Quantitation of secretory group V phospholipase A₂ in human tissues by sandwich enzyme-linked immunosorbent assay

We have developed a sensitive sandwich ELISA (sELISA) for quantitative determination of group V phospholipase A₂ (gVPLA₂). This assay utilizes three monoclonal antibodies (mAbs) directed against human gVPLA₂ (MCL-1B7, MCL-2A5, and MCL-3G1), which recognize specifically different epitopes of gVPLA₂. A mixture of MCL-1B7 and MCL-2A5 was used as the capture mAb, and MCL-3G1 as the detector mAb; purified human gVPLA₂ 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 gVPLA₂, which was 99.7% to 102%, and demonstration of noninterference of the gVPLA₂ 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 gVPLA₂ in cultured human epithelial cells and isolated human eosinophils was determined. Total gVPLA₂ mass in epithelial cells was 2.83±0.33 ng/10⁷ cells; gVPLA₂ was not detected in eosinophils. The presence of high concentration of gVPLA₂ 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 PLA₂s, gVPLA₂, gIIaPLA₂, gIbPLA₂ and gXPLA₂, and accurate quantitation of gVPLA₂ in biological samples.