Volume Requirements

20 ul saliva/replicate

*Can be tested in singlet

(100ul required for testing to account for pipetting needs and sample quality)

Assay Range

0.08 - 6.6 mg/dL

Assay Sensitivity

0.08 mg/dL

Compatible Collection Methods

Passive Drool

Swab

Special Considerations

Researcher may want to exclude data from samples with a trasnferrin concentration above 1 mg/dL. This indicates a break in the blood-oral mucosa barrier, which can have an impact on accuracy of all analyte measures.

Methodology

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 transferrin. Transferrin in standards, controls and unknown samples compete with transferrin-linked horseradish peroxidase (HRP) for available antibody binding sites. Bound transferrin 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 transferrin peroxidase detected, as measured by the intensity of color, is inversely proportional to the amount of transferrin present.

Briefly, 20 µL of standard, controls, and unknown saliva are pipetted onto 96-well microtitre plates and incubated for 45 minutes shaking with antiserum and transferrin conjugated to HRP at room temperature. Plates are washed 4 times followed by addition of a TMB substrate and incubation at room temperature in the dark for 15 minutes. Following incubation, the reaction is stopped with 2M Sulfuric Acid (NH2SO4)and optical density (OD) read at 450nm. Concentrations of controls and samples are calculated from a standard curve generated using a 4-parameter non-linear regression curve fit (Gen5, BioTek).