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Dissociation of immune complexes and inactivation of bound antibodies by reducing agents
lmmunochemistry, 1977.Vol.14, pp. 633-635. PergamonPress. Printedin GreatBritain
DISSOCIATION OF I M M U N E COMPLEXES A N D INACTIVATION OF B O U N D ANTIBODIES BY R E D U C I N G AGENTS OLGA Ya. POPOVA and LUDMILA S. KOSITSKAYA Department of Microbiology and Immunology, Institute of Experimental Medicine of the Academy of Medical Science, Leningrad, U.S.S.R. (First received 7 October 1976; in revised form 1 March 1977)
Abstract--The treatment of antigen-antibody complexes with cysteine-HC1 results in the splitting of these complexes and antigen liberation. Complexes with IgM antibodies are 4-8 times more resistant to cysteine than complexes with IgG antibodies. Dissociation of the immune complex is associated with inactivation of the antibody. It is found that antibodies bound to antigen are more sensitive to cysteine than unbound antibodies: unbound IgM is inactivated only by 31.4 and 15.7mg/ml cysteine; unbound IgG is not sensitive to these concentrations of cysteine. For inactivation of IgM and IgG antibodies complexed with the antigen, 3.75 and 0.45mg/ml cysteine-HC1 is required, respectively, Other reducing agents (glutathion and sodium sulfite) were found to be effective in dissociation of immune complexes too.
INTRODUCTION It is well known that IgM antibodies are sensitive to reducing agents, while IgG antibodies are more resistant to them (Ishizaka et al., 1961; Bauer et al., 1963; Grey, 1963). The present report deals with our findings which show that small doses of cysteine-hydrochloride and some other reducing agents inactivate IgM and IgG antibodies complexed with antigen and dissociate immune complex, but do not affect free IgM and IgG antibodies. The dissociation of the immune complex results in the liberation of antigen identified by serological methods.
CFT was conducted in a volume of 0.5 ml, each component substance contributing 0.1 ml (antigen, antibody, complement, red blood cells, hemolytic serum). Different dilutions of serum with antigen and dilutions of guinea-pig serum, titrated with the same hemolytic system were incubated at 4°C during 18 hr. The results were read after the incubation of all samples with a hemolytic system at 37°C. The positive samples inhibited hemolysis completely with all doses of the complement (Ioffe & Rosental, 1943). Passive hemagglutination test, gel precipitation test and immunosorbent technique were performed according to Boyden (1951), Ouchterlony (1958) and Kuzovleva and Gurvich (1966). The complement binding capacity of immune complexes was evaluated by their ability to absorb the guinea-pig complement (Kabat & Mayer, 1948).
MATERIALS AND METHODS
Cysteine-HC1, glutathion and proteins (HSA, BSA) were purchased by Reanal, Hungary. Sheep red blood cells were washed three times with saline. Antisera against BSA and HSA were harvested from rabbits ten days after the last of 7 weekly subcutaneous injections of 50 mg protein mixed with Freund's complete adjuvant. Anti-RBC and anti-gammaglobulin sera were obtained commercially (Mechnikoff Institute of Vaccines and Sera, Moscow, U.S.S.R.). To inactivate IgM antibodies sera were treated with 31.4rag/m1 of cysteine-hydrochloride (Chernokh*vostova, 1965). The complexes of soluble antigens with antibodies were formed in zones of equivalence (Heidelberger & Kendall, 1935). Immune complexes of sheep RBC with specific antibodies were formed with a minimum antibody concentration that caused complete agglutination of RBC suspension. All the complexes were washed five times with saline and resuspended in saline. Antisera before being formed into complexes were heated to inactivate the complement. Equal volumes of the complex and a reducing agent (pH 7.2) were mixed and kept at 37°C for 5 hr and 4°C for 18 hr followed by dialysis against cold saline for 24 hr. *Abbreviations used: BSA, bovine serum albumin; HSA, human serum albumin; CFT, complement fixation test; PHAT, passive hemagglutination test; RBC, red blood cells. 633
RESULTS AND DISCUSSION The immune complexes of soluble antigens (BSA, HSA) with IgG antibodies exhibited anti-complement properties. The treatment of complexes with cysteine (final concentration 0.45 mg/ml) destroyed their capacity to bind complement, while cysteine concentrations of 0.225-0.112mg/ml produced only partial effect. After treatment with 0.45 mg/ml of cysteine the complex lost its anti-complement properties and the all bound antigen was liberated (Table 1). Larger doses of cysteine (0.9 mg/ml) caused a partial destruction of the antigen, both when it was complexed or unbound. Smaller doses of cysteine liberated the antigen from the complex only partially. The dissoci~ ation of the complex was registered by CFT. The treatment of washed agglutinates of sheep RBC and rabbit IgG antibodies with 0.45--0.9 mg/ml of cysteine resulted in agglutinate disruption. It should be emphasized that treatment of free IgG antibodies with any dose of cysteine ranging from 31.4 to 0.112 mg/ml failed to reduce their antigen-combining activity. The treatment of sheep RBC complexed with IgM antibodies, destroyed these complexes at a cysteine concentration of 3.75 mg/ml only.
OLGA Ya. POPOVA and LUDMILA S. KOSITSKAYA
634
Table 1. Antigen determination in substrates containing cysteine-treated complexes or free antigens (mean value of four samples with S.D.) a Cysteine (mg/ml) 0.9 BSA-anti Unbound HSA-anti Unbound
BSA BSA HSA HSA
50 43.8 43.8 50
0.45
_ 0 _ 6.2 _ 6.2 __+0
87.5 100 87.5 87.5
0.225
_ 3.9 _ 0 _ 3.9 ___3.9
12.5 + 100 _ 15.6 + 87.5 +
0 0 3.1 3.9
0.112 10.9 ~ 1.5 112.5 ___6.3 10.9 ___ 1.5 87.5 ___3.9
aCFT, percentage of original fraction of antigen in cysteine-treated samples.
Table 2. Determination of complement-binding capacity, antigen, antibodies and Ig of antibodies in BSA-anri-BSA immune complexes before and after treatment with 0.45 mg/ml cysteine-HCl a
Total protein #g/ml~
Antigen CFT ~
Antibody CFT d
Complement binding lytic units C'H 100
Complex BSAanti-BSA before treatment
3860 _ 440
N.D.
N.D.
6.36 _ 0.91
908.5 + 19.3
Complex BSAanti-BSA after treatment
3937 _ 63
87.5 _+ 13
<2.8
1.2 + 0.12
1909 _ 8.2
Anti-BSA
2481 _ 707
--
100 _ 27
2.25 ___0.14
1909 _ 8.2
100 _ 0
--
1.33 _ 0.17
--
650 + 119
BSA
Rabbit IgG #g/ml; immunosor bent technique
aAs mean of 4 samples. bBy Lowry method. CAs percentage of original quantity antigen. ~As percentage of original quantity antibody.
Table 3. Anti-complement properties of immune complexes after treatment with reducing agents Complement binding (lyric units C'H 100) Composition of complexes
CysteineHC1 0.45 mg/ml
Sodium sulfite 0.3 mg/ml
Glutathion 0.76 mg/ml
Control
BSA-anti-BSA
1.2 _ 0.1
2.7 _ 0.2
3.2 _ 0.1
6.3 _ 0.9
HSA-anti-HSA
1.3 _ 0.1
2.7 + 0.2
3.2 + 0.1
4.2 + 0.3
O u r experiments also showed that free IgM antibodies were inactivated completely after treatment with 31.4 or 15.7 mg/ml cysteine. The treatment with 7.85 mg/ml reduced the activity of IgM antibodies, while lower doses did n o t affect them. Hence sheep RBC complexes with IgM antibodies are 4-8 times more resistant to a reducing agent t h a n I g G antibodies complexes. W h e n the i m m u n e complexes were destroyed by cysteine the antibodies of the complex were inactivated and lost their antigen combining capacity that was shown by different serological tests (CFT, PHAT, double-diffusion test) in ten samples. Table 2 is a summary of our results. The inactivation of antigen-bind-
ing capacity of antibodies was n o t associated with the changes in the antigenic properties of IgG. This was shown by the i m m u n o s o r b e n t technique: antiglobulin sorbent b o u n d 1909/tg _+ 8.17 protein native anti-BSA serum, only 908.5 __+ 19.34/~g of protein was b o u n d when the antibodies were complexed with antigen in equivalence zone. 1 9 0 9 + 8 . 1 7 # g was sorbed after the treatment of the complex with cysteine. Finally we studied the effect of some other reducing agents o n the i m m u n e complexes. Sodium sulfite a n d glutathion were used. T h e doses of reducing agents were used according to the n u m b e r of active groups in the used dose of cysteine. Table 3 shows that both
Dissociation of Immune Complexes these reducing agents were effective in splitting the immune complexes of IgG antibodies. It should be noted that treatment of free IgM and IgG antibodies with glutathion and sodium sulfite failed to affect their activity. In conclusion, it should be said that free IgG antibodies which are more resistant to reducing agents than free IgM ones, appear to be 8 times more sensitive than IgM antibodies, when both are complexed with antigen.
Acknowledgement--The authors are indepted to Professor Boris N. Sofronov for helpful advice and criticism given during this study.
635 REFERENCES
Bauer D. C., Mathies M. S. & Stavitsky A. B. (1963) J. exp. Med. 117, 889. Boyden S. V. (1951) J. exp. Med. 93, 107. Chernokhvostova E. V. (1965) Lab. Delo U.S.S.R. 6, 323. Grey H. M. (1963) Proc. Soc. exp. Biol. & Med. 113, 963. Heidelberger M. & Kendall F. E. (1935) J. exp. Med. 62, 467. Ioffe V. I. & Rosental K. M. (1943) Zh. Mikrobiol. ff~pidem Immunohiol. 12, 65. Ishizaka K., Ishizaka T. & Sugahara (1961) J. Immun. 87, 548. Kabat E. A. & Mayer M. M. (1948) Experimental lmmunochemistry. Charles Thomas, Springfield, IL. Kuzovleva O. V. & Gurvich A. E. (1966) Vop. med. Khim. Akadl med. Nauk SSSR. 12, 316. Ouchterlony O. (1958) Prog. Allergy 5, 1.
DISSOCIATION OF I M M U N E COMPLEXES A N D INACTIVATION OF B O U N D ANTIBODIES BY R E D U C I N G AGENTS OLGA Ya. POPOVA and LUDMILA S. KOSITSKAYA Department of Microbiology and Immunology, Institute of Experimental Medicine of the Academy of Medical Science, Leningrad, U.S.S.R. (First received 7 October 1976; in revised form 1 March 1977)
Abstract--The treatment of antigen-antibody complexes with cysteine-HC1 results in the splitting of these complexes and antigen liberation. Complexes with IgM antibodies are 4-8 times more resistant to cysteine than complexes with IgG antibodies. Dissociation of the immune complex is associated with inactivation of the antibody. It is found that antibodies bound to antigen are more sensitive to cysteine than unbound antibodies: unbound IgM is inactivated only by 31.4 and 15.7mg/ml cysteine; unbound IgG is not sensitive to these concentrations of cysteine. For inactivation of IgM and IgG antibodies complexed with the antigen, 3.75 and 0.45mg/ml cysteine-HC1 is required, respectively, Other reducing agents (glutathion and sodium sulfite) were found to be effective in dissociation of immune complexes too.
INTRODUCTION It is well known that IgM antibodies are sensitive to reducing agents, while IgG antibodies are more resistant to them (Ishizaka et al., 1961; Bauer et al., 1963; Grey, 1963). The present report deals with our findings which show that small doses of cysteine-hydrochloride and some other reducing agents inactivate IgM and IgG antibodies complexed with antigen and dissociate immune complex, but do not affect free IgM and IgG antibodies. The dissociation of the immune complex results in the liberation of antigen identified by serological methods.
CFT was conducted in a volume of 0.5 ml, each component substance contributing 0.1 ml (antigen, antibody, complement, red blood cells, hemolytic serum). Different dilutions of serum with antigen and dilutions of guinea-pig serum, titrated with the same hemolytic system were incubated at 4°C during 18 hr. The results were read after the incubation of all samples with a hemolytic system at 37°C. The positive samples inhibited hemolysis completely with all doses of the complement (Ioffe & Rosental, 1943). Passive hemagglutination test, gel precipitation test and immunosorbent technique were performed according to Boyden (1951), Ouchterlony (1958) and Kuzovleva and Gurvich (1966). The complement binding capacity of immune complexes was evaluated by their ability to absorb the guinea-pig complement (Kabat & Mayer, 1948).
MATERIALS AND METHODS
Cysteine-HC1, glutathion and proteins (HSA, BSA) were purchased by Reanal, Hungary. Sheep red blood cells were washed three times with saline. Antisera against BSA and HSA were harvested from rabbits ten days after the last of 7 weekly subcutaneous injections of 50 mg protein mixed with Freund's complete adjuvant. Anti-RBC and anti-gammaglobulin sera were obtained commercially (Mechnikoff Institute of Vaccines and Sera, Moscow, U.S.S.R.). To inactivate IgM antibodies sera were treated with 31.4rag/m1 of cysteine-hydrochloride (Chernokh*vostova, 1965). The complexes of soluble antigens with antibodies were formed in zones of equivalence (Heidelberger & Kendall, 1935). Immune complexes of sheep RBC with specific antibodies were formed with a minimum antibody concentration that caused complete agglutination of RBC suspension. All the complexes were washed five times with saline and resuspended in saline. Antisera before being formed into complexes were heated to inactivate the complement. Equal volumes of the complex and a reducing agent (pH 7.2) were mixed and kept at 37°C for 5 hr and 4°C for 18 hr followed by dialysis against cold saline for 24 hr. *Abbreviations used: BSA, bovine serum albumin; HSA, human serum albumin; CFT, complement fixation test; PHAT, passive hemagglutination test; RBC, red blood cells. 633
RESULTS AND DISCUSSION The immune complexes of soluble antigens (BSA, HSA) with IgG antibodies exhibited anti-complement properties. The treatment of complexes with cysteine (final concentration 0.45 mg/ml) destroyed their capacity to bind complement, while cysteine concentrations of 0.225-0.112mg/ml produced only partial effect. After treatment with 0.45 mg/ml of cysteine the complex lost its anti-complement properties and the all bound antigen was liberated (Table 1). Larger doses of cysteine (0.9 mg/ml) caused a partial destruction of the antigen, both when it was complexed or unbound. Smaller doses of cysteine liberated the antigen from the complex only partially. The dissoci~ ation of the complex was registered by CFT. The treatment of washed agglutinates of sheep RBC and rabbit IgG antibodies with 0.45--0.9 mg/ml of cysteine resulted in agglutinate disruption. It should be emphasized that treatment of free IgG antibodies with any dose of cysteine ranging from 31.4 to 0.112 mg/ml failed to reduce their antigen-combining activity. The treatment of sheep RBC complexed with IgM antibodies, destroyed these complexes at a cysteine concentration of 3.75 mg/ml only.
OLGA Ya. POPOVA and LUDMILA S. KOSITSKAYA
634
Table 1. Antigen determination in substrates containing cysteine-treated complexes or free antigens (mean value of four samples with S.D.) a Cysteine (mg/ml) 0.9 BSA-anti Unbound HSA-anti Unbound
BSA BSA HSA HSA
50 43.8 43.8 50
0.45
_ 0 _ 6.2 _ 6.2 __+0
87.5 100 87.5 87.5
0.225
_ 3.9 _ 0 _ 3.9 ___3.9
12.5 + 100 _ 15.6 + 87.5 +
0 0 3.1 3.9
0.112 10.9 ~ 1.5 112.5 ___6.3 10.9 ___ 1.5 87.5 ___3.9
aCFT, percentage of original fraction of antigen in cysteine-treated samples.
Table 2. Determination of complement-binding capacity, antigen, antibodies and Ig of antibodies in BSA-anri-BSA immune complexes before and after treatment with 0.45 mg/ml cysteine-HCl a
Total protein #g/ml~
Antigen CFT ~
Antibody CFT d
Complement binding lytic units C'H 100
Complex BSAanti-BSA before treatment
3860 _ 440
N.D.
N.D.
6.36 _ 0.91
908.5 + 19.3
Complex BSAanti-BSA after treatment
3937 _ 63
87.5 _+ 13
<2.8
1.2 + 0.12
1909 _ 8.2
Anti-BSA
2481 _ 707
--
100 _ 27
2.25 ___0.14
1909 _ 8.2
100 _ 0
--
1.33 _ 0.17
--
650 + 119
BSA
Rabbit IgG #g/ml; immunosor bent technique
aAs mean of 4 samples. bBy Lowry method. CAs percentage of original quantity antigen. ~As percentage of original quantity antibody.
Table 3. Anti-complement properties of immune complexes after treatment with reducing agents Complement binding (lyric units C'H 100) Composition of complexes
CysteineHC1 0.45 mg/ml
Sodium sulfite 0.3 mg/ml
Glutathion 0.76 mg/ml
Control
BSA-anti-BSA
1.2 _ 0.1
2.7 _ 0.2
3.2 _ 0.1
6.3 _ 0.9
HSA-anti-HSA
1.3 _ 0.1
2.7 + 0.2
3.2 + 0.1
4.2 + 0.3
O u r experiments also showed that free IgM antibodies were inactivated completely after treatment with 31.4 or 15.7 mg/ml cysteine. The treatment with 7.85 mg/ml reduced the activity of IgM antibodies, while lower doses did n o t affect them. Hence sheep RBC complexes with IgM antibodies are 4-8 times more resistant to a reducing agent t h a n I g G antibodies complexes. W h e n the i m m u n e complexes were destroyed by cysteine the antibodies of the complex were inactivated and lost their antigen combining capacity that was shown by different serological tests (CFT, PHAT, double-diffusion test) in ten samples. Table 2 is a summary of our results. The inactivation of antigen-bind-
ing capacity of antibodies was n o t associated with the changes in the antigenic properties of IgG. This was shown by the i m m u n o s o r b e n t technique: antiglobulin sorbent b o u n d 1909/tg _+ 8.17 protein native anti-BSA serum, only 908.5 __+ 19.34/~g of protein was b o u n d when the antibodies were complexed with antigen in equivalence zone. 1 9 0 9 + 8 . 1 7 # g was sorbed after the treatment of the complex with cysteine. Finally we studied the effect of some other reducing agents o n the i m m u n e complexes. Sodium sulfite a n d glutathion were used. T h e doses of reducing agents were used according to the n u m b e r of active groups in the used dose of cysteine. Table 3 shows that both
Dissociation of Immune Complexes these reducing agents were effective in splitting the immune complexes of IgG antibodies. It should be noted that treatment of free IgM and IgG antibodies with glutathion and sodium sulfite failed to affect their activity. In conclusion, it should be said that free IgG antibodies which are more resistant to reducing agents than free IgM ones, appear to be 8 times more sensitive than IgM antibodies, when both are complexed with antigen.
Acknowledgement--The authors are indepted to Professor Boris N. Sofronov for helpful advice and criticism given during this study.
635 REFERENCES
Bauer D. C., Mathies M. S. & Stavitsky A. B. (1963) J. exp. Med. 117, 889. Boyden S. V. (1951) J. exp. Med. 93, 107. Chernokhvostova E. V. (1965) Lab. Delo U.S.S.R. 6, 323. Grey H. M. (1963) Proc. Soc. exp. Biol. & Med. 113, 963. Heidelberger M. & Kendall F. E. (1935) J. exp. Med. 62, 467. Ioffe V. I. & Rosental K. M. (1943) Zh. Mikrobiol. ff~pidem Immunohiol. 12, 65. Ishizaka K., Ishizaka T. & Sugahara (1961) J. Immun. 87, 548. Kabat E. A. & Mayer M. M. (1948) Experimental lmmunochemistry. Charles Thomas, Springfield, IL. Kuzovleva O. V. & Gurvich A. E. (1966) Vop. med. Khim. Akadl med. Nauk SSSR. 12, 316. Ouchterlony O. (1958) Prog. Allergy 5, 1.