A PEROXIDASE-LINKED ENZYME-IMMUNOASSAY FOR TUMOR-NECROSIS-FACTOR-ALPHA UTILIZING ALTERNATIVE COLORIMETRIC OR CHEMILUMIMETRIC SUBSTRATES

We present a double antibody immunoassay for tumour necrosis factor alpha (TNFalpha) with a peroxidase dependent endpoint which can be detected by absorbance or chemiluminescence depending on the choice of substrate. The chemilumimetric and colorimetric assays have a detection threshold in human serum of 3.9 pg/ml and 7.8 pg/ml respectively and are able to recognise both rTNFalpha and natural TNFalpha. Concentrations of TNFbeta, interleukin-1alpha (IL-1alpha), IL-beta, IL-2, IL-3, IL-6 or interferon-gamma (IFN-gamma) up to 5 ng/ml failed to show any cross-reactivity. The monoclonal antibody clone 5-2, used in the assays, did not neutralise rTNFalpha in the L929 bioassay. The assay was able to detect rTNFalpha in the presence of excess concentrations of both TNFalpha receptors (p55 and p75). Removal of interference by rheumatoid factor was achieved by the absorbance of the polyclonal antiserum with mouse serum and the inclusion of 10(-2) M dithiothreitol in the buffer containing the TNFalpha polyclonal antiserum. The assay will be useful for the quantitation of endogenous human TNFalpha in serum, other body fluids and culture supernatants, and can also be used to monitor levels of rTNFalpha in clinical trials.