LOS NÚCLEOS CONCEPTUALES DE LA QUÍMICA:

c u ltu re s y s t e m , th e a g a r b ein g r e a d ily p e r m e a b le to m e d iu m ,

C . A s s a y s f o r im m u n o c o m p e te n c e /m a tu r a tio n

1. D e te c tio n of T h y 1 by im m u n o c y to c h e m is tr y

T h e p r e s e n c e of the T c e ll m e m b r a n e a n tig e n T hy 1 w as looked f o r on th e s u r f a c e of d iffe re n t c e ll p o p u la tio n s . B oth

d ir e c t and th e in d i r e c t 's a n d w ic h ' te c h n iq u e w ith sp ecific a n ti s e r u m w as u s e d . B oth th e s e m e th o d s involved u sin g the en zy m e

h o r s e - r a d i s h p e r o x id a s e (H R PO , Sigm a) co u p led to antibody to d e te c t the a n tig e n ,

(a) T h e d i r e c t te c h n iq u e

T h e IgG f r a c t io n of 1 m l of m o u s e a n ti- T h y 1 s e r u m (AKR a n ti-C ^H ) , a k ind gift of D r , G ani (S e a rle R e s e a r c h L a b o r a to r ie s ) w as p u rifie d and c o u p led to h o r s e - r a d i s h p e r o x id a s e by the

follow ing m e th o d :- 1, 0 m l s e r u m

D ia ly se d x 3 w ith O.OIM so d iu m p h o sp h a te (PO^ ) b u ffe r pH 6. 8

L o ad e d onto a D E A E c e llu lo s e (Sigm a) c o lu m n e q u ilib ra te d w ith th e sa m e b u ffe r

E lu te d s e q u e n tia lly w ith 0 .0 4 M N aC l 0 .0 5 M N aC l 0. 075M N aC l 0, 15 M N aC l

T h is w a s n e c e s s a r y a s not enough p ro te in c a m e off at lo w e r ionic s tr e n g th s , a s th e s a m p le w a s p ro b a b ly c o n ta m in a te d w ith o th e r s e r u m p r o te i n s .

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A b s o rb a n c e r e a d at _{10 rnm} and p r o te in c o n c . m e a s u r e d_^

D ia ly se d x 3 a g a in s t 0. 125M P O b u ffe r pH 6 .0 containing 0. 15 M N aC l

C o n c e n tra te d on a 'M in ico n B 125', m e m b r a n e c u t off at 5 0 ,0 0 0 d a lto n s

C e n trifu g e d to r e m o v e p r e c i p ita te

2 80 A b s o rb a n c e r e a d a t E , ^_{1 0m m}

2 ,2 m g of 'IgG ' o b ta in e d in 0 , 6 m l, added 0 ,2 m l PO^ b u ffe r. T o tal volum e = 0 , 8 m l

T h e IgG w as coupled to the e n z y m e by the m e th o d of T e rn y n c k and A v r a m e a s (1976 and 1977), T h e 0, 8 m l of p r o te in so lu tio n w a s added to 0 ,2 m l of a so lu tio n of p -b e n z o q u in o n e (Sigm a) of c o n c , 30 m g / m l (d is s o lv e d in eth an o l). T h e re a c tio n m i x tu r e w as

in c u b a te d f o r 1 h a t ro o m t e m p e r a t u r e on a slow s t i r r e r in the d a r k . T h is w a s th e n lo ad ed onto a S ep h ad ex G, 25 (fine) c o lu m n m a d e up in a 5 m l d is p o s a b le s y rin g e b a r r e l in s a lin e . T he pink c o lo u re d f r a c t io n w a s c o lle c te d and 3 m g of the en zy m e H R P O w as ad d ed .

T h e en z y m e w a s p r e - d i a l y s e d a g a in s t sa lin e to re m o v e c o n ta m in a tin g s a lt s , A 1 /lO th of th e fin a l v o lu m e of. IM N a^C O ^ b u ffe r pH 9 .0 w as added and in c u b a te d f o r 15-24 h in the d a rk a t ro o m t e m p e r a t u r e . T he r e a c tio n w a s stopped by ad ding th e new fin a l vo lu m e of IM ly s in e and in c u b a tin g in the d a r k f o r 2 -3 h. T h e ly s in e so lution w as m a d e by d is s o lv in g ly s in e h y d ro c h lo rid e (BDH) in d is tille d

H^O and a d ju stin g to pH 7. 5 by th e a d d itio n of IM NaOH, 10 m g / m l of bovine s e r u m a lb u m in (BSA, BDH) w as added to h elp p r e s e r v e

the p r e p a r a t io n , th e n d ia ly s e d a g a in s t P B S , aliq u o te d and s to re d at - 2 0 ° C .

T he r e a g e n t p r e p a r e d by th is m eth o d w a s u s e d to d e te c t T h y 1 on c y to c e n tr if u g re p r e p a r a t i o n s of c e l l s . C e lls w e r e spun on a c y to c e n tr if u g re w ith o u t the p r e s e n c e of s e r u m and fix ed in m e th a n o l c o n tain in g 0.2% c o n c . h y d r o c h lo r ic a c id (HCl) to b lo c k endog en o u s p e r o x id a s e a c tiv ity . H R P O c o n ju g ated a n ti- T h y 1 a n ti s e r u m w as a p p lied to the slid e f o r 30 m in a t a ^ / g d ilu tio n . T h e s lid e s w e re w a sh e d in t r i s ( h y d ro x y m e th y lm e th y la m in e , S igm a) b u ffe re d

salin e (TBS) f o r 30 m in and a 0 .0 5 % so lu tio n of d ia m in o b e n z id iin e (DAB, Sigm a) w a s a p p lie d to th e slid e s f o r 5 m in , w a sh ed in

ru n n in g H^O f o r 10 m in and fin a lly sta in e d in M a y e r 's h a e m a lu m fo r 1 m in and m o u n te d in s ty r e n e .

(b) T h e in d i r e c t m e th o d

C y to c e n trifu g e p r e p a r a t io n s w e re m a d e a s d e s c r ib e d a b o v e, fixed in a c ifie d m e th a n o l and tr e a t e d w ith a d ilu tio n of the a n ti- T h y 1 a n ti s e r u m f o r 30 m in . T h e s lid e s w e r e w a sh e d w ith TBS fo r 30 m in , th e n t r e a t e d s e q u e n tia lly w ith ra b b it a n ti- m o u s e Ig (1 /1 0 0 d ilu tio n ), goat a n t i - r a b b i t Xg (1 /2 0 dilution) and fin a lly th e p e r o x i d a s e - a n t i- p e r o x i d a s e c o m p le x (1 /4 0 d ilu tio n ). T h e s e r e a g e n ts w e r e su p p lied by D ako L a b o r a t o r i e s , E a c h a n t is e r u m w as ap p lied fo r 30 m in w ith 30 m in w a s h e s in TB S in b e tw een .

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