Preparation Of Monoclonal Antibodies Against Heavy Mental Lead Ion And Establishment Of ELISA Method

Lead is nacessary in daily life, meanwhile it is one of the most toxic heavy mentals. Thepollution of lead is getting serious in recent years, and this is endangering human. Leadpoisoning has been shown to be toxic to the nervous, cardiovascular, genital, digestive andimmune system. Immunoassays for quantitative measurement of Pb2+offer several advantagesover other traditional methods. Our study get the monoclonal antibody(mAb) to against Pb2+,and used the mAb developed the quantitative indirect ELISAfor the lead ion was used todetect environmental lead content in water sources or other samples.A lead-chelate-protein conjugate was prepared via a bifunctional chelator,1-(4-Isothiocyanobenzyl)ethylenediamine-N, N,N’,N’-tetraacetic acid(ITCBE). The artificialantigens, Pb2+-ITCBE-BSA and Pb2+-ITCBE-OVA, were identified by ultravioletspectroscopy, ICP-AES, SDS-PAGE, the immunogenicity was identified by ELISA. Theartificial antigens were synthesized successfully. The protein concentration ofPb2+-ITCBE-BSA and Pb2+-ITCBE-OVA are5.26mg/mL、6.90mg/mL; the concentration ofPb2+are46.14mg/L、18.60mg/L; the immunogenicity of polyvalent antibody(pAb) is reach to1：1.024×105and the IC50is25.88ng/mL. The pAb has shown a good sensitivity and nocross-reactivity with other heavy mental ions by blocking ELISA.Five6-week old female Balb/C mice were used for immunization, the fifth mouse clearlydemonstrated higher reactivity with Pb2+-EDTA, use its spleen cells to fused with NS0cells,and detective the titer of the hybridoma cells by the indirect ELISA method. Three wells wereselected for subcloning, named them as1B7F4，2G5A2and2B7C11.The produced of mAbsplentiful is achieved by inoculate the hybridoma cells to syngeneic mouse to induce theascites. The immunogenicity of1B7F4，2G5A2and2B7C11were1：2.56×103、1：1.28×103、1：2.56×103, the IC50were measured at37.068ng/mL、45.081ng/mL、34.276ng/mL, and theirisotypes were measured as IgG1/κ、IgG1/λ、IgG2a/κ, and the Mabs of hybridoma cells haveno cross-reactivity with other heavy mental ions by blocking ELISA. The affinity constant(Ka)of2B7C11,which is the most specific,was2.69×109L/mol, and the titer of its ascetic fluidwas1：1.6384×105. To measure the best working concentration of the Pb2+kit, and product the Pb2+kit withthe Mabs of hybridoma cell strian2B7C11. Detect the performance of the kit by the samplerecovery experiment. The sensitivity of the Pb2+kit was5.32ng/mL. The average recoveryrates of Pb2+spiked in pannage, pig urine and serum were82.14、82.76%、82.64%; theaverage variable coefficient of the intra-batch were14.5%、14.2%、13.2%，the averagevariable coefficient of the different batchs were13.23%、11.73%、14.3%. Cross reactities withother mental ions were below1%, and the kits has stability performance.