Detection of Tumour Necrosis Factor from Lipopolysaccharide‐Stimulated Human Mononuclear Cells by Enzyme‐Linked Immunosorbent Assay and Cytotoxicity Bioassay

Abstract

Tumour necrosis factor (TNF) or cachectin it an important mediator of endotoxic activity. To investigate the production of TNF from human mononuclear cells (MNC) in response to lipopolysaccharide (LPS), we developed a sensitive and specific enzyme immunoassay (ELISA) and a cytotoxicity bioassay for TNF. The ELISA utilizes the biotin/avidin system and includes four incubation steps. The detection limit was 25 pg recombinant TNF(rTNF)/100μl. There was no interference of medium, serum, plasma, spinal Fluid, or urine and no cross-reaction with natural or recombinants IL-1-α, IL-1-β, IL-2, IFN-γ, or lymphotoxin (TNF-β). Recovery of TNF added to the media was 83 123% (n=22). The relative standard deviation within and between assays were 7% and 8% respectively. TNF-induced cytotoxicity was measured on actinomycin-D-treated L-M mouse fibroblasts. The detection limit in this bioassay was 0.5 U/30μl or 12.5 pg/30μl of rTNF, IL-1-α and IL-1-β slightly inhibited the cytotoxic activity of rTNF. In this bioassay, cytotoxic activity (50–300 U/ml) was detected only when MNC were stimulated with high concentrations of LPS (100–1000 ng/ml). In contrast, using 0.01–100 ng/ml of LPS, the ELISA detected TNF in a dose-dependent manner (0.25 ng/ml to 40 ng/ml). It is concluded that TNF is liberated from human blood MNC if stimulated with minute amounts of LPS. It is suggested that human TNF may be secreted in a relatively inactive form or that inhibitors of TNF are generated along with the monokine Because of this, and because commonly used bioassays for TNF fail to distinguish between TNF and lymphotoxin, specific ELISA are recommended to supplement TNF bioassays.