Enhanced immunodetection of bovine central nervous tissue using an improved extraction method

- MBP was effectively extracted with the buffers containing divalent salt.
- The concentration of MBP extracted increased with the increase of salt concentration.
- The MBP concentration was higher in heated than unheated samples.
- The LOD of an immunoassay was greatly improved using the improved extraction method.

The infectivity of bovine spongiform encephalopathy (BSE) was mainly associated with bovine central nervous tissue (CNT), which have been banned from food and feed chain in many countries. Detection of bovine CNT is important for the surveillance of BSE occurrence. Myelin basic protein (MBP) has been identified as a thermal-stable maker protein in CNT for the detection of CNT in processed meat and feedstuffs. This study reports an improved extraction method of MBP that substantially enhanced the detectability of bovine CNT.Two different buffers (10 mM phosphate buffered saline 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 (30-180 min) at room temperature. Results from indirect non-competitive enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot show that several factors (buffer type, salt valence and concentration, incubation time, and heat treatment) affected the MBP concentration in the extracts. The concentration of MBP increased with the increase of salt concentration in all extracts, but decreased with the increase of the incubation time in unheated samples. MBP was more effectively extracted with the buffers containing divalent salt (Ca2+ or Mg2+) than that of monovalent salt (Na+). The MBP concentration was higher in heated than unheated samples. 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) CNT samples for 1 h at room temperature. With this sample extraction method, the detection limit of an indirect competitive ELISA can be significantly improved from reported 10%-0.05% (g/g) of bovine brain tissue.