Preparation Of Monoclonal Antibody And Kits For Mono-methyl Phthalate

Phthalate esters (PAEs) is the plasticizer components of PVC and other plastic products, and it is also important industrial raw materials of the textile fiber lubricants and rubber adhesives. With the development of modern industry, the widespread use of plastic products, plastics, textile fibers and adhesives, these materials in our daily lives a major commodity. Therefore, PAEs are everywhere in modern life. PAEs is acess into the environment through the food chain and the water cycle because it is difficult to be decomposited and have the cumulative effect. Taiwan plasticizer storm makes more people concern about the PAEs. The study found PAEs are the environmental endocrine disruptors, it have the reproductive toxicity, carcinogenic, teratogenic. Thus many countries have stepped up efforts to detect the substance.Many methods can be used to detect PAEs such as liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry, GC-MS. These methods can achieve a certain concentration range detection of PAEs, but these methods have many drawbacks:most of the samples need processed, complexed detection procedures, cumbersome and time-consuming, high cost, low sensitivity. Therefore, establishment of a time-saving, low-cost detection methods is imminent.Enzyme-linked immunosorbent assay (ELISA) have a specific detection for the test samples, simple samples processing, low cost detection, multiple samples can be disposable one time and high sensitivity. Thus, enzyme-linked immunosorbent assay (ELISA) can solve the above-mentioned physical and chemical testing of these problems existed, but the key issue is have corresponding antibodies of the PAEs.Mono-methyl phthalate (MMP) is one kind of PAEs. For establishing of MMP ELISA detection method, the study used4-nitro-phthalic anhydride and methanol as raw material to synthesis the4-amino-phthalate ester, obtained artificial antigen MMAP-OVA, MMAP-BSA, MMCP-OVA, MMCP-BSA by chemical coupling methods connected the carrier protein OVA and BSA to the haptens. The artificial antigenes immuned the mice, measured the titer and the inhibition rate of the blood of the mice. We get hybridoma cells by the method of cell fusion, screened and cloned the fused cells, established monoclonal cell lines of MMP. MMP monoclonal antibody prepared and using the antibody to develop the kit.The results show that:synthesis of artificial antigens are successful by using the measured methods UV spectroscopy and SDS-PAGE. Mice serum was measured after the fifth immunization, the group of MMCP-BSA titers reached105or more, achieved the immunity requirements. By indirect competitive ELISA measured the serum of mice get the IC50is115μg/L. These results indicate that the mice immunized well and in line with the requirements of cell fusion. By cell fusion and screening test we obtained one hybridoma strain which can secrete stable antibody. The Chromosome number of the cell lines is about97to103, the result shows the fusion is successful. Identification of the nature of the antibody found protein content is3.75mg/mL. The kit was prepared on the basis of the successful preparation of the antibody, by indirect competition methods we get a good relationship y=0.1646Inx-0.1669, R2=0.9902when MMP concentrations is between10.43-264.72μg/L, the minimum detectable concentration is3.9μg/L. The results showed that the antibody can satisfy trace detection of MMP.This research established monoclonal antibody of MMP, ELISA detection methods for MMP, developed a kit for detection of MMP using the prepared monoclonal antibody. Successful development of the kit can used to detect the residual MMP of the environment and the metabolism largely and rapidly.