T cells have been playing a very important role in the immune response in an organism, which provides a frame of antigen-specific response for infections, diseases, and cancers. A highly selective antigen-specific cells lineage involves and activates that process. The goal of immune diagnosis is to reliably identify a little part of responders and confirm their mode of action as well as the degree of response. The individual antigen is difficult to be recognized by T cells in most cases. Hence, the techniques of measuring T cell immune responses must be sensitive, high-throughput, and widely adopted.
The Enzyme-Linked ImmunoSpot (ELISpot) assay not only measures the magnitude and quality of T cells immunity through detecting antigen-specific T cells that involve in the secretion of cytokines and other utility but also detects the protein secretion from the single cell. Compared to other assays, the ELISpot assay can be utilized to detect the population and frequency of specific proteins such as cytokines and antigen-specific antibodies. T cells ELISpot is much more reproducible and sensitive than other measurements like cytokine measurements in detecting low-frequency antigen-specific CD4+ and CD8+ T cells. More than that, this accurate assay can also validate new T cell epitopes.
Creative Biolabs has developed and optimized the ECIA™ T Cells ELISpot Assay platform. Relying on the excellent sensitivity and resolution, ECIA™ T cells ELISpot assay is able to provide a multidimensional, quantitative assessment of effector function at the single cell level. And it is the first choice for the development of multifunctional T cell analysis in the areas of drug research and preclinical trials.
The ELISpot assay is a kind of technique convergence based on ELISA and Western blotting. The cells are stimulated to produce local cytokines that are captured by specific monoclonal antibody coated onto a PVDF-backed microplate. After the cells are disintegrated, a biotin-labeled antibody specific for the chosen cytokine is added to bind the captured cytokines. Following a wash to remove any unbound biotinylated antibody, the detected cytokine is then visualized using streptavidin conjugated to alkaline phosphatase and a precipitating substrate (e.g., BCIP/NBT). Purple spots will emerge in the PVDF plate. Once the signal is developed, the number of spots can be calculated manually, or by using an image-based spot reader and the accompanying analysis software. By comparing the number of the control wells, the frequency and the total number of responding cells can be determined.
General steps of the ECIA™ T cells ELISpot assay
Fig.1 The Protocol of T Cell ELISpot.