title: Polyclonal B cell activation for accurate analysis of pre-existing antigen-specific memory B cells.
abstract: The enzyme-linked immunospot (ELISPOT) assay is a widely used tool for enumeration of antigen-specific memory B cells in several disciplines, such as vaccination, cancer immunotherapy and transplantation. For the accurate estimation of antigen-specific memory B cell frequencies, a well-defined B cell activation protocol is pivotal. In this study, we aimed to characterize a polyclonal B cell activation protocol to facilitate optimal monitoring of antigen-specific memory B cell frequencies. Total, naive and memory B cells were activated polyclonally with an α-CD40 monoclonal antibody, cytosine-phosphate-guanine (CPG) oligodeoxynucleotide (ODN) 2006, interleukin (IL)-2, IL-10 and IL-21. Polyclonal activation of B cells resulted in equal cell death ratios in naive and memory B cells. When tested in an antigen-specific system, immunoglobulin (Ig)G spots were detected only in the memory fraction. There was no change in B cell polyclonality due to in-vitro activation. Our data show that the current polyclonal activation protocol may be used reliably to estimate the frequency of memory B cells in ELISPOT assays.
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: G E
authors.initials: GE
authors.lastname: Karahan
authors.forename: M
authors.initials: M
authors.lastname: Eikmans
authors.forename: J D H
authors.initials: JD
authors.lastname: Anholts
authors.forename: F H J
authors.initials: FH
authors.lastname: Claas
authors.forename: S
authors.initials: S
authors.lastname: Heidt
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D019013:CD40 Antigens / Q000276:immunology; D002478:Cells, Cultured; D058501:Enzyme-Linked Immunospot Assay; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007156:Immunologic Memory; D007378:Interleukins / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D018655:Lymphocyte Count / Q000379:methods; D016131:Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology*; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; C513533:CpG ODN 2006; D007074:Immunoglobulin G; D007378:Interleukins; D009838:Oligodeoxyribonucleotides
keywords: B cells; ELISPOT; antibodies; antibody-secreting cells