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].