Generation and characterization of a neutralizing rat anti-rmTNF-alpha monoclonal antibody.
A rat anti-recombinant mouse tumour necrosis factor-alpha (rmTNF-alpha) monoclonal IgM antibody (1F3F3) with high specific binding activity for rmTNF-alpha was generated. The 1F3F3 monoclonal antibody (mAb) neutralizes the cytotoxic activity in vitro of rmTNF-alpha on L929 cells and inhibits the binding of radiolabelled rmTNF-alpha to its putative receptor on L929 cells. The 1F3F3 mAb binds to monomeric, dimeric and trimeric rmTNF-alpha and does not bind to reduced rmTNF-alpha, indicating that the recognized epitope is sensitive to denaturation. Using the 1F3F3 mAb as a capturing antibody and a biotinylated anti-rTNF-alpha as a detecting antibody, we have developed a sandwich ELISA that can specifically detect biologically active mTNF-alpha with a detection limit of 10 pg mTNF-alpha/well. This assay correlates well with the classical L929 cristal violet assay for the detection of bioactive rmTNF-alpha in biological fluids. The 1F3F3 mAb inhibits various in vitro biological activities of the rmTNF-alpha, such as the TNF-alpha-mediated tumoricidal activity of activated macrophages, the rmTNF-alpha-dependent stimulation of neutrophil degranulation and the growth-promoting effect of rmTNF-alpha. In vivo the 1F3F3 mAb inhibits lipopolysaccharide (LPS)-induced endotoxic shock. In conclusion, the 1F3F3 mAb is a useful tool to probe rmTNF-alpha activity both in vitro and in vivo.
Lucas
R
R
Unit of Cellular Immunology, Free University of Brussels, Belgium.
Heirwegh
K
K
Neirynck
A
A
Remels
L
L
Van Heuverswyn
H
H
De Baetselier
P
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000627:therapeutic use; D002460:Cell Line; D007075:Immunoglobulin M / Q000276:immunology; D008070:Lipopolysaccharides / Q000633:toxicity; D051379:Mice; D008815:Mice, Inbred Strains; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000276:immunology; D012772:Shock, Septic / Q000517:prevention & control; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha