ELISpot Assay for Quantifying Antigen-Specific and Total Antibody-Secreting Cells in Blood and Bone Marrow of Immunized Rhesus Macaques.

Evaluation of novel vaccine antigens and adjuvants traditionally relies on measuring antibody titers, which require time to develop following immunization. Quantification of antibody-secreting cells (ASCs) after primary and booster immunizations provides an earlier, mechanistically informative measure of vaccine-induced humoral immunity and can facilitate the optimization of vaccine formulations and immunization strategies. Here, a protocol for enumerating ASCs from peripheral blood mononuclear cells (PBMCs) and bone marrow (BM) cells of rhesus macaques is described; rhesus macaques are an important preclinical model for HIV vaccine research. This enzyme-linked immunospot (ELISpot) assay enables the quantification of both antigen-specific and total IgG, IgM, and IgA ASCs by measuring their absolute and relative frequencies. In peripheral blood, the assay captures the peak plasmablast response following immunization, whereas in bone marrow it detects the accumulation of antigen-specific plasma cells of different isotypes, thereby assessing the durability of long-lived ASCs. This protocol offers a robust and reproducible approach for evaluating immunogen and adjuvant combinations in preclinical vaccine studies and provides valuable data to support the translation of promising vaccine candidates into clinical development.