Description
Introduction: Nephrin is a protein necessary for the proper functioning of therenal filtration barrier. The renal filtration barrier consists offenestrated endothelial cells, the glomerular basementmembrane, and the podocytes of epithelial cells. Nephrin is atransmembrane protein involved with the slit diaphragm. A defectin the gene for nephrin is associated with congenital nephroticsyndrome of the Finnish type and causes massive amounts ofprotein to be leaked into the urine, or proteinuria.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated withan antibody specific to nephrin. Standards or samples are thenadded to the appropriate microtiter plate wells with abiotin-conjugated antibody preparation specific for nephrin andAvidin conjugated to Horseradish Peroxidase (HRP) is added toeach microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well.Only those wells that contain nephrin, biotin-conjugated antibodyand enzyme-conjugated Avidin will exhibit a change in color. Theenzyme-substrate reaction is terminated by the addition of asulphuric acid solution and the color change is measuredspectrophotometrically at a wavelength of 450 nm +/- 2 nm. Theconcentration of nephrin in the samples is then determined bycomparing the O.D. of the samples to the standard curve