Hemoglobin interference with an enzyme-linked immunosorbent assay for the 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 performed by using the commercially available ELISA systems Factor-Testttm hTNF-alpha and Predictatm Tumor Necrosis Factor-alpha Kit (Genzyme, Cambridge, MA) in buffered samples containing 0.00 pg ml−1 or 200 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-Testtm hTNF-alpha system. The observed effects were directly proportional to the concentration of Hb ranging from 1 to 20 mg ml−1. It appeared that in freshly spiked samples, Hb cross-reacted with monoclonal anti-TNF-alpha antibodies. When such an interaction occurred, Hb underwent a chemical reaction with hydrogen peroxide to yield a potent oxidant, capable of oxidizing the assayʹs substrates o-phenylenediamine dihydrochloride or 3,3ʹ,5,5ʹ-tetramethylbenzidine dihydrochloride hydrate. The fact that Hb might interact with other proteins and possesses catalytic, peroxidase-like activity, suggests the possibility that this molecule may mimic the action of horseradish peroxidase in peroxidase-based ELISA systems and produce false positive results. It was also found that incubation of the TNF samples with Hb outside the ELISA system may have caused its instability. Non-denaturing size-exclusion chromatography revealed that incubation of recombinant human TNF-alpha with Hb caused its partial dissociation and consequent formation of immunologically less reactive TNF monomers possibly producing false negative results.