Immunological detection of osteocalcin in meat and bone meal: a novel heat stable marker for the investigation of illegal feed adulteration.
A sandwich ELISA was developed for the detection of bovine meat and bone meal (BMBM) in feed, based on polyclonal rabbit antibodies raised against the synthetic N-terminal amino acid sequence 1-9 (YLDHWLGAP) of bovine osteocalcin. To set up a sandwich ELISA pair, a commercial mouse monoclonal capture antibody binding to a highly conserved epitope in the mid-fragment of the peptide was employed. It is shown that the bone marker osteocalcin is immunologically well detectable in BMBM extracts obtained by a simple EDTA-based procedure even in a sample heated up to 145°C. Furthermore, a genus-specific restriction of the major specificity to cattle and horse was possible. The observed bi-specificity is consistent with theoretical predictions. The assay sensitivity with bovine osteocalcin of 1 ng was sufficient to enable the detection of 0.1% BMBM in compound plant feed or fish meal, for which no cross reaction was observed. In general the quantification of osteocalcin in extracts is possible using a standard curve procedure with pure bovine osteocalcin.
Federal Institute for Risk Assessment, D-14195 Berlin, Germany.
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G
Kreuz
J
J
Zagon
H
H
Broll
C
C
Bernhardt
B
B
Linke
A
A
Lampen
D000595:Amino Acid Sequence; D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D000918:Antibody Specificity; D001688:Biological Products / Q000737:chemistry; D015415:Biomarkers; D001842:Bone and Bones / Q000737:chemistry*; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D000939:Epitopes; D005060:Europe; D005506:Food Contamination; D005513:Food Inspection / Q000379:methods*; D006358:Hot Temperature; D007220:Industrial Waste / Q000032:analysis; D057230:Limit of Detection; D008461:Meat Products / Q000032:analysis*; D008462:Meat-Packing Industry; D008903:Minerals / Q000737:chemistry*; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000032:analysis* / Q000737:chemistry; D055550:Protein Stability; D016415:Sequence Alignment
D001688:Biological Products; D015415:Biomarkers; D000939:Epitopes; D007220:Industrial Waste; D008903:Minerals; D015675:Osteocalcin; C027762:bone meal