Analysis of the domain specificity of various murine anti-human IgM monoclonal antibodies differing in human B lymphocyte signaling activity

Abstract

The domain binding specificity of 19 murine anti-human IgM monoclonal antibodies (MoAbs) that have shown considerable heterogeneity in the transduction of stimulatory and inhibitory signals to B lymphocytes was evaluated by competition radioimmunoassays. Through the use of: (i) enzymatic fragments of IgM which each encompass more than a single C H domain, i.e. Fc 5μ and F(ab') 2μ, (ii) isolated single domains, Cμ 2, Cμ 3, and Cμ 4, and (iii) mu heavy chain disease proteins, nine anti-IgM MoAbs were found to have Cμ 1 domain specificity, five to have Cμ 2 specificity, and five others to have Cμ 4 specificity. Ineffective binding to isolated μ chain demonstrated that Cμ 1-specifie MoAbs were directed to epitopes which require light chain for expression. The lack of binding of the Cμ 4-specific MoAbs to CNBr cleavage fragments of Fc 5μ suggest that the determinants recognized by these MoAbs may also be conformational in nature. Cross-inhibition analyses were used to determine the number of unique epitopes recognized by the anti-IgM MoAbs. Results from these experiments showed that: (i) eight of the nine MoAbs specific for Cμ 1 likely bind to a single epitope, or very proximate epitopes, (ii) the five Cμ 2-specifie MoAbs recognize at least three distinct epitopes, and (iii) the five Cμ 4-specific MoAbs each recognize a separate determinant. A comparison of the known B cell activating properties of these MoAbs with their specificity for the various segments of the IgM molecule indicate that mitogenicity cannot be attributed to selective binding to any one domain.