Polymorphonuclear (PMN) Granulocyte Elastase BioAssay(TM) ELISA Kit

The human organism reacts with an inflammatory response to attacks of invading pathogens (microorganisms and viruses) or damaged tissue (after accidents or surgery). Polymorphonuclear (PMN) granulocytes play an important role as primary defense cells in this inflammatory reaction. Different bloodstream mediators (cytokines, leukotrienes, complement factors, bacterial endotoxins, clotting and fibrinolysis factors) attract and stimulate these cells to phagocytize and destroy not naturally occurring agents. PMN granulocytes use proteinases to digest these agents and tissue debris. One of these proteinases is PMN elastase which is localized in the azurophilic granules of the polymorphonuclear granulocytes. During phagocytosis of foreign substances these enzymes are also partially excreted into the extracellular surrounding, where the activity of PMN elastase is regulated by inhibitors (esp. the alpha1- proteinase inhibitor, alpha1-PI). An overwhelming release of PMN elastase, however, can exceed the inhibitory potential of the alpha1-proteinase inhibitor. Thus, enzymatically active PMN elastase, together with simultaneously produced oxidants (O2-radicals, H2O2, OH-radicals), can cause local tissue injury. Due to the bloodstream and lymphatic system, however, alpha1-PI is subsequently delivered and is eventually able to form a complex with all excreted elastase. Therefore, the concentration of the PMN elastase/alpha1-PI complex correlates with the released PMN elastase and can be used as a measure for the activity of granulocytes during an inflammatory response. Primarily, determinations of PMN elastase find its application in observation of the course of trauma, shock and sepsis. Further indications are hemodialysis, infections by obstetrics, joint diseases, effusions of sport injuries, intestinal affection, pancreatitis, cystic fibrosis and male adnex affections. Principle: P4450 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 (egg yolk) against human PMN elastase (antigen). Calibrators, controls and patient samples are pipetted into the antibody-coated microplate. During a 60 minute 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 minute 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'-Tetramethylbenzidine), is added. During a 20 minute 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 intensity 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 450nm. Bi-chromatic measurement with a 600-690nm reference filter is recommended. Kit Components: P4450A: Microtiter Strips, 1x 96 wells Coated with polyclonal antibody anti-human PMN elastase, sealed P4450B: Master Calibrator, 1x2ug PMN elastase/ alpha1-PI complex Supplied as a lyophilized powder. P4450C: Quality Control 1 (Low), 1xvial Supplied as a lyophilized powder. Mean: 29.6ng/ml; Range 23.7-35.5ng/ml P4450D: Quality Control 2 (High), 1xvial Supplied as a lyophilized powder. Mean 240.4ng/ml; Range 192.3-288.5ng/ml P4450E: Dilution Buffer, 1x50ml Ready to use P4450F: Conjugate Solution, 1x16ml Polyclonal rabbit anti-human alpha1-PI complex, enzyme labeled. Ready to use P4450G: Substrate Solution (TMB), 2x 11ml Ready to use P4450H: Stop Solution (2M HCl, 3.5%), 1x11ml Ready to use P4450J: Wash Solution Concentrate (10X), 1x75ml Storage and Stability: Store the kit at 4 degrees C. Under these conditions, assay components are stable for 6 months. Storage conditions for aliquots of standards, quality controls and samples are -20 degrees C. Shelf life of components is 30 days after opening.