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Separation of immunoglobulins from egg yolk using metal chelate interaction chromatography and ion exchange… McCannel, Anne Marie 1988

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SEPARATION OF IMMUNOGLOBULINS FROM EGG YOLK USING METAL CHELATE INTERACTION CHROMATOGRAPHY AND ION EXCHANGE CHROMATOGRAPHY by ANNE MARIE MCCANNEL B . S c , U n i v e r s i t y of Guelph A THESIS SUBMITTED IN PARITIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Department of Food S c i e n c e ) We a c c e p t t h i s t h e s i s as co n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Oct o b e r , 1988 (c) Anne M a r i e McCannel, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 ABSTRACT The immune response of c h i c k e n s immunized w i t h I3-N-a c e t y l g l u c o s a m i n i d a s e was m o n i t o r e d i n the egg y o l k s o£ the b i r d s u s i n g an e n z y m e - l i n k e d immunosorbent a s s a y . S i g n i f i c a n t l y h i g h e r l e v e l s of s p e c i f i c a n t i b o d i e s were d e t e c t e d i n the y o l k s of the b i r d s immunized w i t h the enzyme when compared w i t h the y o l k s of a c o n t r o l b i r d c o l l e c t e d over the same p e r i o d . s i g n i f i c a n t d i f f e r e n c e s a l s o were found i n the response w i t h i n the immunized group of b i r d s , i n d i c a t i n g i n d i v i d u a l v a r i a b i l i t y t o the i n j e c t i o n s . Immunoglobulins were i s o l a t e d from egg y o l k a f t e r a p r e l i m i n a r y p u r i f i c a t i o n u s i n g a l g i n a t e t o p r e c i p i t a t e l i p o p r o t e i n s . A t e n m i l l i l i t r e DEAE-Sephacel i o n exchange chromatography column r e s u l t e d i n a f i n a l p r o d u c t c o n t a i n i n g 16 mg of IgG w i t h a p u r i t y of 60% when 50 mL of an egg y o l k s u p e r n a t a n t was a p p l i e d . S p e c i f i c a n t i b o d y a c t i v i t y toward the a n t i g e n s 8 - l a c t o g l o b u l i n and E_j_ c o l l l i p o p o l y s a c c h a r l d e was h i g h e r i n both cases i n the i s o l a t e d immunoglobulin f r a c t i o n s which c o n t a i n e d lower p u r i t y ( 4 0 % ) . Lower a n t i b o d y a c t i v i t y was observed i n the 60% p u r i f i e d f r a c t i o n s . S e p a r a t i o n of s p e c i f i c a n t i b o d i e s from n o n - s p e c i f i c a n t i b o d i e s appeared t o o c c u r , p o s s i b l y due t o the g i v e n c h a r a c t e r i s t i c s of the s p e c i f i c a n t i b o d i e s , or due t o the d i f f e r e n c e s e x h i b i t e d by c h i c k e n IgG s u b p o p u l a t i o n s . U s i n g m e t a l c h e l a t e i n t e r a c t i o n chromatography, a 10 mL c o p p e r - l o a d e d column was a b l e t o y i e l d 104 mg of IgG w i t h a i i i p u r i t y of 75% when 200 mL egg y o l k s u p e r n a t a n t was a p p l i e d . A g a i n , the heterogeneous n a t u r e of c h i c k e n IgG was i l l u s t r a t e d . A comparison of the two c h r o m a t o g r a p h i c t e c h n i q u e s i n d i c a t e d the advantages o£ m e t a l c h e l a t e i n t e r a c t i o n chromatography over i o n exchange chromatography under the c o n d i t i o n s examined. A p p l i c a t i o n s o£ the c h i c k e n IgG i s o l a t e d by metal c h e l a t e i n t e r a c t i o n chromatography t o an e n z y m e - l i n k e d immunosorbent a s s a y f o r the d e t e c t i o n of 6 - N - a c e t y l g l u c o s a m i n i d a s e was at t e m p t e d . L i n e a r r e l a t i o n s h i p s were o b t a i n e d when s t a n d a r d s o l u t i o n s of the enzyme were used i n the a s s a y . The r e s u l t s i n d i c a t e t h a t M C I C - i s o l a t e d c h i c k e n immunoglobulins have e x c e l l e n t p o t e n t i a l f o r use i n a n a l y t i c a l t e s t s . i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x INTRODUCTION . ... 1 LITERATURE REVIEW 3 A. importance of Immunoglobulins 3 1. Importance of Immunoglobulins i n p r e v e n t i o n of D i s e a s e s 3 2. Immunoglobulins i n D i a g n o s t i c T e s t s 10 B. Egg Y o l k Immunoglobulins 12 1. Egg Y o l k L l v e t i n s 12 2. C h a r a c t e r i s t i c s of C h i c k e n IgG 13 3. Advantages of Yol k as a Source of Immunoglobulins 16 4. Methods of I s o l a t i n g C h i c k e n IgG 19 C. G e n e r a l Methods f o r A n t i b o d y P u r i f i c a t i o n 22 1. Ion Exchange Chromatography 23 2. M e t a l C h e l a t e i n t e r a c t i o n Chromatography 24 MATERIALS AND METHODS 26 A. Immunization of C h i c k e n s 26 1. G e n e r a l Immunization P r o c e d u r e s 26 2. IL_ c o l i c e l l p r e p a r a t i o n 27 3. Ej. c o l i and 8 - l a c t o g l o b u l i n p r e p a r a t i o n 28 4. 6 - N - a c e t y l g l u c o s a m i n i d a s e p r e p a r a t i o n 28 B. Egg Y o l k S u p e r n a t a n t P r e p a r a t i o n 28 C. Chromatography C o n d i t i o n s 28 1. DEAE-Sephacel Ion Exchange Chromatography 28 2. M e t a l C h e l a t e i n t e r a c t i o n Chromaotgraphy 29 D. P r o t e i n D e t e r m i n a t i o n s 30 E. Enzyme-Linked Immunosorbent Assays 31 1. ELISA f o r D e t e c t i o n of A n t i b o d y A c t i v i t y Toward E. c o l l l i p o p o l y s a c c h a r i d e 31 2. ELISA f o r D e t e c t i o n of A n t i b o d y A c t i v i t y Toward G - l a c t o g l o b u l i n 32 3. ELISA f o r D e t e c t i o n of A n t i b o d y A c t i v i t y Toward ( 3 - N - A c e t y l g l u c o s a m l n i d a s e 32 4. ELI SA f o r D e t e c t i o n of ( 3 - N - a c e t y l g l u c o s a m i n i d a s e i n F i s h or Beef Samples 33 F. I m m u n o d i f f u s i o n Techniques 34 1. R a d i a l I m m u n o d i f f u s i o n 34 2. I m m u n o e l e c t r o p h o r e s i s 35 G. E l e c t r o p h o r e s i s 36 1. N o n - d e n a t u r i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s 36 V 2. SDS-PAGE i n homogeneous a c r y l a m i d e c o n c e n t r a t i o n s 37 3. G r a d i e n t SDS-PAGE 38 H. S t a t i s t i c a l methods 39 RESULTS AND DISCUSSION . 40 PART I . Response of C h i c k e n s t o ( 3 - N - A c e t y l g l u c o s a m l n i d a s e I n j e c t i o n s 41 PART I I . S e p a r a t i o n o£ immunoglobulins from Egg Yo l k U s i n g Chromatographic Techniques 47 A. DEAE-Sephacel Chromatography 47 B. M e t a l C h e l a t e I n t e r a c t i o n Chromatography 63 C. A Comparison of DEAE and MCIC Methods f o r Egg Yol k Immunoglobulin S e p a r a t i o n 99 PART I I I . A p p l i c a t i o n of I s o l a t e d Egg Y o l k Immunoglobulins: The D e t e c t i o n of 8 - N - A c e t y l g l u c o s a m i n i d a s e U s i n g ELISA 104 CONCLUSIONS 110 BIBLIOGRAPHY 112 APPENDIX 121 v i LIST OF TABLES Table I . IgG c o n t e n t a t d i f f e r e n t s t a g e s of the i s o l a t i o n of IgG from egg y o l k on a DEAE-Sephacel Column 53 Table I I . IgG c o n t e n t of p o o l e d f r a c t i o n s o b t a i n e d upon i s o l a t i o n of IgG from egg y o l k u s i n g MCIC w i t h 150 mL sample a p p l i c a t i o n 70 Table I I I . IgG c o n t e n t o£ p o o l e d f r a c t i o n s o b t a i n e d upon i s o l a t i o n of IgG from egg y o l k u s i n g MCIC w i t h 50 mL sample a p p l i c a t i o n 76 Table IV. IgG c o n t e n t of f r a c t i o n s e l u t e d by an i m i d a z o l e g r a d i e n t from an MCIC column w i t h 50 mL sample a p p l i c a t i o n 79 Table V. IgG c o n t e n t of p o o l e d f r a c t i o n s o b t a i n e d from a tandem MCIC column system w i t h 150 mL sample a p p l i c a t i o n 82 Table V I . IgG c o n t e n t of p o o l e d unbound f r a c t i o n s o b t a i n e d from an MCIC column w i t h 200 mL sample a p p l i c a t i o n 97 Table V I I . A comparison of the c o s t of MCIC and DEAE-Sephacel r e s i n s 100 Tab l e V l l l . A comparison of the time r e q u i r e m e n t s f o r IgG s e p a r a t i o n from egg y o l k on a 10 mL MCIC or DEAE-Sephacel column 102 Table IX. L e v e l s of S - N - a c e t y l g l u c o s a m i n i d a s e per gram of Chinook Salmon F l e s h as Determined by ELISA and the S p e c t r o p h o t o m e t r i c Method 106 Table X. L e v e l s of 6 - N - a c e t y l g l u c o s a m i n i d a s e i n b o v i n e muscle samples as det e r m i n e d by ELISA and the Spect r o p h o t o m e t r i c method 107 v i i LIST OF FIGURES F i g u r e 1. The change i n s p e c i f i c a n t i b o d y a c t i v i t y toward ( 3 - N - a c e t y l g l u c o s a m i n i d a s e over time i n egg y o l k s c o l l e c t e d from t h r e e c h i c k e n s immunized w i t h NAGase and a c o n t r o l c h i c k e n r e c e i v i n g no i n j e c t i o n s 42 F i g u r e 2. The change i n t o t a l IgG l e v e l s over time i n the egg y o l k of a c h i c k e n immunized w i t h B-N-a c e t y l g l u c o s a m i n l d a s e 45 F i g u r e 3. Reduced SDS-PAGE p r o f i l e s of the s u p e r n a t a n t and unbound f r a c t i o n o b t a i n e d from DEAE-Sephacel chromatography 48 F i g u r e 4. E l u t i o n p r o f i l e of egg y o l k s u p e r n a t a n t on a DEAE-Seph a c e l column u s i n g two l i n e a r g r a d i e n t s of i n c r e a s i n g p o t a s s i u m phosphate c o n c e n t r a t i o n 49 F i g u r e 5. Reduced SDS-PAGE p r o f i l e s of egg y o l k s u p e r n a t a n t f r a c t i o n s o b t a i n e d from a DEAE-Sephacel column 50 F i g u r e 6. SDS-PAGE p r o f i l e s of ( 3 - l a c t o g l o b u l l n used f o r i m m u n i z a t i o n 52 F i g u r e 7, Reduced SDS-PAGE p r o f i l e s of p o o l e d f r a c t i o n s o b t a i n e d from DEAE-Sephacel chromatography 54 F i g u r e 8. I m m u n o e l e c t r o p h o r e s i s of po o l e d samples o b t a i n e d by DEAE-Sephacel chromatography 56 F i g u r e 9. E l u t i o n p r o f i l e of egg y o l k s u p e r n a t a n t on a DEAE-Se p h a c e l column u s i n g two l i n e a r g r a d i e n t s of i n c r e a s i n g phosphate c o n c e n t r a t i o n 58 F i g u r e 10. E l u t i o n p r o f i l e of egg y o l k s u p e r n a t a n t on a cop p e r -loaded MCIC column u s i n g a d e c r e a s i n g pH g r a d i e n t 64 F i g u r e 11. E l u t i o n p r o f i l e of 45 mg IgG i n 5 mL egg y o l k s u p e r n a t a n t on a MCIC column 66 F i g u r e 12. Reduced SDS-PAGE p r o f i l e s of p o o l e d f r a c t i o n s o b t a i n e d when egg y o l k s u p e r n a t a n t c o n t a i n i n g added IgG was e l u t e d from an MCIC column 67 F i g u r e 13. E l u t i o n p r o f i l e of 150 mL egg y o l k s u p e r n a t a n t a p p l i e d t o a c o p p e r - l o a d e d MCIC column 69 F i g u r e 14. E l u t i o n p r o f i l e of 100 mL egg y o l k s u p e r n a t a n t a p p l i e d t o a c o p p e r - l o a d e d MCIC column 71 F i g u r e 15. Reduced SDS-PAGE of f r a c t i o n s o b t a i n e d when 100 mL s u p e r n a t a n t was a p p l i e d t o a MCIC column 72 v i i i F i g u r e 16. E l u t i o n p r o f i l e of 50 mL egg yolk supernatant a p p l i e d to a copper-loaded MCIC column 74 F i g u r e 17. Reduced SDS-PAGE of f r a c t i o n s obtained when 50 mL supernatant was a p p l i e d to a MCIC column 75 F i g u r e 18. E l u t i o n p r o f i l e of 50 mL egg yolk supernatant on a copper-loaded MCIC column 78 F i g u r e 19. Reduced SDS-PAGE of f r a c t i o n s obtained by g r a d i e n t imidazole e l u t i o n on MCIC column 80 F i g u r e 20. E l u t i o n p r o f i l e of egg yolk p r o t e i n s on the second column i n a tandem MCIC system 84 F i g u r e 21. SDS-PAGE of f r a c t i o n s c o l l e c t e d from the second column of a tandem MCIC system 85 F i g u r e 22. Native PAGE of f r a c t i o n s c o l l e c t e d from the second column of a tandem MCIC system 86 F i g u r e 23. E l u t i o n p r o f i l e of egg yolk supernatant a p p l i e d to a MCIC column not loaded with copper 89 F i g u r e 24. Non-reduced SDS-PAGE of samples c o l l e c t e d from an MCIC column not loaded with copper 90 F i g u r e 25. E l u t i o n p r o f i l e of 50 mL egg yolk supernatant a p p l i e d to a copper-loaded MCIC column 92 F i g u r e 26. E l u t i o n p r o f i l e of 100 mL egg yolk supernatant a p p l i e d to a copper-loaded MCIC column 94 F i g u r e 27. E l u t i o n p r o f i l e of 200 mL egg yolk supernatant a p p l i e d to a copper-loaded MCIC column 95 F i g u r e 28. A t y p i c a l 6-N-acetylglucosaminidase (NAGase) standard curve obtained by the ELISA method. L i n e a r r e g r e s s i o n performed on the data 105 i x ACKNOWLEDGEMENTS I w i s h t o extend my s i n c e r e thanks t o Dr. Nakai f o r the guidance he has g i v e n me d u r i n g the cour s e of t h i s p r o j e c t . H i s e t e r n a l o ptimism i s a t r a i t which I have admired, and hope t o mimick i n the f u t u r e i f a t a l l p o s s i b l e . I would a l s o l i k e t o thank my committee members, Dr. B r e n t Skura and Dr. W i l l i a m Powrie of the Food S c i e n c e Department, and Dr. F i t z s i m m o n s of the Animal S c i e n c e Department f o r the e x c e l l e n t a d v i c e and t e c h n i c a l a s s i s t a n c e which t h e y have o f f e r e d t o me. Sherman Yee and V a l Skura a l s o have been v e r y h e l p f u l i n making my time as a graduate s t u d e n t e a s i e r . I e xtend thanks a l s o t o my f a m i l y , as w e l l as t o my b o y f r i e n d , Ben Yee, who have been b o t h p a t i e n t and s u p p o r t i v e t h r o u g h o u t t h i s s t u d y . F i n a l l y , I w i s h t o d e d i c a t e t h i s work t o my dear f o u r t e e n year o l d c o u s i n , Lee Gunson, who l o s t h i s l i f e t h i s year i n a farm a c c i d e n t . We w i l l always remember and miss you. 1 INTRODUCTION Immunoglobulins p l a y a v i t a l r o l e i n the maintenance of h e a l t h (Gough et a l , 1988). The b e n e f i c i a l e f f e c t s of these p r o t e i n s i n the p r e v e n t i o n of i n f a n t i n t e s t i n a l d i s e a s e s have been demonstrated ( B a l l a b r i g a , 1982; F r i e n d e t a l , 1983; Goldblum et a l , 1986; L a r g u i a et a l , 1977). The source of a n t i b o d i e s need not be human; bovine immunoglobulins have been s u c c e s s f u l ( B a l l a b r i g a , 1982). The high l e v e l s of immunoglobulins i n human colostrum o f f e r p a s s i v e immunity to the newborn, whose own immune system does not a c t i v e l y s y n t h e s i z e a n t i b o d i e s d u r i n g the f i r s t few months of l i f e ( B a l l a b r i g a , 1982). The a d d i t i o n of immunoglobulins t o i n f a n t formulae, which c u r r e n t l y c o n t a i n s o n l y low l e v e l s , i s t h e r e f o r e d e s i r a b l e . B e n e f i t s need not be l i m i t e d to the i n f a n t . I n g e s t i o n of bovine immunoglobulins with an a n t a c i d has been e f f e c t i v e i n p r e v e n t i n g e n t e r o t o x i g e n i c c o l l i n f e c t i o n i n human a d u l t s (Tacket et a l , 1988). Immunoglobulins are Important a l s o i n many a n a l y t i c a l t e s t s which are c u r r e n t l y i n use. The enzyme-linked immunosorbent assay (ELISA) i s based on the s p e c i f i c r e a c t i o n which i s e x h i b i t e d by an an t i b o d y toward an a n t i g e n . The ELISA technique i s becoming a prominent t o o l i n a l l areas of s c i e n c e . 2 For a l l a p p l i c a t i o n s , a c o n v e n i e n t , Inexpensive source of a n t i b o d i e s Is d e s i r a b l e . Egg y o l k shows e x c e l l e n t p o t e n t i a l as such a s o u r c e . Egg y o l k has h i g h l e v e l s of immunoglobul ins ( B a r - J o s e p h and M a l k i n s o n , 1980; Rose e t a l , 1974) , and s e v e r a l advantages have been d e s c r i b e d f o r the use of y o l k a n t i b o d i e s ( P o i s o n e t a l , 1980) . S e v e r a l i s o l a t i o n methods have been deve loped f o r y o l k immunoglobul ins ( J e n s e n l u s e t a l , 1981; P o i s o n e t a l , 1980) . A l l r e q u i r e numerous p r e c i p i t a t i o n s t e p s u s i n g components t h a t may not be a c c e p t a b l e f o r human c o n s u m p t i o n . I n d u s t r i a l a p p l i c a t i o n of these methods may not be f e a s i b l e . I t was t h e r e f o r e the o b j e c t i v e of t h i s r e s e a r c h to examine the p o t e n t i a l of egg y o l k immunoglobul in s e p a r a t i o n u s i n g chromatograph ic t e c h n i q u e s . DEAE-Sephace l ion exchange chromatography and meta l c h e l a t e i n t e r a c t i o n chromatography methods were examined and compared. A p p l i c a t i o n o£ the i s o l a t e d c h i c k e n immunoglobul ins i n an ELISA a s s a y f o r the d e t e c t i o n o£ 6 - N - a c e t y l g l u c o s a m i n i d a s e was e v a l u a t e d ; the r e s u l t s i n d i c a t e d e x c e l l e n t p o t e n t i a l f o r use i n a n a l y t i c a l t e s t s . 3 LITERATURE REVIEW A. IMPORTANCE OF IMMUNOGLOBULINS Immunoglobulins are g l y c o p r o t e i n s which o f f e r p r o t e c t i o n a g a i n s t d i s e a s e s by a l l o w i n g the body to i d e n t i f y f o r e i g n substances, such as b a c t e r i a or v i r u s e s , which may attempt to invade the body d u r i n g i n f e c t i o n . The p r o t e c t i v e e f f e c t e x h i b i t e d by immunoglobulins i s due to the i d e n t i f i c a t i o n and b i n d i n g of these f o r e i g n substances, a n t i g e n s . Thus, immunoglobulins are v i t a l i n the maintenance of good h e a l t h . They a l s o have been s u c c e s s f u l l y used as a d i a g n o s t i c t o o l based on the s p e c i f i c r e a c t i o n s with a n t i g e n s , r e s u l t i n g i n h i g h l y s e n s i t i v e a s s ays. A l . The Importance of Immunoglobulins In P r e v e n t i o n of  Diseases Immunoglobulins p l a y a v i t a l r o l e i n combatting d i s e a s e s . A r e c e n t r e p o r t of the s u c c e s s f u l treatment of AIDS r e l a t e d complex through the use of immunoglobulins i n d i c a t e s the importance of these p r o t e i n s i n the maintenance of h e a l t h (Gough et a l , 1988). The b e n e f i c i a l e f f e c t s t o In f a n t s of the i n g e s t i o n of human br e a s t milk over c u r r e n t l y a v a i l a b l e i n f a n t formulae have been d e s c r i b e d by s e v e r a l r e s e a r c h e r s and have been a t t r i b u t e d p r i m a r i l y to immunologic f a c t o r s present i n human milk which are a t low l e v e l s , or absent, i n formulae. 4 R e d u c t i o n i n i n f e c t i o u s , a l l e r g i c , and o ther d i s e a s e s o£ the g a s t r o i n t e s t i n a l and r e s p i r a t o r y t r a c t s (Goldblum et a l , 1986) as w e l l as lowered m o r t a l i t y r a t e s (Welsh and May, 1979) has been r e p o r t e d . Whi le t h i s t r e n d i s apparent i n case s t u d i e s of i n f a n t s i n d e v e l o p i n g c o u n t r i e s , c l o s e e v a l u a t i o n of r e s u l t s examining the e f f e c t of b r e a s t f e e d i n g i n i n d u s t r i a l i z e d c o u n t r i e s i n d i c a t e s , a t b e s t , o n l y a minimal p r o t e c t i v e e f f e c t when numerous f a c t o r s are c o n s i d e r e d (Bauchner e t a l , 1986) . However, the immunoglobul in f r a c t i o n undoubted ly i s of g r e a t s i g n i f i c a n c e . The a b i l i t y of i n f a n t s to endogenous ly s y n t h e s i z e immunoglobul ins t o any s u b s t a n t i a l amount does not b e g i n u n t i l the age of two months has been r e a c h e d . P r i o r to t h i s t i m e , the i n f a n t i s dependent upon the t r a n s m i s s i o n of materna l a n t i b o d i e s b e f o r e b i r t h by the a b s o r p t i o n of IgG through the p l a c e n t a to the f e t u s , and a f t e r b i r t h on immunologic f a c t o r s a v a i l a b l e through b r e a s t f e e d i n g ( B a l l a b r i g a , 1982) . The p o s s i b i l i t y t h a t c e r t a i n components of human mi lk may promote s y n t h e s i s or s e c r e t i o n of immune f a c t o r s in the i n f a n t ' s g a s t r o i n t e s t i n a l t r a c t has r e c e n t l y been sugges ted (Goldblum et a l , 1986; S c h a n l e r e t a l , 1986) . The immunoglobul ins themselves i n the gut may be e f f e c t i v e i n p r e v e n t i n g I n f e c t i o n through s e v e r a l p o s s i b l e modes: n e u t r a l i z i n g t o x i n s ; k i l l i n g v i r u s e s ; f i x i n g complement; a g g r e g a t i n g b a c t e r i a i n the i n t e s t i n e to f a c i l i t a t e removal a n d / o r p reven t b a c t e r i a l c o l o n i z a t i o n of the i n t e s t i n a l l i n i n g ( F r i e n d e t a l , 1983) . I t has been shown 5 t h a t b r e a s t f e d i n f a n t s have a f e c a l f l o r a w i t h a predominance of L a c t o b a c i l l u s b l f i d u s , w h i l e b o t t l e f e d i n f a n t s have a predominance of c o l i f o r m s ( L a r g u i a e t a l , 1977). S i g n i f i c a n t d i f f e r e n c e s e x i s t i n the p a t t e r n s of immunoglobulins s e c r e t e d i n the s a l i v a by f o r m u l a - f e d and b r e a s t f e d i n f a n t s ( G l e e s o n e t a l , 1986). As w e l l , f e c a l e x c r e t i o n of immune f a c t o r s i n c l u d i n g i m m u n o g l o b u l i n s , l a c t o f e r r i n , and lysozyme, has been shown t o be h i g h e r i n i n f a n t s f e d human m i l k ( S c h a n l e r e t a l , 1986; Goldblum e t a l , 1986; Davidson and L o n n e r d a l , 1987). E x a m i n a t i o n of the f e c e s of f u l l term i n f a n t s b e i n g b r e a s t f e d showed the presence of o n l y t h r e e p r o t e i n s a t l e v e l s h i g h enough f o r d e t e c t i o n : s l g A , l a c t o f e r r i n , and a j -a n t i t r y p s i n (Davidson and L o n n e r d a l , 1987). No o t h e r major m i l k p r o t e i n s were d e t e c t e d , s u g g e s t i n g t h a t the i n f a n t s i n the s t u d y had normal p r o t e i n d i g e s t i o n . The s t a b i l i t y of p r o t e i n s which e l i c i t an immunologic e f f e c t i s presumably an i m p o r t a n t f a c t o r i n t h e i r e f f e c t i v e n e s s . S c h a n l e r e t a l (1986) d e t e r m i n e d t h a t a n t i b o d y a c t i v i t y toward E ^ c o l i 0 a n t i g e n s was m a i n t a i n e d d u r i n g p r o c e s s i n g , f e e d i n g and passage t h r o u g h the g a s t r o i n t e s t i n a l t r a c t , based on the i n t a k e and f e c a l l e v e l s of the a n t i b o d i e s . The e f f e c t i v e n e s s of the a d m i n i s t r a t i o n of human c o l o s t r u m i n the r e d u c t i o n of both m o r b i d i t y and m o r t a l i t y caused by E_j_ c o l i i n i n f a n t s has been documented by L a r g u i a e t a l (1977). I n 1971 and 1972, an E^ c o l i d i a r r h e a e p i d e m i c i n the n e o n a t a l u n i t f o r premature i n f a n t s c o u l d not be 6 c o n t r o l l e d . Treatment f o r d i a r r h e a began i n 1973 by a d m i n i s t r a t i o n of f r e s h human colostrum (milk produced one to four days post-partum) a t a dose o£ 5mL/kg/day, along with a conce n t r a t e d i n f a n t formula, over a p e r i o d of 15 months, 280 premature i n f a n t s were t r e a t e d i n t h i s manner, f o r a mean p e r i o d of twenty days. Enteropathogenic c o l i was recovered i n the s t o o l s of 20% of the i n f a n t s , but mil d d i a r r h e a was present i n o n l y three cases. While .the number of cases of d i a r r h e a was d r a m a t i c a l l y reduced d u r i n g treatment, the number of p o s i t i v e c u l t u r e s obtained was u n a f f e c t e d . T h i s may i n d i c a t e t h a t the pathogenic mechanism of the E ^ c o l 1 was i n t e r r u p t e d by the colostrum without b a c t e r i c i d a l e f f e c t s . The p r o t e c t i v e r o l e e x h i b i t e d by human milk (colostrum i n p a r t i c u l a r ) has been suggested by many other r e s e a r c h e r s . Because of the low a v a i l a b i l i t y of human colostrum and milk, other sources of immunologic components have been examined. The predominant immunoglobulin i n human milk i s s e c r e t o r y igA (slgA) (Butte et a l , 1984) with low amounts of IgG and IgM. IgG r e p r e s e n t s the m a j o r i t y of immunoglobulins found i n cow's mil k . In both cases, immunoglobulin l e v e l s , h i g h e s t i n the colostrum, decrease over time (Uechi et a l , 1982) . B a l l a b r i g a (1982) assumed t h a t the IgG found i n bovine milk should be ab l e to p l a y a s i m i l a r r o l e as s l g A i n human milk i f : the bovine immunoglobulin d i s p l a y e d an ant i b o d y s p e c i f i c i t y which i s normally found i n human milk, and i f the a c t i v i t y was r e t a i n e d d u r i n g t e c h n o l o g i c a l p r o c e s s i n g ; i f the 7 pathoraechanisms of the e n t e r o p a t h o g e n i c m i c r o o r g a n i s m s c o u l d be i n t e r r u p t e d by the bovine i m m u n o g l o b u l i n s ; i f the b o v i n e immunoglobulins were r e s i s t a n t to p r o t e o l y t i c d i g e s t i o n ; and I f the p r o p h y l a c t i c and t h e r a p e u t i c v a l u e of m i l k formulae e n r i c h e d w i t h s p e c i f i c b o v i n e m i l k a n t i b o d i e s c o u l d be proven s c i e n t i f i c a l l y . I n o r d e r t o examine t h i s i d e a , B a l l a b r i g a (1982) hyper immunized pregnant cows i n t h e i r f i n a l s t a g e of g e s t a t i o n w i t h a v a c c i n e of E_j_ c o l i s e r o t y p e s t h a t were f r e q u e n t l y r e s p o n s i b l e f o r i n f a n t i l e g a s t r o i n t e s t i n a l i n f e c t i o n s . C o l o s t r u m from the cows was c o l l e c t e d f o r the f i r s t s i x t o e i g h t days p o s t - p a r t u m . The m i l k was skimmed, c a s e i n p r e c i p i t a t e d , l a c t o s e and m i n e r a l s a l t s removed by u l t r a f i l t r a t i o n , s t e r i l e f i l t e r e d , and l y o p h i l i z e d . The m i l k immunoglobulin c o n c e n t r a t e o b t a i n e d had from 30 t o 45% I g p u r i t y , and was c a l l e d c o l o s t r a l whey p r o t e i n s (CWP). T h i s was then used t o t r e a t 152 I n f a n t s up t o f i v e months i n age who were s u f f e r i n g from moderate t o s e v e r e g a s t r o e n t e r i t i s w i t h B_j_ c o l l as the c a u s i t i v e organism. E i t h e r 2g/kg/day of the CWP was added t o the d i e t s f o r f i v e d ays, or l g / k g / d a y was added f o r t e n days. F o u r t y - t h r e e o t h e r i n f a n t s were g i v e n CWP a t s i m i l a r doses from non-immunized cows. The r e s u l t s showed t h a t the CWP from the Immunized cows was a b l e t o s i g n i f i c a n t l y reduce the number of p o s i t i v e c u l t u r e s from s t o o l s of the i n f a n t s compared w i t h those t r e a t e d w i t h the CWP from non-immunized cows ( B a l l a b r i g a , 1982). 8 Heated hyperimmune bo v i n e c o l o s t r u m c o n t a i n i n g a n t i b o d i e s toward c r y p t o s p o r i d l o s i s proved e f f e c t i v e i n p r e v e n t i n g the i n f e c t i o n i n mice. The b o v i n e c o l o s t r u m was then used on a t h r e e y e ar o l d boy, w i t h c o n g e n i t a l hypogammaglobulinaemia, who had been s u f f e r i n g from s e v e r e d i a r r h e a and v o m i t i n g due t o c r y p t o s p o r i d i o s i s f o r t h r e e weeks. A d m i n i s t r a t i o n v i a n a s o g a s t r i c f e e d i n g f o r t w e l v e days was I n i t i a t e d , f o l l o w e d by 50mL c o l o s t r u m o r a l l y f o r f o u r days. Three days a f t e r t r e a t m e n t had begun, v o m i t i n g s t o p p e d , and w i t h i n f i v e days the d i a r r h e a had been c o r r e c t e d . For f o u r months f o l l o w i n g the t r e a t m e n t , he was found t o have no f u r t h e r o c c u r r e n c e of d i a r r h e a , and h i s s t o o l s were f r e e of o o c y s t s ( T z i p o r i e t a l , 1986). T h i s was e f f e c t i v e i n the p a t i e n t w i t h o u t the use of h i s t a m i n e s or a n t a c i d s . B a r t z and coworkers (1980) s u c c e s s f u l l y p r e v e n t e d murine r o t a v i r u s i n f e c t i o n i n mice t h r o u g h o r a l a d m i n i s t r a t i o n of the water s o l u b l e f r a c t i o n o b t a i n e d from the egg y o l k s of immunized c h i c k e n s . The water s o l u b l e f r a c t i o n was p r e p a r e d by d i l u t i o n w i t h PBS (pH 7.4), c e n t r i f u g a t i o n f o r one hour a t 33,750 x g, d i l u t i o n of the s u p e r n a t a n t w i t h sodium c h l o r i d e , f o l l o w e d by c e n t r i f u g a t i o n f o r t w e l v e hours a t 141,650 x g. F u r t h e r p u r i f i c a t i o n of immunoglobulins was a c c o m p l i s h e d t h r o u g h two p r e c i p i t a t i o n s t e p s w i t h 18% sodium s u l f a t e . I n t e r e s t i n g l y , the water s o l u b l e f r a c t i o n showed a h i g h e r t i t e r t h a n the p u r i f i e d f r a c t i o n s based on ELISA r e s u l t s , p o s s i b l y due t o l n a c t i v a t i o n or l o s s of IgG d u r i n g the p u r i f i c a t i o n s t e p . The s u c c e s s f u l t r e a t m e n t of r o t a v i r u s i n 9 mice w i t h egg y o l k immunoglobulins s u g g e s t s p o t e n t i a l a p p l i c a t i o n i n humans as w e l l . The p o t e n t i a l use of bovine immunoglobulins s p e c i f i c f o r e n t e r o t o x i g e n i c c o l l c a u s i n g t r a v e l e r ' s d i a r r h e a i n humans was r e c e n t l y r e p o r t e d ( T a c k et e t a l , 1988). Pregnant cows were immunized d u r i n g the l a s t s t a g e s of g e s t a t i o n w i t h E.  c o l i s e r o g r o u p s commonly c a u s i n g d i a r r h e a i n humans. For the f i r s t t e n days a f t e r c a l v i n g , m i l k c o l l e c t e d from the cows was p a s t e u r i z e d and t r e a t e d t o remove th e f a t , c a s e i n , l a c t o s e and s a l t s . The p r o d u c t was t h e n s t e r i l i z e d and l y o p h i l i z e d . The r e s u l t i n g immunoglobulin p u r i t y was 45% of the t o t a l p r o t e i n . T h i s was t h e n mixed w i t h magnesium and aluminum h y d r o x i d e i n o r d e r t o n e u t r a l i z e stomach a c i d i t y d u r i n g i n g e s t i o n . As a c o n t r o l , an immunoglobulin c o n c e n t r a t e p r e p a r e d i n the same f a s h i o n but o b t a i n e d from cows immunized w i t h r o t a v i r u s was used. Twenty h e a l t h y a d u l t v o l u n t e e r s were t h e n randomly d i v i d e d i n t o a t e s t group ( r e c e i v i n g the t e s t sample p r e p a r e d from the immunized cows) or c o n t r o l group ( r e c e i v i n g the c o n t r o l s a m p l e ) . Immunoglobulin c o n c e n t r a t e (3.55g) and a n t a c i d i n 237 mL of water was a d m i n i s t e r e d t h r e e t i m e s a day, f i f t e e n minutes a f t e r each meal, f o r seven days. On the t h i r d day, t h e s u b j e c t s were c h a l l e n g e d w i t h an e n t e r o t o x i g e n i c s t r a i n of E ^ c o l l . A sodium b i c a r b o n a t e s o l u t i o n was i n g e s t e d j u s t p r i o r t o , and w i t h , t h e Ej_ c o l l s u s p e n s i o n . A dose of Immunoglobulin c o n c e n t r a t e f o l l o w e d f i f t e e n minutes a f t e r the b a c t e r i a s u s p e n s i o n . 10 Nine of the t e n v o l u n t e e r s i n the c o n t r o l group de v e l o p e d d i a r r h e a , w h i l e none of t h o s e r e c e i v i n g the t e s t immunoglobulins showed any symptoms of i n f e c t i o n . In b o t h g r o u p s , however, c o l l was e x c r e t e d i n the s t o o l . No a d v e r s e s i d e e f f e c t s from the b o v i n e immunoglobulins were o b s e r v e d , a l t h o u g h an i n c r e a s e i n serum t r a n s a m i n a s e s o c c u r r e d , p o s s i b l y due t o the h i g h p r o t e i n d i e t . These r e s u l t s I n d i c a t e t h a t the a d m i n i s t r a t i o n of a b o v i n e immunoglobulin c o n c e n t r a t e , i n c o n j u n c t i o n w i t h an a n t a c i d t o reduce stomach a c i d i t y , can be e f f e c t i v e i n the p r e v e n t i o n of d i a r r h e a caused by E. c o l l i n a d u l t s ( T a c k e t e t a l , 1988). The i n c o r p o r a t i o n of an a n t a c i d w i t h immunoglobulins of d e s i r e d s p e c i f i c a c t i v i t y might be e x p e c t e d t o be e f f e c t i v e i n c o m b a t t i n g numerous i n t e s t i n a l i n f e c t i o n s o c c u r r i n g i n a d u l t s . The problem of d i a r r h e a i s s t i l l v e r y r e a l t o d a y , c a u s i n g an e s t i m a t e d f i v e t o t e n m i l l i o n d e aths each year i n d e v e l o p i n g c o u n t r i e s (Fedorak and F i e l d , 1987). The p o t e n t i a l f o r use of i m m u n o g l o b u l i n s , o b t a i n e d from v a r i o u s s o u r c e s , f o r t r e a t m e n t of numerous d i s e a s e s o c c u r r i n g i n people of a l l ages appears q u i t e p r o m i s i n g . A.2 Immunoglobulins i n A n a l y t i c a l T e s t s The use of immunoglobulins f o r a n a l y t i c a l purposes has e v o l v e d r a p i d l y w i t h i n the l a s t few y e a r s t o encompass not o n l y the f i e l d s of m e d i c i n e but v i r t u a l l y a l l a r e a s of s c i e n c e . 11 immunoassays, o£ which enzyme l i n k e d Immunosorbent a s s a y s (ELISA's) a r e an example, make use o£ the h i g h l y s p e c i f i c r e a c t i o n s which t a k e p l a c e between a n t i b o d i e s ( i m m u n o g l o b u l i n s ) and the a n t i g e n toward which t h e y were r a i s e d . S i n c e most compounds h a v i n g a m o l e c u l a r weight of 4000 d a l t o n s or g r e a t e r a r e immunogenic (Vaag and Munck, 1987), a n t i b o d i e s can be o b t a i n e d s p e c i f i c t o numerous compounds. S e v e r a l t y p e s of ELISA's ( d i r e c t ; i n d i r e c t ; s a n d w i c h ) , based on the a n t i b o d y - a n t i g e n r e a c t i o n , have been d e v e l o p e d . A n t i b o d i e s used g e n e r a l l y a r e o b t a i n e d from the b l o o d serum of a n i m a l s immunized w i t h the d e s i r e d a n t i g e n (or immunogen), and somewhere i n the ELISA an enzyme (commonly a l k a l i n e phosphatase or h o r s e r a d i s h p e r o x i d a s e ) i s I n c l u d e d w h i c h , upon a d d i t i o n of the s p e c i f i c s u b s t r a t e , a l l o w s f o r d e t e c t i o n and q u a n t i f i c a t i o n of the a n t i g e n or a n t i b o d y , as d e s i r e d . A p p l i c a t i o n s of ELISA's i n the food s c i e n c e a r e a have been numerous ( A l l e n and S m i t h , 1987; Vaag and Munck, 1987). The s p e c i f i c i t y and s e n s i t i v i t y o f f e r e d by t h e s e immunoassays makes the p o s s i b i l i t y t h a t a method might be d e v e l o p e d f o r the d e t e c t i o n of 6 - N - a c e t y l g l u c o s a m i n l d a s e (NAGase) i n f i s h an e x c i t i n g p r o s p e c t . T h i s enzyme has been shown t o be a u s e f u l i n d i c a t o r of whether f i s h has been f r o z e n or i s f r e s h (Rehbeln e t a l , 1978; Shimomura e t a l , 1987). with c u r r e n t demands from consumers f o r h i g h q u a l i t y , f r e s h food p r o d u c t s , a method of e s t a b l i s h i n g f r e s h n e s s i s d e s i r a b l e . The presence of NAGase appears t o o f f e r such d i s t i n c t i o n , and t h e ELISA method may be a v a l u a b l e t o o l i n I t s d e t e c t i o n . NAGase a l s o 12 has been a s s o c i a t e d w i t h s e v e r a l d i s e a s e s . I t has been shown t o be u s e f u l i n the d e t e c t i o n of m a s t i t i s i n f e c t i o n i n cows ( M a t t i l a e t a l , 1986); l e v e l s i n the u r i n e of newborns have been used as an i n d i c a t o r of r e n a l t u b u l a r damage (Watanabe e t a l , 1987); i t a l s o has been a s s o c i a t e d w i t h d i a b e t e s m e l l i t u s (Skrha e t a l , 1987). Thus, d e t e c t i o n methods of h i g h s e n s i t i v i t y a r e d e s i r a b l e f o r m e d i c a l a p p l i c a t i o n s ( Lim, 1987). B. EGG YOLK IMMUNOGLOBULINS Egg i m m u n o g l o b u l i n s , a r e l a t i v e l y newly i n v e s t i g a t e d s o u r c e of a n t i b o d i e s , a r e found i n the l i v e t i n p r o t e i n f r a c t i o n of y o l k . S e v e r a l advantages of t h e use of c h i c k e n egg y o l k immunoglobulins over c u r r e n t l y used b l o o d s o u r c e s have been d e s c r i b e d (Anonymous, 1988; P o i s o n e t a l , 1980; G o t t s t e i n and Hemmeler, 1985). Methods d e v e l o p e d f o r the s e p a r a t i o n of y o l k IgG have t a k e n advantage of c h a r a c t e r i s t i c s d i f f e r e n t i a t i n g them from the o t h e r p r o t e i n s . B . l Egg Yolk L l v e t l n s L i v e t i n i s a heterogeneous group of w a t e r - s o l u b l e , non-l i p i d , g l o b u l a r p r o t e i n s i n y o l k plasma which has been s e p a r a t e d i n t o t h r e e main f r a c t i o n s by e l e c t r o p h o r e s i s (Shepard and H o t t l e , 1949) and termed a-,6-, and Y - l i v e t i n s ( M a r t i n e t a l , 1957). W i l l i a m s (1962) examined t h e t h r e e l i v e t i n f r a c t i o n s and found t h a t t h e y c o r r e s p o n d t o serum 13 p r o t e i n s : a - l i v e t i n t o serum a l b u m i n ; 6 - l i v e t i n t o an ~ g l y c o p r o t e i n ; and Y - l i v e t i n t o Y - g l o b u l i n . The h e t e r o g e n e i t y of l i v e t i n was demonstrated by s t a r c h g e l e l e c t r o p h o r e s i s , whereby s i x t e e n d i f f e r e n t components were observed by Hui and Common (1966), w h i l e Mclndoe and C u l b e r t (1979) d e t e c t e d t e n p r o t e i n s . The b e t a - l i v e t i n p r o t e i n a l o n e c o u l d be s e p a r a t e d i n t o f o u r e l e c t r o p h o r e t i c a l l y d i f f e r e n t f r a c t i o n s . B u r l e y and Vadehra (1979), u s i n g g e l f i l t r a t i o n , s e p a r a t e d the y o l k s o l u b l e p r o t e i n s i n t o f i v e f r a c t i o n s : a, 6, and Y, as w e l l as a p r o t e i n of h i g h m o l e c u l a r weight (200,000 d a l t o n s ) which was termed < 3 - l i v e t i n , and an a p o v l t e l l e n i n of m o l e c u l a r weight 20,000 d a l t o n s . The Y - l i v e t i n f r a c t i o n of egg y o l k has been shown t o c o r r e s p o n d t o the IgG f r a c t i o n i n serum. I t i s t r a n s f e r r e d t o the y o l k t o c o n f e r p a s s i v e Immunity t o the o f f s p r i n g , i n the event t h a t the egg i s f e r t i l i z e d (Rose and O r l a n s , 1981). T h i s appears t o be a v e r y e x c i t i n g p o t e n t i a l s o u r c e of a n t i b o d i e s . B. 2 C h a r a c t e r i s t i c s of C h i c k e n IgG While Yamamoto and coworkers (1975) r e p o r t e d the d e t e c t i o n of IgA, IgM and IgG i n egg y o l k based on i m m u n o d i f f u s i o n r e s u l t s , e v i d e n c e would c u r r e n t l y s u ggest t h a t the hen's egg y o l k c o n t a i n s o n l y one c l a s s of immun o g l o b u l i n s . E a r l y s t u d i e s of t h i s i mmunoglobulin i n i t i a l l y s u g g e s t e d a mo l e c u l e a n t i g e n i c a l l y d i f f e r e n t from human IgG, c a u s i n g 14 L e s l i e and Clem (1969) to d e s i g n a t e It " IgY" . Ambrosius and Hadge (1987) compared the c h i c k e n "IgY" w i th mammalian IgG and human IgD, and found no c r o s s - r e a c t i v i t y . They c o n c l u d e d tha t the c h i c k e n Ig was not of the same c l a s s . However, c r o s s -r e a c t i v i t y w i th human IgG d e t e c t e d by May and coworkers (1984) s u g g e s t s t h a t the t e r m i n o l o g y of IgG i s most a p p r o p r i a t e and i s the d e s i g n a t i o n used i n t h i s p a p e r . Be ing s i m i l a r t o human IgG, c h i c k e n IgG c o n s i s t s of two heavy c h a i n s and two l i g h t c h a i n s l i n k e d by c o v a l e n t d i s u l p h i d e bonds . The c h a i n s are d i v i d e d i n t o pocke ts (domains) which are s t a b i l i z e d by i n t r a c h a i n d i s u l p h i d e bonds . The l i g h t c h a i n c o n s i s t s of a domain of c o n s t a n t amino a c i d s e q u e n c e , as w e l l as a v a r i a b l e domain . Heavy c h a i n s have t h r e e c o n s t a n t r e g i o n s and one v a r i a b l e domain . The v a r i a b l e r e g i o n s of the mo lecu le a re r e s p o n s i b l e f o r the s p e c i f i c a n t i g e n b i n d i n g p r o p e r t i e s of IgG (Steward , 1984) . C h i c k e n IgG has been r e p o r t e d t o have a m o l e c u l a r weight r a n g i n g from 170,000 d a l t o n s ( L e s l i e and C lem, 1969) to 206,000 d a l t o n s (Tenenhouse and D e u t s c h , 1966) , which i s h i g h e r than t h a t of human IgG (mo lecu la r weight of 150 ,000 ) . T h i s d i f f e r e n c e i s due to a heavy c h a i n of h i g h e r m o l e c u l a r weight a t 70,000 ( L e s l i e and C lem, 1969) , compared t o a human heavy c h a i n of m o l e c u l a r weight 50,000 d a l t o n s . The m o l e c u l a r weight of the l i g h t c h a i n s of c h i c k e n and human IgG are s i m i l a r , a t 22,000 d a l t o n s . The r e p o r t e d v a l u e s f o r the hexose c o n t e n t i n c h i c k e n IgG ranges from 1.5% (Zakay-Rones e t a l , 1972) to 6.0% (Howel l e t a l , 1973) , w i th o ther r e s e a r c h e r s 15 r e p o r t i n g i n t e r m e d i a t e v a l u e s ( L e s l i e and C lem, 1969; Tenenhouse and D e u t s c h , 1966; N iedermeie r e t a l , 1971) . Human IgG v a l u e s range from two to t h r e e p e r c e n t ( N l s o n o f f , 1982) . The r e p o r t e d i s o e l e c t r i c p o i n t s f o r c h i c k e n IgG a l s o v a r i e s c o n s i d e r a b l y . Tenenhouse and Deutsch (1966) r e p o r t e d a v a l u e of 5 . 2 . G a l l a g h e r and Voss (1970) d e t e c t e d a minor p o r t i o n of c h i c k e n IgG h a v i n g a p i of 5 . 3 , w i th the m a j o r i t y a t 6 . 6 . Loeken and Roth (1983) i n d i c a t e d t h a t c h i c k e n IgG i s heterogeneous hav ing i s o e l e c t r i c p o i n t s r a n g i n g from 6.5 to 9 . 0 . The p o s s i b i l i t y of the e x i s t e n c e of s u b c l a s s e s of c h i c k e n IgG has been sugges ted by a few a u t h o r s (Or lans and Rose , 1972; Rose e t a l , 1974; Watanabe and Isayama, 1973; W i l k i n s o n and F r e n c h , 1969) . The r e s u l t s , however, a re u n c l e a r and I n c o n c l u s i v e ( H i g g i n s , 1975) . The most c o n v i n c i n g e v i d e n c e of the e x i s t e n c e of IgG s u b p o p u l a t l o n s i n c h i c k e n s comes from the work of Loeken and Roth (1983) . I s o e l e c t r i c f o c u s s i n g of c h i c k e n IgG showed a heterogeneous r e p r e s e n t a t i o n w i th i s o e l e c t r i c p o i n t s between pH 6.5 and 9 . 0 . Upon i s o e l e c t r i c f o c u s s i n g of the Fc f ragments , four d i s t i n c t bands were o b t a i n e d . U n l e s s the Fc f ragments are a s y m m e t r i c a l l y g l y c o s y l a t e d , each band might r e p r e s e n t one s u b p o p u l a t i o n of Fc m o l e c u l e s . The presence of a t l e a s t one s u b p o p u l a t i o n of c h i c k e n IgG which was r e s i s t a n t to papa in a l s o was d i s c o v e r e d (Loeken and R o t h , 1983) . S u b c l a s s e s of human Immunoglobul ins g e n e r a l l y have been found to e x h i b i t d i f f e r e n c e s i n the h inge r e g i o n 16 s t r u c t u r e due t o v a r y i n g numbers of the i n t e r - d i s u l f i d e bonds, and d i f f e r e n c e s i n t h e i r s u s c e p t i b i l i t y t o p r o t e o l y t i c enzymes ( C a l v a n i c o , 1984). However, the f i n d i n g s of Loeken and Roth (1983) do not n e c e s s a r i l y i n d i c a t e the e x i s t e n c e of s u b c l a s s e s . The h e t e r o g e n e i t y of the c h i c k e n IgG m o l e c u l e however i s a p p a r e n t . F o p p o l i and coworkers (1979) examined a number of c h i c k e n s of Inbred l i n e s , and were a b l e t o d e t e c t t h i r t e e n d i f f e r e n t IgG a l l o t y p e s . The e x i s t e n c e of a t l e a s t two a l l o t y p e s had a l r e a d y been d e t e r m i n e d by Wakeland e t a l (1977), who commented t h a t , a l t h o u g h c h i c k e n IgG s u b c l a s s e s p o s s i b l y e x i s t , numerous a t t e m p t s i n t h e i r l a b o r a t o r y t o d e t e c t t h e s e based on s t r u c t u r a l c r i t e r i a (which were not d e f i n e d ) were u n s u c c e s s f u l . I t has been shown t h a t c h i c k e n IgG does not f i x mammalian complement, does not b i n d S t a p h y l o c o c c u s aureus p r o t e i n A, and does not b i n d t o mammalian Fc r e c e p t o r s ( J e n s e n i u s e t a l , 1981). I t i s apparent t h a t c h i c k e n IgG i s heterogeneous w i t h s u b c l a s s e s p o s s i b l y c o n t r i b u t i n g t o t h i s h e t e r o g e n e i t y . The v a r i e d v a l u e s r e p o r t e d f o r hexose c o n t e n t , p i , and even m o l e c u l a r weight may be due, i n p a r t , t o the s e l e c t i v e p r e p a r a t i o n of a s u b p o p u l a t i o n of m o l e c u l e s which i s i s o l a t e d by the method chosen. B.3 Advantanges of Y o l k as a Source of Immunoglobulins A c o n v e n i e n t s o u r c e of immunoglobulins f o r a d d i t i o n t o 17 i n f a n t f o r m u l a e , as w e l l as f o r the t r e a t m e n t of g a s t r o i n t e s t i n a l i n f e c t i o n s i n a d u l t s , i s c u r r e n t l y r e q u i r e d . The r a p i d development of immunoassays which has t a k e n p l a c e over the l a s t few y e a r s has n e c e s s i t a t e d the a v a i l a b i l i t y of a wide range of p u r i f i e d a n t l s e r a f o r d i a g n o s t i c p u rposes. C o m m e r c i a l l y a v a i l a b l e immunoreagents a r e g e n e r a l l y o b t a i n e d from the b l o o d of immunized h o s t a n i m a l s . The use of hen egg y o l k as an a n t i b o d y s o u r c e , which was f i r s t s u g g e s ted by P o i s o n and coworkers (1980) has many advantages over the use of b l o o d . The most o b v i o u s of t h e s e i s the ease w i t h which eggs can be c o l l e c t e d from l a y i n g hens. w h i l e the b l e e d i n g of a n i m a l s r e q u i r e s employment of s k i l l e d t e c h n i c i a n s , eggs can be c o l l e c t e d by u n t r a i n e d w o r k e r s . Hens can be more e c o n o m i c a l l y f e d and housed t h a n c o n v e n t i o n a l l a b o r a t o r y a n i m a l s , and show l e s s s u s c e p t i b i l i t y t o d i s e a s e s ( P o i s o n e t a l , 1980). A f u r t h e r advantage e x i s t s i n the f a c t t h a t the c h i c k e n n a t u r a l l y w i l l l a y an average of 240 eggs i n a year (Anonymous, 1986), w h i l e b l e e d i n g can o n l y be done p e r i o d i c a l l y w i t h a maximum 50mL b l o o d sample b e i n g t a k e n from a r a b b i t or c h i c k e n w i t h no i l l e f f e c t s (Garvey e t a l , 1977). The l e v e l s of IgG i n y o l k a r e h i g h , r a n g i n g from n i n e mg/raL (Wang e t a l , 1986) t o 25 mg/mL (Rose e t a l , 1974), a t l e a s t e q u a l l i n g ( C a r r o l l and S t o l l a r , 1983), i f not e x c e e d i n g ( P a t t e r s o n e t a l , 1962; Bar- J o s e p h and M a l k i n s o n , 1980; Rose e t a l , 1974), b l o o d l e v e l s . F u r t h e r m o r e , maintenance of h i g h l e v e l s of s p e c i f i c a n t i b o d i e s i n egg y o l k i s r e l a t i v e l y easy ( O r l a n s , 1967). 18 A compar ison of the amount of immunoglobul ins I s o l a t e d from the egg y o l k of a hen Immunized w i th E c h i n o c o c c u s  g r a n u l o s u s , and from the b l o o d of a r a b b i t immunized w i th the same o r g a n i s m , i l l u s t r a t e d the advantage of egg y o l k as an immunoglobul in s o u r c e ( G o t t s t e i n and Hemmeler, 1985) . Eggs c o l l e c t e d a f t e r the s i x t e e n t h day of Immunizat ion , up u n t i l the t h i r t y - n i n t h day were used i n the s t u d y . A s i n g l e b l e e d i n g of the r a b b i t a t day 35 y i e l d e d 40 mL of serum. The method of P o i s o n e t a l (1980) , i n which p o l y e t h y l e n e g l y c o l was used f o r egg y o l k immunoglobul in p r e p a r a t i o n was employed, f o l l o w e d by a f f i n i t y chromatography to p u r i f y those a n t i b o d i e s s p e c i f i c t o g r a n u l o s u s . The r e s u l t s i n d i c a t e d t h a t 298 mg of s p e c i f i c a n t i b o d i e s was o b t a i n e d from the e g g s , wh i le the amount from the r a b b i t ' s b l o o d was e i g h t e e n t imes lower , a t 16.6 mg. Due t o the e v o l u t i o n a r y d i s t a n c e which e x i s t s between b i r d s and mammals, c h i c k e n a n t i s e r a a g a i n s t mammalian a n t i g e n s may be more s p e c i f i c than a n t i s e r a to the same a n t i g e n r a i s e d i n r a b b i t s (Bauwens e t a l , 1987) . H a s s l e t a l (1987) compared i s o l a t e d a n t i b o d i e s o b t a i n e d from egg y o l k w i th those o b t a i n e d from r a b b i t s . Both groups had been immunized w i th a n t i g e n p repared from Toxoplasma g o n d i i . A compar ison of double immunod i f fus ion r e s u l t s i n d i c a t e d t h a t the c h i c k e n IgG p r e c i p i t a t e d d i f f e r e n t components i n the a n t i g e n mix ture when compared w i th the r a b b i t . T h i s i s not s u r p r i s i n g , s i n c e i t i s known t h a t d i f f e r e n c e s may a r i s e between two an ima ls of the same s p e c i e s (Crowle , 1973) . H a s s l e t a l (1987) suggested 19 t h a t the d i f f e r e n c e i n s p e c i f i c i t y e x h i b i t e d by the c h i c k e n IgG might a f f e c t i t s i n c o r p o r a t i o n i n t o an ELISA system. These f e a r s seem unfounded, however, and more p r o b a b l y , the d i f f e r e n c e e x h i b i t e d by c h i c k e n IgG might be advantageous, e l i m i n a t i n g n o n - s p e c i f i c c r o s s - r e a c t i o n s o c c u r r i n g i n c o m p l i c a t e d immunoassays (Anonymous, 1988). A f u r t h e r advantage based on the r e p o r t s t h a t c h i c k e n IgG has an I s o e l e c t r i c p o i n t which i s lower t h a n mammalian IgG has been s u g g e s t e d . In r o c k e t I m m u n o e l e c t r o p h o r e s i s , where p i d i f f e r e n c e s a re e s s e n t i a l f o r good r e s u l t s , t h i s c h a r a c t e r i s t i c ha3 o b v i o u s advantages (Anonymous, 1988). B.4 Methods of I s o l a t i n g C h i c k e n IgG The advantages of egg y o l k as a sour c e of immunoglobulins have r e s u l t e d i n the r e c e n t development of v a r i o u s methods of i s o l a t i o n . V i e i r a e t a l (1984) used egg y o l k a n t i b o d i e s i n a radioimmunoassay f o r d e t e c t i o n of human p a r a t h y r o i d hormone. P r e p a r a t i o n of IgG i n v o l v e d d i l u t i o n of y o l k w i t h t r l s -b u f f e r e d s a l i n e , c e n t r i f u g a t i o n , p r e c i p i t a t i o n w i t h p h o s p h o t u n g s t i c a c i d , c e n t r i f u g a t i o n , f o l l o w e d by p r e c i p i t a t i o n of IgG w i t h ammonium s u l p h a t e , and a f i n a l c e n t r i f u g a t i o n , f o l l o w e d by d i a l y s i s . A mean r e c o v e r y of 63% w i t h an u n d e f i n e d p u r i t y was o b t a i n e d . B a r - J o s e p h and M a l k i n s o n (1980) used c h i c k e n I g from y o l k i n an ELISA a s s a y f o r the d e t e c t i o n of p l a n t v i r u s e s . The p r e p a r a t i o n p r o c e d u r e i n v o l v e d the a d d i t i o n of 20 mL phosphate 20 b u f f e r e d s a l i n e (PBS) t o each y o l k ; s h a k i n g ; and c e n t r i f u g a t i o n a t 12,000 x g f o r 20 mi n u t e s . The r e s u l t i n g s u p e r n a t a n t was then mixed w i t h an e q u a l volume of g l y c e r o l and s t o r e d a t -20<>C. The p u r i t y of the i s o l a t e d f r a c t i o n was not d e s c r i b e d i n the paper, nor was the y i e l d . An I n d i c a t i o n , however, i s g i v e n i n t h a t 200mL of h i g h t i t r e d y o l k e x t r a c t c o u l d be c o l l e c t e d from a s i n g l e hen w i t h i n twenty days f o l l o w i n g i n j e c t i o n . The l a c k of f u r t h e r q u a n t i t a t i o n of t h i s method makes i t d i f f i c u l t t o compare w i t h o t h e r s . Bade and Stegemann (1984) d e s c r i b e d a method of i s o l a t i o n i n which p r o p a n e - 2 - o l , p r e c o o l e d t o -20<>C was used t o p r e c i p i t a t e the p r o t e i n s of the y o l k . L i p i d s were removed by re p e a t e d washings w i t h p r e c o o l e d p r o p a n o l and a c e t o n e . The r e s u l t i n g i s o l a t e d p r o t e i n f r a c t i o n had t e n p r o t e i n bands when examined by p o l y a c r y l a m l d e g e l e l e c t r o p h o r e s i s . F u r t h e r p u r i f i c a t i o n u s i n g DEAE c e l l u l o s e chromatography, or by p r e c i p i t a t i o n of the immunoglobulins w i t h p o l y e t h y l e n e g l y c o l was s u g g e s t e d . Comparison of the a n t i b o d y t i t r e of the e x t r a c t w i t h a n t i b o d i e s o b t a i n e d by t h e method of P o i s o n e t a l (1980) showed as good i f not h i g h e r t i t r e s . Lack of d e t a i l of d e t e c t i o n p r o c e d u r e s , however, makes the comparison of the r e c o v e r y l e v e l s f o r I g i m p o s s i b l e . The d i l u t i o n of y o l k w i t h water ( 1 : 1 0 ) , f o l l o w e d by a f r e e z e - t h a w c y c l e , and then c e n t r i f u g a t i o n , r e s u l t e d i n removal of some l i p i d s ( J e n s e n i u s e t a l , 1981). A d d i t i o n of sodium s u l p h a t e or ammonium s u l p h a t e , and c e n t r i f u g a t i o n , r e p e a t e d t w i c e , gave a r e c o v e r y of IgG of 50 - 70%, w i t h a 21 p u r i t y o£ 90% as determined by p h o t o m e t r i c s c a n n i n g of SDS p o l y a c r y l a m i d e e l e c t r o p h o r e s i s g e l s . A s i m i l a r p u r i t y of immunoglobul ins (90%), w i th a h i g h e r r e c o v e r y (70 - 80%) was a t t a i n e d by the same a u t h o r s ( J e n s e n i u s e t a l , 1981) by d i l u t i n g y o l k w i th t r i s - b u f f e r e d s a l i n e , and u s i n g d e x t r a n s u l p h a t e , f o l l o w e d by sodium s u l p h a t e . The procedure r e q u i r e d s i x c e n t r i f u g a t i o n s t e p s , f o l l o w e d by d i a l y s i s t o remove the s u l p h a t e , and so was t ime consuming . T h i s method was s u c c e s s f u l l y used by Bauwens et a l (1987) f o r p r e p a r a t i o n of c h i c k e n IgG f o r use i n a radio immunoassay f o r d e t e r m i n a t i o n of 1 , 2 5 - d i h y d r o x y v i t a m i n D i n serum. The most f r e q u e n t l y used method f o r Ig e x t r a c t i o n from egg y o l k i s t h a t of P o i s o n e t a l (1980) , which was f u r t h e r m o d i f i e d to g i v e h i g h e r y i e l d ( P o i s o n e t a l , 1985) . The method i n v o l v e s the use of p o l y e t h y l e n e g l y c o l a t a low c o n c e n t r a t i o n (3.5%) t o s e l e c t i v e l y p r e c i p i t a t e v i t e l l i n , and remove some f a t , f o l l o w e d by p r e c i p i t a t i o n of IgG wi th 12% p o l y e t h y l e n e g l y c o l . The p o l y e t h y l e n e g l y c o l then was removed by a d d i t i o n of ammonium s u l p h a t e or p r e c i p i t a t i o n of IgG wi th e t h a n o l a t -20<>c. R e s e a r c h e r s u s i n g the o r i g i n a l p o l y e t h y l e n e g l y c o l method have r e s o r t e d to D E A E - c e l l u l o s e chromatography f o r f u r t h e r Ig p u r i f i c a t i o n ( C a r r o l l and S t o l l a r , 1983) . H a s s l e t a l (1987) compared th ree methods of Ig e x t r a c t i o n from e g g s : p u r i f i c a t i o n by p r o p a n o l p r e c i p i t a t i o n (the method of Bade and Stegemann, 1984); the o r i g i n a l 22 p o l y e t h y l e n e g l y c o l p r e c i p i t a t i o n method ( P o is o n e t a l , 1980); and the improved PEG t e c h n i q u e ( P o is o n e t a l , 1985) . Y i e l d s of s p e c i f i c a n t i b o d i e s o b t a i n e d from a l l th ree methods were about the same. However, o n l y the improved PEG method gave a h i g h p u r i t y IgG sample (which was not q u a n t i f i e d , but was based on g e l f i l t r a t i o n r e s u l t s ) , and was deemed to be the most e f f e c t i v e method. The p u r i f i e d Ig o b t a i n e d from t h i s method was 40 mg per e g g . The e x p r e s s i o n of the p u r i f i e d Ig f r a c t i o n s i n terms of a n t i b o d y t i t r e s determined by d i f f e r e n t methods makes compar ison of the e f f i c i e n c y of the d e s c r i b e d methods d i f f i c u l t . The development of a method u s i n g fewer c e n t r i f u g a t i o n s t e p s would be advantageous f o r i n d u s t r i a l a p p l i c a t i o n s . C . GENERAL METHODS FOR ANTIBODY PURIFICATION S e v e r a l t e c h n i q u e s have been deve loped f o r the s e p a r a t i o n of a n t i b o d i e s from v a r i o u s s o u r c e s . One of the most common methods of p r e p a r a t i o n i n v o l v e s f r a c t i o n a l p r e c i p i t a t i o n of p r o t e i n s w i t h ammonium or sodium s u l p h a t e . O r g a n i c s o l v e n t s such as e t h a n o l have been u s e d , as w e l l as e l e c t r o p h o r e s i s and p r e p a r a t i v e u l t r a c e n t r i f u g a t i o n . Chromatographic methods i n c l u d i n g i o n exchange, g e l f i l t r a t i o n (Steward , 1984) or meta l c h e l a t e i n t e r a c t i o n chromatography (Lee e t a l , 1988) have been s u c c e s s f u l . A f f i n i t y chromatography i s r e q u i r e d f o r p u r i f i c a t i o n of a n t i b o d i e s e x h i b i t i n g s p e c i f i c a c t i v i t y toward a g i v e n a n t i g e n . 23 Egg y o l k immunoglobulin p u r i f i c a t i o n i s c o m p l i c a t e d by the h i g h l i p i d c o n t e n t of the y o l k . A method u s i n g a l g i n a t e , which was o p t i m i z e d f o r maximum e f f i c i e n c y of l i p o p r o t e i n p r e c i p i t a t i o n , r e s u l t e d i n a s u p e r n a t a n t c o n t a i n i n g y o l k l i v e t i n s , i n c l u d i n g immunoglobulins ( H a t t a e t a l , 1988). Use of the a l g i n a t e method as a p r e l i m i n a r y s e p a r a t i o n s t e p f o r egg y o l k immunoglobulin p u r i f i c a t i o n would a l l o w u t i l i z a t i o n of the egg l i p o p r o t e i n s as w e l l as a n t i b o d y r e c o v e r y . C l . Ion Exchange Chromatography A common method of s e p a r a t i n g Immunoglobulins from c o n t a m i n a t i n g p r o t e i n s i s i o n exchange chromatography a f t e r p r e l i m i n a r y p u r i f i c a t i o n by s a l t p r e c i p i t a t i o n (Garvey e t a l , 1977) . Ion exchange chromatography t a k e s advantage of net charge d i f f e r e n c e s e x i s t i n g among p r o t e i n s i n complex m i x t u r e s , and i n v o l v e s the e l e c t r o s t a t i c b i n d i n g of the p r o t e i n s t o a charged r e s i n . B a s i c a l l y , c o u n t e r i o n s p r e s e n t on the s u p p o r t m a t r i x a re a v a i l a b l e f o r exchange w i t h the charged p r o t e i n s . I f the c o u n t e r - i o n i s n e g a t i v e l y c h a r g e d , the s u p p o r t i s termed an a n i o n exchange; p o s i t i v e l y charged i o n s occur on a c a t i o n exchange (Anonymous, 1980). Both a n i o n exchange r e s i n s and c a t i o n exchange r e s i n s have been used s u c c e s s f u l l y f o r i g s e p a r a t i o n . However, the most common c h o i c e has un d o u b t e d l y been the a n i o n exchange r e s i n , d i e t h y l a m i n o e t h y l (DEAE) c e l l u l o s e (Fahey and T e r r y , 1978) . Low c a p a c i t y and poor f l o w p r o p e r t i e s of c e l l u l o s e 24 r e s i n s have been overcome t h r o u g h g e n e r a t i o n of a beaded form, known as S e p h a c e l ( P h a r m a c i a ) . F o l l o w i n g b i n d i n g of p r o t e i n s t o an a n i o n exchange column, e l u t i o n i s p o s s i b l e e i t h e r by d e c r e a s i n g the pH or i n c r e a s i n g the i o n i c s t r e n g t h of the e l u t i n g b u f f e r . A n i o n exchange chromatography has been used f o r the p u r i f i c a t i o n of IgG from s e v e r a l s o u r c e s , and has been a p p l i e d t o c h i c k e n IgG as a f i n a l s t e p i n p u r i f i c a t i o n a f t e r i n i t i a l p r e p a r a t i o n ( C a r r o l l and S t o l l a r , 1983; Loeken and R o t h , 1983). C.2 M e t a l C h e l a t e I n t e r a c t i o n Chromatography i n 1975, P o r a t h and coworkers p u b l i s h e d a s e l e c t i v e method of p r o t e i n s e p a r a t i o n based on v a r y i n g p r o t e i n a f f i n i t y f o r heavy m e t a l i o n s . T h i s t e c h n i q u e , named i m m o b i l i z e d a f f i n i t y chromatography by P o r a t h and O l i n (1983) was l a t e r renamed metal c h e l a t e I n t e r a c t i o n chromatography (MCIC) ( R a s s i and H o r v a t h , 1986). I t e s s e n t i a l l y i s a form of l i g a n d exchange chromatography (Davankov and semechkin, 1977). The system c o n s i s t s of a s u p p o r t m a t r i x , a c h e l a t e - f o r m i n g l i g a n d , and a m e t a l i o n which then donates or a c c e p t s e l e c t r o n s from l i g a n d atoms on the p r o t e i n s . The v a r y i n g b i n d i n g a f f i n i t i e s of p r o t e i n s have been a t t r i b u t e d p r i m a r i l y t o the amounts of exposed h i s t i d i n e , c y s t e i n e , and t r y p t o p h a n on the p r o t e i n s u r f a c e s ( L o n n e r d a l and Keen, 1982). While the s u p p o r t i n g m a t r i x o r i g i n a l l y used was of agarose ( P o r a t h e t a l , 1975), the r e c e n t use of a s i l i c a based m a t r i x has a l l o w e d use of metal c h e l a t e i n t e r a c t i o n 25 chromatography under h i g h performance l i q u i d chromatograph ic c o n d i t i o n s (Belew e t a l , 1987; F l g u e r o a e t a l , 1986) . Numerous p r o t e i n s have been i s o l a t e d from complex systems by MCIC. Among these are a l b u m i n , from a Cohn f r a c t i o n i v e x t r a c t (Hansson and K a g e d a l , 1981); t r a n s f e r r i n and immunoglobul ins from b l o o d serum (Lee e t a l , 1988); and monoc lona l a n t i b o d i e s from mouse a s c i t e s (Be lev e t a l , 1987) . The s u c c e s s f u l use of MCIC f o r s e p a r a t i o n of these complex m i x t u r e s suggested t h a t the t e c h n i q u e a l s o c o u l d be a p p l i e d to the water s o l u b l e egg y o l k p r o t e i n s f o r IgG p u r i f i c a t i o n . 26 MATERIALS AND METHODS A.IMMUNIZATION OF CHICKENS A . l G e n e r a l Immunization P r o c e d u r e s A group o£ l a y i n g w h i t e l e g h o r n hens were o b t a i n e d from the U n i v e r s i t y of B r i t i s h Columbia P o u l t r y U n i t . They were housed and f e d under normal c o n d i t i o n s . The age of most of the b i r d s a t the b e g i n n i n g of the experiment was a p p r o x i m a t e l y t h i r t y weeks. The s t u d y extended over a p e r i o d of two y e a r s , d u r i n g which the same c h i c k e n s were r e t a i n e d and used. Three groups of b i r d s r e c e i v e d i n j e c t i o n s of d i f f e r e n t lmmunogens, d e s c r i b e d below. I n i t i a l i n j e c t i o n s c o n t a i n e d Freund's complete a d j u v a n t ; subsequent i n j e c t i o n s c o n t a i n e d Freund's i n c o m p l e t e a d j u v a n t . A w a t e r - i n - o i l e m u l s i o n was c r e a t e d . The immunogen s u s p e n s i o n was added t o the a d j u v a n t s l o w l y , and e m u l s i f i e d t h o r o u g h l y by r e p e a t e d l y b e i n g drawn i n t o and e j e c t e d from a s t e r i l e 5 mL s y r i n g e ( w i t h a 26G3/8 nee d l e ) ( B e c t o n - D l c k i n s o n , R u t h e r f o r d , N J ) . One drop was a l l o w e d t o f a l l i n t o a beaker of i c e wa t e r ; e m u l s i f i c a t i o n was c o n s i d e r e d t o be complete when d i s p e r s i o n of the drop d i d not o c c u r . I n t r a m u s c u l a r i n j e c t i o n s of one m i l l i l i t r e were g i v e n u s i n g a needle of s i z e 26G3/8 ( B e c t o n - D l c k i n s o n , R u t h e r f o r d , NJ) t o f o u r s i t e s on the c h i c k e n : i n t o each b r e a s t and each t h i g h . I m mediately f o l l o w i n g i m m u n i z a t i o n , the muscle a t the i n j e c t i o n s i t e was massaged b r i e f l y . 27 A2. E. c o l i c e l l p r e p a r a t i o n E^ c o l i (0142:K86:H6) c e l l s (ATCC No. 23985) were i n c u b a t e d i n t r y p t i c a s e soy b r o t h a t 37°C f o r 48h. They were t r e a t e d w i t h 0.5% f o r m a l i n and f r e e z e - d r i e d ( S h i m i z u e t a l , 1988b). L y o p h i l i z e d c e l l s (2.2 mg) were suspended i n a phosphate b u f f e r e d s a l i n e s o l u t i o n (0.01 M sodium phosphate, 0.85% sodium c h l o r i d e , pH 7.2) which had been s t e r i l i z e d by f i l t r a t i o n ( M i l l e x - H A , 0.45 um, M i l l i p o r e Corp., B e d f o r d , MA). T h i s was mixed w i t h an e q u a l volume of a d j u v a n t t o g i v e 8 a p p r o x i m a t e l y 10 c o l o n y f o r m i n g u n i t s ( c f u ) per mL. A.3 E. c o l l and fl-Lactoglobulin p r e p a r a t i o n An E^ _ c o l i s u s p e n s i o n was mixed w i t h 6 - l a c t o g l o b u l l n (Sigma, S t . L o u i s , MO) i n PBS, and then added t o an e q u a l volume of a d j u v a n t t o g i v e a f i n a l c o n c e n t r a t i o n of 2.5 mg 13-l a c t o g l o b u l i n and 10 c f u per mL f o r i n j e c t i o n . A. 4 ( 3 - N - a c e t y l g l u c o s a m i n i d a s e p r e p a r a t i o n 1 3 - N - a c e t y l g l u c o s a m i n i d a s e (NAGase) p u r i f i e d from b o v i n e k i d n e y (Sigma, S t . L o u i s , MO) was o b t a i n e d i n a s o l u t i o n of 5 mg p r o t e i n i n a 3.2 M ammonium s u l p h a t e s o l u t i o n (pH 6 ) . T h i s was added t o an e q u a l amount of a d j u v a n t i n o r d e r t o g i v e a f i n a l p r o t e i n c o n c e n t r a t i o n of 2.5 mg/mL. B. EGG YOLK SUPERNATANT PREPARATION Eggs c o l l e c t e d d a i l y , i d e n t i f i e d by date and c h i c k e n , were s t o r e d a t 4°C u n t i l use. An egg y o l k s u p e r n a t a n t was p r e p a r e d u s i n g the method of H a t t a e t a l (1988). Egg y o l k s were s e p a r a t e d from the egg 28 w h i t e s , r i n s e d w i th d i s t i l l e d water , and r o l l e d on paper towel to remove a d h e r i n g egg w h i t e . P u n c t u r i n g the membrane then a l l o w e d the y o l k c o n t e n t s to be poured i n t o a graduated c y l i n d e r w i thout the v i t e l l i n e membrane. The y o l k was mixed w i th an equa l volume of d e i o n i z e d d i s t i l l e d wa te r , and g e n t l y s t i r r e d . A l g i n ( K e l g i n X L , K e l c o C o . , San D i e g o , CA) (0.15% w/v) i n d e i o n i z e d d i s t i l l e d water was s t i r r e d a t room temperature f o r one h o u r . A volume of four t imes t h a t of the o r i g i n a l egg yo lk was added to the d i l u t e d y o l k , b r i e f l y s t i r r e d , and kept a t room temperature f o r 30 min . C e n t r i f u g a t i o n a t 10,000xg a t 18<>C f o r 15 min r e s u l t e d i n a s u p e r n a t a n t which c o u l d be decanted from the p r e c i p i t a t e . The s u p e r n a t a n t was f i l t e r e d through Whatman No. 1 f i l t e r paper (Whatman L t d . , England) c o n t a i n i n g a s m a l l amount of g l a s s w o o l . Sodium a z i d e ( F i s h e r s c i e n t i f i c C o . , F a i r Lawn, NJ) was added t o g i v e a 0.02% c o n c e n t r a t i o n , and the sample s t o r e d a t 4 ° C . C . CHROMATOGRAPHY CONDITIONS In a l l e x p e r i m e n t s , chromatography was conducted a t room t e m p e r a t u r e . Ten m i l l i l i t r e p l a s t i c s y r i n g e s ( B e c t o n - D l c k s o n , R u t h e r f o r d , NJ) were used as co lumns . Ge l volumes of a p p r o x i m a t e l y 10 mL were u s e d . C l . DEAE-Sephace l Ion Exchange Chromatography DEAE-Sephace l (S igma, S t . L o u i s , MO) was f i r s t e q u i l i b r a t e d w i th a degassed b u f f e r s o l u t i o n of 0.60 M po tass ium phosphate a t pH 8 . 0 . A f t e r s e v e r a l h o u r s , w i th 29 p e r i o d i c d e c a n t i n g and replacement of b u f f e r , the e q u i l i b r a t i n g b u f f e r t o be used i n the experiment r e p l a c e d t h e 0.60 M b u f f e r , and d e c a n t i n g was r e p e a t e d w i t h the new b u f f e r . The column was packed w i t h the g e l , and f u r t h e r e q u i l i b r a t e d w i t h a t l e a s t f i v e bed volumes of e q u i l i b r a t i n g b u f f e r . B u f f e r s were made from d i p o t a s s i u m phosphate t r i h y d r a t e (Sigma, S t . L o u i s , MO). L i n e a r e l u t i o n g r a d i e n t s were c r e a t e d t h r o u g h t h e use of two chambers of e q u a l s i z e c onnected w i t h a s h o r t t u b i n g . E q u a l volumes of s t a r t i n g b u f f e r ( i n f i r s t chamber) and t h e b u f f e r of c a l c u l a t e d r e q u i r e d m o l a r i t y (second chamber) were s l o w l y mixed i n the f i r s t chamber as the experiment p r o g r e s s e d and g r a v i t y f o r c e d the second b u f f e r t o move i n t o the f i r s t . C2. M e t a l C h e l a t e i n t e r a c t i o n chromatography A c t i v a t i o n of Sepharose - 6 B (Pharmacia) was a c c o m p l i s h e d by the method of Sundberg and P o r a t h (1974) . S u c t i o n d r i e d agarose (lOOg), washed w i t h d i s t i l l e d w a t e r , was added t o 100 mL b u t a n e d l o l d i g l y c i d y l e t h e r (Sigma, S t . L o u i s , MO) and 100 mL of 0.6 M sodium h y d r o x i d e c o n t a i n i n g 200 mg sodium b o r o h y d r i d e (Sigma, S t . L o u i s , MO). The m i x t u r e was r o t a t e d g e n t l y i n a r o t a e v a p o r a t o r w i t h o u t vacuum a t room temperature f o r 8 h, f o l l o w e d by washing i n a 500 mL P y r e x s i n t e r e d g l a s s f i l t e r - f u n n e l w i t h l a r g e volumes of water. Attachment of i m i n o d i a c e t i c a c i d was by the method of P o r a t h and O l i n (1983) . To the d r i e d g e l , 125 mL of 2 M sodium c a r b o n a t e , 12 .5 g d i s o d l u m i m i n o d i a c e t a t e (Sigma, S t . 30 L o u i s , MO) and 150 mg sodium b o r o h y d r i d e (Sigma, S t . L o u i s , MO) were added and the mix ture kept a t 60<>C f o r 18 h w i th s low s t i r r i n g . Washing w i th a p p r o x i m a t e l y 2 L of water , and then 2 L of 5% a c e t i c a c i d i n water , was f o l l o w e d by e x t e n s i v e washing w i th d e i o n i z e d d i s t i l l e d water u n t i l the washings were n e u t r a l . The g e l was then s t o r e d i n 0.02% sodium a z i d e a t 4 0 c . G e l s were poured i n t o co lumns , and washed w i th f i v e bed volumes of d e i o n i z e d d i s t i l l e d water . Copper was loaded by a d d i n g 3 mL of 0.05 M c u p r i c c h l o r i d e s o l u t i o n i n water , to g i v e a p p r o x i m a t e l y t w o - t h i r d s s a t u r a t i o n of the co lumn. F u r t h e r washing w i th water ( a p p r o x i m a t e l y two bed volumes) was f o l l o w e d by e q u i l i b r a t i o n w i th a t l e a s t f i v e bed volumes of e q u i l i b r a t i n g b u f f e r b e f o r e sample a p p l i c a t i o n . D. PROTEIN DETERMINATIONS The absorbance a t 280nm of e l u t e d samples was moni tored wi th a Cary 210 Spect rophotometer ( V a r i a n Instrument D i v i s i o n , C A ) . P r o t e i n of p o o l e d f r a c t i o n s was determined w i th the B i o -Rad P r o t e i n Assay ( B l o - R a d , Richmond, C A ) . Samples were d i a l y z e d a g a i n s t 0.02 M sodium phosphate pH 7.2 p r i o r to u s e . A s t a n d a r d curve was prepared u s i n g c h i c k e n IgG (Sigma, S t . L o u i s , MO) i n 0.02 M sodium phosphate b u f f e r , pH 7 . 2 . B lanks of the same b u f f e r were u s e d . One hundred m i c r o l l t r e s of the t e s t samples were p l a c e d i n t e s t - t u b e s . F i v e m i l l l l i t r e s of the B i o - R a d dye reagent d i l u t e d f i v e t imes were added and v o r t e x e d , and the mix ture a l l o w e d to s tand f o r a p p r o x i m a t e l y 31 15 min. Sample c o n c e n t r a t i o n s were c a l c u l a t e d from the l i n e a r s t a n d a r d c u r v e o b t a i n e d w i t h each a s s a y . E. ENZYME-LINKED IMMUNOSORBENT ASSAYS Enz y m e - l i n k e d immunosorbent a s s a y s (ELISA's) were performed on e l u t e d f r a c t i o n s i n or d e r t o o b t a i n r e l a t i v e l e v e l s of s p e c i f i c a n t i b o d y a c t i v i t y . Depending on the a n t i g e n used, the ELISA pro c e d u r e d i f f e r e d s l i g h t l y . E l . ELISA f o r D e t e c t i o n of A n t i b o d y A c t i v i t y Toward E. c o l i  l i p o p o l y s a c c h a r l d e The ELISA method of Stephens (1984), o p t i m i z e d by S h i m i z u e t a l (1988a), was used f o r d e t e c t i o n of a n t i b o d i e s e x h i b i t i n g s p e c i f i c i t y toward the l i p o p o l y s a c c h a r i d e (LPS) p o r t i o n of E.  c o l l . Volumes of samples and s o l u t i o n s added were 100 UL per w e l l u n l e s s o t h e r w i s e s t a t e d . An Immulon I I m i c r o t i t r e p l a t e (Dynatech Lab. I n c . , C h a n t i l l y , VA) was c o a t e d w i t h 0.01% (w/v) LPS (a g i f t from Dr. M. S h i m i z u , e x t r a c t e d by the hot phenol e x t r a c t i o n method of Westphal e t a l (1952)) i n 0.05 M sodium c a r b o n a t e b u f f e r a t pH 9.6. P l a t e s were i n c u b a t e d o v e r n i g h t a t 4°C, then washed t h r e e t i m e s w i t h phosphate b u f f e r e d s a l i n e c o n t a i n i n g 0.05% Tween 20 (PBS-Tween). E l u t e d f r a c t i o n s d i l u t e d w i t h PBS-Tween were added t o the p l a t e s , and i n c u b a t e d f o r t h r e e hours a t room t e m p e r a t u r e . To some of the LPS-coated w e l l s , PBS-Tween w i t h o u t sample was added f o r b l a n k i n g . F o l l o w i n g t h r e e washes w i t h PBS-Tween, a l k a l i n e phosphatase c o n j u g a t e d r a b b i t a n t i - c h i c k e n IgG (Sigma, S t . L o u i s , MO) d i l u t e d 1/2000 i n PBS-Tween was added, and i n c u b a t e d f o r 2 h a t room t e m p e r a t u r e . A f t e r a f i n a l t h r e e 32 washes, 0.1% p - n i t r o p h e n y l phosphate (Sigma, S t . L o u i s , MO) i n 10% d i e t h a n o l a m i n e (Sigma, S t . L o u i s , MO) a t pH 9.8 c o n t a i n i n g 0.02% sodium a z i d e and 0.01% magnesium c h l o r i d e was added, and Incu b a t e d a t room tem p e r a t u r e f o r 30 min. The r e a c t i o n was stopped by a d d i t i o n of 50 U L of 2.5 N sodium h y d r o x i d e . The absorbance a t 405 nm was read u s i n g an SLT L a b i n s t r u m e n t ( A u s t r i a ) ELISA r e a d e r . E2. ELISA f o r t h e D e t e c t i o n of A n t i b o d y A c t i v i t y Toward  1 3 - L a c t o g l o b u l i n For the d e t e c t i o n of 6 - l a c t o g l o b u l i n s p e c i f i c a n t i b o d i e s , the method d e v e l o p e d f o r the LPS s p e c i f i c a n t i b o d i e s was used as d e s c r i b e d above, e x c e p t t h a t p l a t e s were i n i t i a l l y c o a t e d w i t h a s o l u t i o n of 0.01% (w/v) 6 - l a c t o g l o b u l i n i n c a r b o n a t e b u f f e r r a t h e r t h a n LPS. E3. ELISA f o r the D e t e c t i o n of A n t i b o d y A c t i v i t y Toward  6 - N - A c e t y l q l u c o s a m l n i d a s e S l i g h t m o d i f i c a t i o n s were made t o o p t i m i z e the ELISA f o r d e t e c t i o n of a n t i b o d i e s h a v i n g s p e c i f i c i t y f o r I3-N-a c e t y l g l u c o s a m i n i d a s e (NAGase) (Sigma, S t . L o u i s , MO). P l a t e s were I n i t i a l l y c o a t e d w i t h an NAGase s o l u t i o n of 20 ug/mL ca r b o n a t e b u f f e r , and l e t s t a n d f o r 2 h a t 37<>C. P l a t e s were washed t h r e e t i m e s w i t h t a p water, and a 2% B l o t t o s o l u t i o n ( B l o t t o : 10% C a r n a t i o n s k i m m i l k powder i n d e i o n i z e d d i s t i l l e d w a t e r , w i t h 0.02% sodium a z i d e ) added f o r 30 min a t 37<>C. F o l l o w i n g washing w i t h t a p water, d i l u t e d samples were added and Incubated f o r 2 h a t 37<>C. Washing was f o l l o w e d by a d d i t i o n of a l k a l i n e phosphatase c o n j u g a t e d i l u t e d 1/2000 i n 33 PBS-Tween and I n c u b a t i o n f o r 90 min a t 37 ° c . A f i n a l wash was f o l l o w e d by the a d d i t i o n of s u b s t r a t e as d e s c r i b e d above. The absorbance a t 405 nm was read a f t e r a r e a c t i o n time of a p p r o x i m a t e l y 45 m i n u t e s . E4. ELISA f o r t h e D e t e c t i o n of fl-N-Acetylglucosamlnldase i n  F i s h or Beef Samples When the l e v e l of NAGase i n f i s h or bovine samples was examined, the ELISA procedure was as d e s c r i b e d above, except t h a t p l a t e s were c o a t e d w i t h the t e s t sample r a t h e r t h a n t h e pure NAGase s o l u t i o n . As w e l l , 1/350 d i l u t e d egg y o l k IgG o b t a i n e d by MCIC was used r a t h e r t h a n the e l u t e d samples. Chinook salmon muscle samples were a n a l y z e d on the day of c o l l e c t i o n and a f t e r one week of f r o z e n s t o r a g e a t -20<>c. Beef samples were examined on the day o b t a i n e d , and a f t e r 24 h s t o r a g e a t -20°C. F r o z e n samples were a l l o w e d t o thaw a t room temp e r a t u r e b e f o r e p r e p a r a t i o n . Meat was ground u s i n g a K i t c h e n A i d meat g r i n d e r (Hobart M a n u f a c t u r i n g Co., Troy, OH), f o r c i n g meat t h r o u g h a h o l e of 5.0 mm d i a m e t e r . Ground sample (5 g) was p l a c e d i n the upper p a r t of a f i l t e r - f u g e (model 2427, i n t e r n a t i o n a l Equipment Co., Needham H e i g h t s , MA). C e n t r i f u g a t i o n a t 9,600 x g f o r 30 min a t 5<>c a l l o w e d j u i c e t o be f i l t e r e d and c o l l e c t e d i n the bottom of the f i l t e r - f u g e . T h i s l i q u i d was d i l u t e d t o 5 mL w i t h 1% sodium c h l o r i d e (w/v) and termed the p r e s s e d j u i c e ( P J ) . The r e s u l t i n g r e s i d u e was weighed i n grams, and a volume of 1% sodium c h l o r i d e of t e n times the r e s i d u e weight was added. The m i x t u r e was homogenized f o r 30 s e c . w i t h the U l t r a - T u r r a x I k a - T r o n (Janke 34 and K u n k e l , S t a u f e n , W. Germany) u s i n g an 18N probe and g e n e r a t o r model 30/2G a t 12,000 rpm. F o l l o w i n g s t a n d i n g f o r 30 min a t 4°C, c e n t r i f u g a t i o n a t 22,500 x g f o r 10 min a t 5<>C r e s u l t e d i n a s u p e r n a t a n t which was d i l u t e d t o 40 mL w i t h 1% sodium c h l o r i d e . T h i s was termed the f i s h e x t r a c t ( Y o s h i o k a , 1988). A s p e c t r o p h o t o m e t r l c method f o r d e t e r m i n i n g NAGase i n the meat samples was performed by Ms K. Y o s h i o k a . P r e p a r e d meat samples ( p r e s s j u i c e or e x t r a c t ) (1 mL) were added t o 2 mL 0.2 M c i t r a t e b u f f e r (pH 4 .5) and 1 mL 6 mM p - n i t r o p h e n y l - 2 -a c e t o a m i d o - 2 - d e o x y - 6 - D - g l u c o p y r o n o s i d e . F o l l o w i n g i n c u b a t i o n f o r 60 min a t 37<>c, 4 mL 0.4 M b o r i c a c i d (pH 10.0) was added. The s o l u t i o n was f i l t e r e d t h r o u g h Whatman No. 1 f i l t e r paper and the absorbance a t 405 nm r e a d . The method was termed the s p e c t r o p h o t o m e t r l c method. U n i t s of enzyme a c t i v i t y were c a l c u l a t e d based on a s t a n d a r d c u r v e . F. IMMUNODIFFUSION TECHNIQUES F l . R a d i a l I m m u n o d i f f u s i o n I m m u n o d i f f u s i o n p l a t e s were p r e p a r e d f o r IgG q u a n t i t a t i o n as d e s c r i b e d by M i l f o r d - W a r d (1981) w i t h m o d i f i c a t i o n s . B r i e f l y , 0 .35 mL of a n t i - c h i c k e n IgG (Sigma, S t . L o u i s , MO, or ICN, C l e v e l a n d , OH) was mixed w i t h 1.65 mL b a r b i t a l b u f f e r (50 mM sodium b a r b i t a l , 10 mM b a r b i t a l , pH 8 .6 ) (Sigma, S t . L o u i s , MO) i n a t e s t - t u b e and p l a c e d i n a 56°C water b a t h . Agarose (0.07 g) (Sigma, S t . L o u i s , MO) mixed w i t h 4 .6 mL of the b a r b i t a l b u f f e r and 0.4 mL of 0.35% sodium a z i d e ( F i s h e r S c i e n t i f i c Co., F a i r Lawn, NJ) was p l a c e d i n a b o i l i n g water 35 b a t h , heated u n t i l the agarose d i s s o l v e d , and the n was t r a n s f e r r e d t o the 56<>c water b a t h . The two s o l u t i o n s were mixed w e l l , and poured i n t o r a d i a l i m m u n o d i f f u s i o n p l a t e s (ICN, C l e v e l a n d , OH). The p l a t e s were a l l o w e d t o s o l i d i f y 15 min a t room t e m p e r a t u r e , and the n t r a n s f e r r e d t o a moi s t chamber a t 4°C f o r s t o r a g e p r i o r t o use. Three mm di a m e t e r h o l e s were c u t i n the agarose u s i n g a t e m p l a t e (ICN, C l e v e l a n d , OH). S i x m i c r o l i t r e samples, p r e v i o u s l y d i a l y z e d a g a i n s t 20 mM sodium phosphate b u f f e r pH 7.0, l y o p h i l i z e d , and r e c o n s t i t u t e d i n b a r b i t a l b u f f e r , were a p p l i e d t o the w e l l s . A s t a n d a r d c u r v e u s i n g c h i c k e n IgG (Sigma, S t . L o u i s , MO) of f i v e d i f f e r e n t c o n c e n t r a t i o n s r a n g i n g from 0.12 mg/mL t o 1.20 mg/mL were used i n each p l a t e . D i f f u s i o n i n a m o i s t chamber a t room temperature was deemed complete when the l a r g e s t r i n g f a i l e d t o i n c r e a s e based on d a i l y measurements ( a p p r o x i m a t e l y t h r e e d a y s ) . C a l c u l a t i o n of the IgG s t a n d a r d c u r v e was made by t a k i n g the square of the d i a m e t e r of the p r e c i p i t i n r i n g v e r s u s the IgG c o n c e n t r a t i o n . F2. I m m u n o e l e c t r o p h o r e s i s I m m u n o e l e c t r o p h o r e s i s was c a r r i e d out a c c o r d i n g t o Garvey et a l (1977) w i t h m o d i f i c a t i o n s . Nine m i l l i l i t r e s of one p e r c e n t agarose (Sigma, S t . L o u i s , MO) i n a 50 mM sodium b a r b i t a l and 10 mM b a r b i t a l b u f f e r a t pH 8.6 (Sigma, S t . L o u i s , MO) was mixed and p l a c e d In a b o i l i n g water b a t h , when the agarose had d i s s o l v e d , t h e s o l u t i o n was poured onto the h y d r o p h i l i c s i d e of Gelbond f i l m (FMC C o r p o r a t i o n , R o c k l a n d , MA) of dim e n s i o n s 3.9 cm by 3.0 cm. A f t e r s o l i d i f i c a t i o n a t 36 room temperature f o r 15 min, 6 U.L of sample was added t o a punched w e l l of 3 mm d i a m e t e r and I m m u n o e l e c t r o p h o r e s i s c a r r i e d out f o r 45 min a t 100V a t room t e m p e r a t u r e . The b a r b i t a l e l e c t r o d e b u f f e r used was as d e s c r i b e d f o r g e l p r e p a r a t i o n . S i x t y m i c r o l i t r e s of a n t i s e r u m ( e i t h e r r a b b i t a n t i - c h i c k e n IgG (Sigma, S t . L o u i s , MO), or r a b b i t a n t i - e g g y o l k ( g i f t from Dr. S. A l - M a s h i k h i ) ) was added t o a t r o u g h of 1.0 mm w i d t h and 2.6 mm l e n g t h , and d i f f u s i o n s a l l o w e d t o t a k e p l a c e a t room temperature f o r 18 h i n a m o i s t chamber. D e p r o t e i n i z a t i o n was t h e n a c c o m p l i s h e d by two changes of 0.3 M sodium c h l o r i d e and two changes of 0.15 M sodium c h l o r i d e over a p e r i o d of 24 h. A p i e c e of m o i s t f i l t e r paper the s i z e of the s l i d e was p l a c e d onto i t , and a l l o w e d t o d r y a t room t e m p e r a t u r e . S t a i n i n g w i t h 0.2% bromophenol b l u e (BDH, P o o l e , England) i n 10% a c e t i c a c i d , 50% methanol, f o l l o w e d by d e s t a i n i n g i n the same s o l u t i o n w i t h o u t the dye a l l o w e d v i s u a l i z a t i o n of p r e c i p i t i n a r c s . G. ELECTROPHORESIS G . l N o n - d e n a t u r i n g P o l y a c r y l a m l d e G e l E l e c t r o p h o r e s i s P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) conducted under n o n - d e n a t u r i n g sample c o n d i t i o n s was termed n a t i v e PAGE. Samples were d i a l y z e d , l y o p h i l i z e d , and r e c o n s t i t u t e d w i t h 0.02 M sodium phosphate b u f f e r . Phast g e l s , b u f f e r s t r i p s , and the e l e c t r o p h o r e s i s system used were o b t a i n e d from Pharmacia ( U p p s a l a , Sweden). G e l s of 8 - 25% a c r y l a m i d e g r a d i e n t w i t h 2% c r o s s - l i n k i n g were r u n a t 400V, 10mA, 15<>c f o r 280 v o l t h o u r s . These g e l s were then f i x e d w i t h a 20% t r i c h l o r o a c e t i c 37 a c i d s o l u t i o n , washed w i t h a s o l u t i o n of 30% methanol and 10% a c e t i c a c i d , s t a i n e d w i t h 0.02% Phast G e l Blue R, and d e s t a l n e d w i t h 30% methanol, 10% a c e t i c a c i d . G2. S D S - P o l y a c r y l a m l d e G e l E l e c t r o p h o r e s i s i n Homogeneous  A c r y l a m i d e C o n c e n t r a t i o n s Sodium d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS-PAGE) was performed u s i n g the method of Weber and Osborn ( 1 9 6 9 ) . E i g h t p e r c e n t a c r y l a m i d e g e l s were p r e p a r e d by m i x i n g 28 mL of a 0.2 M sodium phosphate b u f f e r a t pH 7.0 c o n t a i n i n g 2% SDS, 17 mL of a 22.2% a c r y l a m i d e (Sigma, S t . L o u i s , MO) s o l u t i o n c o n t a i n i n g 0.6% b i s a c r y l a m i d e (Sigma, S t . L o u i s , MO), and t h r e e mL of 1.2% ammonium p e r s u l f a t e (Sigma). The a d d i t i o n of 60UL of N , N , N • , N ' - t e t r a m e t h y l e t h y l e n e d iamine (TEMED) (B l o - R a d , Richmond, CA) c a t a l y z e d p o l y m e r i z a t i o n . The m i x t u r e was poured i n t o a s l a b type v e r t i c a l g e l of d i m e n s i o n s 0.2cm x 11cm x 13.5 cm. L y o p h i l i z e d samples ( 2 - 4 mg) were added t o one m i l l i l i t r e of 4% SDS i n 0.01 M sodium phosphate, pH 7.0, c o n t a i n i n g 10% g l y c e r o l and 1% m e r c a p t o e t h a n o l ( f o r n o n - r e d u c i n g SDS-PAGE, m e r c a p t o e t h a n o l was o m i t t e d ) , and p l a c e d i n a b o i l i n g water b a t h f o r 5 min. An e l e c t r o d e b u f f e r of 0.1 M sodium phosphate a t pH 7.0 was used. G e l s were ru n a t room temperature a t 90 mA f o r 8 h ( A t t o Co., Tokyo, J a p a n ) . S t a i n i n g was w i t h 0.05% Coomassie B l u e R250 (ICN, C l e v e l a n d , OH) and 0.05% Coomassie B l u e G250 ( B i o - R a d , Richmond, CA) i n 30% methanol, 10% a c e t i c a c i d o v e r n i g h t a t room t e m p e r a t u r e . D e s t a i n i n g was a c c o m p l i s h e d w i t h 30% methanol, 10% a c e t i c a c i d . 38 G3. G r a d i e n t S D S - P o l y a c r y l a m l d e G e l E l e c t r o p h o r e s i s Sample p r e p a r a t i o n f o r g r a d i e n t SDS-PAGE was the same as d e s c r i b e d f o r the homogeneous method. Pha s t e l e c t r o p h o r e s i s g e l s of 10 t o 15% a c r y l a m l d e g r a d i e n t ( w i t h 2% c r o s s - l i n k i n g ) were ru n on the P h a s t e l e c t r o p h o r e s i s u n i t u s i n g b u f f e r s t r i p s c o n t a i n i n g 0.55% SDS, pH 7.5 (Pharmacia, U p p s a l a , Sweden). G e l s were ru n a t 250 V, 10 mA, 150C, f o r 60 v o l t h o u r s . Coomassie s t a i n i n g or s i l v e r s t a i n i n g a c c o r d i n g t o the P h a s t p r o t o c o l was then performed. Coomassie s t a i n i n g i n v o l v e d the use of a 0.1% P h a s t G e l B l u e R s o l u t i o n i n 30% methanol, 10% a c e t i c a c i d . D e s t a i n i n g was a c c o m p l i s h e d w i t h 30% methanol, 10% a c e t i c a c i d . A p r e s e r v i n g s o l u t i o n of 10% a c e t i c a c i d and 5% g l y c e r o l completed the p r o c e s s . S i l v e r s t a i n i n g i n v o l v e d a wash of the g e l w i t h 50% e t h a n o l , 10% a c e t i c a c i d , f o l l o w e d by f u r t h e r washings of 10% e t h a n o l and 5% a c e t i c a c i d . For s e n s i t i z a t i o n , 8.3% g l u t a r a l d e h y d e (BDH, T o r o n t o , Ont.) i n water was used. A wash w i t h d e i o n i z e d d i s t i l l e d water was f o l l o w e d by s t a i n i n g w i t h 0.25% s i l v e r n i t r a t e ( F i s h e r S c i e n t i f i c , Canada) i n water. Development w i t h 0.04% formaldehyde ( F i s h e r S c i e n t i f i c , Canada) i n 2.5% sodium c a r b o n a t e , f o l l o w e d by a s t o p bath of 5% a c e t i c a c i d , and a p r e s e r v i n g s o l u t i o n of 10% a c e t i c a c i d and 5% g l y c e r o l completed the s i l v e r s t a i n i n g p r o c e d u r e . 39 H . S T A T I S T I C A L M E T H O D S O n e w a y a n a l y s i s o f v a r i a n c e w a s p e r f o r m e d t o d e t e r m i n e i f s i g n i f i c a n t d i f f e r e n c e s i n s p e c i f i c a n t i b o d y l e v e l s w e r e d e t e c t a b l e i n t h e y o l k o f I m m u n i z e d b i r d s b y t h e t w e l f t h d a y f o l l o w i n g i n j e c t i o n o f N A G a s e . T h e l e a s t s i g n i f i c a n t d i f f e r e n c e t e s t w a s u s e d t o d e t e r m i n e w h i c h b i r d s d i f f e r e d s i g n i f i c a n t l y . o n e w a y a n a l y s i s o f v a r i a n c e a l s o w a s u s e d t o e v a l u a t e d i f f e r e n c e s i n t o t a l I g G d u r i n g t h e p e r i o d o f t h e s t u d y i n t h e o n e c h i c k e n e x a m i n e d . I n t h e d e v e l o p m e n t o f a n E L I S A m e t h o d f o r t h e d e t e c t i o n o f f 3 - N - a c e t y l g l u c o s a m i n l d a s e , l i n e a r r e g r e s s i o n w a s p e r f o r m e d o n t h e s t a n d a r d c u r v e . T h e P e a r s o n ' s p r o d u c t m o m e n t c o r r e l a t i o n c o e f f i c i e n t ( r ) w a s c a l c u l a t e d a n d t h e s t a t i s t i c a l s i g n i f i c a n c e o f t h e r e l a t i o n s h i p d e t e r m i n e d b y c o m p a r i n g t h e c a l c u l a t e d r v a l u e w i t h t h e c r i t i c a l r v a l u e ( K v l z a n d K n a f l , 1 9 8 0 ) . RESULTS AND DISCUSSION 41 PART I . RESPONSE OF CHICKENS TO 6-N-ACETYLGLUCOSAMINIDASE INJECTIONS Eggs were sampled over a f i f t y day p e r i o d from the t h r e e c h i c k e n s immunized w i t h 6 - N - a c e t y l g l u c o s a m i n i d a s e (NAGase). The eggs were s t o r e d a t 4<>C u n t i l b e i n g c o l l e c t i v e l y examined f o r a n t i b o d y a c t i v i t y toward NAGase. The eggs of a f o u r t h c h i c k e n , which was of the same age and housed under the same c o n d i t i o n s as the t e s t b i r d s , were examined as a c o n t r o l . The ELISA method was used t o d e t e c t the presence of a n t i b o d i e s h a v i n g s p e c i f i c a c t i v i t y toward NAGase. The response of the c h i c k e n s t o the i n j e c t i o n s i s shown ( F i g u r e 1 ) . The l e v e l of s p e c i f i c a n t i b o d i e s d e t e c t e d i n the y o l k s of the c o n t r o l c h i c k e n remained q u i t e low d u r i n g the p e r i o d of s t u d y . The t h r e e immunized b i r d s showed s i g n i f i c a n t l y h i g h e r l e v e l s of s p e c i f i c a n t i b o d i e s than the c o n t r o l (a=0.01) by the t w e l f t h day of the s t u d y . The l e v e l s c o n t i n u e d t o c l i m b a f t e r the second i n j e c t i o n which was a d m i n i s t e r e d on the f o u r t e e n t h day. A p p r o x i m a t e l y two weeks a f t e r the second i n j e c t i o n (days 35 t o 4 0 ) , a s l i g h t d e c r e a s e i n a c t i v i t y was d e t e c t e d i n the t h r e e b i r d s . However, by the f o r t i e t h day, the a n t i b o d y l e v e l s had i n c r e a s e d a g a i n . The apparent d e c l i n e i n l e v e l of s p e c i f i c a n t i b o d i e s which o c c u r r e d a t a p p r o x i m a t e l y day 35 i s i n t e r e s t i n g . S i n c e the second i n j e c t i o n was a d m i n i s t e r e d o n l y two weeks a f t e r the f i r s t , the i n t e r p r e t a t i o n of the r e s u l t s i s d i f f i c u l t . I n humans, an i n j e c t i o n of an immunogen i n i t i a l l y e l i c i t s a " p r i m a r y " a n t i b o d y r e s p o n s e , which i s g e n e r a l l y of the igM c l a s s (Steward, 1984). A peak may be reached w i t h i n two weeks 42 F i g u r e 1. The change i n s p e c i f i c a n t i b o d y a c t i v i t y toward fl-N - a c e t y l g l u c o s a m i n i d a s e over time i n egg y o l k s c o l l e c t e d from t h r e e c h i c k e n s immunized w i t h NAGase and from a c o n t r o l c h i c k e n r e c e i v i n g no i n j e c t i o n s . Arrows i n d i c a t e time of I n j e c t i o n s . Y o l k s were d i l u t e d 1/500 i n PBS-Tween f o r the a s s a y . 43 f o l l o w e d by a d e c l i n e i n l e v e l , s i m i l a r l y , i n c h i c k e n s , a s i n g l e i n j e c t i o n of E. c o l l has been shown t o r e s u l t i n e l e v a t e d serum IgM a n t i b o d i e s ( S h i m i z u e t a l , 1988b). The t r a n s m i s s i o n of IgM a n t i b o d i e s t o the y o l k does not occur (Rose e t a l , 1974). S i n c e the immunoglobulins a r e t r a n s f e r r e d t o the y o l k t o p r o t e c t the unborn c h i c k a g a i n s t i n f e c t i o n s , i t seems c o n c e i v a b l e t h a t some mechanism may a l l o w the t r a n s f e r of IgG t o the y o l k d u r i n g the p r i m a r y r e s p o n s e . While an o v e r l a p of the p r i m a r y and secondary r e s p o n s e s i s e x p e c t e d due t o the i m m u n i z a t i o n p r o c e d u r e s chosen, the d e c r e a s e d e t e c t e d a t day 35 i n the c h i c k e n s may i n d i c a t e the end of the p r i m a r y r e s p o n s e , w i t h the s e c o n d a r y response then t a k i n g over t o m a i n t a i n the s p e c i f i c a n t i b o d y l e v e l s . I t may a l s o be of s i g n i f i c a n c e t h a t the i n i t i a l i n j e c t i o n of NAGase was w i t h Freund's complete a d j u v a n t (FCA), w h i l e the second i n j e c t i o n used Freund's i n c o m p l e t e a d j u v a n t ( F I A ) . S i n c e i t i s known t h a t FCA s t i m u l a t e s T-lymphocytes ( S h i m i z u e t a l , 1988b), the r e d u c t i o n i n s p e c i f i c a n t i b o d y l e v e l a t day 35 may r e f l e c t lowered T-lymphocyte s t i m u l a t i o n . The response of the t h r e e c h i c k e n s , w h i l e a l l s i m i l a r , d i d d i f f e r s i g n i f i c a n t l y w i t h r e g a r d t o the l e v e l of the r e s p o n s e . At day 13, c h i c k e n s 2 and 3 had s i g n i f i c a n t l y h i g h e r a n t i b o d y l e v e l s d e t e c t e d i n the y o l k t h a n c h i c k e n 1 (a=0.05). T h i s i s not s u r p r i s i n g , as i t has been shown t h a t i n d i v i d u a l r e s p o n s e s t o Immunogens o f t e n d i f f e r s c o n s i d e r a b l y . w h i l e the i n j e c t i o n dose was not a d j u s t e d t o g i v e a c o n s t a n t dose per g i v e n body w e i g h t , t h i s i s e x p e c t e d t o c o n t r i b u t e t o the v a r i a b i l i t y o n l y s l i g h t l y . D i f f e r e n c e s were n o t e d , however, i n whole egg s i z e , which may 44 p a r t i a l l y e x p l a i n the v a r i e d response l e v e l s . A l s o , w h i l e two of the c h i c k e n s l a i d f a i r l y r o u t i n e l y d u r i n g the s t u d y , one ( c h i c k e n 1) l a i d u n u s u a l l y l a r g e eggs i r r e g u l a r l y . Thus, i n d i v i d u a l v a r i a b i l i t y may be due t o s e v e r a l f a c t o r s , and I t i s not s u r p r i s i n g t o see t h i s o c c u r r i n g i n the t h r e e b i r d s . E x a m i n a t i o n of the t o t a l egg y o l k IgG l e v e l s d e t e r m i n e d by r a d i a l i m m u n o d i f f u s i o n of c h i c k e n 3 over the i m m u n i z a t i o n p e r i o d showed l e v e l s r a n g i n g from 8.98 t o 10.8 mg/mL ( F i g u r e 2 ) . A n a l y s i s of v a r i a n c e i n d i c a t e d a s i g n i f i c a n t d i f f e r e n c e between the days (a=0.05) w i t h a t r e n d s i m i l a r t o t h a t observed w i t h r e s p e c t t o the s p e c i f i c a n t i b o d y r e s p o n s e . T h i s s u p p o r t s the f i n d i n g s by P o i s o n e t a l (1980) t h a t v i r a l i m m u n i z a t i o n of b i r d s r e s u l t e d i n an i n c r e a s e i n t o t a l IgG. Burger e t a l (1985) i s o l a t e d 14 mg of IgG per egg from unimmunized b i r d s , w h i l e 55 mg per egg was i s o l a t e d from immunized b i r d s . The l e v e l of 14 mg per egg i s e x t r e m e l y low, s u g g e s t i n g a y o l k c o n c e n t r a t i o n of l e s s than 1 mg IgG per mL. The d i f f e r e n c e i n IgG l e v e l s r e s u l t i n g from i m m u n i z a t i o n was not n e a r l y as d r a m a t i c i n t h i s c u r r e n t s t u d y . However, i n t e r p r e t a t i o n of the work by Burger e t a l (1985) i s d i f f i c u l t because o n l y one immunized c h i c k e n was used and no d e s c r i p t i o n of the non-immunized sou r c e of eggs was g i v e n . The d i f f e r e n c e t h e y d e s c r i b e d perhaps cannot be s o l e l y a t t r i b u t e d t o i m m u n i z a t i o n , as serum I g l e v e l s i n c h i c k e n s have been shown t o d i f f e r not o n l y w i t h i n groups of s i m i l a r b i r d s , but a l s o are a f f e c t e d by the age and breed (Wolfe e t a l , 1957). Thus, i t would be e x p e c t e d t h a t egg y o l k IgG l e v e l s would a l s o be a f f e c t e d F i g u r e 2. The change i n t o t a l IgG l e v e l s over time i n the egg y o l k of C h i c k e n 3 ( o f F i g . 1) immunized w i t h 6-N-a c e t y l g l u c o s a m l n i d a s e . Arrows i n d i c a t e time of i n j e c t i o n s . 46 by such f a c t o r s . The c u r r e n t r e s u l t s showed t h a t the i m m u n i z a t i o n of the t h r e e c h i c k e n s w i t h the enzyme G - N - a c e t y l g l u c o s a m i n i d a s e r e s u l t e d i n i n c r e a s e d a n t i b o d y p r o d u c t i o n showing s p e c i f i c a c t i v i t y toward the a n t i g e n . No e l e v a t e d l e v e l s were d e t e c t e d i n the eggs of an unimmunized b i r d . The t o t a l IgG l e v e l s i n the y o l k of C h i c k e n 3 i n c r e a s e d s l i g h t l y w i t h i m m u n i z a t i o n . 47 PART I I . SEPARATION OF IMMUNOGLOBULINS FROM EGG YOLK USING CHROMATOGRAPHIC TECHNIQUES A. DEAE-Sephacel Ion Exchange Chromatography V a r i o u s e q u i l i b r a t i n g c o n d i t i o n s were examined f o r DEAE-Sephacel chromatography. The b u f f e r of 30 mM p o t a s s i u m phosphate a t pH 8.0 gave s a t i s f a c t o r y b i n d i n g of IgG t o the column w h i l e e l i m i n a t i n g some c o n t a m i n a t i n g p r o t e i n s , as shown by e l e c t r o p h o r e s i s of the p o o l e d unbound f r a c t i o n s ( F i g u r e 3 ) . Subsequent use of a l i n e a r p o t a s s i u m phosphate g r a d i e n t i n c r e a s i n g from 30 mM t o 42 mM phosphate caused a s m a l l amount of p r o t e i n t o be e l u t e d from the column, based on the absorbance a t 280 nm ( F i g u r e 4 ) . These p r o t e i n s were p r i m a r i l y f 3 - l i v e t l n a t the b e g i n n i n g of the g r a d i e n t , and a m i x t u r e of p r o t e i n s , i n c l u d i n g i m m u n o g l o b u l i n s , i n the second p o r t i o n , as shown by e l e c t r o p h o r e s i s ( F i g u r e 5 ) . By f u r t h e r i n c r e a s i n g the p o t a s s i u m phosphate c o n c e n t r a t i o n , IgG c o n t i n u e d t o be e l u t e d . The eggs used i n t h i s s t u d y had been o b t a i n e d from a c h i c k e n immunized w i t h formaldehyde t r e a t e d E_;_ c o l l c e l l s . ELISA r e s u l t s showed the presence of a n t i b o d i e s w i t h s p e c i f i c i t y toward E_j_ c o l i l i p o p o l y s a c c h a r i d e (LPS) ( F i g u r e 4 ) . The i n i t i a l g r a d i e n t , g o i n g from 30 t o 42 mM phosphate r e s u l t e d i n e l u t e d f r a c t i o n s of low p r o t e i n c o n t e n t w i t h a h i g h l e v e l of s p e c i f i c a c t i v i t y toward the LPS. The second g r a d i e n t y i e l d e d f r a c t i o n s of h i g h e r p r o t e i n c o n c e n t r a t i o n , but lower s p e c i f i c a c t i v i t y . P o s s i b l e e x p l a n a t i o n s f o r the observed r e s u l t s were e x p l o r e d . 48 - a - l i v e t i n - IgG H.C. - ( 3 - l i v e t l n - IgG L.C. 1 2 F i g u r e 3. Reduced SDS-PAGE p r o f i l e s of the s u p e r n a t a n t and unbound f r a c t i o n o b t a i n e d from DEAE-Sephacel chromatography. Lane 1 , s u p e r n a t a n t ; l a n e 2 , unbound f r a c t i o n ; HC, IgG heavy c h a i n ; L . C , l i g h t c h a i n . Eluant Volume (mL) F i g u r e 4. E l u t i o n p r o f i l e of egg y o l k s u p e r n a t a n t from a DEAE-Sephacel column u s i n g two l i n e a r g r a d i e n t s of i n c r e a s i n g p o t a s s i u m phosphate c o n c e n t r a t i o n . Eggs were o b t a i n e d from c h i c k e n s immunized w i t h E ^ c o l i c e l l s . ELISA r e s u l t s i n d i c a t e a n t i b o d y s p e c i f i c i t y toward E_;_ c o l l l l p o -p o l y s a c c h a r i d e . 50 F i g u r e 5. Reduced SDS-PAGE p r o f i l e s of egg y o l k s u p e r n a t a n t from DEAE-Sephacel chromatography. Lane 1, f i r s t p a r t of f i r s t g r a d i e n t ; l a n e 2, second p a r t of f i r s t g r a d i e n t ; l a n e 3, main peak o£ second gradient; lane 4, second peak of second g r a d i e n t . 51 While i t g e n e r a l l y i s b e l i e v e d t h a t o n l y one c l a s s of IgG e x i s t s i n egg y o l k (Rose and O r l a n s , 1981), the p o s s i b i l i t y t h a t IgM or IgA might be p r e s e n t i n the e l u t e d f r a c t i o n s was examined. U s i n g double i m m u n o d i f f u s i o n , p o o l e d f r a c t i o n s were t e s t e d a g a i n s t a n t i - c h i c k e n IgG, a n t i - c h i c k e n IgM, and a n t i -c h i c k e n IgA. R e s u l t s were p o s i t i v e a g a i n s t the a n t i - c h i c k e n IgG a n t i s e r u m . However, n e g a t i v e r e s u l t s were o b t a i n e d u s i n g the IgM and IgA a n t i s e r a . To d e t e r m i n e whether the r e s u l t s o b t a i n e d were unique t o the response o b t a i n e d when E. c o l i c e l l s were used as the immunogen, b i r d s were immunized w i t h a m i x t u r e of 6-l a c t o g l o b u l i n and E. c o l i c e l l s , and the ex p e r i m e n t s r e p e a t e d . S i l v e r s t a i n i n g of the 6 - l a c t o g l o b u l i n sample r u n on an e l e c t r o p h o r e s i s g e l showed two p r o t e i n bands ( F i g u r e 6 ) . A n t i b o d i e s s p e c i f i c toward not o n l y 6 - l a c t o g l o b u l i n , but a l s o toward u n i d e n t i f i e d p r o t e i n s o c c u r r i n g i n the p r o t e i n and E. c o l i m i x t u r e would be e x p e c t e d . P r o t e i n and r a d i a l i m m u n o d i f f u s i o n (RID) d e t e r m i n a t i o n s on the p o o l e d f r a c t i o n s were performed, and the p u r i t y and y i e l d of IgG c a l c u l a t e d (Table I ) . The unbound f r a c t i o n s , c o n t a i n i n g low l e v e l s of IgG based on SDS-PAGE ( F i g u r e 7 ) , gave a double r i n g upon i m m u n o d i f f u s i o n . T h i s would I n d i c a t e t h a t the a n t i s e r u m used f o r the RID p l a t e p r e p a r a t i o n c o n t a i n e d a n t i b o d i e s s p e c i f i c f o r two d i f f e r e n t components p r e s e n t i n the unbound f r a c t i o n s . T h i s might have d e t e c t e d d i f f e r e n c e s i n the c h i c k e n IgG p o p u l a t i o n i t s e l f , or might have r e v e a l e d the presence of an o t h e r t o t a l l y s e p a r a t e contam-52 F i g u r e 6 . D S - P J O . f * ^ ^ % V ^ T i i S t - S S - i -\T»-lai% c h a r i n t U x f G Lqc'., i f f l i g h t c h a i n . 53 Table I . IgG Content a t D i f f e r e n t s t a g e s of the I s o l a t i o n of IgG From Egg Yol k on a DEAE-Sephacel column IgG IgG P r o t e i n Cone. IgG Cone. P u r i t y Recovery sample mg/mL mg/mL % % Supe r n a t a n t ( C o n t r o l ) 6.17 2.05 33.2 Unbound 1.32 0.276 0.0990 20.9 7.5 23.8 8.5 Grad.1 0.464 0.186 40.1 20.2 Grad.2a 0.487 0.291 59.8 15.8 Grad.2b 0.516 0.0930 18.0 11.2 54 - IgG H . C . - IgG L . C . • 1 2 3 4 5 6 Figure 7. Reduced SDS-PAGE p r o f i l e s of pooled fractions obtained from DEAE-Sephacel chromatography. Lane 1, unbound; lane 2, f i r s t gradient; lane 3, f i r s t part of second gradient (G2a); lane 4, second part of second gradient (G2b); lane 5, 0.30 M phosphate wash; lane 6, standard chicken IgG. 5 5 i n a t i n g p r o t e i n f o r which the a n t i s e r u m c o n t a i n e d some s p e c i f i c a n t i b o d i e s . In o r d e r t o t e s t whether the p r o t e i n which was p r o d u c i n g the second r i n g behaved s i m i l a r l y t o IgG upon n a t i v e e l e c t r o p h o r e s i s , i m m u n o e l e c t r o p h o r e s i s of the p o o l e d unbound f r a c t i o n was c a r r i e d out ( F i g u r e 8 ) . I t would be expected t h a t i f the second p r o t e i n was not of the same e l e c t r o p h o r e t i c m o b i l i t y as i n t a c t IgG, a second p r e c i p i t a t i o n r i n g would be obs e r v e d . T h i s was not the c a s e , however, s u g g e s t i n g t h a t the double r i n g r e s u l t e d from the r e a c t i o n of the a n t i s e r u m w i t h two p r o t e i n s of s i m i l a r e l e c t r o p h o r e t i c m o b i l i t y . The a r c t h a t was produced from the r e a c t i o n of the unbound f r a c t i o n s w i t h a n t i - c h i c k e n IgG was b l u r r e d , however, and t h i s may be an i n d i c a t i o n of h e t e r o g e n e i t y . Whether t h i s i s due t o the presence of a s u b c l a s s of c h i c k e n IgG cannot be d e t e r m i n e d c o n c l u s i v e l y based on the i m m u n o d i f f u s i o n r e s u l t s . The c l a s s and s u b c l a s s of immunoglobulins i s d e f i n e d by the amino a c i d sequence of the c o n s t a n t r e g i o n of the heavy c h a i n ( N i s o n o f f , 1982), and may be i d e n t i f i e d based on r e a c t i o n w i t h a n t i s e r a s p e c i f i c f o r the g i v e n c l a s s or s u b c l a s s . However, a n t i s e r a i d e n t i f y i n g s u b c l a s s e s a r e d i f f i c u l t t o o b t a i n . While a b s o l u t e i d e n t i f i c a t i o n of the p r o t e i n c a u s i n g the double r i n g upon i m m u n o d i f f u s i o n was not p o s s i b l e , the r e s u l t s seemed t o I n d i c a t e the presence of a s u b p o p u l a t i o n of c h i c k e n IgG which d i d not b i n d t o the DEAE-Sephacel column under the c o n d i t i o n s chosen, and t h e r e f o r e , was found i n the unbound f r a c t i o n s . 56 Anti-EY A n t i - I g Anti-EY Ant i - I g Anti-EY ^^^^ s UB Gl G2a G2b F i g u r e 8. Immunoelectrophoresis of samples obtained by DEAE-Sephacel chromatography. Anti-EY, a n t i - e g g yolk antiserum; A n t i - I g , a n t i - c h i c k e n IgG antiserum; S, supernatant; UB, unbound f r a c t i o n s ; G l , f i r s t g r a d i e n t f r a c t i o n s ; G2a, f r a c t i o n s from the f i r s t p a r t of second g r a d i e n t ; G2b, f r a c t i o n s from the second p a r t of the second g r a d i e n t . 57 The r e s u l t s o£ e l u t i o n of the bound p r o t e i n s are i l l u s t r a t e d i n F i g u r e 9, w i t h the e l e c t r o p h o r e s i s r e s u l t s shown In F i g u r e 7. u s i n g f 3 - l a e t o g l o b u l i n as the a n t i g e n i n ELISA gave s i m i l a r r e s u l t s t o the LPS c u r v e , but w i t h much h i g h e r absorbance r e a d i n g s ( F i g u r e 9), I t may be t h a t (3-l a c t o g l o b u l l n was a b e t t e r lmmunogen than the E^. c o l l c e l l s under the c o n d i t i o n s chosen; or perhaps the ( 3 - l a c t o g l o b u l i n ELISA a s s a y was more s e n s i t i v e than the LPS a s s a y . However, i t Is a l s o p o s s i b l e t h a t the response t o the ( 3 - l a c t o g l o b u l l n may have been enhanced t h r o u g h the combined i n j e c t i o n s of the p r o t e i n w i t h E^ c o l l , s i n c e i t i s known t h a t b a c t e r i a l l i p o p o l y s a c c h a r i d e s o f t e n e x h i b i t an a d j u v a n t e f f e c t (Mota, 1986). i n any c a s e , r e s u l t s p a r a l l e l e d t h o s e o b t a i n e d when E.  c o l l was the s i n g l e lmmunogen. The r e s u l t i n g p a t t e r n of i n i t i a l h i g h s p e c i f i c a c t i v i t y c o r r e s p o n d i n g t o low p r o t e i n l e v e l s , f o l l o w e d by h i g h e r p r o t e i n l e v e l s of lower s p e c i f i c a c t i v i t y was a p p arent w i t h both 6 - l a c t o g l o b u l i n and c o l i a n t i g e n s . F r a c t i o n s from the second g r a d i e n t were p o o l e d i n t o two s e p a r a t e groups (G2a or G2b) s i n c e the f i r s t p o r t i o n of the second g r a d i e n t showed h i g h e r a n t i b o d y s p e c i f i c a c t i v i t y t h a n the l a t t e r . E x a m i n a t i o n of the e l e c t r o p h o r e t o g r a m i n d i c a t e d t h a t the unbound p o r t i o n s c o n t a i n e d b oth IgG and o t h e r p r o t e i n s . A p r o t e i n band of the m o l e c u l a r w e i g ht of the IgG heavy c h a i n was then d e t e c t e d i n the f i r s t g r a d i e n t , a l o n g 58 F i g u r e 9. E l u t i o n p r o f i l e of egg y o l k s u p e r n a t a n t from DEAE-Sephacel column u s i n g two l i n e a r g r a d i e n t s of i n c r e a s i n g phosphate c o n c e n t r a t i o n . C h i c k e n s had been immunized w i t h a m i x t u r e of E^. c o l l c e l l s and 6-l a c t o g l o b u l i n . E l u t e d f r a c t i o n s were d i l u t e d 1/100 f o r the ELISA a s s a y . The f l o w r a t e was 0.8 mL/min. 59 w i t h a c o n t a m i n a t i n g p r o t e i n , w h i l e a p p a r e n t l y v e r y h i g h l e v e l s of IgG p u r i t y were shown i n both p o r t i o n s of the second g r a d i e n t . I n t e r e s t i n g l y , RID r e s u l t s showed the f i r s t p a r t of the second g r a d i e n t t o have the h i g h e s t p u r i t y of I g (59.8%) (Table I ) ; y e t lower l e v e l s of s p e c i f i c a n t i b o d y a c t i v i t y t h a n the f i r s t g r a d i e n t ( F i g u r e 9) i n which o n l y 40.1% of the p r o t e i n was IgG. While i t has p r e v i o u s l y been shown t h a t the i s o l a t i o n method chosen f o r o b t a i n i n g IgG from human plasma may a f f e c t the b i n d i n g a b i l i t i e s of the p u r i f i e d a n t i b o d i e s (McCue e t a l , 1988), the change i n the e l u t i n g b u f f e r c o n d i t i o n s from 30 mM t o 200 mM, a t the c o n s t a n t pH of 8.0, would not be e x p e c t e d t o induce the observed changes i n b i n d i n g a b i l i t y . W h ile s e p a r a t i o n of bovine IgG i n t o v a r i o u s f r a c t i o n s on DEAE i o n exchange chromatography has been used as a c r i t e r i o n f o r d e s i g n a t i o n of bov i n e IgG s u b c l a s s e s , t h i s can l e a d t o erroneous c o n c l u s i o n s . Duncan e t a l (1972) showed t h a t , w h i l e b o v i n e I g G l c o u l d be e l u t e d from a DEAE-Sephadex column a t l e v e l s of h i g h e r c o n d u c t i v i t y , IgG2 was e l u t e d t h r o u g h the e n t i r e spectrum s t u d i e d , and so co n t a m i n a t e d I g G l . I n o r d e r t o d e f i n e s u b c l a s s e s , the a n t i g e n i c d i f f e r e n c e s must be shown. Schlamowitz e t a l (1975) were a b l e t o s e p a r a t e r a b b i t IgG i n t o t h r e e f r a c t i o n s u s i n g DEAE c e l l u l o s e chromatography. E x a m i n a t i o n of the t h r e e samples i n d i c a t e d c l e a r l y an i n c r e a s e In s i a l i c a c i d w i t h i n c r e a s i n g o r d e r of e l u t i o n . D i f f e r e n c e s i n t he s u s c e p t i b i l i t y of the d i s u l p h i d e bonds t o r e d u c t i o n 60 were n o t e d , as w e l l as s u s c e p t i b i l i t y t o d i g e s t i o n w i t h p a p a i n . W h i l e t h e s e d i f f e r e n c e s suggest t h a t the t h r e e f r a c t i o n s r e p r e s e n t e d s e p a r a t e s u b c l a s s e s , such d e s i g n a t i o n was not p o s s i b l e w i t h o u t f u r t h e r a n t i g e n i c d e t e r m i n a n t work. S e p a r a t i o n of the c h i c k e n a n t i b o d i e s based on a n t i g e n s p e c i f i c i t y i s a l s o p o s s i b l e . The s e p a r a t i o n of b o v i n e IgG2a i n t o s u b p o p u l a t l o n s u s i n g DEAE chromatography ( B u t l e r e t a l . , 1987) was a t t r i b u t e d t o charge d i f f e r e n c e s i n the v a r i a b l e r e g i o n s of the IgG heavy c h a i n s . T h i s r e g i o n i s known t o d etermine the s p e c i f i c i t y of the a n t i b o d i e s . E x a m i n a t i o n of a c t i v i t y of the e l u t e d f r a c t i o n s toward seven d i f f e r e n t a n t i g e n s i n d i c a t e d d i f f e r e n c e s based on the a n t i g e n i c s p e c i f i c i t y . I t has been shown t h a t , i n humans, immunogenic s t i m u l a t i o n may r e s u l t i n the i n c r e a s e i n c e r t a i n s u b c l a s s e s p r e f e r e n t i a l l y over o t h e r s u b c l a s s e s . T h i s appears t o be dependent upon the n a t u r e of the a n t i g e n . P r o t e i n and T-dependent a n t i g e n s r e s u l t i n e l e v a t e d l e v e l s of i g G l , w h i l e c a r b o h y d r a t e s and b a c t e r i a l a n t i g e n s promote the p r o d u c t i o n of lgG2 ( S c o t t e t a l , 1986). I t has been shown t h a t i n f e c t i o n w i t h Candida a l b i c a n s i n humans r e s u l t s i n e l e v a t e d s a l i v a r y I g A l l e v e l s w h i l e i n h e a l t h y i n d i v i d u a l s IgA2 predominates (Jeganathan e t a l , 1987). F u r t h e r m o r e , g u i n e a p i g s immunized w i t h ovalbumin and complete a d j u v a n t produced IgG2 a n t i b o d i e s . When i n c o m p l e t e a d j u v a n t was used, I g G l was the predominant immunoglobulin s y n t h e s i z e d (Mota, 1986). 61 Immunization of c h i c k e n s was shown t o cause an i n c r e a s e i n a n t i b o d i e s w i t h s p e c i f i c a c t i v i t y toward the a n t i g e n . The b e h a v i o u r of c h i c k e n IgG on DEAE-Sephacel was the same whether the immunogen was whole c o l l c e l l s or 8 - l a c t o g l o b u l i n . I t i s p o s s i b l e t h a t t h e s e s p e c i f i c a n t i b o d i e s were p r i m a r i l y of one s u b c l a s s , and charge d i f f e r e n c e s e x i s t i n g between s u b c l a s s e s r e s u l t e d i n e a r l y e l u t i o n i n the f i r s t g r a d i e n t . I t i s p o s s i b l e , however, t h a t no s u b c l a s s e s e x i s t , but t h a t s e p a r a t i o n of s p e c i f i c from n o n - s p e c i f i c a n t i b o d i e s was due t o amino a c i d d i f f e r e n c e s o c c u r r i n g In the v a r i a b l e r e g i o n of the heavy c h a i n . T h i s c o u l d not be c o n f i r m e d , but i t i s c l e a r t h a t the c h i c k e n IgG was a heterogeneous group of m o l e c u l e s which e l u t e d a t v a r i o u s i o n i c s t r e n g t h s from the DEAE-Sephacel column, w i t h s p e c i f i c a n t i b o d y a c t i v i t y b e i n g d e t e c t e d p r i m a r i l y i n the e a r l y e l u t e d f r a c t i o n s . E x a m i n a t i o n of the reduced SDS-PAGE p r o f i l e I n d i c a t e d t h a t the second p o r t i o n of the second g r a d i e n t from the DEAE chromatography was of a p parent h i g h IgG p u r i t y ( F i g u r e 7 ) . A s i n g l e p r o t e i n band of a m o l e c u l a r weight s i m i l a r t o the heavy c h a i n of IgG was d e t e c t e d . However, RID showed v e r y low amounts of IgG i n t h i s f r a c t i o n (Table I ) . E x a m i n a t i o n of the I m m u n o e l e c t r o p h o r e s i s p a t t e r n ( F i g u r e 8) r e v e a l e d t h a t the f r a c t i o n showed a f a i n t c u r v e i d e n t i f i e d as IgG when compared w i t h the a n t i - c h i c k e n IgG a n t i s e r u m . However, p r e c i p i t a t i o n a g a i n s t the a n t i - c h i c k e n egg y o l k a n t i s e r u m showed two o t h e r a r c s a t p o s i t i o n s of h i g h e r e l e c t r o p h o r e t i c m o b i l i t y . The p a t t e r n seemed t o i n d i c a t e t h a t the egg y o l k s u p e r n a t a n t 62 c o n t a i n e d a p r o t e i n of m o l e c u l a r weight s i m i l a r t o the heavy c h a i n of IgG. T h i s a l s o may e x p l a i n why e l e c t r o p h o r e t o g r a m s o f t e n were observed t o show s t a i n i n g of the c h i c k e n IgG "heavy c h a i n " , w i t h o u t the appearance of the l i g h t c h a i n . The e s t i m a t i o n of p u r i t y based on e l e c t r o p h o r e s i s r e s u l t s of reduced samples, o f t e n used i n the l i t e r a t u r e , i s t h e r e f o r e c a u t i o n e d a g a i n s t . Over e s t i m a t i o n of y o l k IgG p u r i t y may r e s u l t . 63 B. METAL CHELATE INTERACTION CHROMATOGRAPHY I n i t i a l l y , a b u f f e r of 0.05 M t r i s - a c e t a t e , 0.5 M sodium c h l o r i d e , pH 8.0 was used f o r e q u i l i b r a t i o n of the copper-loaded M C I C column. These e q u i l i b r a t i n g c o n d i t i o n s a l l o w e d s u c c e s s f u l b i n d i n g of immunoglobulins from b l o o d serum (Lee e t a l , 1988). A p p l i c a t i o n of egg y o l k s u p e r n a t a n t w i t h pH a d j u s t e d t o 8.0, c o n t a i n i n g 0.05 M t r i s and 0.5 M sodium c h l o r i d e a l l o w e d some p r o t e i n t o b i n d t o the column, which was then e l u t e d by a pH g r a d i e n t d e c r e a s i n g from 8.2 t o 2.8 ( F i g u r e 1 0 ) . However, r a d i a l i m m u n o d i f f u s i o n of the e l u t e d p r o t e i n f r a c t i o n s i n d i c a t e d t h a t o n l y f o u r p e r c e n t of the bound p r o t e i n was IgG. E l i m i n a t i o n of the sodium c h l o r i d e r e s u l t e d i n even l e s s p r o t e i n b i n d i n g , s u p p o r t i n g Hemdan and P o r a t h ' s (1985a) f i n d i n g t h a t amino a c i d i n t e r a c t i o n w i t h MCIC columns was promoted when sodium c h l o r i d e was i n c l u d e d . G e n e r a l l y , sodium c h l o r i d e i s i n c l u d e d i n the e q u i l i b r a t i n g b u f f e r i n o r d e r t o p r e v e n t n o n - s p e c i f i c e l e c t r o s t a t i c i n t e r a c t i o n s ( S u l k o w s k i , 1985). The a p p l i c a t i o n of b l o o d serum t o a column of s i m i l a r c o n d i t i o n s has been shown t o r e s u l t i n b i n d i n g of IgG which can t h e n be e l u t e d i n a p u r i f i e d form by a b u f f e r of pH 4.0 (Lee e t a l , 1988). The i n a b i l i t y of c h i c k e n IgG t o b i n d t o the c o p p e r - l o a d e d MCIC column under th e s e c o n d i t i o n s may be due i n p a r t t o the s t r u c t u r a l d i f f e r e n c e s which e x i s t between i t and bov i n e IgG, as w e l l as t o the presence of o t h e r p r o t e i n s i n the egg y o l k s u p e r n a t a n t which may, under the chosen c o n d i t i o n s , p r e f e r e n t i a l l y b i n d t o the column. 64 a F i g u r e 10. E l u t i o n p r o f i l e of adsorbed p r o t e i n s from c o p p e r - l o a d e d MCIC column u s i n g a d e c r e a s i n g pH g r a d i e n t Column e q u i l i b r a t i o n and wash was w i t h 0.05 M t r i s - a c e t a t e , ' £;! " S O d i u ? c h l o r i d e , pH 8.0. The f l o w r a t e was 1.2 mL/min F r a c t i o n volumes were 6.4 mL. 65 S i n c e i t has been shown t h a t a n t i c h a o t r o p i c s a l t s (such as phosphates) te n d t o promote p r o t e i n a d s o r p t i o n t o MCIC columns (Hemdan and P o r a t h , 1985b; P o r a t h , 1987), the e q u i l i b r a t i n g b u f f e r c o n s i s t i n g of 0.02 M sodium phosphate, 0.5 M sodium c h l o r i d e , a t pH 7.0, was examined. I n o r d e r t o de termine whether c h i c k e n immunoglobul ins would b i n d t o an MCIC column under t h e s e e q u i l i b r a t i n g c o n d i t i o n s , egg y o l k IgG p u r i f i e d by the p o l y e t h y l e n e g l y c o l method of P o i s o n e t a l (1985) was added t o f i v e mL of egg y o l k s u p e r n a t a n t t o g i v e a s o l u t i o n c o n t a i n i n g a p p r o x i m a t e l y 45 mg of c h i c k e n IgG. To t h i s sample, sodium phosphate and sodium c h l o r i d e were added t o c o n c e n t r a t i o n s of 0.02 M and 0.5 M, r e s p e c t i v e l y , and the pH a d j u s t e d t o 7.0. From the p r o t e i n e l u t i o n c u r v e ( F i g u r e 1 1 ) , i t can be seen t h a t o n l y a v e r y s m a l l amount of p r o t e i n d i d not b i n d t o the column. A pH 4.0 b u f f e r t h e n removed some p r o t e i n from the column, w i t h most b e i n g e l u t e d w i t h 10 mM i m i d a z o l e . The e l e c t r o p h o r e t o g r a m of the p o o l e d samples ( F i g u r e 12) d i d not a l l o w i d e n t i f i c a t i o n of the p r o t e i n s i n the unbound or pH 4.0 e l u t e d f r a c t i o n s due t o low p r o t e i n c o n c e n t r a t i o n s ; however, i t was a p p a r e n t t h a t c h i c k e n IgG was e l u t e d w i t h 10 mM i m i d a z o l e . Thus, c h i c k e n IgG was bound t o the c o p p e r - l o a d e d MCIC column under e q u i l i b r a t i o n c o n d i t i o n s c r e a t e d by the phosphate b u f f e r a t pH 7.0, and c o u l d be e l u t e d w i t h 10 mM i m i d a z o l e . 66 F i g u r e 11. E l u t i o n p r o f i l e of 45 mg IgG i n 5 mL egg y o l k s u p e r n a t a n t on a MCIC column. E q u i l i b r a t i n g and washing b u f f e r of 0.02 M phosphate, 0.5 M sodium c h l o r i d e , a t pH 7.0. Sample a p p l i c a t i o n ( S ) , wash (W), e l u t i o n w i t h 0.05 M t r i s - a c e t a t e , 0.05 M sodium c h l o r i d e , pH 4 ( E l ) , and e l u t i o n w i t h 0.01 M i m i d a z o l e (E2) are i n d i c a t e d . Flow r a t e was 0.4 mL/min. 67 F i g u r e 12. R e d u c e d S D S - P A G E p r o f i l e s o f p o o l e d f r a c t i o n s o b t a i n e d w h e n e g g y o l k s u p e r n a t a n t c o n t a i n i n g a d d e d I g G w a s e l u t e d f r o m a n M C I C c o l u m n . L a n e 1, s u p e r n a t a n t ; l a n e 2, u n b o u n d ; l a n e 3, p H 4 e l u t e d f r a c t i o n ; l a n e s 4 a n d 5, i m i d a z o l e e l u t e d f r a c t i o n s ; l a n e 6, E D T A e l u t e d f r a c t i o n . 68 Not a l l of the p r o t e i n was e l u t e d by i m i d a z o l e , however; a s m a l l amount of p r o t e i n was d e t e c t a b l e i n the EDTA f r a c t i o n upon e l e c t r o p h o r e s i s ( F i g u r e 1 2 ) . The mechanism by which i m i d a z o l e e f f e c t i v e l y e l u t e s bound p r o t e i n i s t h r o u g h c o m p e t i t i o n w i t h h i s t i d i n e of the p r o t e i n s , which a r e b e l i e v e d t o be p r i m a r i l y r e s p o n s i b l e f o r p r o t e i n a d s o r p t i o n ( P o r a t h e t a l , 1975). The s t r o n g b i n d i n g of t h i s p r o t e i n f r a c t i o n must be a t t r i b u t e d t o mechanisms o t h e r than t h a t of s u r f a c e h i s t i d i n e amino a c i d s i n t e r a c t i n g w i t h the copper. The o b s e r v a t i o n t h a t exposed t h i o l groups on p r o t e i n s a l s o c o n t r i b u t e t o p r o t e i n a d s o r p t i o n ( P o r a t h e t a l , 1975) may e x p l a i n the i n a b i l i t y of i m i d a z o l e t o remove a l l of the p r o t e i n . The a p p l i c a t i o n of a s u p e r n a t a n t sample s i z e of 150 mL r e s u l t e d i n a l a r g e amount of s p e c i f i c a n t i b o d i e s b e i n g d e t e c t e d i n the unbound f r a c t i o n s ( F i g u r e 1 3 ) . RID r e s u l t s ( T a b le I I ) showed t h a t s i x t y - s i x p e r c e n t of the immunoglobulins a p p l i e d d i d not b i n d t o the column. R e d u c t i o n i n sample s i z e t o 100 mL a l s o r e s u l t e d i n a c o n s i d e r a b l e amount of IgG b e i n g d e t e c t e d i n the unbound f r a c t i o n s . ELISA r e s u l t s showed the presence of IgG h a v i n g s p e c i f i c a c t i v i t y i n the e l u a n t a f t e r a p p l i c a t i o n of 42 mL of sample ( F i g u r e 1 4 ) . E l e c t r o p h o r e s i s of the unbound f r a c t i o n s ( F i g u r e 15) showed a - l i v e t i n and IgG t o be e l u t e d i n i t i a l l y , w i t h 6 - l i v e t i n s a p p e a r i n g i n the l a t t e r p o r t i o n w i t h o v e r l o a d i n g of sample. 69 Volume (mL) S W El E2 F i g u r e 13. E l u t i o n p r o f i l e of 150 mL egg y o l k s u p e r n a t a n t a p p l i e d t o a c o p p e r - l o a d e d MCIC column. E q u i l i b r a t i n g and washing b u f f e r was w i t h pH 7.0 phosphate b u f f e r , sample a p p l i c a t i o n ( S ) , wash (W), e l u t i o n w i t h 0.01 M I m i d a z o l e ( E l ) and e l u t i o n w i t h pH 4 t r i s - a c e t a t e b u f f e r (E2) are i n d i c a t e d . Flow r a t e was 0.4 mL/min. ELISA r e s u l t s i n d i c a t e s p e c i f i c a n t i b o d y a c t i v i t y toward 6 - l a c t o g l o b u l i n i n which samples were d i l u t e d 1/50. 70 Table I I . IgG Content of P o o l e d F r a c t i o n s O b t a i n e d Upon I s o l a t i o n of IgG From Egg Yolk on MCIC U s i n g 150 mL Sample A p p l i c a t i o n S i z e IgG IgG P r o t e i n Cone. IgG Cone. P u r i t y Recovery Sample mg/mL mg/mL % % Sup e r n a t a n t 8.04 3.15 39.0 Unbound 3.86 2.23 57.6 65.8 I m i d a z o l e 6.48 1.89 29.2 8.0 71 F i g u r e 14. E l u t i o n p r o f i l e of 100 mL egg y o l k s u p e r n a t a n t a p p l i e d t o a c o p p e r - l o a d e d MCIC column. E q u i l i b r a t i n g and washing b u f f e r was pH 7 phosphate b u f f e r . Sample a p p l i c a t i o n ( S ) , wash (W), e l u t i o n w i t h pH 4.0 t r i s - a c e t a t e b u f f e r ( E l ) and e l u t i o n w i t h i m i d a z o l e g r a d i e n t (E2) a r e i n d i c a t e d . The f l o w r a t e was 0.4 mL/min. ELISA r e s u l t s i n d i c a t e s p e c i f i c a n t i b o d y a c t i v i t y toward ( 3 - l a c t o g l o b u l l n i n which samples were d i l u t e d 1/50. 72 I t -J U L_J L_J L J LJ B B B * t f « I - a - l i v e t i n -IgG H.C. - G - l i v e t i n -IgG L.C. 1 2 3 4 5 8 9 10 F i g u r e 15. Reduced SDS-PAGE of f r a c t i o n s o b t a i n e d when 100 mL s u p e r n a t a n t was a p p l i e d t o a MCIC column. Lane 1, s u p e r n a t a n t ; l a n e s 2 t o 6, unbound f r a c t i o n s c o l l e c t e d d u r i n g e l u t i o n ; l a n e 7, pH 4 e l u t e d f r a c t i o n ; l a n e s 8 and 9, i m i d a z o l e e l u t e d f r a c t i o n s ; l a n e 10, s t a n d a r d IgG. The pH 4.0 b u f f e r r e s u l t e d i n a p r o t e i n peak ( F i g u r e 14) which c o n t a i n e d b oth IgG and 6 - l i v e t i n s ( F i g u r e 15) and showed the presence of s p e c i f i c a n t i b o d y a c t i v i t y . An i m i d a z o l e g r a d i e n t t h e n r e s u l t e d i n f u r t h e r e l u t i o n of IgG as d e t e r m i n e d by e l e c t r o p h o r e s i s ( F i g u r e 1 5 ) . An a p p l i c a t i o n of 50 mL s u p e r n a t a n t r e s u l t e d i n some p r o t e i n not b i n d i n g t o the MCIC column, i n c l u d i n g some IgG, as de t e r m i n e d by ELISA ( F i g u r e 16) and e l e c t r o p h o r e s i s ( F i g u r e 1 7 ) . R a d i a l i m m u n o d i f f u s i o n of the p o o l e d unbound f r a c t i o n s , however, i n d i c a t e d t h a t o n l y 0.8% of the i g a p p l i e d t o the column d i d not b i n d (Table I I I ) . S i n c e e l u t i o n w i t h pH 4.0 i n p r e v i o u s e x p e r i m e n t s had r e s u l t e d i n combined e l u t i o n of IgG and G - l i v e t i n , a l e s s s e v e r e t r i s - a c e t a t e b u f f e r a t pH 5.0 was chosen. E l e c t r o p h o r e s i s of the s e e l u t e d f r a c t i o n s I n d i c a t e d the pH 5.0 removed p r i m a r i l y a- and 6 - l i v e t i n s , w i t h a s m a l l amount of IgG b e i n g e l u t e d ( F i g u r e 1 7 ) . R a d i a l i m m u n o d i f f u s i o n showed t h a t o n l y one p e r c e n t of the t o t a l Ig a p p l i e d t o the column was e l u t e d a t pH 5.0 (Table I I I ) . However, a s i g n i f i c a n t amount of o t h e r c o n t a m i n a t i n g p r o t e i n s were s u c c e s s f u l l y removed. E l u t i o n w i t h 10 mM i m i d a z o l e t h e n caused e l u t i o n of 46% of the a p p l i e d I g , w i t h a p u r i t y of 65%. A n t i b o d i e s s p e c i f i c t o the a n t i g e n were p r e s e n t i n t h e s e f r a c t i o n s , as de t e r m i n e d by ELISA ( F i g u r e 1 6 ) . A p p r o x i m a t e l y seven p e r c e n t of the I g a p p l i e d t o the column was not e l u t e d by the i m i d a z o l e , but was removed w i t h the copper by the EDTA s o l u t i o n (Table I I I ) . The presence of 74 F i g u r e 16. E l u t i o n p r o f i l e of 50 mL egg y o l k s u p e r n a t a n t a p p l i e d t o a c o p p e r - l o a d e d MCIC column. E q u i l i b r a t i o n and washing w i t h pH 7.0 phosphate b u f f e r . Sample a p p l i c a t i o n ( S ) , wash (W), e l u t i o n w i t h pH 5.0 t r i s - a c e t a t e b u f f e r ( E l ) , and e l u t i o n w i t h 0.01 M I m i d a z o l e a r e I n d i c a t e d . ELISA r e s u l t s i n d i c a t e d s p e c i f i c a n t i b o d y a c t i v i t y toward 0-l a c t o g l o b u l i n i n which samples were d i l u t e d 1/100. The f l o w r a t e was 0.4 mL/min. 75 MuysaB - ( 3 - l i v e t i n - IgG L . C . 10 11 12 F i g u r e 1 7 . R e d u c e d S D S - P A G E o f f r a c t i o n s o b t a i n e d w h e n 50 m L s u p e r n a t a n t w a s a p p l i e d t o a M C I C c o l u m n . L a n e 1 , s u p e r n a t a n t ; l a n e 2 , s t a n d a r d I g G ; l a n e s 3 t o 5 , u n b o u n d f r a c t i o n s ; l a n e s 6 t o 8, p H 5 . 0 e l u t e d f r a c t i o n s ; l a n e s 9 t o 1 1 , i m i d a z o l e e l u t e d f r a c t i o n s ; l a n e 1 2 , E D T A e l u t e d f r a c t i o n . Table I I I . IgG Content of D i f f e r e n t F r a c t i o n s Of The I s o l a t i o n of IgG From Egg Yolk on MCIC Column U s i n g 50 mL Sample A p p l i c a t i o n IgG IgG P r o t e i n Cone. IgG Cone. P u r i t y Recovery Sample mg/mL mg/mL % % Supe r n a t a n t 8.04 3.15 39.0 Unbound 1.15 0.0161 1.40 0.83 pH 5.0 1.20 0.0340 2.83 1.12 I m i d a z o l e 3.86 2.52 65.0 46.3 EDTA 2.88 1.08 37.6 6.85 77 IgG i n t h i s f r a c t i o n was a l s o a p parent based on e l e c t r o p h o r e s i s ( F i g u r e 1 7 ) . The erroneous c o n c l u s i o n s which may r e s u l t when c h i c k e n IgG p u r i t y i s e s t i m a t e d based on reduced SDS-PAGE e l e c t r o p h o r e s i s was i l l u s t r a t e d i n an experiment where a 50 mL sample was a p p l i e d as d e s c r i b e d above w i t h a pH 5.0 e l u t i o n t o remove some c o n t a m i n a t i n g p r o t e i n s , but w i t h an i m i d a z o l e g r a d i e n t a t t e m p t e d i n o r d e r t o det e r m i n e whether b e t t e r IgG s e p a r a t i o n c o u l d be o b t a i n e d . The g r a d i e n t r e s u l t e d i n an apparent t h r e e peaks based on the absorbance a t 280 nm ( F i g u r e 18) w i t h ELISA d e t e c t i n g a n t i b o d y a c t i v i t y o n l y i n the l a t t e r p o r t i o n of the g r a d i e n t . RID a l s o I n d i c a t e d t h a t IgG was e l u t e d o n l y i n the t h i r d peak of the i m i d a z o l e g r a d i e n t (Table I V ) . However, e x a m i n a t i o n of the e l e c t r o p h o r e t o g r a m i n d i c a t e d c l e a r l y a p r o t e i n h a v i n g a s i m i l a r m o l e c u l a r weight of the IgG heavy c h a i n b e i n g e l u t e d t h r o u g h o u t the g r a d i e n t ( F i g u r e 1 9 ) . S t a i n i n g of the l i g h t c h a i n , however, was o n l y a p p a r e n t i n the t h i r d peak of the i m i d a z o l e g r a d i e n t , c o r r e s p o n d i n g w i t h the ELISA and RID r e s u l t s . The presence of the p r o t e i n h a v i n g a m o l e c u l a r weight s i m i l a r t o the heavy c h a i n may e x p l a i n these r e s u l t s . Based on r e s u l t s o b t a i n e d from the egg y o l k s u p e r n a t a n t from the c h i c k e n s immunized w i t h ( 3 - l a c t o g l o b u l i n , i t was app a r e n t t h a t w i t h s m a l l amounts of s u p e r n a t a n t b e i n g l o a d e d t o the column, the Ig was a b l e t o b i n d . E l u t i o n w i t h a t r i s -a c e t a t e b u f f e r a t pH 5.0 the n was a b l e t o remove some of the c o n t a m i n a t i n g p r o t e i n s from the column. I m i d a z o l e e l i c i t e d 78 F i g u r e 18. E l u t i o n p r o f i l e of 50 mL egg y o l k s u p e r n a t a n t on MCIC. ELISA r e s u l t s I n d i c a t e s p e c i f i c a c t i v i t y toward 6-l a c t o g l o b u l i n . E q u i l i b r a t i o n and washing b u f f e r was pH 7.0 phosphate. Sample a p p l i c a t i o n ( S ) , wash (W), e l u t i o n w i t h pH 5.0 t r l s - a c e t a t e ( E l ) , and e l u t i o n w i t h an I m i d a z o l e g r a d i e n t (E2) a r e i n d i c a t e d . 79 Table IV, i g o c o n t e n t of the F r a c t i o n s E l u t e d by an i m i d a z o l e G r a d i e n t from an MCIC Column F o l l o w i n g 50 mL Sample A p p l i c a t i o n IgG IgG Sample P r o t e i n Cone. IgG Cone. P u r i t y Recovery (mg/mL) (mg/mL) (%) (%) I m i d a z o l e 1.97 0* ( f i r s t peak) I m i d a z o l e 2.71 0* (second peak) I m i d a z o l e 3.72 2.06 55.5 31.4 * While a v i s i b l e p r e c i p i t a t i o n r i n g was observed i n t h e s e f r a c t i o n s , l e v e l s were too low f o r q u a n t i t a t i o n (below 0.12 mg/mL) 80 - H.C. - L.C, 1 2 3 4 5 6 7 8 9 F i g u r e 19. Reduced SDS-PAGE of f r a c t i o n s obtained by gr a d i e n t imidazole e l u t i o n on MCIC column. Lanes 1 and 2, f i r s t peak, imidazole g r a d i e n t ; lanes 3 to 5, second peak, imidazole g r a d i e n t ; lanes 6 to 9, t h i r d peak, imidazole g r a d i e n t . H.C, IgG heavy c h a i n ; L . C , IgG l i g h t c h a i n . 81 e l u t i o n of most of the a p p l i e d immunoglobulins. However, I t was a l s o noted t h a t , upon o v e r l o a d i n g of the MCIC column, p u r i f i c a t i o n of the c h i c k e n IgG o c c u r r e d i n the unbound f r a c t i o n s . T h i s s u g g ested t h a t some c o n t a m i n a t i n g p r o t e i n s ( i d e n t i f i e d t o be p r i m a r i l y ( 3 - l i v e t i n s , based on e l e c t r o p h o r e s i s ) were p r e f e r e n t i a l l y bound t o the column. Thus, two d i f f e r e n t methods of a n t i b o d y p u r i f i c a t i o n appeared p o s s i b l e by MCIC - t h r o u g h b i n d i n g of I g , f o l l o w e d by e l u t i o n ; or by o v e r l o a d i n g and c o l l e c t i o n of the unbound. These r e s u l t s suggested t h a t a tandem MCIC column system might be s u c c e s s f u l i n o b t a i n i n g p u r i f i e d IgG. O v e r l o a d i n g of the f i r s t column was ex p e c t e d t o r e s u l t i n removal of some of the c o n t a m i n a t i n g p r o t e i n s . A p p l i c a t i o n of the unbound f r a c t i o n t o the second column would t h e n a l l o w b i n d i n g of the IgG t o the column, s i n c e most . of the more s t r o n g l y b i n d i n g competing p r o t e i n s would have been removed. To examine t h i s i d e a , a sample s i z e of 150 mL was a p p l i e d t o a c o p p e r - l o a d e d column, and the e n t i r e unbound f r a c t i o n s p o o l e d and r e a p p l i e d t o a second. R a d i a l i m m u n o d i f f u s i o n and p r o t e i n d e t e r m i n a t i o n s of the r e s u l t i n g f r a c t i o n s were completed ( T a b l e V ) . The amount of IgG a p p l i e d t o the second column was c a l c u l a t e d t o be a p p r o x i m a t e l y 180 mg. While some of the IgG d i d b i n d t o the column, and then was e l u t e d w i t h the subsequent b u f f e r s , some s t i l l d i d not b i n d t o the second column. A double r i n g d e v e l o p e d upon i m m u n o d i f f u s i o n of the unbound f r a c t i o n s . T h i s i n d i c a t e d t h a t the a n t i s e r u m was r e a c t i n g w i t h two components found i n the unbound f r a c t i o n s 82 Table V. IgG Content of P o o l e d F r a c t i o n s O b t a i n e d From A Tandem MCIC column system with 150 mL s u p e r n a t a n t A p p l i c a t i o n F r a c t i o n s P r o t e i n Cone. mg/mL IgG Cone. mg/mL IgG P u r i t y IgG Recovery Unbound 2.87 ( f i r s t column) Unbound 0.371 (second column) pH 5.0 2.9 4 I m i d . l 4.87 ( f r a c t i o n 50) lmi d . 2 5.96 ( f r a c t i o n 51-52) l m i d . 3 5.03 ( f r a c t i o n s 53-57) 1.25 0.445 0.274 0.629 1.06 2.32 2.82 43.6 >100 21.4 21.7 38.9 56.0 58.9 26.2 7.53 1.62 6.86 21.1 EDTA 0.341 0.441 >100 1.32 83 c o l l e c t e d from the second column. Comparison of the IgG c a l c u l a t e d l e v e l s w i t h the t o t a l p r o t e i n showed an e s t i m a t e d p u r i t y of IgG of g r e a t e r than one hundred p e r c e n t . The i m p l i c a t i o n s of a double r i n g i n i m m u n o d i f f u s i o n have been d i s c u s s e d i n the DEAE-Sephacel r e s u l t s s e c t i o n . Here i t appeared t h a t the second component, when p r e c i p i t a t e d w i t h the a n t i s e r u m , had h i g h e r m o b i l i t y t h a n the c h i c k e n IgG s t a n d a r d used. Thus, c a l c u l a t e d IgG l e v e l s were h i g h e r t h a n e x p e c t e d . I t a l s o was c l e a r t h a t t h i s p r o t e i n was c o n c e n t r a t e d i n the unbound p o r t i o n s of the MCIC column. Whether t h i s o c c u r r a n c e was due t o the presence of IgG s u b p o p u l a t i o n s , or s i m p l y a c o n t a m i n a t i n g p r o t e i n t o which the a n t i s e r u m was s p e c i f i c , was not d e t e r m i n e d . I t was a l s o i n t e r e s t i n g t o observe c l o s e l y the e l u t e d f r a c t i o n s c o l l e c t e d from the second column. S p e c i f i c a c t i v i t y toward ( 3 - l a c t o g l o b u l i n was d e t e c t e d i n the pH 5.0, i m i d a z o l e , and EDTA f r a c t i o n s ( F i g u r e 20). While reduced SDS-PAGE e l e c t r o p h o r e s i s i n d i c a t e d the presence of a p r o t e i n w i t h a m o l e c u l a r weight of the heavy c h a i n of IgG i n a l l the e l u t e d f r a c t i o n s ( F i g u r e 2 1 ) , n a t i v e PAGE ( F i g u r e 22) i n d i c a t e d p r o t e i n s of v a r y i n g charge d e n s i t i e s i n the r e g i o n e x p e c t e d t o be IgG. Whether the e l u t i o n procedure caused t h e s e changes i n IgG, or the IgG e l u t e d i n the d i f f e r e n t f r a c t i o n s because of the e x i s t e n c e of t h e s e d i f f e r e n c e s , was not known. In any c a s e , the use of a tandem column was not as s u c c e s s f u l as e x p e c t e d . T h i s appeared t o be due t o the heterogenous n a t u r e of c h i c k e n IgG, i n which immunoglobulins 84 F i g u r e 20. E l u t i o n p r o f i l e of p r o t e i n s on the second column i n a tandem MCIC system. E q u i l i b r a t i n g and washing b u f f e r was pH 7.0 phosphate. A p p l i c a t i o n of the unbound from the f i r s t column ( S ) , wash (W), e l u t i o n w i t h pH 5.0 t r i s - a c e t a t e b u f f e r ( E l ) , and e l u t i o n w i t h i m i d a z o l e (E2) a r e i n d i c a t e d . The f l o w r a t e was 0.4 mL/min. 85 - a - l i v e t i n - IgG H.C. - fl-livetin 1 2 3 4 5 6 7 8 F i g u r e 21. SDS-PAGE of f r a c t i o n s c o l l e c t e d from the second column of a tandem MCIC system. Lane 1, unbound from the f i r s t column which was a p p l i e d t o the second; l a n e 2, unbound; lan e 3, pH 5.0 e l u t e d f r a c t i o n ; l a n e s 4 t o 7, I m i d a z o l e e l u t e d f r a c t i o n s : l a n e 4, f r a c t i o n 49, l a n e 5, f r a c t i o n 50, lane 6, f r a c t i o n s 51 t o 52, lane 7, f r a c t i o n s 53 t o 57; l a n e 8, EDTA e l u t e d f r a c t i o n . 8 6 - IgG 5 4 3 2 1 F i g u r e 22. N a t i v e PAGE of f r a c t i o n s c o l l e c t e d from the second column of a tandem MCIC system. Lane 1, s t a n d a r d IgG; l a n e 2, unbound from f i r s t column; l a n e s 3 t o 5, i m i d a z o l e e l u t i o n : l a n e 3, f r a c t i o n 50, l a n e 4, f r a c t i o n s 51 and 52, l a n e 5, f r a c t i o n s 53 t o 57. 87 were e l u t e d a t d i f f e r e n t t i m e s depending on the e l u t i o n b u f f e r . I n MCIC e l u t i o n , v a r y i n g h i s t i d i n e c o n t e n t s of s u b p o p u l a t i o n s may e x p l a i n the heterogeneous e l u t i o n . Wang e t a l (1986) i s o l a t e d a "mannose-binding p r o t e i n " from egg y o l k which was l a t e r i d e n t i f i e d as IgG. A comparison of the amino a c i d c o m p o s i t i o n of the p r o t e i n (which t h e y i s o l a t e d by a f f i n i t y chromatography u s i n g a mannose column) w i t h a y o l k IgG s t a n d a r d (method of p u r i f i c a t i o n not g i v e n ) showed s i m i l a r l e v e l s i n most of the amino a c i d s examined. However, the h i s t i d i n e c o n t e n t of the mannose b i n d i n g p r o t e i n was d e t e r m i n e d t o be 5.8 h i s t i d i n e r e s i d u e s per 100 amino a c i d r e s i d u e s . T h i s was c o n s i d e r a b l y h i g h e r than the y o l k IgG s t a n d a r d , a t 1.6 r e s i d u e s per 100 r e s i d u e s . S i n c e d i f f e r e n t l e v e l s of h i s t i d i n e are e xpected t o a f f e c t p r o t e i n b i n d i n g t o a m e t a l l o a d e d MCIC column, the h e t e r o g e n i c e l u t i o n of IgG may be due t o v a r i a t i o n s i n h i s t i d i n e c o n t e n t . The p o s s i b l e d i f f e r e n c e s i n h i s t i d i n e c o n t e n t due t o s p e c i f i c i t y f o r d i f f e r e n t a n t i g e n s , or t o the presence of d i f f e r e n t s u b p o p u l a t i o n s , may e x p l a i n the h e t e r o g e n e i t y o b s e r v e d . I n o r d e r t o determine whether the h e t e r o g e n i c e l u t i o n p r o f i l e might be due t o i n t e r a c t i o n s of the p r o t e i n s w i t h the p o r t i o n of the MCIC column which was not l o a d e d w i t h copper, an MCIC column was p r e p a r e d as b e f o r e , but w i t h o u t copper l o a d i n g . The e l u t e d f r a c t i o n s were th e n c o l l e c t e d and m o n i t o r e d f o r absorbance a t 280 nm (A280). The r e s u l t i n g 88 e l u t i o n p a t t e r n i s shown i n F i g u r e 23. I t can be seen t h a t pH 5.0 r e s u l t e d i n no e l u t i o n of p r o t e i n s based on A280. A peak was o b t a i n e d , however, by p a s s i n g i m i d a z o l e t h rough the column, w i t h a f u r t h e r s m a l l peak o c c u r r i n g w i t h EDTA e l u t i o n . In o r d e r t o t e s t whether the A280 was g i v i n g apparent p r o t e i n peaks due t o i n t e r f e r e n c e of o t h e r s u b s t a n c e s , the p o o l e d I m i d a z o l e and p o o l e d EDTA f r a c t i o n s were d i a l y z e d , l y o p h i l i z e d , and r e c o n s t i t u t e d t o t e n t i m e s c o n c e n t r a t i o n f o r f u r t h e r a n a l y s i s . SDS-Page was then conducted on the samples. Whi l e s t a i n i n g w i t h Coomassie Blue showed no p r o t e i n bands, s i l v e r s t a i n i n g d i d d e t e c t a s m a l l amount of p r o t e i n ( F i g u r e 2 4 ) . Thus, i t appeared t h a t a v e r y s m a l l amount of p r o t e i n was e l u t e d w i t h the i m i d a z o l e and EDTA s o l u t i o n s f o l l o w i n g sample a p p l i c a t i o n t o the unloaded MCIC column. One p o s s i b l e e x p l a n a t i o n f o r t h i s may be t h a t , a l t h o u g h d e i o n i z e d d i s t i l l e d water was used i n a l l sample and b u f f e r p r e p a r a t i o n s , sample a p p l i c a t i o n might have r e s u l t e d i n " l o a d i n g " of the column w i t h i o n s from the egg y o l k . These i n t u r n may i n t e r a c t w i t h p r o t e i n s , thus g i v i n g a m e t a l - l o a d e d MCIC column. T h i s may c o n t r i b u t e t o the h e t e r o g e n i c e l u t i o n of IgG m o l e c u l e s , s i n c e p r o t e i n s w i l l e x h i b i t v a r y i n g a f f i n i t i e s f o r d i f f e r e n t m e t a l s . However, i n t h i s case o n l y a s m a l l amount of p r o t e i n was found t o b i n d when no copper was l o a d e d , and thus i s not l i k e l y a s i g n i f i c a n t f a c t o r . The n e c e s s i t y of the use of d e i o n i z e d d i s t i l l e d water f o r sample and b u f f e r p r e p a r a t i o n s i s emphasized, however. 89 F i g u r e 23. E l u t i o n p r o f i l e of egg y o l k s u p e r n a t a n t a p p l i e d t o a MCIC column not l o a d e d w i t h c opper. Wash w i t h e q u i l i b r a t i n g b u f f e r (W), e l u t i o n w i t h pH 5.0 b u f f e r ( E l ) , e l u t i o n w i t h i m i d a z o l e ( E 2 ) , and e l u t i o n w i t h EDTA (E3) a r e i n d i c a t e d . 90 w • -Hi H I r — — 4 3 2 1 F i g u r e 24. Non-reduced SDS-PAGE o£ samples c o l l e c t e d from an MCIC column not lo a d e d w i t h copper. Lane 1, I m i d a z o l e e l u t e d f r a c t i o n ; l a n e 2, i m i d a z o l e e l u t e d f r a c t i o n ; l a n e 3, EDTA e l u t e d f r a c t i o n ; l a n e 4, non-reduced s t a n d a r d IgG. The g e l was 10 t o 15% a c r y l a m i d e g r a d i e n t , and was s i l v e r s t a i n e d . 91 F i g u e r o a e t a l (1986) examined a d s o r p t i o n of p r o t e i n s t o a MCIC column p r e p a r e d u s i n g s i l i c a as a s u p p o r t . They c o n c l u d e d t h a t h y d r o p h o b i c i n t e r a c t i o n c o u l d occur as a r e i n f o r c e m e n t of the b i n d i n g of the p r o t e i n s t o the m e t a l -l o a d e d column. T h i s was a l s o observed by R a s s i and Horvath (1986), who found t h a t a d e c r e a s i n g s a l t g r a d i e n t r e s u l t e d i n e l u t i o n of p r o t e i n s based on h y d r o p h o b i c i n t e r a c t i o n w i t h the s i l i c a - b a s e d s u p p o r t . S i n c e the pH 5.0 b u f f e r used i n the c u r r e n t experiment c o n t a i n e d 0.5 M sodium c h l o r i d e , w h i l e the i m i d a z o l e d i d n o t , the de c r e a s e i n i o n i c s t r e n g t h may p l a y a r o l e i n the observed p r o t e i n e l u t i o n . I t i s g e n e r a l l y agreed t h a t the f u l l mechanism by which c o p p e r - l o a d e d MCIC columns o p e r a t e t o s e p a r a t e p r o t e i n s i s not w e l l u n d e r s t o o d ( R a s s i and H o r v a t h , 1986). However, the r e t e n t i o n of p r o t e i n s due t o i n t e r a c t i o n w i t h the s u p p o r t m a t r i x used i n t h i s experiment (IDA-Sepharose) was v e r y low, w i t h p r o t e i n s o n l y d e t e c t a b l e by s i l v e r s t a i n i n g of the e l e c t r o p h o r e s i s g e l . These f i n d i n g s were th e n a p p l i e d t o the p u r i f i c a t i o n of c h i c k e n IgG u s i n g eggs c o l l e c t e d from c h i c k e n s immunized w i t h 1 3 - N - a c e t y l g l u c o s a m i n i d a s e (NAGase). V a r i o u s sample s i z e s were a p p l i e d t o a c o p p e r - l o a d e d MCIC column, and the absorbance a t 280 nm of the e l u t e d f r a c t i o n s m o n i t o r e d , as w e l l a s , the s p e c i f i c a c t i v i t y toward NAGase. E l u t i o n p r o f i l e s of a 50 mL sample s i z e a p p l i e d t o a c o p p e r - l o a d e d column showed no s p e c i f i c a n t i b o d y a c t i v i t y i n the unbound or the pH 5.0 f r a c t i o n s ( F i g u r e 2 5 ) . 92 F i g u r e 25. The e l u t i o n p r o f i l e of 50 mL egg y o l k s u p e r n a t a n t a p p l i e d t o a MCIC column. ELISA r e s u l t s i n d i c a t e s p e c i f i c a c t i v i t y toward NAGase In which samples were d i l u t e d 1/200. Flow r a t e was 0.4 mL/min. Sample a p p l i c a t i o n ( S ) , wash (W), e l u t i o n w i t h pH 5.0 b u f f e r ( E l ) and e l u t i o n w i t h i m i d a z o l e (E2) a r e i n d i c a t e d . 93 With an i n c r e a s e i n sample s i z e t o 100 mL, a n t i b o d i e s of s p e c i f i c a c t i v i t y were d e t e c t e d i n the unbound f r a c t i o n s ( F i g u r e 26). A n t i b o d i e s were f u r t h e r e l u t e d w i t h the pH 5.0 e l u t i o n b u f f e r , f o l l o w e d by i m i d a z o l e e l u t i o n . By i n c r e a s i n g the sample s i z e t o 200 mL, more s p e c i f i c a n t i b o d i e s were d e t e c t e d i n the unbound f r a c t i o n s ( F i g u r e 2 7 ) . I t was i n t e r e s t i n g t o note t h a t w h i l e the pH 5.0 e l u t i o n s t r i p p e d more p r o t e i n s from the column, ELISA i n d i c a t e d e x t r e m e l y low l e v e l s of s p e c i f i c a n t i b o d i e s , w h i l e w i t h the 100 mL sample a p p l i c a t i o n , h i g h s p e c i f i c a c t i v i t y was d e t e c t e d i n the pH 5.0 f r a c t i o n s . T h i s p a t t e r n s u g g e s t s t h a t , w i t h the 200 mL sample a p p l i e d t o the column, the a n t i b o d i e s t h a t c o u l d be e l u t e d w i t h the pH 5.0 b u f f e r had been p r e f e r e n t i a l l y d i s p l a c e d from the column by o t h e r p r o t e i n s , and no a n t i b o d i e s c o n t a i n i n g s p e c i f i c a c t i v i t y f o r NAGase were then d e t e c t e d . A n t i b o d i e s e l u t e d o n l y by i m i d a z o l e , however, were s t i l l d e t e c t a b l e by ELISA i n the l a t t e r p o r t i o n of the i m i d a z o l e e l u t i o n p a t t e r n ( F i g u r e 2 7 ) . These a n t i b o d i e s had not been d i s p l a c e d by the c o n t a m i n a t i n g p r o t e i n s and remained adsorbed t o the column u n t i l the i m i d a z o l e e l u t i o n . Upon e x a m i n a t i o n of the p a t t e r n of p r o t e i n and s p e c i f i c a n t i b o d y e l u t i o n i n the unbound p o r t i o n s of the 100 mL and 200 mL sample s i z e s , i t became apparent t h a t the e l u t e d p r o t e i n s d i f f e r e d depending on the s a t u r a t i o n p o i n t of the column. The f i r s t 50 mL a p p l i e d t o the column appeared t o r e s u l t i n t o t a l b i n d i n g of s p e c i f i c a n t i b o d i e s . I n c r e a s i n g the sample s i z e above 50mL r e s u l t e d i n an o v e r l o a d and a p o r t i o n 94 F i g u r e 26. The e l u t i o n p r o f i l e of 100 mL egg y o l k s u p e r n a t a n t a p p l i e d t o a MCIC column. ELISA r e s u l t s i n d i c a t e s p e c i f i c a c t i v i t y toward NAGase i n which samples were d i l u t e d 1/200. Flow r a t e was 0.4 mL/min. Sample a p p l i c a t i o n ( S ) , wash (W), e l u t i o n w i t h pH 5.0 b u f f e r ( E l ) , and w i t h I m i d a z o l e are i n d i c a t e d . 95 F i g u r e 27. The e l u t i o n p r o f i l e of 200 mL egg y o l k s u p e r n a t a n t a p p l i e d t o a MCIC column. ELISA r e s u l t s I n d i c a t e s p e c i f i c a c t i v i t y toward NAGase i n which samples were d i l u t e d 1/200. Flow r a t e was 0.4 mL/min. Sample a p p l i c a t i o n ( S ) , wash (W), e l u t i o n w i t h pH 5.0 b u f f e r ( E l ) , and w i t h I m i d a z o l e are i n d i c a t e d . 96 of the s p e c i f i c a n t i b o d i e s d i d not b i n d t o the column. The t o t a l p r o t e i n c o n t e n t of the unbound f r a c t i o n s became c o n s t a n t a t a p p r o x i m a t e l y 120 mL (based on A280), i n d i c a t i n g column s a t u r a t i o n . The unbound f r a c t i o n s c o l l e c t e d from the 200 mL sample a p p l i c a t i o n were p o o l e d i n t o t h r e e groups f o r f u r t h e r a n a l y s i s : f r a c t i o n s 3 t o 8 (which e x c l u d e d the v e r y f i r s t 20 mL e l u t e d which had low A280 and no s p e c i f i c a c t i v i t y ) were p o o l e d ; f r a c t i o n s 9 t o 11, showing peak l e v e l s of s p e c i f i c a n t i b o d y a c t i v i t y ; and f r a c t i o n s 12 t o 22, which c o n t a i n e d the peak l e v e l s of p r o t e i n as w e l l as the f i r s t p o r t i o n of the wash f r a c t i o n s . The l e v e l s of IgG p u r i t y and r e c o v e r y i n each of the f r a c t i o n s was t h e n d e t e r m i n e d (Table V I ) . The p o o l e d m i d d l e p o r t i o n c o n t a i n e d the h i g h e s t p u r i t y of IgG o b t a i n e d , a t 74.8%. The p u r i t y of the p o o l e d f r a c t i o n s appeared t o r e f l e c t the v a r i o u s b i n d i n g a f f i n i t i e s of the egg y o l k s u p e r n a t a n t p r o t e i n s . Other p r o t e i n s w i t h s t r o n g e r a f f i n i t y f o r the column d i s p l a c e d IgG, r e s u l t i n g i n i n c r e a s e d p u r i f i c a t i o n of IgG i n the unbound. Upon column s a t u r a t i o n , however, the more s t r o n g l y bound p r o t e i n s were then e i t h e r d i s p l a c e d or passed t h r o u g h w i t h f u r t h e r sample a p p l i c a t i o n , so t h a t IgG p u r i t y was t h e n a g a i n reduced. The a c t u a l amount of IgG o b t a i n e d a t 75% p u r i t y was 105 mg from 200 mL egg y o l k s u p e r n a t a n t . ELISA of t h e s e e l u t e d f r a c t i o n s i n d i c a t e d a r e l a t i v e l y h i g h l e v e l of s p e c i f i c a n t i b o d y a c t i v i t y . Thus, the method of s e l e c t i v e p o o l i n g of 97 Table V I . IgG Content of D i f f e r e n t P o o l e d Unbound F r a c t i o n s O b t a i n e d When 200 mL Sup e r n a t a n t was A p p l i e d t o a MCIC Column IgG IgG P r o t e i n Cone. IgG Cone. P u r i t y Recovery F r a c t i o n s mg/mL mg/mL % % 2 t o 8 0.844 0.433 51.3 8.77 9 t o 11 4.16 3.11 74.8 27.1 12 t o 22 5.19 1.65 31.8 38.0 98 the unbound f r a c t i o n s o b t a i n e d from o v e r l o a d i n g the copper-l o a d e d MCIC column gave q u i t e s a t i s f a c t o r y r e s u l t s . 99 C. A COMPARISON OF THE USE OF DEAE-SEPHACEL CHROMATOGRAPHY WITH COPPER-LOADED METAL CHELATE INTERACTION CHROMATOGRAPHY FOR EGG YOLK IMMUNOGLOBULIN SEPARATION With the DEAE-Sephacel chromatography, a maximum IgG p u r i t y of 60% was o b t a i n e d , w i t h 16 mg of IgG b e i n g i s o l a t e d from 50 mL s u p e r n a t a n t . By combining t h i s f r a c t i o n w i t h a f r a c t i o n of 40% p u r i t y , a combined p u r i t y of 47% was o b t a i n e d , and the amount of IgG p u r i f i e d was 37 mg. In c o m p a r i s o n , u s i n g m e t a l c h e l a t e i n t e r a c t i o n chromatography, a p u r i t y of 75% c o u l d be o b t a i n e d by s e l e c t i v e l y p o o l i n g the middl e p o r t i o n s of the unbound f r a c t i o n s . T h i s r e p r e s e n t e d 104 mg IgG from 200 mL egg y o l k s u p e r n a t a n t . By p o o l i n g t h i s w i t h the i n i t i a l unbound f r a c t i o n s , 133 mg of IgG w i t h a p u r i t y of 68% was o b t a i n e d . The 10 mL me t a l c h e l a t e i n t e r a c t i o n chromatography column was a b l e t o p u r i f y more IgG t o a h i g h e r p u r i t y t h a n the DEAE-Sep h a c e l (133 mg a t 68% p u r i t y v s . 16 mg a t 60% p u r i t y ) . S i n c e the m etal c h e l a t e i n t e r a c t i o n chromatography i s e x p e n s i v e , a comparison of the two methods was at t e m p t e d . A rough e s t i m a t e of the c o s t s was made based on the method of g e l p r e p a r a t i o n method used i n these e x p e r i m e n t s (Table V I I ) . C o n s i d e r i n g the c o s t of the g e l , i t was e s t i m a t e d t h a t , under the c o n d i t i o n s d e s c r i b e d i n t h i s e x p e r i m e n t , one m i l l i g r a m of IgG c o u l d be p u r i f i e d a t the c o s t of $0,031 u s i n g MCIC; w i t h DEAE-Sephacel, the c o s t of one m i l l i g r a m was h i g h e r a t $0,046. 100 T a b l e V I I . A Comparison o£ the Cost of one l i t r e of MCIC g e l and one l i t r e of DEAE-Sephacel r e s i n R e s i n M a t e r i a l s R e q u i r e d S u b t o t a l T o t a l Cost ($) ($ ) * DEAE-Sephacel Sep h a c e l g e l 169.00 169.00 MCIC Sepharose-6B 218.00 1,4 b u t a n e d i o l - 49.45 d i g l y c i d y l e t h e r 0.6 M sodium h y d r o x i d e 0.30 Sodium b o r o h y d r i d e 0.32 Sodium c a r b o n a t e 3.66 Disodium 80.56 i m i n o d i a c e t a t e Sodium b o r o h y d r i d e 0.24 5 hours p r e p a r a t i o n 75.00 time @ $15/h 485.53 *Cost based on Sigma 1988 c a t a l o g u e p r i c e s , i n U.S. d o l l a r s . 101 While the t o t a l c o s t o£ b u f f e r p r e p a r a t i o n s was not c a l c u l a t e d , t h e y appeared t o be r o u g h l y e q u a l f o r the two methods. With the r e q u i r e m e n t f o r maximum throughput f o r i n d u s t r i a l a p p l i c a t i o n s , MCIC o f f e r s a f u r t h e r advantage. S i n c e s i m p l e c o l l e c t i o n of the unbound appears t o be the method of c h o i c e f o r IgG s e p a r a t i o n u s i n g MCIC, time r e q u i r e m e n t s and l a b o u r c o s t s are s l i g h t l y r educed. The number of s t e p s i n v o l v e d u s i n g MCIC and DEAE-Sephacel was the same; however, w i t h MCIC, two of the s t e p s s i m p l y r e q u i r e washes w i t h water, w h i l e a l l of the DEAE-Sephacel s t e p s r e q u i r e d b u f f e r p r e p a r a t i o n . A comparison of approximate time r e q u i r e d i s g i v e n w i t h the method f l o w c h a r t s (Table V I I I ) . F i n a l l y , i n both the i o n exchange and m e t a l c h e l a t e i n t e r a c t i o n e x p e r i m e n t s , t e n m i l l i l i t r e column s i z e s were used. However, i n the case of the MCIC column, o n l y s i x m i l l i l i t r e s of the t e n was l o a d e d w i t h copper, and, t h e r e f o r e , i n r e a l i t y , o n l y 6 mL was a c t u a l l y used f o r the I g s e p a r a t i o n . T h i s p r a c t i c e of not l o a d i n g the bottom p o r t i o n of the column i s t a k e n i n o r d e r t o p r e v e n t l e a k a g e of i o n s from the column. Under the e x p e r i m e n t a l c o n d i t i o n s chosen, however, leakage of copper was never v i s u a l l y a p p a r e n t . I t seems t h a t i t may not be n e c e s s a r y f o r such a l a r g e amount of the column t o be u n s a t u r a t e d i n the case of egg y o l k s u p e r n a t a n t s e p a r a t i o n . The amount of i o n leakage would have t o be d e t e r m i n e d i n o r d e r t o f i n d out i f lowered volumes of u n s a t u r a t e d MCIC g e l c o u l d 102 T a b l e V I I I . A Comparison of the Time Requirements f o r IgG S e p a r a t i o n From Egg Yol k on a 10 mL MCIC or DEAE-Sephacel Column Under C o n d i t i o n s Developed i n the Study MCIC Time DEAE Time ( f l o w rate:0.4mL/min) (min) ( f l o w rate:0.8mL/min) (min) Copper L o a d i n g 7 Wash w i t h Water 50 E q u i l i b r a t e w i t h phosphate b u f f e r 125 A p p l y 150 mL sample 375 Wash w i t h EDTA 25 Wash w i t h water 50 E q u i l i b r a t e w i t h 63 phosphate b u f f e r A p p l y sample 63 Wash w i t h phosphate 40 E l u t i o n w i t h f i r s t 206 g r a d i e n t E l u t i o n w i t h second 250 g r a d i e n t Wash w i t h 0.6 M phos. 40 T o t a l 628 662 103 be used. I f t h i s i s s o , however, MCIC becomes even more c o s t e f f e c t i v e t h a n the DEAE-Sephacel. In any c a s e , under the c o n d i t i o n s examined, the MCIC method of IgG s e p a r a t i o n from egg y o l k was more e f f e c t i v e t h a n the DEAE-Sephacel method. A t t e m p t i n g t o compare th e s e r e s u l t s w i t h o t h e r p u b l i s h e d methods was d i f f i c u l t . H a s s l e t a l (1987) compared t h r e e methods, and the b e s t gave a " h i g h " p u r i t y (which was not d e f i n e d ) , w i t h a y i e l d of 40 mg IgG per egg. In the c u r r e n t e x p e r i m e n t s , a p p r o x i m a t e l y 35 mg IgG was o b t a i n e d per egg. The advantages t h a t a c h r o m a t o g r a p h i c method may have over the numerous p r e c i p i t a t i o n s t e p s r e q u i r e d i n the o t h e r methods cannot be de t e r m i n e d w i t h o u t a thorough s t u d y of c o s t and time r e q u i r e m e n t s . 104 PART I I I . APPLICATION OF ISOLATED EGG YOLK IMMUNOGLOBULINS: THE DETECTION OF (3-N-ACETYLGLUCOSAMINIDASE USING ELISA V a r i o u s c o n d i t i o n s f o r an ELISA method f o r a n a l y s i s of 6-N - a c e t y l g l u c o s a m i n i d a s e (NAGase) were examined u s i n g s t a n d a r d s o l u t i o n s of bov i n e k i d n e y NAGase. Three s e p a r a t e ELISA t e s t s were r u n on t h r e e d i f f e r e n t days u s i n g s t a n d a r d NAGase c o n c e n t r a t i o n s r a n g i n g between 1 ug/mL and 10 ug/mL. A s t a t i s t i c a l l y s i g n i f i c a n t (ot=0.01) l i n e a r r e l a t i o n s h i p was o b t a i n e d between the absorbance a t 405 nm and the l o g of the s t a n d a r d c o n c e n t r a t i o n s on each day. A t y p i c a l s t a n d a r d c u r v e i s i l l u s t r a t e d ( F i g u r e 2 8 ) . A p p l i c a t i o n of t h i s i n f o r m a t i o n was then extended t o f i s h meat from which p r e s s e d j u i c e (PJ) or e x t r a c t (Ex) was o b t a i n e d . The c o n c e n t r a t i o n of 6 - N - a c e t y l g l u c o s a m i n i d a s e i n each sample was c a l c u l a t e d based on the l i n e a r r e g r e s s i o n e q u a t i o n o b t a i n e d from the s t a n d a r d c u r v e . Three d i l u t i o n s of the f i s h samples were examined: no d i l u t i o n , h a l f d i l u t i o n and one q u a r t e r d i l u t i o n . The c a l c u l a t e d NAGase c o n c e n t r a t i o n of the samples was found t o be v e r y d i f f e r e n t depending on the sample d i l u t i o n used (Table I X ) . C o n s i s t e n t l y , use of the u n d i l u t e d f i s h samples i n the ELISA method r e s u l t e d i n lower p r e d i c t e d NAGase c o n c e n t r a t i o n s than the v a l u e s c a l c u l a t e d f o r the more d i l u t e d samples. T h i s p r o b a b l y I n d i c a t e s t h a t o t h e r s u b s t a n c e s b e i n g c o a t e d t o the p l a t e i n t e r f e r e w i t h the NAGase b i n d i n g . D i l u t i o n of f i s h samples would d i l u t e not o n l y the NAGase, but a l s o the i n t e r f e r i n g s u b s t a n c e s , and thus a l l o w e d f o r b i n d i n g of NAGase t o the p l a t e . F i g u r e 28. A t y p i c a l ( 3 - N - a c e t y l g l u c o s a m i n i d a s e (NAGase) s t a n d a r d curve o b t a i n e d by the ELISA method. L i n e a r r e g r e s s i o n was performed on the d a t a . 106 Table IX. L e v e l s o£ 6 - N - A c e t y l g l u c o s a m i n l d a s e per gram o£ Chinook Salmon F l e s h as Determined by ELISA and the S p e c t r o p h o t o m e t r i c Method a ELISA, Ug/g Spec. Method, 5 , c A c t . U n i t s / g Sample Sample D i l u t i o n 1:0 1:1 1:3 A F r e s h P . J . 1.27 C 1.25 E 1.28 G 1.23 B F r o z e n P . J . 0.17 D 0.12 F 0.09 H 0.10 A F r e s h E x t r a c t 5.30 C 9.44 E 10.9 G 8.96 B F r o z e n E x t r a c t 0.73 •D 0.64 F 0.64 H 0.64 6.54 4.79 0.9055 4.16 6.03 1.7090 3.57 6.73 1.1210 3.47 5.43 0.9420 0.99 2.27 3.2032 0.33 1.06 3.6632 0.41 1.16 3.2912 0.35 0.99 2.8296 18.5 31.1 4.8785 19.4 31.8 5.2015 19.5 30.5 3.6905 16.4 29.1 3.6270 2.81 3.44 6.8848 1.76 2.48 4.2160 1.92 2.32 2.1040 1.84 2.40 2.9512 a Y o s h i o k a , 1988 b U n i t s per gram f i s h f l e s h , where one u n i t w i l l h y d r o l y z e 1.0 umole of p - n i t r o p h e n y l - N - a c e t y l - 6 - D - g l u c o s a m i n i d e t o p - n i t r o p h e n y l and N - a c e t y l - D - g l u c o s a m l n e per min a t pH 4.25 a t 250c c D i l u t i o n of sample t o c a r b o n a t e b u f f e r , pH 9.6 d Samples of the same l e t t e r r e p r e s e n t the same f i s h sample; p a i r e d l e t t e r s (eg. A and B, C and D, e t c . ) r e p r e s e n t d i f f e r e n t samples o b t a i n e d from the same f i s h 107 T h i s f i n d i n g t h a t more d i l u t e d samples g i v e s a h i g h e r ELISA r e a d i n g t h a n a l e s s d i l u t e d sample i s c o n s i s t e n t w i t h the f i n d i n g s of P a t t e r s o n and Jones (1985), who used a s i m i l a r ELISA method f o r the i d e n t i f i c a t i o n of s p e c i e s i n meat samples. They emphasized t h e r e f o r e t h a t the optimum d i l u t i o n had t o be a c h i e v e d e x p e r i m e n t a l l y i n o r d e r t o overcome t h i s problem. P o s s i b l y f u r t h e r p u r i f i c a t i o n of the f i s h samples p r i o r t o a p p l i c a t i o n t o the ELISA p l a t e would reduce the i n t e r f e r i n g s u b s t a n c e s and r e s u l t i n more a c c u r a t e d e t e r m i n a t i o n s of the NAGase i n the u n d i l u t e d samples. Comparing the ELISA r e s u l t s f o r f r e s h and f r o z e n salmon samples, the f r o z e n samples c o n s i s t e n t l y gave lower r e s u l t s t han the f r e s h (Table I X ) . However, r e s u l t s u s i n g the p-n i t r o p h e n y l s p e c t r o p h o t o m e t r l c method showed the f r o z e n samples t o c o n t a i n c o n s i d e r a b l y h i g h e r l e v e l s t h a n the f r e s h ( Y o s h i o k a , 1988). The r e s u l t s comparing the f r o z e n and f r e s h samples were o b t a i n e d on d i f f e r e n t days and on d i f f e r e n t ELISA p l a t e s , i n * o r d e r t o t e s t whether the low f r o z e n sample v a l u e s might be due t o p l a t e t o p l a t e v a r i a t i o n , i t was d e s i r a b l e t o p repare samples t o be t e s t e d on one p l a t e . S i n c e the immunogen used t o s t i m u l a t e a n t i b o d y p r o d u c t i o n was o b t a i n e d from bovine k i d n e y , and not from f i s h , the p o s s i b i l i t y e x i s t e d t h a t reduced s e n s i t i v i t y o c c u r r e d due t o low c r o s s - r e a c t i v i t y between the a n t i b o d i e s s p e c i f i c f o r b o v i n e k i d n e y NAGase and the f i s h NAGase. T h e r e f o r e , f r e s h b o v i n e muscle was o b t a i n e d from a r e t a i l s t o r e and d i v i d e d 108 i n t o two p o r t i o n s . One p o r t i o n was s t o r e d a t 4°C w h i l e the o t h e r was f r o z e n o v e r n i g h t . The next day, p r e s s j u i c e and e x t r a c t were p r e p a r e d i n a manner s i m i l a r t o the f i s h . I n t e r e s t i n g l y , the u n d i l u t e d samples i n the case of the bovine k i d n e y gave v e r y h i g h ELISA r e a d i n g s (Table X ) , w h i l e f u r t h e r d i l u t i o n gave q u i t e low r e s u l t s . S i m i l a r t r e n d s were noted i n d a t a o b t a i n e d u s i n g b o v i n e k i d n e y ( A p p e n d i x ) . O b v i o u s l y , o p t i m i z a t i o n of the d i l u t i o n of bovine samples a l s o Is n e c e s s a r y . Comparing the f r e s h and f r o z e n r e s u l t s , the p - n i t r o p h e n y l method i n d i c a t e d i n c r e a s e d l e v e l s of NAGase i n the p r e s s j u i c e due t o f r e e z i n g , and no change was d e t e c t e d i n the e x t r a c t a b l e NAGase ( Y o s h i o k a , 1988). A g a i n , ELISA r e s u l t s i n d i c a t e d the o p p o s i t e - lower NAGase l e v e l s were d e t e c t e d both i n the f r o z e n p r e s s j u i c e and e x t r a c t samples. While the a n t i g e n i c d e t e r m i n a n t s of NAGase have not y e t been d e s c r i b e d , s t u d i e s of myoglobin have shown t h a t the a n t i b o d y response depends on the t h r e e d i m e n s i o n a l s t r u c t u r e of the p r o t e i n ( A t a s s i , 1984). T h e r e f o r e , p a r t i a l d e n a t u r a t i o n of the n a t i v e NAGase s t r u c t u r e t h r o u g h f r e e z i n g might e x p l a i n the reduced r e c o g n i t i o n of the a n t i b o d i e s s p e c i f i c f o r NAGase used i n the ELISA system. While a comparison of NAGase l e v e l s i n f r e s h and f r o z e n samples may be c o m p l i c a t e d by t h i s , the use of an ELISA t e s t u t i l i z i n g c h i c k e n IgG appears t o be p r o m i s i n g i n the a r e of a n a l y t i c a l t e s t s . 109 Table X. L e v e l s of G - N - A c e t y l g l u c o s a m i n i d a s e In Bovine Muscle Samples as Determined by ELISA and the S p e c t r o p h o t o m e t r i c Method a ELISA, Ug/q Spec. Method, c A c t . U n i t s / g b Sample Sample D i l u t i o n 1:0 1:1 1:3 F r e s h P . J . F r o z e n P . J . F r o z e n P . J . / F r e s h P . J . F r e s h E x t r a c t F r o z e n E x t r a c t F r o z e n P . J . / F r e s h P . J . 64.0 3.35 35.2 2.92 0.550 0.872 201 14.0 95.4 14.4 0.471 1.03 3.57 1.2620 3.41 3.4135 0.955 2.7048 29.2 3.6056 34.8 3.9048 1.19 1.0830 Y o s h i o k a , 1988 b U n i t s per gram f i s h f l e s h , where one u n i t w i l l h y d r o l y z e 1.0 Umole of p - n i t r o p h e n y l - N - a c e t y l - 6 - D - g l u c o s a m i n i d e t o p - n i t r o p h e n y l and N - a c e t y l - D - g l u c o s a m l n e per min at pH 4.25 a t 25<>C c D i l u t i o n of sample t o c a r b o n a t e b u f f e r , pH 9.6 110 CONCLUSIONS The immunogenic response of i n j e c t i o n s of G-N-a c e t y l g l u c o s a m i n i d a s e t o t h r e e c h i c k e n s was c l e a r l y d emonstrated u s i n g an ELISA a s s a y f o r m o n i t o r i n g s p e c i f i c a n t i b o d y a c t i v i t y i n y o l k s . I n c r e a s e s i n s p e c i f i c a n t i b o d i e s o c c u r r e d i n the b i r d s immunized w i t h the enzyme, w h i l e no i n c r e a s e was d e t e c t e d i n a c o n t r o l b i r d r e c e i v i n g no i n j e c t i o n s . The h e t e r o g e n i c n a t u r e of c h i c k e n egg y o l k IgG was appa r e n t t h r o u g h o u t the s t u d i e s . I t seems p r o b a b l e t h a t s u b p o p u l a t i o n s of IgG h a v i n g m o l e c u l a r w e i g h t s a l l v e r y s i m i l a r , e x i s t i n the y o l k and r e a c t w i t h c o m m e r c i a l l y a v a i l a b l e a n t i s e r u m s p e c i f i c f o r the IgG c l a s s . D i f f e r e n c e s both i n i o n i c charge as w e l l as h i s t i d i n e c o n t e n t appear l i k e l y . The s p e c i f i c a n t i b o d i e s were i s o l a t e d from egg y o l k by two d i f f e r e n t methods: DEAE-Sephacel i o n exchange chromatography and metal c h e l a t e i n t e r a c t i o n chromatography. A comparison of the two methods c l e a r l y i n d i c a t e d the advantages of MCIC over DEAE-Sephacel. A maximum immunoglobulin p u r i t y of 75% was o b t a i n e d by the MCIC method. The M C I C - i s o l a t e d c h i c k e n immunoglobulins c o n t a i n i n g a n t i b o d i e s s p e c i f i c f o r 6 - N - a c e t y l g l u c o s a m i n l d a s e were s u c c e s s f u l l y used i n an ELISA f o r d e t e c t i o n of the enzyme i n a s t a n d a r d s o l u t i o n . A p p l i c a t i o n of the t e c h n i q u e f o r the q u a n t i t a t i o n of the enzyme i n meat samples appears p r o m i s i n g . 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