Elispot
Enzyme-linked immunospot, or Elispot, assays were first described two decades ago as a way to detect a specific immune response at the single cell level. The assay is regularly used to monitor the effectiveness of a vaccine during clinical trials.
The assay works by coating a membrane with antibodies. For example, a treated patient’s PBMCs (i.e., T-cells) and target cells (i.e., viral antigen) are added to the coated membrane and incubated. The responding T-cells produce cytokines. Biotinylated antibodies to the cytokine and an avidin-enzyme conjugate are added to identify the responding cells. Each responding cell produces one spot on the membrane. The spots on the membrane are then counted to determine the vaccine effectiveness.
The Elispot assay can have variable and often misleading results if each step is not carefully optimized (e.g., type of antibodies used, subjectivity of spot interpretation, cell enrichment techniques, sample integrity, manual cell counts/viability).
The Guava ViaCount® Assay, with its automated cell counts, viability, and apoptotic fraction, is known to greatly improve the reliability of Elispot results. The assay provides improved accuracy and precision over manual trypan blue because it automates cell counts and viability assessments and uses a superior approach to assess viabilities using DNA staining dyes. The assay allows you to easily monitor PBMC sample integrity for the apoptotic fraction: Apoptotic cells will not respond in an Elispot assay.
The recent Elispot Harmonization
Guidelines also recommend: A counting
method for apoptotic cells for determining adequate cell dilution for plating
(Janetzki S, et al. Cancer Immunol.
August 2007.).
The ViaCount Assay is the only automated method that also provides an apoptotic fraction.
The platelet fraction, obtained by measuring light scatter, is also important to monitor as secretion of inhibitors from platelets may adversely affect T-cell function, and debris index freeze, thaw methods may affect sample integrity.
The Guava ViaCount Assay is a validated method within many laboratories such as PAVE (AIDS Vaccine Evaluation), HVTN (HIV Vaccine Trials Network), other non-AIDS Elispot users.
For more information about how a Guava System can meet your cell counting needs, please visit the following links.
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