Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses

Abstract

The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, p<0.05). The precision results from the automated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

Fig. 1

Triplicate mean IFN-γ responses to limiting dilutions of CEF (10⁻⁷ to 2 μg/mL) for six healthy controls measured by manual (◇) and automated (●) ELISpot assays. Assay results were comparable (p>0.05 for all dilutions, Wilcoxon signed-rank test of paired differences in triplicate means), with greater variation at weaker dilutions for both processes (Spearman’s correlation between replicate CV and concentration: r=−0.77 manual; r=−0.64 automated; p < 0.0001).

Fig. 2

(a) V-formation of 96-well plates set up to assess plate variation in automated ELISpot assays measuring IFN-γ responses to 2 μg/mL of CEF and anti-CD3 antibody. (b) Responses from Three healthy individuals (plate 1: △, plate 2: ○) to either anti-CD3 (Control 1) or CEF (Controls 2 and 3) chosen according to measurability (range of 100–500 SFUs/10⁶ PBMCs). Intra-assay CVs are provided for each plate, together with dotted lines indicating 2 standard deviations above and below the plate means. The average inter-assay CV was 23%.