| Malachite Green (MG) is a kind of anti-parasitic and anti-bacterial drug that has been widely used in the aquaculture for the prevention and the treatment of diseases of aquatic animals, such as water mildew, gill phthiriasis, etc, and gets good results. As the MG and its derived product Leuco Malachite Green (LMG) are of high residual, high toxic, carcinogenic, teratogenic, mutagenic and other side effects, the residue of this drug in the food has received considerable attractions, and thus the MG is prohibited in the aquaculture by many countries.Currently, the world has established the physical and chemical analysis as well as microbiological methods for the detection of MG, such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), high-performance liquid chromatography - fluorescence detection (HPL-CFLD ) and so on, However, these methods have the disadvantages of high price and low efficiency for detection, which cannot be used for large-scale screening. The competitive inhibition enzyme-linked immunosorbent assay(ciELISA) method has the advantages of celerity, nicety and low pollution, which has been widely used in the analysis of pesticide residue. The acquisition of monoclonal antibody(McAb) with high quality is the core of immunoassay. Compared with the polyclonal antibodies(PcAb), the McAb has the advantages of high specificity, good heterogeneity and easiness for standardization. The commercial ELISA for MG and LMG is available in the international market. However, the ELISA reagent for the detection of MG used in our country is largely depended on import. Therefore, this study focused on the preparation of McAb with high quality, which layed a solid foundation for the research and preparation of ELISA with independent intellectual property rights.The 6 to 8-week-old healthy Balb/c mice were immunized with synthesized Leuco-Malachite Green -bovine serum albumin(LMG -BSA). Weak immunogenicity of soluble antigen needed the addition of Freund's adjuvant, which contained complete Freund's adjuvant and incomplete Freund's adjuvant. After four times of immunization, the effect of blood serum reached above 1:10000 and the mice produced the antibody with high potency, which proved that the good immunogenicity of the synthesized LMG-BSA and enabled the body to have a strong immune response.Immuned spleen cells were syncretized with SP2 / 0 myeloma cell by taking 50% PEG to promote syncretize. After 3-5 days, the grape-shaped small collective clones of syncretized cells with different cell numbers were visible. The research results showed that the numbers of chromosome of the hybridoma cell were 99.12±3.6, the numbers of chromosome of mice spleen cells were 40, while the numbers of SP2 / 0 myeloma cells were 62.24±2.8, indicating that the 2E6 was the hybridoma cell of SP2 / 0 myeloma cells and immuned spleen cells. The syncretic rate was 86.32% and the positive rate of antibody produced by a single clone cell was 4.56%. After choosing and cloning, 6 hybridoma cells were obtained. One stable LMG single clone hybridoma cell with high potency and high specificity was picked out as 2E6. After 3 times of subcloning, clone-positive rate reached 100%. The hybridoma cell could still maintain the ability of producing antibody after many times of cryopreservation and recovery.ciELISA analysis showed that the 50% inhibitory concentration of detection of LMG was 20.39ng/mL, and the antibody potency of inducing mouse ascites reached 1:2.3×106. The ciELISA calculation results of the cross-reaction rate between 2E6 McAb with other compounds showed that the 2E6 McAb had cross-reaction with MG and LMG in the test, but had no reaction with other common prohibited hormones and antibacterial drugs in aquaculture, such as estrostilben, chloromycetin, pararosaniline, methyl orchid, tetracycline. This result showed that the McAb had good sensitivity and specificity, which layed a solid basis for the research and development of reagent kits. |
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