Positive controls in ELISpot with the addition of a polyclonal T-cell activator

Positive controls in ELISpot

When measuring antigen-specific T-cell responses in ELISpot, relevant controls are vital. Apart from the negative control which normally consists of cells in medium without antigen or other stimuli, the addition of a polyclonal T-cell activator may serve as a positive control both for cell viability and the functionality of the immunoassay. For polyclonal activation, certain antibodies specific for CD3 are ideally used. A multitude of T-cell cytokines including IFN-γ, IL-2, IL-4, IL-5, IL-10 and IL-13 are elicited by this stimulation, and this control can thus be used in a variety of ELISpot assays. Other commonly used stimulators include phytohemagglutinin (PHA) and concanavalin A (ConA), which are both potent T-cell activators and can be used in a similar way as anti-CD3 antibodies for activation of CD4+ and CD8+ T cells.



IFN-γ CD3 stimulation 50 000 cells	IFN-γ PHA stimulation 50 000 cells	IL-4 CD3 stimulation 100 000 cells	IL-4 PHA stimulation 100 000 cells
ELISpot analysis after stimulation of human PBMC with the anti-CD3 antibody CD3-2 (available from Mabtech) or with phytohemagglutinin (PHA). The spots generated by stimulation with anti-CD3 antibody are generally more distinct than those obtained with PHA which facilitates spot counting.

Another useful positive control is based on the addition of antigens to which most people have specific T cells. With IFN-γ production by antigen-specific human CD8+ T cells being a major area of use for the ELISpot, a positive antigen-specific control, the CEF peptide pool, has been developed particularly for this application. This pool contains 23 different class-I restricted peptides, all defined as common CD8+ T-cell epitopes derived from cytomegalovirus, Epstein-Barr virus and influenza virus. It efficiently induces IFN-γ production by virus-specific CD8+ T cells in almost 90% of Caucasians and also elicits Perforin, Granzyme B and MIP-1β responses in many individuals. In addition to serving as a positive control, the CEF peptidepool is useful for standardization procedures.