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Species-specific detection of meat and bone meal in feed by affinity peptides identified by phage display

Project

Food and consumer protection

This project contributes to the research aim 'Food and consumer protection'. Which funding institutions are active for this aim? What are the sub-aims? Take a look:
Food and consumer protection


Project code: BfR-LMS-08-1322-524
Contract period: 01.03.2012 - 31.12.2013
Purpose of research: Experimental development

To prevent spreading of BSE (bovine spongiforme encephalopathy), feeding of Meat and Bone Meals (MBM) to farmed animals has been prohibited in the European Union (Regulation (EC) No. 999/2001). Furthermore it is forbidden to feed a species to the same species again (“cannibalism ban”; Regulation (EC) No. 1069/2009). The sole exception from the strict rule is fishmeal which so far is allowed to be used for feeding pigs and fowl but also mixed up with milk replacer for young ruminants. The official method for the detection of components of animal origin in feed is microscopy (Regulation (EC) Nr. 152/2009). However, classical light microscopy does not allow differing between different species. Alternative detection methods like e.g. polymerase chain reaction (PCR), immunologically based methods or mass spectroscopy suffer from different drawbacks respecting their specificity, selectivity or sensitivity (Fumière et al. 2009). Thus at time the existing intraspecies-feeding ban cannot be surveyed sufficiently. As a consequence there is further need for research activities to develop and validate alternative methods to fill the present gaps. The goal of the following project is the development of specific proteins which bind with high affinity to the bone marker protein osteocalcin. These proteins might be suited to be used in the same way as traditional antibodies gained by animal immunisation to establish Enzyme Linked Immunological Assays (ELISA) or to develop Dipstick Tests. To yield such “affinity peptides” (respectively peptide aptamers) the methodology of Phage Display will be applied. By this technique peptides binding specifically to a target molecule (in our case osteocalcin) will be selected and augmented in an in-vitro process. Finally an affinity peptide pair should be generated which is the basis to establish a detection system which will be comparable to an antibody-based sandwich ELISA for species specific detection of MBM in feed. Most advantageous the method will be completely independent from commercially available antibodies, respectively ELISA kits. If the approach is successful the isolation of anti-osteocalcin binding affinity peptides against species of relevance (cattle/sheep/goat, pig, chicken or fish might be tackled to develop efficient and routine suited screening methods for the simultaneous investigation of animal species in MBM and feed mixtures. The possiblility for quantitative measurement is given.

Three different heat-stable proteins with different size and ionic properties (fibrinopeptid b, osteocalcin und myoglobin) were applied in phage-display experiments with commercial M13-phage libraries (New England, Biolabs). The aim was the identification of peptid-binder (‚peptide aptamers‘) against specific processed animal proteins as target molecules in feed which could be used in the same way like antibodies in ELISA. This goal was not reached since all identified sequences obtained after biopanning were unspecific with respect to the target molecules. Possible reasons could be not enough sequenced clones, a panning design requiring further optimization but also internal aberrations (bias) of the phage libraries used in these experiments.

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Subjects

Framework programme

BMEL Frameworkprogramme 2008

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