The muscle specific intermediate filament protein, desmin, was shown to be a potentially useful insoluble antigen for the production of pork specific monoclonal antibodies and in the development of poultry meat specific immunoassays. Differential extraction and chromatographic purification procedures were optimised for the recovery of desmin from smooth and skeletal muscles in forms suitable for antibody production. Desmin of sufficient purity for the production of monoclonal antibodies was obtained using differential extractions followed by an anion exchange step and then a gel filtration step. Very pure desmin necessary for the production of polyclonal antibodies was obtained using electro-elution from SDS-PAGE of desmin enriched samples prepared by differential extraction only. Although smooth muscle was a rich source for desmin it was found that pork specific monoclonal antibodies produced with this immunogen were also tissue specific in simple assay formats and thus did not react with desmin enriched extracts of pork steak. Those antibodies that did react with extracts of pork steak also reacted with similar extracts of beef skeletal muscle. As a result of these findings, improvements to the extraction and purification procedures were made and applied to the recovery of pure desmin from pork skeletal muscle. Anti-desmin antibodies were recovered from the single successful fusion performed but none were found to be pork specific. Although outside the remit of this project it was noted that the tissue specificity of some of the anti-pork desmin antibodies may suggest a hitherto unrecognised tissue dependent post-translational modification of desmin. Polyclonal sera obtained from rabbits immunised with pure smooth muscle desmin obtained from pig stomach cross-reacted extensively with similar smooth and skeletal muscle extracts of beef, lamb and chicken. Although not species specific, these sera could be used as revealing antibodies in two site assays should a pork specific antiskeletal muscle antibody be created. Immunoassays for the detection and quantitation of chicken in beef were developed using the poultry specific monoclonal antibody 4B4/B2 produced in this laboratory by Dr. Ruth Bevan. An indirect antibody capture type ELISA was developed capable of detecting 10% w/w contamination of minced beefsteak with chicken meat along with a indirect Western blot technique capable of detecting 2% w/w contamination of minced beefsteak with chicken meat
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:263939 |
| Date | January 1998 |
| Creators | Gibbons, Brian |
| Publisher | Nottingham Trent University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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