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 cotinine. Cotinine in standards, controls and unknown samples compete with cotinine linked horseradish peroxidase (HRP) for available antibody binding sites. Bound cotinine peroxidase 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 cotinine peroxidase detected, as measured by the intensity of color, is inversely proportional to the amount of cotinine present.
Briefly, 20 µL of standard, controls, and unknown saliva are pipetted in duplicate onto 96-well microtitre plates and incubated for 1.5 hours shaking at 37⁰C with cotinine conjugated to HRP and cotinine antiserum. If a participant reports nicotine use, the saliva is diluted 10-fold prior to adding to the plate. 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 450 nm with a 620nm 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).