Abstract
Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor immunity, it is also advisable to account for determinant spreading by testing multiple epitopes. Efforts for comprehensive immune monitoring would require substantial numbers of PBMC to run the above tests systematically, which in most test cases is limiting. Immune monitoring with ELISPOT assays have been performed, thus far, in a 96-well format. In this study we show that one can increase cell utilization by performing the assay in 384-well plates whose membrane surface area is one third that of 96-well plates. Systematic testing of PBMC for antigen-specific T cell response in the two formats demonstrated that the 384-well assay corresponds to a one-in-three miniaturization of the 96-well assay. The lowest number of cells that can be used in the 384-well format, while allowing for sufficient contact with APC, is 33,000 PBMC/well. Therefore, with one million PBMC typically obtained from 1 mL of blood, a 30 well T cell ELISPOT assay can be performed in a 384-well format.
Highlights
T-lymphocytes are major players in the body’s defense against cancer and various infections
In an effort to reduce the number of PBMC used, recent studies have introduced ELISPOT assays in which the cells are first pre-activated with antigen in 384-well and 1536-well plates, and transferred into regular
Human IFN-γ ELISPOT assays were performed in parallel in 96- and 384-well plates, using the CEF peptide pool to stimulate CD8+ cells in PBMC
Summary
T-lymphocytes are major players in the body’s defense against cancer and various infections. It has become clear that only if all these classes are assessed, will the true nature of T cell immunity be revealed [1] Another challenge is that many times a multitude of antigens/peptides are targeted by T cells, and only if these are all tested will one obtain reliable information on the magnitude of T cell immunity. In an effort to reduce the number of PBMC used, recent studies have introduced ELISPOT assays in which the cells are first pre-activated with antigen in 384-well and 1536-well plates, and transferred into regular. 96-well ELISPOT plates for establishing the numbers of cytokine producing cells [8] Since such cell transfers are error prone and labor intensive, we verified whether ELISPOT assays could be performed by plating the PBMC directly in dedicated 384-well ELISPOT plates that have a PVDF membrane surface, similar to 96-well ELISPOT plates. (APC), we hypothesized that 384-well assays could be performed with exactly one-third the amount of reagents, including cell material, as would be required for 96-well assays
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