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International Immunology Advance Access originally published online on March 29, 2004
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International Immunology, Vol. 16, No. 5, pp. 675-684, May 2004
© 2004 Japanese Society for Immunology

Affinity maturation of secreted IgM pentamers on B cells

Takeyuki Shimizu1, Yuko Kozono1,2, Haruo Kozono1, Masayuki Oda3 and Takachika Azuma1

1 Division of Biosignaling, Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba 278-0022, Japan 2 Present address: Clock Cell Biology, IMCB, National Institute of Advanced Industrial Science and Technology, Ibaragi 305-8566, Japan 3 Present address: Department of Cellular Macromolecule Chemistry, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522, Japan

The first two authors contributed equally to this work
Correspondence to: T. Azuma; E-mail: tazuma{at}rs.noda.tus.ac.jp
Transmitting editor: K. Okumura

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-bio29 or NP7-BSA-bio23) 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-bio23 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.

Keywords: antibody, B lymphocyte, Fc receptor, memory


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