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

Abstract

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 10⁴ 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 10⁵ PBMCs with 3 x 10⁶ 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.