Preparation Of Monoclonal Antibodies Against Malachite Green And Development Of An ELISA Method

Malachite green (MG), a triphenylmethane compound, is used for paper, leather, silk, wool, ceramic staining and for cell and tissue stains in biology, it was also used as food additive in the past. MG was used in aquaculture industry in the1930s and has effects on the fungus, mold and parasites in vivo. In addition, during transport or market’s temporary storage process it is the preservative to prevent fungal infections and death after collision and to keep fish bright color. However, MG has the characteristics of teratogenic, carcinogenic and mutagenic, its main metabolite is Leucomalachite green (LMG), a fat-soluble substance and having strong biological toxicity and long residual time. Many countries and regions have banned the use of MG in every link of aquaculture, our country also rules MG and its metabolites should not appear in any animal product.Now methods for detection of MG and its metabolite residues mainly have high performance liquid chromatographic method (HPLC), chromatography and other technical combination assay, enzyme-linked immune technology(ELISA), colloidal gold immunoassays (CGIA), Raman spectroscopy, electrochemical method, fluorescence resonance scattering method and molecular imprinting method, etc. Although some methods can be used for detection of MG residue and can satisfy the state testing standards, but they have the disadvantage of complicated sample preparation, high cost, long detection time and the operators of higher professional and technical requirements to can not get and be not suitable for the actual production of MG and its metabolite residues detection in the life. The ELISA method is operation simple, low cost, strong specificity, high efficient and suitable for rapid detection of large quantities of samples, easy to be popularized.This research is based on the preparation of monoclonal antibody and enzyme-linked immunosorbent detection technology, we synthesized artificial antigen by small molecule and protein connection technology, prepared the monoclonal antibody for MG and its main metabolites of LMG and established the ELISA detection method of MG and LMG residual.1. Synthesis and identification of LMG artificial antigens. Synthesize immunogen LMG-BSA by structure modification and diazotization and coating antigen LMG-OVA through the chemical reaction and EDC method. We judged synthesis of artificial antigens successful by UV scanning absorption peak shifts and immunological method. The protein concentrations of LMG-BSA and LMG-OVA were2.4mg/mL and2.0mg/mL by CBB method.2. Preparation of the anti-LMG monoclonal antibodies. Female BALB/c mice were immunized with LMG-BSA as routine immunization, after the fifth immunization the titer of blood serum reached1:30,000. Take the spleen cells of immunized mice fusion with SP2/0on the third day after the strengthened immune. We used HAT medium and enzyme-linked immune technology to screening positive hybridoma. By limited dilution method we got3strains hybridoma cells of secreting anti-LMG and anti-MG monoclonal antibodies, they were named1B11,1E5and4B7. The titers of1B11antibodies and1E5antibodies were1:16,000and1:32,000, and the protein concentrations were31.69mg/mL and28.89mg/mL.3. The establishment of ELISA detection method. Use1E5hybridoma cells to product monoclonal antibodies and establish indirect competitive ELISA detection method of MG and LMG remaining. The best dilution concentration of coating antigen is8,000, and the best working concentration of antibody is1:16,000. Draw LMG inhibition standard curve, with the linear equation:y=0.4586x-0.1801(R2=0.9954), IC5030.4ng/mL, LOD2.5ng/mL, LOQ5.17ng/mL. The cross experiment shows that the monoclonal antibody has100%inhibition rate for LMG, the cross reaction rate of MG was71.4%, and it has no cross reaction with Pararosaniline, Methyl blue, Crystal violet, Chloramphenicol and Rhodamine B.4. Simulation experiment. Obtain the residual LMG samples by simulating the use of MG in actual production and set up blank group meanwhile. Disposed the sample, the interference of sample matrix mainly disappeared when the sample solution diluted6times. Within5～200ng/mL we established CiELISA standard curve with linear equationfor y=0.4628x-0.191(R2=0.9947), the LMG content in the experimental sample was0.501mg/kg.