EFFECT OF VALENCY ON BINDING-PROPERTIES OF THE ANTIHUMAN IGM MONOCLONAL-ANTIBODY-202

A murine monoclonal IgG(2a) antibody, 202, specific for human IgM, was produced and immunochemically characterized, Binding features of MAb 202, epitope localization, and its accessibility at the quaternary str ucture of polymeric IgM were investigated, Direct and competitive ELIS A with fragments of IgM molecule demonstrated that the epitope recogni zed by MAb 202 lies on the Fc(5) portion of IgM, Sandwich ELISA with M Ab 202, which could be used simultaneously to capture and to detect bo und IgM, indicated that more than one 202 epitope is present on the Ig M molecule, MAb 202 did not precipitate IgM in solution, whereas good precipitation lines were obtained in agarose gel, Binding of MAb 202 t o the J chain, C-terminal tailpiece and C mu 2 peptide, which remain a ttached to the C mu 3 domain of the Fc(5) fragment, was excluded by a number of experimental results and structural reasons, Therefore a pot ential candidate for epitope 202 expression was the C mu 3 domain, MAb 202 did not react with isolated mu chain, which is expected since epi tope 202 is of a conformational type, Furthermore, the reaction with m onomeric IgM was almost undetectable as was demonstrated by a number o f methods (ELISA, immunofluorescence, Western blotting), Since monoval ent Fab portions of MAb 202 weakly reacted with polymeric IgM, we conc luded 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 K-d = 2.14 X 10(-9) M(-1), This implie d 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 re act with monomeric IgM.