HEMOGLOBIN INTERFERENCE WITH AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY FORTHE DETECTION OF TUMOR-NECROSIS-FACTOR-ALPHA

The purpose of this study was to evaluate the effect of hemoglobin (Hb ) on an enzyme-linked immunosorbent assay (ELISA) for the detection of tumor necrosis factor-alpha (TNF-alpha). Detection of TNF-alpha was p erformed by using the commercially available ELISA systems Factor-Test (TM) hTNF-alpha and Predicta(TM) Tumor Necrosis Factor-alpha Kit (Genz yme, Cambridge, MA) in buffered samples containing 0.00 pg ml(-1) or 2 00 pg ml(-1) of recombinant human TNF-alpha, spiked with human Hb. The results suggest that Hb interferes with the detection of TNF-alpha by ELISA. A more pronounced effect was observed with the Factor-Test(TM) hTNF-alpha system. The observed effects were directly proportional to the concentration of Hb ranging from 1 to 20 mg ml(-1). It appeared t hat in freshly spiked samples, Hb cross-reacted with monoclonal anti-T NF-alpha antibodies. When such an interaction occurred, Hb underwent a chemical reaction with hydrogen peroxide to yield a potent oxidant, c apable of oxidizing the assay's substrates o-phenylenediamine dihydroc hloride or 3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate. The fact that Hb might interact with other proteins and possesses catalyt ic, peroxidase-like activity, suggests the possibility that this molec ule may mimic the action of horseradish peroxidase in peroxidase-based ELISA systems and produce false positive results. It was also found t hat incubation of the TNF samples with Hb outside the ELISA system may have caused its instability. Non-denaturing size-exclusion chromatogr aphy revealed that incubation of recombinant human TNF-alpha with Hb c aused its partial dissociation and consequent formation of immunologic ally less reactive TNF monomers possibly producing false negative resu lts.