Purification of antibodies with immunoadsorbents prepared using bromoacetyl cellulose

Immunochemistry. Pergamon Press 1967. Vol. 4, pp. 11-22. Printed in Great Britain

PURIFICATION OF ANTIBODIES WITH IMMUNOADSORBENTS PREPARED USING BROMOACETYL CELLULOSE* J. B. ROBBINS,tJ. HAIMOVICHand M. SELA Section of Chemical Immunology, The Weizmarm Institute of Science, Rehovoth, Israel (Received 10 June 1966)

Al~tractmConjugates prepared by reacting bromoacetyl cellulose with proteins, synthetic polypeptides, and hapten derivatives of proteins and synthetic polypeptides have been studied for their immunoadsorbent properties. Each antigen showed differences in the extent of attachment to the cellulose and the ability of the antigen-cellulose conjugate to adsorb antibody. These conjugates had high capacity for extracting antibodies of the IgM and IgG classes of immunoglobulins from antisera. There was almost no non-specific interaction with other serum proteins at conditions at which the antibodies were bound to the immunoadsorbent. The immunoadsorbents were stable for several months and could be reused repeatedly without significant loss of their antibody binding capacity. The antigencellulose conjugates described may be useful as analytical reagents for the detection of antibodies as well as for isolation of specific immunoglobulins. INTRODUCTION THE immunospeeifie purification of antibodies is an essential step in efforts to better understand immunological phenomena on a molecular level. Methods for the isolation of antibodies utilize either antigen-antibody precipitates or immunoadsorbents. The use of antigens, insolubilized by chemical binding to a solid carrier, namely the diazotization product of p-aminobenzyl cellulose, for purification of antibodies was introduced by Campbell et al. ~1~ Since then there has been much effort directed towards the preparation and study of other immunoad.qorbent~, c~-4~ The advantage of using an insoluble immunoadsorbent to purify antibodies from protein mixtures is that antibodies are removed from solution by their primary interaction with the antigen. In contrast, isolation procedures which depend upon precipitation of the antibody with the antigen utilize a secondary property of antigen-antibody interaction, There are several examples of antibodies that do not form insoluble complexes after they interact with the antigen. ~5-7~ In addition, there are many experimental situations in which the concentration of antibody is too low for the formation of a precipitate with the antigen.~S, 9~ The use of immunoadsorbent~ in the latter two instances would permit the isolation of the antibody based upon its primary activity, that of binding to the antigen. An ideal immunoadsorbent should: (1) be highly insoluble: (2) bind all the specific antibodies under conditions at which it will not retain other proteins: (3) release quantitatively the adsorbed antibodies without significant change in their activity: (4) be stable with respect to time. • This investigation was supported in part by a U.S. Public Health research grant (AI-04715) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. t Recipient of a N.I.H. Career Development Award HD-053-00384. 11

12

J. B. ROBBINS, J. HAIMOVICHand M. SELA

The synthesis of bromoacetyl cellulose (BAC) tl°~ permitted the preparation of highly substituted protein-cellulose conjugates which retained much of the biological activity of the protein, c11~We wish to report in this paper the successful use of conjugates, prepared using BAC, as immunoadsorbents. These immunoadsorbents included conjugates of proteins, synthetic polypeptides and haptens attached to proteins and synthetic polypeptides.

Antigens

MATERIALS AND METHODS

The proteins used in these experiments were obtained from commercial sources: papain, egg white lysozyme and egg white albumin, 2 × crystallized (Worthington Biochemicals, Freehold, N.J.), bovine pancreatic ribonuclease A, 5 × crystallized (Sigma Chemicals, St. Louis, Mo.), diphtheria toxoid (Cutler Laboratories, Los Angeles, Calif.), bovine serum albumin (BSA) and rabbit serum albumin (RSA) (Armour, Chicago, Ill.), human serum albumin (HSA) (Magen David Adorn Blood Bank, Jaffa, Israel). Hapten-protein conjugates were prepared according to published methods: (a) dinitrophenyl (DNP) derivatives of ovalbumin and HSA, containing approximately 13 moles of DNP/mole protein were prepared according to Sangertm: (b) the p-azobenzenearsonate conjugate of HSA cm (30 moles/mole protein) was a gift from Dr. F. Borek; (c) the polypeptidyl protein conjugates, poly-DL-analyl and poly-DL-phenylalanyl RSA, having respectively 287 and 57 amino acid residues attached per mole of protein, t14~ were a gift from Dr. I. Schechter. Poly-Ltyrosyl gelatin, ttS~ enriched with 14 per cent of tyrosine residues, was a gift from Dr. A. Rimon. The multichain synthetic polypeptide antigen, 353, p(Tyr, GIu)-pDL-Ala-pLys, to be denoted (T, G)-A - - L, was a sample with a tool. wt. of 165,000, prepared as described previously ~16~and was a gift from Miss Y. Stupp. The linear synthetic polypeptide antigen, 1004, Ala40Glu 15Tyr 12, to be denoted (T, G, A), was a sample with a mol. wt. of 17,200, prepared as described previously, t16~ and was a gift from Miss S. Bauminger. The uridine conjugate of multichain poly-DLalanine tm had a molecular weight of 80,000 and contained 9 per cent uridine. The pyridoxal conjugate of multichain poly-DL-alanine had a mol. wt. of 80,000 and contained 9 per cent pyridoxal, tts~ The last two antigens were a gift from Dr. H. Ungar-Waron.

Antisera The antisera were elicited by immunization of rabbits with either one or two injections of 5-10 mg of antigen emulsified in complete Freund's adjuvant (Difco, Michigan) injected intramuscularly or intradermally. Some of the antisera were kindly supplied by colleagues in the Section of Chemical Immunology. For purification of antibodies with p-azobenzenearsonate specificity, antisera to a conjugate of hexa-L-tyrosine c19~ were used. Antisera to various dinitrophenyl conjugates were described previously. ~z0~ Goat antisera, specific for rabbit IgG and IgM proteins were prepared by immunization with the Fc fragment and IgM respectively. Goat serum specific for the t,-chain was obtained after absorption of the anti-IgM with purified rabbit IgG.

Immunoadsorbents Prepared with Bromoacetyl Cellulose

13

Bromoacetyl cellulose Bromoacetyl cellulose was prepared according to Patehornik, c1°~ with several modifications. Powdered cellulose (Whatman) was washed with acetone and dioxane and then dried in vacuo over phosphorus pentoxide to a constant weight. A solution of 100 g of bromoacetic acid in 30 ml of absolute dioxane, was added to 10 g of cellulose powder and the suspension stirred at room temperature for 20 hr in a tightly stoppered 1 I. round bottom flask. Bromoacetyl bromide (75 ml) was added and the flask connected to a sodium hydroxide trap which prevented the entrance of water vapour and neutralized the hydrobromic acid which was evolved. This solution was stirred vigorously for 10-12 hr and then slowly poured, with vigorous stirring into 7 1. of deionized water which had previously been cooled to 4°C. The stirring was continued uninterrupted for 15 rain. The precipitated cellulose was washed exhaustively with deionized water and then in turn with 0-1 M sodium bicarbonate and deionized water over a sintered glass funnel, it was found that if the BAC had been dried extensively, its physical properties were changed and the extent of conjugation with the antigens was reduced. The BAC was, therefore, stored at 4°C in a moist state. To determine the extent of substitution, a sample of the cellulose was dried to a constant weight over phosphorus pentoxide in vacuo and the content of bromide analysed according to Schoniger. Izl~ The average degree of substitution in 6 preparations was 1.3 m-equiv. Br/g cellulose (range 1.0-1 "4). The BAC could be used for at least 3 months after preparation.

Preparation of the antigen-cellulose conjugates The physical adsorption of the antigen to bromoacetyl cellulose was done in 0.15 M sodium phosphate citrate buffers at the pH values given in Table 1. In those cases in which the physical adsorption of the antigen to the BAC was investigated as a function of pH, it was found that the optimum hydrogen ion concentration for the adsorption was unique for each conjugate. The use of a homogenizer ~11~facilitated this analysis. When the optimum pH was determined, 300-500 mg of the antigen was dissolved in the appropriate phosphate-citrate buffer and added to 1 g (dry weight) of the BAC. This suspension was stirred with maximum agitation for 30 hr at room temperature using a magnetic stirrer. To prevent foaming and bacterial contamination, a drop of Dow Corning anti-foam and toluene was added to the reaction mixture. The reaction mixture was then centrifuged at 10,000 × g for 10 min, resuspended in 30 ml of 0.1 M sodium bicarbonate buffer, pH 8.9, and allowed to stand at 4°C for 24 hr with occasional stirring. At this pH the chemical bonds between the BAC and the antigen were formed. The suspension was centrifuged at 10,000 x g for 10 min, the supernatant fluid discarded, and the cellulose resuspended in 0.05 M 2-aminoethanol-0.1 M sodium bicarbonate buffer, pH 8.9, to block any unreacted bromine, and allowed to stand at 4°C for another 24 hr. The unbound antigen was removed by centrifugation and resuspension of the conjugate in 0.15 M NaCI until there was no antigen in the supernatant fluid as measured by light absorbance at 280 m/z. In order to remove any antigen bound non-covalently to the cellulose, the sample was then resuspended in 30 ml of 8 M urea free of cyanate ions, tz2~stirred slowly for 24 hr at room temperature, centrifuged and washed with 8 M urea until there was no more

14

J . B . ROBBINS, J. HAIMOVICH a n d M . SELA

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Immunoadsorbents Prepared with Bromoacetyl Cellulose

15

antigen in the supernatant fluid as measured by light absorbance at 280 rap. The conjugate was resuspended in 0.15 M NaCI and centrifuged. To simulate the conditions of elution of the antibodies from the immunoadsorbent, the conjugates w e r e stirred in 0.1 M acetic acid at 37°C for 1 hr. No antigen was eluted from the cellulose conjugate under these conditions. Following centrifugation and resuspension the conjugates were stored in 0.15 M phosphate buffer, pH 7.4-, at 4-°C. The conjugate could be kept for at least 6 months without appreciable loss of antibodybinding capacity. The total amount of antigen bound to the cellulose was determined by nitrogen analysis (Dumas) of extensively dried samples.

Purification of the antibodies The serum was clarified by centrifugation at 20,000 × g for 1 hr at 4-°C. The lipid material that floated to the top was removed and the immunoadsorbent dispersed in the serum. The suspension was stirred (magnetic stirring) for 2 hr at 4-°C, and the cellulose conjugate was removed by centrifugation at 20,000 × g for 20 rain. The centrifuge tubes were inverted and drained over filter paper to insure good removal of the supernatant serum. The adsorbent was resuspended in 0.15 M NaCI and recentrifuged at 20,000 × g for 10 min, and this was repeated (usually 3-4 times) until the absorbance at 280 m~ of the washing fluid was less than 0.08. To elute the antibody from the adsorbent, two methods were used: (1) the antibodyimmunoadsorbent complex was resuspended in 0.1 M acetic acid, pH 2"8, and stirred at 37°C for 1 hr. The suspension was centrifuged at 20,000 × g for 30 min and the supernatant fluid dialysed against 350-700 volumes of 0.1 i NaC1-0.01 M Tris HC1, pH 7-0. (2) In two experiments, the adsorbed anti-hapten antibody was dissociated using the specific hapten. For the anti-DNP system, 0.1 M DNP-c-aminocaproic acid or dinitrophenol brought to pH 8.0 with NaOH was used. The anti-p-azobenzenearsonate antibodies were dissociated from the immunoadsorbent with 0.3 M sodium arsanilate, pH 8.0. Incubation of 1 g of the antibody-immunoadsorbent complex with 100 ml of the hapten stirred at 37°C released the antibody from the conjugate. The hapten was removed by chromatography/~a~ in the case of the DNP system, and by exhaustive dialysis against 0.15 M NaCI-0.01 M phosphate buffer, pH 7.4, for the arsanilate. The antibody solution was centrifuged again at 20,000 × g for 30 min to remove the slight amount of precipitate, concentrated by ultra-filtration to 10-20 mg/ml and then dialysed against 0-1 M NaCI-0.01 M Tris HCI, pH 7.0. The purified antibody solutions were stored at --20°C.

Other methods Quantitative precipitin reactions, immunoelectrophoresis and analytical ultracentrifugation were done according to published methods summarized by Kabat.C~ Gel filtration, using Sephadex G-200 (Pharmacia, Uppsala) was done as described.~5~ To determine the amount of protein that was precipitable by the antigen, the antibody concentration was adjusted to 1 "0 mg/ml using 14.0 as the EI~'~ 280 m# ~2e)and reacted with an equal volume of the specific antigen. RESULTS

Composition and antibody-binding capacity of the immunoadsorbents The reaction between the antigens and BAC proceeded in two steps. In the first

16

J. B. ROBBINS, J. HAIMOVICH a n d M . SELA

step, that of a physical adsorption, the antigens bound to a maximum extent of approximately 1.0-2.2/~ M/1 g of the BAC. However, in the second step, that of covalent binding, not all antigens, once physically adsorbed, reacted equally well with the BAC at pH 8 '9. This difference in the extent of covalent interactions of the various antigens resulted in a great variation in the amount of the antigen in the immunoadsorbents (Table 1). Thus, the lysozyme derivative contained a high ratio of antigen to cellulose, whereas the cellulose conjugates of (T, G, A) and of ribonuclease had comparatively low antigen to cellulose ratios. The antibodybinding capacity also differed from one immunoadsorbent to another. For example, the lysozyme-cellulose conjugate adsorbed only 0.46 mg antibody/1 mg antigen bound to cellulose, whereas the ribonuclease-cellulose conjugate adsorbed 3 mg antibody/1 mg antigen. The overall recovery was calculated from the total amount of precipitable antibody adsorbed from the serum and the total amount of antibody protein in the TABLE 2. PURIFICATION OF ANTI-DNP ANTIBODIES ELICITED BY VARIOUS DNP DERIVATIVES~ MAKING USE OF DNP-HSA-CELLULOSE

Immunogen DNP-poly-L-lysine DNP-(T, G)-A- - L DNP-(T, G)-A - - L* DNP-(T, G, L) DNP-poly-L-lysyl RSA DNP-ovalbumin

Antibody in the Antibody serum isolated Yield (mg/ml) (mg) (per cent) 0"10 0"35 0'08 0"26 0"43 2.4

72 105 28 35 70 250

48 68 88 67 81 95

Anti-DNP antibodies elicited by the various DNP derivatives were purified by adsorption and elution from DNP-HSAcellulose. The yield was determined by dividing the total amount of purified antibody by the total amount of precipitable antibody in the serum, assuming the latter to be 100 per cent. * Anti-DNP antibody was eluted in this case with 0"1 M DNP-~-aminocaproic acid. final concentrated solution. Following the elution with 0.1 M acetic acid approximately 3-5 mg of the antigen/1 g of immunoadsorbent was released into solution. This soluble antigen formed a precipitate when the eluted antibody was neutralized and concentrated. Thus, the overall recovery was directly related to the total amount of antibody for each experiment. Similar results have been reported by other workers. ~27-30~In experiments using elution with hapten at neutral pH, this loss of antibody was considerably reduced (Tables 2 and 3). In later experiments it was found that the soluble antigen released into solution from the immunoadsorbent could be almost entirely removed upon passage of the 0.1 M acetic acid solution through membrane filters (pore size 0.22 /~, Millipore Corp., Mass.), and is, therefore, most probably a conjugate of the antigen with some chemically degraded cellulose. In view of several reports 12°,31~ which indicated incomplete removal of bound antibodies in the acid pH range used in these experiments, the extent of dissociation

Immunoadsorbents Prepared with Bromoacetyl Cellulose

17

of the antibody from the antigen-cellulose conjugate in 0.1 M acetic acid (pH 2.8) at 37°C, as a function of time was further investigated. As seen in Fig. 1, complete release of the bound antibody from the immunoadsorbent was accomplished with one extraction within 45-60 min, with two different antigen systems. No significant amounts of antibodies were eluted upon reextraction with 0.1 M acetic acid. Further treatment of the adsorbent with solutions of HCI, pH 1-0 at 37°C did not release more protein into solution. The versatility of immunoadsorbents in this study is illustrated by the data in Table 2. Antisera with DNP specificity were elicited by various DNP conjugates of synthetic polypeptides, of a protein and of a polylysyl protein. These anti-DNP antisera were described recently ~z0~in connection with the observation that there is a correlation between the net electrical charge on an antigen and the electrophoretic mobility of the specific antibody. As seen in Table 2, D N P - H S A cellulose is an effective immunoadsorbent for anti-DNP antibodies elicited by various DNP derivatives. The concentration of anti-DNP antibodies elicited by TABLE 3. PURIFICATION OF ANTI-p-AZOBENZENEAIKSONATEANTIBODIES ELICITED BY p-AZOBENZENEARSONATEDERIVATIVES,MAKINGUSE OF p-AZOBENZENEARSONATE-HSA-cELLULOSE

Immunogen p-azobenzenearsonatehexa-L-tyrosine p-azobenzenearsonate-RSA

Antibody in the serum (mg/ml)

Setalln volume used (ml)

Antibody isolated (mg)

Yield (per cent)

not detectable 0"26

I O0 100

13 25

96

Anti-p-azobenzenearsonate antibodies were purified by adsorption and elution from p-azobenzenearsonate-HSA-Cellulose. T h e antibodies were dissociated from the immunoadsorbent by incubation for 1 ha" with 0"3 M sodium arsanilate, p H 8.0, at 37 °. T h e hapten was removed by exhaustive dialysis against 0-15 M NaC1-0.01 M phosphate buffer, p H 7"4. T h e yield was determined by dividing the total amount of purified antibody by the total amount of precipitable antibody in the serum, assuming the latter to be 100 per cent.

DNP conjugates of synthetic polypeptides was considerably lower (0"08--0.35 mg/ml antiserum) than the level of serum antibody achieved after immunization with DNP conjugates of proteins such as ovalbumin (2.4 mg/ml antiserum). Nevertheless, it was possible to isolate the anti-DNP antibodies elicited by immunization with DNP-poly-r-lysine. One and a half 1. of serum which contained 150 mg of anti-DNP antibody, representing approximately 0.2 per cent of the total protein in the reaction mixture, was mixed with the DNP-HSA-cellulose adsorbent. The eluted antibody (72 mg) represented a highly specific extraction from a mixture of proteins, with a relatively good recovery (48 per cent). An interesting use of the immunoadsorbents is illustrated in Table 3. In this table it is shown that it was possible to isolate, by means of an immunoadsorbent, antibodies to a p-azobenzenearsonate conjugate of hexa-L-tyrosine from an early rabbit antiserum, which was negative by the tests of precipitin and passive cutaneous anaphylaxis.~32~ In a recent study, concerned with the role of the molecular size in determining its immunogenicity, it was shown that this hexatyrosine conjugate

18

J. B. ROBBINS, J. HAIMOVICHand M. Sm.~ •i -i ~ 1. 1 1 I~,.._ I00

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FIG. 1. Effect of time on the release of antibodies from immunoadsorbents, in 0.1 M acetic acid at 37 °. Aliquots of 1 '0 ml were taken at various intervals. The samples were centrifuged at 20,000 x g for 10 min at 4 °, and the protein content of the supernatant fluids was determined from absorbance at 280 mt~. The antibody release (per cent) was calculated by dividing the amount of protein in each sample by the amount of antibody released into solution after 2 hr. Further exposure of the immunoadsorbents to 0.1 N HC1 for 30 min did not result in the release of a significant amount of protein into the supernatant fluid. • • , anti-lysozyme: O . . . . O, anti-(T, G) - A - - L. elicited both delayed sensitivity and antibody formation in guinea pigs. c19~ Its immunogenicity has now been demonstrated in rabbits. T h e specific antibodies elicited in rabbits b y this antigen which were isolated with the i m m u n o a d s o r b e n t were I g G and I g M proteins.

Specificity of the antigen-cellulose conjugates T h e specificity of the i m m u n o a d s o r b e n t s investigated was further confirmed b y experiments shown in T a b l e 4. T h e D N P - H S A and BSA immunoadsorbents, mixed with a variety of non-specific serum and treated under the same experimental conditions as described before failed to yield more than 0.5-1 "4 m g of protein in the eluate. T h i s result, indicating very low non-specific binding of s e r u m c o m ponents to the cellulose conjugates, was probably due to the hydrophilic properties TABLE 4. SPECIFICITY OF IMMUNOADSORBENTS PREPARED USING BROMOACETYL CELLULOSE

Immunoadsorbent DNP-HSA.-Cellulose DNP-HSA-CelIulose BSA-Cellulose BSA-Cellulose

Serum

Volume of serum (ml)

Eluted protein (mg)

normal rabbit serum anti-BSA anti-DNP-ovalbumin normal rabbit serum

500 500 200 500

1"4 0"8 0.5 0"9

Amounts of 1.0 g (dry weight) of the immunoadsorbents were mixed for 2 hr with the various sets, washed with 0.15 M NaC1, and suspended in 0'1 r¢ acetic acid for 1 hr at 37 °. The eluted protein was neutralized and concentrated to 5.0 ml by ultra-filtration. The protein was evaluated from absorbanee at 280 mt~, assuming an arbitrary E ~ of 7"0.

FIc. 2. Analytical ultra-centrifugation of purified anti-(T, G) - A - - L antxbodies. The conditions were: rotor temp., 22°: rotor speed, 59,790 rev/min: solvent, 0"1 M NaC1-0.01 M Tris HC1 buffer, pH 7"0: concentration of the purified antibody preparation, 20"0 mg/ml: concentration of the isolated IgG antibody, 18"0 mg/ml: concentration of the isolated IgM antibody, 3"0 mg/ml.

(Facing p. 18)

Immunoadsorbent~ Prepared with Bromoacetyl Cellulose

19

of cellulose. Thus, the cellulose as a base for immunoadsorbents formed a conjugate that had the characteristics of being quite insoluble, and yet permitted the release of non-specifically bound proteins under conditiom which did not dissociate the antibody.

Purity and activity of isolated antibodies The purity of the eluted antibodies was analysed by several criteria. Table 1 shows the results obtained by quantitative precipitin analysis expressed as the percentage of antibody that was precipitable by the antigen. Approximately 70-94 per cent of the proteins in the various antibody preparations was precipitable with the specific antigen. These values are in good agreement with results using antibodies purified by other methods.~,97,ze,a0,as) Analytical ultra-centrifugation of the purified antibodies (1-2 per cent solutions) revealed a major component with 6.6 S and a minor component with an s20.,, of 15 -8-18.0 S (Fig. 2). In two anti(T, G)-A - - L preparations, a minor component of 9.4 S was detected. Aside from some contamination with lipoproteins in early experiments, no other component was observed in the schlieren patterns of 1-2 per cent antibody solutions. Immunoelectrophoresis showed antibodies of the IgM and IgG classes of immunoglobulins in most of the preparations. These two classes of immunoglobulins were separable by gel filtration using Sephadex G-200 ~9'5) (Fig. 3). 2"5

2.0

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/

d I.o 0.5 F I O0

1.50

200

250

300

350

400

VOL. EFFLUENT (ml.)

purified anti-(T, (3) - A o - L antibody on Sephadex G-200. A 5"0 ml aliquot, containing 50 mg of anti-(T, G) - A - - L antibody, F I ( ; . 3. G e l f i l t r a t i o n o f

was applied to a 2.2 x 100 cm column equilibrated with 1"0 M NaC1-0.01 M Tris HC1 buffer, pH 8.1. The flow rate of the effluent was 15"0 rnl/h and 5"0 ml fractions were collected. The 9.4 S component, present in two of the anti-(T, G)-A - - L preparations, was tentatively identified as an immunoglobulin of the IgAclass. This identification was based upon the reaction of this protein with goat anti-rabbit light chain serum and failure to react with goat anti-Fc and anti-/, chain antisera in immunoelectrophoresis. DISCUSSION Conjugates prepared by reacting various antigens with bromoacetyl cellulose proved to be useful immunoadsorbents for the purification of antibodies to haptens, proteins, and synthetic polypeptides. The amount of serum proteins non-specifically

20

J. B. Roeems, J. HAIMOVICH and M. SBLA

bound to these conjugates was low (Table 4). The capacity of most of the antigencellulose conjugates tested to specifically bind antibodies was high. Elution of the bound antibody, using either 0.1 M acetic acid at 37°C or excess of specific hapten, was complete for all the antisera studied. The adsorbent was stable during storage for as long as 6 months and could be reused at least 5 times without significant loss of antibody-binding activity. Thus, bromoacetyl cellulose seems to be a useful reagent for coupling to a wide variety of antigens to prepare immunoadsorbents. The antibodies prepared with the use of such cellulose conjugates were immunoglobulins of the IgG and IgM classes as detected by analytical ultracentrifugation (Fig. 2), gel filtration (Fig. 3) and immunoelectrophoresis, and were not contaminated by other serum proteins. As an assay of the activity of the purified antibodies, the extent of their precipitability by the specific antigen was determined. The values obtained, in the range of 70-94 per cent, compare favorably with those reported for purified antibodies isolated by other techniques.~2-4,2a,24,27-31,33,a4~ Analysis of the preeipitability, as a criterion for activity of purified antibodies, may not be valid for immunoglobulins extracted by immunoadsorbents. Antibodies that fail to precipitate with their antigen may still bind specifically to the immunoadsorbent and thus be extracted by this procedure. Following covalent attachment of the antigen to the modified cellulose, the binding capacity of the conjugates varied considerably. For the ease of egg white lysozyme, 285 mg of the antigen/g cellulose was capable o f binding only 131 mg of anti-lysozyme antibody. For this anti-lysozyme serum, a combining ratio of antibody to antigen was 2.7 at the equivalence zone of precipitation. From adsorbtion experiments using excess of antibody, we infer that the cellulose-bound lysozyme was 30 times less efficient in binding the antibodies than was the free antigen. Nevertheless, successive absorptions of this serum extracted all precipitable antibody. We concluded, therefore, that lysozyme was covalently attached at different sites to the modified cellulose with a significant loss of overall activity, but that most of the different types of antigenic determinants on this protein were still available for interaction with the serum antibody. Using these same assumptions, the antigenic activities of the conjugates of ribonuclease, BSA, and (T, G)-A - - L were 10, 12, and 32 per cent of the free antigens in the equivalence zone, respectively. These conjugates were capable of removing all the precipitable antibodies fi'om hyperimmune sera. Gurvich et al. t85~ showed that the state of dispersion of the cellulose immunoadsorbents was a critical factor in determining the antibody-binding capacity of protein-cellulose conjugates. When their cellulose-based conjugate was reprecipitated from an ammoniacal copper solution it contained the same amount of nitro groups as the original material but was capable of combining with more antigen, and thus had a greater antibody binding capacity. In the synthesis of bromoacetyl cellulose, the product was obtained in a non-aqueous solution. When this organic solution was poured into cold water, the bromoacetyl cellulose was precipitated as a fine powder. We infer that this precipitated BAC had a high surface area to weight ratio. Extensive drying of this powder resulted in a significant decrease in the ability to wet the derivative as well as in the total amount of antigen that it could covalently bind, even though the dry BAC had the same bromine content as the wet material. It would seem, thus, that effective cellulose immunoadsorbents should, indeed, be

ImmunoadsorbentsPrepared with BromoacetylCellulose

21

in the form of well dispersed suspensions as only under such conditions is an efficient covalent interaction with the antigen obtained.C~.~s~ The overall yield of antibody varied from 48 to 96 per cent of the predpitable antibody removed from the serum. Some antibody was lost, following neutralization of the e h t e d protein, in each experiment. This loss was due to precipitation of the antibody with a rather constant amount of antigen (3-5 mg/g conjugate) that was released into solution during the elution step. Similar findings were reported previously~ sv-a°~ (most probably this antigen was bound to chemically degraded cellulose). Thus, the overall yield was directly related to the total amount of antibody in the eluate. The use of specific haptens, rather than acidic conditions, to dissociate the antibody from the immunoadsorbent, considerably reduced this loss of antibody. An interesting application of the use of these immunoadsorbents was illustrated by the purification of antibodies to a simple, low tool. wt. antigen. This antigen, a conjugate of azobenzenearsonate group with hexa-L-tyrosine, elicited low levels of antibodies. Nevertheless, the high binding capacity and low non-specific interaction with other serum proteins of the specific immunoadsorbent prepared for this purpose permitted the isolation of antibodies to this antigen. Were it not for the property of the immunoadsorbent to bind the antibody in dilute solution and permit its extraction and concentration, it might have been concluded that the compound investigated was not immunogenic. Antibodies of the IgA and IgM, as well as the IgG class of immunoglobulins have been isolated using immunoadsorbents. ~°,a°,se~ The IgM and IgA immunoglobulins comprise about 15 per cent of the total pool of immunoglobulins in the rabbit.~8v,as~ With the use of immunoadsorbents prepared with bromoacetyl cellulose, immunoglobulins of the IgM as well as the IgG classes were isolated in most of the antibody preparations investigated. Analysis of the immunoglobulin components of the purified antibodies using specific goat antisera has permitted a quantitative estimation of the relative amounts of IgM and IgG in normal and immune sera. ~38~ Acknowledgements--The capable technical assistance of Miss M. Borenstein is gratefully acknowledged. We also wish to thank Mrs. S. Ehrlich-Rogozinski for the mierochemical analyses and Mr. A. Lustig for analytical ultra-centrifugation.

REFERENCES 1 CAMPBELLD. H., LUESCHERE. and LI~MAN L. S., Proc. natn. Acad. Sd., U.S.A. 37, 575 (1951). 2 WELIKYN. and W~'rALL H. H., lmmunochemistry 2, 293 (1965). 3 SmaoNA. H., Br. reed. Bull. 19, 183 (1963). 4 SILMANI. H. and KATCHALSKIE., Ann. Rev. Biochem. (In press). 5 HEIDELBERGER M. and KENDALL F. E., .7. exp. Med. 62, 697 (1935). 6 PAPPENHVIMmaA. M., JR., j~. exp. Med. 71, 263 (1940). 7 KLXNMANN. R., RocrmYJ. H. and K~usH F., Science 146, 401 (1964). s STANWORTHD. R., Advances in Immunology (Edited by DIXONF. J., JR. and HUMPHREY J. H.), Vol. 3, p. 181. Academic Press (1963). 9 NEZLINR. S., Molecular and Cellular Basis of Antibody Formation, p. 595 (Edited by STmaZLJ.), Publishing House of the Czechoslovak Academy of Science (1965). 10 PATCHOm~IKA., Israel Patent Application No. 18207 (1962). ix JAOENDORFA. T., PATCHOI~UKA. and SELA M., Biochim. Biophys..4eta 78, 516 (1963). 12 SANCERI., Biochem. 3. 39, 507 (1945). 18 BoI~K F. and STUPPY., Immunochemistry 2, 323 (1965).

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J. B. RoemNs, J. HAIMOVICHand M. S~LA

14 SCHECH~ I., Biochim. Biophys. Acta 104, 303 (1965). Is SvJ~AM. and A ~ o N R., Biochem. 07. 75, 91 (1960). ~e Sm.n M., FUCHS S. and AI~NONR., Biochem. 07. 85, 223 (1962). 17 SELA M., UNGAR-W~oN H. and SrmcH~m Y., Proc. natn. Acad. Sci., U.S.A. 52, 285 (1964-). is UNG~m-WPalON H. and SELA M., Biochim. Biophys. Acta 124, 147 (1966). 19 BOPJ~KF., STuPP Y. and SELn M., Science 150, 1177 (1965). 2o SELA M. and MOZES E., Proc. hath. Acad. Sci., U.S.A. 55, 44.5 (1966). et SCHONIOImW., Mikrochim. Acta 1, 869 (1956). 32 ST~atK G. R., STEIN W. H. and MOORE S., 07. biol. Chem. 236, 4.36 (1961). 3a F ~ H F. S., Kam_~ M. and EZSENH. N., 07. exp. Med. 112, 1195 (1960). K~AT E. A., Kabat and Mayer's Experimontal Immunochemistry (2nd ed.). C. C. Thomas (1961). 35 FLODI~ P. and K I L L ~ D ~ J., Biochim. Biophys..4cta 63, 403 (1962). 3s P o R ~ R. R., Biochem. 07. 73, 119 (1959). 37 GIVOL D., FucHs S. and SELA M., Biochim. Biophys. Acta 63, 222 (1962). 3s MOUDQALN. R. and PORTERR. R., Biochim. Biophys. Acta 71, 185 (1963). s3 SL~G]m S. J., FOTHlmOILLJ. E. and SHAXNO~ J. R., 07. Am. chem. Soc. 82, 565 (1960). s00Notrs K., YAGI Y. and PSmSMAN D., Immunochemistry 2, 181 (1965). sl V~J~Nx~ W. E., BRY~a~W. P. and C~PmSLL D. H., Immunochemistry 2, 1 (1965). s3 BoP~K F., STUPP Y. and SELA M. (unpublished data). 8a SLOB~ L. I. and SELA M., Biochem. Biophys. Acta 107, 593 (1965). SCHLOSS~.N S. F. and K~AT E. A., 07. exp. Med. 116, 535 (1962). 3s GtmVICH A. E., KuzovI~VA O. B. and TUMANOVAA. E., Biokhimiya 26, 803 (1962). ~e ONOUS O., YAG~Y. and P l t m S ~ D., 07. exp. Med. 123, 173 (1966). ~7 C ~ I ~ J. J. and ROlliNS J. B., 07. Immun. (in press). as ROBmNS J. B. and R~MO~ A. (manuscript in preparation).