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 C-Reactive protein. C-Reactive protein in standards, controls and unknown samples bind to the anti-CRP antibodies on the plate. Anti-CRP antibody enzyme linked to horseradish peroxidase(HRP) binds to the bound standard, control and sample CRP. Anti-CRP antibody enzyme conjugate 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 CRP antibody enzyme conjugate peroxidase detected, as measured by the intensity of color, is directly proportional to the amount of CRP present.
Briefly, samples are first diluted at a 1:2 dilution by added 150ul of saliva in to 150ul 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 2 hours shaking at room temperature. 100ul of anti-CRP antibody enzyme conjugate is added to each well and incubated for 2 hours shaking at room temperature. Plates are washed 4 times followed by addition of a TMB substrate and incubation at room temperature in the dark for 30 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).