144
mmmmdogr lesda),, A ugu.t! 1981
Studies with monoclonal antibodies to complement components M o n o c l o n a l a n t i b o d i e s are a newly available tool for dissecting t h e c o m p l e x i n t e r a c t i o n s t h a t go to m a k e u p t h e c o m p l e m e n t p a t h w a y s . C e n t r a l to all t h e s e p a t h w a y s is t h e third c o m p o n e n t of c n m p l e m e n t , C3, a n d it is to C3 t h a t o u r initial m o n o c l o n a l a n t i b o d i e s were r a i s e d I, a l t h o u g h m o n o c l o n a l a n t i b o d i e s to factor B have also b e e n p r e p a r e d a n d are b e i n g a n a l y s e d ( R u d d y , S. a n d L a c h m a n n , P . J . ) .
Specificity of anti-C3 T h r e e distinct a n t i - C 3 m o n o c l o n a l s h a v e b e e n raised a n d their r e a c t i o n s s t u d i e d b o t h w i t h C3 in s o l u t i o n a n d w i t h fixed C3 b o u n d nn cells. T h e table s h o w s t h e reactivities of the t h r e e m o n o c l o n a l a n t i b o d i e s a n d of bovine c o n g l u t i n i n with C3 a n d with its v a r i o u s b r e a k d o w n p r o d u c t s . C l o n e 3 b e h a v e s as a n a n t i - C 3 d a n d clone 4 as a n a n t i - C 3 c . C l o n e 9, however, s h o w s a n u n u s u a l reactivity, r e a c t i n g best with C3bi b u t r e a c t i n g with n e i t h e r of the g e n e r a l l y r e c o g n i z e d b r e a k d o w n p r o d u c t s of C3bi: C 3 c a n d C3d.
T h e b r e a k d o w n of C 3 b i h is a p p a r e n t t h a t t h e b r e a k d o w n of C3bi is m o r e c o m p l e x t h a n h a s previously been believed a n d t h e n e w view t h a t arises from this a n a l y s i s is s h o w n in Fig. 1. C 3 b i J3(75K)
| /
[
acdc
C3bi (186K)
ab(43K) (68K)
{
'
that splits C3bi into C3c a n d a2d in vivo is not c e r t a i n l y known. P l a s m i n h a s t h e correct specificity b u t carries o u t the reaction inefficiently a n d s t r e p t o k i n a s e activation of whole s e r u m fails to accelerate t h e split.
I n h i b i t i o n of alternative p a t h w a y c o n v e r t a s e by a n t i b o d i e s to C3 and to factor B T h e use of m o n o c l o n a l a n t i b o d i e s to inhibit C3 f u n c t i o n confirms t h a t the site for factor B is in t h e C3c f r a g m e n t b u t only m o n o c l o n a l a n t i b o d i e s to factor B c a n inhibit the pref o r m e d C3 c o n v e r t a s e ( C 3 b , B b ) . T h i s is not u n e x p e c t e d in view of t h e fact t h a t t h e e n z y m a t i c site is located in factor B. TABLE I Reactivity of monoclonal antibodies and bovine conglutinin with C3 and its breakdown products Reactivity In .rolu6mz (by c.o- C3 preczplta- C3b tion) C3bi C3c cL2d C3d C3e (,?[[-b~m~ld (by uptake C3b of~2qC3bi anti-rat Ig c~2d C3d
Clone 3 Clone 4 Clone 9 Bovine a n t i ' d ' a n t i ' c ' a n t i ' e ' conglutinin + + ++ ~ + 0 ++ ++
mmmmdogr lesda),, A ugu.t! 1981
Studies with monoclonal antibodies to complement components M o n o c l o n a l a n t i b o d i e s are a newly available tool for dissecting t h e c o m p l e x i n t e r a c t i o n s t h a t go to m a k e u p t h e c o m p l e m e n t p a t h w a y s . C e n t r a l to all t h e s e p a t h w a y s is t h e third c o m p o n e n t of c n m p l e m e n t , C3, a n d it is to C3 t h a t o u r initial m o n o c l o n a l a n t i b o d i e s were r a i s e d I, a l t h o u g h m o n o c l o n a l a n t i b o d i e s to factor B have also b e e n p r e p a r e d a n d are b e i n g a n a l y s e d ( R u d d y , S. a n d L a c h m a n n , P . J . ) .
Specificity of anti-C3 T h r e e distinct a n t i - C 3 m o n o c l o n a l s h a v e b e e n raised a n d their r e a c t i o n s s t u d i e d b o t h w i t h C3 in s o l u t i o n a n d w i t h fixed C3 b o u n d nn cells. T h e table s h o w s t h e reactivities of the t h r e e m o n o c l o n a l a n t i b o d i e s a n d of bovine c o n g l u t i n i n with C3 a n d with its v a r i o u s b r e a k d o w n p r o d u c t s . C l o n e 3 b e h a v e s as a n a n t i - C 3 d a n d clone 4 as a n a n t i - C 3 c . C l o n e 9, however, s h o w s a n u n u s u a l reactivity, r e a c t i n g best with C3bi b u t r e a c t i n g with n e i t h e r of the g e n e r a l l y r e c o g n i z e d b r e a k d o w n p r o d u c t s of C3bi: C 3 c a n d C3d.
T h e b r e a k d o w n of C 3 b i h is a p p a r e n t t h a t t h e b r e a k d o w n of C3bi is m o r e c o m p l e x t h a n h a s previously been believed a n d t h e n e w view t h a t arises from this a n a l y s i s is s h o w n in Fig. 1. C 3 b i J3(75K)
| /
[
acdc
C3bi (186K)
ab(43K) (68K)
{
'
that splits C3bi into C3c a n d a2d in vivo is not c e r t a i n l y known. P l a s m i n h a s t h e correct specificity b u t carries o u t the reaction inefficiently a n d s t r e p t o k i n a s e activation of whole s e r u m fails to accelerate t h e split.
I n h i b i t i o n of alternative p a t h w a y c o n v e r t a s e by a n t i b o d i e s to C3 and to factor B T h e use of m o n o c l o n a l a n t i b o d i e s to inhibit C3 f u n c t i o n confirms t h a t the site for factor B is in t h e C3c f r a g m e n t b u t only m o n o c l o n a l a n t i b o d i e s to factor B c a n inhibit the pref o r m e d C3 c o n v e r t a s e ( C 3 b , B b ) . T h i s is not u n e x p e c t e d in view of t h e fact t h a t t h e e n z y m a t i c site is located in factor B. TABLE I Reactivity of monoclonal antibodies and bovine conglutinin with C3 and its breakdown products Reactivity In .rolu6mz (by c.o- C3 preczplta- C3b tion) C3bi C3c cL2d C3d C3e (,?[[-b~m~ld (by uptake C3b of~2qC3bi anti-rat Ig c~2d C3d
Clone 3 Clone 4 Clone 9 Bovine a n t i ' d ' a n t i ' c ' a n t i ' e ' conglutinin + + ++ ~ + 0 ++ ++
