ENHANCED DETECTION OF HUMAN IL-5 IN BIOLOGICAL FLUIDS UTILIZING MURINE MONOCLONAL ANTIBODIES WHICH DELINEATE DISTINCT NEUTRALIZING EPITOPES Richard R. Dickason,i Marilyn M. Huston,r David P. Hustonr,* Interleukin 5 (IL-S) is a homodimeric cytokine arranged in a head-to-tail configuration covalently linked by two disulfide bonds. IL-5 has pleiotropic effects on murine and human leukocytes and has been implicated in the pathogenesis of many inflammatory disorders. To facilitate the study of functionally relevant IL-5 domains involved in receptor binding and to develop a highly sensitive and specific ELISA capable of detecting IL-5 in biological fluids, a library of murine anti-human IL-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 hIL-5 biological activity in the BClr proliferation assay. By competitive ELISA, the mAb were divided into two binding groups. Utilizing comparative’analysis with TRFK-5, a rat anti-mIL-5 mAb crossreactive with hIL-5, at least three hIL-5 neutralizing epitopes were defined. By ELISA and Western analysis, each epitope was shown to be present as a conformationally identical pair on the hIL-5 dimer. Various combinations of mAb in sandwich ELISA were used to predict the relative proximity of each epitope pair. Utilizing mAb binding characteristics, highly sensi- tive and specific sandwich ELISA were developed with a minimum detection limit of 6.25 pg hILJ/ml (P < 0.05). Quantitation of hIL-5 in both serum and bronchoalveolar lavage (BAL) fluid demonstrated the utility of these anti-hIL-5 mAb for investigating the role of hIL-5 in inflammation. These mAb should also serve as useful reagents for epitope mapping of functional hIL-5 domains. IL-5 is a homodimeric cytokine secreted predomi- nantly by activated Th2 lymphocytes.1-4 The homo- dimer is covalently linked by two disulfide bonds in a head-to-tail configuration.5-7 Dimer formation is essential for biological activity as monomeric IL-5 does not bind to its receptor.5,6,s’9 IL-5 is hetero- geneously glycosylated with both N- and O-linked residues. Although these residues may provide some thermal stability,’ they are not required for biological activity. “,il Murine IL-5 and hIL-5 share 73% amino From the Departments of ‘Microbiology and Immunology and 2Medicine, Baylor College of Medicine, Houston, Texas 77030, USA. Correspondence to: David P. Huston, MD, Dept. of Medicine, Immunology Section, Baylor College of Medicine, 6565 Fannin, M.S. F501 Houston, TX 77030, USA. Received 28 December 1993; revised and accepted for publication 7 May 1994 @ 1994 Academic Press Limited 1043-4666/94/060647+ 10 $08.00/O KEY WORDS: conformational epitopelhuman interleukin 51 monoclonal antibody/sandwich ELISA/Western CYTOKINE, Vol. 6, No. 6 (November), 1994: pp 647-656 acid homology,i2 which enables cross-species reacti- vity in various bioassays. However, while hIL-5 and mIL-5 have comparable activity in human cell assays, mIL-5 is greater than loo-fold more potent in murine cell assays.i3 Using murine/human chimeric con- structs of IL-5, species specific activity has been local- ized to the C-terminal 36 amino acid residues.i2 Recently, hIL-5 crystal structure was solved revealing two four-a-helix bundle motifs situated about a two- fold axis of symmetry.14 Although a single four-ol- helix bundle motif is characteristic of many cyto- kines,i4 IL-5 contains two identical motifs. Even more unique, each IL-5 bundle is composed of helices from both chains. IL-5 has been well characterized for its effects on growth, differentation, and activation of murine B- cells I5 Its effects on human B-cells remains contro- versial. 16-i9 IL-5 has also been shown to enhance IL-2 induced differentiation of human and murine thy- mocytes to cytotoxic T-cells,z0,21 augment human IL-2-dependent T-cell proliferation2’ and enhance IL-2 mediated murine lymphokine-activated killer 647