Granulocyte contamination dramatically inhibits spot formation in AIDS virus-specific ELISpot assays: analysis and strategies to ameliorate.

The interferon-gamma (IFNgamma) ELISpot assay has become the most critical tool for HIV vaccine evaluation. External factors affecting ELISpot results must be minimized for the data to be reliably used in vaccine research and development processes. In pre-clinical pigtail macaque studies analyzing HIV/SIV vaccine studies, we detected a strong correlation between levels of granulocytes contaminating PBMC preparations and reduction in the quality and quantity of spots in the IFNgamma ELISpot assay. Acute SHIV infection of macaques worsened granulocyte contamination of PBMC fractions and made the assay much less reliable in detecting SIV-specific T cell immunity compared to intracellular cytokine staining (ICS). This problem could be ameliorated by using an F(ab)(2) form of the MD-1 IFNgamma capture antibody, presumably reflecting that activation of granulocytes in the well by the Fc portion of the standard capture antibody disrupts spot formation. Improving the standard ELISpot assay by using an F(ab)(2) capture antibody should make it more reliable for use in critical vaccine development studies.