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Neutralization of diphtheria toxin by human immunoglobulin classes and subunits
hmmm,,,hcmrsl~l. 19;4. ~ol. I I Pl' 223 225
Pclgalnol~ Press.
Prmlcd in G r e a t Britain
NEUTRALIZATION OF DIPHTHERIA TOXIN BY H U M A N I M M U N O G L O B U L I N CLASSES AND SUBUNITS D O N A L D D. O U R T H Department of Microbiology, Harvard University School of Public Health, Boston, Massachusetts 02115, U.S.A.
(Receired 5 July 1973) This investigation found that the human antibody class of importance in neutralizing diphtheria toxin was IgG, and that toxin neutralization was retained by the F(ab')2 and Fab' subunits of IgG. IgM and IgA did not neutralize diphtheria toxin, although these two antibody classes reacted with diphtheria toxoid in the indirect hemagghltination test. Abstract
INTRODUCTION In immunization for diphtheria with toxoid, prevention depends on the ability of immunoglobulins to neutralize bacterial exotoxin. This investigation was therefore directed to determining which human antibody class (IgG, IgM, IgA) was most capable of protecting against diphtheria toxin. At the same time, subunits of human IgG and IgM were isolated by pepsinization, reduction and alkylation to determine if antibody subunits could neutralize diphtheria toxin. In addition, indirect hemagglutination (HA) was used to investigate whether antibodies could bind diphtheria toxoid, even though not capable of neutralizing diphtheria toxin.
MATERIALS AND METHODS
Preparation of human IyG, F(ab') 2 and Fah' suhunits. A pseudoglobulin supernatant was isolated from Cohn fraction II (Cohn et al., 1946) human immune serum globulin* (ISG) having a diphtheria antitoxin titer of 40 AU/ml by dialysis against distilled water for 72 hr at 4 (2 Tile supernatam was then applied to Scphadex DEAE A-50 in 0.0175 M NaH2PO4 buffer, pH 6"9 (Palmer and Nisonoff, 1963). After digestion with pepsin (2 mg pepsin/100 mg IgG), F(ab')~ was isolated bv twice passing the digest through Scphadcx (i-150 in 0"(11 M borate saline buffer pH 8 (BSB) (Ni~,onol]cl a/.. 1960). Fab' was prepared b~ redaction of the F(ab'), with 0.05 M 2-mercaptoethanol (redistilled) (2ME) in a nitrogen atmosphere at room temp followed by alkylation with a 201~; molar excess of iodoacetic acid (redistilled) at 4:'C and then Sephadex G-150 filtration in BSB (Nisonoff, 1960). Immunoelectrophoresis (IEP) was used to check for specificity of the lgG, F(ab'), and Fab'. Preparation (?.]human 19M. To isolate lgM, the euglobulin from ISG* after dialysis against distilled water was applied to Sephadex DEAE A-50 in 0.1 M NaH2PO~, buffer, pH 6"3. After elution at this molarity and pH, a salt gradient of
0'1 M NaH2PO 4 buffer, pH 6.3 to 0.45 M NaH2PO 4 buffer, pH 4-2 followed (Chaplin et al., 1965). The ascending portion of the first peak eluted was applied to Sephadex G-200 in BSB to obtain IgM as indicated with specific antiserum by IEP. Reduction with 2-mercaptoethanol. To obtain subunits, IgM was reduced in a nitrogen atmosphere with 0.1 M 2ME for 2 hr at room temperature followed by alkylation with an equal volume of 0.12 M iodoacetic acid (redistilled) for 2 hr at 4°C. This was followed by dialysis for 72 hr in BSB at 4°C. Preparation of human IgA. To isolate IgA, the pseudoglobulin supernatant of Cohn Fraction It (Cohn et al., 1946) ISG* was applied to Sephadex DEAE A-50 in 0.0175MNaH2PO 4 buffer, pH 6.9. After elution at this molarity and pH, a second fraction was eluted with 0.2 M NaH2PO 4 buffer, pH 5.8. To this second fraction, zinc glycinate was added slowly with stirring at 25°C to a final concentration of 20 mM and then stirred for l hr (Saravis, unpublished data). After centrifugation at 8000g at room temp for 1 hr, EDTA was added to the supernatant with stirring to a final concentration of 20 mM (Saravis, unpublished data). This procedure was a modification of Heremans et al. (1959). The supernatant was then applied to a Bio-Gel P-200 column in BSB. IgA (most likely the polymer form) was found in the first peak after exclusion of the void volume and IgG in a second peak as evidenced with specific antiserum by IEP. The first peak was also adsorbed with an ethylchloroformate prepared polymer (Avrameas and Ternynck, 1967)of rabbit antihuman lgG (absorbed at equivalence by the quantitative precipitin reaction with human F(ab')2 before polymerization). Diphtheria antitoxin determination. Antitoxin unitage (AU/ml) was determined by intradermal toxin neutralization in guinea pigs (Fraser, 1931) by comparison with standard horse antitoxin from NIH. Two-fold dilutions were titrated beginning at the level of 0-01 AU/ml, the lowest level readily detectable. The toxin levels used in titration were L R/100. Indirect hemagglutination. The method of Oalazka and Abgarowicz (1967) was employed using formalinized and tannin-treated horse red blood cells sensitized with diphtheria toxoid.* A standard horse antitoxin from NIH was used as a control.
* Provided by Biologic Laboratories, Massachusetts Department of Public Health. Boston, Mass. 223 I M M Vol. I I. No. 5
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DONALD D. OURTH
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Table 1. Intradermal neutralization of diphtheria toxin by human immunoglobulin classes and subunits
Antibody ISG ISG IgG F(ab')e Fab' IgM IgM-2ME IgA
(mg/ml) 160 5 5 5 5 5 5 2
moL wt
155,000 105,000 44,000
Antibody binding sites
Antibody binding sites/AU
Neutralization units (AU/ml) 40'0 1'3 1"3 1'3 0.32 <0.01 <0.01 <0.01
3"9 x 1016 5"7 × 1 0 1 6 6.9 x 10~°
3"0 x 10l~ 4"4 × 101~ 2"2 × 101~
ISG = Immune serum globulin. 2ME = 2-mercaptoethanol. hmmmoeh,ctrophoresis. Electrophoresis for specificity of IgG, antibody subunits, IgM and IgA was performed in 1% agar gel, followed by precipitation with rabbit antihuman serum, rabbit antihuman IgG and rabbit antihuman IgM (BehringDiagnostics,Woodbury, N.Y.) and goat antihuman IgA (Kallestad Lab., Minneapolis, Minn.). Protein determination. 1SG* nitrogen concentration tconverted to protein) was determined by the microKjeldahl technique (Hiller et al., 1948). Protein concentrations of IgG, IgM, IgA, F(ab')2 and Fab' were from optical density readings at 280nm using E2~onm t% = 15 (Mandy et al., 1961). Globulins were applied to DEAE columns at 15 mg protein/ml packed DEAE.
RESULTS It was found that, besides IgG, F(ab')2 and Fab' subunits of IgG were capable of neutralizing diphtheria toxin. However, there was a decrease in ability of the subunits to neutralize toxin when compared with undigested IgG (Table 1). Approximately 5 and 7.3 times the number of antibody binding sites/AU of FaN were needed to neutralize toxin when compared, respectively, with F(ab')2 and IgG. About 1-5 times the number of antibody binding sites/AU of F(ab')2 were needed to neutralize toxin when compared with IgG. lgG, F(ab')2, lgM and IgA were capable of HA with diphtheria toxoid (Table 2). However, it was found that IgM and IgA, though able to bind diphtheria toxoid by the HA test (Table 2), were unable to neutralize diphtheria toxin at the 0-01 AU/ml level by intradermal skin testing in guinea pigs (Table 1). Reduced and alkylated IgM also did not neutralize diphtheria toxin (Table 1). DISCUSSION
that rabbit IgM diphtheria antitoxin did not neutralize diphtheria toxin, although it combined with toxoid in the HA test. IgG was capable of both HA and neutralization of diphtheria toxin. In the horse, diphtheria antitoxin was found predominantly in what Raynaud (1959, 1967) defined as IgA but also in IgG. Pepsinization and reduction of horse IgG yielded 3S fragments that still retained their avidity (Raynaud, 1967; Raynaud and Relyveld, 1959; Relyveld, 1959; Iscaki and Raynaud, 1961). Peptic digestion of horse IgA to obtain 5S fragments and reduction to 3S fragments yielded subunits that were capable of neutralizing diphtheria toxin (Raynaud, 1967; Raynaud and Iscaki, 1964). Heremans et al. (1963) found that a greater concentration of human IgM than IgG was needed in HA for diphtheria antitoxin. However, Newcomb and Ishizaka (1967) were not able to demonstrate human IgM diphtheria antitoxin but were able to demonstrate high IgG and low IgA activities by HA, intradermal toxin neutralization, and radioactive toxoid-binding methods. They also found that IgA activity was mainly associated with the polymer rather than with the monomer form of IgA. They were unable to detect human IgM diphtheria antitoxin even by radioimmunodiffusion. However, Raynaud (1967) found human diphtheria antitoxins present in IgG, IgM and IgA by radioimmunoelectrophoresis. Table 2. Indirect hemagglutination of diphtheria toxoid by human immunoglobulin classes and subunits Antibody ISG IgG
(mg/ml) 160 5 5 5 5 5 2
Bauer and Stavitsky (1961) found by intradermal toxin neutralization that rabbit 19S diphtheria antitoxin neutralized diphtheria toxin after incubation at 5'C but not at 23°C. However, Robbins (1965) found
F(ab')2 Fab' IgM IgM-2ME IgA
* Provided by Biologic Laboratories, Massachusetts Department of Public Health, Boston. Mass.
ISG = Immune serumglobulin. 2ME = 2-mercaptoethanol.
Reciprocal HA titer 256,000 32,000 4000 64 8 32
Neutralization of Diphtheria Toxin F r o m this investigation, it is not understood why IgM and IgA are able to bind diphtheria toxoid by the HA test yet, at the same time, are unable to neutralize diphtheria toxin. These two antibody classes may be directed toward toxoid sites which produce a non-neutralizing antibody response to the toxin. Or possibly IgM and IgA molecules are too large to combine readily with presumed poorly displayed or hidden antigenic sites on the toxin molecule. However, reduced and alkylated IgM did not neutralize diphtheria toxin. This may indicate that subunits of IgM also do not neutralize, since Deutsch and Morton (1957, 1958) found that human macroglobulins are dissociated to 6.5 S gamma globulins following treatment with sulfhydryl compounds. Solheim (1972) has found that divalent 8S subunits of human IgM following 2ME treatment were still active in HA. This investigation found that the human antibody class of importance in neutralizing diphtheria toxin was IgG, and that toxin neutralization was retained by the F(ab')2 and Fab' subunits of IgG. However, a decrease was noted, as indicated by the number of antibody binding sites/AU, in the stability of the toxin-antitoxin union with F(ab')2 and Fab' when compared with IgG. Acknowledgements--The author is grateful to Dr. A. B.
MacDonald of Harvard University for providing the ethylchloroformate-prepared polymer of rabbit antihuman IgG and to Mrs. Nancy Blake of the Biologic Laboratories for performing the toxin neutralization test. This investigation was supported by NIH Training Grant T01 AI-00221. REFERENCES Avrameas S, and Ternynck J. (1967) J. biol. Chem. 242, 1651. Bauer D. C. and Stavitsky A. B. (1961) Proc. natn. Acad. Sci. U.S.A. 4% 1667.
225
Chaplin H., Cohen S. and Press E. M. (1965) Biochem. J. 95, 256. Cohn E. J., Strong L. E., Hughes W. L., Jr., Mulford D. J., Ashworth J. N., Melin M. and Taylor H. L. (1946) J. Am. chem. Soc. 68, 459. Deutsch H, F. and Morton J. I. (1957) Science 125, 600. Deutsch H, F. and Morton J. I. (1957) Fedn Proc. 16, 172. Deutsch H, F. and Morton J. I. (1958) J. biol. Chem. 231, 1107. Fraser D. T. (1931) Trans. R. Soc. Canada 25, 175. Galazka A. and Abgarowicz A. (1967) Epid. Rev. 21,237. Heremans J. F., Heremans M.-Th. and Schultze. H. E. (1959) Clinica chim. Acta 4, 96. Heremans J. F., Vaerman J. P. and Vaerman C. ( 1963} J. 1mmun. 91, 11. Hiller A., Plazin J. and Van Slyke D. D. (1948) J. biol. Chem. 176, 1401. Iscaki S. and Raynaud M. (1961) C. r. Acad. Sci. 253, 2286. Mandy W. J., Rivers M. M. and Nisonoff A. (1961) J. biol. Chem. 236, 3221. Newcomb R. W. and Ishizaka K. (1967) J. lmmun. 99, 40. NisonoffA. (1960) Biochem. biophys. Res. Comm. 3,466. NisonoffA., Wissler F. C., Lipman L. N. (1960) Science 132, 1770. Palmer J. L. and NisonoffA. (1963) J. biol. Chem. 238, 2393. Raynaud M. (1959) Mechanisms of Hypersensitivity (Edited by Shaffer J. H.. Logrippo G. A. and Chase M W.I, p. 27. Vol. 1. Little. Brown, Boston. Raynaud M. (1967) Antibodies to Biolo~tically AetiLe Molecules (Edited by Cinader B.), p. 197. Pergamon Press, Oxford. Raynaud M. and Relyveld E. H. (1959) Ann, Inst. Pasteur 97, 636. Raynaud M. and Iscaki S. (1964) Nature, Lond. 203, 758. Relyveld E. H. (1959) Toxine et Antitoxine Diphterique~s. Etude Immunologique, Vol. 1. Hermann, Paris. Robbins J. B. (1965) Molecular and Celhdar Basis o]" Antibody Formation (Edited by Sterzl J.), p. 241. Academic Press, New York. Saravis C. A. Unpublished data. Solheim B. G. (1972) Scand. J. hnmun. I, 179.
Pclgalnol~ Press.
Prmlcd in G r e a t Britain
NEUTRALIZATION OF DIPHTHERIA TOXIN BY H U M A N I M M U N O G L O B U L I N CLASSES AND SUBUNITS D O N A L D D. O U R T H Department of Microbiology, Harvard University School of Public Health, Boston, Massachusetts 02115, U.S.A.
(Receired 5 July 1973) This investigation found that the human antibody class of importance in neutralizing diphtheria toxin was IgG, and that toxin neutralization was retained by the F(ab')2 and Fab' subunits of IgG. IgM and IgA did not neutralize diphtheria toxin, although these two antibody classes reacted with diphtheria toxoid in the indirect hemagghltination test. Abstract
INTRODUCTION In immunization for diphtheria with toxoid, prevention depends on the ability of immunoglobulins to neutralize bacterial exotoxin. This investigation was therefore directed to determining which human antibody class (IgG, IgM, IgA) was most capable of protecting against diphtheria toxin. At the same time, subunits of human IgG and IgM were isolated by pepsinization, reduction and alkylation to determine if antibody subunits could neutralize diphtheria toxin. In addition, indirect hemagglutination (HA) was used to investigate whether antibodies could bind diphtheria toxoid, even though not capable of neutralizing diphtheria toxin.
MATERIALS AND METHODS
Preparation of human IyG, F(ab') 2 and Fah' suhunits. A pseudoglobulin supernatant was isolated from Cohn fraction II (Cohn et al., 1946) human immune serum globulin* (ISG) having a diphtheria antitoxin titer of 40 AU/ml by dialysis against distilled water for 72 hr at 4 (2 Tile supernatam was then applied to Scphadex DEAE A-50 in 0.0175 M NaH2PO4 buffer, pH 6"9 (Palmer and Nisonoff, 1963). After digestion with pepsin (2 mg pepsin/100 mg IgG), F(ab')~ was isolated bv twice passing the digest through Scphadcx (i-150 in 0"(11 M borate saline buffer pH 8 (BSB) (Ni~,onol]cl a/.. 1960). Fab' was prepared b~ redaction of the F(ab'), with 0.05 M 2-mercaptoethanol (redistilled) (2ME) in a nitrogen atmosphere at room temp followed by alkylation with a 201~; molar excess of iodoacetic acid (redistilled) at 4:'C and then Sephadex G-150 filtration in BSB (Nisonoff, 1960). Immunoelectrophoresis (IEP) was used to check for specificity of the lgG, F(ab'), and Fab'. Preparation (?.]human 19M. To isolate lgM, the euglobulin from ISG* after dialysis against distilled water was applied to Sephadex DEAE A-50 in 0.1 M NaH2PO~, buffer, pH 6"3. After elution at this molarity and pH, a salt gradient of
0'1 M NaH2PO 4 buffer, pH 6.3 to 0.45 M NaH2PO 4 buffer, pH 4-2 followed (Chaplin et al., 1965). The ascending portion of the first peak eluted was applied to Sephadex G-200 in BSB to obtain IgM as indicated with specific antiserum by IEP. Reduction with 2-mercaptoethanol. To obtain subunits, IgM was reduced in a nitrogen atmosphere with 0.1 M 2ME for 2 hr at room temperature followed by alkylation with an equal volume of 0.12 M iodoacetic acid (redistilled) for 2 hr at 4°C. This was followed by dialysis for 72 hr in BSB at 4°C. Preparation of human IgA. To isolate IgA, the pseudoglobulin supernatant of Cohn Fraction It (Cohn et al., 1946) ISG* was applied to Sephadex DEAE A-50 in 0.0175MNaH2PO 4 buffer, pH 6.9. After elution at this molarity and pH, a second fraction was eluted with 0.2 M NaH2PO 4 buffer, pH 5.8. To this second fraction, zinc glycinate was added slowly with stirring at 25°C to a final concentration of 20 mM and then stirred for l hr (Saravis, unpublished data). After centrifugation at 8000g at room temp for 1 hr, EDTA was added to the supernatant with stirring to a final concentration of 20 mM (Saravis, unpublished data). This procedure was a modification of Heremans et al. (1959). The supernatant was then applied to a Bio-Gel P-200 column in BSB. IgA (most likely the polymer form) was found in the first peak after exclusion of the void volume and IgG in a second peak as evidenced with specific antiserum by IEP. The first peak was also adsorbed with an ethylchloroformate prepared polymer (Avrameas and Ternynck, 1967)of rabbit antihuman lgG (absorbed at equivalence by the quantitative precipitin reaction with human F(ab')2 before polymerization). Diphtheria antitoxin determination. Antitoxin unitage (AU/ml) was determined by intradermal toxin neutralization in guinea pigs (Fraser, 1931) by comparison with standard horse antitoxin from NIH. Two-fold dilutions were titrated beginning at the level of 0-01 AU/ml, the lowest level readily detectable. The toxin levels used in titration were L R/100. Indirect hemagglutination. The method of Oalazka and Abgarowicz (1967) was employed using formalinized and tannin-treated horse red blood cells sensitized with diphtheria toxoid.* A standard horse antitoxin from NIH was used as a control.
* Provided by Biologic Laboratories, Massachusetts Department of Public Health. Boston, Mass. 223 I M M Vol. I I. No. 5
A
DONALD D. OURTH
224
Table 1. Intradermal neutralization of diphtheria toxin by human immunoglobulin classes and subunits
Antibody ISG ISG IgG F(ab')e Fab' IgM IgM-2ME IgA
(mg/ml) 160 5 5 5 5 5 5 2
moL wt
155,000 105,000 44,000
Antibody binding sites
Antibody binding sites/AU
Neutralization units (AU/ml) 40'0 1'3 1"3 1'3 0.32 <0.01 <0.01 <0.01
3"9 x 1016 5"7 × 1 0 1 6 6.9 x 10~°
3"0 x 10l~ 4"4 × 101~ 2"2 × 101~
ISG = Immune serum globulin. 2ME = 2-mercaptoethanol. hmmmoeh,ctrophoresis. Electrophoresis for specificity of IgG, antibody subunits, IgM and IgA was performed in 1% agar gel, followed by precipitation with rabbit antihuman serum, rabbit antihuman IgG and rabbit antihuman IgM (BehringDiagnostics,Woodbury, N.Y.) and goat antihuman IgA (Kallestad Lab., Minneapolis, Minn.). Protein determination. 1SG* nitrogen concentration tconverted to protein) was determined by the microKjeldahl technique (Hiller et al., 1948). Protein concentrations of IgG, IgM, IgA, F(ab')2 and Fab' were from optical density readings at 280nm using E2~onm t% = 15 (Mandy et al., 1961). Globulins were applied to DEAE columns at 15 mg protein/ml packed DEAE.
RESULTS It was found that, besides IgG, F(ab')2 and Fab' subunits of IgG were capable of neutralizing diphtheria toxin. However, there was a decrease in ability of the subunits to neutralize toxin when compared with undigested IgG (Table 1). Approximately 5 and 7.3 times the number of antibody binding sites/AU of FaN were needed to neutralize toxin when compared, respectively, with F(ab')2 and IgG. About 1-5 times the number of antibody binding sites/AU of F(ab')2 were needed to neutralize toxin when compared with IgG. lgG, F(ab')2, lgM and IgA were capable of HA with diphtheria toxoid (Table 2). However, it was found that IgM and IgA, though able to bind diphtheria toxoid by the HA test (Table 2), were unable to neutralize diphtheria toxin at the 0-01 AU/ml level by intradermal skin testing in guinea pigs (Table 1). Reduced and alkylated IgM also did not neutralize diphtheria toxin (Table 1). DISCUSSION
that rabbit IgM diphtheria antitoxin did not neutralize diphtheria toxin, although it combined with toxoid in the HA test. IgG was capable of both HA and neutralization of diphtheria toxin. In the horse, diphtheria antitoxin was found predominantly in what Raynaud (1959, 1967) defined as IgA but also in IgG. Pepsinization and reduction of horse IgG yielded 3S fragments that still retained their avidity (Raynaud, 1967; Raynaud and Relyveld, 1959; Relyveld, 1959; Iscaki and Raynaud, 1961). Peptic digestion of horse IgA to obtain 5S fragments and reduction to 3S fragments yielded subunits that were capable of neutralizing diphtheria toxin (Raynaud, 1967; Raynaud and Iscaki, 1964). Heremans et al. (1963) found that a greater concentration of human IgM than IgG was needed in HA for diphtheria antitoxin. However, Newcomb and Ishizaka (1967) were not able to demonstrate human IgM diphtheria antitoxin but were able to demonstrate high IgG and low IgA activities by HA, intradermal toxin neutralization, and radioactive toxoid-binding methods. They also found that IgA activity was mainly associated with the polymer rather than with the monomer form of IgA. They were unable to detect human IgM diphtheria antitoxin even by radioimmunodiffusion. However, Raynaud (1967) found human diphtheria antitoxins present in IgG, IgM and IgA by radioimmunoelectrophoresis. Table 2. Indirect hemagglutination of diphtheria toxoid by human immunoglobulin classes and subunits Antibody ISG IgG
(mg/ml) 160 5 5 5 5 5 2
Bauer and Stavitsky (1961) found by intradermal toxin neutralization that rabbit 19S diphtheria antitoxin neutralized diphtheria toxin after incubation at 5'C but not at 23°C. However, Robbins (1965) found
F(ab')2 Fab' IgM IgM-2ME IgA
* Provided by Biologic Laboratories, Massachusetts Department of Public Health, Boston. Mass.
ISG = Immune serumglobulin. 2ME = 2-mercaptoethanol.
Reciprocal HA titer 256,000 32,000 4000 64 8 32
Neutralization of Diphtheria Toxin F r o m this investigation, it is not understood why IgM and IgA are able to bind diphtheria toxoid by the HA test yet, at the same time, are unable to neutralize diphtheria toxin. These two antibody classes may be directed toward toxoid sites which produce a non-neutralizing antibody response to the toxin. Or possibly IgM and IgA molecules are too large to combine readily with presumed poorly displayed or hidden antigenic sites on the toxin molecule. However, reduced and alkylated IgM did not neutralize diphtheria toxin. This may indicate that subunits of IgM also do not neutralize, since Deutsch and Morton (1957, 1958) found that human macroglobulins are dissociated to 6.5 S gamma globulins following treatment with sulfhydryl compounds. Solheim (1972) has found that divalent 8S subunits of human IgM following 2ME treatment were still active in HA. This investigation found that the human antibody class of importance in neutralizing diphtheria toxin was IgG, and that toxin neutralization was retained by the F(ab')2 and Fab' subunits of IgG. However, a decrease was noted, as indicated by the number of antibody binding sites/AU, in the stability of the toxin-antitoxin union with F(ab')2 and Fab' when compared with IgG. Acknowledgements--The author is grateful to Dr. A. B.
MacDonald of Harvard University for providing the ethylchloroformate-prepared polymer of rabbit antihuman IgG and to Mrs. Nancy Blake of the Biologic Laboratories for performing the toxin neutralization test. This investigation was supported by NIH Training Grant T01 AI-00221. REFERENCES Avrameas S, and Ternynck J. (1967) J. biol. Chem. 242, 1651. Bauer D. C. and Stavitsky A. B. (1961) Proc. natn. Acad. Sci. U.S.A. 4% 1667.
225
Chaplin H., Cohen S. and Press E. M. (1965) Biochem. J. 95, 256. Cohn E. J., Strong L. E., Hughes W. L., Jr., Mulford D. J., Ashworth J. N., Melin M. and Taylor H. L. (1946) J. Am. chem. Soc. 68, 459. Deutsch H, F. and Morton J. I. (1957) Science 125, 600. Deutsch H, F. and Morton J. I. (1957) Fedn Proc. 16, 172. Deutsch H, F. and Morton J. I. (1958) J. biol. Chem. 231, 1107. Fraser D. T. (1931) Trans. R. Soc. Canada 25, 175. Galazka A. and Abgarowicz A. (1967) Epid. Rev. 21,237. Heremans J. F., Heremans M.-Th. and Schultze. H. E. (1959) Clinica chim. Acta 4, 96. Heremans J. F., Vaerman J. P. and Vaerman C. ( 1963} J. 1mmun. 91, 11. Hiller A., Plazin J. and Van Slyke D. D. (1948) J. biol. Chem. 176, 1401. Iscaki S. and Raynaud M. (1961) C. r. Acad. Sci. 253, 2286. Mandy W. J., Rivers M. M. and Nisonoff A. (1961) J. biol. Chem. 236, 3221. Newcomb R. W. and Ishizaka K. (1967) J. lmmun. 99, 40. NisonoffA. (1960) Biochem. biophys. Res. Comm. 3,466. NisonoffA., Wissler F. C., Lipman L. N. (1960) Science 132, 1770. Palmer J. L. and NisonoffA. (1963) J. biol. Chem. 238, 2393. Raynaud M. (1959) Mechanisms of Hypersensitivity (Edited by Shaffer J. H.. Logrippo G. A. and Chase M W.I, p. 27. Vol. 1. Little. Brown, Boston. Raynaud M. (1967) Antibodies to Biolo~tically AetiLe Molecules (Edited by Cinader B.), p. 197. Pergamon Press, Oxford. Raynaud M. and Relyveld E. H. (1959) Ann, Inst. Pasteur 97, 636. Raynaud M. and Iscaki S. (1964) Nature, Lond. 203, 758. Relyveld E. H. (1959) Toxine et Antitoxine Diphterique~s. Etude Immunologique, Vol. 1. Hermann, Paris. Robbins J. B. (1965) Molecular and Celhdar Basis o]" Antibody Formation (Edited by Sterzl J.), p. 241. Academic Press, New York. Saravis C. A. Unpublished data. Solheim B. G. (1972) Scand. J. hnmun. I, 179.