[Modification of a disulfide in the hinge region of rabbit IgG and study of the interaction of polyvalent ferritin antigen, protein A, and anti-IgG].
Since immunoglobulins are used in a vast variety of immunoassays, the problem of obtaining antibodies with enhanced antigen-binding activity is of great importance. In order to discriminate between putative approaches to activating antibody modification, some functional characteristics of rabbit IgG modified at the hinge disulfide by three reagents: iodoacetamide, N-ethylmaleimide, and 2.2'-dipyridyl disulfide, have been studied. As can be judged from gel-permeation chromatography data, the molecular sizes of modified rabbit IgG were slightly increased in comparison with the native protein. Using enzyme immunoassay, it was shown that modification by each of the above reagents results in the same degree of activation of the antibody binding to the protein polyvalent antigen-human ferritin, due to the increase in segmental flexibility, i.e., Fab motion around the Fo fragment of IgG. The type of concentration dependencies of antigen binding suggest that another determinant stimulating the antigen binding in addition to the increase in segmental flexibility, can be attributed to intra- or interdomain flexibility of domains constituting the Fab fragments. Using protein A and anti-IgG as conformational probes for the antibody Fo fragment, the conformation and conformational dynamics of both CD2 domain epitopes and the switch region between the CD2 and CH3 domains have been shown to be essentially unaffected by modification.
Lepesheva
G I
GI
Martsev
S P
SP
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D002850:Chromatography, Gel; D004220:Disulfides / Q000737:chemistry* / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005293:Ferritins / Q000276:immunology*; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D007460:Iodoacetamide / Q000494:pharmacology; D011817:Rabbits; D013205:Staphylococcal Protein A / Q000276:immunology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000888:Antibodies, Anti-Idiotypic; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D013439:Sulfhydryl Reagents; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D005293:Ferritins; D005033:Ethylmaleimide; D007460:Iodoacetamide