Description
Introduction: Myeloperoxidase (MPO) is a peroxidase enzyme most abundantly present in neutrophil granulocytes. It is a lysosomal protein stored in azurophilic granules of the neutrophil. MPO has a heme pigment, which causes its green color in secretions rich in neutrophils, such as pus and some forms of mucus. The 150 kDa MPO protein is a dimer consisting of two 15kDa light chains and two variable-weight glycosylated heavy chains bound to a prosthetic heme group. Three isoforms have been identified, differing only in the size of the heavy chains. It contains a calcium binding site with 7 ligands forming a pentagonal pyramid conformation. One of the ligands is the carbonyl group of Asp 96. Calcium binding is important for structure of the active site because of Asp 96's close proximity to the catalytic His95 side chain. MPO produces hypochlorous acidfrom hydrogen peroxide and chloride anion during the neutrophil's respiratory burst. It requires heme as a cofactor. Furthermore, it oxidizes tyrosine to tyrosyl radical using hydrogen peroxide as oxidizing agent. Hypochlorous acid and tyrosyl radical are cytotoxic, so they are used by the neutrophil to kill bacteria and other pathogens. Myeloperoxidase deficiency is a hereditary deficiency of the enzyme, which predisposes to immune deficiency.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to MPO. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for MPO 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 MPO, 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 MPO in the samples is then determined by comparing the O.D. of the samples to the standard curve