Affinity maturation of secreted IgM pentamers on B cells

We prepared a series of hapten-BSA conjugates with varying ratios of biotin to measure ligand-receptor interactions on B cells by flow cytometry using avidin for detection. Surface plasmon resonance measurements of the interaction with a monoclonal anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody suggested that NP5-BSA or NP7-BSA harboring 29 or 23 biotin molecules (NP5-BSA-bio(29) or NP7-BSA-bio(23)) would be suitably sensitive for flow cytometric analysis. By using NP-BSA-bio, we analyzed NP-binding cells in immunized mice. Unexpectedly, 30-40% of spleen cells expressing IgM could bind to NP5-BSA or NP7-BSA after immunization of mice with NP40-chicken gamma-globulin. The proteins binding to NP7-BSA-bio(23) on the cell surface were analyzed by immunoprecipitation and western blotting. Surprisingly, most of the proteins binding NP-BSA-bio on the cell surface were not the membrane form of IgM monomer, but a secreted IgM pentamer. It is likely that the IgM pentamer bound through Fc receptors for polymeric IgA or IgM and contributed to antigen binding. Comparison of the binding ratio of NP0.9-BSA:NP5-BSA between B cells of primary and secondary immunization suggested that the affinity of IgM matured during immunization.