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Synthesis of Antigen and Preparation of Monoclonal Antibody for Heavy Metal Pb2+

Author: ZhangZhiQiang
Tutor: YangZhiQiang
School: Gansu Agricultural University
Course: Clinical Veterinary Medicine
Keywords: Pb2 Antigen synthesis Indirect competitive ELISA Monoclonal antibodies
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 49
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Abstract


With the continuous improvement of people's living standards and awareness of environmental protection, heavy metal pollution is particularly concerned about the residual detection urgent need to strengthen. In recent years, heavy metal pollution causes many reports of poisoning, particularly lead. Body prolonged intake of excessive levels of lead can cause decline in immune function and induce a variety of diseases, lead poisoning in children can severely affect IQ development, can also lead to stunted reproductive system. In this study, the use of the chelator chelate Pb 2 , coupling to a carrier protein characteristics, in the synthetic Pb in vitro 2 immune antigen and its identification and assessment, through animal immunization antibodies; then use the new technology to provide a simple, fast, high specificity monoclonal antibody preparation techniques initially established ELISA for the detection of lead ions; heavy metal residue detection. Results: (1) When the DTPA with KLH, BSA coupling and complexed Pb 2 , the UV absorption spectrum with the carrier protein KLH, BSA, compared to the absorption curve has changed, near 280nm a maximum absorption peak, and the location of the maximum absorption peak absorbance values ??increase, indicating the successful synthesis of the antigen. (2) by graphite furnace atomic absorption assay to detect antigen Pb 2 concentration 115.2μg/mL, coupling rate of 77.84% direct instructions antigen synthesized successfully; (3) Pb 2 mice the 200μg / dose immunized mice, the antiserum titer was significantly higher than 50,100 μg / dose immunization only 5 Free antiserum titers in 1:128000 above; (4) with BSA-DTPA-Pb and BSA-DTPA was detected in cell supernatants were screened the 2 hole positive hybridoma 1C7 and 3H12, the positive rate was 0.27%; positive rate of 100% clone only anti-Pb 2 hybridoma cell lines 3H12. (5) the use of established indirect competitive ELISA detected the 3H12 cell supernatant titer of 1:1 024 or more, titers higher than 1:256 000. (6) The ascites protein concentration of 12.14 mg / mL, and the monoclonal antibody is IgG1. Conclusion: (1) DTPA for heavy metal of Pb 2 immune antigen detect antigen synthesis chelator. (2) antigen heavy metal Pb 2 coupling rate of 77.84%, the direct instructions antigen synthesis successfully. (3) reasonable immunization programs help produce high titers of anti-serum titer after immunization of more than 128,000, to achieve the desired effect. (4) Although the process for the preparation of anti Pb 2 monoclonal antibodies, but the fusion rate is low, the clone is not stable enough, and the the antigen synthesis process requires further optimization.

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