ELSEVIER Quantitation of the cytosolic phospholipase A2 ( type IV) in isolated human peripheral blood eosinophils by sandwich-ELISA zyxwvutsrqpon JOURNAL OF lMMUNOLOGlCAL METHODS Journal of Immunological Methods 199 (1996) I 19- 126 Xiangdong Zhu, Nilda M. Mufioz, Noel Rubio, Anja Herrnreiter, Diane Mayer, Ivor Douglas, Alan R. Leff * zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM Section ofPu/monar~ and Critical Care Medicine. Department of Medicine nnd Department zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ qf Phurmacological and Physiological Sciences. Pediatrics. Anesthesia and Critical Care and Committees on Clinical Pharmncologv and Cell Physiology. Dil,ision oj’the Biological Sciences. The Unil~ersi@ of Chicago. Chicqo. IL 60637, USA Received 15 April 1996; revised 5 August 1996; accepted 5 August 1996 zyxwvutsrqponmlkjihgfedcbaZYXWV Abstract Sandwich enzyme-linked immunosorbent assay (SELLSA) was developed for precise quantitation of cytosolic phospholi- pase AZ (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-hu- man cPLA, antiserum was used as the capture antibody, polyclonal rabbit anti-human cPLAz antiserum as the secondary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG as the tertiary, reporter antibody. Purified human cPLA r (o-1000 ng/ml) dissolved in Tris-HCl buffered saline was used as the standard protein. The detection limit for cPLA, in IO’ 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, (sPLA, types I-III) either from porcine pancreas, human synovial fluid, or bee venom. In separate studies, the recovery of cPLA, was > 83% when eosinophil lysate was supplemented exogenously with two different concentrations of cPLA,. From a total protein content of 22.3 + 1.7 pg/106 cells. the baseline concentration of cPLA 2 was 0.38 + 0.18 ng/ I O6 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 cPLAZ in nanogram quantities per million cells, which has not previously been possible by immunoblotting analysis. Keyword.s: ELISA, sandwich: Cytosolic phospholipase Al: Secretory phospholipase A,; Eosinophil, human Abbreviations: BSA. bovine serum albumin: cPLA2. cytosolic phospholipase AZ; EDTA. ethylenediaminetetraacetic zyxwvutsrqponmlkjihgfe acid; &LISA. sandwich enzyme-linked immunosorbent assay; HBSS. Hanks‘ balanced salt solution; LTC,. leukotriene C,; TBS. Tris-buffered saline: PMSF, phenylmethylsulfonyl fluoride: sPLA :, secretory cytosolic phospholipase A?. * Corresponding author. At: Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC6076. University of Chicago, 5841 S. Maryland Ave.. Chicago, IL 60637. USA. Tel.: 312-702-1859; Fax: 312-702-9181. 002?- 1759/96/$15.00 Copyright 0 1996 Published by Elsevier Science B.V. All rights reserved PII SOO21- 1759(96)00 166-4