Quantifying Protein Levels in Saliva

Highly sensitive assay kits (R&D Systems) were used to determine protein concentrations in saliva samples. The tests were performed according to the manufacturer’s recommended protocols. The microtiter plate provided in the kits was pre-coated with a monoclonal antibody specific to the analysed protein. Standards and samples were added to the appropriate microtiter plate wells. Following incubation at room temperature, an enzyme-linked polyclonal antibody was added. Then, the microplate wells were aspirated and washed four times. Next, a substrate solution was added to each well. The enzyme–substrate reaction was terminated by addition of a stop solution and the colour change was measured spectrophotometrically at 450 ± 2 nm. The antigen concentration in the samples was determined by comparing the O.D. to the standard curve.