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A sensitive and precise ELISA has been developed for the quantitation of recombinant Tumor Necrosis Factor-Alpha (rTNF-alpha) in undiluted sera. Affinity purified rabbit antibody was used as capture antibody and mouse monoclonal antibody labelled with horseradish peroxidase was used as the second antibody in a sandwich ELISA. The assay range was from 50 to 2000 pg/ml and the relative standard deviation was 8% or less for both interassay and intra-assay precision studies. Recovery of rTNF-alpha added to 10 different human and 10 different monkey sera ranged from 81 to 102% and 100 to 120% of the expected value, respectively. This ELISA has been used to measure serum rTNF-alpha levels in over 60 patients in Phase I Clinical Trials treated with rTNF-alpha. The levels in a representative, pharmacokinetic study showed low variability between 8 patients receiving intravenous bolus administration of 100 mu rTNF-alpha/m(2). The ELISA results correlated well with TNF bioassay data with a mean specific activity of 2.5 x 10(7) U/mg.