Description
Introduction: Platelet-activating factor, also known as a PAF, PAF-acether or AGEPC is a potent phospholipid activator and mediator of many leukocyte functions, including platelet aggregation, inflammation, and anaphylaxis. It is produced in response to specific stimuli by a variety of cell types, including neutrophils, basophils, platelets, and endothelial cells. PAF is biosynthesized from lysophosphatidylcholine (LPC) and acetyl CoA by the enzyme LPC acetyltransferase (LPCAT). It is degraded by a group of enzymes called PAF acetylhydrolases (PAFAHs) which are related to phospholipase A2. Several molecular species of platelet-activating factor have been identified which vary in the length of the O-alkyl side chain. Its alkyl group is connected by an ether linkage at the C1 carbon to a sixteen carbon chain. The acyl group at the C2 carbon is an acetate unit (as opposed to a fatty acid) whose short length increases the solubility of PAF, allowing it to function as a soluble signal messenger. The C3 has a phosphocholine head group, just like standard phosphatidylcholine. It is an important mediator of bronchoconstriction. It causes platelets to aggregate and blood vessels to dilate. Thus it is important to the process of hemostasis. At a concentration of 10^-12 M, PAF causes life threatening inflammation of the airways to induce asthma like symptoms.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to PAF. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for PAF 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 PAF, 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 PAF in the samples is then determined by comparing the O.D. of the samples to the standard curve