Simple enzyme immunoassay methods for recombinant human tumor necrosis factor α and its antibodies using a bacterial cell wall carrier

Abstract We have developed simple methods for measuring recombinant human tumor necrosis factor α (rHu-TNFα) and antibodies to rHu-TNFα in the sera of animals intravenously injected with rHu-TNFα. rHu-TNFα was measured by a competitive binding enzyme immunoassay (C-EIA) using standard rHu-TNFα, β-galactosidase labeled rHu-TNFα as enzyme-labeled antigen (E-Ag) and anti-rabbit IgG goat immunoglobulins coupled to bacterial cell walls (insolubilized second antibody). In contrast, anti-rHu-TNFα antibodies were measured by a sandwich EIA (S-EIA) using purified anti-rHu-TNFα rabbit IgG as standard, β-galactosidase labeled rHu-TNFα as E-Ag, and rHu-TNFα coupled to bacterial cell walls as insolubilized antigen. C-EIA permits the determination of serum rHu-TNFα within the range of 2–150 U/ml (about 0.7–52 ng/ml) with a CV of below 7.6% and 99% recovery. S-EIA permits the determination of anti-rHu-TNFα antibodies within the range of 70–1000 ng/ml with a CV of less than 4% and 94.8–106.9% recovery.