抗重金属铅离子mAb的制备及ELISA检测方法的建立

发布时间：2018-05-20 06:20

本文选题：重金属Pb2+ + 人工抗原　；参考：《河南科技学院》2015年硕士论文

【摘要】：铅是的用处极广，同时也是对人类危害最大的重金属。近年来，铅的污染问题日益严重，遭受其危害的人数也越来越多。铅对机体毫无益处，对神经系统、心血管系统、生殖系统、消化系统、免疫系统等都有毒害作用。由于重金属传统检测方法的诸多弊端，本论文利用免疫学分析技术制备出抗铅离子单克隆抗体，研制出铅离子检测试剂盒，建立了重金属铅离子快速检测体系。通过异硫氰酯法将铅离子和蛋白分子（BSA/OVA）偶联到双功能偶联剂ITCBE上，制备出人工抗原Pb2+-ITCBE-BSA和Pb2+-ITCBE-OVA，并通过包括紫外分光光度法、ICP-AES法、SDS-PAGE、免疫鼠多抗血清ELISA鉴定，证明人工抗原制备成功。其结果为：Pb2+-ITCBE-BSA和Pb2+-ITCBE-OVA的蛋白浓度分别为5.26mg/mL、6.90mg/mL，其Pb2+含量分别为46.14mg/L、18.60mg/L；Pb2+-ITCBE-BSA免疫小鼠后效价达到1：1.024×105，，阻断ELISA检测到多抗血清对Pb2+的半数抑制浓度IC50为25.88ng/mL，与其他重金属离子基本上无交叉反应。用Pb2+-ITCBE-BSA免疫5只Balb/C小鼠，通过ELISA检测多抗血清的方法挑选出最优小鼠，取其脾脏与NS0细胞进行融合。通过ELISA法对杂交瘤细胞上清液进行检测后，筛选出1B7F4，2G5A2，2B7C11三株对Pb2+敏感特异的单克隆杂交瘤细胞株。运用体外诱生腹水法大量制备抗重金属Pb2+单克隆抗体。其结果为：1B7F4，2G5A2，2B7C11三株单抗细胞株上清液ELISA检测效价分别为1：2.56×103、1：1.28×103、1：2.56×103，IC50分别为37.068ng/mL、45.081ng/mL、34.276ng/mL，其同种型鉴定结果分别为IgG1/κ、IgG1/λ、IgG2a/κ，与其他重金属基本上无交叉反应。其中特异性最好的2B7C11单抗细胞株亲和常数Ka为2.69×109，腹水效价为1：1.6384×105. 对试剂盒最佳工作浓度的选择进行测定，用2B7C11株产生的单抗组装Pb2+检测试剂盒，并对其性能进行测定。该试剂盒的灵敏度为5.32ng/mL.饲料样、猪尿样、猪血清样的平均添加回收率分别为82.14、82.76%、82.64%，平均批内变异系数分别为14.5%、14.2%、13.2%，平均批间变异系数分别为13.23%、11.73%、14.3%，与其他重金属基本上无交叉反应，且试剂盒性能稳定。
[Abstract]:Lead is widely used and is the most harmful heavy metal to human beings. In recent years, the pollution of lead is becoming more and more serious. Lead is not good for the body. It is toxic to nervous system, cardiovascular system, reproductive system, digestive system, immune system, etc. Due to the disadvantages of traditional methods of heavy metal detection, monoclonal antibody against lead ion was prepared by immunological analysis technique, lead ion detection kit was developed, and a rapid detection system of heavy metal lead ion was established. The artificial antigens Pb2-ITCBE-BSA and Pb2-ITCBE-OVA were prepared by coupling lead ion and protein molecule BSA / OVA to the bifunctional coupling agent ITCBE by isothiocyanyl ester method. The results showed that the artificial antigens Pb2-ITCBE-BSA and Pb2-ITCBE-OVA were identified by ICP-AES and immunized multiantiserum ELISA. It was proved that the artificial antigen was successfully prepared. The results showed that the protein concentrations of: PB2 -ITCBE-BSA and Pb2 -ITCBE-OVA were 5.26 mg / mL, 6.90 mg / mL, respectively, and their Pb2 contents were 46.14 mg / L ~ 18.60 mg / L ~ (-1) Pb ~ (2) -ITCBE-BSA respectively, the titer reached 1: 1.024 脳 10 ~ (5). The half inhibitory concentration (IC50) of blocking ELISA to Pb2 was 25.88 ng 路mL ~ (-1), which had no cross reaction with other heavy metal ions. Five Balb/C mice were immunized with Pb2-ITCBE-BSA. The optimal mice were selected by the method of ELISA detection of polyantiserum, and the spleen of the mice was fused with NS0 cells. After the supernatant of hybridoma cells was detected by ELISA method, three monoclonal hybridoma cell lines, 1B7F4, 2G5A2B7C11, which were sensitive to Pb2, were selected. Monoclonal antibodies against heavy metal Pb2 were prepared by in vitro induced ascites. The results showed that the ELISA detection titer of supernatant of three McAb cell lines: 1: 1B7F4G5A2B7C11 was 1: 2.56 脳 10 3: 1.28 脳 10 3: 1: 2.56 脳 10 3, IC50 was 37.068 ng / mL 45.081ng / mL 34.276ngmL, the homotypic results were IgG1/ 魏 IgG 1 / 位 T IgG2a / 魏 and no cross reaction with other heavy metals. The affinity constant Ka of the best specific 2B7C11 McAb cell line was 2.69 脳 10 ~ 9, and the ascites titer was 1: 1.6384 脳 10 ~ 5. The optimal working concentration of the kit was determined, and the Pb2 test kit was assembled with the monoclonal antibody produced by 2B7C11 strain, and its performance was determined. The sensitivity of the kit is 5.32 ng / mL. The average recovery rate of feed sample, pig urine sample and pig serum sample were 82.14 ~ 82.76 and 82.64, respectively. The average coefficient of variation was 14.5and 14.2cm, and the average coefficient of variation between batches was 13.23 and 11.733.There was basically no cross reaction with other heavy metals, and the performance of the kit was stable.
【学位授予单位】：河南科技学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.8

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