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 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,kappa) 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 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-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-1 alpha, and RANTES and the C-X-C chemokines IL-8, gro alpha and NAP-2. In culture supernatants the sensitivity of the sandwich ELISA was approximately equal to 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.
Peri
G
G
Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
Milanese
C
C
Matteucci
C
C
Ruco
L
L
Zhou
D
D
Sozzani
S
S
Coletta
I
I
Mantovani
A
A
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D018932:Chemokine CCL2; D002630:Chemotactic Factors / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007124:Immunoenzyme Techniques; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D018932:Chemokine CCL2; D002630:Chemotactic Factors; D011994:Recombinant Proteins