Immunoblotting with monoclonal antibodies: loss of immunoreactivity with human immunoglobulins arises from polypeptide chain separation.
Immunoblotting has been used to study the antigen binding characteristics of 5 monoclonal antibodies (Mc/Abs) against human Ig (1 anti-kappa, 2 anti-gamma and 2 anti-delta chain. Of the 4 Mc/Abs only 1 (the anti-kappa chain Mc/Ab) reacted with its antigen when blotted from reducing SDS polyacrylamide gels. However, the 4 Mc/Abs which recognise immunoglobulin heavy chains were able to bind their antigens when blotted from native or non-reducing SDS gels. The lack of reactivity of the latter Mc/Abs in blots from reduced SDS gels may be attributed to the separation of Ig which occurs during electrophoresis after the -S-S- bonds are broken. It may be concluded that the conformation of Ig heavy chains is considerably altered when Ig molecules are disrupted and Ig chains separated, and several heavy chain determinants are lost during this process. Therefore determinants recognised by the anti-heavy chain Mc/Abs are most likely to be of the 'conformational' type whereas the anti-light chain Mc/Ab may well recognise a purely sequential determinant.
D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D006801:Humans; D007072:Immunoglobulin D / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000276:immunology; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007072:Immunoglobulin D; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D046911:Macromolecular Substances