Quantitation of secretory group V phospholipase A 2 in human tissues by sandwich enzyme-linked immunosorbent assay

Abstract

We have developed a sensitive sandwich ELISA (sELISA) for quantitative determination of group V phospholipase A 2 (gVPLA 2). This assay utilizes three monoclonal antibodies (mAbs) directed against human gVPLA 2 (MCL-1B7, MCL-2A5, and MCL-3G1), which recognize specifically different epitopes of gVPLA 2. A mixture of MCL-1B7 and MCL-2A5 was used as the capture mAb, and MCL-3G1 as the detector mAb; purified human gVPLA 2 was used as the standard protein. The limit of detection of the sELISA is 2 ng/ml; the intra- and inter-coefficients of variation were 4.97±0.81% and 8.42±3.4%. The validity of the sELISA was assured by the recovery of exogenous recombinant gVPLA 2, which was 99.7% to 102%, and demonstration of noninterference of the gVPLA 2 assay by a high concentrations of other protein from murine lung and heart. To assess the usefulness of this sELISA for tissue measurements, the amount of gVPLA 2 in cultured human epithelial cells and isolated human eosinophils was determined. Total gVPLA 2 mass in epithelial cells was 2.83±0.33 ng/10 7 cells; gVPLA 2 was not detected in eosinophils. The presence of high concentration of gVPLA 2 in epithelial cells was confirmed by immunoprecipitation/Western blot analysis and by flow cytometry. This assay allows for convenient differentiation between the highly homologous 14-kDa secretory PLA 2s, gVPLA 2, gIIaPLA 2, gIbPLA 2 and gXPLA 2, and accurate quantitation of gVPLA 2 in biological samples.