Xd. Zhu et al., QUANTITATION OF THE CYTOSOLIC PHOSPHOLIPASE A(2) (TYPE-IV) IN ISOLATED HUMAN PERIPHERAL-BLOOD EOSINOPHILS BY SANDWICH-ELISA, Journal of immunological methods, 199(2), 1996, pp. 119-126
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 al
ternative to semiquantitative chemiluminescent assay employing immunop
recipitation/Western blot analysis. In this assay, monoclonal mouse an
ti-human cPLA, antiserum was used as the capture antibody, polyclonal
rabbit anti-human cPLA(2) antiserum as the secondary antibody, and alk
aline phosphatase-conjugated goat anti-rabbit IgG as the tertiary, rep
orter antibody. Purified human cPLA, (0-1000 ng/ml) dissolved in Tris-
HCl buffered saline was used as the standard protein. The detection li
mit 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%, respectivel
y. There was no cross-reactivity with other (secretory) isoforms of PL
A(2) (sPLA(2) types I-III) either from porcine pancreas, human synovia
l fluid, or bee venom. In separate studies, the recovery of cPLA(2) wa
s >83% when eosinophil lysate was supplemented exogenously with two di
fferent 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 dono
rs. 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 immunoblottin
g analysis.