An enzyme linked immunosorbent assay for the determination of cyromazine and melamine residues in animal muscle tissues

Cyromazine and its metabolite melamine are toxic to animal and human. Herein this paper, an indirect competitive enzyme linked immunosorbent assay (ELISA) was first developed for the simultaneous determination of cyromazine and melamine in animal meats. The antigen of melamine was prepared by using of mixed anhydride method and the polyclonal antibody was obtained by immunising three rabbits with the conjugate. The antibody obtained was specific for melamine and showed 59% of crossreactivity toward cyromazine. Muscle samples were extracted with ammoniacal acetonitrile and purified by passing through a Millipore filter before ELISA analysis. The limits of detection for cyromazine and melamine were 4.5 and 1.8 ng/g, respectively. Recoveries for cyromazine and melamine from the fortified blank samples at fortification levels of 20, 50 and 100 ng/g were in the range of 87.0-98.6% and 84.6-94.3%, respectively, with coefficients of variation (CV) lower than 12%. The ELISA method was used for detection of the two analytes in some unknown samples and the results were further confirmed with a HPLC-UV method.