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Poorly soluble peptides can mimic authentic ELISPOT responses.

R Karl Karlsson1, Wim Jennes, Kimberly Page-Shafer

  • 1Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141, USA.

Journal of Immunological Methods
|February 12, 2004
PubMed
Summary

False-positive ELISPOT results can occur due to improperly prepared HIV-1 peptides. Careful peptide preparation and controls are crucial for accurate assessment of cell-mediated immunity using the interferon-gamma ELISPOT assay.

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Area of Science:

  • Immunology
  • Virology
  • Biotechnology

Background:

  • The ELISPOT assay is a key tool for measuring cell-mediated immunity.
  • Assessing immune responses to HIV-1 peptides is critical for understanding viral control and vaccine development.

Purpose of the Study:

  • To investigate the cause of unexpected positive results in an interferon-gamma ELISPOT assay using HIV-1 peptides.
  • To identify methods to prevent false-positive ELISPOT responses.

Main Methods:

  • Performed interferon-gamma ELISPOT assays using peripheral blood mononuclear cells (PBMC) from HIV-1 exposed seronegative (ESN) individuals.
  • Analyzed an HIV Gag peptide for solubility and potential interactions with assay components.
  • Tested the effect of peptide filtering on ELISPOT assay results.

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Main Results:

  • An HIV Gag peptide, incompletely dissolved, induced non-specific spot formation in ELISPOT assays, even without cells.
  • The peptide interacted with avidin and the ELISPOT membrane, contributing to false positives.
  • Filtering the peptide successfully prevented non-specific spot formation.

Conclusions:

  • Improper peptide preparation, including incomplete dissolution and interaction with assay components, can lead to false-positive ELISPOT results.
  • Rigorous quality control of peptide preparation and the use of appropriate controls are essential for reliable ELISPOT assay data.
  • These findings highlight the importance of meticulous experimental procedures in immunological assays.