The Preparation Of Monoclonal Antibody Of Three Kinds Of Heavy Metal,Sequence Analysis Of Antibody And The Establishment Of Immunodetection Methods

Heavy metal pollution incidents have been emerging endlessly and increasing fiercely. The poultry products existed heavy metal in different degree are in excess of the standards, which are jeopardizing the health of human and animals. Monitoring heavy metals in the environment rapidly and inexpensively is a prerequisite to minimize harm for human and animals. The development of immunoassays detecting heavy metals residues has been a promising trend because of its speediness and cheapness. The aim of this study was to establish hybridoma cell lines that can steadily secrete high affinity and specificity monoclonal antibodies (McAbs) against mercury, copper and zinc respectively, and clone the variable region genes of the McAb to develop and apply indirect competitive enzyme linked i m m u n o s o r b e n t a s s a y ( I C - E L I S A ) f o r m e r c u r y, c o p p e r a n d z i n c .In this study, heavy metal (mercury, copper and zinc) was coupled to carrier proteins such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) via bifunctional chelator. Carrier protein of immunizing antigen and detection antigen was KLH and BSA respectively. The artificial antigens of mercury, copper and zinc were identified with the methods of bicinchoninic acid (BCA), ultraviolet spectrophotometry, sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and graphite furnace atomic absorption spectrometry. BALB/c mice were immunized with immunizing antigen and the hybridoma cell lines that can steadily secrete high affinity and specificity monoclonal antibodies against mercury, copper and zinc respectively were established by hybridoma technique. And the chromosomes of hybridoma cell were analyzed. Additionally, the class and subclass of McAbs were determined by Mouse Monoclonal Antibody Isotyping Kit. Purified McAbs were prepared after ascites fluid was produced in BALB/c mice. The titer and affinity of McAbs and the stability of the hybridoma cell lines secreting McAbs were determined by indirect ELISA, the specificity of McAbs was determined by competitive inhibition ELISA. The total RNA of hybridoma cell lines was isolated, and the variable region genes were amplified by RT-PCR to analyze the variable region genes. The optimal working parameter of IC-ELISA was determined through the research of working concentration of antigen and antibody, blocking agent, pH and ionic strength. The results showed that the synthesis of immune and detection antigen of mercury, copper and zinc was successful. Three hybridoma cell lines producing stably specific MAbs against mercury, copper and zinc were obtained, which were named G4-G5, 9D11-F3 and Z1A5 respectively. The class and subclass of all McAbs were IgM and kappa respectively. The affinity constant of antibodies was 10⁹L/mol¹0¹⁰L/mol. The IC50 values of 3G4-G5, 9D11-F3 and Z1A5 was 1.15μM, 0.257μg/mL and 0.628μg/mL respectively. The cross-reactivities were lower than other metal ions. The variable region genes of the McAbs against mercury, copper and zinc respectively were amplified from 3G4-G5, 9D11-F3 and Z1A5. With the optimization of working parameter, the standard curves of IC-ELISA for mercury, copper and zinc were established. The detection range (IC20^IC80) was 0.15μM⁹.0μM, 0.055μg/mL¹.198μg/mL and 0.062μg/mL⁶.341μg/mL respectively. The lowest detection limit was 3.94×10^-5mM, 0.0227μg/mL and 0.015μg/mL respectively. Recoveries of mercury, copper and zinc from the analyte-fortified into tap water and ultrapure water were in the range of 88.9%¹07.3%, 87.4%¹06.8% and 88.6%¹09.4% respectively.The synthesis of antigen of mercury,copper and zinc was successful. Three hybridoma cell lines producing stably MAbs with high affinity and specificity against mercury, copper and zinc were obtained. The variable region genes of the McAbs that are against mercury, copper and zinc respectively were amplified, which laid a foundation for transformation antibody with genetic engineering. The IC-ELISA for mercury, copper and zinc was established. The immunoassays method set the stage of the development of immunoassay test kits for determining residues of mercury, copper and zinc, which could provide an important tool for farm products as well as environmental safety test, so it would have the realistic significance to improve the efficiency and quality of risk assessment.