Development Of The ELISA Kit For Rapid Detection Of Melamina

Food safety has already become the global focus of attention. In China, melamine has beenone of the conventional detection items for milk and related products. Melamine is a kind oftriazine kind of nitrogen heterocyclic organic chemical intermediates, due to low price, widelyused in building materials and paints, unscrupulous businessmen because its nitrogen content ishigher in molecular, illegally added to food, increase the nitrogen content.In recent ten years, alot ofnews about food poisoning resulting from melamine residues in food has been reported.Enzyme-linked immunosorbent assay （ELISA） must be the convenient enzyme immunoassaymethods for large samples. But now, mainly depending on expensive foreign kits hinder theapplication of the methods, so it is urgent to develop ELISA fast kit for clenbuterol with ownindependent property right.Based on the analysis of melamine, the study, on the basis of immunogenicity molecularand it’s structure, through two ways: one is the modified melamine analogues synthesis carboxylstructure melamine hapten, melamine hapten with carrier protein accidentally into artificialantigen. Two is due to the melamine has amino directly with carrier protein accidentally unitedinto artificial antigen of melamine. Synthesis with ultraviolet identification of artificial antigens,combined with high than the artificial antigen of immune rabbit polyclonal antibody formelamine, melamine polyclonal antibody to purified for direct ELISA and its conditions wereoptimized, thus establish a direct ELISA method of rapid detection of melamine, kit and kitvalidation, the main research results are as follows:1. Immunogen using various chemical methods of synthesis.2-Chloro-4,6-diamino-1,3,5-triazine will be synthetic carboxylic MEA, formic acid and chlorine by activated estermethod coupling carboxyl butyl ester method, MEA immunized original MEA-BSA and thepackage is the original （MEA-OVA）.Using melamine amine by glutaraldehyde method directlywith BSA synthesis of artificial antigen （MEL-BSA1and MEL-BSA₂）. By the simpledetermination of EDC method of hapten with carrier protein MEL-BSA coupling ratio was29.35:1. Glutaraldehyde was prepared with melamine artificial immunogen coupling ratio of:Though the EDC was29.35:1; MEL-BSA1was7.25:1; MEL-BSA₂was9.06:1ï¼› Aftertransforming synthesis of melamine artificial immunogen coupling ratio: MEA-BSA was29.63:1.2. After animal immunition with immunogen four times, indirect ELISA indicated that theantiserum titer was1:2000, and antiserum IC₅₀was45.92ng/mL. 3. Using immune polyclonal antibody affinity chromatography for purification, throughultraviolet spectrophotometry of purified polyclonal antibody concentration is8.76mg/mL.4. On the basis of the selection of package is liquid, package is condition, selection ofsealing solution, closed condition, competition time, optimized, the optimized conditionsre-established after MEL standard curve, standard curve equation Y=-46.77X+97.27, R²=0.997.IC₅₀of calculation of MEL is10.24ng/mL.5. To assemble kits and the kits of the assessment of accuracy and precision conform to therequirements, and use HPLC to make sure this methods.