The Preparation Of Malachite Green Polyclonal Antibody And The Research Of Its Rapid Detection Methods

Malachite green is a kind of toxic compounds that synthesis of triphenyl methane. For itsinexpensive and easy to operate, as well as its function in the prevention and treatment of aquaticanimal fungal diseases, malachite green is widely used in aquaculture. However, clinical andexperiments reported that malachite green has high toxicity, high residue, teratogenic,carcinogenic and mutagenic effects on organs. Thus, an ELISA method that can instantly detectmalachite green has practical significance to inspect the safety of aquatic products.The study, based on the analysis of the molecular structure of malachite green andimmunogenicity, synthesized carboxylate haptens malachite green, and hapten coupled withcarrier protein to artificial malachite green antigen. This artificial antigen was used to immunerabbit to obtain polyclonal antibodies of malachite green. Purified malachite green polyclonalantibodies were used to indirect ELISA and its conditions optimization. Hence indirect ELISAmethod was established for detection rapidly of malachite green. The main research results wereas follows:1. Using chemical methods, carboxylated recessive malachite green （CLMG） wassynthesized with4-formyl benzoic acid and N, N-dimethyl aniline. Then CLMG was oxidized ascarboxylate malachite green （CMG）. By activated ester method and carbodiimide method, CMGwas coupled to artificial immunogen （CMG-BSA） and coating antigen （CMG-OVA）,respectively, and UV spectrophotometry indicated that the coupling ratio of hapten and carrierprotein were13.8:1（CMG-BSA） and17.1:1（CMG-OVA）.2. After animal immunition with immunogen four times, indirect ELISA indicated that theantiserum titer was1:2000, and antiserum IC₅₀was40.44ng/mL.3. Immune serum was purified by caprylic acid-ammonium method, SDS-PAGE identifiedthat purified polyclonal antibody with higher purity, and UV spectrophotometry showed that theantibody concentration was11.02mg/mL. The best dilution concentration of coating antigen andpurified antibody, determined by Phalanx titration method, was1:1000,1：8000.4. On the basis that mentioned above, conditions were optimized, such as the coatingsolution and its conditions, sealing solution and its condition, competition time, volume ratiobetween MG standard solution and polyclonal antibodies, as well as antibody formula. MGstandard curve of optimized conditions was y=-42.44x+71.08, R²=0.985, the half inhibitionrate (IC₅₀) and the detection limit (IC₉₀) were3.2ng/mL and0.35ng/mL, respectively. Crossreaction rates between malachite green（MG） and structural analogues that lecomalachite green （LMG）, crystal violet （CV） and leuco-crystal violet （LCV） were13.3%,100%,11.4%. Thesample recovery rate of fish and water was both more than70%, the coefficient of variation wereless than10%. The high recovery rate of the sample and high repeatability lays a foundation forapplication of malachite green ELISA test kit.