Polyclonal B cell activation for accurate analysis of pre‐existing antigen‐specific memory B cells
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.