ENHANCED DETECTION OF HUMAN IL-5 IN BIOLOGICAL-FLUIDS UTILIZING MURINE MONOCLONAL-ANTIBODIES WHICH DELINEATE DISTINCT NEUTRALIZING EPITOPES

Interleukin 5 (IL-5) is a homodimeric cytokine arranged in a head-to-t ail configuration covalently linked by two disulfide bonds. IL-5 has p leiotropic effects on murine and human leukocytes and has been implica ted in the pathogenesis of many inflammatory disorders. To facilitate the study of functionally relevant IL-5 domains involved in receptor b inding and to develop a highly sensitive and specific ELISA capable of detecting IL-5 in biological fluids, a library of murine anti-human I L-5 (hIL-5) mAb was generated to baculovirus expressed recombinant hIL -5 (rhIL-5). Fifteen subclones of seven hybridomas were characterized. All. mAb bound hIL-5, but not murine IL-5 (mIL-5), and neutralized hI L-5 biological activity in the BCl1 proliferation assay. By competitiv e ELISA, the mAb were divided into two binding groups. Utilizing compa rative analysis with TRFK-5, a rat anti-mIL-5 mAb crossreactive with h IL-5, at least three hIL-5 neutralizing epitopes were defined. By ELIS A and Western analysis, each epitope was shown to be present as a conf ormationally identical pair on the hIL-5 dimer. Various combinations o f mAb in sandwich ELISA were used to predict the relative proximity of each epitope pair. Utilizing mAb binding characteristics, highly sens itive and specific sandwich ELISA were developed with a minimum detect ion limit of 6.25 pg hIL-5/ml (P < 0.05). Quantitation of hIL-5 in bot h serum and bronchoalveolar lavage (BAL) fluid demonstrated the utilit y of these anti-hIL-5 mAb for investigating the role of hIL-5 in infla mmation. These mAb should also serve as useful reagents for epitope ma pping of functional hIL-5 domains.