Preparation Of Lead Artificial Antigen And Establishment Of Immune Detection System Of Lead Abzyme

Lead is a Physiological and nerve toxicant with high accumulating property and multiple affinities, and every country has close restraints on the residual quantity of lead in food and environment. The preparation of lead antigen and lead antibody is the basis and key point to build up quick testing kit for lead-enzyme immunity. This research considers adopting antigen and antibody reaction with high-specificity, along with the immune competition binding principle, to bridge and synthesize lead and BSA (Bovine Serum Albumin) into holoantigen immune in Balb/c mouse by using difunctional chelant, optimizing the testing method toward lead antibody, and to prepare anti-lead monoclonal antibody through cell fusion and other steps as content below.1 st. Preparation for lead holoantigenUsing difunctional chelant (P-NH2-Bn-Chx-A"-Dtpa) to prepare heavy metal lead holoantigen, respectively adopting glutaraldehyde method and diazotization titration method to link chelant with BSA, and then chelate synthetic protein chelant compound with lead ion to get two types of lead holoantigen BSA-P-NH2-Bn-Chx-A"-Dtpa-Pb, with respective coupling ratio of 1:15.64 and 1:42.95 between protein and lead. In addition, this research will optimize the glutaraldehyde method by applying two steps'chelating between protein chelant and lead ion BSA-P-NH2-Bn-Chx-A"-Dtpa-Pb, and the coupling ratio will be up to above 1: 20.2nd. Founding testing method towards lead antibody ELISATo found efficient and convenient testing method towards lead antibody by optimizing experimental conditions of indirect testing method towards ELISA. On the basis of initiating operational procedures, optimizing the types of buffer solution, Ph of buffer solution, time of antibody peridium, temperature of antibody peridium and other parameters, it ensures to use HBS with Ph of 7.4 as buffer solution, take the temperature and time of peridium as 37 degrees Celsius per hour or 4 degrees Celsius overnight. Through assurance of parameters above, it draws up the testing system of lead antibody ELISA.3rd. Preparation and appraisal of anti-lead monoclonal antibodyApplying the antibody prepared in the 1 st to the immune experiment of mouse and examining the immune effect through ELISA, the research separately conducts contrast of these two doses of 50 u g and 100 u g in the immune procedures designing, and properly increases immune times, with such a result as the titer of mice after immune reaches highest level of over:1:102400, and the variance ratio of different peridium antigens is up to 128%, which proves that antibody aiming at lead actually emerges in the mice.The fusion rate turns out to reach 86.04% after combination of positive mice spleen cells and myeloma cells, and the monoclonal rate to 40.6%. The two cell strains 15E8,20C11 with relatively better specificity and sensitivity survived after five times subcloning, and ascites model monoclonal antibody comes out through body-tempt-body method.