Zonulin Stool ELISA

This assay is based on the method of a competitive ELISA. As a first preparation step, biotinylated zonulin family peptide 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-zonulin family peptides antibodies. During the incubation, the free target antigen in the samples competes with the biotinylated zonulin family peptides for the binding of the polyclonal anti-zonulin family peptides 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 zonulin family peptides 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 zonulin family peptides concentration in the sample. A dose response curve of absorbance units (optical density, OD at 450 nm) versus concentration is generated using the values obtained from the standards.