Saliva is tested at IISBR in duplicate using a commercially-available enzyme-linked immunosorbent assay kit (ELISA) from Salimetrics following manufacturer’s protocol without modification. This test employs a 96-well microtitre plate coated with a monoclonal antibody (Ab) to IL-6. IL-6 in standards, controls and unknown samples bind to the antibodies on the plate. Anti-IL6 goat antibody enzyme linked to biotin binds to the bound standard, control and sample IL-6. Streptavidin-HRP (horse radish peroxidase) is added and binds to the biotin conjugated goat IL-6. Bound streptavidin-HRP is measured by the reaction of the peroxidase enzyme on the substrate tetramethylbenzidine (TMB) producing a blue color. The reaction is stopped and the resultant yellow color is read at 450nm on a spectrophotometer (PowerWave HT, BioTek Instruments). The amount of streptavidin-HRP detected, as measured by the intensity of color, is directly proportional to the amount of IL-6 present.
Briefly, samples are first diluted at a 1:5 dilution by added 60ul of saliva in to 240ul sample diluent. This is mixed thoroughly. 100 µL of standard, controls, and diluted unknown saliva are pipetted in duplicate onto 96-well microtitre plates and incubated for 1 hour shaking at room temperature. Plates are washed 4 times. 100ul of antibody conjugate is added to each well and incubated for 2 hours shaking at room temperature. Plates are washed 4 times. 100ul of streptavidin-HRP conjugate solution is added to each well and incubated for 20minutes at room temperature. Plates are washed 4 times followed by addition of a TMB substrate and incubation at room temperature in the dark for 20 minutes. Following incubation the reaction is stopped with 2M Sulfuric Acid (NH2SO4) and optical density (OD) read at 450nm with a 630nm correction filter. Concentrations of controls and samples are calculated from a standard curve generated using a 4-parameter non-linear regression curve fit (Gen5, BioTek).