Quantitation of human tissue and immune cell type II 14 kDa phospholipase A2 by enzyme immunoassay

Abstract

The metabolism of arachidonic acid into inflammatory mediators (e.g. prostaglandin, leukotrienes) is dependent upon the rate-limiting enzyme phospholipase A(2). Localization and quantification of type II 14 kDa phospholipase A(2) (PLA(2)) in cells or tissue preparations has historically been accomplished through activity measurements, a process that can provide variable results due to interference by exogenous substances with hydrolysis assessment. Others have reported on the use of sandwich enzyme immunoassays (EIA) to measure 14 kDa PLA(2) by mass in serum and exudate fluids, e.g. synovial fluid. Herein, we report the utilization of a human recombinant type II 14 kDa PLA(2) sandwich EIA to directly measure cell or tissue-residing 14 kDa PLA(2). It is known that type II 14 kDa PLA(2) resists acid treatment, and this technique was applied to cell fractions which liberated the enzyme from cellular membrane components prior to quantitation by EIA. Two human immune cell populations were assessed and shown to contain measurable levels of 14 kDa PLA(2). Neutrophil or monocyte cytosolic fractions contained no measurable levels whereas the respective 100 000g particulate fractions contained 2.6+/-0.8 pg (neutrophil) and 2.1+/-0.6 pg (monocyte) 14 kDa PLA(2)/mug protein. Human placenta cytosolic fractions contained no measurable levels while 100 000g particulate contained approximately 25 ng 14 kDa PLA(2)/mg protein. This EIA, in conjunction with acid extraction, provides an easy and reproducible assay to identify and quantify this enzyme in cells and whole tissues, expanding our ability to study the relationship of this enzyme to inflammatory processes.