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

Giuseppe Peri, Claudio Milanese, Cristian Matteucci, Luigi Ruco, Zhou Dan, Sozzani Silvano, Coletta Isabella, Mantovani Alberto

Research output: Contribution to journalArticlepeer-review

Abstract

Chemokines are a superfamily of structurally related cytokines involved in leukocyte recruitment in normal and neoplastic tissues. The availability of non-cross-reacting reagents specific for each member of the C-C and C-X-C family is important for careful characterization of their in vitro and in vivo production and relevance. Here we describe a novel, highly specific, mAb against monocyte chemotactic protein-1 (MCP-1). The 5D3-F7 mAb (IgG1,κ) recognizes human recombinant and natural MCP-1 in ELISA, immunoprecipitation and immunoblot analysis. As a source of natural MCP-1 we used the 8387 human sarcoma line which produces spontaneously MCP-1 and responds to INF 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-MCP-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 closely related C-C chemokines MCP-1, MCP-2, MCP-3, MIP-1α, and RANTES and the C-X-C chemokines IL-8, groα and NAP-2. In culture supernatants the sensitivity of the sandwich ELISA was {equal to or succeeds} 30 pg/ml. The sandwich ELISA permitted detection of MCP-1 in resting or cytokine-stimulated endothelial, mesothelial and Kaposi's sarcoma cells. Preliminary immunohistochemical analysis revealed production of MCP-1 by macrophage-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.

Original languageEnglish
Pages (from-to)249-257
Number of pages9
JournalJournal of Immunological Methods
Volume174
Issue number1-2
DOIs
Publication statusPublished - Sep 14 1994

Keywords

  • Chemokine
  • Chemotaxis
  • Monocyte
  • Monocyte chemotactic protein

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

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