Production technology of IL-2 enzyme-linked immunosorbent assay / Монголын Анагаах Ухаан

ABSTRACT

Background@#Enzyme-linked immunosorbent assay (ELISA) kit is used abundantly in order to measure the quantity of agents and their antibodies. Introducing the production of the kit in our own country for research and diagnostic uses will be beneficial for economy and time.@*Goal@#Producing enzyme-linked immunosorbent assay for the determination of IL-2 cytokine in biological fluids.@*Materials and Methods@#Elisa kits made by R&D systems in USA were used in our study. Anti-IL-2 monoclonal antibody was diluted with concentrations of 0.5-3μg/ml in polystyrene well, and then incubated with conjugate solution of pH 7.2-7.4 for 12 hours with the purpose of measuring the optimal starting concentration for the conjugate antibody. Later, the reaction was blocked with PBS blocking buffer with 1% BSA (Bovine serum albumin) for an hour. After washing the wells, 100ul of standard solutions with concentrations of 7.81-2000pg/ul were added and incubated for 60 or 120 minutes. Subsequently, secondary antibody and streptavidin-HRP were added as substrate, and the reaction was terminated with stop solution. Absorbance was measured immediately at 450 nm optical densities, and then regression equation was calculated using standard curve.@*Result@#The light absorbance was insufficient when then dilution of starting concentration for the conjugate antibody was 0.5μg/ml and the light absorbance decreased as the concentration of conjugate antibody reduced. The light absorbance increased when the concentration was 2-4μg/ml, but the standard curve was constructed nonlinear. When standard curve was constructed with 1 ug/ml, the regression equation had R2=0.96, which identifies the optimal concentration. The results were analogous when the incubation times were 60 and 120 minutes with the concentration of 4μg/ml.@*Conclusion@#The optimal condition for determining IL-2 cytokine was when ELISA conjugate concentration was 1μg/ml (sample and conjugate) and incubation time was 120 minutes, and the regression equation from the standard curve was y=0.0013x+0.1901, R²=0.96.