Short communication

Enzyme-linked immunosorbent assay (ELISA) for human tumor necrosis factor (hTNF)

Immune checkpoint inhibitor (ICI) monoclonal antibody drugs are an important interface of immunology and cancer biology with the intended goal to create cancer specific treatments with less systemic toxicity. Recognition of immune-related adverse events is critical and these include significant cardiovascular toxicity and myocarditis. Compared with other immune-related events, ICI associated myocarditis is rare but is associated with high mortality. The majority of cases present early in the course of therapy and patients can rapidly progress to fulminant myocarditis. Initially, the mainstay of treatment in patients with ICI-associated myocarditis is immunosuppressive therapy with glucocorticoids. For those who do not respond to steroids, the optimal treatment is unclear. This review summarizes the potential adjunctive treatment options for patients with steroid-refractory myocarditis by illustrating a case of myocarditis that was treated with Thymoglobulin and immunoglobulin.

Tumor necrosis factor α (TNF-α) 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-α 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-α/ml of the sample. The assay is calibrated to the World Health Organization (WHO) standard for hTNF-α (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-α ELISA) correlated well (n = 20, r = 0.956).

Tumor necrosis factor-alpha (TNF-α) was demonstrated in human normal and atherosclerotic aorta, iliac and femoral arteries by immunohistochemistry using monoclonal and polyclonal antibodies. TNF was present in the cells of the arterial wall and as granular and diffuse extracellular deposits in the connective tissue matrix. Quantitative determinations of TNF by ELISA showed mean values of 21.7 ± 0.7 ng/100 mg total extracted protein in normal intima, 38.2 ± 0.5 in intimal thickenings, 25.5 ± 1.1 in fibrous plaques and 16.8 ± 0.2 ng/100 mg total extracted protein in media. Intimal thickenings presented the highest amounts of TNF with a statistically significant difference when compared to normal intima (P < 0.05) and media (P < 0.01). TNF-α concentrations in arterial eluates were about 200 times higher than in the corresponding serum samples. Western blotting analysis confirmed TNF-α eluted from the arterial wall to be about 17 kDa similar to human recombinant TNF-α. TNF-α in human atherosclerotic wall could be actively involved in the inflammatory events associated with atherosclerosis.