Effect of valency on binding properties of the antihuman IgM monoclonal antibody 202.
A murine monoclonal IgG2a antibody, 202, specific for human IgM, was produced and immunochemically characterized. Binding features of MAb 202, epitope localization, and its accessibility at the quaternary structure of polymeric IgM were investigated. Direct and competitive ELISA with fragments of IgM molecule demonstrated that the epitope recognized by MAb 202 lies on the Fc3 portion of IgM. Sandwich ELISA with MAb 202, which could be used simultaneously to capture and to detect bound IgM, indicated that more than one 202 epitope is present on the IgM molecule. MAb 202 did not precipitate IgM in solution, whereas good precipitation lines were obtained in agarose gel. Binding of MAb 202 to the J chain, C-terminal tailpiece and C mu 2 peptide, which remain attached to the C mu 3 domain of the Fc5 fragment, was excluded by a number of experimental results and structural reasons. Therefore a potential candidate for epitope 202 expression was the C mu 3 domain. MAb 202 did not react with isolated mu chain, which is expected since epitope 202 is of a conformational type. Furthermore, the reaction with monomeric IgM was almost undetectable as was demonstrated by a number of methods (ELISA, immunofluorescence, Western blotting). Since monovalent Fab portions of MAb 202 weakly reacted with polymeric IgM, we concluded that intrinsic affinity of their interaction is low but greatly enhanced by bivalent binding. Antipolymeric IgM binding specificity of MAb 202 was demonstrated only in the case of bivalent binding with a functional affinity constant of Kd = 2.14 x 10(-9) M-1. This implied up to a 10(4) difference between intrinsic and functional affinity, as in the range of concentration used in this study MAb 202 did not react with monomeric IgM.
Cirić
B
B
Immunology Research Center, University of Belgrade, Yugoslavia.
Radulović
M
M
Dimitrijević
L J
LJ
Jankov
R M
RM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000276:immunology; D004220:Disulfides / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007075:Immunoglobulin M / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D011233:Precipitin Tests; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007075:Immunoglobulin M