Preparation Of Specific Monoclonal Antibodies And Establishment Of Immunoassay Of Mercury

Heavy metals are toxic and persistent environmental contaminants. Unlike carbon-based contaminants that can be completely degraded to relatively harmless products, metal ions can be transformed in only a limited number of ways by biological or chemical remediation processes. While such transformations are intended to limit the toxicity or solubility of a given metal species, there are usually competing processes in nature that will eventually recycle at least some of the metal ions back into their original highly toxic state. Metals often persist in the environment for long periods of time bound to soils or sediments; such bound metals are relatively non-toxic except to bottom-feeding animals. Unfortunately, changes in weather, in the pH of the soil or water, or in other combinations of environmental factors can mobilize bound metals and greatly increase their availability and effective toxicity. Mercury is one of the most toxic element of heavy metals, Detection of trace mercury still depend on traditional instruments. But immunoassays have the characteristics of quick, inexpensive, simple to perform, and reasonably portable, highly sensitive and selective and so on., So immunoassays have been focused in home and abroad in recent years.In our study, mercuric ions was coupled to Keyhole Limpet Hemocyanin(KLH) via p-Isothiocyanate-benzyl-EDTA to gain the complete antigen (KLH-ITCBE-Hg(â…¡)). Several BALB/C mice were immunized with this immunoconjugate. Spleen cells of an immunized mouse was fused with myeloma cells and the hybridomas were screened by ELISA and cloned by the limiting dilution, Five hybridoma cells lines E/H₉, E/B₂, 1/F₁₁, 1/H₇ and B/B₁₁ that produced MAbs with high selectivity and sensitivity were obtained. The five hybridoma cell lines were found to secrete antibodies of IgM kappa type. The titers of the culture supernatant of three hybridoma (E/H₉,1/F₁₁ and B/B₁₁) were 1:640,1:320, 1:160 and ascites titers reached 1:6400, 1:3200 and 1:6400, respectively. The Affinity constant of B/B₁₁ is 2.981×l0⁸L/mol, the specificity test showed that the McAbs was highly specific to mercury, because no apparent cross reactivity to other fourteen metal ions were found. Specific monoclonal antibodies of mercury ions were obtained successfully and established a foundation for further study on technology of measuring trace mercury in natural materials.One of antibody secreted from hybridoma cell lines was selected to be used for further study. By the study of the three factors, including the condition of the icon concentration, the block, the pH, it is eventually fixed for the ELISA assay conditions: The concentration of BSA-GSH-Hg(II) is 0.9μg/mL; the culture supernatants is diluted two multiple; the block is 1% gelatin; in the reaction system HBS, the NaCl concentrayion is 0.15mol/L, the suitable pH is 5.5. An assay curse is formed by Logit(B/B₀) and the negative logarithm of the mercuric ions concentration (mM) as the two influencing factors, in the range of 0.00001-0.01mmol/L, the two factors represent an apparent linear relation, the equation is as follows: Y=1.6723X-4.6806, the R² reaches 0.994 and the sensitivity reaches 9.105×10^-6 mmol/L, the intra-assay CV is 1.2%, the inter-assay CV is 4.7%, the recovery rate from ultrapure water, tap water, pool water and greengrocery samples is 102.21%, 96.92%, 103.23% and 87.12%%, respectively.