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 NP(5)-BSA or NP(7)-BSA harboring 29 or 23 biotin molecules (NP(5)-BSA-bio(29) or NP(7)-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 NP(5)-BSA or NP(7)-BSA after immunization of mice with NP(40)-chicken gamma-globulin. The proteins binding to NP(7)-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 NP(0.9)-BSA:NP(5)-BSA between B cells of primary and secondary immunization suggested that the affinity of IgM matured during immunization.
Shimizu
Takeyuki
T
Division of Biosignaling, Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba 278-0022, Japan.
Kozono
Yuko
Y
Kozono
Haruo
H
Oda
Masayuki
M
Azuma
Takachika
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D001360:Avidin / Q000032:analysis; D001402:B-Lymphocytes / Q000276:immunology*; D001710:Biotin / Q000737:chemistry; D002460:Cell Line; D005434:Flow Cytometry; D015870:Gene Expression; D007114:Immunization; D007075:Immunoglobulin M / Q000276:immunology*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D051379:Mice; D008805:Mice, Inbred A; D009596:Nitrophenols / Q000737:chemistry / Q000276:immunology*; D010648:Phenylacetates; D011485:Protein Binding; D011961:Receptors, Fc; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013154:Spleen / Q000166:cytology; D005719:gamma-Globulins / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D009596:Nitrophenols; D010648:Phenylacetates; D011961:Receptors, Fc; D005719:gamma-Globulins; C026242:4-hydroxy-5-nitrophenyl acetic acid; D001360:Avidin; D012710:Serum Albumin, Bovine; D001710:Biotin