| 3-(4,6-diamino-1,3,5-triazin-2-ylthio) propionic acid was Synthesized as the hapten of melamine in the study. The hapten was respectively conjugated to carrier protein bovine serum albumin (BSA) and egg ovalbumin (OVA) to generate immunizing antigen and coating antigen. And the preparation of high-titer anti-melamine serum was achieved by immunizing animals with the artificial immunogen. The serum was purified after confirmatory and competitive indirect ELISA methord was established to detecte melamine. The antiserum was coupled to CNBr-activated Sepharose4B to prepare the immunoaffinity chromatography column. The capacity and recovery rate of melamine immunoaffinity chromatography column were evaluated preliminarily and IAC-HPLC which detect melamine residues in animal-origin food was established. The results are as follows.1Preparation of melamine Cyanamid specificity antibodyAs a kind of Melamine structural analogue,2-chloro-4,6-diamino-1,3,5-triazine was reacted with3-mercaptopropionic acid under alkaline conditions to get hapten3-(4,6-diamino-1,3,5-triazin-2-ylthio) propionic acid. The production was identified by the infrared spectrum and mass spectrometric assay and the results showed the reaction was successful. The hapten was respectively conjugated to Bovine Serum Protein (BSA), ovalbumin (OVA) to prepare immunizing antigen Mel-BSA and coating antigen Mel-OVA and they were identified by UV spectroscopy. The result was successful and the hapten binding ratio were12.6:1and6.5:1.2. Establishment of competitive indirect enzye-linked immunosorbent assay.Mel-BSA was used as the immunogen to immunize rabbits of the experiment to prepare the anti-melamine serum. With the coating antigen Mel-OVA, indirect ELISA was developed to detect the titer of antiserum and the titer is1:23×103. The bset working concentration of coating antigen and the antiserum were1μg/ml and1:2000. With anti-melamine serum, the indirect competitive ELISA standard curve of melamine was established. The linear regression equation of the standard curve was y=-0.3494x+1.1872. The50%inhibition concentration was92.64ng/mL. The limit of detection was3.89ng/mL. The linear range was3.89～500ng/mL.Its sensitivity can meet the requirements of the current detection of melamine. The specificity of the antibody was detected, cyanuric acid, tetracycline, ammelid,ammeline and ampicillin did not have cross reaction except cyromazine, indicating good specificity of the antiserum. The detection precision of the method meet the requirements of detection and the coefficient of variation was less than5.23%in intra-assay and10.13%in inter-assay. Adding melamine in the range of10～400ng/ml, the recovery of melamine in milk and eggs separately reached78.60%～92.38%and79.43～90.82%and reached the accuracy requirements of ELISA method.3. IAC-HPLC detection method for melamine residues in food of animal originThe prepared melamine-specific antiserum was purified by saturated ammonium sulfate method. The purified protein was coupled with CNBr-activated Sepharose4B and the coupling rate reached90.8%. The melamine-specific IAC column was prepared and evaluated by HPLC. Its melamine dynamic column capacity was1046.3±17.2ng/mL and the absolute column capacity was121.5ng/mg IgG. IAC column was used as sample purification meathod in IAC-HPLC detecting melamine. In the range of50-5000ng/ml, melamine working standard solution had the good linear relationship (R2>0.999). The limit of detection of melamine in milk and egg samples was20ng/ml, and the limit of quantification was30ng/ml. Adding the concentration of of30,400,1000ng/mL melamine in milk and egg, the recovery rated from86.92～95.29%and82.83～95.82%. |
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