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Biogel-absorbed insoluble protein derivatives for the isolation of antibody
BIOCHIMIE, 1972, 54, 203-208.
Biogel-absorbed insoluble protein derivatives for the isolation of antibody. Marc STANISLAWSKIa n d Ghislaine C(EUR-J'oLY.
Unit~ d ' I m m u n o d i f f ~ r e n c i a t i o n , Institut de Biologie Mol~culaire, Universit~ Paris VII, 2, Place Jussieu, 75 - P a r i s 5% (11/2/1972). Summary. - - Highly dispersed glutaraldehyde-insolubilized protein antigens were adsorbed on Biogel, packed into chromatographic columns and used as immunoadsorbents for the isolation of antibody. The preparation of Biogel-lmmunoadsorbent complex is simple and rapid. Such derivatives possess an elevated adsorbing capacity for homologous antibody. Antibody recovery is quantitative. Due to the elevated flow rates of such columns the adsorption and elution steps can be accomplished with relative speed. INTRODUCTION. Some of the advantages of i m m u n o a d s o r b e n t s as used for the isolation of a n t i b o d y have a l r e a d y been well d o c u m e n t e d [1, 2, 3]. It must again be emphasized that such methods offer p r e p a r a t i o n s of specific a n t i b o d y often i n q u a n t i t a t i v e yield a n d free of detectable soluble a n t i g e n - a n t i b o d y complexes ; the later is due to the insoluble nature of the antigen. Isolation w o r k w i t h glutaraldehyde p r e p a r e d i m m u n o a d s o r b e n t s is usually p e r f o r m e d w i t h the batchwise p r o c e d u r e w h e r e the constituents i n solution are separated from the insoluble derivative by consecutive c e n t r i f u g a t i o n s [4]. W h e n the derivative is i n t e n d e d to be p a c k e d into chromatographic columns, the fine i m n m n o a d s o r b e n t particles must be e l i m i n a t e d i n o r d e r to avoid clogging d u r i n g s u b s e q u e n t w a s h i n g a n d elution steps. This m a y lead to appreciable loss of the i m m u n o a d s o r b e n t a n d is p a r t i c u l a r l y crucial w h e n prepar i n g derivatives w i t h p u r e antigens. I n the p r e s e n t w o r k g l u t a r a l d e h y d e - i n s o l u b i l i z e d derivatives [4] were finely dispersed w i t h a high shear apparatus, adsorbed on Biogel beads a n d packed into c h r o m a t o g r a p h i c columns. This method has been applied to the isolation of a n t i b o d y i n several a n t i g e n - a n t i b o d y systems. An cvaiuation of this modified t e c h n i c was sought on the basis of two p a r a m e t e r s : 1) a n t i b o d y a d s o r b i n g capacity of an i m m u n o a d s o r b e n t a n d 2) a n t i b o d y recovery. MATERIALS AND M~THODS.
hnmunesera. Rabbit i m m u n e s e r a were used throughout. Each r a b b i t received 3 mg of the clonal IgG 2a from
MOPC 173 m u r i n e plasmocytoma, a d m i n i s t e r e d i n 3 sub-cutaneous i n j e c t i o n s w i t h Complete F r e u n d ' s A d j u v a n t and spaced two weeks apart. Boosters of 200 to 400 ~g were p e r f o r m e d b y the i n t r a - v e n o u s route two weeks after the last F r e u n d a n d regularly at 6 weeks i n t e r v a l thereafter. The a n i m a l s were bled 50 ml at 10-20 days after each i n t r a venous booster. P r e c i p i t i n titers of the sera r a n ged from 2 to 5 mg of a n t i b o d y / m l . Anti-Fab a n d a n t i - F c i m m u n e s e r a of the same Ig were o b t a i n e d i n an i d e n t i c a l fashion except that 1.5 to 2 mg were injected. A n t i b o d y titers r a n g e d from 3 to 5 m g / m l . Anti-lysozyme was p r e p a r e d w i t h 3 mg of enzyme (egg w h i t e Lysozyme, 3 x crystallized, Sigma). The i m m u n e s e r u m used in the p r e s e n t w o r k h a d a titer of 470 tLg/ml.
Antigens. The clonal IgG 2a i m m u n o g l o b u l i n was o b t a i n e d by p r e p a r a t i v e electrophoresis i n starch block [5]. The F a b a n d Fc p o r t i o n s of the molecule were o b t a i n e d by p r e p a r a t i v e electrophoresis i n Difco Agar [6] following controlled p a p a i n cleavage ]7] of IgG 2a. P u r i t y controls were systematically done by i m m u n o e l e c t r o p h o r e s i s [8] a n d by electrophoresis on 5 p. cent a c r y l a m i d e discs [9]. These constituents were p u r e w h e n tested at 1 to 3 mg of p r o t e i n / m l . Insoluble protein derivatives. The method of p r e p a r i n g i n s i l u b l e p r o t e i n derivatives was that described by Avrameas a n d T e r n y n c k [4], u s i n g glutaraldehyde (Schuchardt, Mfinchen) at 10 mg/100 mg of p r o t e i n . All proteins were co-polymerized w i t h Bovine Serum Alb u m i n (BSA, Sigma) i n the p r o p o r t i o n of 1 part of 14
Marc S t a n i s l a w s k i and Ghislaine Cveur-Jolg.
204
specific antigen w i t h 4 parts of BSA ( w / w ) i n 0,2 M acetic acid-Na acetate buffer, pH 5.0. Crossp o l y m e r i z a t i o n was allowed to take place d u r i n g 3 hrs. at room t e m p e r a t u r e for Fab, a n d 4 1/2 hrs. for IgG 2a, Fc a n d Lysozyme.
Preparation of Immunoadsorbent columns. The g l u t a r a l d e h y d e - i n s o l u b i l i z e d derivatives were first dispersed into small pieces w i t h a sharp i n s t r u m e n t , s u s p e n d e d i n 20 m l of 0.1 M p h o s p h a t e buffer pH 7.0 a n d t h e n homogenized w i t h a Polytron d i s i n t e g r a t o r (Block, Strasbourg) at 13000 r p m for 2 to 3 min. The m i l k y s u s p e n s i o n was diluted to 1 liter of the same buffer a n d Biogel P-30,0, 50-150 mesh ((:Bio Rad) from w h i c h fine beads were t h o r o u g h l y removed) was added dropwise u n d e r vigorous stirring. Adsorption of the insoluble p r o t e i n was i m m e d i a t e and lead to an agglutination of the Biogel beads. Addation of Bio gel is c o n t i n u e d unti]l all visible t u r b i d i t y has d i s a p p e a r e d from the s u p e r n a t a n t . I n our practice 50 to 70 ml of g r a v i t y - s e d i m e n t e d gel is sufficient to adsorb out 109 mg of insoluble protein.
washed w i t h phosphate buffer, 0.2 N glycine-HC1 buffer, pH 2.2 a n d b r o u g h t back to n e u t r a l i t y w i t h the first buffer. All buffers c o n t a i n 0.02 p. cent sodium azide as preservative.
Procedure for the isolation of antibody. An a p p r o p r i a t e volume of i m m u n e s e r u m is filtered t h r o u g h a 0 . 4 5 9 Selectron m e m b r a n e and is applied to the i m m u n o a d s o r b e n t column at room t e m p e r a t u r e ; the flow rate is adjusted so that the time of contact with the i m m u n o a d s o r b e n t is 1 to 2 hrs. W a s h i n g with 0.1 M p h o s p h a t e buffer pH 7.0 is c o n t i n u e d u n t i l the elnate shows no optical density at 280 nm. The c o l u m n is then transferred to a cold room ( + 4°C) a n d left there 30 nlin. to cool. A n t i b o d y is desorbed w i t h icecold 0.2 N glycine-HC1 buffer, pH 2.8 a n d collected as 5 to 6 ml fractions into a volume of 1 M phosphate buffer (K2HPO 4- KH2PO4) pH 7.0 calculated to give a n e a r n e u r a l final pH. Each i n d i vidual f r a c t i o n is t h o r o u g h l y a d m i x e d w i t h the n e u t r a l i z i n g buffer i m m e d i a t e l y after collection. The pooled fractions are dialysed at + 4°C
FIG. 1. - - Magnified view (1O0 ×) of Biogel P-300, beads (50-100 mesh) with an adsorbed, insolubilized mouse elonal IgG 2a-SAB co-polymer, (left) and virgin beads, (right). The protein was stained with Amido Black 10B.
The s l u r r y c o r r e s p o n d i n g to this q u a n t i t y is p o u r e d into a 2.5 X 30 cm siliconized chromatographic column, the flow rate b e i n g slowed to 25 to 30 m l / h r . The i m m u n o a d s o r b e n t is successively
BIOCHIMIE, 1972, 54, n ° 2.
against two changes of 5 1 0.15 M NaC1 buffered with 0.01 M phosphate, pH 7.0. A n t i b o d y r e c o v e r y is estimated at this step. W h e n e v e r desired the isolated a n t i b o d y is c o n c e n t r a t e d to 2-4 m g / m l by
Biogel-adsorbed insoluble proteins for antibody isolation. p r e s s u r e f i l t r a t i o n u n d e r N 2 (Diaflo, XM-50 m e m brane).
Protein estimation. T h e c o n t e n t of c l o n a l IgG 2a w a s e s t i m a t e d e i t h e r by a n m d i f i e d B i u r e t m e t h o d [10] w i t h H u m a n IgG ( C o h n F r a c t i o n II, N. B. Co.) as r e f e r e n c e s t a n d a r d o r by r e a d i n g o p t i c a l d e n s i t y f r o m E 1~-/° 280 n m = 14.6 [11] E i t h e r e s t i m a t i o n ¢m g a v e a l m o s t i d e n t i c a l values. F o r F c , F a b a n d Lys o z y i n e t h i s c o e f f i c i e n t w a s t a k e n as 10.0 [7] ; 15.0 [12], a n d 20..0 [13] at 281 nm, r e s p e c t i v e l y .
Antibody estimation. T h e c o n t e n t of p r e c i p i t a t i n g a n t i b o d y in w h o l e immunesera was estimated either by quantitative p r e c i p i t a t i o n [14] o r b y e l e c t r o i m m u n o d i f f u s i o n [15]. A n t i F c a n d anti F a b a n t i b o d y w e r e e s t i m a t e d e x c l u s i v e l y by e l e c t r o i m m u n o d i f f u s i o n u s i n g p e r o x i d a s e - l a b e l l e d c l o n a l IgG 2a [16! in t h e gel a n d a n t i - c l o n a l IgG 2a as t h e r e f e r e n c e s t a n d a r d . T h e a n t i b o d y c o n t e n t of i m m u n o a d s o r b e n t - i s o l a t e d a n t i b o d y p r e p a r a t i o n s w a s e s t i m a t e d by t h e m o d i fied B i u r e t m e t h o d [10]. RESULTS.
Interaction of Biogel with insoluble protein derivatives. T h e a d s o r p t i o n of g l u t a r a l d e h y d e - i n s o l u b i l i z e d d e r i v a t i v e s o n t o B i o g e l P-300 w a s i n s t a n t a n e o u s . T w o o t h e r Biogels t r i e d as a d s o r b i n g m a t r i x , t h e
P6 a n d P10, w e r e i n e f f i c i e n t . M i c r o s c o p i c o b s e r v a t i o n of v a r i o u s b e a d s at 100 x m a g n i f i c a t i o n s h o w e d d i s t i n c t s u r f a c e d i f f e r e n c e s . T h e P-300 used here possessed a rough surface, whereas the unadsorbing varieties were smooth-surfaced. The e x a c t n a t u r e of t h e r e a c t i o n w i t h i m m u n o a d s o r b e n t p a r t i c l e s has not b e e n i n v e s t i g a t e d h e r e . T h e s p a t i a l a r r a n g e m e n t of the g l u t a r a l d e h y d e - i n s o l u b i l i z e d p a r t i c l e s on B i o g e l b e a d s is s h o w n in F i g u r e 1. E t h y l c h l o r o f o r m a t e - i n s o l u b i l i z e d d e r i v a t i v e s [17] b e h a v e in an i d e n t i c a l f a s h i o n [18]. M o r e o v e r , the g l u t a r a l d e h y d e - i n s o l u b i l i z e d p l a n t l e c t i n c o n c a n a v a l i n - A has b e e n s u c c e s s f u l l y a d s o r b e d o n t o Bio gel a n d u s e d f o r t h e i s o l a t i o n of glyc o p r o t e i n s [19].
Antibody adsorbing capacity o[ immunoadsorbent columns. T a b l e 1 gives the m a x i m u m a d s o r b i n g c a p a c i t y figures f o r a n t i b o d y of s e v e r a l i m m u n o a d s o r b e n t c o l m n n s . In all cases t h e m a x i m u m a m o u n t of a n t i b o d y r e t a i n e d on a n y p a r t i c u l a r c o l u m n e x c e e d e d on a w / w basis t h e a m o u n t of t h e i n s o lubilized specific antigen. Not all of o n c e - i s o l a t e d a n t i b o d y w a s r e t a i n e d f o l l o w i n g a s e c o n d r e a d s o r p t i o n ( T a b l e 2). It is conceivable that such unadsorbed antibody consists of m o l e c u l e s w h o s e b i n d i n g sites f o r a n t i g e n h a v e b e e n d e n a t u r e d b y p r e v i o u s c o n t a c t w i t h glyc i n e - H C l b u f f e r at p H 2.8 d u r i n g t h e i r e l u t i o n . S u c h a n t i b o d y a c c o u n t s f o r 4-12 p. c e n t for an isolated antibody preparation, the remainder being
TABLE I.
Adsorbing capacity of Biogel immm~oadsorbent columns (*). Antigen-antibody system
CIonal IgG-anti IgG . . . . . . . . Clonal IgG-anti Fc . . . . . . . . . Fe.anti Fe . . . . . . . . . . . . . . . . . ! Fab-anti Fab . . . . . . . . . . . . . . . Lysozyme-anti Lysozyme . . . . i
iMaximum amount r AntigenAntigen ! of antibody antibody ('"} insolubilized (") i adsorbed (w/w) (mg) ! (mg) 18
36
112
18 15 16.6 16.3
22 28.9 18.3 66
1/1.22 1/1.92 1/1.1 1/4.0
(*) Successive aliquots of an immuneserum were run through the column and the presence of non-adsorbed antibody in the effluent was detected by the ring test. The column is considered fully saturated w i t h antibody when the effluents give a positive reaction w i t h i n 10 ~min at room temperature. Control ring tests were done simultaneously w i t h the starting immuneserum diluted to the same optical density as measured a 440 rim. All controls were positive within 20 sec to 5 min. In our hands the limit of detection of antibody ~vas 50 to 60 ~ g / m l of immuneserum, against a standard antigen solution at 1'0 min. (**) The Fab-SAB co-polymer was insolubilized at a final protein concentration of 40 mg/ml. The other specific antigens were insolubilized at a final concentration of 25 mg/ml. (***) The antigen-antibody at equivalence in quantitative precipitation was 1/5.4 for clonal IffG-anti IgG, and 1/1 for Lysozyme-anti Lysozyme.
BIOCHIM1E, 1972, 54, n ° 2.
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Marc Stanislawski and Ghislaine Coeur-Jolg.
206
bio]ogically active in so far as b i n d i n g w i t h antigen is c o n s i d e r e d .
Antibody recovery. A r e p r e s e n t a t i v e a n t i b o d y elution profile is s h o w n in F i g u r e 2. A n t i b o d y elutes as a single, w e l l - r e s o l v e d p e a k w i t h only a slight drag effect. T h e flow rates can be so adjusted that the acid elution step is c o m p l e t e d in one h o u r thus avoi-
d i n g excessive contact w i t h the low p H glycineHC1 buffer. A n t i b o d y r e c o v e r y values are p r e s e n t e d in Table 3. F o r the a n t i g e n - a n t i b o d y systems p r e s e n ted the values r a n g e d f r o m 53-100 p. cent after elution at p H 2.8. An a d d i t i o n a l 1.0.-26 p. cent is o b t a i n e d by eluting at pH 2.2. T h e r e f o r e , the total r e c o v e r i e s at both p H values r a n g e f r o m 63 to 126 p. cent. E s t i m a t i o n of r e c o v e r y w i t h all the
TABLE I[.
Antibody recovery following reisolation of once-isolated antibody. Antigenantibody system
Antibody adsorbed (mg)
pH 2.8
pH 2.2
P. cent recovered at pH 2.8
16
14.3
13.7
0.62
96
1.08
89
! Clonal IgG-I anti IgG.
Fab--anti Fab!
Antibody recovered (my)
Antibody added (mg)
9.6
9.06
8.14
i m m u n o a d s o r b e n t c o l u m n s w e r e done utilizing only 30-70 p. cent of t h e i r m a x i m u m a d s o r b i n g c a p a c i t y . W h e n u t i l i z i n g t h e i r full c a p a c i t y or surpassing it, substantially l o w e r r e c o v e r i e s of antib o d y w e r e o b t a i n e d (Table 3).
,2 o 2.0
Since only the a m o u n t of p r e c i p i t a t i n g a n t i b o d y a d d e d onto a c o l u m n is d e t e r m i n e d , w h e r e a s calculated r e c o v e r y is r e p r e s e n t a t i v e of both p r e c i p i t a t i n g and n o n - p r e c i p i t a t i n g antibody, r e c o v e r y figures t e n d to be overestimated. A c c o r d i n g l y , once-isolated a n t i b o d y w a s reisolated on the same i m m u n o a d s o r b e n t column. T h e values of t w o e x p e r i m e n t s are given in Table 2. The r e c o v e r y of clonal anti IgG w a s 9'6 p. cent and anti F a b w a s 8<9 p. cent after elution at p H 2.8. E l u t i o n at p H 2.2 gave an a d d i t i o n a l 4.3 p. cent, and 12 p. cent, r e s p e c t i v e l y . U n d e r these c o n d i t i o n s the true recov e r y values are 10,0 p. cent.
I.$
I0
0.5
DISCUSSION.
0 I0
5
15
20
Fractions
FIG. 2. - - Antibody elution profiles from a Bioge]adsorbed Lysozyme-SAB immunoadsorbent column (2.5 × 37 em). Two consecutive isolations of anti Lysozyme antibody were done with this column : one, ( • $-- ) from 52.5 ml, the other, ( - A - - A - ) from 15 ml of rabbit anti Lysozyme immunesernm, respectively. Elution with the pH 2.8 glycine-ttC1 buffer was begun at Fraction 1 following washing of unadsorbed proteins with 9.1 M phosphate buffer, pH 7.0. Fractions of 5.5 ml were collected into 2 ml of 1 M phosphate buffer pH 7.0. The flow rate was 80 ml/hr.
BIOCHIMIE, 1972, 54, n ° 2.
The Biogel-insoluble d e r i v a t i v e c o m p l e x is h i g h l y stable ; n e i t h e r v i g o r o u s s h a k i n g n o r successive w a s h i n g w i t h acid or neutral buffers leads to a detectable release of the adsorbed insoluble particles. No loss of the specificity of the i m m u n o a d s o r bent has been o b s e r v e d f o l l o w i n g a d s o p t i o n on Biogel. This could be e x p e c t e d since any modification of the specific antigen w o u l d result f r o m the p r o c e d u r e used for its insolubilization, not on
Biogel-adsorbed insoluble proteins for antibody isolation.
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TABLE ] [l.
Antibody recovery from Biogel immunoadsorbent columns.
Antigen_antibody system
Clonal lg-anti IgG . . . . Clonal Ig-anti F e . Fc-anti Fe . . . . . . . . . . F a b - a n t i Fab . . . . . . . . .
~ .
Lysozyme- anti L y s o zyme . . . . . . . . . . . . . . . . i
Antibody recovered (mg)
P. cent recovered
pH 2 . 8
pH 2.2
at pH 2.8
13.2 36 14.85 27 12.5 18.4
9.3 16.3 7.9 9.3 9.2 10
3.1 0.814 1.48 Not done Not done Not done
70 45 53 34 73 54
20.6
20.72
5.4
100
Antibody added (mg)
Antibody absorbed (rag)
13.2 36 14.85 27 12.5 22 20.6
t h e a c r y l a m i d e w h i c h s e r v e s m a i n l y as a s u p p o r t i n g m a t r i x a n d i n t h i s r e s p e c t is e s s e n t i a l l y n e u tral. Nevertheless, in several experiments not r e p o r t e d h e r e , a d s o r p t i o n of a n t i b o d y w a s h i g h l y s p e c i f i c as r e p o r t e d w i t h g l u t a r a l d e h y d e - i n s o l u b i l i z e d p r o t e i n d e r i v a t i v e s [4]. O n e e x c e p t i o n w a s n o t e d i n t h e c a s e of t h e I . y s o z y m e i m m u n o a d s o r b e n t . F r o m 15 m l of s e r u m of a n u n i m m u n i z e d r a b b i t 1.7 m g of p r o t e i n w a s e l u t e d at p H 2.8. T h e s a m e a m o u n t of t h e s p e c i f i c i m m u n e s e r u m g a v e 2,0.72 m g of e l u t e d a n t i b o d y p r o t e i n ( T a b l e 3). A s i m i l a r n o n - s p e c i f i c a d s o r p t i o n of i n s o l u b i l i z e d L y s o z y m e h a s b e e n r e p o r t e d [20]. T h e a n t i b o d y a d s o r b i n g c a p a c i t y of t h e c o l u m n s w a s m o r e e l e v a t e d t h a n is r e p o r t e d f o r o t h e r syst e m s w h e r e e i t h e r p r o t e i n - p r o t e i n p o l y m e r s [43 o r p r o t e i n - a c r y l a m i d e p o l y m e r s [21] w e r e u s e d . T h i s relatively more elevated capacity can reasonably b e a t t r i b u t e d to t h e h i g h d i s p e r s i o n of t h e i n s o luble derivative which thus provides a greater surface area for contact with antibody. The capacity figures given in Table 1 are undere s t i m a t e d . T h i s is so, s i n c e o n l y t h e a m o u n t of p r e cipitating antibody was used to calculate capacity. Non-precipitating antibody present in varying proportions in the different immunesera, however, also c o m b i n e s w i t h t h e i m m u n o a d s o r b e n t and t a k e s u p a c o r r e s p o n d i n g a m o u n t of its c a p a c i t y . R e c o v e r y v a l u e s of a n t i b o d y a r e i n t h e s a m e r a n g e as t h o s e r e p o r t e d f o r g l u t a r a l d e h y d e - i n s o l u b i l i z e d d e r i v a t i v e s [4]. I s o l a t i o n of a n t i b o d y f r o m c o l u m n s s a t u r a t e d to their maximmn capacity gives substantially lower r e c o v e r y v a l u e s . T h i s h a s b e e n o b s e r v e d w i t h glut a r a l d e h y d e - i n s o l u b i l i z e d p r o t e i n d e r i v e s [22].
BIOCHIMIE, 1972, 54, n ° 2.
Acknowledgments. This work was supported by the ¢ Direction G6n6rale la Recherche Scientifique et Technique ~, Convention n ~' 70.02.270. The a u t h o r s are indebted to Dr. S. A v r a m e a s for his c o n s t a n t interest a n d criticism of this work. P~SUMg. Des polym6res de prot~ines o b t e n u s p a r i n s o l u b i l i sation avee la g l u t a r a l d e h y d e ont ~t6 disperses e n particu]es de faible d i m e n s i o n , adsorh~s sur Biogel et uti]is~s eomme i m m u n o a d s o r b a n t s p o u r l'iso]ement d'anticorps. De tels d~riv~s ont une h a u t e eapaeit~ d'adsorption p o u r les anticorps. Des r e n d e m e n t s q u a n t i t a t i f s d ' a n t i c o r p s sont o b t e n u s apr6s 61ution avec un t a m p o n Glycocolle-HCi h pH 2.8. Utilis6s sur colonne de c h r o m a t o g r a p h i c , de tels d6riv6s poss~dent des vitesses d'~coulement ~lev6s et p e r m e t t e n t d'effectuer l ' i s o l e m e n t d ' a n t i c o r p s avec u n e rapidit6 relative. ZUSAMMENFASSUNG. P r o t e i n p o l y m e r e , die d u r e h I n s o l u b i l i s a t i o n m i t Glut a r a l d e h y d e r h a l t e n w u r d e n , w u r d e n hoeh dispergiert, a u f Bio-Gel a d s o r b i e r t u n d als I m m u n o a d s o r b e n t e n z u r Isolierung der A n t i k 6 r p e r v e r w a n d t . Derartige Derivate h a b e n den A n t i k 6 r p e r n gegeniiber eine h o h e Adsorptionskapazit~it. Q u a n t i t a t i v e Ertr~ige der AntikSrper w u r d e n n a e h E l u i e r u n g m i t einem Glykoko]l- HC1-Puffer bet pH 2,8 erhalten. Uber e i n e r Chromatographiesi~ule hesitzen diese Derivate eine hohe Flussgeschwindigkeit u n d erlauben, die T r e n n u n g der A n t i k 6 r p e r m i t r e l a t i v e r Schnelligkeit durchzufiihren. REFERENCES. 1. Campbell, D. H. & Weliky, N,. (19~7) in Methods in I m m u n o l o g y and I m m u n o c h e m i s t r y , Vol. 1, C. A. W i l l i a m s and A. H. Chase ed., Academic Press, p. 365. 2. Silman, I. H. & Katehalski, E. (1966) Ann. Rep. Biochem., 35, 873.
208
Marc Stanislawski
and
3. Centeno, E. R. ,~ Sehon, A. H. (1971) Immunochemistry, 8, 887. 4. Avrameas, S. ~ Ternynck, T. (196,9) Immunochemistry, 6, 53. 5. Edelman, G. M., Herem,ans, J. F., Heremans, M. T. Kunkcl, H. G. (196'01) J. Exp. Med., 112, 203. 6. Stanislawski, M., u n p u b l i s h e d results. 7. Porter, R. R. (1959) Biochem. J., 73, 119. 8. Grabar, P. ~ Williams, C. A. jun. (1953) Biochim. Biophys. Acta, 10, 1'93. 9. Davis, B. J. (1964~) Ann. N. Y. Acad. Sci., 121, 404. 10. Kabat, E. A., in E x p e r i m e n t a l I m m u n o c h e m i s t r y , 2nd Ed., E. A. Kabat and M. M. Mayer (1961) ed., Charles C. Thomas, Springfi.eld, I l l . 1I. Onoue, K., Yagi, Y., Grossberg, A. L. • P r e s s m a n , D. (1965) Immunochemistry, 2, 401. 12. S.temke, J. W. P h . D . Thesis (1963) University of Illinois, U.S.A.
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Ghislaine
Ceeur-Joly.
13. Biochemist's Handbook, Cyril Long ed., E. and F. N. Spon Ltd., London, p. 82. 14. Heidelberg~er, M. • Kendall, F. E. (1935) J. Exp. Med., 62, 697. 15. Lopez, M., Tsu, T., Hysiop, N. E. (19'69) l m m u n o chemistry, 6, 513. 16. Stanislawski, M. (1970) C. R. Acad. Sci., 271, 1452. 17. Avrameas, S. ~ Ternynck, T. (1967) J. Biol. Chem., 242, 1651. 18. Rouze, P., personal communication. 19. Donnelly, E. H. • Goldstein, I. J. (1970) Biochem. J., 118, 679. 2~0. Cazenave, P.-A. (197{)) Th~se de Doctorat 3Q Cycle, Paris, France. 21. Carrel, S. ~ Barandun, S. (1971) lmmunoehemistry, 8, 39. 22. Ternynck, T. (1969) Th6sc de Doctoral d'Universit6, Paris, France.
Biogel-absorbed insoluble protein derivatives for the isolation of antibody. Marc STANISLAWSKIa n d Ghislaine C(EUR-J'oLY.
Unit~ d ' I m m u n o d i f f ~ r e n c i a t i o n , Institut de Biologie Mol~culaire, Universit~ Paris VII, 2, Place Jussieu, 75 - P a r i s 5% (11/2/1972). Summary. - - Highly dispersed glutaraldehyde-insolubilized protein antigens were adsorbed on Biogel, packed into chromatographic columns and used as immunoadsorbents for the isolation of antibody. The preparation of Biogel-lmmunoadsorbent complex is simple and rapid. Such derivatives possess an elevated adsorbing capacity for homologous antibody. Antibody recovery is quantitative. Due to the elevated flow rates of such columns the adsorption and elution steps can be accomplished with relative speed. INTRODUCTION. Some of the advantages of i m m u n o a d s o r b e n t s as used for the isolation of a n t i b o d y have a l r e a d y been well d o c u m e n t e d [1, 2, 3]. It must again be emphasized that such methods offer p r e p a r a t i o n s of specific a n t i b o d y often i n q u a n t i t a t i v e yield a n d free of detectable soluble a n t i g e n - a n t i b o d y complexes ; the later is due to the insoluble nature of the antigen. Isolation w o r k w i t h glutaraldehyde p r e p a r e d i m m u n o a d s o r b e n t s is usually p e r f o r m e d w i t h the batchwise p r o c e d u r e w h e r e the constituents i n solution are separated from the insoluble derivative by consecutive c e n t r i f u g a t i o n s [4]. W h e n the derivative is i n t e n d e d to be p a c k e d into chromatographic columns, the fine i m n m n o a d s o r b e n t particles must be e l i m i n a t e d i n o r d e r to avoid clogging d u r i n g s u b s e q u e n t w a s h i n g a n d elution steps. This m a y lead to appreciable loss of the i m m u n o a d s o r b e n t a n d is p a r t i c u l a r l y crucial w h e n prepar i n g derivatives w i t h p u r e antigens. I n the p r e s e n t w o r k g l u t a r a l d e h y d e - i n s o l u b i l i z e d derivatives [4] were finely dispersed w i t h a high shear apparatus, adsorbed on Biogel beads a n d packed into c h r o m a t o g r a p h i c columns. This method has been applied to the isolation of a n t i b o d y i n several a n t i g e n - a n t i b o d y systems. An cvaiuation of this modified t e c h n i c was sought on the basis of two p a r a m e t e r s : 1) a n t i b o d y a d s o r b i n g capacity of an i m m u n o a d s o r b e n t a n d 2) a n t i b o d y recovery. MATERIALS AND M~THODS.
hnmunesera. Rabbit i m m u n e s e r a were used throughout. Each r a b b i t received 3 mg of the clonal IgG 2a from
MOPC 173 m u r i n e plasmocytoma, a d m i n i s t e r e d i n 3 sub-cutaneous i n j e c t i o n s w i t h Complete F r e u n d ' s A d j u v a n t and spaced two weeks apart. Boosters of 200 to 400 ~g were p e r f o r m e d b y the i n t r a - v e n o u s route two weeks after the last F r e u n d a n d regularly at 6 weeks i n t e r v a l thereafter. The a n i m a l s were bled 50 ml at 10-20 days after each i n t r a venous booster. P r e c i p i t i n titers of the sera r a n ged from 2 to 5 mg of a n t i b o d y / m l . Anti-Fab a n d a n t i - F c i m m u n e s e r a of the same Ig were o b t a i n e d i n an i d e n t i c a l fashion except that 1.5 to 2 mg were injected. A n t i b o d y titers r a n g e d from 3 to 5 m g / m l . Anti-lysozyme was p r e p a r e d w i t h 3 mg of enzyme (egg w h i t e Lysozyme, 3 x crystallized, Sigma). The i m m u n e s e r u m used in the p r e s e n t w o r k h a d a titer of 470 tLg/ml.
Antigens. The clonal IgG 2a i m m u n o g l o b u l i n was o b t a i n e d by p r e p a r a t i v e electrophoresis i n starch block [5]. The F a b a n d Fc p o r t i o n s of the molecule were o b t a i n e d by p r e p a r a t i v e electrophoresis i n Difco Agar [6] following controlled p a p a i n cleavage ]7] of IgG 2a. P u r i t y controls were systematically done by i m m u n o e l e c t r o p h o r e s i s [8] a n d by electrophoresis on 5 p. cent a c r y l a m i d e discs [9]. These constituents were p u r e w h e n tested at 1 to 3 mg of p r o t e i n / m l . Insoluble protein derivatives. The method of p r e p a r i n g i n s i l u b l e p r o t e i n derivatives was that described by Avrameas a n d T e r n y n c k [4], u s i n g glutaraldehyde (Schuchardt, Mfinchen) at 10 mg/100 mg of p r o t e i n . All proteins were co-polymerized w i t h Bovine Serum Alb u m i n (BSA, Sigma) i n the p r o p o r t i o n of 1 part of 14
Marc S t a n i s l a w s k i and Ghislaine Cveur-Jolg.
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specific antigen w i t h 4 parts of BSA ( w / w ) i n 0,2 M acetic acid-Na acetate buffer, pH 5.0. Crossp o l y m e r i z a t i o n was allowed to take place d u r i n g 3 hrs. at room t e m p e r a t u r e for Fab, a n d 4 1/2 hrs. for IgG 2a, Fc a n d Lysozyme.
Preparation of Immunoadsorbent columns. The g l u t a r a l d e h y d e - i n s o l u b i l i z e d derivatives were first dispersed into small pieces w i t h a sharp i n s t r u m e n t , s u s p e n d e d i n 20 m l of 0.1 M p h o s p h a t e buffer pH 7.0 a n d t h e n homogenized w i t h a Polytron d i s i n t e g r a t o r (Block, Strasbourg) at 13000 r p m for 2 to 3 min. The m i l k y s u s p e n s i o n was diluted to 1 liter of the same buffer a n d Biogel P-30,0, 50-150 mesh ((:Bio Rad) from w h i c h fine beads were t h o r o u g h l y removed) was added dropwise u n d e r vigorous stirring. Adsorption of the insoluble p r o t e i n was i m m e d i a t e and lead to an agglutination of the Biogel beads. Addation of Bio gel is c o n t i n u e d unti]l all visible t u r b i d i t y has d i s a p p e a r e d from the s u p e r n a t a n t . I n our practice 50 to 70 ml of g r a v i t y - s e d i m e n t e d gel is sufficient to adsorb out 109 mg of insoluble protein.
washed w i t h phosphate buffer, 0.2 N glycine-HC1 buffer, pH 2.2 a n d b r o u g h t back to n e u t r a l i t y w i t h the first buffer. All buffers c o n t a i n 0.02 p. cent sodium azide as preservative.
Procedure for the isolation of antibody. An a p p r o p r i a t e volume of i m m u n e s e r u m is filtered t h r o u g h a 0 . 4 5 9 Selectron m e m b r a n e and is applied to the i m m u n o a d s o r b e n t column at room t e m p e r a t u r e ; the flow rate is adjusted so that the time of contact with the i m m u n o a d s o r b e n t is 1 to 2 hrs. W a s h i n g with 0.1 M p h o s p h a t e buffer pH 7.0 is c o n t i n u e d u n t i l the elnate shows no optical density at 280 nm. The c o l u m n is then transferred to a cold room ( + 4°C) a n d left there 30 nlin. to cool. A n t i b o d y is desorbed w i t h icecold 0.2 N glycine-HC1 buffer, pH 2.8 a n d collected as 5 to 6 ml fractions into a volume of 1 M phosphate buffer (K2HPO 4- KH2PO4) pH 7.0 calculated to give a n e a r n e u r a l final pH. Each i n d i vidual f r a c t i o n is t h o r o u g h l y a d m i x e d w i t h the n e u t r a l i z i n g buffer i m m e d i a t e l y after collection. The pooled fractions are dialysed at + 4°C
FIG. 1. - - Magnified view (1O0 ×) of Biogel P-300, beads (50-100 mesh) with an adsorbed, insolubilized mouse elonal IgG 2a-SAB co-polymer, (left) and virgin beads, (right). The protein was stained with Amido Black 10B.
The s l u r r y c o r r e s p o n d i n g to this q u a n t i t y is p o u r e d into a 2.5 X 30 cm siliconized chromatographic column, the flow rate b e i n g slowed to 25 to 30 m l / h r . The i m m u n o a d s o r b e n t is successively
BIOCHIMIE, 1972, 54, n ° 2.
against two changes of 5 1 0.15 M NaC1 buffered with 0.01 M phosphate, pH 7.0. A n t i b o d y r e c o v e r y is estimated at this step. W h e n e v e r desired the isolated a n t i b o d y is c o n c e n t r a t e d to 2-4 m g / m l by
Biogel-adsorbed insoluble proteins for antibody isolation. p r e s s u r e f i l t r a t i o n u n d e r N 2 (Diaflo, XM-50 m e m brane).
Protein estimation. T h e c o n t e n t of c l o n a l IgG 2a w a s e s t i m a t e d e i t h e r by a n m d i f i e d B i u r e t m e t h o d [10] w i t h H u m a n IgG ( C o h n F r a c t i o n II, N. B. Co.) as r e f e r e n c e s t a n d a r d o r by r e a d i n g o p t i c a l d e n s i t y f r o m E 1~-/° 280 n m = 14.6 [11] E i t h e r e s t i m a t i o n ¢m g a v e a l m o s t i d e n t i c a l values. F o r F c , F a b a n d Lys o z y i n e t h i s c o e f f i c i e n t w a s t a k e n as 10.0 [7] ; 15.0 [12], a n d 20..0 [13] at 281 nm, r e s p e c t i v e l y .
Antibody estimation. T h e c o n t e n t of p r e c i p i t a t i n g a n t i b o d y in w h o l e immunesera was estimated either by quantitative p r e c i p i t a t i o n [14] o r b y e l e c t r o i m m u n o d i f f u s i o n [15]. A n t i F c a n d anti F a b a n t i b o d y w e r e e s t i m a t e d e x c l u s i v e l y by e l e c t r o i m m u n o d i f f u s i o n u s i n g p e r o x i d a s e - l a b e l l e d c l o n a l IgG 2a [16! in t h e gel a n d a n t i - c l o n a l IgG 2a as t h e r e f e r e n c e s t a n d a r d . T h e a n t i b o d y c o n t e n t of i m m u n o a d s o r b e n t - i s o l a t e d a n t i b o d y p r e p a r a t i o n s w a s e s t i m a t e d by t h e m o d i fied B i u r e t m e t h o d [10]. RESULTS.
Interaction of Biogel with insoluble protein derivatives. T h e a d s o r p t i o n of g l u t a r a l d e h y d e - i n s o l u b i l i z e d d e r i v a t i v e s o n t o B i o g e l P-300 w a s i n s t a n t a n e o u s . T w o o t h e r Biogels t r i e d as a d s o r b i n g m a t r i x , t h e
P6 a n d P10, w e r e i n e f f i c i e n t . M i c r o s c o p i c o b s e r v a t i o n of v a r i o u s b e a d s at 100 x m a g n i f i c a t i o n s h o w e d d i s t i n c t s u r f a c e d i f f e r e n c e s . T h e P-300 used here possessed a rough surface, whereas the unadsorbing varieties were smooth-surfaced. The e x a c t n a t u r e of t h e r e a c t i o n w i t h i m m u n o a d s o r b e n t p a r t i c l e s has not b e e n i n v e s t i g a t e d h e r e . T h e s p a t i a l a r r a n g e m e n t of the g l u t a r a l d e h y d e - i n s o l u b i l i z e d p a r t i c l e s on B i o g e l b e a d s is s h o w n in F i g u r e 1. E t h y l c h l o r o f o r m a t e - i n s o l u b i l i z e d d e r i v a t i v e s [17] b e h a v e in an i d e n t i c a l f a s h i o n [18]. M o r e o v e r , the g l u t a r a l d e h y d e - i n s o l u b i l i z e d p l a n t l e c t i n c o n c a n a v a l i n - A has b e e n s u c c e s s f u l l y a d s o r b e d o n t o Bio gel a n d u s e d f o r t h e i s o l a t i o n of glyc o p r o t e i n s [19].
Antibody adsorbing capacity o[ immunoadsorbent columns. T a b l e 1 gives the m a x i m u m a d s o r b i n g c a p a c i t y figures f o r a n t i b o d y of s e v e r a l i m m u n o a d s o r b e n t c o l m n n s . In all cases t h e m a x i m u m a m o u n t of a n t i b o d y r e t a i n e d on a n y p a r t i c u l a r c o l u m n e x c e e d e d on a w / w basis t h e a m o u n t of t h e i n s o lubilized specific antigen. Not all of o n c e - i s o l a t e d a n t i b o d y w a s r e t a i n e d f o l l o w i n g a s e c o n d r e a d s o r p t i o n ( T a b l e 2). It is conceivable that such unadsorbed antibody consists of m o l e c u l e s w h o s e b i n d i n g sites f o r a n t i g e n h a v e b e e n d e n a t u r e d b y p r e v i o u s c o n t a c t w i t h glyc i n e - H C l b u f f e r at p H 2.8 d u r i n g t h e i r e l u t i o n . S u c h a n t i b o d y a c c o u n t s f o r 4-12 p. c e n t for an isolated antibody preparation, the remainder being
TABLE I.
Adsorbing capacity of Biogel immm~oadsorbent columns (*). Antigen-antibody system
CIonal IgG-anti IgG . . . . . . . . Clonal IgG-anti Fc . . . . . . . . . Fe.anti Fe . . . . . . . . . . . . . . . . . ! Fab-anti Fab . . . . . . . . . . . . . . . Lysozyme-anti Lysozyme . . . . i
iMaximum amount r AntigenAntigen ! of antibody antibody ('"} insolubilized (") i adsorbed (w/w) (mg) ! (mg) 18
36
112
18 15 16.6 16.3
22 28.9 18.3 66
1/1.22 1/1.92 1/1.1 1/4.0
(*) Successive aliquots of an immuneserum were run through the column and the presence of non-adsorbed antibody in the effluent was detected by the ring test. The column is considered fully saturated w i t h antibody when the effluents give a positive reaction w i t h i n 10 ~min at room temperature. Control ring tests were done simultaneously w i t h the starting immuneserum diluted to the same optical density as measured a 440 rim. All controls were positive within 20 sec to 5 min. In our hands the limit of detection of antibody ~vas 50 to 60 ~ g / m l of immuneserum, against a standard antigen solution at 1'0 min. (**) The Fab-SAB co-polymer was insolubilized at a final protein concentration of 40 mg/ml. The other specific antigens were insolubilized at a final concentration of 25 mg/ml. (***) The antigen-antibody at equivalence in quantitative precipitation was 1/5.4 for clonal IffG-anti IgG, and 1/1 for Lysozyme-anti Lysozyme.
BIOCHIM1E, 1972, 54, n ° 2.
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bio]ogically active in so far as b i n d i n g w i t h antigen is c o n s i d e r e d .
Antibody recovery. A r e p r e s e n t a t i v e a n t i b o d y elution profile is s h o w n in F i g u r e 2. A n t i b o d y elutes as a single, w e l l - r e s o l v e d p e a k w i t h only a slight drag effect. T h e flow rates can be so adjusted that the acid elution step is c o m p l e t e d in one h o u r thus avoi-
d i n g excessive contact w i t h the low p H glycineHC1 buffer. A n t i b o d y r e c o v e r y values are p r e s e n t e d in Table 3. F o r the a n t i g e n - a n t i b o d y systems p r e s e n ted the values r a n g e d f r o m 53-100 p. cent after elution at p H 2.8. An a d d i t i o n a l 1.0.-26 p. cent is o b t a i n e d by eluting at pH 2.2. T h e r e f o r e , the total r e c o v e r i e s at both p H values r a n g e f r o m 63 to 126 p. cent. E s t i m a t i o n of r e c o v e r y w i t h all the
TABLE I[.
Antibody recovery following reisolation of once-isolated antibody. Antigenantibody system
Antibody adsorbed (mg)
pH 2.8
pH 2.2
P. cent recovered at pH 2.8
16
14.3
13.7
0.62
96
1.08
89
! Clonal IgG-I anti IgG.
Fab--anti Fab!
Antibody recovered (my)
Antibody added (mg)
9.6
9.06
8.14
i m m u n o a d s o r b e n t c o l u m n s w e r e done utilizing only 30-70 p. cent of t h e i r m a x i m u m a d s o r b i n g c a p a c i t y . W h e n u t i l i z i n g t h e i r full c a p a c i t y or surpassing it, substantially l o w e r r e c o v e r i e s of antib o d y w e r e o b t a i n e d (Table 3).
,2 o 2.0
Since only the a m o u n t of p r e c i p i t a t i n g a n t i b o d y a d d e d onto a c o l u m n is d e t e r m i n e d , w h e r e a s calculated r e c o v e r y is r e p r e s e n t a t i v e of both p r e c i p i t a t i n g and n o n - p r e c i p i t a t i n g antibody, r e c o v e r y figures t e n d to be overestimated. A c c o r d i n g l y , once-isolated a n t i b o d y w a s reisolated on the same i m m u n o a d s o r b e n t column. T h e values of t w o e x p e r i m e n t s are given in Table 2. The r e c o v e r y of clonal anti IgG w a s 9'6 p. cent and anti F a b w a s 8<9 p. cent after elution at p H 2.8. E l u t i o n at p H 2.2 gave an a d d i t i o n a l 4.3 p. cent, and 12 p. cent, r e s p e c t i v e l y . U n d e r these c o n d i t i o n s the true recov e r y values are 10,0 p. cent.
I.$
I0
0.5
DISCUSSION.
0 I0
5
15
20
Fractions
FIG. 2. - - Antibody elution profiles from a Bioge]adsorbed Lysozyme-SAB immunoadsorbent column (2.5 × 37 em). Two consecutive isolations of anti Lysozyme antibody were done with this column : one, ( • $-- ) from 52.5 ml, the other, ( - A - - A - ) from 15 ml of rabbit anti Lysozyme immunesernm, respectively. Elution with the pH 2.8 glycine-ttC1 buffer was begun at Fraction 1 following washing of unadsorbed proteins with 9.1 M phosphate buffer, pH 7.0. Fractions of 5.5 ml were collected into 2 ml of 1 M phosphate buffer pH 7.0. The flow rate was 80 ml/hr.
BIOCHIMIE, 1972, 54, n ° 2.
The Biogel-insoluble d e r i v a t i v e c o m p l e x is h i g h l y stable ; n e i t h e r v i g o r o u s s h a k i n g n o r successive w a s h i n g w i t h acid or neutral buffers leads to a detectable release of the adsorbed insoluble particles. No loss of the specificity of the i m m u n o a d s o r bent has been o b s e r v e d f o l l o w i n g a d s o p t i o n on Biogel. This could be e x p e c t e d since any modification of the specific antigen w o u l d result f r o m the p r o c e d u r e used for its insolubilization, not on
Biogel-adsorbed insoluble proteins for antibody isolation.
207
TABLE ] [l.
Antibody recovery from Biogel immunoadsorbent columns.
Antigen_antibody system
Clonal lg-anti IgG . . . . Clonal Ig-anti F e . Fc-anti Fe . . . . . . . . . . F a b - a n t i Fab . . . . . . . . .
~ .
Lysozyme- anti L y s o zyme . . . . . . . . . . . . . . . . i
Antibody recovered (mg)
P. cent recovered
pH 2 . 8
pH 2.2
at pH 2.8
13.2 36 14.85 27 12.5 18.4
9.3 16.3 7.9 9.3 9.2 10
3.1 0.814 1.48 Not done Not done Not done
70 45 53 34 73 54
20.6
20.72
5.4
100
Antibody added (mg)
Antibody absorbed (rag)
13.2 36 14.85 27 12.5 22 20.6
t h e a c r y l a m i d e w h i c h s e r v e s m a i n l y as a s u p p o r t i n g m a t r i x a n d i n t h i s r e s p e c t is e s s e n t i a l l y n e u tral. Nevertheless, in several experiments not r e p o r t e d h e r e , a d s o r p t i o n of a n t i b o d y w a s h i g h l y s p e c i f i c as r e p o r t e d w i t h g l u t a r a l d e h y d e - i n s o l u b i l i z e d p r o t e i n d e r i v a t i v e s [4]. O n e e x c e p t i o n w a s n o t e d i n t h e c a s e of t h e I . y s o z y m e i m m u n o a d s o r b e n t . F r o m 15 m l of s e r u m of a n u n i m m u n i z e d r a b b i t 1.7 m g of p r o t e i n w a s e l u t e d at p H 2.8. T h e s a m e a m o u n t of t h e s p e c i f i c i m m u n e s e r u m g a v e 2,0.72 m g of e l u t e d a n t i b o d y p r o t e i n ( T a b l e 3). A s i m i l a r n o n - s p e c i f i c a d s o r p t i o n of i n s o l u b i l i z e d L y s o z y m e h a s b e e n r e p o r t e d [20]. T h e a n t i b o d y a d s o r b i n g c a p a c i t y of t h e c o l u m n s w a s m o r e e l e v a t e d t h a n is r e p o r t e d f o r o t h e r syst e m s w h e r e e i t h e r p r o t e i n - p r o t e i n p o l y m e r s [43 o r p r o t e i n - a c r y l a m i d e p o l y m e r s [21] w e r e u s e d . T h i s relatively more elevated capacity can reasonably b e a t t r i b u t e d to t h e h i g h d i s p e r s i o n of t h e i n s o luble derivative which thus provides a greater surface area for contact with antibody. The capacity figures given in Table 1 are undere s t i m a t e d . T h i s is so, s i n c e o n l y t h e a m o u n t of p r e cipitating antibody was used to calculate capacity. Non-precipitating antibody present in varying proportions in the different immunesera, however, also c o m b i n e s w i t h t h e i m m u n o a d s o r b e n t and t a k e s u p a c o r r e s p o n d i n g a m o u n t of its c a p a c i t y . R e c o v e r y v a l u e s of a n t i b o d y a r e i n t h e s a m e r a n g e as t h o s e r e p o r t e d f o r g l u t a r a l d e h y d e - i n s o l u b i l i z e d d e r i v a t i v e s [4]. I s o l a t i o n of a n t i b o d y f r o m c o l u m n s s a t u r a t e d to their maximmn capacity gives substantially lower r e c o v e r y v a l u e s . T h i s h a s b e e n o b s e r v e d w i t h glut a r a l d e h y d e - i n s o l u b i l i z e d p r o t e i n d e r i v e s [22].
BIOCHIMIE, 1972, 54, n ° 2.
Acknowledgments. This work was supported by the ¢ Direction G6n6rale la Recherche Scientifique et Technique ~, Convention n ~' 70.02.270. The a u t h o r s are indebted to Dr. S. A v r a m e a s for his c o n s t a n t interest a n d criticism of this work. P~SUMg. Des polym6res de prot~ines o b t e n u s p a r i n s o l u b i l i sation avee la g l u t a r a l d e h y d e ont ~t6 disperses e n particu]es de faible d i m e n s i o n , adsorh~s sur Biogel et uti]is~s eomme i m m u n o a d s o r b a n t s p o u r l'iso]ement d'anticorps. De tels d~riv~s ont une h a u t e eapaeit~ d'adsorption p o u r les anticorps. Des r e n d e m e n t s q u a n t i t a t i f s d ' a n t i c o r p s sont o b t e n u s apr6s 61ution avec un t a m p o n Glycocolle-HCi h pH 2.8. Utilis6s sur colonne de c h r o m a t o g r a p h i c , de tels d6riv6s poss~dent des vitesses d'~coulement ~lev6s et p e r m e t t e n t d'effectuer l ' i s o l e m e n t d ' a n t i c o r p s avec u n e rapidit6 relative. ZUSAMMENFASSUNG. P r o t e i n p o l y m e r e , die d u r e h I n s o l u b i l i s a t i o n m i t Glut a r a l d e h y d e r h a l t e n w u r d e n , w u r d e n hoeh dispergiert, a u f Bio-Gel a d s o r b i e r t u n d als I m m u n o a d s o r b e n t e n z u r Isolierung der A n t i k 6 r p e r v e r w a n d t . Derartige Derivate h a b e n den A n t i k 6 r p e r n gegeniiber eine h o h e Adsorptionskapazit~it. Q u a n t i t a t i v e Ertr~ige der AntikSrper w u r d e n n a e h E l u i e r u n g m i t einem Glykoko]l- HC1-Puffer bet pH 2,8 erhalten. Uber e i n e r Chromatographiesi~ule hesitzen diese Derivate eine hohe Flussgeschwindigkeit u n d erlauben, die T r e n n u n g der A n t i k 6 r p e r m i t r e l a t i v e r Schnelligkeit durchzufiihren. REFERENCES. 1. Campbell, D. H. & Weliky, N,. (19~7) in Methods in I m m u n o l o g y and I m m u n o c h e m i s t r y , Vol. 1, C. A. W i l l i a m s and A. H. Chase ed., Academic Press, p. 365. 2. Silman, I. H. & Katehalski, E. (1966) Ann. Rep. Biochem., 35, 873.
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Marc Stanislawski
and
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