甲基对硫磷特异性单克隆抗体和单链抗体的制备及应用

发布时间：2018-05-14 09:47

本文选题：甲基对硫磷 + 单克隆抗体　；参考：《山东农业大学》2017年硕士论文

【摘要】：甲基对硫磷(O,O-dimethyl-O-4-nitrophenyl phosphorothioate,PM)是一种高毒有机磷酸酯类农药,主要用于防治棉花、水稻、果树害虫。甲基对硫磷在食品中的残留会通过食物链进入人体,对神经系统、内分泌系统、免疫系统及生殖系统等产生毒害作用。为保障食品安全,建立快速、简单、准确的甲基对硫磷残留检测方法是非常必要的。相对于传统的酶抑制法、仪器分析法等,酶联免疫吸附测定(ELISA)具有操作简单、成本低廉、特异性强、可批量检测多个样品等优势。本研究制备了甲基对硫磷特异性单克隆抗体和单链抗体,并使用制备的单克隆抗体和单链抗体分别建立了甲基对硫磷的免疫检测方法。主要研究结果如下:(1)首先,使用甲基对硫磷特异性免疫原免疫BALB/C小鼠,通过细胞融合,杂交瘤细胞筛选及腹水的制备和纯化获得了甲基对硫磷特异性单克隆抗体(PM-4G6)。(2)利用改进的高碘酸钠法将单克隆抗体(PM-4G6)与辣根过氧化物酶(HRP)偶联,获得酶标抗体(PM-4G6-HRP)。分别利用PM-4G6和PM-4G6-HRP建立了甲基对硫磷的的间接竞争酶联免疫吸附测定法(IC-ELISA)和直接竞争酶联免疫吸附测定法(DC-ELISA)。在最佳分析条件下,IC-ELISA的检测限(LOD,IC10)为0.6ng/m L,半抑制浓度(IC50)为6.6ng/m L,线性检测范围是1.4-28.0ng/mL;DC-ELISA的检测限为0.6ng/m L,IC50为3.6ng/m L,线性检测范围是1.2-12.4ng/mL。交叉反应测定结果表明所建立的ELISAs除了与杀螟硫磷、对硫磷、甲基对氧磷、对氧磷这4种农药有一定的交叉反应外,与其余用于分析的农药无明显交叉反应,对甲基对硫磷的特异性较好。(3)采用样品添加回收实验对建立的检测方法进行评价。对面粉和大米样品的甲基对硫磷添加回收实验结果显示,IC-ELISA的加标回收率在85.2%-128.9%之间,变异系数范围为2.6%-7.1%;DC-ELISA的加标回收率在101.9%-116.3%之间,变异系数范围为3.8%-10.6%。结果证明了这两种方法在样品中应用具有较高的可靠性。(4)使用甲基对硫磷特异性免疫原免疫BALB/C小鼠,成功构建了甲基对硫磷的噬菌体单链抗体库。通过固相淘选和噬菌体单克隆筛选,获得两种不同的甲基对硫磷单链抗体PM-30和PM-25,其中PM-30的灵敏度较高。将PM-30的基因插入到pET-28a(+)载体,并转化E.coli BL21(DE3),进行原核表达。最佳表达条件为25℃,0.1mmol/L IPTG条件下诱导表达8h。表达后利用生物素连接酶(BirA)对单链抗体进行生物素化,经纯化,生物素化单链抗体产量可达59.2±3.7mg/L。(5)使用获得的生物素化PM-30单链抗体建立了甲基对硫磷的IC-ELISA检测方法。经测定,在最佳分析条件下,IC-ELISA的检测限为0.9ng/m L,IC50为14.5ng/m L,线性检测范围是5.9-191.4ng/m L。交叉反应测定结果显示该方法除了与对硫磷、杀螟硫磷、甲基对氧磷3种农药有较弱的交叉反应外,与其余用于分析的农药无明显交叉反应,对甲基对硫磷的特异性较好。
[Abstract]:Methyl parathion (O, O-dimethyl-O-4-nitrophenyl phosphorothioate, PM) is a highly toxic organophosphate pesticide, which is mainly used to prevent cotton, rice and fruit tree pests. Methyl parathion residues in food will enter the human body through food chain, and have toxic effects on the nervous system, the internal secretion system, the immune system and the reproductive system. In order to ensure food safety, it is very necessary to establish a rapid, simple and accurate methyl parathion residue detection method. Relative to traditional enzyme inhibition method, instrument analysis method, enzyme linked immunosorbent assay (ELISA) has the advantages of simple operation, low cost, strong specificity, and batch detection of multiple samples. This study prepared methyl parathion Specific monoclonal antibodies and single chain antibodies were used to establish an immunoassay for methyl parathion using the monoclonal antibody and single chain antibody prepared. The main results are as follows: (1) first, using methyl parathion specific immunogenic immunization BALB/C mice, screening of hybridoma cells, preparation and purity of ascites through cell fusion, hybridoma cells screening. The methyl parathion specific monoclonal antibody (PM-4G6) was obtained. (2) using the improved sodium iodate method, the monoclonal antibody (PM-4G6) and horseradish peroxidase (HRP) were coupled to obtain the enzyme labeled antibody (PM-4G6-HRP). The indirect competitive enzyme linked immunosorbent assay (IC-ELISA) for methyl parathion (IC-ELISA) was established by PM-4G6 and PM-4G6-HRP respectively. Direct competitive enzyme linked immunosorbent assay (DC-ELISA). Under the optimal analysis conditions, the detection limit of IC-ELISA (LOD, IC10) is 0.6ng/m L, and the semi inhibitory concentration (IC50) is 6.6ng/m L, and the linear detection range is 1.4-28.0ng/mL. The ELISAs, which is established in Ming Dynasty, has a certain cross reaction to 4 kinds of pesticides, parathion, methyl parathion, methyl parathion, and oxygen and phosphorus. There is no obvious cross reaction with the other pesticides used for analysis, and the specificity of methyl parathion to methyl parathion is better. (3) using sample adding recovery experiment to evaluate the established detection methods. The results of the methyl parathion recovery experiment showed that the recovery rate of IC-ELISA was between 85.2%-128.9%, the range of variation coefficient was 2.6%-7.1%, the recovery rate of DC-ELISA was 101.9%-116.3%, and the range of variation coefficient was 3.8%-10.6%.. The results showed that the two methods were highly reliable in the application of the samples. (4) the use of armour was used. The phage single chain antibody library of methyl parathion was constructed successfully in BALB/C mice based on the specific immunogenic immunogenicity of parathion. Through solid-phase selection and phage screening, two different methyl parathion single chain antibodies PM-30 and PM-25 were obtained, in which the sensitivity of PM-30 was higher. PM-30 gene was inserted into pET-28a (+) vector and E. was transformed into E.. Coli BL21 (DE3) was used for prokaryotic expression. The optimum expression condition was 25 C, 0.1mmol/L IPTG induced expression of 8h. expression by biotin ligase (BirA) for biotinylation of single chain antibody. After purification, the production of biotinylated single chain antibody could reach 59.2 + 3.7mg/L. (5), and a methyl pair was established by using the obtained biotinylated PM-30 single chain antibody. IC-ELISA detection method of phosphorous phosphorus. Under the optimum analysis conditions, the detection limit of IC-ELISA is 0.9ng/m L, IC50 is 14.5ng/m L, and the linear detection range is 5.9-191.4ng/m L. cross reaction determination results show that the method has a weak cross reaction to 3 kinds of pesticides, such as parathion, Pho, methyl parathion, and the rest of the method. The pesticides had no obvious cross reaction, and the specificity for methyl parathion was good.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S481.8

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