Study On The Methods For Detection Of Melamine In Animal Feeds And Animal Derived Foods

In this study, the hapten of melamine was synthesized and the hapten was then coupled to bovine serum albumin and ovalbumin by use of mixed anhydride method to prepare the immunogen and coating antigen. The coupling ratio of melamine to bovine serum albuin was 10:1, and that of to ovalbumin was 8:1. The immunogen was used to immunize the rabbits and the obtained serum was purified by the saturated ammonium sulfate-octylic acid precipitation method.Then, an indirect competitive ELISA method was developped for simultaneous detection of melamine and cyromazine. Optimum concentrations of coating antigen and antibody were determined by board titration as 40,000 and 1,000-fold dilution. The antibody showed high specificity to melamine, and showed a crossreactivity of 59.7% toward cyromazine. The standard inhibition curves for melamine and cyromazine were developped by using of inhibition rate as longitudinal coordinates and logarithm of concentrations as abscissa. The linear equations for melamine and cyromazine were Y=-24.938X+106.53 and Y=-23.856X+109.46, respectively. The IC50 values for melamine and cyromazine were 185 ng/mL and 310 ng/mL, and the limit of detection for melamine and cyromazine was 4.6 ng/mL and 6.5 ng/mL, respectively.After fortification of melamine in the blank feed, milk, at levels of 1-125ng/g(mL), the samples were determined by the ELISA. Recoveries for melamine were in the range of 84.5%-98.03% and 61.6 %-78.72% with coefficients of variation of 2.0%-2.9% and 4.2%-9.6%, after fortification of cyromazine in chicken, eggs at levels of 1-125ng/g(mL), the samples were determined by the ELISA. Tnd recoveries for cyromazine were in the range of 80.44-97.39% and 75.64-93.25% with coefficients of variation of 1.0%-4.9% and 1.4%-3.3%.An affinity chromatography column (IAC) was prepared based on the antibody and the conditions of loading, washing, eluting and regeneration were evaluated. The dynamic column capacity for melamine was 1250 ng per mL of gel and the absolute column capacity was 125 ng/mg IgG. The column could be used successively for 30 times with column capacity still up to 320 ng/mL per mL of gel. The IAC-HPLC procedure for determination of melamine was firstly developed with the limit of detection of 15ng/mL and the limit of quantification of 25 ng/mL, respectively. At fortified levels of 15, 30,105,and 150μg/kg, the recovery of melamine in bank blank feed, milk, chicken, eggs, were 90.7%-101.4%, 86.0%-97.3%, 84.7%-93.6% with coefficients of variation of 11.1%-16.5%, 8.8%-17.9%, 11.9%-19.7%. Furthermore, the purification performance of IAC was better than that of the conventional SPE.Therefore, the ELISA method present in this study could be used as rapid screening method and the IAC-HPLC could be used as confirmation method for the routine determination of melamine residues in animal derived products.