Immunodetection of Bovine Central Nervous System Tissue in Processed Products

The infectivity of bovine spongiform encephalopathy is mainly associated with bovine central nervous system (CNS) tissue, which has been banned from food and feed chain worldwide. Current methods for the detection of CNS tissue are not effective in processed meat and feedstuffs due to lacking of a suitable thermostable biomarker in CNS. Myelin basic protein (MBP), the major CNS myelin protein, was reported as thermostable. The overall goal of this research was to develop an enzyme immunoassay for the detection of bovine CNS tissue in processed meat and feedstuffs using MBP as the marker in CNS tissue. The specific objectives were to: a) study the stability of bovine MBP under various experimental conditions; b) develop an improved method for MBP extraction; and c) develop a reliable MBP-specific enzyme-linked immunosorbent assay (ELISA) for the detection of bovine CNS tissue in meat and feedstuffs. To study the stability of bovine MBP, the 18.5-kDa MBP was purified from bovine brain by cation exchange chromatography. The effects of pH (3.0, 7.2 and 10.5), temperature (unheated and heated [100°C, 121°C, and 133°C for 30 min]) and storage time (0 – 19 days at 4°C) on the stability of bovine MBP were investigated by using indirect non-competitive ELISA, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Results show that bovine MBP was most stable at pH 7.2. MBP started to degrade after heating at 121°C for 30 min at pH 7.2. Storage up to 19 days at 4°C did not significantly affect the immunoreactivity of unheated and heated samples (pH 7.2). No degradation was observed in heated samples over the storage time. To improve the extraction efficiency of the target analyte (MBP), two different buffers (10 mM PBS and 20 mM Tris) at neutral pH containing different salt concentrations (150 mM – 1 M) were used to extract proteins from unheated and heated (100°C for 30 min) bovine brain. The mixtures were extracted with different time periods (5 – 180 min) at room temperature. Results from indirect non-competitive ELISA, SDS-PAGE and WB show that several factors (buffer type, salt valence and concentration, incubation time, and heat treatment) affected the MBP concentration in the extracts. The optimal method for MBP extraction was using 20 mM Tris-HCl containing 600 mM MgCl2 (pH 7.4) to extract heated (100°C for 30 min) CNS samples for 1 h at room temperature. With this sample extraction method, the detection limit of an indirect competitive ELISA was improved from reported 10% to 0.1% (g/g) of bovine brain tissue. Subsequently an anti-bovine MBP antibody (Ab3E3) along with a new MBP-specific ELISA was developed to further improve the detectability of CNS tissue in processed meat and feed samples. Ab3E3 was produced against bovine 18.5-kDa MBP using hybridoma technique. Ab3E3 was characterized using indirect ELISAs (competitive and non-competitive) and WB. Results from the epitope mapping against polypeptide segments of the 18.5-kDa MBP and dot blot show Ab3E3 to be a pool of monoclonal antibodies, which confers a strong binding of the antigen. The optimized indirect competitive ELISA employing Ab3E3 was ruminant CNS-selective without cross-reactivity with meat (beef and pork) and soy proteins. The five-parameter logistic model was used to establish the assay standard curve. This assay had low intra-assay (%CV [coefficient of variation] ≤ 3.5) and inter-assay variability (%CV ≤ 3.3); and low detection limits for bovine MBP (6.4 ppb) and CNS tissue spiked in both meat (0.05%, g/g) and feed (0.025%, g/g) samples. This assay is therefore suitable for the quantitative detection of low levels of contaminated ruminant CNS proteins in processed food and feed products. This research offers an efficient tool not only for improving the safety of animal feed, but also for enforcing the food labeling laws.