Establishment And Application Of Rapid Detection Technology To Determinate The Residues Of Leuco Malachite Green

Malachite Green（MG）, chemical name is four methyl generation two amino threebenzene methane, molecular formula is C23H25ClN2, belongs to three benzene methanekind of dyes. In the aquatic animals’body, MG will be metabolic into leucomalachitegreen（LMG）. As fishery drugs, MG has a very strong toxic side. In the water, MG cansolute into lots of zinc（Zn）,which can cause aquatic animals including edible fishes acutezinc poisoning, also cause gills organization and skin epithelium of aquatic fish mildinflammation. The toxicity test of mammals which is feeding LMG to mouse for104weeks shows that tumor in the mouse liver tissue increases significantly. The otheranimals’ experiment shows that MG can cause lots of viscera and tissue toxication.Therefore, MG and LMG become into great food safety problems. Now, MG has beenlisted as a disabled fishery drug in China. In1992, Canada has prohibited MG and LMG asfisheries fungicide; The United States government gives the strict regulation, banne LMGdetecting in edible aquatic products;In June2002, the European Union governmentpromulgated the related laws, banned using MG in any fish farms. In May2002, theChinese government listed MG in<Food animal bans veterinary medicine and itscompound list>, banned MG in all food animal breeding and transportation. At present,methods of testing for LMG mainly include thin layer chromatography （TLC）,spectrophotometry, high performance liquid chromatography （HPLC）, etc. However, thesemethods are complex to operate, need professional technology personnel, and its operationinstruments are expensive, which are not suitable for the large-scale testing. The rapiddetection method basing on immunological method is rapid, sensitive, price cheap, suitablefor the large-scale testing. It is recognized as one of the most suitable method for screeningdrug residue. Therefore, researching and developing the rapid, sensitive has a veryimportant practical significance and technological value,which is a rapid, sensitive, and noneeds for any instrument can be used as an important early screening means to field testMG and LMG drug residues. This experiment synthesizes the artificial antigens of LMG,immunes Balb/c mouse with it. Collect the spleen cells of the immunity mouse and use toprepare a monoclonal antibody that can detect LMG. using the specificity monoclonalantibody prepareFirst of all, using mixed acid nitrification reduction method to transform LMG, getamino LMG （NH₂-LMG）; At the same time, using the analogue----parafuchsin （PA）, thenusing improved carbodiimide method to prepare the two different LMG complete antigensLMG-BSA and PA-BSA. Through the ultraviolet spectrophotometer scanning, SDS-PAGE gel electrophoresis, mass spectrum analysis to evaluate LMG complete antigen LMG-BSAand PA-BSA. The results show that: the ultraviolet scan had discovered drift of the peak;mass spectrogram of the PA-BSA coupling material show there is a polymer compoundwhose molecular weight is78180.With compare the molecular weight of BSA and PA, wecan consider the coupling as success and the coupling ratio is about37:1, LMG-BSAcoupling material show there is a polymer compound whose molecular weight is68759.With compare the molecular weight of BSA and NH2-LMG, we can consider the couplingas success and the coupling ratio is about7:1. Using LMG-BSA and PA-BSA to immunemouse can get serum whose titer is over105. This shows that LMG-BSA and PA-BSAcoupling objects can be used for preparation of MG and LMG artificial antigens.UsingLMG-BSA and PA-BSA coupling objects to immune5mice, obtain the polyclonaldntibody. Respectively use LMG-OVA and PA-BSA as coating to do the serum titerdetermination. The results show that each antibody serum titer is higher than105. In theexperiment, select BALB/C mice cells whose serum titer is the highest to do cell fusion.Using to do fusion of spleen and SP2/0myeloma. Then use the indirect ELISA and indirectcompetition ELISA to screening positive cloned hybridoma cells, and use limited dilutionmethod to do three times sub-cloning, final screening four hybridoma cells strains that canstably secrete specific monoclonal antibody, which respectively are D5, E9B8and H-6.Prepare a great quantity monoclonal antibody in ascites, through the saturated ammoniumpersulfate method for purification monoclonal antibodies. characteristics of monoclonalantibodies identification results show that: affinity of four monoclonal antibodies strainscan reach to10⁸L/mol, all belong to high affinity monoclonal antibodies, throughestablishing standard curve, minimum detection limit of four monoclonal antibodies strainsD5, E9B8H6respectively are3.98ug/L,5.61ug/L,4.62ug/L and12.04ug/L, all belongto a high sensitive antibody; At the same time monoclonal antibody specificity is good, allcan be used in development of testing aquatic products Enzyme league immune test kit todetect LMG.This experiment used sodium periodate method to mark on horseradish peroxidase ofthe synthetic complete artificial antigens PA-BSA, successfully prepare PA-BSA markedby horseradish peroxidase, through the uv spectrophotometer and ELISA, identify HRPlabelledantigen. The identification results show HRP labelledantigen is in good effect, canbe used for ELISA kit establishment. Base on the horseradish peroxidase markedcompletely antigen, successful established the enzyme league immune detection kit, andmeasured the detection performance. At the condition of laboratory processing, thisenzyme league immune detection kit minimum detection limit is370ng/L, which can be used in residue detection of LMG in aquatic products.In summary, this Enzyme league immune test kit to detect LMG which is preparethough the experiment can be used for field residues detection of LMG in aquatic products.