B-cell ELISpot assay to quantify antigen-specific antibody-secreting cells in human peripheral blood mononuclear cells

Abstract

Peripheral blood is commonly used to assess the cellular and humoral immune responses in clinical studies. It is a convenient sample to collect for immunological research as compared to the surgically excised and biopsied lymphoid specimens. To determine the functional status of immune system from peripheral blood, the enzyme-linked immunospot (ELISpot) assay is a popular method of choice owing to its high sensitivity, great accuracy, and easy performance. The ELISpot allows detection and quantification of cellular functionality at the single-cell level. Therefore, ELISpot assay is commonly applied to detect cytokines and cytotoxic granules released from T cells as well as to measure antibodies secreted from B cells. Because the ELISpot assay has been increasingly used for evaluation of the vaccine efficacy in clinical trials, standardization and reproducibility are crucial to minimize assay variability amongst samples from different sources. Here we introduce methods to isolate human peripheral blood mononuclear cells (PBMCs) for quantification of the antigen-specific antibody-secreting cells using the ELISpot assay.