Detection of ruminant meat and bone meal in feeds by sandwich ELISA with monoclonal antibodies

Bovine spongiform encephalopathy (BSE) is a neurodegenerative disease caused by prions, which are disease-causing forms of normal proteins. Prions are transmitted through contaminated animal feeds containing meat-and-bone meal (MBM) that originated from infected animals [5, 11, 12, 15]. MBM supplements are manufactured by autoclaved, heat-dried or pulverized residual parts of livestock. To prevent BSE transmission, the Japanese government amended The Feed Safety Law in 2001 that prohibited the addition of any animal protein, including bovine, swine or poultry MBM to feeds. Guidelines to prevent cross-contamination of feed with animal proteins urged manufacturers to separate milling equipment and production lines from equipment and lines used for non-ruminant feeds that contained proteins prohibited in feeds [8]. Legislation was revised in 2005 and permitted the addition of swine and poultry MBM in swine and poultry feeds, respectively; however, the restriction remained unchanged for ruminant feeds. The eight major policies implemented by the Japanese government since 2001 were discussed in various reviews; 1) Surveillance in farm by veterinarian, 2) Prion test at healthy 1.3 million cows/year, by veterinarian, 3) Elimination of specified risk material (SRM), 4) Ban of MBM for production, sale use, 5) Prion test for fallen stocks, 6) Transparent information and traceability, 7) New Measures, such as Food Safety Basic Law, and 8) Establish of Food Safety Commission in the Cabinet Office [3, 10, 16]. The official methods of feed analysis for animal protein contamination were reviewed by Kusama et al. [7]. The Official Method of Feed Analysis stipulates that the results of three different tests have to be taken into consideration: (1) Microscopic and morphological examinations for the presence of bone debris, (2) a polymerase chain reaction (PCR) test for the presence of a DNA sequence specific to the target animal species and (3) an enzyme-linked immunosorbent assay (ELISA) for the presence of a protein reactive to an antibody specific to the target animal species. Our group previously developed the ELISA kit, which was listed in 2003 as an Official Method of Feed Analysis. The conventional ELISA kit employed a rabbit polyclonal antibody against heat-denatured bovine serum albumin (BSA); BSA was selected as the target protein, because it is universally distributed in cow’s bodies and remains soluble even after heat-denaturation. Additionally, the amino acid sequence of BSA has significant homology with serum albumin from swine. The advantage of the conventional ELISA kit was its ability to detect bovine and swine MBM with no cross-reactivity to fish meal, chicken meal or mixed botanical feeds. The disadvantage was that the detection of milk BSA and the reaction with gelatine produced false-positive results. Kotoura et al. [6] produced hybridoma cell lines secreting monoclonal antibodies specific to bovine myoglobin and developed an ELISA for quantifying bovine meat in food, in order to reduce the risk of food-allergies. However, Kotoura’s method has been developed to determine the beef content in model processed foods and some commercial foods. The objective of this study was to adapt the ELISA system for detecting bovine MBM in feed, thereby circumventing the disadvantages of the conventional ELISA kit, which detect bovine serum albumin.