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J Immunol Methods. 2004 Feb 1;285(1):89-92.

Poorly soluble peptides can mimic authentic ELISPOT responses.

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Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141, USA.


The ELISPOT assay is a specific, sensitive, quantitative assay for assessing cell-mediated immune responses to a variety of antigens including HIV-1 peptides. In an interferon (IFN)-gamma-ELISPOT assay, peripheral blood mononuclear cells (PBMC) from two HIV-1 exposed seronegative (ESN) individuals appeared to respond strongly to an HIV Gag peptide. Analysis of this peptide revealed that it was incompletely dissolved and induced non-specific spot formation, even in the absence of cells. In subsequent experiments, the peptide was found to interact with avidin and the ELISPOT membrane. Filtering the peptide prevented non-specific spot formation. These findings underscore the need for appropriate controls and proper peptide preparation in order to reduce the risk of false-positive ELISPOT responses.

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