Evaluation of IFN-γ producing cells using ELISPOT assay
An ELISPOT assay was used to quantify IFN-γ production by PBMC isolated
from the study subjects in response to nine HCV genotype 4a isolate ED43
peptide antigen pools composed 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 custom synthesized
by Mimotopes (Australia). Approximately 15ml of whole blood were
collected into EDTA vacutainer tubes (Becton Dickinson Biosciences, NJ,
USA). PBMC were isolated by Ficoll-Hypaque density gradient
centrifugation and 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. Negative and
positive controls included medium containing DMSO alone and 0.1μg/ml of
SEB or other polyclonal stimuli, respectively. At the end of the
incubation period, the assay was developed until the appearance of
spots, and then the wells were rinsed with tap water to stop the
reaction. The number of spots per well was counted using an automated
ELISpot reader (Cellular Technology Ltd., Cleveland, USA) as described
[18]. The average number of spot forming cells (SFC) in control
wells were subtracted from the average number of peptide-stimulated
wells to correct for background cytokine production and are expressed as
SFC/million PBMC. A positive HCV antigen-specific response was
considered if the SFC in the presence of antigen were at least
three-fold the number of SFC in the medium control and if there was
>55 SFC/million PBMC, as we previously reported [19].