PMN Elastase ELISA Assay

Additional Information

Assay Background

The human organism reacts with an inflammatory response to attacks of invading pathogens (micro-organisms 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 α1-proteinase inhibitor, α1-PI). An overwhelming release of PMN elastase, however, can exceed the inhibitory potential of the α1-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, α1-PI is delivered subsequently and eventually able to form a complex with all excreted elastase. Therefore, the concentration of the PMN elastase/ α1-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 the areas of hemodialysis, infections by obstetrics, joint diseases, and effusions of sport injuries, intestinal affection, pancreatitis, cystic fibrosis and male adnex affections.

Assay Procedure

1. 1. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it.
   2. Add 100 μl of standards, controls and samples in duplicate into wells.
   3. Cover the wells and incubate for 1 hour at RT.
   4. Aspirate each well and wash, repeating the process 3 times for a total 4 washes. Wash by filling each well with 1× Wash Buffer (350 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating, decanting or blotting against clean paper towels.
   5. Add 150 μl of Enzyme-conjugated Antibody to each well. Cover wells and incubate for 1 hour at RT.
   6. Aspirate each well and wash as step 4.
   7. Add 200 μl of TMB Reagent to each well. Incubate for 20 minutes at room temperature in dark.
   8. Add 50 μl of Stop Solution to each well.
   9. Read the OD with a microplate reader at 450 nm immediately.