發(fā)布時間：2018-05-20 06:20

  本文選題：重金屬Pb2+ + 人工抗原　； 參考：《河南科技學(xué)院》2015年碩士論文

【摘要】：鉛是的用處極廣，同時也是對人類危害最大的重金屬。近年來，鉛的污染問題日益嚴(yán)重，遭受其危害的人數(shù)也越來越多。鉛對機(jī)體毫無益處，對神經(jīng)系統(tǒng)、心血管系統(tǒng)、生殖系統(tǒng)、消化系統(tǒng)、免疫系統(tǒng)等都有毒害作用。由于重金屬傳統(tǒng)檢測方法的諸多弊端，本論文利用免疫學(xué)分析技術(shù)制備出抗鉛離子單克隆抗體，研制出鉛離子檢測試劑盒，建立了重金屬鉛離子快速檢測體系。 通過異硫氰酯法將鉛離子和蛋白分子（BSA/OVA）偶聯(lián)到雙功能偶聯(lián)劑ITCBE上，制備出人工抗原Pb2+-ITCBE-BSA和Pb2+-ITCBE-OVA，并通過包括紫外分光光度法、ICP-AES法、SDS-PAGE、免疫鼠多抗血清ELISA鑒定，證明人工抗原制備成功。其結(jié)果為：Pb2+-ITCBE-BSA和Pb2+-ITCBE-OVA的蛋白濃度分別為5.26mg/mL、6.90mg/mL，其Pb2+含量分別為46.14mg/L、18.60mg/L；Pb2+-ITCBE-BSA免疫小鼠后效價達(dá)到1：1.024×105，，阻斷ELISA檢測到多抗血清對Pb2+的半數(shù)抑制濃度IC50為25.88ng/mL，與其他重金屬離子基本上無交叉反應(yīng)。 用Pb2+-ITCBE-BSA免疫5只Balb/C小鼠，通過ELISA檢測多抗血清的方法挑選出最優(yōu)小鼠，取其脾臟與NS0細(xì)胞進(jìn)行融合。通過ELISA法對雜交瘤細(xì)胞上清液進(jìn)行檢測后，篩選出1B7F4，2G5A2，2B7C11三株對Pb2+敏感特異的單克隆雜交瘤細(xì)胞株。運(yùn)用體外誘生腹水法大量制備抗重金屬Pb2+單克隆抗體。其結(jié)果為：1B7F4，2G5A2，2B7C11三株單抗細(xì)胞株上清液ELISA檢測效價分別為1：2.56×103、1：1.28×103、1：2.56×103，IC50分別為37.068ng/mL、45.081ng/mL、34.276ng/mL，其同種型鑒定結(jié)果分別為IgG1/κ、IgG1/λ、IgG2a/κ，與其他重金屬基本上無交叉反應(yīng)。其中特異性最好的2B7C11單抗細(xì)胞株親和常數(shù)Ka為2.69×109，腹水效價為1：1.6384×105. 對試劑盒最佳工作濃度的選擇進(jìn)行測定，用2B7C11株產(chǎn)生的單抗組裝Pb2+檢測試劑盒，并對其性能進(jìn)行測定。該試劑盒的靈敏度為5.32ng/mL.飼料樣、豬尿樣、豬血清樣的平均添加回收率分別為82.14、82.76%、82.64%，平均批內(nèi)變異系數(shù)分別為14.5%、14.2%、13.2%，平均批間變異系數(shù)分別為13.23%、11.73%、14.3%，與其他重金屬基本上無交叉反應(yīng)，且試劑盒性能穩(wěn)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.
【學(xué)位授予單位】：河南科技學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S859.8

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