Description
Introduction: Neutrophil elastase is a serine protease in the same family as chymotrypsin and has broad substrate specificity. Secreted by neutrophils during inflammation, it destroys bacteria and host tissue. As with other serine proteases it contains a charge relay system composed of the catalytic triad of histidine, aspartate, and serine residues that are dispersed throughout the primary sequence of the polypeptide but that are brought together in the three dimension conformation of the folded protein. The gene encoding neutrophil elastase consists of five exons. It is one of the two human forms of elastase. The neutrophil form of elastase is 218 amino acids long, with two asparagine-linked carbohydrate side chains and four intramolecular disulfide bridges. It is present in azurophil granules in the neutrophil cytoplasm. There appear to be two forms of neutrophil elastase, termed IIa and IIb. Neutrophil elastase is an important protease enzyme that when expressed aberrantly can cause emphysema or emphysematous changes. This involves breakdown of the lung structure and increased airspaces. Mutations of the NE gene cause severe congenital neutropenia, which is a failure of neutrophils to mature.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to NE. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for NE 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 NE, 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 NE in the samples is then determined by comparing the O.D. of the samples to the standard curve