Enzyme-linked immunospot assays for direct ex vivo measurement of vaccine-induced human humoral immune responses in blood

Abstract

The enzyme-linked immunospot (ELISPOT) assay was originally developed to enumerate antigen-specific antibody-secreting cells (ASCs), and has subsequently been adapted for various applications, including the detection cytokine-secreting cells. Owing to its exceptionally high sensitivity, the ELISPOT has proven to be especially useful for detecting discrete populations of active cells (e.g., antigen-specific cells). Because of its versatility, the ELISPOT assay is used for a wide range of applications, including clonal analyses of immune responses after vaccination or after immunotherapy. Here we describe standard protocols for the detection of human ASCs specific to virtually any vaccine antigen after enrichment of circulating plasmablasts. In addition, a protocol is described for the measurement of mucosal ASC responses after prior immunomagnetic enrichment of mucosally derived blood lymphocytes. The protocols described allow rapid (~6-8 h) detection of specific ASCs in small (1-2 ml) samples of blood and can be performed in resource-poor settings.

Circulating Gut-Homing (α 4 β 7 + ) Plasmablast Responses against Shigella Surface Protein Antigens among Hospitalized Patients with Diarrhea

“…Assays were carried out in nitrocellulose-bottomed 96-well plates (Millipore, USA; catalog no. MSHAN4510) using a recently described whole-blood ELISPOT method (16). Wells were coated individually with five different types of purified Shigella-specific antigens (PSSP1, PSSP2, IpaD20, IpaD120, and IpaC) applied in 50-l volumes at a final concentration of 5 g/ml in phosphate-buffered saline (PBS).…”

“…Migration of mature lymphocytes from mucosal inductive sites to the gut via the systemic circulation occurs soon after vaccination or infection (13)(14)(15)(16). These homing lymphocytes include a contingent of antibody-secreting cells (ASCs) that are transiently circulating and whose frequency peaks in blood as early as 1 week after the onset of infection or after stimulation of the gut-associated lymphoid tissue (16)(17)(18)(19). In these studies, mucosal immunity was determined by enzyme-linked immunosorbent spot (ELISPOT) assay using peripheral blood specimens collected about a week after antigen stimulation.…”

Impaired Antibody-mediated Protection and Defective IgA B-Cell Memory in Experimental Infection of Adults with Respiratory Syncytial Virus

“…Observed end point titers were corrected for dilution using the ratio of serum to nasal lavage urea before analysis as described (21). Antibody-secreting cells (ASCs) were detected using enzyme-linked immunospot (ELISpot) as previously described (22), using whole RSV M37 lysate from HEp-2 cells and recombinant F protein based on the RSV A2 strain (see online supplement for detailed methods). For memory B cells (MBCs), additional plates were coated with 10 mg/ml measles antigen (Meridian Lifesciences, Memphis, TN), and 5 mg/ml HEp-2 antigen, 2.5 mg/ml keyhole limpet hemocyanin (Sigma, Dorset, UK), and media as negative controls.…”

Alternative administration routes and delivery technologies for polio vaccines

“…IPV has been shown to boost mucosal immunity among recipients who have earlier received OPV [75,76]. Further clinical studies on heterogeneous prime-boost vaccination schedules, but also of other administration strategies, mucosal immunity will be more and better addressed by modern techniques [77].…”