The use of desmin as an insoluble antigen in the immunochemical identification of pork and chicken meats.
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