Monoclonal Antibody–Based Sandwich Enzyme-Linked Immunosorbent Assay for Sensitive Detection of Prohibited Ruminant Proteins in Feedstuffs

Abstract

Regulations aimed to control the epidemic of bovine spongiform encephalopathy have banned the use of certain animal products, i.e., ruminant meat and bone meals, in ruminant animal feeds. A sensitive enzyme-linked immunosorbent assay has been developed to detect prohibited bovine and ovine muscles in feedstuffs. The assay utilizes a pair of monoclonal antibodies (MAbs) against skeletal troponin I (TnI). MAb 5G9, specific to bovine and ovine TnI, was used as the capture antibody and the biotin-conjugated MAb 2G3, reacting to all heterologous TnI, was used as the detection antibody. Quantitative procedures were applied to samples containing 5, 0.5, and 0.05% (wt/wt) of heat-treated (132°C/2 bar, 2 h) bovine and ovine meat meals in three different feeds, coexisting with porcine, chicken, or turkey meat meal. The presence of these nonprohibited species did not affect the detection of bovine and ovine meat meals in the feed samples (P > 0.05). Quantitative determinations of extractable bovine and ovine TnI, with a detection limit of 5.0 and 4.0 ng/ml, respectively, were achieved when the matching feed matrixes were used in the calibration curves. This new assay provides a rapid and reliable way to detect animal protein products containing a trace amount of bovine or ovine muscle tissue in feedstuffs.