Memory B cells drive the adaptive humoral immune response following reexposure to antigen and are important for eliminating antigen not cleared by preexisting circulating antibodies. Measuring antigen-specific memory B cells would be useful for predicting long-term vaccine efficacy and a person's level of immunity, which often may not correlate well with circulating antibody titers. We quantified antibody-secreting B cells (ASCs) from peripheral blood mononuclear cells (PBMCs) from healthy donors stimulated with pokeweed mitogen, Staphylococcus aureus Cowan strain 1, and the Toll-like receptor 9 agonist, CPG. By ELISPOT, goat antihuman IgG-, IgM-, and IgA were used as capture antibodies for total Ig isotype and tetanus toxoid (TT) and 23-valent pneumococcal polysaccharide vaccine (PVAX) for antigen-specific ASCs. Enzyme-conjugated isotype-specific antibodies served as detector antibodies. ASCs were counted, and antibody levels were measured using a kinetic ELISA. Autologous and heterologous soluble competitive inhibitor antigens were added to TT and PVAX wells. We observed strong total IgM, IgG, and IgA responses. Large numbers of IgM ASCs were observed in the TT and PVAX wells, with fewer IgG and IgA responses to TT. Antigen adsorption data suggested that the majority of IgM ASCs reacting with TT and PVAX were nonspecific and did not reflect true antigen-specific responses. Moreover, the frequencies of TT- and PVAX-reactive IgG and IgA ASCs in subjects without recent immunization were low. Thus, methods to elucidate the frequency of antigen-specific memory cells should include the isotype and specificity of cells reactive with specific antigens.
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