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An absolute protein requirement for the acid elution of some antibodies
Journal of ImmunologicalMethods 1 (1971) 101-106. North-Holland Publishing Company
AN ABSOLUTE PROTEIN REQUIREMENT FOR THE ACID ELUTION OF SOME ANTIBODIES * A.L. LUZZATI **, A.O. CARBONARA and R.M. TOSI *** Istituto di Genetica Medica, Universitb di Torino, Torino, Italy
Received 21 June 1971 Instances are reported in which radiolabelled antibodies can be purified by acid elution with good yield only if protein is added to the buffer used to wash the antigen-antibody complex. The same effect is obtained if the protein is added directly to the acid buffer.
1. INTRODUCTION The acid elution is a commonly used procedure to obtain purified antibodies from a variety of immunocomplexes (Kabat and Mayer, 1961). We have observed a very marked difference in the elution recovery from bacteria, depending upon the presence or absence of bovine serum albumin (BSA) during the washings of the antigen-antibody complex. We decided to study this protein requirement under experimental conditions in which the antibody elution yield can be reliably and easily determined by very sensitive methods, i.e., by using radioactively labelled antibody. The results reported in this communication refer to elution of specific antibodies from: (a) Salmonella enteritidis bacterial bodies; (b)Group C Streptococcus bacterial bodies and isolated cell walls; (c) Rabbit immunoglobulin, insolubilized by cross-linking. The antibodies were radioactively labelled either with 12sI or with 14C. The latter were obtained from in vitro cultures of stimulated lymph node fragments, as previously described (Carbonara et al., 1969). The following absorption-elution procedure was used: 1)Absorption was performed by incubating the antigen with labelled antibody at * This research was supported in part by Grant No. AI-08896 from the National Institutes of Health, in part by Consigtio Nazionale delle Ricerche, Centro di Studio per l'Immunogenetica e l'Istocompatibflit~. ** Present address: Clinica del Lavoro 'L. Devoto', Via San Barnaba 8, Milano, Italy. *** Present address: Laboratorio di Biologia Cellulare, Via Carrara 18, Roma, Italy.
102
A.L.Luzzati et aL, Proteins required in antibody elution
37°C for one hour and then overnight at 4°C. The tubes were finally centrifuged at 4°C at 10,000g for 10 min and the supernatant removed. The amount of antigen in each system was chosen on the basis of quantitative absorption experiments, by incubating the same volume of antibody with increasing amounts of antigen. The amount of antigen sufficient to bind 90-95% of antibody was selected. 2) Washings were performed by resuspending the pellet in ten times the original reaction volume of cold phosphate-buffered saline (PBS) (0.15 M NaCI, 0.015 M sodium phosphate buffer, pH 8.0) either containhlg or not containing BSA at the concentration of 1%. Three to five washings were necessary to remove all non-bound or non-specifically bound radioactive material. 3) Elution was obtained by resuspending the antigen-antibody complex in half the initial volume (i.e. 0.2-0.4 ml) of glycine HCI buffer 0.2 M, pH 2.2. After 5 rain at room temperature the preparation was centrifuged in the cold for 10 rain at 10,000g and the supernatant was immediately neutralized with 1 M Tris HC1 buffer, pH 10.0. For the elution from Salmonella enteritMis bacterial bodies the following procedure, originally described by Robbins et al. (1965) and shown to be more efficient, was used. The antigen-antibody complex was resuspended in half the initial volume of 0.1 M sodium acetate buffer, pH 3.9. After 90 min at 37°C with frequent stirring, the preparation was centrifuged in the cold as above and tile supernatant dialyzed overnight against PBS. 2. RESULTS In all experiments the proportion of antibody molecules recovered was determined from the radioactivity of the eluate. The antibody activity of the eluted material was tested by reabsorbing it on the specific antigen. Generally around 70% of the eluted counts could be reabsorbed. In addition, in the case of anti-Salmonella antibodies, the eluates exhibited agglutinating activity when tested with sheep red blood cells coated with Salmonella enteritidis somatic antigen (Landyoet al., 1965). Table 1 shows the different yields obtained by using or not using 1% BSA during the washings of the Salmonella anti-Salmonella complex. While with 1% BSA 40-50% of the counts absorbed on the bacteria could be eluted, the counts recovered when BSA was omitted from the washing fluid were negligible. Repeated elutions did not markedly increase the recovery in either case. Practically all the counts which could not be eluted were found to be still bound to the antigen. Radioactive proteins did not non-specifically stick to the walls of the plastic tubes. Superimposable results were obtained with ~4C-labelled antibodies synthesized in vitro by lymph nodes from rabbits stimulated with Salmonella enteritidis endotoxin (Carbonara et al., 1969). Table 2 shows the results obtained with anti-Group C Streptococcus antibodies. Both bacterial bodies and isolated cell walls (Bleiweis et al., 1964) were tested and a striking influence of BSA was apparent in either case.
Table 1 Elution of specific 125I-labelled antibody from Salmonella enteritidis bacterial bodies, after washing with or without 1% BSA. With BSA
Without BSA
cpm of 125I-labelled globulin fraction employed
7.4 × 106
7.4 × 106
cpm absorbed on bacterial bodies (after washings)
12,751
14,262
Proportion of counts absorbed cpm eluted by the first acid treatment Proportion of counts eluted
0.17% 5050 39.6%
0.19% 220 1.5%
cpm eluted by the second acid treatment Proportion of counts eluted
855 6.7%
249 1.75%
Proportion of eluted counts (first treatment) reabsorbed on bacteria
68.5%
Not done
Anti-Salmonella serum was obtained by injecting intravenously into a normal adult rabbit 1 ug SalmoneUa enteritidis endotoxin purified by the procedure of Ribi et al. (1961). 0.5 ml of antiserum was precipitated with 2 M ammonium sulphate and the globulin fraction (5.5 mg) was labelled with 1251 by the method of McFarlane (1958). The specific activity of the labelled protein was about 7000 cpm/~g. 5 X l0 s bacterial bodies were used in each test. Absorption and elution of the labelled antibody were performed as described in the text.
Table 2 Proportion of specific 12Sl-labelled antibodies eluted from Group C streptococcal cell walls under different conditions.
1 2 3 4 5 6 7 8 9 10
Washing
Elution
% antibody elu ted
PBS BSA 1% PBS PBS PBS PBS PBS PBS PBS PBS
Glycine buffer Glycine buffer Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer +
5 44 48 40 20 49 42 1.6 2.3 1.9
1% BSA 0.1% BSA 0.01% BSA 1% human hemoglobin 1% horse ferritin 1% dextran 500,000 MW 1% dextran 200,000 MW 1% dextran 40,000 MW
A normal adult rabbit was immunized with pepsin-treated Group C Streptococci, following the method described by Braun and Krause (1968). Under these conditions a high serum concentration of antibodies of restricted heterogeneity is produced. IgG were purified by ammonium sulphate precipitation followed by DEAE chromatography. The DEAE column was eluted with phosphate buffer 0.02 M, pH 8.0. About 70% of the purified IgG possessed specific antibody activity, as judged by quantitative binding experiments using streptococcal cell walls. The latter were prepared following the method of Bleiweis et al. (1964). The purified IgG was labelled with 12 s I by the Chloramin T method (Greenwood et al., 1963). The specific activity obtained was about 500,000 cpmh~g. Approximately 20,000 cpm were used in each test, with 0.5 mg (dry weight) of streptococcal cell wails. Absorption and elution of the labelled antibody were performed as described in the text.
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A.L.Luzzati et al., Proteins required in antibody elution
Table 3 Elution of specific 125Iqabelled anti-Ab9 rabbit allotype antibody from antigen insolubilized by cross-linking.
cpm of 12St-labelledanti-Ab9 IgG employed cpm absorbed on cross-linked serum Proportion of counts absorbed cpm eluted by acid treatment Proportion of counts eluted
With BSA
Without BSA
7734 2310 30.0% 1820 78.5%
7676 2330 30.5% 1810 77.8%
The serum from a normal rabbit homozygous for the Ab9 allotype was insolubilized with ethyl chloroformate (Avrameas et al., 1967). This material was used to purify (by absorption and subsequent acid elution) the specific anti-Ab9 antibody produced immunizing an Ab4 homozygous rabbit by the procedure described by Dubiski (Dubiski et al., 1959). The eluted material was applied on a G-200 Sephadex column (2.5 X 100cm) and the resulting 7S fractions were pooled and concentrated. The anti-Ab9 IgG thus obtained was labelled with 12 sI by the chloramin T method (Greenwood et al., 1963). The specific activity was about 500,000 cpm/pg. Although initially this labelled preparation was 70% reabsorbable on the specific antigen, at the time of the experiment, after prolonged storage, only 30% of the counts could be absorbed. The experiment was performed with the same immunoadsorbent used for the purification of anti-Ab9 antibodies.
As shown in table 2, BSA does not need to be present during the washing step, but is critically required during the acid treatment. Therefore it can be directly dissolved in the eluting buffer. The minimal concentration of BSA required for maximal recovery is around 0.1%. Since similar results are obtained either including 0.1% BSA directly in the acid buffer or washing with 1% BSA before the acid treatment, it may be concluded that the amount of BSA remaining in the pellet after the last washing, although diluted by the buffer, is still adequate. Proteins other than BSA exert the same effect, when used in comparable amounts. Non proteinic substances, even of high molecular weight, such as dextran, do not influence the elution yield (table 2). Table 3 shows similar experiments performed using as immunoadsorbent rabbit serum (from an Ab9 homozygous animal) insolubilized by crosslinking with ethyl chloroformate (Avrameas et al., 1967). Partially purified ~2SI-labelled rabbit IgG with anti-allotype 9 activity was used as antibody. In this instance no effect at all was obtained by addition of BSA. Little, if any, effect was observed also in the case of elution of anti-HL-A isoantibodies from platelets. A quantitative evaluation of this system is in progress.
A.L.Luzzati et al., Proteins required in antibody elution
105
3. DISCUSSION From the results reported above it appears that the presence of a protein is a necessary requirement to obtain a relatively high yield of purified antibody by acid elution, at least in some a n t i g e n - a n t i b o d y systems. Although the effect is very striking, no generalization can be made from the three systems that we have investigated. However, it may be relevant that in both cases in which an absolute protein requirement is shown, antibodies against bacterial polysaccharide determinants are involved. In the case of Salmonella, the antibodies are directed against Group 0 somatic antigens, and in the case o f Streptococcus, against Group C specific determinants. The single system in which protein addition was without effect was with antibodies against a protein determinant (the Ab9 allotypic determinant of rabbit IgG). To clarify this issue it will be necessary to extend this investigation to systems involving non-bacterial polysaccharide determinants (e.g., blood group antigens, dextran, etc.) and bacterial proteins (e.g., Salmonella flagellin). No explanation can be offered of the mechanism by which BSA influences the detachment o f antibody from bacterial surfaces. However, BSA must be present at the moment in which the elution occurs: previous exposure of the bacterial bodies to BSA, either before or after antibody attachment, is without effect if all protein is removed by extensive washings before elution. Since other proteins work as well, this effect cannot be attributed to some specific property of BSA. In our experimental conditions the presence of protein seems to be an absolute requirement (the recovery in absence of BSA is indistinguishable from the background level). Such an extreme situation could be due to the very small amount of labelled antibody used in our system and may not be found when the starting material is total immune serum and in large amount. However by including BSA in the elution procedure an unusually good reproducibility of the amount of antibody recovered was also observed. Therefore we feel that under all conditions a careful control over the protein environment is essential for the standardization of any elution system.
ACKNOWLEDGEMENTS We want to thank Prof. Ruggero Ceppellini for helpful discussions and criticism of the manuscript. Streptococcal vaccines and streptococcal cell walls were kindly provided by Dr. D.G. Braun. The expert technical assistance of Miss Cleide Boccazzi is gratefully acknowledged.
REFERENCES Avrameas, S. and T. Ternynck, 1967, J. Biol. Chem. 242, 1651. Bleiweis, A.S., W.W. Karakawa and R.M. Krause, 1964, J. Bacteriol. 88, 1198.
106
A.L.Luzzati et al., Proteins required in anbitody elution
Braun, D.G. and R.M. Krause, 1968, J. Exptl. Med. 128, 969. Carbonara, A.O., A.L. Luzzati, R.M. Tosi, G. Mancini and R. Ceppellini, 1969, in: Lymphatic tissue and germinal centers in immune response (Plenum Publishing Corp., New York) 5,441 Dubiski, S., Z. Dudziak, D. Skalba and A. Dubiska, 1959, Immunology 2, 84. Greenwood, F.C., W.M. Hunter and J.S. Glover, 1963, Biochem. J. 89, 114. Kabat, E.A. and M.M. Mayer, 1961, Experimental immunochemistry (Charles C Thomas, Springfield, Illinois). Landy, M., R.P. Sanderson and A.L. Jackson, 1965, J. Exptl. Med. 122,483. McFarlane, A.S., 1958, Nature 182, 53. Ribi, E., W.T. Askins, M. Landy and K.C. Mllner, 1961, J. Exptl. Med. 114,647. Robbins, J.B., K. Kenny and E.J. Surer, 1965, J. Exptl. Med. 122,385.
AN ABSOLUTE PROTEIN REQUIREMENT FOR THE ACID ELUTION OF SOME ANTIBODIES * A.L. LUZZATI **, A.O. CARBONARA and R.M. TOSI *** Istituto di Genetica Medica, Universitb di Torino, Torino, Italy
Received 21 June 1971 Instances are reported in which radiolabelled antibodies can be purified by acid elution with good yield only if protein is added to the buffer used to wash the antigen-antibody complex. The same effect is obtained if the protein is added directly to the acid buffer.
1. INTRODUCTION The acid elution is a commonly used procedure to obtain purified antibodies from a variety of immunocomplexes (Kabat and Mayer, 1961). We have observed a very marked difference in the elution recovery from bacteria, depending upon the presence or absence of bovine serum albumin (BSA) during the washings of the antigen-antibody complex. We decided to study this protein requirement under experimental conditions in which the antibody elution yield can be reliably and easily determined by very sensitive methods, i.e., by using radioactively labelled antibody. The results reported in this communication refer to elution of specific antibodies from: (a) Salmonella enteritidis bacterial bodies; (b)Group C Streptococcus bacterial bodies and isolated cell walls; (c) Rabbit immunoglobulin, insolubilized by cross-linking. The antibodies were radioactively labelled either with 12sI or with 14C. The latter were obtained from in vitro cultures of stimulated lymph node fragments, as previously described (Carbonara et al., 1969). The following absorption-elution procedure was used: 1)Absorption was performed by incubating the antigen with labelled antibody at * This research was supported in part by Grant No. AI-08896 from the National Institutes of Health, in part by Consigtio Nazionale delle Ricerche, Centro di Studio per l'Immunogenetica e l'Istocompatibflit~. ** Present address: Clinica del Lavoro 'L. Devoto', Via San Barnaba 8, Milano, Italy. *** Present address: Laboratorio di Biologia Cellulare, Via Carrara 18, Roma, Italy.
102
A.L.Luzzati et aL, Proteins required in antibody elution
37°C for one hour and then overnight at 4°C. The tubes were finally centrifuged at 4°C at 10,000g for 10 min and the supernatant removed. The amount of antigen in each system was chosen on the basis of quantitative absorption experiments, by incubating the same volume of antibody with increasing amounts of antigen. The amount of antigen sufficient to bind 90-95% of antibody was selected. 2) Washings were performed by resuspending the pellet in ten times the original reaction volume of cold phosphate-buffered saline (PBS) (0.15 M NaCI, 0.015 M sodium phosphate buffer, pH 8.0) either containhlg or not containing BSA at the concentration of 1%. Three to five washings were necessary to remove all non-bound or non-specifically bound radioactive material. 3) Elution was obtained by resuspending the antigen-antibody complex in half the initial volume (i.e. 0.2-0.4 ml) of glycine HCI buffer 0.2 M, pH 2.2. After 5 rain at room temperature the preparation was centrifuged in the cold for 10 rain at 10,000g and the supernatant was immediately neutralized with 1 M Tris HC1 buffer, pH 10.0. For the elution from Salmonella enteritMis bacterial bodies the following procedure, originally described by Robbins et al. (1965) and shown to be more efficient, was used. The antigen-antibody complex was resuspended in half the initial volume of 0.1 M sodium acetate buffer, pH 3.9. After 90 min at 37°C with frequent stirring, the preparation was centrifuged in the cold as above and tile supernatant dialyzed overnight against PBS. 2. RESULTS In all experiments the proportion of antibody molecules recovered was determined from the radioactivity of the eluate. The antibody activity of the eluted material was tested by reabsorbing it on the specific antigen. Generally around 70% of the eluted counts could be reabsorbed. In addition, in the case of anti-Salmonella antibodies, the eluates exhibited agglutinating activity when tested with sheep red blood cells coated with Salmonella enteritidis somatic antigen (Landyoet al., 1965). Table 1 shows the different yields obtained by using or not using 1% BSA during the washings of the Salmonella anti-Salmonella complex. While with 1% BSA 40-50% of the counts absorbed on the bacteria could be eluted, the counts recovered when BSA was omitted from the washing fluid were negligible. Repeated elutions did not markedly increase the recovery in either case. Practically all the counts which could not be eluted were found to be still bound to the antigen. Radioactive proteins did not non-specifically stick to the walls of the plastic tubes. Superimposable results were obtained with ~4C-labelled antibodies synthesized in vitro by lymph nodes from rabbits stimulated with Salmonella enteritidis endotoxin (Carbonara et al., 1969). Table 2 shows the results obtained with anti-Group C Streptococcus antibodies. Both bacterial bodies and isolated cell walls (Bleiweis et al., 1964) were tested and a striking influence of BSA was apparent in either case.
Table 1 Elution of specific 125I-labelled antibody from Salmonella enteritidis bacterial bodies, after washing with or without 1% BSA. With BSA
Without BSA
cpm of 125I-labelled globulin fraction employed
7.4 × 106
7.4 × 106
cpm absorbed on bacterial bodies (after washings)
12,751
14,262
Proportion of counts absorbed cpm eluted by the first acid treatment Proportion of counts eluted
0.17% 5050 39.6%
0.19% 220 1.5%
cpm eluted by the second acid treatment Proportion of counts eluted
855 6.7%
249 1.75%
Proportion of eluted counts (first treatment) reabsorbed on bacteria
68.5%
Not done
Anti-Salmonella serum was obtained by injecting intravenously into a normal adult rabbit 1 ug SalmoneUa enteritidis endotoxin purified by the procedure of Ribi et al. (1961). 0.5 ml of antiserum was precipitated with 2 M ammonium sulphate and the globulin fraction (5.5 mg) was labelled with 1251 by the method of McFarlane (1958). The specific activity of the labelled protein was about 7000 cpm/~g. 5 X l0 s bacterial bodies were used in each test. Absorption and elution of the labelled antibody were performed as described in the text.
Table 2 Proportion of specific 12Sl-labelled antibodies eluted from Group C streptococcal cell walls under different conditions.
1 2 3 4 5 6 7 8 9 10
Washing
Elution
% antibody elu ted
PBS BSA 1% PBS PBS PBS PBS PBS PBS PBS PBS
Glycine buffer Glycine buffer Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer + Glycine buffer +
5 44 48 40 20 49 42 1.6 2.3 1.9
1% BSA 0.1% BSA 0.01% BSA 1% human hemoglobin 1% horse ferritin 1% dextran 500,000 MW 1% dextran 200,000 MW 1% dextran 40,000 MW
A normal adult rabbit was immunized with pepsin-treated Group C Streptococci, following the method described by Braun and Krause (1968). Under these conditions a high serum concentration of antibodies of restricted heterogeneity is produced. IgG were purified by ammonium sulphate precipitation followed by DEAE chromatography. The DEAE column was eluted with phosphate buffer 0.02 M, pH 8.0. About 70% of the purified IgG possessed specific antibody activity, as judged by quantitative binding experiments using streptococcal cell walls. The latter were prepared following the method of Bleiweis et al. (1964). The purified IgG was labelled with 12 s I by the Chloramin T method (Greenwood et al., 1963). The specific activity obtained was about 500,000 cpmh~g. Approximately 20,000 cpm were used in each test, with 0.5 mg (dry weight) of streptococcal cell wails. Absorption and elution of the labelled antibody were performed as described in the text.
104
A.L.Luzzati et al., Proteins required in antibody elution
Table 3 Elution of specific 125Iqabelled anti-Ab9 rabbit allotype antibody from antigen insolubilized by cross-linking.
cpm of 12St-labelledanti-Ab9 IgG employed cpm absorbed on cross-linked serum Proportion of counts absorbed cpm eluted by acid treatment Proportion of counts eluted
With BSA
Without BSA
7734 2310 30.0% 1820 78.5%
7676 2330 30.5% 1810 77.8%
The serum from a normal rabbit homozygous for the Ab9 allotype was insolubilized with ethyl chloroformate (Avrameas et al., 1967). This material was used to purify (by absorption and subsequent acid elution) the specific anti-Ab9 antibody produced immunizing an Ab4 homozygous rabbit by the procedure described by Dubiski (Dubiski et al., 1959). The eluted material was applied on a G-200 Sephadex column (2.5 X 100cm) and the resulting 7S fractions were pooled and concentrated. The anti-Ab9 IgG thus obtained was labelled with 12 sI by the chloramin T method (Greenwood et al., 1963). The specific activity was about 500,000 cpm/pg. Although initially this labelled preparation was 70% reabsorbable on the specific antigen, at the time of the experiment, after prolonged storage, only 30% of the counts could be absorbed. The experiment was performed with the same immunoadsorbent used for the purification of anti-Ab9 antibodies.
As shown in table 2, BSA does not need to be present during the washing step, but is critically required during the acid treatment. Therefore it can be directly dissolved in the eluting buffer. The minimal concentration of BSA required for maximal recovery is around 0.1%. Since similar results are obtained either including 0.1% BSA directly in the acid buffer or washing with 1% BSA before the acid treatment, it may be concluded that the amount of BSA remaining in the pellet after the last washing, although diluted by the buffer, is still adequate. Proteins other than BSA exert the same effect, when used in comparable amounts. Non proteinic substances, even of high molecular weight, such as dextran, do not influence the elution yield (table 2). Table 3 shows similar experiments performed using as immunoadsorbent rabbit serum (from an Ab9 homozygous animal) insolubilized by crosslinking with ethyl chloroformate (Avrameas et al., 1967). Partially purified ~2SI-labelled rabbit IgG with anti-allotype 9 activity was used as antibody. In this instance no effect at all was obtained by addition of BSA. Little, if any, effect was observed also in the case of elution of anti-HL-A isoantibodies from platelets. A quantitative evaluation of this system is in progress.
A.L.Luzzati et al., Proteins required in antibody elution
105
3. DISCUSSION From the results reported above it appears that the presence of a protein is a necessary requirement to obtain a relatively high yield of purified antibody by acid elution, at least in some a n t i g e n - a n t i b o d y systems. Although the effect is very striking, no generalization can be made from the three systems that we have investigated. However, it may be relevant that in both cases in which an absolute protein requirement is shown, antibodies against bacterial polysaccharide determinants are involved. In the case of Salmonella, the antibodies are directed against Group 0 somatic antigens, and in the case o f Streptococcus, against Group C specific determinants. The single system in which protein addition was without effect was with antibodies against a protein determinant (the Ab9 allotypic determinant of rabbit IgG). To clarify this issue it will be necessary to extend this investigation to systems involving non-bacterial polysaccharide determinants (e.g., blood group antigens, dextran, etc.) and bacterial proteins (e.g., Salmonella flagellin). No explanation can be offered of the mechanism by which BSA influences the detachment o f antibody from bacterial surfaces. However, BSA must be present at the moment in which the elution occurs: previous exposure of the bacterial bodies to BSA, either before or after antibody attachment, is without effect if all protein is removed by extensive washings before elution. Since other proteins work as well, this effect cannot be attributed to some specific property of BSA. In our experimental conditions the presence of protein seems to be an absolute requirement (the recovery in absence of BSA is indistinguishable from the background level). Such an extreme situation could be due to the very small amount of labelled antibody used in our system and may not be found when the starting material is total immune serum and in large amount. However by including BSA in the elution procedure an unusually good reproducibility of the amount of antibody recovered was also observed. Therefore we feel that under all conditions a careful control over the protein environment is essential for the standardization of any elution system.
ACKNOWLEDGEMENTS We want to thank Prof. Ruggero Ceppellini for helpful discussions and criticism of the manuscript. Streptococcal vaccines and streptococcal cell walls were kindly provided by Dr. D.G. Braun. The expert technical assistance of Miss Cleide Boccazzi is gratefully acknowledged.
REFERENCES Avrameas, S. and T. Ternynck, 1967, J. Biol. Chem. 242, 1651. Bleiweis, A.S., W.W. Karakawa and R.M. Krause, 1964, J. Bacteriol. 88, 1198.
106
A.L.Luzzati et al., Proteins required in anbitody elution
Braun, D.G. and R.M. Krause, 1968, J. Exptl. Med. 128, 969. Carbonara, A.O., A.L. Luzzati, R.M. Tosi, G. Mancini and R. Ceppellini, 1969, in: Lymphatic tissue and germinal centers in immune response (Plenum Publishing Corp., New York) 5,441 Dubiski, S., Z. Dudziak, D. Skalba and A. Dubiska, 1959, Immunology 2, 84. Greenwood, F.C., W.M. Hunter and J.S. Glover, 1963, Biochem. J. 89, 114. Kabat, E.A. and M.M. Mayer, 1961, Experimental immunochemistry (Charles C Thomas, Springfield, Illinois). Landy, M., R.P. Sanderson and A.L. Jackson, 1965, J. Exptl. Med. 122,483. McFarlane, A.S., 1958, Nature 182, 53. Ribi, E., W.T. Askins, M. Landy and K.C. Mllner, 1961, J. Exptl. Med. 114,647. Robbins, J.B., K. Kenny and E.J. Surer, 1965, J. Exptl. Med. 122,385.