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. 1990 Oct;71(2):218–223.

Generation and characterization of a neutralizing rat anti-rmTNF-alpha monoclonal antibody.

R Lucas 1, K Heirwegh 1, A Neirynck 1, L Remels 1, H Van Heuverswyn 1, P De Baetselier 1
PMCID: PMC1384307  PMID: 2228022

Abstract

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.

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Selected References

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