Quantitation of the cytosolic phospholipase A(2) (type IV) in isolated human peripheral blood eosinophils by sandwich-ELISA

Sandwich enzyme-linked immunosorbent assay (sELISA) was developed for precise quantitation of cytosolic phospholipase A(2) (cPLA(2) type IV) concentration in isolated human peripheral blood eosinophils as an alternative to semiquantitative chemiluminescent assay employing immunoprecipitation/Western blot analysis. In this assay, monoclonal mouse anti-human cPLA, antiserum was used as the capture antibody, polyclonal rabbit anti-human cPLA(2) antiserum as the secondary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG as the tertiary, reporter antibody. Purified human cPLA, (0-1000 ng/ml) dissolved in Tris-HCl buffered saline was used as the standard protein. The detection limit for cPLA(2) in 10(6) eosinophils was 0.109 ng/ml, and coefficients of inter- and intra-assay variation were 4.23% and 7.07%, respectively. There was no cross-reactivity with other (secretory) isoforms of PLA(2) (sPLA(2) types I-III) either from porcine pancreas, human synovial fluid, or bee venom. In separate studies, the recovery of cPLA(2) was >83% when eosinophil lysate was supplemented exogenously with two different concentrations of cPLA,. From a total protein content of 22.3+/-1.7 mu g/10(6) cells, the baseline concentration of cPLA(2) was 0.38+/-0.18 ng/10(6) cells in eosinophils obtained from mildly atopic donors. Immunoblotting studies confirmed the complete specificity for the type IV isoform as detected by sELISA. This sELISA method permits the precise quantitative assessment of cPLA(2) in nanogram quantities per million cells, which has not previously been possible by immunoblotting analysis.