Analysis of antigenic substances corresponding to sperm immobilizing monoclonal antibodies

P 11 ANALYSIS OF ANTIGENIC SUBSTANCES CORRESPONDING TO SPERM IMMOBILIZING MONOCLONAL ANTIBODIES

K. KAMEDA, Y. TSUJI, K. KOYAMA AND S. ISOJIMA, Dept. of OB/GYN, Hyogo Medical College, Nishinomiya 663, Japan S e v e r a l sperm i m m o b i l i z i n g monoclonal a n t i b o d i e s (Mab) 2C6, 2E5 and 2B6 were s u c c e s s f u l l y p r o d u c e d i n our l a b o r a t o r y from hybridomas which were e s t a b l i s h e d by f u s i n g mouse myeloma c e l l s and s p l e e n c e l l s o f immunized mice w i t h human sperm or s e m i n a l p l a s m a . These Mabs r e c o g n i z e d c l o s e r e l a t e d a n t i g e n e p i t o p e s o f sperm, b e c a u s e b i n d i n g s o f t h e s e Mabs t o human sperm were p a r t i a l l y i n h i b i t e d each o t h e r . A n t i g e n i c components e x t r a c t e d from human sperm were a p p l i e d t o SOSPAGE and a n a l y z e d by Western b l o t t i n g . A n t i g e n i c components were d e t e c t e d i n 1S-2SKd m o l e c u l e s on n i t r o c e l l u r o s e membrane u n d e r r e d u c i n g e i t h e r n o n - r e d u c i n g c o n d i t i o n s and no d i f f e r e n t s t a i n i n g p a t t e r n was r e c o g n i z e d when t h e s e t h r e e d i f f e r e n t Mabs were u s e d . Chemical p r o p e r t i e s o f a n t i g e n s c o r r e s p o n d i n g t o t h e s e Mabs were s t u d i e d , and i t was found t h a t t h e r e was no d e t e c t a b l e p r o t e i n s t a i n i n g band a t t h e a n t i g e n i c p o r t i o n p r o v e d by i m m u n o s t a i n i n g o f Western b l o t t i n g . P r o t e a s e s , t h e r m o l y s i n , t r y p s i n and V8 f a i l e d t o change immunogenicity o f a n t i g e n s c o r r e s p o n d i n g t o t h e s e Mabs, but d e g l y c o s i l a t i o n t r e a t m e n t s as NaI04 or TFMS c o m p l e t e l y d e s t r o y e d t h e a n t i g e n s . T h e r e f o r e , i t i s assumed t h a t t h e a n t i g e n i c components might be h i g h l y g l y c o s i l a t e d and have e x t r e m e l y a smal~ amount o f p e p t i d e c h a i n o r n o n - p e p t i d e s u b s t a n c e . sperm imanobilizing a n t i b o d i e s ;

c a r b o h y d r a t e a n t i g e n ; monoclonal a n t i b o d i e s

P 12 PUBIFICATION OF RUMINANT SPERM MEMBRANE AUTOANTIGENS FOR INDUCING INFERTILITY IN MALES

S.KINGER, S.P. AHUJA, and P.P.GUPTA, Department of Biochemistry, Punjab Agric.University, Ludhiana, India Immunization of buffalo bull, cow bull and goat buck with their own spermatozoa induced sperm autoantibodies (AAb) highly specific to their sperm membrane (SM) glycoproteins (GP). These AAb were purified by FPLC and could pick up GP/peptide (PP) SM autoantigens (AAg) as demonstrated by FPLC and PAGE. Very high mol.wt. GP were AAg in buffalo bull, cow bull and goat buck. In the latter two species low mol.wt. GP were also AAg. Experimental autoimmunization of individuals of the 3 species with their own sperms produced complete aspermatogenesis. A comparison of the immunoprecipitates of AAg with the SM detergent extracts showed selectiv removal of AAg by AAb. The FPLC, PAGE patterns of deoxycholate (BOC) and sodium dodecyl sulfate (SOS) extracts showed that the PAGE mobilities (for mol.v~c, determination) of the ionic detergent extracted AAg were not comparable with those by FPLC. Extraction of the ruminant SM AAg with detergents selectively extracted the AAg and also brought about detachment of tails from the heads. Sperm autoentigens purificetion

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