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A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies directed against a synthetic peptide with an amino-acid sequence related to the C-terminus of bovine myoglobin and the whole molecule of sodium dodecyl sulphate (SDS)-denatured bovine myoglobin was adapted for detecting bovine myoglobin in contaminated feeds. The ELISA employed bovine meat extract of a known myoglobin concentration as a calibration standard and had an limit of detection (LOD) of 3.54 ng/ml and an limit of quantification (LOQ) of 11.0 ng/ml corresponding to 0.022% and 0.067% (wt/wt) bovine meat-and-bone-meal (MBM) mixed in 20-fold-diluted feed extracts, respectively. A cut-off threshold of 20.6 ng/ml bovine myoglobin was set to simplify ELISA and facilitate quick assessment of test results without a tedious calibration process. The ELISA was able to detect bovine MBM in artificially prepared model feeds, mixed botanical feeds, mixed botanical feeds with skimmed milk, fish meal, pork meal and pork/chicken meal at 0.1% (wt/wt). It was also able to detect sheep MBM in test feeds, but showed no reactivity to swine MBM, chicken MBM, skimmed milk or gelatine of bovine origin. The advantages of this method are the quick and easy extraction protocol of proteins from test feeds, using 100 mM sodium sulphide and 0.6% sodium dodecyl sulphate in the extraction solution and the effective detection of bovine and sheep MBM at 0.1% (wt/wt).

Two kinds of polyclonal antibodies against beef myoglobin were produced by immunizing rabbits with denatured myoglobin or with a peptide having the amino acid sequence unique to beef myoglobin. The latter peptide antibody reacted specifically with beef myoglobin without cross-reacting with other food proteins or with pork and chicken myoglobin. A sandwich ELISA, in which this peptide antibody was used as a capture antibody and the anti-denatured myoglobin antibody as an enzyme-labeled antibody, was also specific against beef myoglobin. When the beef content was determined in pork-or chickenbased model foods by this sandwich ELISA, the recoveries of beef protein agreed well with the actual mixing ratio of beef. Moreover, it could be confirmed by this method that the labeling of beef usage in several commercially processed foods was reasonable. Thus, a sandwich ELISA suitable for the evaluation of beef content in various foods was established.