A new technique for enhanced cell counting precision with the ELISPOT assay

The evaluation of the response of T-Cells to a prospective vaccine is a critical aspect of any study of the effectiveness of vaccine candidates, such as prospective anti-cancer vaccines or vaccines against infectious diseases. The T-Cell response is usually monitored by techniques such as the ELISPOT assay, or an intracellular cytokine-staining assay. The ELISPOT assay involves the enumeration of the spots on a membrane after incubating peripheral blood mononucleated cells (PBMCs) with specific antigens in a 96-well plate. Each spot on the membrane represents a cell that can produce an appropriate cytokine in response to the specific antigen stimulus. The response to the antigen is typically reported as Spot Forming Cells per million peripheral blood mononucleated cells (SFC/PBMC). The number of cells that are input for the assay is a vital parameter and the accuracy and precision of the overall assay is clearly very dependent on the accuracy and precision of the counting of the number of cells in the sample. Several different technologies are commonly used to count the number of viable cells, including staining with Trypan blue or with a fluorescence substrate, followed by visual counting methods. In our laboratories, it has been found that these techniques lead to a high variation when the counting is performed at different times by the same operator, as well as when the counting is performed by different operators on the same sample. This does, of course, lead to uncertainty in the number of cells in the sample, and thereby leads to an uncertainty in the overall evaluation of the efficacy of the vaccine candidate.