Preparation Of Lead Artificial Antigen And Establishment Of Antibody Detection System

Lead has continuous toxicity to human bodies, which is hard to degrade by biological and chemical ways. Thus, countries all over the world clarify the limit of lead residual in the environment or food. Compared with atomic absorption method and Inductance coupling, ELISA is economical, easy and fast and high specificity. This study aimed to purify the preparation of lead artificial antigen and improve the effects of antibody. We used the Balb/c mouse to prepare the anti-lead monoclonal antibody. The results are as follows:1) The preparation of anti-lead monoclonal antibodyIn this study, P-NH2-Bn-Chx-A"-Dtpa was used as bifunctional chelating agent and we compared the methodologies of diazotization, traditional glutaraldehyde and improved glutaraldehyde. Diazotization method had high antigen coupling rate (1:47), but with too much precipitation. Besides, the reaction of diazotization method was vigorous, which tend to cause the denaturation of the carrier protein and thus reduce the immunogenicity. Although traditional glutaraldehyde method reacted tenderly, the antigen coupling rate is relatively low(1:14). Hence, researchers has improved the traditional glutaraldehyde method to increase the antigen coupling rate at1:20.2) purification of lead artificial antigenThe purity of antigen is one of the most important factors for immunogenicity. The higher purity of the antigen, the higher possibility of the body to produce specific antibody. The phenomenon of protein linking protein tended to happen in the preparation of artificial antigen, which could seriously affect the determination of artificial antigen. Our study used ultracentrifugation tube with bigge rmembrane pore size to remove cross-linking protein with larger molecular. The purity of artificial antigen increased using SDS-PAGE and the antigen coupling rate increased from1:20to1:23.3) establishment of lead antibody detection systemThe effects of buffer compositions, pH, time and temperature of ELISA were investigated and high efficiency was achieved:HBS as buffer, pH at7.4,1h at37℃ or HBS as buffer, pH at7.4,12h at4℃.4) Preparation and determination of heavy metal lead monoclonal antibodyOur study used5-8-week-old, female mouse to perform the experiments. The anti-lead monoclonal antibody was injected into the mouse and after the fourth immunization, the titer of the mouse reached between1:102400and1:20480. The Difference in rates of OD arrived at131%, which demonstrated the occurrence of antigen-specific immune response and antibodies. We integrated the spleen of the Positive mice and myeloma cells of the normal ones and found that its infusion rate reached85.91%, with the percentage of Monoclone at41.1%. After fifth immunization, we obtained15E8and20C11with satisfactory specificity and sensitivity. Besides, we obtained ascitic monoclonal antibody by in vivo method.