Development of ELISA systems for measurement of human tumor necrosis factor-alpha (TNF-alpha )

The aim of this study was to develop a human tumor necrosis factor alpha (hTNF-alpha) ELISA system because specific and sensitive measurement of low levels of circulating TNF-alpha is very important for enlightening the immunopathologial mechanisms associated with TNF-alpha. Monoclonal antibodies 6A4c and 8A6 were produced against hTNF-alpha and were used as the capture antibody and the tracer antibody respectively for the first hTNF-alpha ELISA system (6A4c/biotin-8A6). Murine polyclonal IgG was used as the tracer antibody in the second hTNF-alpha ELISA system (6A4c/biotin-polyclonal IgG). Both systems could detect both recombinant hTNF-alpha and native hTNF-alpha. The detection limits defined as minimal concentration of hTNF-alpha were less than 4 pg/ml for the first and less than 12 pg/ml for the second ELISA systems. 6A4c/biotin-8A6 system resulted in some non-specific reactions to some extent with human sera; however, 6A4c/biotin-polyclonal IgG system produced acceptable background levels with human sera. A prominent inhibitory effect of TNF receptors-I and -II did not occur in any of the ELISA systems at physiological concentrations. Two different types of ELISA systems with high sensitivity and specificity were developed to measure hTNF-alpha level both in human serum and cell culture supernatant.

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