- Email: [email protected]
Molecular heterogeneity effects of immunoglobulin classes in radial immunodiffusion
Journal of Immunological Methods 1 (1972) 211-213. © North-Holland Publishing Company
Short communication
MOLECULAR HETEROGENEITY EFFECTS OF IMMUNOGLOBULIN CLASSES IN RADIAL IMMUNODIFFUSION * J. MULDER, L.C.E. SLOOTS and M.A.T. VERHAAR Departmen t of Clinical Chemistry, Free University Hospital, Amsterdam, the Netherlands Received 30 June 1971 Mancini et al. (1965) have reported a method for immunological quantitation of proteins by means of single radial diffusion, which is now used extensively in immunochemistry for measurement of immunoglobulin concentrations in sera. Using this method, in instances of monospecific antibodies, to quantitate immunoglobulinsleads in some cases to difficulties because of multiple ring precipitations. In this communication we report multiple precipitation in single radial diffusion, using recently commercially available plates (Tri-partigen plates, Behringwerke, Marburg, Germany), as an indication of molecular heterogeneity in different immunoglobulinclasses.
lgMglobulin: Several sera developed one main precipitation ring as well as a second faint precipitation ring on radial quantitative immunodiffusion. In most of these cases the sera were obtained from patients with lymphocytic neoplasm diseases. Each serum which gave a double precipitation on radial diffusion was screened by 7% agarose immunodiffusion (Soloman, 1969) to detect low-molecular weight IgM. All of the sera which gave double precipitation rings were also positive on immunodiffusion in 7% agarose. Treatment of the sera with cysteine followed by alkylation~(according to Miller and Metzger, 1965) gives extremely large and faint single rings on radial immunodiffusion. This seems to indicate that double precipitation is related to the presence of low-molecular weight IgM which lacks certain antigenic determinants characteristic for high-molecular weight lgM. Thus, depending on the relative concentration, low-molecular weight IgM can be detected as a second ring on single radial immunodiffusion in agar gel containing monospecific antiserum to human 19S lgM. IgG globulin: As expected on the basis of the balance of immunoglobulin synthesis, most of the double precipitation rings of IgG on radial immunodiffusion were found in association with multiple myeloma. Immunoelectrophoresis in these * Correspondence: J. Mulder, Immunochemical Laboratory, Free University Hospital, De Boelelaan 1117, Amsterdam, The Netherlands. 211
212
J. MULDER et al.
cases showed a heterogeneity of tile lgG are due to precipitation of monoclonal and polyclonal IgG. When one of the populations is absent, as in sera of myeloma patients with extremely diminished polyclonal IgG or in sera of normal persons without monoclonal IgG, double precipitation does not exist. Since myeloma IgG belongs to only one of the four subclasses of IgG globulin (Bernier, 1967), differing from each other in the primary amino acid sequence of their Fc-fragments (Prahl, 1967; Frangione, 1969) and therefore in the antigenic determinants (Longmire, 1971), it seems to be quite reasonable that double precipitation arises from the absence of antigenic determinants in myeloma IgG in comparison to non-myeloma lgG, if there is a high relative concentration of myeloma IgG.
IgA globulin: Persons, especially children, with a deficiency of lgA can give double or even triple precipitation of IgA on radial immunodiffusion in agar gel containing rabbit antiserum to human IgA. In a recent publication Leikola and Vyas (1971) mentioned double precipitation of IgA due to antibodies reacting to a component in normal and immune sera of goat in the case of a normal donor. In our study immunoelectrophoretic analyses of patient sera failed to show precipitation arcs when reacted with normal rabbit sera. At this moment the reason for the existence of multiple precipitation of IgA on radial immunodiffusion is rather obscure. Multiple precipitation in single radial immunodiffusion using monospecific antibodies can be explained by the assumption that different antigens with overlapping antigenicity exist under some pathological conditions. Fick's law in these cases becomes very complicated when terms such as concentration and diffusion coefficients of the respective antigens must be a part of the final concentration expression. A mathematical expression for the different antigenic determinants cannot be given, since at this moment nothing is known about the total number of antigenic groups in each antigen. If a solution containing antigens A, with antigenic determinants 1, 3, 5, ..., m, and B, with antigenic determinants 1, 2, 3 .... , n, is placed in an agar gel containing antibody C, with antibody determinants 1 , 2 , 3, ..., n, then a double precipitation can occur, depending on the relative concentration and diffusion coefficient of the respective antigens. If the speed of diffusion of antigen A is greater than that of antigen B, antigen A's front diffuses into the agar, sweeping out all antibody determinants 1, 3, 5 ..... m, until enough of these antibodies have been combined to precipitate the antigen A. The slower-moving antigen B can react afterwards only with antibody determinants 2, 4, 6, ..., n inside the precipitating A ring, since the antibody determinants 1, 3, 5, ..., m are removed by antigen A. Outside the precipitating A ring, antigen B can react with antibody determinants 1,2, 3, ..., n. Depending on the relative concentration of antigen B the B precipitation ring can be precipitated inside, outside, or at the same position as the A ring. In any case
Immunoglobulin classes in radial immunodiffusion
213
it is clear that the area of the ring formed by antigen B will be larger than it would have been if antigen A was not present. Therefore, to calculate the concentrations by ring size measurements in cases of mixed antigens with overlapping antigenicity can lead to errors (Lietze et al., 1970), because the areas are not only related to the concentration and diffusion coefficients, but also on the overlapping antigenic groups. In view o f our data radial immunodiffusion can reflect qualitative changes in immunoglobulins if there is a great heterogeneity and concentration difference within the concerning immunoglobulin class. A detailed discussion of the double precipitation o f |gG in association with multiple myeloma and IgM in association with lymphoproliferative disorders will be published later. REFERENCES Bernier, G.M., R.E. Ballieux, K.T. Tominaga and F.W. Putnam, 1967, J. Exptl. Med. 125,303. Frangione, B., C. Milstein and J.R.L. Pink, 1969, Nature 221,145. Leikola, J. and G.N. Vyas, 1971, J. Lab. Clin. Med. 77,629. Lietze, A., C. Sinclair and A.H. Rowe, 1970, Clin. Biochem. 3,335. Longmire, R.L., R.S. Smith, R. McMiUan and R.T. Reid, 1971, Immunochemistry 8,153. Mancini, G., A.O. Carbonara and J.F. Heremans, 1965, Immunochemistry 2, 235. Miller, F. and H. Metzger, 1965, J. Biol. Chem. 240, 3325, 4740. Prahl, J.W., 1967, Biochem. J. 105, 1019. Soloman, A., 1969, J. Immunol. 102,496.
Short communication
MOLECULAR HETEROGENEITY EFFECTS OF IMMUNOGLOBULIN CLASSES IN RADIAL IMMUNODIFFUSION * J. MULDER, L.C.E. SLOOTS and M.A.T. VERHAAR Departmen t of Clinical Chemistry, Free University Hospital, Amsterdam, the Netherlands Received 30 June 1971 Mancini et al. (1965) have reported a method for immunological quantitation of proteins by means of single radial diffusion, which is now used extensively in immunochemistry for measurement of immunoglobulin concentrations in sera. Using this method, in instances of monospecific antibodies, to quantitate immunoglobulinsleads in some cases to difficulties because of multiple ring precipitations. In this communication we report multiple precipitation in single radial diffusion, using recently commercially available plates (Tri-partigen plates, Behringwerke, Marburg, Germany), as an indication of molecular heterogeneity in different immunoglobulinclasses.
lgMglobulin: Several sera developed one main precipitation ring as well as a second faint precipitation ring on radial quantitative immunodiffusion. In most of these cases the sera were obtained from patients with lymphocytic neoplasm diseases. Each serum which gave a double precipitation on radial diffusion was screened by 7% agarose immunodiffusion (Soloman, 1969) to detect low-molecular weight IgM. All of the sera which gave double precipitation rings were also positive on immunodiffusion in 7% agarose. Treatment of the sera with cysteine followed by alkylation~(according to Miller and Metzger, 1965) gives extremely large and faint single rings on radial immunodiffusion. This seems to indicate that double precipitation is related to the presence of low-molecular weight IgM which lacks certain antigenic determinants characteristic for high-molecular weight lgM. Thus, depending on the relative concentration, low-molecular weight IgM can be detected as a second ring on single radial immunodiffusion in agar gel containing monospecific antiserum to human 19S lgM. IgG globulin: As expected on the basis of the balance of immunoglobulin synthesis, most of the double precipitation rings of IgG on radial immunodiffusion were found in association with multiple myeloma. Immunoelectrophoresis in these * Correspondence: J. Mulder, Immunochemical Laboratory, Free University Hospital, De Boelelaan 1117, Amsterdam, The Netherlands. 211
212
J. MULDER et al.
cases showed a heterogeneity of tile lgG are due to precipitation of monoclonal and polyclonal IgG. When one of the populations is absent, as in sera of myeloma patients with extremely diminished polyclonal IgG or in sera of normal persons without monoclonal IgG, double precipitation does not exist. Since myeloma IgG belongs to only one of the four subclasses of IgG globulin (Bernier, 1967), differing from each other in the primary amino acid sequence of their Fc-fragments (Prahl, 1967; Frangione, 1969) and therefore in the antigenic determinants (Longmire, 1971), it seems to be quite reasonable that double precipitation arises from the absence of antigenic determinants in myeloma IgG in comparison to non-myeloma lgG, if there is a high relative concentration of myeloma IgG.
IgA globulin: Persons, especially children, with a deficiency of lgA can give double or even triple precipitation of IgA on radial immunodiffusion in agar gel containing rabbit antiserum to human IgA. In a recent publication Leikola and Vyas (1971) mentioned double precipitation of IgA due to antibodies reacting to a component in normal and immune sera of goat in the case of a normal donor. In our study immunoelectrophoretic analyses of patient sera failed to show precipitation arcs when reacted with normal rabbit sera. At this moment the reason for the existence of multiple precipitation of IgA on radial immunodiffusion is rather obscure. Multiple precipitation in single radial immunodiffusion using monospecific antibodies can be explained by the assumption that different antigens with overlapping antigenicity exist under some pathological conditions. Fick's law in these cases becomes very complicated when terms such as concentration and diffusion coefficients of the respective antigens must be a part of the final concentration expression. A mathematical expression for the different antigenic determinants cannot be given, since at this moment nothing is known about the total number of antigenic groups in each antigen. If a solution containing antigens A, with antigenic determinants 1, 3, 5, ..., m, and B, with antigenic determinants 1, 2, 3 .... , n, is placed in an agar gel containing antibody C, with antibody determinants 1 , 2 , 3, ..., n, then a double precipitation can occur, depending on the relative concentration and diffusion coefficient of the respective antigens. If the speed of diffusion of antigen A is greater than that of antigen B, antigen A's front diffuses into the agar, sweeping out all antibody determinants 1, 3, 5 ..... m, until enough of these antibodies have been combined to precipitate the antigen A. The slower-moving antigen B can react afterwards only with antibody determinants 2, 4, 6, ..., n inside the precipitating A ring, since the antibody determinants 1, 3, 5, ..., m are removed by antigen A. Outside the precipitating A ring, antigen B can react with antibody determinants 1,2, 3, ..., n. Depending on the relative concentration of antigen B the B precipitation ring can be precipitated inside, outside, or at the same position as the A ring. In any case
Immunoglobulin classes in radial immunodiffusion
213
it is clear that the area of the ring formed by antigen B will be larger than it would have been if antigen A was not present. Therefore, to calculate the concentrations by ring size measurements in cases of mixed antigens with overlapping antigenicity can lead to errors (Lietze et al., 1970), because the areas are not only related to the concentration and diffusion coefficients, but also on the overlapping antigenic groups. In view o f our data radial immunodiffusion can reflect qualitative changes in immunoglobulins if there is a great heterogeneity and concentration difference within the concerning immunoglobulin class. A detailed discussion of the double precipitation o f |gG in association with multiple myeloma and IgM in association with lymphoproliferative disorders will be published later. REFERENCES Bernier, G.M., R.E. Ballieux, K.T. Tominaga and F.W. Putnam, 1967, J. Exptl. Med. 125,303. Frangione, B., C. Milstein and J.R.L. Pink, 1969, Nature 221,145. Leikola, J. and G.N. Vyas, 1971, J. Lab. Clin. Med. 77,629. Lietze, A., C. Sinclair and A.H. Rowe, 1970, Clin. Biochem. 3,335. Longmire, R.L., R.S. Smith, R. McMiUan and R.T. Reid, 1971, Immunochemistry 8,153. Mancini, G., A.O. Carbonara and J.F. Heremans, 1965, Immunochemistry 2, 235. Miller, F. and H. Metzger, 1965, J. Biol. Chem. 240, 3325, 4740. Prahl, J.W., 1967, Biochem. J. 105, 1019. Soloman, A., 1969, J. Immunol. 102,496.