Evaluation of IFN-γ producing cells using ELISPOT assay
An ELISPOT assay was used to quantify IFN-γ synthesis by PBMC separated from the study subjects in response to nine HCV genotype 4a isolate ED43 peptide antigen pools consisting of 15 (15mer) and overlapping by 10 amino acids. These were 600 peptides combined in nine pools corresponding to all the HCV proteins. These synthetic peptides were specially manufactured by Mimotopes (Australia). About 15ml of blood was collected in EDTA vacutainer tubes (Becton Dickinson Biosciences, NJ, USA). PBMC were separated from whole blood using Ficoll-Hypaque density gradient centrifugation. Cellular viability was determined by trypan blue exclusion method. Briefly, 2x105 PBMC (200µl/well) were incubated in triplicate cultures in the ELISpot plates (Whatman Unifilter, USA) coated with anti-human IFNγ antibody and incubated for ~16 hours with or without recombinant HCV antigens at 3μg/ml of each single peptide in complete RMPI-1640 medium. A medium containing DMSO alone and 0.1μg / ml of SEB (or other polyclonal stimuli) represented the negative and positive controls, respectively. The assay was developed at the end of the incubation period until the spots appeared. The wells were, then, rinsed with tap water to stop the reaction. The number of spots/well was calculated using an automated ELISpot reader (Cellular Technology Ltd., Cleveland, USA) as reported [18]. The mean number of spot forming cells (SFC) in control wells were subtracted from the mean number of peptide-stimulated wells to correct for background cytokine production and are expressed as SFC/million PBMC. A positive antigen-specific HCV response was considered if the SFC in the presence of antigen was at least three times the number of SFCs in the medium control and > 55 SFC / million PBMC were present, as previously reported [19].