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In this work, new analytical methods for the detection of animal proteins in animal feed and by-products are described. Due to the enforcement of legislation against transmissible spongiform encephalopathy (TSE) of the European Union and of the planned relaxation of this, it needs urgent development of species-specific detection methods, in particular to prevent the entry of bovine material in the feed chain.
The aim of this study was the development and validation of immunological methods for species-specific detection of processed animal proteins in feed as alternative methods of microscopy. Therefore, first a number of possible tissue markers of animal origin have been identified and tested for their potential suitability on the basis of sequence comparisons and literature searches. Due to the extreme stability and heat resistance, the species-specific differences in sequence and the abundant presence in meat and bone meal the bone protein osteocalcin has proved to be particularly suitable and was therefore selected as the target protein for the detection systems to be developed.
One focus of this work was the development and optimization of an enzyme-linked immunosorbent assay (ELISA), which should enable the detection of bovine material.
To limit the assay specificity on cattle, both species- and sequence-specific polyclonal antibodies have been generated against bovine osteocalcin and tested for their suitability. In combination with a commercial monoclonal antibody directed against osteocalcin a sandwich ELISA for the detection of bovine osteocalcin has been developed.
Based on the methodology of the elaborated ELISA, a real-time immuno-PCR (RT-iPCR) should be developed as another strategy for sequence-specific detection of osteocalcin.
It should be verified whether a higher sensitivity of the osteocalcin-detection compared to the developed sandwich ELISA might be achieved.
Finally, it was investigated whether an immunological detection system for bovine osteocalcin is developable using phage display methodology. Therefore, based on a sequence analysis, a specific amino acid sequence of bovine osteocalcin was chosen for the isolation of specifically binding phage. Isolated phage with positive binding affinity against bovine osteocalcin were used in an ELISA for the detection of bovine osteocalcin and the interacting peptide was characterizes by the use of DNA sequencing.
After optimization of individual work steps, the efficiency for those systems was verified. The possibilities and limitations of the developed detection methods based on reference materials and commercial samples were investigated. It could be demonstrated, that both the developed ELISA, as well as the RT-iPCR enable the detection of bovine osteocalcin with a sensitivity of 1 ng osteocalcin with distinctive specificity. For both systems a remarkable detection limit of 0.1 % bovine meat and bone meal could be determined in plant feed. Therefore a bovine meat and bone meal was used, which was even treated at 145 °C, which is above the statutory requirements. Moreover, it was demonstrated that due to the use of individual in vitro selected phage clones in a direct ELISA both pure bovine osteocalcin, as well as a high processed bovine meat and bone meal could be detected.
The results demonstrate the possibility of species-specific, selective detection of animal proteins in animal feed, based on known osteocalcin sequences. The described osteocalcin assay could be the basis for a validated detection method for ensuring the compliance of the feed ban and therefore lead to an improvement in the fight against transmissible spongiform encephalopathy. A far-reaching application of the developed ELISA method for the species-specific detection of animal proteins in feed for other species is possible.