title: A peroxidase-linked enzyme immunoassay for tumour necrosis factor alpha utilising alternative colorimetric or chemilumimetric substrates.
abstract: We present a double antibody immunoassay for tumour necrosis factor alpha (TNF alpha) 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 rTNF alpha and natural TNF alpha. Concentrations of TNF beta, interleukin-1 alpha (IL-1 alpha), 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 rTNF alpha in the L929 bioassay. The assay was able to detect rTNF alpha in the presence of excess concentrations of both TNF alpha 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 TNF alpha polyclonal antiserum. The assay will be useful for the quantitation of endogenous human TNF alpha in serum, other body fluids and culture supernatants, and can also be used to monitor levels of rTNF alpha in clinical trials.
authors.affiliation: North West Immunology Service, St. Mary's Hospital, Manchester, UK.
authors.forename: W R
authors.initials: WR
authors.lastname: Lamb
authors.forename: R S
authors.initials: RS
authors.lastname: Pumphrey
authors.forename: P E
authors.initials: PE
authors.lastname: Brenchley
mesh_terms: D000293:Adolescent; D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D003124:Colorimetry; D003429:Cross Reactions; D016207:Cytokines / Q000032:analysis / Q000276:immunology; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D008297:Male; D008875:Middle Aged; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D016207:Cytokines; D014409:Tumor Necrosis Factor-alpha