Saliva is tested at IISBR in duplicate using a commercially-available enzymatic assay kit (ELISA) from Salimetrics following manufacturer’s protocol without modification. This test employs an un-treated 96-well microtitre plate. Alpha Amylase in controls and unknown samples reacts with a 2-chloro-p-nitrophenol bound to maltitriose in an amylase substrate reagent to produce a yellow chromogen. The resultant yellow color is read at 405 nm on a spectrophotometer (PowerWave HT, BioTek Instruments). The amount of alpha amylase, as measured by the intensity of color, is directly proportional to the amount of alpha amylase present. 10ul of each sample is diluted in a series of two dilutions, resulting in a final 1:200 diluted sample to be used for testing.
Briefly, 8 µL of controls, and 1:200 diluted unknown saliva samples were pipetted in duplicate onto 96-well microtitre plates, followed by 190ul preheated amylase substrate at 37⁰C. The plates are immediately placed in the plate reader, and the optical density (OD) read at 405 nm. Two OD readings are taken at 1 minute and 3 minutes, following a period of shaking in the plate reader. Concentrations of controls and samples are calculated (Gen5, BioTek).