Development Of Enzyme-linked Immunosorbent Assay For The Detection Of Malachite Green

In this paper the immune detection methods of malachite green is studied .The content of this paper included the hapten design and synthesis,preparation and purification of polyclonal antibody, establishment of ELISA immune detection method ,Actual sample studies and verification of HPLC-MS/MS.In this study, 4-Carboxybenzaldehyde and N,N-dimethylaniline as starting materials,get carboxyl leucomalachite green by the catalysis of anhydrous ZnCl2. With two methods synthesized malachite green haptens:the first method is interacted with P-chloranil and glacial acetic acid:The second method is generated under the oxidation of PbO2 . The two haptens were conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin(BSA) to get four immunogens. Then male rabbits were immunized with four immunogens and Malachite green polyclonal antibody was prepared. Results indicated that the titer of serum is 1:64000 and the special antiserum to malachite green(1000 ?g/L) showed 93%inhibition. The antibody was Purified through Protein A-Sepharose 4B.In established icELLISA, the plate was coated with 0.1?g per well, while the antibody was diluted in 1:8000. The sensitivity of the method (middle inhibition concentration) was IC50= 2.17±0.32 ?g/L,and the detection limit (IC15) was 0.061 ±0.009 ?g/L,and the cross reactivity with crystal violet is 93%.The study selected the grass carp, carp, perch, cod, shrimp, 5 kinds of common aquatic products test the actual samples.. The limit of detection in fish samples was 2.4?g/kg. The recovery of IcELISA was 71.4%- 86.8%, and coefficient of variation was less than 12.3%.The established ELISA test results verified by HPLC-MS/MS method,the results indicated that the method established in this experiment had high accuracy and precision to meet the requirements of rapid detection.