Modified ELISpot to detect viral proteins

In a study by Puertas et al., the ELISpot method was used in a modified setup in order to detect antigens secreted by HIV-infected cells. To quantify the frequency of CD4 T cells reversing from latency to the production of viral antigens, the authors chose ELISpot due to its sensitivity and adapted the method to their needs.

The authors’ ELISpot setup included coating the plate with three antibodies, against CD3 and CD28 (to reactivate the infected cells) and against HIV-1 p24 to capture the viral antigen. Before adding the cells to the ELISpot plate, CD4 cells were enriched from cryopreserved PBMCs by negative immunomagnetic separation. In each well, the secreted antigen was captured cumulatively over a three-day in vitro culture. The final detection steps followed a typical ELISpot protocol using a biotinylated anti-p24 detection antibody.

The linearity of the assay was evaluated using serial dilutions of PBMCs. Additionally, the assay was validated through comparison to several assays including intracellular p24 staining and SIMOA (ELISA-based). The authors called their adapted ELISpot viral protein spot (VIP-SPOT) assay.