Determination of human tumor necrosis factor alpha by a highly sensitive enzyme immunoassay

Tumor necrosis factor alpha (TNF-alpha) is a polypeptide produced primarily by monocytes and macrophages. It is involved in a wide variety of immune reactions. A simple and sensitive microplate enzyme-linked immunosorbent assay for the detection of hTNF-alpha in serum, plasma, and cell culture supernatants is described. The method is based on the use of horseradish peroxidase in biotin-streptavidin amplification system which is performed in Nunc StarWell. This system has enabled us to achieve a sensitivity of 0.1 pg hTNF-alpha/ml of the sample. The assay is calibrated to the World Health Organization (WHO) standard for hTNF-alpha (87/650). The within-run coefficient of variation ranged from 3.7 to 5.9 and the between-run coefficient of variation ranged from 8.0 to 9.9. The results obtained by the proposed method and by a commercially available kit (DRG hTNF-alpha ELISA) correlated well (n = 20, r = 0.956).