Description
Principle of the assay: The test kit is a solid phase enzyme immunometric assay (ELISA) in the microplate format, designed for the quantitative measurement of the complex of human PMN elastase and alpha1 proteinase inhibitor (alpha1-PI) in plasma. The microplate is coated with a first polyclonal antibody against human PMN elastase (antigen). Calibrators, controls and patient samples are pipetted into the antibody coated microplate. During a 60 minutes incubation present antigens in the sample bind to the antibodies fixed on the inner surface of the wells. Non-reactive sample components are removed by a washing step. Afterwards, a second polyclonal antibody against alpha1-PI, which is labeled with horseradish peroxidase, is added. During a 60 minutes incubation, the PMN elastase/alpha1-PI complex bound to the first antibody is specifically recognized by the enzyme labeled antibodies, and a sandwich complex is formed. An excess of enzyme conjugate is washed out. A chromogenic substrate, TMB (3,3',5,5'-Tetra-Methyl-Benzidine), is added. During a 20 minutes incubation, the substrate is converted to a colored endproduct (blue) by the fixed enzyme. Enzyme reaction is stopped by dispensing of hydrochloric acid as stop solution (change from blue to yellow). The color intensitiy is direct proportional to the concentration of PMN elastase present in the sample. The optical density of the color solution is measured with a microplate reader at 450 nm. Bi-chromatic measurement with a 600 - 690 nm reference filter is recommended