Zonulin Serum ELISA

This assay is based on the method of competitive ELISA. As a first preparation step, a biotinylated zonulin family peptides (ZFP) tracer is added to the samples, standards, and controls. Afterwards, aliquots of the treated samples, standards, and controls are transferred and incubated in microtiter plate wells coated with polyclonal anti-ZFP antibodies. During the incubation, the free target antigen in the samples competes with the biotinylated ZFP tracer for the binding of the polyclonal anti-ZFP antibodies immobilized on the microtiter plate wells. The unbound components are removed by a washing step. During a second incubation step, peroxidase-labeled streptavidin, which binds to the biotinylated ZFP tracer, is added into each microtiter well. After a washing step to remove the unbound components, the peroxidase substrate tetramethylbenzidine (TMB) is added. Finally, the enzymatic reaction is terminated by an acidic stop solution. The color changes from blue to yellow and the absorbance is measured in the photometer at 450 nm. The intensity of the yellow color is inversely proportional to the ZFP concentration in the sample; this means, high ZFP concentration in the sample reduces the concentration of the biotinylated ZFP tracer bound to the immobilized anti-ZFP antibodies and lowers the photometric signal. A dose response curve of absorbance unit (optical density, OD at 450 nm) versus concentration is generated using the values obtained from the standard. Zonulin family peptide levels in the samples are determined directly from this curve.