Description
The assay utilises the two-site sandwich technique with two selected antibodies that bind to S100A8/S100A9. Standards, controls and diluted samples which are assayed for S100A8/S100A9 are added to the microtiter wells coated with high affinity anti-S100A8/S100A9 anti-bodies. During the first incubation step, S100A8/S100A9 in the samples is bound by the immobilised antibodies. In a next incubation step, a monoclonal anti-S100A8/S100A9 antibody is added to each microtiter well. Then a peroxidase labeled anti-mouse conjugate is pipetted into each well and the following complex is formed: capture antibodies – S100A8/S100A9 – detection antibody – peroxidase conjugate. Tetramethylbenzidine is used as a substrate for peroxidase. Finally, an acidic stop solution is added to terminate the reaction. The colour changes from blue to yellow. The intensity of the yellow colour is directly proportional to the S100A8/S100A9 concentration of the sample. A dose response curve of the absorbance unit (optical density, OD at 450 nm) vs. concentration is generated, using the values obtained from the standard. S100A8/S100A9 present in the samples is determined directly from this curve