinsed 23 exhaustively with pyrogen-free distilled water to reduce the possibility of endotoxin contamination. Twenty ml of sterile dBHI was inoculated with a loopful of S. aureus MN8 and this seed culture was grown for 24 h with shaking at 175 RPM at 37°C in ambient atmosphere. One ml of this culture was aseptLcally transferred to 250 ml of sterile, pyrogen-free dBHI in each 1000 ml pyrogen-free Erlenmeyer flasks. These large cultures were then grown aerohically for 24 h with shaking at 240 RPM in ambient atmosphere. Culture supernatants were harvested by centrifugation in 250 ml plastic centrifuge bottles at 15,000 x g for 30 min in an ultracentrifuge (Beckman Instruments Inc, Palo Alto, California) at 4°C. Supernatants were decanted, filter-sterilized and stored in pyrogen free bottles until use. c. Ion exchange with SP-Sephadex C-25 Following a modification of the method of Igarashi et al (35), ion exchange chromatography was used to separate proteins in the culture supernatant. Seven liters cf culture supernatant were diluted with 21 liters of pyrogen free water and the pH adjusted with 5 N HCl to pH 5.0. Approximately 600 ml of SP-Sephadex C-25 (Phamacia Fine Chemicals, Uppsala, Sweden), equilibrated with phosphate-citrate buffer (PCB) (0.01 M, pH 5.0), was added to the diluted culture supernatant and the inixture was stirred for 2 h at room temperature. The Sephadex was allowed to settle and the liquid above was removed by decanting. This resin was layered onto 600 ml of PCB-equilibrated (pH 5.0) SP-Sephadex C-25 in a 5 cm diameter column. The column was then washed with 1.5 liters of PCB, pH 5.0. To elute the TSST-1 from the resin, an increasing linear gradient was produced with 1 liter cf 0.01 M PCB, pH 5.0, and 1 liter 0.02 M PCB, pH 8.7. The flow rate was adjusted to 240 ml/h and 20 ml fractions were collected. Fractions were monitored by absorption at 280 nm and 24 were assessed for TSST-1 content by the ELISA previously described. Fractions containing greater than 1 ug/ml (TSST-1) were pooled in a pyrogen-free container. A second ion exchange was performed with SP-Sephadex C-25 in order to concentrate the TSST-1. Pooled fractions were filter-sterilized, treated with Thimerosol (0.005%) and adjusted to pH 5.0. This was then layered onto a 5 cm diameter column of 600 ml SP-Sephadex C-25 previously equilibrated with 0.01 M PCB, pH 5.0, and allowed to drain into the gel bed. TSST-1 was eluted with 0.02 M PCB, pH 8.7, at a flow rate of 50 ml/h while 10 ml fractions were collected. Fractions were assessed for TSST-1 by ELISA and those fractions with greater than 100 ug/ml TSST-1 were pooled. Pooled TSST-1 was dialyzed against pyrogen-free water for 24 h with 2 changes and lyophilized. d. Gel filtration with Sephadex G-75 Sur^rfine Fifteen g of dry Sephadex G-75 Superfine (Pharmacia) were swollen in 0.1 M NH^HCO-j and 200 ml of gel was used to pack a 2.5 x 90 cm column. Lyophilized TSST-1 from fractions containing the peak of the second ion exchange profile was reconstituted with 10 ml of 0.1 M NH^CO^ and applied to the top of the gel bed. TSST-1 was eluted with 0.1 M NH^CO^ at a flow rate of 15 ml/h, collecting 15 ml fractions. TSST-1 was assessed in fractions by ELISA. At all stages of purification, aliquots of TSST-1 were saved and stored at -70°C for analysis of TSST-1 and protein content, endotoxin contamination, and for purity by SDS-PAGE, Ouchterlony and immunoblotting analyses. e. Protein and endotoxin measurements Protein concentrations at different stages of the purification procedure were estimated by the method of Lowry. et al (42). During chromatographic procedures, absorbance at 280 nm was used to estimate protein peaks in the 25 eluted fractions. Endotoxin contamination was determined by Limulus amoeoocyte lysate gelation according to the method of Levin et al (41). The lysate sensitivity was 0.25 endotoxin units per ml and Polymyxin B (1 mg/ml) neutralizahle activity was reported. f. SDS-PAGE Analysis of products Polyacrylamide gel electrophoresis (PAGE) in 0.1% sodium dodecyl sulphate (SDS) was performed to assess purity and molecular weight of the purified TSST-1 and was carried out according to the method of Laemmli et al (39). Prior to electrophoresis, samples to be tested were desalted by dialysis against distilled water, and concentrated as necessary by lyophihzation and reconstitution in a smaller volume. Samples were treated with 1% mercaptoethanol (2-ME) at 95°C for 5 min. Gels were 1.5 mm thick and 16 cm long and consisted of a 14% acrylamide running gel and a 4% acrylamide stacking gel portion. Standard low molecular weight protein markers (phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and lysozyme, Bio-Rad Laboratories, Richmond, California) were used to estimate molecular weights of protein in samples. Protein bands in polyacrylamide gels were visualized by staining with CoomassLe Brilliant Blue (0.25%), then destained with methanohacetic acidrwater (5:1:5), or alternatively, gels were stained with Bio-Rad silver stain reagents (Bio-Rad Laboratories) according to manufacturers instructions. g. Isoelectric focusing of partially purified TSST-1 The isoelectric point (pD of our partially purified TSST-1 was determined by analytical isoelectric focusing in horizontal polyacrylamide gel according to the Bio-Rad standard method (Bio-Rad Laboratories). Briefly, 5% polyacrylamide containing Bio-Lyte 3/10 ampholytes (Bio-Rad Laboratories) was cast into a 1.6 mm thick slab and photopolymerized. Five ul samples of TSST-1 preparations, 2 6 containing 2% ampholytes, were applied to filter paper ovals placed 1 cm from the basic (cathode) portion of the geL Isoelectric focusing standards (Bio-Rad Laboratories, pi 4.6-9.6, cat. no. 161-0310) were applied similarly in 5 ul volumes and run concurrently. Gels were focused for 30 min at 2 W followed by 2.5 h at 12 W. 2. Immunoblot analysis with human serum a. Immunoblot procedure TSST-1 preparations were electrophoresed in 14% polyacrylamide gels as previously described. Following electrophoresis, gels were transferred to nitrocellulose using a Bio-Rad Trans-Blot cell (Bio-Rad Laboratories, Richmond, CA) overnight at 20V, followed by 2 h at 60V. The nitrocellulose was then treated overnight in skim milk buffer containing 2% human serum to be tested. Nitrocellulose was then washed 3 times with Tris-buffered saline (TBS) with 0.05% Tween 20 (TBS-T). BiotLnated goat anti-human IgG (BRL, Life Technologies, Inc., Burlington, Ont., Cat. # 9580SA), diluted 1:500 in TBS-BSA (0.5%), was added and the nitrocellulose was incubated for 2 h at room temperature on a rotating platform. After washing 3 times with TBS-BSA, streptavidin horseradish peroxidase (BRL, Cat. # 9534SA) diluted 1:500 was added, followed by 30 min incubation and washing twice with TBS-T and once with TBS. Immunoreactive bands were visualized with 4-chloronapthol substrate (BRL, Cat. # 5979SA) for 15 min and the reaction was stopped by rinsing with distilled water. b. TSS patient sera used for probing Acute and convalescent phase sera from 12 TSS patients were used for immunoblot analyses. Of these 12 patients, 7 were menstrual TSS women, 3 were nonmenstrual TSS women and 2 were male TSS patients. Patient age and duration after onset of illness when each serum sample was obtained are 27 summarized in Table 7. Acute sera were obtained less than or equal to 10 days after onset and convalescent sera were obtained between 10 days and 3 months. If multiple sera were available from the convalescent phase as outlined, the first serum specimen obtained 3 months after onset of illness was used. 3. Immunoblot analysis with rabbit serum Transblots were prepared as described above, except biotinated goat anti-rabbit IgG (BRL, cat. # 9586SA) was used to detect bound rabbit antibody. Immune and nonimmune rabbit serum used were ammonium sulphate precipitated preparations, as described earlier, and were used at various dilutions. D. STATISTICAL METHODS Regression coefficients reported were calculated by the least squares method. Statistical comparisons between different groups were analyzed using Mam-Whitney rank sum test (2-tailed) for continuous variables which are non normal in distribution, and Student t-test for values with a normal distribution. Fisher's exact test was used for discrete variables. 28 IV. RESULTS A. TSST-1 DETECTION BY ELISA 1. Standardization of the ELISA procedure for TSST-1 Detec±Lon The Immulon I (Dynatech Laboratories) polystyrene microtiter plate was chosen over several other brands, including two other polystyrene plates (Linbro Titertek, Flow Laboratories, MacLean, Va., and Nunc Immunoplate I, Vangard International, Neptune, N.J.), and a polyvinyl plate (Immulon H, Dynatech Laboratories). The Immulon I was found to be superior for its maximal specific binding of antitoxin, and for minimal nonspecific binding of other immunoreactants (data not shown). Binding of specific antibody was determined using goat anti-rabbit IgG conjugated to alkaline phosphatase. Optimal dilutions of coating antibody and conjugate were determined by a series of checkerboard titrations in order to achieve greatest sensitivity and specificity while requiring the least amount of immunoreactants. Optimal working conditions were 1:10,000 for coating antibody (0.33 ug protein per well) and 1:750 for the antibody conjugate. At these dilutions of antibody and in the presence of 16 ng/ml TSST-1 reference standard, a constant rate of hydrolysis for the p-nitrophenyl phosphate substrate could be demonstrated over 90 min incubation at 37 °C (data not shown). 2. Reproducibility of the ELISA assay for TSST-1 detection A typical standard curve generated with reference TSST-1 over the range of 0.5 to 16 ng/ml is shown in Figure 1. The regression coefficient (r) obtained from 20 separate experiments was 0.985 + 0.007 (95% confidence liniits). Considerable within-day, and day-to-day variations due to plate^to-plate differences in binding characteristics were observed (data not shown). These results indicate that test samples and reference TSST-1 standards must be 29 2.0 1.8 -1 . 6 -1 . 4 -Ln o cr 1 . 2 -cu o 1 . 0 -cr o . Q 0 . 8 -i_ o C O . Q <C 0.6-G.4 -G.2-4 4 / 4 / 4 4 4 y 0.5 1.0 2.0 4.0 8.0 16.0 TSST-1 (ng/ml) F i g u r e 1. R e p r o d u c i b i l i t y a n d s p e c i f i c i t y o f E L I S A a s s a y . T y p i c a l c u r v e (•—-——•) g e n e r a t e d w i t h s t a n d a r d r e f e r e n c e TSST -1 o v e r t h e c o n c e n t r a t i o n r a n g e o f 0.5 t o 16.0 ng/ml i n w e l l c o a t e d w i t h r e f e r e n c e r a b b i t a n t i - T S S T - 1 a n t i b o d y , r e g r e s s i o n c o e f f i c i e n t ( r ) = O.985 0.007 (95% c o n f i d e n c e l i m i t s f o r 20 e x p e r i m e n t s ) . S p e c i f i c i t y c f E L I S A was d e m o n s t r a t e d i n i d e n t i c a l e x p e r i m e n t s i n w h i c h m i c r o t i t e r w e l l s w e r e c o a t e d w i t h 1:10,000 d i l u t i o n r a b b i t s e r u m d e v o i d o f a n t i - T S S T - 1 ( 30 determined on the same plate to obtain reliable results. The coefficient of variation from repeated testing of the same TSST-1 was consistently less than 10%, confirming the reproducibility of the ELISA assay. 3. Specificity of the ELISA for TSST-1 detection The specificity of the ELISA was demonstrated in additional experiments in which microtiter wells were coated with a 1:10,000 dilution of ammonium sulphate precipitated, nonimmune rabbit serum that was shown to be negative for anti-TSST-1 by Ouchterlony immunoduffusion (Figure 1). The fact that no dose-response relationship was produced with this control, as was demonstrated in wells coated with TSST-1 immune rabbit serum, further demonstrated the specificity of the ELISA for TSST-1. Other negative controls included wells with no toxin added, and wells with toxin that had been pretreated with antitoxin. Furthermore, no false positive results were obtained using purified preparations of other S. aureus enterotoxins intruding A, B, C^, Cy D and E at concentrations ranging from 10-1000 ng/ml. A crude preparation of enterotoxin D at a concentration of 1800 ng/ml did demonstrate false positive results equivalent to 1.29 ng/ml TSST-1 (0.07%). This preparation did not immunoprecipitate with rabbit antL-TSST-1 by Ouchterlony immunodiffusion. The false positive reactivity in the ELISA method, presumed to be protein A, was completely eliminated when crude enterotoxin D preparation was pretreated with 10% normal rabbit serum. The specificity of the ELISA for TSST-1 detection is dependent on the use of coating rabbit antiserum which is monospecific for TSST-1. To further examine this aspect, reference TSST-1 (Bergdoll SEF, 1.25 ug/well) was separated by SDS-PAGE (14% separating gel) and transblotted to nitrocellulose. Blots were probed with ammonium sulphate precipitated reference rabbit anti-^rsST-1 at dilutions of 1:70, 1:700, 1:7000 (working dilution of rabbit 31 F i g u r e 2a. Immunoblot a n a l y s i s o f TSST-1 w i t h r e f e r e n c e r a b b i t a n t i - T S S T - 1 . T r a n s b l o t s o f TSST-1 ( B e r g d o l l SEF, 1.25 u g / w e l l ) were probed w i t h ammonium s u l p h a t e p r e c i p i t a t e d r a b b i t a n t i -TSST-1 a t k 1 0-fold d i l u t i o n s . MW) low m o l e c u l a r p r o t e i n s t a n d a r d s , s t a i n e d w i t h amido b l a c k . D i l u t i o n s o f serum: 1) 1:70, 2) 1:700, 3) 1:7000 (working d i l u t i o n o f anti-TSST - 1 i n ELISA f o r TSST-1 d e t e c t i o n ) , and h) 1:70,000. 32 F i g u r e 2b. Immunoblot a n a l y s i s o f TSST-1 and p r o t e i n A w i t h immune and normal r a b b i t serum. Ammonium s u l p h a t e p r e c i p i t a t e d r e f e r e n c e r a b b i t anti-TSST-1 (IRS) and normal r a b b i t serum (NRS) were used to probe: lane l ) TSST-1 ( B e r g d o l l SEF, 1.25 jug/ml), and lane 2) p r o t e i n A (6 ug/ml). T r a n s b l o t s were a l s o s t a i n e d f o r t o t a l p r o t e i n ( P r o t e i n ) w i t h amido b l a c k t o v i s u a l i z e m o l e c u l a r w e i g h t s t a n d a r d s (MW) as w e l l as p o s i t i o n s o f TSST-1 and p r o t e i n A. 33 aritibody for coating of microtiter walls in ELISA for TSST-1 detection), and 1:70,000. Development of nitrocellulose strips probed with 1:70 and 1:700 dilutions of serum (Figure 2a) revealed many immunoreactive bands in addition to TSST-1. However, at the working dilution (1:7000) and at 1:70,000, reactivity was ranfined to TSST-1 as well as to some higher molecular weight material in the range of 40-60 kd. Interestingly, reactivity to the high molecular weight region did not decrease proportionally with increasing dilution of rabbit sera, indicating that this reactivity may have been due to the presence of protein A in the TSST-1 preparation. To demonstrate the likelihood of protein A being responsible for this pattern of immunoreactivity, blots of protein A (Sigma, Cat. # P-6650, 6 ug/well) and TSST-1 (Bergdoll SEF, 1.25 ug/well) were probed with ammonium sulphate precipitated rabbit anti-TSST-1 CERS) and normal rabbit serum (NRS), at dilutions of 1:100 and 1:7000 (Figure 2b). Staining of transblots with amido black revealed the presence of several protein bands in the SEF preparation in addition to TSST-1 (24 kd). As expected (32a), protein A did not migrate in a discrete band in the gel, but instead, appeared as a diffuse band corresponding to molecular weights ranging between 40-60 kd. Probing of transblots with 1:100 diluted IRS resulted in reactivity to many bands in addition to TSST-1, but probing with 1:7000 diluted serum resulted in reactivity only to TSST-1 and the high molecular weight region corresponding to the position of protein A. Reactivity to protein A was very strong with both dilutions of IRS. Probing with NRS did not result in immunoreactivity to TSST-1 at either dilution while it displayed reactivity to protein A at both dilutions, although not to the extent observed with IRS. These results indicate that, at the dilution of rabbit anti-TSST-1 used in the ELISA for TSST-1 detection, the antiserum is monospecific for TSST-1. 34 4. EUminatLon of protein A effect by normal rabbit serum The interfering effect of protein A and its elimination by pretreatment of test samples with 10% normal rabbit serum were further characterized (Figure 3). Standard concentrations of reference TSST-1 (0.5 - 16 ng/ml, final concentrations) were prepared in BHI broth, and pretreated either with protein A (1000 ng/ml, Sigma Diagnostics, cat # P-6650), protein A plus 10% normal rabbit serum, or 10 % normal rabbit serum alone. Negative controls included BHI without TSST-1 added. Quantitation of TSST-1 in these different experimental groups were measured in parallel by our ELISA procedure as described earlier and compared to positive controls in which reference TSST-1 standards were prepared in PBS-T. The results clearly demonstrate: a) the interfering effect of protein A in the assay; b) the complete eliinination cf this effect by the addition of 10 % normal rabbit serum, without loss of sensitivity or specificity over the range of reference TSST-1 standard concentrations studied. 5. Detection of TSST-1 in culture supernatants of S. aureus isolates The results of testing for TSST-1 production in culture supernatants of 95 S. aureus isolates are shown in Figure 4. Among the 60 isolates confirmed to be negative for TSST-1 by immunodiffusion, 59 also had undetectable toxin by ELISA. Among the 35 isolates confirmed positive for TSST-1 by immunodiffusion (24 were isolated from TSS patients), all had detectable toxin by ELISA. Negative controls included uninoculated BHI broth treated in identical fashion. The necessity of pretreating the culture supernatants with 10% normal rabbit serum to absorb protein A produced by isolates is clearly Mustrated. Forty-4:wo of 60 isolates negative for TSST-1 production as tested by immuncdiffusion would be falsely positive by ELISA if culture supernatants were not pretreated with 10% normal rabbit serum to remove the mterferLng effect of protein A. Similarly, all 35 isolates that were shown bo be positive for toxin production by 35 2.OH I I I i I I 0.5 1.0 2.0 4.0 8.0 16.0 TSST-1 (ng/ml) F i g u r e 3 . C o m p a r i s o n o f TSST-1 d e t e c t i o n by ELISA i n P B S - T ( O ) o r b r a i n h e a r t i n f u s i o n b r o t h (BHI) a l o n e ( A ) , o r i n BHI p r e t r e a t e d w i t h e i t h e r 1000 ng /ml p r o t e i n A ( • ) , 10% normal r a b b i t serum ( • ) , o r p r o t e i n A p l u s 10% normal r a b b i t serum (d). The i n t e r f e r i n g e f f e c t o f p r o t e i n A was c o m p l e t e l y e l i m i n a t e d by the a d d i t i o n o f 10% normal r a b b i t serum w i t h o u t l o s s o f s e n s i t i v i t y when compared t o s t a n d a r d c u r v e s g e n e r a t e d i n PBS o r BHI a l o n e . 36 F i g u r e h . E f f e c t o f 10% n o r m a l r a b b i t s e r u m (NRS) o n e l i m i n a t i o n o f i n t e r f e r e n c e by p r o t e i n A d u r i n g d e t e c t i o n o f T S S T - 1 i n c u l t u r e s u p e r n a t a n t s o f a u r e u s b y E L I S A . S^. a u r e u s i s o l a t e s w e r e p r e v i o u s l y d e t e r m i n e d b y O u c h t e r l o n y i m m u n o d i f f u s i o n t o b e t o x i g e n i c ( A ) o r n o n - t o x i g e n i c ( • ) . E L I S A r e s u l t s o f c u l t u r e s u p e r n a t a n t s w e r e c o m p a r e d t o u n i n o c u l a t e d m e d i a c o n t r o l s ( • ) w i t h o r w i t h o u t a d d i t i o n o f N R S . 37 N o n t o x i g e n i c a u r e u s (n=60) T o x i g e n i c S .^ a u r e u s (n=35) M e d i a C o n t r o l (n=17) 100 —I 50 H AA A A A A A AA AA A A AA AA AA AA AA A AA A A 10 —1 A AA A A AA AAA AA at s—l co CO AA AA AA A A AA AA A 1 —\ • •••• • •••• • •• • • 0.5 •• ••••• •••••• B • • • a • B fl ••••••• • i IB • B ••«•••••• • i ••••••• • • BBBB • ••• • BI IB B B BB • • • •• • W i t h o u t W i t h W i t h o u t W i t h W i t h o u t W i t h NRS NRS NRS NRS NRS NRS PRETREATMENT OF CULTURE SUPERNATANTS 38 100 50-10-oo 5-0. oo L L J A A AA A A — A-A A A A A A A A A A A A A A A 1.0-0.5-1 TSS Assoc i a ted _S. au reus (n=24) Non-TSS Control S. aureus (n=1l) Figure 5. Quant i ta t ive TSST-1 production as measured by ELISA among toxigenic S_. aureus vaginal i so lates from TSS patients ( A ) and 11 toxigenic S. aureus vaginal i so lates from non-TSS healthy women ( A ) ( p < 0 . 0 5 , Mann-Whitney rank sum t e s t ) . 39 immunodiffusion yielded higher values of TSST-1 in untreated compared to treated culture supernatants. Of interest, isolates from TSS patients produced significantly higher concentrations of TSST-1 in culture supernatants compared to toxm-producing strains from non-TSS patients (p<0.05, Mann-Whitney 2-tailed rank sum test) (Figure 5). 6. Detection of TSST-1 in vaginal washings of TSS patients and healthy  women A total of 36 vaginal washings from 9 menstrual TSS patients and 14 healthy control women were available for quantitation of TSST-1 by ELISA (Table 3). Three of 4 specimens collected within 3 days of hospitalization from 3 women with acute TSS had detectable TSST-1, compared to 0 of 17 washings from 9 TSS women collected after 3 days of lxispitalization (range 6-63 days), median 30 days) (p=0.003, Fisher's exact test), and 1 of 15 washings from 14 healthy control women (p=0.016). Specific TSST-1 concentrations, detected in vaginal washings from the two women with acute TSS and a single control subject, are shown in Table 4. TSST-1 concentrations from vaginal washings of a 29 year old woman with typical menstrual TSS (subject A) were highest on day 1 of her hospitalization and rapidly declined thereafter, coincident with anti-staphylococcal therapy and ehmination of S. aureus in the vaginal flora. Vaginal washings from a 22 year old healthy control woman (subject C) was iMtially negative for TSST-1 concurent with negative vaginal culture for isolation of S. aureus. A subsequent washing obtained one month later was positive for TSST-1 when her concurrent vaginal culture was also positive for TSST-1 producing s . aureus. Her serum antibody titer was within the 69th percentile of normal anti-TSST-1 titers as determined from 87 healthy women. 40 Table 3. Detection of TSST-1 in Vaginal Washings of Menstrual TSS Patients and Healthy Control Women No. P o s i t i v e / T o t a l Tested Women (P)* Washings ( P ) » TSS Patients 2/12 - 3/21 < 3 days i l l n e s s 2/3 - 3A > 3 days i l l n e s s 0/9 (0.045) 0/17 (0.003) Control Subjects 1/14 (0.063) 1/15 (0.016) * P value by F i s h e r ' s exact t e s t , compared with TSS pat ients tested wi th in 3 days of acute i l l n e s s 41 Table 4. Quant i tat ion of TSST-1 in vaginal washings of TSS and  control women. TSST-1 Detected (ng/ml) With NRS+ Without NRS TSS Washings Subject A (Day 1)* 15.8 51.2 (Day 3) 2.2 2.1 (Day 7) 0 0 Subject B (Day 1) 2.5 2.5 (Day 60) 0 0 Control Washings Subject C (Apri1 83) 0 1 .9 (May 83) 3.7 8.7 + Pretreatment with 10% normal rabbit serum (NRS) to e l iminate any i n t e r f e r i n g e f f ec t by prote in A * Day a f ter h o s p i t a l i z a t i o n when specimen was c o l l e c t e d k2 7. Detection of TSST-1 in human urine The ELISA assay was standardized for detection of TSST-1 in human urine. Reference TSST-1, diluted in normal pooled urine, was used to generate the standard curve which was found to be identical to that generated with reference TSST-1 diluted in PBS-T (r = 0.978 + 0.024, 95% confidence limits for 10 experiments, data not shown). Control urine from 12 healthy women and all five urine specimens from TSS patients (obtained on days 1, 2, 4, 10 and 23 of illness, respectively) were negative for TSST-1. 8. Standardization of ELISA for detection of TSST-1 in human serum All attempts to detect TSST-1 in human serum spiked with TSST-1 were unsuccessful, presumably due to the presence of TSST-1 antibodies in the sera used. However, normal rabbit serum, shown to be free of anti-TSST-1 antibodies, was spiked with reference TSST-1 from 0.5 to 16 ng/ml and tested in parallel to standard TSST-1 in PBS-T. The coefficient of correlation (r) for TSST-1 standard curves generated with normal rabbit serum was 0.966 + 0.034 (95% confidence Umits, n = 3, data not shown). B. SEROLOGIC STUDIES 1. Standardization of ELISA for anti-TSST-1 detection in serum Specificity of the ELISA for anti-TSST-1 was determined by neutralization of antL-TSST-1 activity following pretreatment of three positive sera with graded concentrations of TSST-1 (ranging from 10 to 1000 ng/ml in 1:1000 dilutions of each serum) (Figure 6). In contrast, no neutralization of the reference sera was observed after preincubation with 10 ug/ml of staphylococcal enterotoxins A, B, C-^ C^, and C^ (Toxin Technology Inc., Madison, Wisconsin) (data not shown). The relative antibody titers were expressed as percent of standard activity (POSA), and obtained from a standard curve by log transformation of the relative OD values (Figure 7). The linearity 43 2.0-1.8-0.2-H 10 100 1000 TSST-1 (ng/ml) F i g u r e 6• N e u t r a l i z a t i o n o f TSST-1 s p e c i f i c IgG f o l l o w i n g p r e t r e a t m e n t of h i gh ( • ) , med i urn ( • ), and low ( • ) t i t e r r e f e r e n c e s e r a wi th graded doses o f TSST-1. 1/  I > ' > I I I I I —I I 2.0 2.5 3.0 Log R e l a t i v e OD Figure 7• Standard curve for predicting TSST-1 s p e c i f i c r e l a t i v e ELISA t i t e r s (POSA). Regression c o e f f i c i e n t (r) = 0.998 (d.f. = 28, p< 0.001). h5 of the POSA standard curve was confirmed in repeated experiments (mean regression coefficient (r) = 0.996, d.f.=14, p<0.001). By converting the relative OD of the test sera to log values, a corresponding log POSA was predicted, expressed as a percent of the anti-TSST-1 activity relative to the medium level internal standard. As the relative OD values served to standardize each test serum against the internal standard, it was not necessary to generate the POSA curve in each experiment. With this procedure, values tested in different experiments could be more readily compared. Reproducibility of the POSA values were confirmed by repeated testing of a low level positive serum (19.0 + 2.7, mean POSA + S.D. in 24 separate experiments; coefficient of variation (c.v.) = 14 %) and a high level positive serum (278 + 44 in 43 experiments; c.v. = 16%). 2. Detection of TSST-1 specific IgG in serum of TSS patients and healthy  controls Sera from TSS patients were grouped according to the duration of illness when collected. Sera collected less than or equal to 10 days after onset were considered "acute", greater than 10 days and less than 3 months were considered "early convalescent", and greater than 3 months were considered 'late convalescent". Male and female nonmenstrual TSS patients were separately analysed. The distribution of anti-^rsST-1 ELISA titers (POSA) in acute sera obtained from menstrual and nonmenstrual TSS patients, and from 87 female and 66 male healthy controls are shown in Figure 8. Acute titers from menstrual TSS patients were significantly lower than those from control women (p< 0.01, Mann-Whitney rank sum test). However, acute titers from nonmenstrual TSS women were not significantly different from control women, but were significantly higher than those of menstrual TSS women (p<0.05). Acute titers from male TSS patients were also significantly lower than the male control Figure 8. TSST-1 s p e c i f i c r e l a t i v e ELISA t i t e r s (POSA) in acute ( <^ 10 days) , ear ly convalescent (11 days - 3 months) and late convalescent (> 3 months) sera of menstrual TSS pa t i en t s , male and female nonmenstrual TSS pa t i en t s , and in healthy male and female c o n t r o l s . hi 300 -co o o 200 a •o o 4—1 CO £ i o o -cz a> o s_ CD A • • AA A\ A A A A A A A ••w : r **• A V A A 8k A A A a ec 1 c (8) (10) (5) Menstrua 1 TSS Women a ec Ic (8) (k) (3) Nonmenstrual TSS Women (87) Control Women a ec Ic (6) (.3) (D. Male TSS (66) Control Men group (p<0.05). Acute titers of female nonmenstrual TSS women were also significantly higher than those of the male TSS patients (p<0.05). An interesting observation was that the titers of the female control group were sLgnificantly lower than those of the male control group. This difference was not due to age differences between the two groups as no correlation of age and POSA was demonstrated and no interaction effect of sex and age on ELISA titers was found by two-way analysis of variance test. Antibody titers from nonmenstrual and menstrual TSS patients were further analysed according to whether S. aureus strains positive for TSST-1 in vitro were isolated during their acute illness (Table 5). The acute titers of all 14 individuals who had associated TSST-1 positive S. aureus were sLgnificantly lower than the titers of the 8 patients who had either nontoxLgenic or no S. aureus isolated (p<0.01). Acute as well as early and late convalescent sera from menstrual TSS women were compared to female controls (Figure 8). No significant differences in titers were observed between each phase of illness, and titers during all phases were sLgnificantly lower than those of control women (p< 0.05). Similarly, in male TSS patients, no significant differences were observed between acute and convalescent titers, and titers from both acute and convalescent phases were significantly lower than controls (p< 0.05). In nonmenstrual TSS women, titers were not significantly different from control women during acute or convalescent phases of the illness. 3. Standardization for detection of TSST-1 specific IgG and IgA in vaginal washings Linear regression analysis was performed on the standard curves by plotting (-Log Dilution) (Y-axis) versus (-log Absorbance + 1.0) (X-axis) (Figure 9). Absorbance values from test vaginal washings were used to predict h3 T a b l e 5. Compar ison o f ELISA t i t e r s (POSA) in a c u t e s e r a f rom TSS  p a t i e n t s w i t h and w i t h o u t a s s o c i a t e d S . a u r e u s . Mean POSA in A c u t e S e r a + SD TSS Type Menst rua1 Nonmenstrua l Men No. TSA 8/8 3/8 3/6 A l 1 S e r a 26.2 + 1:4.9 71.8 + 52.7 37.0 + 38.2 TSA I s o l a t e d 26.2 + 14.9 36.7 +9-9 22.0 + 11.3 TSA not I s o l a t e d 93.0 + 56.9 52.0 + 53.5 T o t a l 14/22 45.5+42.3 27-3+14.5 77.6+55.8 * TSA = T o x i g e n i c S t a p h y l o c o c c u s a u r e u s . + A l l nonmenst rua l p a t i e n t s a r e women. 50 X o CM c o O) o 5.0 ID 3 4.0 (0 c cn fO 3.0 2.0 1 .0 J r=0.998 / I g A / A r=0.989 ' * .6 .8 1.0 1.2 1.4 1.6 1.8 - Log A b s o r b a n c e +1.0 F i g u r e 9. D e m o n s t r a t i o n o f dose-response r e l a t i o n s h i p i n the d e t e c t i o n o f TSST-1 s p e c i f i c IgG and IgA i n v a g i n a l w ashings. 51 corresponding standard dilutions which, when compared to the actual dilution tested, gave a percent of standard activity (POSA). Reproducibility testing of a single vaginal wash sample resulted in TSST-1 specific IgG levels (mean POSA + S.D.) of 153 + 29.2, (n=7, coefficient of variation = 19%) and IgA levels of 4.0 + 0. 52, (n=3, coefficient of variation = 13%). C.IMMUNOBLOT ANALYSES 1. Purification of TSST-1 Seven liters of culture supernatant prepared from S. aureus strain MN8 were harvested. The mean concentration of TSST-1 in the supernatant was 15 ug/ml as determined by ELISA; thus, the total estimated TSST-1 at this stage was approximately 106 mg. Results of the first ion exchange procedure with SP-sephadex C-25 is shown in Figure 10. Protein determination by ultraviolet absorption (280 nm) and TSST-1 quantitation by ELISA showed the maximum yield in fractions 30-60. These fractions were pooled and rechromatographed with SP-sephadex C-25 using only 0.02 M PCB, pH 8.7, to elute the toxin (Figure 11). Almost 100% of the toxin was eluted between pH 5.5 and 6.7, with the main fraction containing approximately 300 ug/ml TSST-1. Following the gel filtration step (Figure 12), one fraction of approximately 16 ml volume contained 80% of the eluted toxin at a concentration of 1.02 mg/ml, or 15.8% of the initial, toxin amount. Protein, TSST-1 and endotoxin analyses were performed on preparations at all stages of the purification and are summarized in Table 6 . The advantage of dialyzed BHI is illustrated in the reduction of total protein by approximately 50%, compared to nondialyzed medium. The relative degree of purity was determined by comparing TSST-1 (estimated by ELISA) to total protein (w/w). Thus, in the culture supernatant, TSST-1 made up just 0.21% of total protein, 14% after the first ion exchange, 32% after the second ion exchange, and 94.4% 52 0.8-1 0.7-1 0..6J o oo CM CJ e_> cr a s_ o CO j Q c n co r i i T 1 1 I 1 ' ~ 1 10 20 30 kO 50 60 70 80 90 100 110 120 F r a c t i o n no. (20 m l / f r a c t i o n ) F i g u r e 1 0 . Ion exchange ch roma tog raphy o f TSST - 1 f rom c u l t u r e s u p e m a t a n t s on S P - S e p h a d e x C - 2 5 . 53 30CT 16 20 24 28 32 36 ko kk 48 52 56 F r a c t i o n no. (10 m l / f r a c t i o n ) Figure 11 . Rechromatography of TSST-1 on SP-Sephadex C-25, ind ica t ing pH of eluted f r a c t i o n s . 54 1 2 3 4 5 6 7 8 9 10 F r a c t i o n no. (15 m l / f r a c t i o n ) F i g u r e 12. Gel f i l t r a t i o n o f TSST-1 through Sephadex G-75 S u p e r f i n e . The f i r s t f r a c t i o n c o n t a i n i n g g r e a t e r than 10 ug/ml TSST-1 was a r b i t r a r i l y d e s i g n a t e d 1. 55 Table 6. Re la t ive pur i ty of TSST-1 in d i f f e r e n t f rac t ions during the p u r i f i c a t i o n procedure LAL* P r o t e i n + TSST-1® TSST-1 /Prote in % Recovery Fract i ons (ng/ml) (mg) (mg) (mg/mg) of TSST-1 Crude Culture Supernatant 0.05 51,100 106.4 26.8 23-6 16.8 0 0.0021 0.141 0.320 F i r s t Ion - 190 exchange Second Ion - 73-8 exchange Gel f i l t r a t i o n - 17.8 Media C o n t r o l : BHI broth 0.09 117,600 (non dialyzed) BHI broth 0.05 5.9,850 (d ia lyzable) « Limulus amoebocyte lysate test for LPS contamination (41) + Prote in determined by the method of Lowry et al (42) @ TSST-1 determined by ELISA (61) 100 25 22 15.8 56 after the gel filiation step, for a final purification factor of 430 times over that of the culture supernatant. Purity was assessed at all stages of the procedure by SDS-PAGE analysis (Figure 13). Increased purification and concentration of TSST-1 with each purification step is evident. Protein was concentrated 20 times by lyophihzation prior to electrophoresis in all preparations, except for the final one which did not require concentration. Our TSST-1 preparation was examined by Ouchterlony immunodiffusion against several other TSST-1 preparations obtained from investigators in the field, inciuding: a) R. Reiser (Toxin Technology, Madison, Wisconsin), b) M. Melish (University of Hawaii, Honolulu), c) J. Kirkland (Proctor and Gamble Inc, Cincinnati) and reference TSST-1 (SEF-50) from M.S. Bergdoll (University of Wisconsin) (Figure 14). All toxin preparations demonstrated a line of identity with SEF-50 against a reference rabbit antiserum (M.S. Bergdoll). The molecular weight of the above TSST-1 preparations were estimated by SDS-PAGE to be 24 kd, and the isoelectric point was estimated to be 7.0 for all preparations. 2. Comparison of four TSST-1 preparations by immunoblots probed with normal  human serum Immunoreactivity was also demonstrated by immunoblots probed with pooled normal human serum. Pooled human serum was prepared from equal aliquots of serum from 10 healthy women. Four different TSST-1 preparations were subjected to SDS-PAGE under reducing and non-reducing (xinditions, and transferred to nitrocellulose. Pooled human serum was used as the first antibody. After development of immunoreactLve bands (Figure 15a) several additional proteins were visible in the immunoblots which were not observed in the polyacrylamide gels stained with CoomassLe Blue (Figure 15b). There was no effect in either the number or apparent molecular weights of proteins seen in 57 Figure 13- SDS-PAGE ana lys i s of p a r t i a l l y p u r i f i e d TSST-1 at each stage of the p u r i f i c a t i o n procedure. Gel is s tained with Coomassie Blue. Lanes: 1) low molecular weight prote in standards ( k i l o d a l t o n s ) , 2) TSST-1 (Toxin Technology Inc . ; 6 ug p r o t e i n ) , 3) cu l ture super-natant (5 ug p r o t e i n ) , k) Pooled f rac t ions from 1st ion exchange (7.5 ug p r o t e i n ) , 5) pooled f rac t ions from 2nd ion exchange (10 ug p r o t e i n ) , 6) main f r a c t i o n from gel f i l t r a t i o n (6.4 ug p r o t e i n ) , 7) low molecular weight standards as in lane 1. 58 1 2 3 4 5 6 7 kd 92.5> 66 • 45 -31 • TSST-1• 21. 5K 14.5 59 F i g u r e 14. D e m o n s t r a t i o n o f immunolog ic i d e n t i t y o f p a r t i a l l y p u r i f i e d TSST-1 w i t h r e f e r e n c e TSST-1 and t h r e e a d d i t i o n a l p r e p a r a t i o n s . C e n t e r w e l l c o n t a i n s r e f e r e n c e r a b b i t a n t i - T S S T - 1 1:4 d i l u t i o n . TSST-1 p r e p a r a t i o n s were a l l t e s t e d at 50 u g / m l . From t o p , c l o c k w i s e : SEF 50 (M .S . B e r g d o l l , U n i v e r s i t y o f W i s c o n s i n ) , Ros ten ( U n i v e r s i t y o f B . C . ) , R e i s e r ( T o x i n T e c h n o l o g y I n c . ) , SEF 50, M e l i s h ( U n i v e r s i t y o f H a w a i i ) , K i r k l a n d ( P r o c t o r and G a m b l e ) . 60 Figure 15. Immunoblot ana lys i s of four d i f f e r e n t TSST-1 preparat ions , probing with pooled normal human serum. TSST-1 preparations (6 ug /we l l ) : lane 1) Rosten (Univers i ty of B . C . ) , 2) Reiser (Toxin Technology I n c . ) , 3) Mel ish (Univers i ty of Hawaii) , h) K irk land (Proctor and Gamble). MW; low molecular weight prote in s tandards . 15a. Immunoblot resu l t s 15b. SDS-PAGE of same preparations (non-reduced), s ta ined with Coomassie Blue. 15c. SDS-PAGE of same preparations (non-reduced), s ta ined f i r s t with Coomassie Blue, followed by s i l v e r s t a i n i n g . 15d. SDS-PAGE of same preparations (non-reduced), s ta ined with s i l v e r without p r e - s t a i n i n g with Coomassie Blue . 61 Non-Reduced Reduced Figure 15a 62 MW kd 92.5 • 66 • 45 • 31 > TSST-1 • 21.5 • 14.5 > r Figure 15b 63 MW 1 2 3 k MW Figure 15d 65 non-reduced gels compared to reduced gels. Prominent protein bands observed in the toxin preparations following SDS-PAGE and staining with CoomassLe Blue included a major 24 kd protein common in all preparations, a minor 32 kd band in our preparation, and a minor 28 kd band in the toxin from Proctor and Gamble. Reduction also did not seem to alter the number or position of bands in the immunoblot, but the variety of additional bands seen by immunoblot among the 4 different TSST-1 preparations was very striking. The 24 kd TSST-1 band was very prominent in all preparations. These data indicate that none of the TSST-1 preparations including our own were absolutely pure, and that CoomassLe Blue staining may not be sensitive enough for detecting minute amounts of other protein bands present. Subsequently, gels previously stained with CoomassLe Blue were silver stained (Figure 15c). Silver staining after CoomassLe Blue staining demonstrated protein bands corresponding to those observed by immunoblot. Interestingly, when SDS-PAGE of the same preparations was repeated under identical conditions, sLlver staining of gels not prestained with CoomassLe Blue, revealed fewer bands than gels pre-stained with Coomassie Blue (Figure 15d). 3. Comparison of Immunoblots of TSST-1 preparations probed with acute and  convalescent sera from 12 TSS patients Immunoblots of our partially purified TSST-1 preparation as well as another preparation (Reiser, Toxin Technology) revealed many immunoreactive bands when probed with TSS patient acute and convalescent sera (Figures 16a-d). TSST-1 preparations were not reduced prior to electrophoresis. When paired sera were compared, seroconversion to TSST-1 (24 kd band) was observed in 7 of 10 patients with TSST-1 positive S. aureus, 0 of 2 patients with TSST-1 negative or no S. aureus (Table 7). Of the 7 menstrual TSS women studied, 5 demonstrated seroconversion to TSST-1; 1 failed to seroconvert when examined 1 month after acute illness, despite isolation of TSST-1 positive S. aureus 66 (Patient 7), while 1 had preexisting antibodies to TSST-1 in her acute serum (Patient 4). Of the 3 nonmenstrual TSS women studied, only one had associated TSST-1 positive S. aureus, but all 3 women had preexisting anti-TSST-1 in their acute sera (Patients 8, 9, 10). The two male TSS patients both had TSST-1 positive S. aureus, and both seroconverted to TSST-1 during convalescence (Patients 11 and 12). Of great interest were seroconversions to several staphylococcal exoproteins (21, 28, 32 and 49 kd antigens) besides the TSST-1 (24 kd) antigen observed in the 12 patients. Reactivity to these antigens are also summarized in Table 7. Among 7 patients who seroconverted to TSST-1, 4 patients had concurrent seroconversions to other proteins (4 to the 49 kd antigen, and 1 each to the 21, 28 and 32 kd antigens), while 3 seroconverted exclusively to TSST-1. Among 5 patients who did not demonstrate seroconversion to TSST-1, 3 seroconverted to other antigens (1 to the 32 kd antigen and 2 to the 21 kd antigen), while 2 did not demonstrate seroconversions to any of the identifiable proteins in the partially purified TSST-1 preparations. Thus, multiple seroconversions to different staphylococcal products was not uncommon (5 of 12 patients studied). 67 MW PAGE Acute Conv Control 1 2 1 2 1 2 1 2 «TSST-1 F i g u r e 16a. Immunoblot a n a l y s i s o f two TSST-1 p r e p a r a t i o n s . Lane 1: p a r t i a l l y p u r i f i e d TSST-1 ( R o s t e n ) , lane 2: c o m m e r c i a l l y o b t a i n e d TSST-1 ( T o x i n T e c h n o l o g y , I n c . ) . MW: low m o l e c u l a r we igh t p r o t e i n s t a n d a r d s s t a i n e d w i t h amido b l a c k . PAGE: t r a n s b l o t o f p o l y a c r y l a m i d e g e l , i d e n t i c a l to t h a t used f o r immunoblo ts , but s t a i n e d f o r t o t a l p r o t e i n w i t h amido b l a c k . A c u t e : a c u t e p a t i e n t serum. Conv: c o n v a l e s c e n t p a t i e n t serum. C o n t r o l : p o o l e d normal human se rum. P a t i e n t S- t y p i c a l m e n s t r u a l TSS woman (see T a b l e 7 ) . 68 TSST-1 F i g u r e 16b. Immunoblot a n a l y s i s as d e s c r i b e d in F i g u r e 17a. P a t i e n t 8: nonmenst rua l TSS woman (see T a b l e 7)-69 Patient 11 PAGE Acute Conv Control 1 2 1 2 1 2 1 2 «TSST-1 F i g u r e 16c. Immunoblot a n a l y s i s as d e s c r i b e d in F i g u r e 17a. P a t i e n t 11: Male TSS p a t i e n t (see T a b l e 7). 70 «TSST-1 F i g u r e 16d. Immunoblot a n a l y s i s as d e s c r i b e d in F i g u r e 17a. P a t i e n t 7: T y p i c a l m e n s t r u a l TSS woman who f a i l e d to s e r o c o n v e r t (see T a b l e 7). 71 T a b l e 7- I m m u n o r e a c t i v i t y o f a c u t e and c o n v a l e s c e n t s e r a o f TSS p a t i e n t s t o f i ve d i f f e r e n t m o l e c u l a r w e i g h t b a n d s . P a t i e n t * Sex Age TSA JL Days A f t e r Onse t Immunoreac t i ve Bands 21 2 4 @ 28 32 49 1 F 29 + 1 60 + + + + + 2 F 19 + 2 k2 + + + + + 3 F 24 + 1 20 + + + -4 F 37 + 6 14 + + + + + + + + + 5 F 29 + 6 300 + + + 6 F 29 + 1 60 + -+ + + 7 F 19 + 1 29 + + + + + + 8 F 2k - 2 70 + + + + + + + + 9 F 2k + 7 46 + + + + + + + + 10 F 63 - 10 40 + + + + + + + + 11 M 7 + 1 120 + - -12 M 6 + 5 15 + + + + + + P a t i e n t s 1-7 i n c l u s i v e a r e m e n s t r u a l TSS women, p a t i e n t s 8-10 a r e n o n m e n s t r u a l TSS women and p a t i e n t s 11 and 12 a r e m a l e . * TSA = T o x i g e n i c S t a p h y l o c o c c u s a u r e u s . @ T S S T - 1 . 72 V. DISCUSSION Several assay techniques have been developed to demonstrate and quantitate TSST-1 in S. aureus culture supernatants _in vitro, including analytic isoelecrtric focusing (72), Ouchterlony immunoprecipitation (54), immunoblotting (85), passive latex agglutination (34), radioimmunoassay (M.E. Melish, F.S. Chen, M.S. Murata. Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, B21, p.27), and most recently a competitive ELISA using TSST-1 as an enzyme conjugate (51). Analytic isoelectric focussing and immunoblotting are both qualitative assays, and the former lacks specificity. Ouchterlony immunodiffusion techniques including several modifications such as the microslide immunodiffusion test (13), the cptimal-sensitivity plate method (57), and the single gel diffusion tube method (57), can be used for quantitation of TSST-1, but these generally lack sensitivity, require excessive use of reagents, and cannot be adapted for batch testing of larger samples. Although use of radioimmunoassay should improve both sensitivity and specificity, this method requires expensive equipment and handling of radioactive materials. A reversed passive latex agglutination assay has been developed by Igarashi at al (34), and can detect nanogram quantities of TSST-1. Although promising, its specificity and possible interference by protein A, however, will require additional evaluation. Most recently, Parsonnet et al (51) described a competitive ELISA capable of quantitating 0.03 ug/ml TSST-1 in S. aureus culture supernatants. This assay was not influenced by protein A. but did cross-react miriimalLy with staphylococcal enterotoxLns A, D and E. Furthermore, a competitive ELISA using enzyme-labelled antigen requires purified TSST-1 in relatively large amounts for preparation of the enzyme-antigen conjugate. A more important disadvantage cf this method is that the competitive ELISA might not be readily adapted for quantitation of TSST-1 in biologic fluids or tissue 73 extracts. This is due to the need to incubate enzyme-labelled TSST-1 directly with biologic fluids or tissue extracts which themselves may contain proteases or enzyme inhibitors, and may sLgnigicantly alter the kinetics and activity of the enzyme conjugated to TSST-1, thus severely affecting the accuracy of the test (29). The noncompetitive ELISA described here has a number of advantages over the other methods described above. It is sensitive, specific, simple, economical, and does not require handling and disposal of radioactive materials. It obviates the necessity of repeated and elaborate procedures for purification of TSST-1. Importantly, it can be readily adapted to q^antitate TSST-1 in biologic fluids and tissue extracts, including urine, serum and vaginal washings. Its lower limit of sensitivity, 0.5 ng/ml, appears to be adequate for TSST-1 detection in culture supematants as well as vaginal washings. It does not cross-react with other staphylococcal enterotoxins, and the potential interference by protein A is readily eliminated by pretreatment of samples with 10 % rabbit serum. Protein A is produced by most strains of S. aureus , but the amount released in culture may vary from strain to strain. Since protein A has the unique ability to bind the Fc portion of immunoglobulins, primarily IgG, its presence can readily interfere with the ELISA by binding to both the coating as well as the enzyme-conjugated antitoxin, as is demonstrated in Figure 2. Berdal et al (6) determined that presence of protein A in concentrations greater than 500 ng/ml could affect the detection of staphylococcal enterotoxins A, B and C from culture supematants using a similar ELISA technique. These investigators employed affinity chromatography to remove protein A prior to the enterotoxin assays. Our method of protein A adsorption with 10% rabbit serum appears equally effective but much more simple. Since both the sensitivity and specificity of our ELISA method appear satisfactory, we did not feel that use of monoclonal antibodies to TSST-1 for coating or antibody enzyme conjugation, as was recently described (D.E.Wells, M.W.Reeves, L.M.Graves, and R.M.McKinney, Abstr. Annu.Meet. Am. Soc. Microbiol. 1985, V17, p.391), or use of immunoglobulin subclasses or Fab-specific preparations, will offer any significant advantage. Indeed, use of polyclonal antitoxin may have provided an element of amplification in the sensitivity of our assay by possibly perndtting several different enzyme-labelled antibody epitopes to bind a single TSST-1 molecule with different antigenic domains. Use of polyclonal antitoxin has also obviated the need for tissue culture facilities. Our finding that toxigenic S. aureus strains isolated from TSS patients produced sLgnificantly more TSST-1 in vitro compared to toxigenic control strains is of interest. The ability to produce large quantites of TSST-1 _in vivo may be an important virulence factor. Conversely, local and mucosal host factors which permit optimal production of TSST-1 in vivo by colonizing or infecting strains of S. aureus may be important risk factors of TSS. In this regard, Mills et al (46) have noted the important role of magnesium ion on TSST-1 production in vitro. They postulated that use of certain tampon brands could have precipitated menstrual TSS since these tampon fibers may avidly bind magnesium ions _in vivo and cause a striking increase in TSST-1 production. Alternatively, since our studies indicate that TSST-1 can be detected in vaginal washings of some healthy women, presence of neutralizing systemic or local antibodies to TSST-1 may be an important protective mechanism in normal individuals. It is clear that avail ability of a simple, sensitive, specific and reproducible assay as described here, capable of detecting TSST-1 in concentrations as low as 0.5 ng/ml both _in vitro and in vivo, should greatly facilitate future studies which are urgently needed to further our understanding of the biologic effects 75 of TSST-1 and the pathogenesis of toxic shock syndrome. The feasibility of proceeding with standardization of the ELISA for the detection of TSST-1 in human serum is high. Several human sera have been identified which are devoid of TSST-1 specific antibodies, and can be used to generate standard curves for TSST-1 c^antitation. The ability to demonstrate TSST-1 in the sera of acutely i l l TSS patients would greatly strengthen support for its role in the systemic manifestations of the syndrome. We were not able to detect TSST-1 in patient urine, even in the urine specimen obtained from a menstrual TSS woman on day 1 of her illness. It is possible that the TSST-1 may be metabolized in some manner, making i t undetectable in the urine. Consideration must be given to potential pitfalls concerning the ELISA for TSST-1 detection. An important potential source of error is in the specificity of the rabbit antiserum used to coat microtiter wells and to which alkaline phosphatase is conjugated. The purity of the reference TSST-1 preparation used is also a major concern. The greater the number of extraneous proteins in the standard toxin preparation, the greater the chances are of obtaining an inaccurate standard curve. Although possible interference by other staphylococcal enterotoxins was addressed and disproved, a much more definitive method of deterrnining either the presence of these enterotoxins in the TSST-1 standard, or their respective antibodies in the reference antiserum, would be by immunoblotting analyses. With respect to the antitoxin reference, ideally one desires a preparation with specificity only for the TSST-1 antigen; monoclonal antibodies are not necessary, and in fact, polyclonal antibodies may be better suited because of the possibility of having specificities for several different antigenic determinants on the TSST-1 molecule. In reality, use of a monospecific antibody preparation is all that is necessary to achieve a high level of specificity in ELISA assays. In the case of the ELISA described here 76 for TSST-1 detection, the reference TSST-1 preparation is not as pure as it could be but the reference antiserum is indeed monospecific for TSST-1 at the working dilution, as demonstrated by immunoblot analyses. In addition, many standardization steps were performed to rule out obvious sources of interference including protein A and staphylococcal enterotoxins A through E. The serologic data presented here lend further support to the etiologic role of TSST-1 in the pathogenesis of menstrual toxic shock syndrome and also provide new and exciting information concerning the pathogenesis of nonmenstrual TSS in women and TSS in men. In menstrual TSS patients, our ELISA data suggest that presence of TSST-1 antibodies confers immunity while absence or low titers may be a marker for susceptibility to TSS in these patients. In addition, the weak immunologic response to TSST-1 among convalescent TSS cases may also explain the high risk for recurrence in menstrual TSS. The reason why some women with menstrual TSS fail to seroconvert and develop lasting immunity against TSST-1 is unknown and clearly warrants further investigation. Possible explanations include: a) the nature and duration of intravaginal exposure to TSST-1 may have been too brief and inadequate to stimulate specific antibody formation systenucally; and b) a selective and preexisting local immunologic defect existed during menses in these individuals which precluded sustained local and systemic antibody responses after intravaginal exposure to TSST-1. In this regard, Chow et al have ob s e r v e d marked intermenstrual c y c l i c variation in cervicovaginal immunoglobulins among healthy and TSS women (23). Wira and Sullivan (86) demonstrated in the rat that uterine secretory IgA (slgA) and IgG were lowest at diestrus and highest at estrus. In light of this, future studies involving the quantitation of TSST-1 specific slgA and IgG in cervicovaginal secretions, using 77 the ELISA method designed for this purpose, should provide better understanding of the role of local immune responses to TSST-1 in TSS. Of great interest in this study are the marked differences observed between nonmenstrual TSS women and menstrual TSS women. The high antibody titers in acute sera of some women with nonmenstrual TSS would suggest that TSST-1 was not the cause of illness in these women. These findings are consistent with the findings of Garbe et al (31) who demonstrated that factors other than TSST-1 could be implicated in some cases of nonmenstrual TSS. Another interesting finding is that acute anti-TSST-1 titers in male TSS patients were significantlty lower than in male control sera, suggesting that TSST-1 was the etiologic agent in these patients, similar to women with menstrual TSS. Previous reported studies have failed to separately analyze male and female nonmenstrual TSS patients. In failing to do this, possible significant differences as observed here may easily be masked and conclusions may be inadvertently biased. The reason why male control sera were higher in titer than female controls is unclear, but may be an incidental finding, especially since no age-dependent effect on titers was observed. Of great significance was the demonstration of an inverse relationship of recovery of TSST-1 positive S. aureus and antibody titers to TSST-1 in acute sera of TSS patients, regardless of sex or whether the illness was associated with menstruation in women. In patients who presented with a symptom-complex consistent with TSS, and in whom TSST-1 positive S. aureus could be isolated in conjunction with low antibody titers to TSST-1 in acute sera, the likelihood of TSST-1 mediated illness appears highly plausible. Conversely, in patients with identical clinical manifestations but high antibody titers to TSST-1, or from whom TSST-1 positive S. aureus could not be isolated, the etiology and pathogenesis of TSS may be attributable to agents other than TSST-1. 78 The method of antibody titration employed in this study is quite different from those used by other investigators. In spite of the unconventional nature cf the POSA method, there are several advantages for its use. The POSA compares the specific antibody activity of the test serum to that of a reference serum standard in a quantitative manner and the result is more linearly proportional to the actual antibody titer than is the relative OD value. The subjective nature of determining negative or endpoint titers that are encountered in conventional systems is thus etiminated. The sensitivity of POSA is very high and was developed under highly controlled conditions to ensure specificity and reproducibility. In addition, the method of testing at one dilution makes determination less labour intensive, inexpensive and allows screening of many sera at one time. Possible sources of error also exist with the ELISA for TSST-1 specific IgG determination in serum. Although it was shown that activity was not decreased by the preincubation of serum with enterotoxins A through E, nonspecific activity may not be obvious but may be present. Again, the key is to use an extremely pure preparation of TSST-1 with which to coat microtiter wells so that only TSST-1 specific immunoglobulins will be captured. Because the reference TSST-1 employed in our ELISA is lacking in purity, two main results occur: 1) the positive/negative cutoff values are not obvious. Only after immunoblot analyses were performed could the 95 % upper confidence limit for negative sera titers be determined (see below). 2) Single serum values provide very limited information. Much more information can be derived from paired or sequential, sera from TSS patients to observe changes in antibody response. Another possible pitfall of the method is the use of a single serum dilution to calculate antibody titers. It is possible that testing a single dilution of serum may fail to reflect the actual titer due to prediction from a nonlinear portion 79 of a standard curve. This is true in many cases, especially if only O.D. or relative O.D. values are reported. However, the linearity of the standard POSA curve employed here was clearly demonstrated after log transformation and POSA values even in low titers can be reliably predicted from the standard curve. The logical questions which follow these serological studies are related to the vaginal immunological response to TSST-1 in both healthy individuals and in menstrual TSS patients. Prehminary experiments indicate that TSST-1 specific IgG and secretory IgA are present in vaginal secretions of normal healthy women. It would be most interesting to study cyclic variations in these immunoglobulins as well as to determine which immunoglobulin may be of greater clinical importance against TSST-1. Studies of seroprevalence of TSST-1 specific igM and IgA were also attempted but were unsuccessful. Immunoblotting analyses performed here have provided exciting new evidence for the implication of toxins or factors other than TSST-1 in TSS and in particular, nonmenstrual TSS. These data further support the etiologic role of TSST-1 in the pathogenesis of TSS, particularly for those from whom TSST-1 positive S. aureus could be isolated, or in whom seroconversion against TSST-1 could be demonstrated. However, imunoblots also demonstrate that a number of other staphylococcal exoproteins could be implicated in TSS. In addition, multiple seroconversions to different staphylococcal products were not uncommon, and it is very possible that these agents could interact additively or synergistically in producing the clinical manifestations of TSS. Interactions of different staphylococcal products may explain the variable spectrum of clinical illness seen in humans, and the variable results observed in animal studies of the syndrome, particularly since standardization of stringent criteria for assessment 80 of purity of TSST-1 preparations used in these studies has not been implemented. Scott et al (74) have identified a 30 kd protein, produced by some TSS-associated S. aureus, which cross-reacts immunologically with TSST-1, and have tentatively named this protein TSST-2. In our studies, among the non-TSST-1 proteins MentifLed by immunoblots with acute and convalescent patient sera, the 32 and 21 kd antigens are tentatively named TSST-3 and TSST-4, since seroconversions to these proteins were observed in 3 patients who had preexisting antibodies to TSST-1 in their acute sera (Patients 4,8 and 10). It is also of interest to speculate whether the 49 kd protein is a dimer formed by the complex of two TSST-1 moities, since seroconversion to this protein was always concurrent with that against TSST-1 (Patients 1,5,6,12). i t is also unknown whether these non-TSST-1 proteins were staphylococcal enterotoxins. Crass and Bergdoll (25) noted that although TSST-1 was the major exoprotein produced in TSS-associated S. aureus (over 90% of strains), additional enterotoxins (especially SEA and SEC) were produced by greater than 60% of these isolates. Among 55 isolates associated with nonmentrual TSS, only 12 (22%) produced TSST-1 alone, while 34 (62%) produced SEA or SEC as well as TSST-1, 8 (14%) produced SEB alone, and 1 produced neither TSST-1 nor enterotoxins. More recently, Garbe et al (31) and Scott et al (74) independently demonstrated that TSST-1 negative strains of S. aureus associated with nonmenstrual TSS produced death and illness in rabbits, similar to that produced by TSST-1 positive strains of S. aureus. Scott et al (74) also described a 30 kd staphylococcal protein produced by a TSST-1 negative TSS-associated isolate, which caused TSS-like illness in rabbits and was partially cross-reactive with TSST-1. There is a strong possibility that this 30 kd protein is a precursor of 81 TSST-1. Previous work by Cohen and Falkow (24) reported 2 protein antigens (30 and 33 kd) produced by TSS-associated S. aureus which reacted with human convalescent TSS sera in immunoblots. These proteins were found in 23 of 26 TSS and in only 4 of 18 control S. aureus strains (p< 0.001, Fisher's exact test). Unfortunately, acute sera were not studied in immunoblots against these proteins. The relationship of these proteins and that described by Scott et al (74) or those in our immunoblot studies is unknown. In our partially purified TSST-1 preparation, immunoblot analyses demonstrated two low molecular weight bands at 14.5 and 13 kd with 11 of 12 TSS patient acute sera as well as with pooled control sera. The probable identity of these bands is staphylococcal enterotoxin B. SDS-PAGE analysis of SEB illustrated these same two bands (data not shown). Although SEB is not a dimer, i t has been shown that SEB is separated by SDS-PAGE into two fragments of the size observed here (7). The trace amounts of SEB present in the partially purified TSST-1 should have been removed by the gel filtration step if they were of the size observed in the gels. However, the nonfragmented molecular weight of SEB is approximately 28 Kd and the fragments stay non-covalently associated except in SDS-PAGE. Thus, its molecular weight is similar enough to TSST-1 to allow it to be co-purified. A chromatofocusing step should allow better separation of SEB from TSST-1 as the isoelectric point of SEB is 8.6 compared to 7.0 for TSST-1. A very important observation made in this study relates to the apparent impurity of the TSST-1 preparations that were used. By protein analysis, our TSST-1 preparation was determined to be almost 95% pure, and several of the other preparations have been claimed to be at least as pure. Thus, the discovery of such a number and variety of co-purified substances by silver staining and immunoblot was most alarming. Others have not tested their TSST-1 82 preparations against human serum, as we did, but only against rabbit serum. One could not realistically expect to observe antibodies to as many staphylococal products in young laboratory rabbits due to their sheltered environment. The significance of impurities in TSST-1 preparations is that these types of preparations are being used to determine biologic effects of TSST-1 in animal models and in in vitro systems. It is obvious that more stringent criteria for toxin purity will need to be developed. Observations reported with TSST-1 preparations whose purity has not been critically evaluated must be interpreted with caution. The immunoblotting results are also not without pitfalls, although their qualitative nature makes their interpretation somewhat easier. The data presented are preliiiunary and several additional controls need to be performed. The streptavidin method used is, according to manufacturers, much less subject to nonpecific binding than egg white avidin. In addition, bands in the TSST-1 preparations may be probed with monospecific rabbit antiserum against other staphylococcal enterotoxins in order to identify their nature and relationship to known staphylococcal enterotoxins. It is also possible that the TSST-1 and non-TSST-1 proteins described here, may have been the same protein but with varying degrees of glycosylation, thus reflecrting varying degrees of post^transcriptional modification. It is possible that both the method of growth of organisms and production of TSST-1 as well as the method of purification may influence the final TSST-1 form in this manner. If this were true, one would expect to have seen seroconversions to all of such glycoproteins, not to only some, as was observed. However, when these proteins are further characterized, determination of the degree of glycosylation would be informative. 83 We were also able to use immunoblotting results to validate the ELISA for TSST-1 specific IgG in serum, as the two assays correlated quite well. Six of 7 patients with associated TSST-1 producing S. aureus who demonstrated seroconversion against TSST-1 by immunoblot, also demonstrated rises in ELISA titers (POSA). In addition, POSA values of sera that were shown to be negative for TSST-1 IgG by immunoblotting were S L g n i f i c a n t l y lower than those that were positive by immunoblot (p< 0.02, Mann Whitney rank sum test). The negative cutoff value for POSA was found to be 20.2 + 8.6 (95% confidence limits) based on sera known to be negative for TSST-1 by immunoblot analysis. Finally, immunoblotting techniques were also employed to determine the specificity of the reference rabbit antL-TSST-1 used as the coating antibody in the ELISA for TSST-1 detection. It was demonstrated that this antibody preparation is monospecific at the dilution used in the ELISA for TSST-1 detection. The presence of a small amount of protein A was also demonstrated in the TSST-1 standard used for both the TSST-1 and anti-TSST-1 detection ELISA assays. The presence of protein A was not evident in SDS-PAGE analysis of this TSST-1 preparation; thus, its concentration must be very low. Berdal et al (6) reported that greater than 500 ng/ml of protein A is required to affect the detection of other staphylococcal enterotoxins, so the the possibility of this small amount cf protein A interfering with the TSST-1 detection ELISA is small. It is also possible for this protein A to affect the ELISA for anti-TSST-1 detection, but the effect should be constant for all sera tested. However, the influence of protein A contamination in immunoblot systems can be very great, particularly in systems where whole staphylococcal culture filtrates are used. In such cases, an absorption step with purified goat Fc fragments of IgG, prior to treatment of nitrocellulose with antiserum, should be effective in blocking any protein A which may be present. 8k VL CONCLUSIONS A very comprehensive study of the role of TSST-1 in toxic shock syndrome has been performed. In conclusion: 1) The role cf TSST-1 in TSS has been strengthened by our demonstration of the ability to measure TSST-1 in vaginal washings of menstrual TSS women from whom toxigenic S. aureus was isolated. Previously, positive detection cf TSST-1 in vaginal washings c£ acutely i l l TSS patents has not been published. 2) The serologic studies performed support the etiologic role of TSST-1 in menstrual TSS and in patients from whom toxigenic S. aureus could be cultured, but not in nonmenstrual TSS women from whom toxigenic S. aureus was not isolated. This study is the first to recognize the need to separate nonmenstrual TSS patients, not just according to sex, but also with respect to microbiological findings. 3) Jjnmunoblotting analyses against TSST-1 with acute and convalescent sera from TSS patients and controls, not only add further support for the role of TSST-1 in patients from whom toxigenic S. aureus could be isolated, but also indicate that there may be several staphylococcal products implicated in toxic shock syndrome. Immunoblotting with acute and convalescent human serum has not been reported by others, but the practicality and relevence, with respect to future studies of TSS are enormous. A logical step from the study performed here is to study the acute and convalesent sera of nonmenstrual TSS patients against culture filtrates of homologous S. aureus isolates, in order to identify seroconversions to staphylococcal products that were encountered during illness. It would also be of interest to test acute and convalescent sera against S. epidermidis strains isolated from nonmentrual TSS patients from whom no S. aureus were found. In addition, i t would be interesting to explore the TSST-1 molecule for the presence of immunogenic regions by examining fragments of TSST-1 in immunoblots following proteolytic cleavage , probing with immune rabbit serum, 85 monoclonal antibodies and human serum. 4) Finally, results of this study have indicated that TSST-1 preparations to be used for future TSS studies must be more highly controlled for purity to ensure the validity of results. Immunoblotting in conjunction with silver slzaining of gels appear highly sensitive, and therefore may be an appropriate method for assessing purity of TSST-1 preparations and other proteins. 86 REFERENCES 1. Altmeier WA, Lewis S, Schlievert PM, Bjornson HS. 1981. Studies of the staphylococcal causation of toxic shock syndrome. Surg Gyn Ob 153:481-485. 2. 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