Preparation Of Monoclonal Antibodies And Establishment Of ELISA For Detection Of Against Heavy Metal Mercury Ion

Heavy metal mercury is not only one of the required important elements in people’s production and life, but also a kind of element which causes the great damage to the environment. With the widespread using of mercury, the pollution of the environment is becoming more and more serious. It’s a threat to human health through the cyclic enrichment of the food chain. Up to now, the traditional instrument detection methods had been established such as AAS, FAS, ICP-AES and so on, but these methods had too many faults such as high cost and requirement of experiment to be used widely. Therefore, the immunoassay was necessary to be developed. The monoclonal antibodies against mercury ions were prepared by using cell fusion technique and ELISA for Detection of Heavy Metal Mercury Ions was established, these could further improve the method of the detection for mercury ions and provide support for clinical environment or animal products in batch in the study.The mercury ion was modified and formed the hapten. The artificial immunogen Hg²⁺-ITCBE-BSA was synthisied by the chelator of ITCBE and the coating antigen Hg²⁺-ITCBE-OVA was obtained in the same way. The Hg²⁺-ITCBE-BSA and Hg²⁺-ITCBE-OVA were identified by UV, ICP-AES, SDS-PAGE and ELISA of polyclonal antibody（pAb）, the BALB/C mice were immunized with Hg²⁺-ITCBE-BSA, the titres of pAb was detected by indirect ELISA, the sensitivity of pAb was identified by blocking ELISA and the specificity of pAb was identified by cross-reaction. The results showed that immunogen of the Hg²⁺-ITCBE-BSA and the coating antigen Hg²⁺-ITCBE-OVA were synthetised successfully.The concentration of BSA and Hg²⁺ in Hg²⁺-ITCBE-BSA were 9.09 g/L and 45.88 mg/L. The Hg²⁺-EDTA pAb of Balb/C mice had good imunological properties with titers of 1∶1.024×105 by indirect ELISA, a good sensitivity with the 50% inhibitive concentration（ IC50） of Hg²⁺-EDTA 45.49 g/L to Hg-EDTA by blocking ELISA. The artificial complete immunogen of heavy mercury ion was successfully prepared.Artificial immune original Hg²⁺-ITCBE-BSA immuned the BALB/c mices and the mice which have highest titer by indirect ELISA and the lowest titer by blocking ELISA was chosen,then, spleen cells were obtained sterilely, spleen cells and myeloma cells fusion together. Two hybridoma cell lines with high sensitivity were chosen and named 3B1E5 and 2G6C3 by detecting the culture supernatant. Through the method of induced ascites, a large number of monoclonal antibodies were obtained. The results showed that the 3B1E5 was the best with indirect ELISA titers of 1∶5.12 × 105 in ascites. Its isotype was Ig G2/κ and its Ka was 9.1 × 1010 L/mol. It had good sensitivity with IC50 of Hg²⁺-EDTA 29.99 ng/ mL and it had good specificity with little or no CR with other compounds. The results showed that high specificity monoclonal antibody was successfully obtained.The blocking ELISA kit for determination of Hg²⁺ were established with anti-Hg²⁺-EDTA mAb of 3B1E5. First, the parameters of coating concentration, working concentration of monoclonal antibody and working concentration of enzyme were optimized. Second, ELISA for detection of heavy metal mercury ions was established and the standard curve was draught, the linear regression equation was deduced. Third, its sensitivity and detection limit were determined,its performances were measured.The results showed that it had good sensitivity with IC50 of Hg²⁺-EDTA 12.42 ng/mL, the detection limit of 4.97 ng/mL and it had good precision and accuracy with the coefficient variation（CV%） were below 15% by inter-assay and intra-assay, these show that the accuracy and precision meet the requirements, and had no significantly cross reaction with other heavy metals, kit had stable performance.