Nephrin ELISA is an indirect competitive ELISA designed to measure nephrin in urine specimens of rodent or human origin. Nephrin, a transmembrane protein expressed in epithelial cells (podocytes) of the renal glomerulus, regulates passage of plasma proteins across the glomerular filtration barrier and may be shed into the urine in diseases affecting the glomerulus such as diabetes mellitus.
To complete the assay, standard or sample containing nephrin in the soluble phase is added to microtiter wells that contain nephrin antigen immobilized onto the solid phase, and to which rabbit anti-nephrin antibody is added in the fluid phase. The antibody binds to either the immobilized nephrin or the soluble nephrin, which compete for this binding. After a one hour incubation at room temperature, the plates are washed and a goat anti-rabbit – HRP conjugate is used to detect bound antibody. Plates are washed and only the antibody conjugate bound to the stationary phase antigen remains in the well, which is detected with a chromogenic reaction. Color intensity is inversely proportional to the concentration of nephrin in the fluid phase. Concentrations in experimental samples are determined from a standard curve.