Description
Introduction: Cyclophilins, abbreviated Cyp, are proteins that bind to cyclosporine (cyclosporin A), an immunosuppressant which is usually used to suppress rejection after internal organ transplants. These proteins have peptidyl prolyl isomerase activity, which catalyzes the isomerization of peptide bonds from trans form to cis form at proline residues and facilitates protein folding. Cyclophilin A (CyPA) is an abundant intracellular protein that is considered to be the main target of the immunosuppressive drug cyclosporine A. Human Cyclophilin A exists in cytosol, has a beta barrel structure with two alpha helices and a beta-sheet. Other cyclophilins have similar structures to cyclophilin A. The cyclosporin A-cyclophilin A complex inhibits a calcium/calmodulin-dependent phosphatase, calcineurin, the inhibition of which is thought to suppress organ rejection by halting the production of the pro-inflammatory molecules TNF alpha and interleukin 2.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to CyPA. Standards or samples are then added to the appropriate microtiter plate wells with a biotin conjugated antibody preparation specific for CyPA and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each 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 CyPA, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of CyPA in the samples is then determined by comparing the O.D. of the samples to the standard curve