Equine anti-hapten antibody—VII. Anti-lactoside antibody induced by a bacterial vaccine

Immunochemistry,1973, Vol. 10, pp. 365-37 i. pergamon Press. Printed in Great Britain

EQUINE ANTI-HAPTEN A N T I B O D Y - V I I . ANTI-LACTOSIDE A N T I B O D Y I N D U C E D BY A BACTERIAL VACCINE* Y U N G D. KIM and FRED K A R U S H t Department of Microbiology,School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. (Received 15 November 1972) Almtraet- Immunization of a horse with a bacterial vaccine of Streptococcus faecalis, strain N led to the sustained and concomitant production of IgM and 7 S antibody with anti-lactoside specificity. Restriction of heterogeneity was apparent in both tool. wt groups with the IgM population exhibiting three major components when analyzed by isoelectric focusing. Equilibrium dialysis studies of the specific binding of a lactoside hapten, p-(p-dimethylaminophenylazo)-phenyl-~-lactoside (Lac dye), were carried out with three preparations of purified IgM antibody. The intrinsic association constants (Ko) ranged between 1 × 10eM-1 and 2× 10SM-1 for bleedings taken from 6 weeks to 14 weeks after the initiation of immunization. The availability of 10 binding sites per IgM molecule was clearly evident with no indication of intramolecular heterogeneity although substantial deviation from homogeneous binding was observed. Conversion of the pentameric IgM molecule to the monomeric form resulted in identical binding behavior. The 7 S antibody fraction gave a maximum value of K0 of 8 × 105M-L The binding of lactose by IgM yielded a value of K0 which was 24-fold smaller than that for the Lac dye. Thus, the nonspecificcontribution of the aglycoside,arising presumablyfrom its hydrophobic character, to the free energy of complex formation was- 1.8 kcal/mole of bapten. The temperature dependence of the lgM binding of the Lac dye demonstrated that the enthalpy contribution was primarily responsible for the affinity. From this result it was ird'erred that the specific binding of lactose involves multiple hydrogen bonds, perhaps as many as 10. INTmODUCTION During the past few years bacterial vaccines have proven of inestimable value for the induction of antibody of restricted heterogeneity with specificity for carbohydrate antigens. Most of the recent studies using bacterial vaccines have been done with the rabbit and have provided homogeneous fractions of 7 S antibody (Krause, 1970; Pincus et al., 1970). It has been known for several decades, however, that pneumococcal vaccines induce high levels of anti-carbohydrate antibody of the macrogiobulin variety in the horse and the cow (Heidelberger and Pederson, 1937; Kabat, 1961). In view of the restrictions observed in the 7 S response it appeared of interest to examine IgM antibodies induced with a bacterial vaccine for a similar selective stimulation. The simultaneous production of 7 S antibodies which often occurs in these species would allow the comparison of homogeneous fractions of each class of antibody for *Supported in part by Public Health Service research grant AI-09492 from the National Institute of Allergy and Infectious Diseases and in part by Public Health Service training grant 5-TI-AI-204from the National Institute of Allergy and Infectious Diseases. 1"Recipient of a Public Health Service research career award (5-K6-AI 14,012) from the National Institute of Allergy and Infectious Diseases.

possible idiotypic identity. Common idiotypic determinants of rabbit IgG and IgM anti-Salmonella antibodies have been inferred from lines of identity observed by double diffusion in agar gel (Oudin and Michel, 1969). The utility of equine anti-pneumococcal polysaccharide serum for the quantitative study of the affinity of IgM antibody has recently been demonstrated (Pappenheimer et al., 1968). The recent characterization of the immunological specificity of a cell wall glycan of Streptococcus faecalis, strain N has served as the starting point for the study described in this paper (Pazur et al., 1971, 1972). The cell wall of this strain contains a diheterogiycan of glucose and galacrose as well as a tetraheteroglycan of rhamnose, glucose, galactose and N-acetyl-galactosamine. The lactosyl moiety of the diheteroglycan is the immunodominant group as shown by agar gel double diffusion reactions with the mixture of glycans and the effectiveness of lactose as an inhibitor of the precipitin reaction with the diheteroglycan. Earlier studies with a lactosyl-containing haptenic group (Lac)* in the rabbit (Karush, 1957) had yielded measurements of the affinity of lactose and some ~-lactoside derivatives for antibody induced with the Lac group (p-azophenyl/3-1actoside) coupled to a protein carrier. Later studies with equine anti-Lac sera not only pro-

365

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YUNG D. KIM and FRED KARUSH

vided additional affinity data but also demonstrated that a multiplicity of molecular forms of 7 S antiLac antibody were synthesized by the horse (Klinman and Karush, 1967; Rockey et al., 1964). The use of the vaccine prepared from strain N coupled with the large quantity of equine serum available opened up the possibility that homogeneous populations of IgM and 7 S antibody could be isolated by preparative isoelectric focusing applied to specifically purified IgM and 7 S antibody fractions. As is indicated below both varieties of anti-lactosyl antibody were produced simultaneously in the horse over several months. In this paper we describe the methods for the induction and purification of the antibody, the characterization of the IgM antibody and the interaction of the antibody with a lactoside-conjugated azo dye in terms of the valence of the antibody and its thermodynamic characteristics. btATERIAI~ AND METIlOD$ Preparation of antisera Equine antisera were obtained from a horse which was immunized by intravenous injection with a sterile heatkilled suspension of S.faecalis in PBS*. The first bleeding was made 6 weeks after the immunization was started and was preceded by a series of three injections per week for 4 weeks. The second bleeding was made 4 weeks later following an additional five injections over a 2-week period. The third bleeding occurred 4 weeks later with an additional six injections over a 2-week period. Quantitative precipitin analysis of anti-lactose antibody was performed with the diheteroglycan prepared according to Pazur et al. (1971).

Purification of anti-lactose antibody The anti-lactose antibody was purified by the use of an immunoadsorbent prepared by the reaction of paminopbenyl-jS-lactoside (PAPL) with activated Sepharose (Wofsy and Burr, 1969). The hapten-coupled Sepharose was obtained by the addition of 4 × 10-4 mole of PAPL to 100mi of CNBr-activated Sepharose (Axen et al., 1967). The mixture was stirred gently at 4"C for 24 hr (pH 9.0) and washed thoroughly with phosphatebuffered saline (PBS; 0.02 M phosphate, pH 7.5, 0.15 M NaCI). Analysis of the supernatant for unreacted PAPL showed that 62 per cent of the hapten was coupled to the Sepharose. The equine anti-serum (300 mi) was passed through the Sepharose column ( 1.5 x 42 era) and the column was washed with PBS until the eluent, monitored by absorbarite at 280 nm, appeared free of non-specific protein. The absorbed antibody was then eluted with 0-2M lactose in PBS. It had previously been established that 0.2 M lactose was sufficient to dissociate the adsorbed antibody. The fractions containing the antibody were pooled and dialyzed against PBS (15 × volume) at 4"C *Abbreviations- PBS, 0.15 M NaCI, 0.02 M phosphate, pH 7-5; PBS-EDTA, PBS containing2 × 10-3 Mdisodium salt of ethylenediaminetetmacetic acid; PAPL, p-aminophenyl-~-lactoside; Lac dye, p-(p-dimethylaminophenylazo)-pbenyl-~-lactoside.

for 16hr. The dialysate was then replaced by 0.5M galactose in PBS to facilitate the displacement of lactose bound to antibody. The antibody solution was further dialyzed against PBS for 72 hr, changing the buffer solution every 12 hr. The purified anti-lactose antibody solution was concentrated under negative pressure before being subjected to fractionation. A Sephadex G-200 column (3 × 66cm) equilibrated with PBS containing 2 × 10-3M EDTA (PBS-EDTA) was utilized to fractionate the antibody.

Analysis of antibody concentration Protein concentrations were determined by optical density at 280 nm using extinction coefficients (EZ~) of 13-5 for IgM and 13-8 for the 7 S fraction. These extinction coefficients were determined by microkjeldahl analysis assuming N contents of 14.5% for IgM (Metzger, 1970) and 16.0% for the 7 S fraction. U ltracentrOeugation Analytical ultracentrifugation was carried out in a Spinco Model E centrifuge at 52,640 rev/min. Sedimentation constants were calculated from schlieren patterns obtained at 20°12with protein concentrations ranging from 2.5 to 5.2 mg/mi in PBS-EDTA. Electrophoresis lmmunoelectrophoresis was performed according to the microtechnique of Scheidegger (1955). The immunoelectrophoresis patterns were developed with rabbit anti-whole horse serum obtained from Baltimore Biological Laboratories, Baltimore, Md. A Beckman Model R-101 Microzone Electrophoresis Cell was utilized for the analytical electrophoresis experiments. For analysis by isoelectric focusing an apparatus similar to that described by Wrigley (1968) was used with ampholine (pH5-8) supplied by LKB-Produkter, Sweden. The protein bands were stained with 0.2~ bromphenoi blue. Preparation of lgM monomers The purified anti-lactose IgM molecules were subject to very mild reduction and alkylation to dissociate the pentameric IgM into monomers. To 70 mg of IgM in 10ml of 0.2M Tris-HCl (pH8.0), 8.5× 10-~mole of dithiothreitol (Sigma Chemical Company) were added and the mixture was stirred under a nitrogen atmosphere for 1 hr at room temperature. The free thiol groups of the protein were then alkylated with iodoacetamide (1.27 × 10-Smole, Sigma Chemical Company). The reaction products were dialyzed against 0.02 M Tris-HC1 (pH 8.0) solution for 2 days at 4"C and fractionated at room temperature with a Sephadex G-200 column (2.5 x 65 cm) previously equilibrated with 0-02 M Tris-HCl (pH 8"0) solution. Two elution peaks were observed by absorbance measurement at 280 nm. The major peak was the second one containing more than 80 per cent of the total protein. It exhibited a very narrow tool. wt distribution with a sedimentation constants of 6.6 S. Hence, the protein component in the second peak was taken as the monomer of anti-lactose IgM. Measurement of hapten binding Binding of hapten by the anti-lactose antibody was measured by equilibrium dialysis using micro-dialysis cells (Eisen. 1971), fabricated by the Drummond Scientific Company, Broomall, Pennsylvania, as previously des-

Equine Anti-Lactoside Antibody

367

cribed (Gopalakrishnan et al., 1973). Most of the binding experiments were done with p-(p-dimethylaminophenylazo)-phenyI-B-lactoside radiolabeled with tritium (3H-Lac dye) whose preparation has been described in an earlier publication (Oopalakrishnan et aL, 1973.) Some binding experiments were also carried out with 14C-lactose (AmershardSearle Company). RESULTS

Purification of antibody Three sera, collected 6, 10 and 14 weeks after immunization was initiated, were found to contain 1.7 (I), 2.9 (II) and 2.3 (III)mg of antibody per ml of serum, respectively, when purified diheteroglycan was used as the antigen in the quantitative precipitin analysis. The purification procedure described above allowed quantitative recovery of the precipitable antibody.

Fractionation of the purified antibody into lgM and 7 S fraction Figure 1 shows an elution pattern of the purified

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Fig. 2. Analytical uitracentrifugation at 52,640 revlmin. The samples were in 0.02 M phosphate, 0.15 M NaCI, 0.002 M EDTA, pH 7.5 at 20°C. (A) Equine anti-lactose antibody, purified by the hapten-coupled Sepharose column, after 32rain. Protein concentration was 5.3 mg/ml. (B) Equine anti-lactose IgM, purified by Sephadex G-200 column chromatography, after 12 rain. Protein concentration was 3.0 mg/ml.

Fraction number

Fig. 1. Elution patterns of purified equine anti-lactose antibody from Sephadex G-200 column chromatography eluted with 0.02M phosphate, 0.15M NaCI, 0.002 M EDTA, pH 7-5 at room temperature. The dotted fines represent the elution patterns from the repeated G-200 column chromatography using the initially separated, pooled IgM and 7 S fraction. anti-lactose antibody from a Sephadex G-200 column. A relatively sharp peak appeared near the void volume, followed by a shallow broader peak. The fact that the antibody components of the two peaks possess distinctly different mol. wt could be also demonstrated when the same sample was analyzed by analytical ultracentrifugation (Fig. 2). The fractions for the first peak and those for the second peak were pooled separately, concentrated and rechromatographed on the same 0-200

column. The purified antibody, eluted under the first peak of Fig. 1, showed a sedimentation constant of 19 S and was identified by its electrophoretic mobility as IgM. The antibody species emerging under the second peak appeared to consist of more than five molecularly different components, as judged from their electrophoretic properties (see the following section) and, therefore, they will be collectively called the 7 S fraction. The sedimentation characteristics of the purified antibody and the separated IgM and the ratio of IgM to the 7 S fraction for each of the three antibody preparations are included in Table I. It is of particular interest that the ratio of IgM to the 7 S fraction had not changed during the course of 3 months.

Electrophoretic properties The immunoelectrophoretic patterns demonstrate a single IgM component and a 7 S fraction

368

YUNG D. KIM and FRED KARUSH Table I. Sedimentation characteristics of purified anti-lactose antibody* Serum number I II III

Purified antibody* Major peak Minor peak 17.9S 17.9S 17.9S

Purified IgMa

Ratio of IgM to 7 S

18.3S 19.3S 19.5 S

3.3:1 3.0:1 3.4:1

7.1S 7.1S 7.1S

aThe protein concentrations of the total purified antibody and the purified IgM were 5 and 3 mg/ml respectively. All experiments were performed with 0.02 M phosphate, 0.15 MNaCI, 0.002 M EDTA, pH 7.5 at 20°C. ~I'he values were calculated by comparing the area under each peak of the schlieren patterns and also of the G-200 column elution patterns.

with 5 electrophoretically distinguishable components in the purified equine anti-lactose antibody (Fig. 3). This finding is substantiated by the results of microzone electrophoresis in which one fast moving strong band followed by a faint band were observed with the purified IgM. With the 7 S fraction the microzone pattern could be resolved into five closely spaced bands. T h e degree of homogeneity o f the I g M sample was further investisated by the isoelectric focusing technique. Since the IgM molecule is too large to migrate through the pores o f polyacrylamide gel (Hagland, 1970) experiments were carried out with the IgM monomer. T h e resulting pattern showed three strong bands at the position corresponding to the p H value of 5 . 7 _ 0.2.

Hapten binding by anti-lactose antibody Figure 4 illustrates the result of ~H-Lac dye binding by I g M antibody measured by the equilibrium dialysis method. In Table 2 the binding properties of IgM, IgM monomer and the 7 S fraction are expressed in terms of the average intrinsic association constant and the index of heterogeneity (a) derived from the Sips distribution function (Karush, 1962). These values are based on the assumption o f 10 combining sites per IgM molecule. This assumption is justified on the basis of the fact that an r value of 8.5 could be experimentally demonstrated. Also included in Table 2 are the binding properties of the antibody samples

Fig. 3. The immunoelectrophoresis patterns of purified equine anti-lactose antibody developed with rabbit antiwhole horse serum. The top pattern is the horse antiserum before purification. The second pattern shows the purified antibody after the hapten-coupled Sepharosecolumn treatment. The third and fourth patterns are of IgM and the 7 S fraction obtained from the repeated Sephedex G-200 treatment of the purified antibody.

Table 2. Binding of SH-Lac dye by purified anti-lactose antibody at 25°C a Serum number I II III

IgM Ko × 10-5 (M -l) 1.21 ~-0.22 1.74-4-0.20 1.96±0.19

a 0.66 0.80+-0-02 0-79-4-0.02

7 S fraction Ko × 10-s (M-1) 6-26 +_ 1.06 1.47±0.07 8-04+0.02

a

0-62 ~-0-07 0-49__.0-05 0.46±0-03

Ig.M Submit K0 x 10-s (M -1) 1-70±0.82

0-82"4-0.03

~The errors indicated are the arithmetic mean deviation between two samples purified independently from the serum. A moi. wt of 180,000 was assumed for the IgM subunit and 150,000 for the 7 S fraction. The notations Ko and a represent the average intrinsic association constant and the index of heterogeneity derived from the Sips distributions function, respectively. The equilibrium dialysis experiments were carried out in PBS-EDTA buffer (pH 7-5) at 25.0-4-0. I°C.

Equine Anti-Lactoside Antibody

tion due to the p-(p-dimethyl-amino-phenylazo) phenyl group of ~H-Lac dye on the antibody binding by using ~ - l a c t o s e as a hapte~ The binding experiments performed with 'lgM antibody # 3 at 4°(3 produced the following parameters: K o = 1.95 (_+0-42) × 10*M -~ and a -- 0-58_+0.02. Thus, the Lac dye was bound with a value of Ko 24-fold larger than that for lactose. Control experiments were carried out with normal IgM prepared from equine serum, obtained from a prebleeding, by (NH4)= SO4 precipitation and repeated Sephadex G-200 treatment. Binding of 3H-Lac dye by the normal IgM (at 25°(= and under the same experimental conditions noted in Table 2) was insignificant.

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Fi~ 4. The binding of 3H-Lac dye by purified equine anti-lactose IgM (No. 3) at 25°C where r represents the number of moles of =H-Lac dye bound per 180,000 g of antibody at the equilibrium free dye concentration c. The calculation and the theoretical analysis of the data, obtained by liquid scintillation counting, were executed with a programmable calculator (Model 720, Wang Laboratories) linked to a plotting output writer. The procedure is described in detail elsewhere (Oopalakrishnan et al., 1973). purified from sera collected over a 3-month period. Some relationships noticeable from Table 2 are that the IgM samples show lower K0 values but apparently higher a values than the 7 S fractions and the binding properties of IgM were not changed when it was dissociated into monomers. The temperature dependence of binding has been examined with SH-Lac dye and IgM # 3 and the results, including the thermodynamic parameters, are presented in Table 3. The attempt was made to estimate the contribuTable 3. Temperature dependence of the binding of SHLac dye by IgM and thermodynamic parameters assocmted with the bindings Temp. °C 4 25 37 ~F= (kcal/mole) --9.7~-0.1

Ko x 10-s (M -1) 4.7 ±0.3 1.96~_0.19 0.59- 0-00

a 0-56±0.05 0.79__.0-03 0.77 +_0-03 •

~/(kcal/mole) ~S= (cal/mole de,g) --11.0-----0.2

--4.7±1.1

=The binding experiments were carried out at pH 7-5 (PBS-EDTA) with IgM (III). The unitary free energy, AF,, is given for 25"(= and the thermodynamic values refer m the binding of one mole of hapten. The error indicated for ~ / w a s estimated from the van't Hoffplot.

DISCUS'ION

The immune response of the horse to the streptococcal vaccine is characterized by prolonged production of IgM antibody accompanied by the simultaneous appearance of 7 S antibody. The weight ratio of these components remained quite constant, approximately 3:1, during the interval of 6-14 weeks following the initial injection of vaccine. More recent observations have demonstrated the continued synthesis of IgM antibody for at least 28 weeks with the maintenance of its predominant role in the humoral response. With respect to heterogeneity the IgM antibody appears, from the results of isoelectric focusing experiments, to be highly restricted. This inference is based on the finding of only three bands when the monomeric form of the IgM antibody was analyzed. It is noteworthy that the isoelectric points of the three bands were very similar, falling within the range of pH 5.7_+0.2. There remains the reasonable possibility of obtaining homogeneous IgM antibody preparations by further fractionation. In the case of the 7 S antibody some restriction of heterogeneity is also evident from the microzone pattern. The multiplicity of bands in the pattern arises in part, undoubtedly, from the participation of several genetic loci controlling the constant regions of the heavy chains associated with 7 S products. This genetic complexity was previously noted in the variety of 7 S antibodies with antilactoside activity produced in the horse (Rockey et al., 1964). Preliminary results of the chromatographic fractionation by DEAE-cellulose of the 7 S antibody induced by the vaccine indicate that a similar variety of classes and subclasses is present. The possibility that these possess common variable regions is of great interest and probably subject to experimental analysis in this system. The specific interaction of the IgM antibody with the Lac dye (Table 2) clearly demonstrates the decavalence of the antibody and the virtual constancy of the intrinsic affinity during the 8-week immunization interval encompassed by the three

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YUNG D. KIM and FRED KARUSH

sera. The availability of 10 binding sites per IgM molecule is in agreement with several published studies bearing on this issue (Metzger, 1970). In particular our results substantiate for a conventionally induced anti-hapten antibody the finding of decavalence for a Waldenstr6m macroglobulin capable of binding to its Fab region nitrophenyl-containing ligands (Ashman and Metzger, 1969). Other studies with induced IgM antibody against small ligands (Clem and Small, 1968; Voss and Eisen, 1968), however, led to the conclusion that the molecule was functionally pentavalent although Onoue et al, (1968) inferred that there were five weak and five strong binding sites per molecule. The binding curves of the antilactosyl IgM antibody (Fig. 4), on the other hand, not only exhibit decavalence but also demonstrate that the affinities cluster closely around a single average value, as indicated by the figure for the Sips heterogeneity index (a -- 0.80). Some observations regarding the temporal dependence of the IgM and 7 S responses are suggested by the results of Table 2. Thus, with respect to the lgM response, there appears to be no significant increase of average affinity over the 8-week period involved, indicating the absence of maturation in this response. Earlier observations with rabbit anti-hapten IgM antibody have led to a similar conclusion (Sarvas and MgkelL 1971). In the case of the 7 S response the unexpected decrease of average affinity between the first and second bleedings and the subsequent increase reflect a complexity in the multiple clones involved which does not permit the simple and usual interpretation of selection of higher affinity clones. It is, nevertheless, quite apparent that the 7 S population is capable of exhibiting higher affinity than that of the IgM fraction, in agreement with the results found with rabbit antibodies (M~ikel~i et al., 1970). This difference, in turn, indicates that the 7 Sproducing clones which are stimulated utilize variable region genes different from those involved in the activ/ty of the IgM-producing clones. In the former case there is also a greater tendency for the selection of clones with maximum affinity. Finally, it may be noted that the pentameric structure of the IgM molecule does not appear to influence its intrinsic binding properties since the monomeric form (Table 2) exhibits the same association constant and heterogeneity index as the parent population. The comparison of the affinities for the binding of Lac dye and lactose showed a 24-fold greater value of the association constant for the dye, corresponding to an incremental value in AF o f - 1.8 kcal/mole of hapten. This increment represents the non-specific contribution of the /t-linked aglycoside, p-(p-dimethylaminophenylazo)benzene, to the total free energy. The substantial magnitude of this contribution is undoubtedly related to the

hydrophobic character of the aglycoside, It indicates furthermore that probably most or all of it can be accommodated within the cavity or cleft which comprises the region of interaction of the antibody site (PoUak et al., 1972). The temperature dependence for the binding of the Lac dye (Table 3) demonstrates that the affinity is due to the AH contribution ( - I 1.0 kcai) reduced somewhat by a slight decrease in unitary entropy. The source of the AH contribution is largely due to the specific interaction of the lactosyl moiety because of the minor contribution to the affinity of the aglycoside. It may be deduced, therefore, that the lactosyl group is held to the contact amino acids by hydrogen bonds numbering perhaps as many as l0 (Karush, 1962). There is a striking similarity with respect to the AH contribution in the equine system to that observed with rabbit antibody previously (Karush, 1957). In the latter case the antibody was induced with a carrier protein conjugated with the pazophenyl-/i-lactoside group. Nevertheless, the rabbit 7 S antibody purified from early bleedings and the equine IgM exhibited similar affinities, about 1 × 10s M - ' at 2S°C for Lac dye and about 1 x 104 M - ' at 25°C for lactose. The binding of the Lac dye by the rabbit antibody was characterized by a AH of 10 kcal/mole of hapten and a negligible change in unitary entropy. It was inferred that the AH contribution was the consequence of multiple hydrogen bonds. In retrospect it would appear that this early 7 S antibody, obtained 4 weeks after the initial administration of antigen, was characterized by a specific reactivity limited to the lactose group and a non=specific reaction with the aglycoside, The speculative implication may be drawn from this comparison that the IgM and early IgG responses may involve the recognition of smaller antigenic determinants than those associated with later IgG antibody since such IgG antibody exhibited an association constant of about 1 × 107 M - ' (Klinman and Karush, 1967). Acknowledgement-We are indebted to the School of Veterinary Medicine of the University of Pennsylvania for invaluable assistance in the immunization and bleeding of the horse. We are also indebted to Patricia Gearhart for preparation of the bacterial vaccine and to Dana Wontorsky for technical aid. RF,FER~NCF_~ Ashman R. F. and Metzger H. (1969)d. biol. Chem. 244, 3405. Axen R., Porath J. and Eruback S. (1967) Nature, Lend. 214, 1302. Clem L. W. and Small P. A., Jr. (1968) Fedn Prec. 27, 684. Eisen H. N. (1971) Methods in Immunology and lmmunochemistry (edited by Williams C. A. and Chase M. W.), Vol. III, p. 393. Academic Press, New York Gopalakrishnan P. V., Hughes W. S., Kim Y. D. and Karush F. (1973) immunochemistry 10, 191. Haglund H. (1970) Meth. biochem. Analysis 19, 1.

Equine Anti-Lactoside Antibody Heidelberger M. and Pedersen K. O. (1937)J. exp. Med. 65,393. Kabat E. A. (1961) Kabat and Mayer's Experimental Immunochemistry, 2nd Ed. Thomas, Springfield,Ill. Karush F. (1957)J.Am. chem. Soc. 79, 3380. Karush F. (1962)Adv. Immunol. 2, I. Klinman N. R. and Karush F. (I967) Immunochemistry 4, 387. Krause R. M. (1970)Adv. Immunol. 12, I. M~kel~i O., Ruoslahti E. and Sepp~I~ I. J. T. (1970) Immunochemistry 7, 917. Metzger H. (1970) Adv. Immunol. 12, 57. Onoue K., Orossberger A. L., Yagi Y. and Pressman D. (1968) Science 162, 574. Oudin J. and Michel M. (1969)J. exp. Med. 130, 619. Pappenheimer A. M., Jr., Reed W. P. and Brown R. (1968)J. Immun. 100, 1237.

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Pazur J. H., Anderson J. S., and Karakawa W. W. (197 I) J. biol.Chem. 246, 1793. Pazur J. H., Cepure A., Kane J. A. and Hellerquist C. G. (1973) J. biol.Chem. (in press). Pincus J. H., Jaton J. C.. Bloch K. J. and Haber E. (1970) J. Immun. 104, 1143. Poljak R. J., Amzel L. M., Avey H. P., Becka L. N. and NisonoffA. (1972) Nature, N e w Biol. 23& 137. Rockey J. H., Klinman N. R. and Karush, F. (1964) J. exp. Med. 120, 589. Sarvas H. and M~ikel~i O. (1970) Immunochemistry 7, 933. Scheidegger J. J. (1955) Int.Archs Allergy 7, 103. Voss E. W., Jr. and Eisen H. N. (1968) Fedn Proc. 27, 684. Wofsy L. and Burr B. (1969) J. Immun. 103, 380. Wringley C. (1968) Science Tools 15, 17.