Preparation Of Monoclonal Antibody Against Heavy Metal Mercury And Establishment Of Enzyme-linked Immunosorbent Assay

In this paper,the mercury Immunogen was successfully synthesized using 6-mercaptonicotinic acid,and the monoclonal antibody against mercury was prepared to establish an indirect enzyme-linked immunosorbent assay.The method was optimized to detect heavy metal mercury in tap water and lake water.6-mercaptonicotinic acid as a bifunctional chelating agent and bovine serum albumin(BSA)as a carrier protein was prepared mercury immunogen by carbodiimide method.The complete antigen was qualitative appraised by ultraviolet spectroscopic spectroscopy and SDS-PAGE electrophoresis,and quantitative identificated by ICP-MS.Then coupling rate of mercury complete antigen was calculated 13:1;After five immunizations of five mice with immune antigen,two mice No.2 and No.5 were screened for sprint immunity;The cell fusion was carried out by polyethylene glycol method,and a hybridoma cell line 4B12,stably secreting mercury monoclonal antibody,was obtained after three subclonin;A large amount of antibody were got by cell strain cultured in mice to purify and store.The antibody subtype was IgGl by the kit;the affinity constant of 5.109 X 107 L/mol demonstrates the anti-mercury antibody belongs to high affinity antibody;the crossover rate of Au+and Cu2+was 3.36%and 0.84%,respectively.The crossover ratios of the remaining metal ions Cr3+?Cr6+?Mg2+?Ni+?Ee2+?Ca2+?Mn2+?Zn2+?As3+?Ca2+?Sn2+?Cd2+?Pb2+?Ba2+?Al3+ and Sr2+ are all lower than 0.84%.The study found that the anti-mercury monoclonal antibody recognizes Hg2+without a chelating agent,and based on this,established an ic-ELISA method.After parameters of ic-ELISA were optimized,including coating solution,blocking solution,antibody dilution and astandard dilution,the method has a half-inhibition rate(IC50)of 1.68 ng/mL for mercury,a half-inhibition rate(IC50)for methyl mercury of 12.8 ng/mL,and a detection limit of 0.079 ng/mL for mercury.And the detection range(IC20-IC80)is 0.22-12.67 ng/mL.The method was used for the detection of negative spiked lake water and tap water.The recovery rate was 91%-1 16%,and the coefficient variation was 5.6%-16.6%.In comparison with ICP-MS,it was determined that the optimized ic-ELISA can be used for the detection of actual samples.