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

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. Fc₅μ and F(ab')₂μ, (ii) isolated single domains, Cμ₂, Cμ₃, and Cμ₄, and (iii) mu heavy chain disease proteins, nine anti-IgM MoAbs were found to have Cμ₁ domain specificity, five to have Cμ₂ specificity, and five others to have Cμ₄ specificity. Ineffective binding to isolated μ chain demonstrated that Cμ₁-specifie MoAbs were directed to epitopes which require light chain for expression. The lack of binding of the Cμ₄-specific MoAbs to CNBr cleavage fragments of Fc₅μ 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 Cμ₁ likely bind to a single epitope, or very proximate epitopes, (ii) the five Cμ₂-specifie MoAbs recognize at least three distinct epitopes, and (iii) the five Cμ₄-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.