A NEW MONOCLONAL-ANTIBODY (5D3-F7) WHICH RECOGNIZES HUMAN MONOCYTE-CHEMOTACTIC PROTEIN-1 BUT NOT RELATED CHEMOKINES - DEVELOPMENT OF A SANDWICH ELISA AND IN-SITU DETECTION OF PRODUCING CELLS

Chemokines are a superfamily of structurally related cytokines involve d in leukocyte recruitment in normal and neoplastic tissues. The avail ability of non-cross-reacting reagents specific for each member of the C-C and C-X-C family is important for careful characterization of the ir in vitro and in vivo production and relevance. Here we describe a n ovel, highly specific, mAb against monocyte chemotactic protein-1 (MCP -1). The 5D3-F7 mAb (IgG1,kappa) recognizes human recombinant and natu ral MCP-1 in ELISA, immunoprecipitation and immunoblot analysis. As a source of natural MCP-1 we used the 8387 human sarcoma line which prod uces spontaneously MCP-1 and responds to TNF with increased expression and release. The 5D3-F7 mAb inhibited the chemotactic activity of MCP -1 for monocytes. Using the 5D3-F7 mAb and a polyclonal rabbit anti-MC P-1 serum, a sandwich ELISA was developed. In both the direct and the sandwich ELISA, the 5D3-F7 mAb recognized human MCP-1, but not the clo sely related C-C chemokines MCP-1, MCP-2, MCP-5, MIP-1a, and RANTES an d the C-X-C chemokines IL-8, gro alpha and NAP-2. In culture supernata nts the sensitivity of the sandwich ELISA was congruent to 30 pg/ml. T he sandwich ELISA permitted detection of MCP-1 in resting or cytokine- stimulated endothelial mesothelial and Kaposi's sarcoma cells. Prelimi nary immunohistochemical analysis revealed production of MCP-1 by macr ophage-like cells at sites of inflammation. The 5D3-F7 mAb provides a novel, highly specific reagent with which to investigate the in vitro and in vivo production and role of MCP-1.