Secretory Immunoglobulin A (SIgA)
Saliva is tested at IISBR in duplicate using a commercially-available indirect competitive enzyme immunoassay kit from Salimetrics following manufacturer’s protocol without modification. This test employs a co-incubation with diluted sample or standard with a constant concentration of monoclonal antibody (Ab) specific to SIgA conjugated with an enzyme, horseradish peroxidase (HRP). The SIgA in the sample or standard will bind with the enzyme linked antibody. The co-incubated solution is added to a 96-well microtitre plate coated with human SIgA. The unbound antibody specific to SIgA conjuagated to HRP will bind the SIgA adsorbed on the 96-well plate. The sample or standard SIgA that bound during the initial co-incubation step to the conjugated SIgA antibody is washed away resulting in the conjugated antibody remaining is bound to the plate adsorbed SIgA. The resulting bound SIgA antibody HRP conjugate 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 SIgA peroxidase detected, as measured by the intensity of color, is inversely proportional to the amount of SIgA present.
Briefly, 10 µL of saliva samples are pipetted into 40 µL of sample diluent to make a 1:5 dilution. 10 µL of the diluted samples, standards and controls are added individually to 4mL of SIgA diluent and co-incubated with SIgA antibody conjugated to HRP for 1.5 hours at room temperature. 50 µL co-incubated solution is plated in duplicate onto 96-well microtitre plates and incubated for 1.5 hours shaking constantly at room temperature. Plates are washed 6 times followed by addition of a TMB substrate and incubation at room temperature in the dark for 45 minutes. Following incubation the reaction is stopped with 2M Sulfuric Acid (NH2SO4) and optical density (OD) read at 450nm with a 490nm 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).