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Cellulose-fixed immunoglobulin subunits
Iramunochemistry. Pergamon Press 1967. Vol. 4, pp. 269-272. Printed in Great Britain
RESEARCH NOTE Cellulose-fixed immunoglobul/n subun/ts
(Received I March 1967) IN RECENT years antigens fixed on insoluble carriers have been often used for detection and isolation of antibodies. ¢1-6~ In all these experiments attachment of whole protein molecules was carried out. However, to solve a number of problems, it was found preferable to use fixed immunoglobulin subunits. By means of these immunoadsorbents it proved possible to detect antibodies to given parts of immunoglobulin molecules. The complexes of fixed subunits and corresponding antibodies were used for identification of a radioactive piece of molecule. Methods. Rabbit ~,G-immunogiobulin (RGG) was isolated from serum on DEAE-Sephadex A50. ~7~ After S-sulphonation of RGG in 0.25 M NazSO3 and 0.005 M CuSO4, peptide chains were fractionated on Sephadex G100 in 6 M urea and 0.05 M formic acid. ~s~The last fractions of the first peak (heavy chains) and all fractions of the second peak (light chains) were filtered through Sephadex G25 in borate buffer, pH 8. Fab-fragments were isolated from RGG papain hydrolysate by chromatography on CM-cellulose ~9~and F(ab')2-fragments were isolated from RGG peptic hydrolysate according to Mandy et al. ~1o~The sedimentation coefficients (S20.w) of RGG, Fab and F(ab')z were found to be 6.6 S, 3.6 S and 4.8 S respectively. The isolated subunits were conjugated to small cellulose particles through diazo groups. ~8~ Absorbing properties of fixed subunits. At first we investigated the adsorption of antibodies from anti-RGG donkey antiserum on different conjugated immunoglobulin subunits. Adsorbent (4 mg, dry weight) was added to serum (1 ml) and the suspension was centrifruged after 10 min of stirring. A new portion of the same adsorbent was then added to the supernatant and the procedure was repeated three times. The sediments were washed five times with saline (pH 7) and fixed antibodies were eluted with 0.1 N HCI. The protein concentration in the eluates was determined by Lowry's method. Non-specific protein adsorption was detected in control experiments with non-specific adsorbent (fixed human serum albumin). The adsorption of antibodies in all experiments was almost quantitative as shown by the fact that the protein concentration in the last eluates was close to zero. Control experiments with radioactive 14C anti-HSA antibodies showed that the efficiency of the elution procedure was about 90 per cent. From the data obtained (Table 1) it may be concluded that the cellulose-fixed subunits of RGG retain their ability to react with antibodies. The sum of antibodies adsorbed by fixed heavy chains and by fixed light chains is smaller than the quantity of antibodies bound to fixed RGG. There may be two explanations for this fact. First, new antigenic determinants may be formed on association of light and heavy chains. Secondly, after isolation and fixation of chains some of the determinants may be lost. It is possible to evaluate quantitatively the immunogenicity of different parts of the protein molecule. Thus, the amount of anti-light chain antibodies is about one 269
270
R . S . NEZLIN
fourth of the amount of anti-RGG antibodies. The amount of anti-F(ab')2 antibodies is only slightly higher than that of anti-Fab antibodies. The Fc-fragment has a smaller part of the antigenic determinants of the R G G molecule since 74 per cent of anti-RGG antibodies were adsorbed on fixed Fab (Table 1).
TABLE 1. ADSORPTION OF ANTIBODIES FROM ANTt-RGG DONKEY ANTISERUM BY CELLULOSE-FIXEDRGG AND StmUNITSoF RGG Fixed proteins
RGG Heavy peptide chains Light peptide chains F(ab')z-fragments Fab-fragrnents
Antibody adsorbed from 1 ml of antiserum and eluted with 0"1 N HCI* (~g) 1278 733 334 967 939
* The efficiency of the elution procedure was of the order of 90 per cent.
It may be convenient to use fixed subunits for the complete removal of the corresponding antibodies from antisera. In the experiments using 1 ml of the same antiR G G serum (Table 1) two subsequent adsorptions with 4 mg of fixed light chains were sufficient to eliminate all anti-light chain antibodies. This helps to avoid the contamination of antiserum by an excess of added antigen. Use offixed subunitsfor the identification of immunoglobulinfragments. It has been shown that a complex of fixed antigen and antibodies (referred to as antibodyimmunoadsorbent) can specifically bind free antigen in a solution .tn,m The use of the complexes of fixed subunits and corresponding antibodies is of interest for the study of immunoglobulin synthesis. The antibody-immunoadsorbents may be useful for the identification of radioactive subunits: we have investigated the nature of microglobulin synthesized in vitro by rabbit spleen ceils and isolated specifically with other t4C immunoglobulins. The radioactive protein with a molecular weight of the order of 25,000 was separated from other t4C immunoglobulins by filtration through Sephadex G200 in 1 N acetic acid.t13,14~ After being passed through a Sephadex G25 column in borate buffer, pH 8, the microglobulin solution was divided into two equal parts. Non-specific adsorbent (fixed HSA) was added to one part of the solution for the detection of non-specific adsorption. After centrifugation a washed complex of the fixed light chains and the corresponding donkey antibodies was added to the supernatant. The adsorption of radioactive microglobulin on a complex of the fixed heavy chains and corresponding antibodies was studied. The bound radioactive protein was eluted with 0.1 N HC1 and measured with a scintillation counter. In control experiments with 14C R G G the yield of the elution procedure was found to be about 84 per cent. As shown in Table 2 practically all microglobulin had adsorbed on the complex of the fixed light chains and antibodies.
Research
TABLE2. ADSORPTION OF THE
Note
271
MICROGLOBULIN SYNTHESIZED IMMUNOADSORBENTS
in ~tro
BY DIFFERENT ANTIBODY-
Aliquot No.
Total radioactivity of aliquot (counts/min)
1
752
HSA Light chains of RGG Heavy chains of RGG
not treated treated treated$
0 727 7
2
752
HSA Heavy chains of RGG
not treated treated$
0 95
Fixed proteins (4 mg)
Treatment of fixed proteins with 0"4 ml of anti-RGG antiserum*
Adsorbed activityt (counts/min)
* Donkey anti-RGG antiserum as described in Table 1. t Total adsorbed radioactivity (eluted (84%) and non-eluted with 0"1 N HCI). All antibodies against light chains were previously adsorbed with fixed light chains. To the second half of microglobulin solution a complex of the fixed heavy chains and corresponding antibodies was added after the non-specific adsorbent. In contrast to the previous experiment about 13 per cent of the total radioactivity was adsorbed on the complex of the fixed heavy chains and antibodies. From these data it is evident that the main part of microglobulin represents free light chains synthesized in excess. Recently the synthesis of excess light chains had also been described by Shapiro et al. ~15~A small quantity of protein with antigenic properties of heavy chains may also be present in microglobulin preparations (at pH 8 in the form of complexes with light chains). A contamination of fixed heavy chains by light chains cannot account for these findings since the last fractions of the heavy chain peak was fixed in which light chains are present either only in trace amount or are totally absent. Furthermore, the fixed heavy chains used for the preparation of the antibody-immunoadsorbent were treated with anti-RGG antiserum from which anti-light chains antibodies were all absorbed. It must, of course, be borne in mind that the purity of fixed protein cannot be ignored, since it has a strong effect on the experiments using the immunoadsorbent technique. Fixed heavy chains which were not treated with antiserum did not bind the microglobulin radioactivity. It is therefore obvious that after being fixed on cellulose, heavy chains lose their ability to recombine with light chains. In conclusion, it can be stated that fixed fragments of protein molecules may be useful for the investigations of the structure and synthesis of immunoglobulins and other proteins. Institute of Molecular Biology Academy of Sciences of the U . S . S . R . Moscow B-312
R . S . NEZLIN L . M . KULPINA
REFERENCES 1 CAMPBELLm. H., LUESCHERF. and LERMANL. S., Proc. natn. Acad. Sci. Wash. 37, 575 (1951). 2 ISLIKERH., Adv. Protein Chem., 12, 387 (1957). 3 GURVlCHA. E., Immunological Methods (Edited by ACK~OYDJ. F.), p. 113. Blackwell (1964) 4 SEHONA. H., Br. reed. Bull. 19, 183 (1963).
272
R . S . NI~ZLIN
5 NSZLIN R. S., Biochemistry of Antibodies. Nauka, Moscow (1966). e SXLMANJ. H., KATCHALSXIE., A. Rev. Biochem. 35, 873 (1966). ? BAUMSTARKJ. S., LAFFIN R. J. and BARDAWILW. A., Archs. Biochem. Biophys. 108, 514 (1964). s FRANi~KF. and ZIKAN J., CoUn Czech. chem. Commun. 29, 1401 (1964). e MAGS R. G., RSlS~J~D R. A. and DRAYS., Immunochemistry 3, 299, (1966). 10 MANVY W. J., RIVERS M. M. and NISONOFF A., ~. biol. Chem. 236, 3221 (1961). 11 WILLIAMS R. R. and STONE S. S., Archs Biochem. Biophys. 71, 377 (1957). 1~ GURVXCHA. E. and DmZLIKH G. I., Nature 203, 648 (1964). 13 NEZLIN R. S. and KULPINA L. M., Nature 212, 845 (1966). 14 NEZLIN R. S. and KULPXNAL. M., Biokhimiya 31, 1257 (1966). 15 SHAPIROA. L., SCHARFFM. D., MAIZEL J. V. and UHR J. W., Nature 211, 243 (1966).
RESEARCH NOTE Cellulose-fixed immunoglobul/n subun/ts
(Received I March 1967) IN RECENT years antigens fixed on insoluble carriers have been often used for detection and isolation of antibodies. ¢1-6~ In all these experiments attachment of whole protein molecules was carried out. However, to solve a number of problems, it was found preferable to use fixed immunoglobulin subunits. By means of these immunoadsorbents it proved possible to detect antibodies to given parts of immunoglobulin molecules. The complexes of fixed subunits and corresponding antibodies were used for identification of a radioactive piece of molecule. Methods. Rabbit ~,G-immunogiobulin (RGG) was isolated from serum on DEAE-Sephadex A50. ~7~ After S-sulphonation of RGG in 0.25 M NazSO3 and 0.005 M CuSO4, peptide chains were fractionated on Sephadex G100 in 6 M urea and 0.05 M formic acid. ~s~The last fractions of the first peak (heavy chains) and all fractions of the second peak (light chains) were filtered through Sephadex G25 in borate buffer, pH 8. Fab-fragments were isolated from RGG papain hydrolysate by chromatography on CM-cellulose ~9~and F(ab')2-fragments were isolated from RGG peptic hydrolysate according to Mandy et al. ~1o~The sedimentation coefficients (S20.w) of RGG, Fab and F(ab')z were found to be 6.6 S, 3.6 S and 4.8 S respectively. The isolated subunits were conjugated to small cellulose particles through diazo groups. ~8~ Absorbing properties of fixed subunits. At first we investigated the adsorption of antibodies from anti-RGG donkey antiserum on different conjugated immunoglobulin subunits. Adsorbent (4 mg, dry weight) was added to serum (1 ml) and the suspension was centrifruged after 10 min of stirring. A new portion of the same adsorbent was then added to the supernatant and the procedure was repeated three times. The sediments were washed five times with saline (pH 7) and fixed antibodies were eluted with 0.1 N HCI. The protein concentration in the eluates was determined by Lowry's method. Non-specific protein adsorption was detected in control experiments with non-specific adsorbent (fixed human serum albumin). The adsorption of antibodies in all experiments was almost quantitative as shown by the fact that the protein concentration in the last eluates was close to zero. Control experiments with radioactive 14C anti-HSA antibodies showed that the efficiency of the elution procedure was about 90 per cent. From the data obtained (Table 1) it may be concluded that the cellulose-fixed subunits of RGG retain their ability to react with antibodies. The sum of antibodies adsorbed by fixed heavy chains and by fixed light chains is smaller than the quantity of antibodies bound to fixed RGG. There may be two explanations for this fact. First, new antigenic determinants may be formed on association of light and heavy chains. Secondly, after isolation and fixation of chains some of the determinants may be lost. It is possible to evaluate quantitatively the immunogenicity of different parts of the protein molecule. Thus, the amount of anti-light chain antibodies is about one 269
270
R . S . NEZLIN
fourth of the amount of anti-RGG antibodies. The amount of anti-F(ab')2 antibodies is only slightly higher than that of anti-Fab antibodies. The Fc-fragment has a smaller part of the antigenic determinants of the R G G molecule since 74 per cent of anti-RGG antibodies were adsorbed on fixed Fab (Table 1).
TABLE 1. ADSORPTION OF ANTIBODIES FROM ANTt-RGG DONKEY ANTISERUM BY CELLULOSE-FIXEDRGG AND StmUNITSoF RGG Fixed proteins
RGG Heavy peptide chains Light peptide chains F(ab')z-fragments Fab-fragrnents
Antibody adsorbed from 1 ml of antiserum and eluted with 0"1 N HCI* (~g) 1278 733 334 967 939
* The efficiency of the elution procedure was of the order of 90 per cent.
It may be convenient to use fixed subunits for the complete removal of the corresponding antibodies from antisera. In the experiments using 1 ml of the same antiR G G serum (Table 1) two subsequent adsorptions with 4 mg of fixed light chains were sufficient to eliminate all anti-light chain antibodies. This helps to avoid the contamination of antiserum by an excess of added antigen. Use offixed subunitsfor the identification of immunoglobulinfragments. It has been shown that a complex of fixed antigen and antibodies (referred to as antibodyimmunoadsorbent) can specifically bind free antigen in a solution .tn,m The use of the complexes of fixed subunits and corresponding antibodies is of interest for the study of immunoglobulin synthesis. The antibody-immunoadsorbents may be useful for the identification of radioactive subunits: we have investigated the nature of microglobulin synthesized in vitro by rabbit spleen ceils and isolated specifically with other t4C immunoglobulins. The radioactive protein with a molecular weight of the order of 25,000 was separated from other t4C immunoglobulins by filtration through Sephadex G200 in 1 N acetic acid.t13,14~ After being passed through a Sephadex G25 column in borate buffer, pH 8, the microglobulin solution was divided into two equal parts. Non-specific adsorbent (fixed HSA) was added to one part of the solution for the detection of non-specific adsorption. After centrifugation a washed complex of the fixed light chains and the corresponding donkey antibodies was added to the supernatant. The adsorption of radioactive microglobulin on a complex of the fixed heavy chains and corresponding antibodies was studied. The bound radioactive protein was eluted with 0.1 N HC1 and measured with a scintillation counter. In control experiments with 14C R G G the yield of the elution procedure was found to be about 84 per cent. As shown in Table 2 practically all microglobulin had adsorbed on the complex of the fixed light chains and antibodies.
Research
TABLE2. ADSORPTION OF THE
Note
271
MICROGLOBULIN SYNTHESIZED IMMUNOADSORBENTS
in ~tro
BY DIFFERENT ANTIBODY-
Aliquot No.
Total radioactivity of aliquot (counts/min)
1
752
HSA Light chains of RGG Heavy chains of RGG
not treated treated treated$
0 727 7
2
752
HSA Heavy chains of RGG
not treated treated$
0 95
Fixed proteins (4 mg)
Treatment of fixed proteins with 0"4 ml of anti-RGG antiserum*
Adsorbed activityt (counts/min)
* Donkey anti-RGG antiserum as described in Table 1. t Total adsorbed radioactivity (eluted (84%) and non-eluted with 0"1 N HCI). All antibodies against light chains were previously adsorbed with fixed light chains. To the second half of microglobulin solution a complex of the fixed heavy chains and corresponding antibodies was added after the non-specific adsorbent. In contrast to the previous experiment about 13 per cent of the total radioactivity was adsorbed on the complex of the fixed heavy chains and antibodies. From these data it is evident that the main part of microglobulin represents free light chains synthesized in excess. Recently the synthesis of excess light chains had also been described by Shapiro et al. ~15~A small quantity of protein with antigenic properties of heavy chains may also be present in microglobulin preparations (at pH 8 in the form of complexes with light chains). A contamination of fixed heavy chains by light chains cannot account for these findings since the last fractions of the heavy chain peak was fixed in which light chains are present either only in trace amount or are totally absent. Furthermore, the fixed heavy chains used for the preparation of the antibody-immunoadsorbent were treated with anti-RGG antiserum from which anti-light chains antibodies were all absorbed. It must, of course, be borne in mind that the purity of fixed protein cannot be ignored, since it has a strong effect on the experiments using the immunoadsorbent technique. Fixed heavy chains which were not treated with antiserum did not bind the microglobulin radioactivity. It is therefore obvious that after being fixed on cellulose, heavy chains lose their ability to recombine with light chains. In conclusion, it can be stated that fixed fragments of protein molecules may be useful for the investigations of the structure and synthesis of immunoglobulins and other proteins. Institute of Molecular Biology Academy of Sciences of the U . S . S . R . Moscow B-312
R . S . NEZLIN L . M . KULPINA
REFERENCES 1 CAMPBELLm. H., LUESCHERF. and LERMANL. S., Proc. natn. Acad. Sci. Wash. 37, 575 (1951). 2 ISLIKERH., Adv. Protein Chem., 12, 387 (1957). 3 GURVlCHA. E., Immunological Methods (Edited by ACK~OYDJ. F.), p. 113. Blackwell (1964) 4 SEHONA. H., Br. reed. Bull. 19, 183 (1963).
272
R . S . NI~ZLIN
5 NSZLIN R. S., Biochemistry of Antibodies. Nauka, Moscow (1966). e SXLMANJ. H., KATCHALSXIE., A. Rev. Biochem. 35, 873 (1966). ? BAUMSTARKJ. S., LAFFIN R. J. and BARDAWILW. A., Archs. Biochem. Biophys. 108, 514 (1964). s FRANi~KF. and ZIKAN J., CoUn Czech. chem. Commun. 29, 1401 (1964). e MAGS R. G., RSlS~J~D R. A. and DRAYS., Immunochemistry 3, 299, (1966). 10 MANVY W. J., RIVERS M. M. and NISONOFF A., ~. biol. Chem. 236, 3221 (1961). 11 WILLIAMS R. R. and STONE S. S., Archs Biochem. Biophys. 71, 377 (1957). 1~ GURVXCHA. E. and DmZLIKH G. I., Nature 203, 648 (1964). 13 NEZLIN R. S. and KULPINA L. M., Nature 212, 845 (1966). 14 NEZLIN R. S. and KULPXNAL. M., Biokhimiya 31, 1257 (1966). 15 SHAPIROA. L., SCHARFFM. D., MAIZEL J. V. and UHR J. W., Nature 211, 243 (1966).