A preparation of Fc fragment of normal mouse IgG1 for production of rabbit anti mouse IgG1 serum

Journal of Immunological Methods, 8 (1975) 203--212 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

A P R E P A R A T I O N O F Fc F R A G M E N T O F N O R M A L M O U S E IgG1 F O R P R O D U C T I O N O F R A B B I T A N T I M O U S E IgG1 S E R U M

T.N. HARRIS, SUSANNA HARRIS and EDWARD M. HENRI From the Joseph Stokes, Jr., Research Institute of the Children's Hospital of Philadelphia, and the Department of Pediatrics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. (Received 4 December 1974, accepted 14 March 1975)

Ascitic fluid globulins obtained by peritoneal irritation from normal CBA mice were digested with papain, and the digests were examined by gradient chromatography on CM cellulose. The classical three peaks of OD2s 0 found by Porter with papain digests of rabbit IgG were obtained. Fraction II contained material which reacted in immunodiffusion against mouse anti-IgG. The ascending part of this peak reacted againt anti-IgG l and not against anti-IgG2, anti-IgG2 reacting material appearing at detectable levels in the descending part of this peak. A pool from the ascending part of this peak, containing Fc of IgG1 and not visibly contaminated with Fc of IgG2, was injected into rabbits. The resulting antisera were free enough of anti-IgG2 to be effective in removing IgG 1 from alloantibody-containing globulins without appreciable loss of antibodies of IgG2 class, thus allowing the complement-dependent IgG2-class antibodies full expression of their titer, without competition by IgG1 class antibodies. Chromatography of native mouse globulin did not produce a similar degree of separation of IgG 1 from IgG2.

INTRODUCTION A r e c e n t s t u d y o f t h e e f f e c t s o f anti H-2 a l l o a n t i b o d i e s on t h e t i m e o f r e j e c t i o n o f p r i m a r y skin allografts in n o r m a l m i c e y i e l d e d a range of e f f e c t s including, at o n e e x t r e m e , a c c e l e r a t e d r e j e c t i o n , and at t h e o t h e r , p r o l o n g e d r e t e n t i o n (Harris et al., 1 9 7 2 ) . T h e a n t i b o d y - c o n t a i n i n g globulins w e r e all e v a l u a t e d b y t h e i r t i t e r o f suppressive a l l o a n t i b o d y - - an a n t i b o d y w h i c h can p r e v e n t t h e s y n t h e s i s o f a n t i b o d y b y allogeneic spleen cells o f m i c e previously i n j e c t e d w i t h an antigen (Harris et al., 1 9 5 8 ) . T h e t e s t e m p l o y e d was an a d a p t a t i o n (Harris a n d Harris, 1 9 6 6 ) o f t h e h e m o l y t i c a n t i b o d y p l a q u e test ( J e r n e a n d N o r d i n , 1 9 6 3 ; I n g r a h a m and Bussard, 1 9 6 4 ) , and all t h e globulin p r e p a r a t i o n s w e r e w i t h i n a given, high, range o f t i t e r o f t h e suppressive effect. T h e o b s e r v a t i o n o f such d i f f e r e n t results f r o m t h e use o f various globulin pools, essentially 7S p r e p a r a t i o n s , i n d i c a t e d t h e i m p o r t a n c e o f det e r m i n i n g t h e d i s t r i b u t i o n o f t h e anti B a l b / C a n t i b o d i e s o f o u r globulin p r e p a r a t i o n s b e t w e e n sub-classes IgG1 and I g G 2 , b e c a u s e of t h e k n o w n diff e r e n c e s in biologic e f f e c t s o f a n t i b o d i e s o f these sub-classes in t h e guinea pig

204 (Bloch et al., 1963) and the mouse (Nussenzweig et al., 1964). Because of the great difficulty of separating IgG1 and IgG~ in the mouse (Fahey et al., 1964) it was decided to approach the problem by removing one of the 7S immunoglobulins from portions of our globulin preparations, by the use of heterologous sera vs individual sub-classes of mouse IgG, leaving the alloantibodies of the other sub-class. For the injection of rabbits to produce the necessary reagents, the Fc fractions of the respective immunoglobulins would be the materials of choice. Also, Coe (1966) has shown a greater difference in electrophoretic mobility between the Fc's of IgG~ and IgG2 than between the original immunoglobulins. A corresponding difference in the chromatographic behavior of the two Fc fragments was sought, in the present study, as a method of preparing the materials for injection into rabbits. The present paper describes a method of preparing a part of the Fc fragment of mouse IgG~, from papain digests of normal mouse globulins, with only slight contamination by Fc of IgG2, based on the earlier appearance of the Fc of IgG~ than that of IgG2 on gradient elution from CM cellulose. Also, such preparations of the Fc of mouse IgG1 are shown to cause the production of rabbit anti mouse IgG~ with a sufficiently low level of contamination by anti-IgG2 to permit use of the serum for the removal of IgGl from mouse globulin without significant loss of the IgG2 -class antibody. MATERIALS AND METHODS

Preparation of normal CBA globulins Normal CBA mice were given two i.p. injections, at a two-week interval, of 0.25 ml of equal parts of arlacel (Hill Top Research Inc., Miamiville, Ohio) and bayol (Humble Refining Company, Bala Cynwyd, Pa.), the mixture also containing oleic acid at a final concentration of 0.5%, as previously described (Harris et al., 1971). Alternatively, where the oleic acid appeared to be toxic to the mice, 3 weekly injections of Freund's incomplete adjuvant (Difco, Detroit, Mich.) were given. At appropriate intervals after the second injection of Bayol--Arlacel, usually in the second to fourth week, when the ascitic fluid was present in the greatest amounts, these fluids were collected and clarified by stirring for 60 min in the cold with an equal volume of fluorocarbon (Freon TF, Du Pont Chemical Company, Wilmington, Del.). From the cleared preparations, protein precipitable with ammonium sulfate at 27% of saturation was removed. The supernate was brought to 50% of saturation, and the protein thus precipitated was collected as the globulin, dialyzed, and stored at 4× its original concentration in the ascitic fluid.

Papain digest To 20 ml of ascitic fluid globulin (OD2 s 0 = 20) were added 16 mg of papain 2× crystallized (Worthington Biochemical Co., Freehold, N.J.); L-

205 cysteine HC1, 80 mg in 0.5 ml, adjusted to pH 7; EDTA, 4 mg; and 0.5 ml of 0.1 M PO4 buffer, pH 8. After 4 hr at 37 °C the reaction was stopped by adding 0.5 ml of 0.1 N Na iodoacetate, pH 7. The mixture was cleared b y centrifugation and the supernatant was then dialyzed against 4 1 of the desired buffer.

Chromatography CM cellulose (Standard grade, Schleicher and Schuell, Keene, N.H.) 0.77 meq/g, was suspended in 1 M NaC1 containing 0.5 N NaOH. The suspended cellulose was stirred for 30 min, washed repeatedly with de-ionized water on a filter, resuspended in 2 1 for settling. After adequate removal of fines, the sediment was resuspended in 0.01 M acetate buffer, pH 5.5. The suspension was de-aerated and packed in a K 25/45 Pharmacia column packed to 37 cm. The column was equilibrated in the cold by washing overnight with the starting buffer. After application of the sample, addition of 0.01 M acetate buffer was continued until all unadsorbed material had been recovered. Gradient elution to 0.6 M acetate buffer pH 5.5 (total vol 900 ml) was n o w begun, at a column flow rate of 30--40 ml/hr under a pressure of 15--25 cm. Column fractions were examined or pooled as will be described below, and concentrated by pervaporation, dialysis, lyophilization and solution at appropriate concentrations for examination by immunodiffusion.

Immunodiffusion (IDF) Microscope slides, 3 in. × 1 in., were precoated twice with 0.2 ml agarose solution and dried overnight at 37°C. A solution of 0.85% agarose in phosphate-buffered saline (PBS) containing 0.02% Na azide was distributed by allowing 3 ml to flow onto the slides. After hardening of the agar in a humid b o x in the refrigerator, sets of 7 wells, comprising a central well and 6 radially distributed wells around it, were cut with a die, and the cut-out agar was removed with the tip of a 19-gauge needle. Solutions to be tested were introduced into the wells in 0.005 ml vol. The slides were then allowed to develop overnight at r o o m temperature in a humid box, and photographed the next day with the Cordis immunodiffusion camera.

Radial immunodiffusion (RID) This was done essentially by the m e t h o d described by Fahey and McKelvey (1965) with 0.6 ml antiserum--agarose solution on a 22-mm square cover slip precoated with 0.2% agarose. The antiserum--agarose solution was prepared by mixing equal volumes of 1.7% agarose in PBS with anti mouse IgG1 or anti mouse IgG: serum (Meloy Laboratories, Springfield, Va.) diluted 1 : 12 in PBS. The same die was used, employing only 3 of the outer circle of 6 wells of each set.

206 RESULTS

Chromatography of papain digests of CBA ascitic fluid globulin Papain digests of normal CBA ascitic fluid globulin, prepared as described above, were applied to columns of CM cellulose equilibrated to 0.01 M acetate buffer pH 5.5. After collecting OD2 s 0 -absorbing material not adsorbed to the column, a linear gradient to 0.6 M acetate buffer, pH 5.5, was begun, and further fractions were collected until the OD2 s 0 approached 0. A typical elution pattern is shown in fig. 1. Because of the similarity of the elution to the classical pattern originally described for papain digests of rabbit IgG under quite similar conditions of chromatography by Porter (1959), these peaks were designated fractions I, II, III, with the additional designation of fractions IIa and IIb for the ascending and descending parts of fraction II, as will be indicated below.

Immunodiffusion of fractions of the eluate for IgG1 and IgG2 antigenic material In preliminary examinations of the fractions by immunodiffusion (IDF) against anti-IgG~ and anti-IgG2 sera, material reacting both with anti-IgGl and anti-IgG2 was found in pooled fraction III, and neither reactivity was found in fraction I. However, pooled fraction II also gave evidence of the presence of Fc, by giving bands with anti-IgG1 and anti-IgG2 sera. Since fraction II appeared to contain the first portions of Fc, sub-fractions in that region were tested to see whether the earliest Fc to appear would give

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e v i d e n c e o n l y o f IgG~ activity. This was, in fact, f o u n d to be t h e case. Figure 2 s h o w s t h e r e a c t i o n s in I D F vs anti-IgG~ a n d anti-IgG2 o f various f r a c t i o n s o f such a run, t h e c e n t r a l well c o n t a i n i n g in each case anti-IgGl ( u p p e r r o w ) or anti-IgG2 ( l o w e r r o w ) . T h e materials b e i n g e x a m i n e d in t h e r e s p e c t i v e p h o t o g r a p h s are, f r o m l e f t t o right, p o o l e d f r a c t i o n I, p o o l e d fraction IIa (ascending part of fraction II), pooled fraction IIb (descending part o f f r a c t i o n II), and p o o l e d f r a c t i o n I I I . In e a c h circle t h e m a t e r i a l is used at 12 o ' c l o c k at a s t a r t i n g O D level (see fig. legend) a n d t h e n in a series o f 2-fold dilutions, c l o c k w i s e , t h r o u g h 4 m o r e wells. Well No. 6 is in each case a s t a n d a r d p r e p a r a t i o n o f m o u s e IgG~ f o r t h e u p p e r r o w , or m o u s e IgG2 f o r t h e lower. I t can be seen t h a t n e i t h e r o f t h e F c ' s was in e v i d e n c e in fract i o n I, in t h e range o f c o n c e n t r a t i o n used, and b o t h w e r e f o u n d in f r a c t i o n s I I b a n d I I I . I n f r a c t i o n IIa, IgG~ -reacting m a t e r i a l was clearly d e m o n s t r a t e d in a series o f 2-fold dilutions. I g G 2 - r e a c t i n g m a t e r i a l did n o t p r o d u c e a p r e c i p i t i n line even at t h e highest c o n c e n t r a t i o n . At this c o n c e n t r a t i o n t h e r e is, h o w e v e r , an i n d i c a t i o n o f such m a t e r i a l in t h e s h o r t e n i n g o f t h e r e f e r e n c e line at well 6.

Comparison of eluted fractions of digest with analogous fractions of undigested globulin T o s h o w t h a t t h e d i f f e r e n c e in t h e p o i n t o f a p p e a r a n c e o f the IgGl and IgG2 r e a c t i n g m a t e r i a l was a result o f a greater charge d i f f e r e n c e b e t w e e n t h e Fc f r a g m e n t s , similar c h r o m a t o g r a p h y was p e r f o r m e d o n u n d i g e s t e d n o r m a l CBA ascitic fluid globulin. T h e e l u t i o n p a t t e r n o n CM cellulose in a series of

208

4 runs was remarkably similar to that of a papain digest of such globulin, with only minor differences in the positions of peaks II and III. However, material reacting with anti IgG2 appeared in the first fraction of peak II. (Such material was already barely detectable in peak I). In contrast, on CM chromatography of a papain digest of the same preparation of normal CBA ascitic fluid globulin, IgG2 material was not found even in the peak II until the second descending fraction, although IgG~ material was found in the first portion of eluate comprising peak II. Radial immunodiffusion vs anti-IgG~ and anti-IgG2 Fractions of digested mouse globulin and native globulin were also examined against these t w o antisera by radial immunodiffusion (RID). A set of such data is shown in fig. 3. In each of the four sets of wells a standard solution of IgG1 or IgG~ from m y e l o m a t u m o r preparations (Potter, 1967) at OD2 a 0 0.5 is placed in the 8 o'clock position. The other t w o wells used in each group, at 12 and 4 o'clock, contain fractions of runs of the digested or undigested globulin in a pool of the ascending part of fraction II, and the peak OD fraction. RID vs anti-IgGl is shown in the upper row, and vs anti-IgG~ in the lower. The digest is shown in the left column and the Digest

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209 undigested on the right. It can be seen that whereas these fractions of both digested and undigested protein gave evidence of containing IgG~ reacting material, IgG2 material is absent from the two fractions of the digest but is present in the fractions of the undigested globulin.

Immunization of rabbits with fraction IIa and use of the antiserum to permit full expression of IgG2 -class antibody in CBA anti Balb/C globulin We have recently shown that following immunization with Balb/C spleen cells, CBA or C3H anti-Balb/C globulin contains anti-Balb/C antibody of both 7S sub-classes, and that the full titer of the suppressive or other complement fixing antibody present could be revealed only by treating the antiBalb/C globulin with rabbit anti mouse IgG1 (Harris and Harris, 1972). In this procedure, rabbit anti mouse IgG1 was added in increasing amounts to constant amounts of CBA or C3H anti-Balb/C globulin, and the supernates of such precipitin mixtures were examined for their titer of suppressive antibody. It was f o u n d that the antibody titer rose with increasing amounts of anti-IgG1 a d d e d until a plateau of suppressive titer was attained, and remained at that level through further increases in the a m o u n t of anti-IgG~ added. This was taken as the true level of the IgG2 -class antibody present. It was f o u n d there that a condition for the attainment of this plateau of titer was an absence of contaminating anti-IgG2 in the anti-IgG1 serum, or a contamination of sufficiently low level to permit the titer to remain at the plateau level through several further increases in a m o u n t of anti-IgG1 serum added before any reduction in titer due to contaminating anti-IgG~. Although the IIa fraction of our papain digest gave no evidence of contaminating IgG2 material at our threshold of detection by IDF, it was decided, since antibodies induced by contaminating protein provide a substantially more sensitive test than the detection of such contamination in the antigen preparation, to immunize rabbits and seek evidence of a sufficiently pure preparation of anti IgGl in those sera. Rabbits were injected in the hind footpads with approx. 1 mg of the IIa fraction combined with an equal volume of complete Freund's adjuvant. Four weeks later serum obtained from these rabbits was examined by IDF vs IgG1 and IgG2 and then by their ability to yield plateau titers of anti-Balb/C suppressive antibody in CBA anti-Balb/C globulins. In a few of the rabbits so injected the sera showed faint indications of anti-IgG2 at our threshold of detection by IDF. The sera of the remaining rabbits were pooled and these were used in a test for purity of anti-IgG~ . T o 0.1-ml quantities of CBA anti-Balb/C globulin were added the pooled antiserum in amounts of 0.1, 0.2, 0.3 ml, and so on. The precipitin mixtures were incubated for 1 hr at 37°C and overnight at 4°C. After centrifugation the supernates were examined for suppressive titer for Balb/C spleen cells. Data of two such CBA anti-Balb/C globulins are shown in fig. 4. It can be seen that from original titers of the anti-Balb/C globulin, with no anti-IgG~ Serum added, the titer rose with addition of increasing amounts of

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Fig. 4. Suppressive titers vs B a l b / C spleen cells of s u p e r n a t e s of p o r t i o n s of C B A antiB a l b / C g l o b u l i n t r e a t e d w i t h increasing a m o u n t s o f r a b b i t anti-IgG 1 , t h e a n t i s e r u m being o b t a i n e d b y i n j e c t i o n o f F r a c t i o n IIa p r e p a r e d as d e s c r i b e d here. F o r each a l l o a n t i b o d y p r e p a r a t i o n , t h e t i t e r of t h e t r e a t e d g l o b u l i n is seen to rise f r o m its original level to a p l a t e a u level o f suppressive titer, a t w h i c h it r e m a i n s c o n s t a n t t h r o u g h c o n t i n u i n g increases in a m o u n t o f a n t i IgG l s e r u m used to t r e a t t h e p o r t i o n s of CBA a n t i - B a l b / C globulin.

anti-IgGl serum until, after 0.2 ml or 0.3 ml, respectively, a plateau of elevated titer was reached at which no further increase occurred with further increase in a m o u n t of anti IgG~ added. This indicated so high a ratio of anti IgG1 to anti IgG2 in these sera, that the complete removal of IgGl-class immunoglobulins from CBA anti-Balb/C preparations did not remove detectable amounts of the suppressive, IgG2 -class antibody. DISCUSSION

The well-known differences in biologic function among antibodies of different 7S sub-classes, notably between antibodies of IgG1 and IgG2 in several species of rodents, gives special importance to the problem of separating these, or to the feasibility of examining the effects of each of these separately. However, the very small difference in the net charge of these sub-classes of immunoglobulins makes physical separation, as by electrophoresis or chromatography, essentially impossible. This requires the use of some other approach to the study of individual sub-classes of antibodies, such as the use of a reagent like heterologous anti-IgG1 or anti-IgG2 serum. However, even in the amounts of the order required for immunization for such antisera, the preparation of either sub-class virtually uncontaminated by the other is itself a problem, especially in the mouse, where the charge difference between the two sub-classes is particularly small. The present study describes the preparation, from normal mouse globulin, of a fraction of papain-digested globulin

211 containing Fc of IgG~ sufficiently u n c o n t a m i n a t e d by that of IgG2 to be suitable for immunization to produce an effective anti-IgG~ reagent. To achieve this degree of separation use was made of the observation by Coe (1966) that the difference in electrophoretic mobility between the Fc fragments of mouse IgG1 and IgG2 is greater than the difference between the whole Ig molecules, indicating a greater difference in net surface charge between the Fc's than the parent Ig molecules. This led to a chromatographic m e t h o d for collecting some of the Fc fragments of mouse IgG~ with so little contamination with Fc of IgG: as to permit their use in the immunization of rabbits for anti-IgG~ serum. That the greater difference in charge between the Fc's than the original Ig's was involved in this chromatographic behavior was clearly shown by the failure of analogous fractions from chromatography of whole Ig to show this separation, as shown in fig. 3. Although the amounts of material required for immunization are small, the criterion of contamination of one Fc by the other is especially strict when this is judged by the antibodies produced, since immunization amplifies the effect of trace contamination of the antigen. A recent observation on the effect of anti-mouse IgG1 on the naturally occurring mixture of the sub-classes of 7S in sera of immunized mice has provided us with a sensitive indicator of the degree of the contamination of anti-IgGl with anti-IgG2, and thus an especially sensitive indicator of the contamination of IgG~ with IgG2 in the preparation injected into the rabbits. In that study (Harris and Harris, 1972) it was found that if an anti-IgG~ serum virtually free of contamination by anti-IgG2 was added in increasing amounts to constant portions of an alloanfibody-containing mouse serum or globulin, the titer of a complement-dependent antibody in the treated portions of the serum rose with increasing amounts of the anti-IgG1 added until it attained a plateau level at which it expressed the true titer of the complement-dependent, IgG2-class antibody present in the serum. However, contamination by antiIgG2 in such sera gave the expected result of combining with the IgG2 -class antibody and prevented the titer from reaching the plateau level on such treatment. The clear attainment and 'holding' of the plateau titer in the globulin preparations shown in fig. 4 indicates that if any contaminating anti-IgG2 was present in our anti-IgG~ serum it was so low in concentration that even two or three times the a m o u n t of rabbit antiserum required to remove all the mouse IgG1 present failed to affect appreciably the titer of the IgG2 -class antibody. This, then, provides an especially critical test of the freedom from contamination of the chromatographic fraction of mouse globulin originally used in immunizing the rabbit for production of the anti-IgG~ reagent. Since the purpose of the present study was to examine the feasibility of obtaining Fc of IgG1 sufficiently free of contaminating IgG2 material for immunization of rabbits, for the purposes noted, no study was made of fraction I and II in terms of Fab effects. However, the substantial resemblance to the chromatographic pattern obtained by Porter (1959) with rab-

212

bit globulin would strongly suggest t hat the fraction II here contains Fab fragments. If this is so, then it is a reflection of the m uch greater antigenicity of the Fc than the Fab fragments, t hat the single relatively small a m o u n t injected into the rabbits did n o t p r o d u c e enough anti light-chain a n t i b o d y to precipitate IgG2 by their c o m m o n light chains. No absorption of the antisera with mouse IgG2 or light chains was required for the effective use of the anti-IgG~ serum in bringing mouse alloantisera to a plateau titer. A p o in t should be made here as to the possibilities of broader application of this m e t h o d , to sera of o t h e r species. In the case of the mouse, the IgG's of which are the subject of this paper, we have also used preparations of Fc of IgG1 obtained f r o m IgGl -class m y e l o m a protein, with similar results, in that the antisera could be used effectively in bringing our alloantibodycontaining mouse globulins to plateau levels of c o m p l e m e n t - d e p e n d e n t antib o d y effect. However, the m e t h o d of obtaining heterologous anti IgGl serum described here, and its application to revealing the true level of a c o m p l e m e n t - d e p e n d e n t a n t i b o d y in an antiserum of that species, can find a wider application in that it may well be applicable to the p r o d u c t i o n of heterologous anti-IgG1 serum for species in which m y e l o m a proteins of k n o w n IgG sub-class are n o t available. It was in one such species, in fact, the guinea pig, th at Kourilsky et al. (1963) f o u n d evidence of internal competition between antibodies of the 7S sub-classes in c o m p l e m e n t fixation. ACKNOWLEDGEMENT This study was supported by U.S. Public Health Service Grants CA 14487 and AI 11466, and by Grant IM-3 of the American Cancer Society.

REFERENCES Bloch, K.J., F. Kourilsky, Z. Ovary and B. Benacerraf, 1963, J. Exp. Med. 117,965. Coe, J.E., 1966, Immunochemistry 3, 427. Fahey, J.L. and E.M. McKelvey, 1965, J. Immunol. 94, 84. Fahey, J.L.: J. Wunderlich and R. Mishell, 1964, J. Exp. Med. 120, 223. Harris, S. and T.N. Harris, 1966, J. Immunol. 96,478. Harris, S., T.N. Harris and M.B. Farber, 1958, J. Exp. Med. 108,411. Harris, T.N. and S. Harris, 1972, J. Immunol. 109, 1096. Harris, T.N., S. Harris, M.H. Bocchieri, M.B. Farber and C.A. Ogburn, 1972, Transplantation 14, 495. Harris, T.N., S. Harris and C.A. Ogburn, 1971, Transplantation 12, 448. Ingraham, J.S. and A. Bussard, 1964, J. Exp. Med. 119, 667. Jerne, N.K. and A.A. Nordin, 1963, Science 140, 405. Kourilsky, F.M., K.J. Bloch, B. Benacerraf and Z. Ovary, 1963, J. Exp. Med. 118, 699. Nussenzweig, R.S., C. Merryman and B. Benacerraf, 1964, J. Exp. Med. 120, 315. Porter, R.R., 1959, Biochem. J. 73, 119. Potter, M., 1967, in: Methods in Cancer Research, Vol. 2, ed. H. Busch (Academic Press, New York) p. 105.