Evaluation of musk by enzyme-linked immunosorbent assay

We report the development of enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of a unique musk protein (MP-1) in musk samples. Musk defatted with ethyl acetate/methanol (9 : 1, v/v) was dipped in cold water and ammonium sulfate was added to the supernatant up to 85% saturation. The resulting precipitate was applied to a Bio-Gel P-100 chromatography. The fraction eluted at the void region was collected and it was consecutively purified by affinity chromatography on a DEAE Affi-Gel Blue and on anion-exchange columns containing DEAE-Sepharose CL-6B. This protein was determined to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions with an apparent molecular weight of 35000 Da and was called as musk protein-1 (MP-1). Polyclonal antibodies of MP-1 were produced by injecting it into a rabbit. These antibodies were reactive to the aqueous extract of musk and the pure antigen. The ELISA could be applied to detect nano gram quantities of the antigen in musk samples. This method made it possible to distinguish musk samples from different origins.