Optimization of Enzyme-linked Immunospot Assay for quantitative analysis of interferon gamma producing cells

Enzyme-Linked Immunospot (ELISPOT) is the most sensitive assay for the enumeration of IFN -g producing cells at single cell level. This study aimed to optimize the ELISPOT assay to study the IFN -g production in response to B. pseudomallei and dengue virus in healthy individuals. The optimal peripheral blood mononuclear cells (PBMCs) concentrations, incubation time and concentration of each stimulator to maximize IFN -g production were examined. The results showed that using PBMCs at 2.5 x 10 4 cells and stimulating with a positive control stimulator, phytohemagglutinin (PHA), at 3 µg/ml was the optimal condition for IFN -g responses. The number of IFN -g -Spot Forming Cell (IFN -g -SFC) was appropriate for enumeration by Stereomicroscope. In contrast, incubation of 5 x 10 5 PBMCs with 3 x 10 6 cfu/ml heat-killed B. pseudomallei or dengue virus at a dilution of 1:2700 was the optimal condition. These optimal conditions were then used to study the IFN -g production in 8 healthy volunteers. The results clearly showed that B. pseudomallei and dengue virus could stimulate all healthy PBMCs to produce IFN -g at higher levels than those of medium control. However, the degree of IFN -g responses was different for each individual. Moreover, linear regression analysis showed that the number of IFN -g -SFC obtained from B. pseudomallei stimulation was correlated with the dengue virus stimulation. In conclusion, in this study, the ELISPOT for IFN -g production using B. pseudomallei and dengue virus stimulations have been optimized. Our results of IFN -g production in response to B. pseudomallei and dengue virus provides basic information for further study on the ability of B. pseudomallei or Dengue’s peptides in IFN -g induction in screening for vaccine candidate.