Saliva is tested at IISBR 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 IgG. IgG in standards, controls and unknown samples bind to available antibody binding sites on the plate. A secondary antibody linked to horseradish peroxidase is added and binds to the bound IgG from standards, controls and samples. Bound 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 IgG peroxidase detected, as measured by the intensity of color, is directly proportional to the amount of IgG present.
Briefly, 100 µL of standard, controls, and unknown, diluted saliva are pipetted onto 96-well microtitre plates and incubated for 2 hours shaking at room temperature. Plates are washed 4 times followed by addition the prepared IgG enzyme conjugated to HRP. Plate is incubated for 2 hours shaking at room temperature, followed by the addition of a TMB substrate. Plate is incubated shaking 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 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).