抗赭曲霉毒素A单克隆抗体的制备及初步应用

发布时间：2018-05-26 21:27

本文选题：赭曲霉毒素A + 单克隆抗体　；参考：《山东大学》2010年硕士论文

【摘要】： 目的制备抗赭曲霉毒素A(ochratoxin A, OA)的单克隆抗体(monoclonal antibody, McAb)并对其进行初步鉴定,在此基础上建立竞争抑制酶联免疫吸附试验(competitive inhibition enzyme linked immunosorbent assay, ci-ELISA)用于OA的检测。优化小米和玉米中OA的提取方法,应用建立的ci-ELISA检测济南市五个区的集市和超市出售的小米和玉米样品中OA的含量,初步了解污染情况。方法采用小剂量长周期的免疫方案,OA-牛血清白蛋白(bovine serum albumin, BSA)偶联物免疫雌性BALB/c小鼠,分别以OA-BSA. BSA为包被抗原,用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测小鼠血清中抗体的效价,观察该McAb与BSA的交叉反应情况。采用细胞融合的方法,以OA-BSA作为包被抗原,同时以BSA作对照,用ELISA法检测杂交瘤细胞培养上清中的McAb,获得分泌抗OA的McAb的杂交瘤细胞株,研究该McAb与BSA的交叉反应情况,以评价McAb的特异性。以有限稀释法对特异性分泌抗OA McAb的杂交瘤细胞进行克隆化,以ci-ELISA法进一步确定McAb的特异性,腹水诱生法大量制备McAb,采用辛酸-饱和硫酸铵法纯化腹水中的McAb,以ELISA检测纯化前和纯化后腹水效价,同时以BSA作包被抗原对照,研究交叉反应情况,以OA为竞争抗原,建立ci-ELISA方法用于OA的检测。选用不同浓度甲醇和氯化钠的溶液作提取溶剂提取样品中的OA,比较不同提取溶剂的加标回收率,观察提取溶剂中甲醇和氯化钠的浓度对小米、玉米样品中OA提取效果的影响,优化OA提取方法。采集济南市市中、历下、槐荫、天桥、历城五个区的集市出售的小米和玉米、济南市区超市出售的小米和玉米各50份,用所建立的ci-ELISA,采用标准加入法,对这些小米、玉米样品中OA含量进行了检测,计算检出率,并依照《中国国家标准GB2715—2005(粮食卫生标准)》中对谷类、豆类的OA的限量要求,计算超标率。所有数据均经SPSS软件包处理。结果OA-BSA免疫的BALB/c小鼠血清抗体效价为1：512000,与BSA有强烈的交叉反应。细胞融合后,ELISA筛选抗体分泌阳性的杂交瘤细胞株,抗OA-BSA的McAb与BSA的交叉反应率仅为3.50%,对分泌抗OA-BAS特异的McAb的细胞株经3轮克隆化,抗体分泌阳性率达到100%,建立了1株能稳定分泌抗OA-BSAMcAb的杂交瘤细胞株,ci-ELISA进一步证明了该抗体是特异针对OA的。腹水诱生法制备了大量的McAb,纯化后腹水抗体效价为1：512000,而且与BSA无交叉反应。ci-ELISA线性范围为0.04ng／ml-1250.OOng／ml,标准曲线的线性方程y=-0.1368x+0.6796,相关系数r=0.9965,检测下限为0.04ng／ml。当提取溶剂中甲醇含量为60%且含有5%氯化钠时对OA的提取效果较为理想,OA加标量为50.OOng／g、25.OOng／g、12.50ng／g时,以该提取溶剂进行确证实验,小米各加标量的平均回收率(%)分别为93.52%、96.86%、95.66%；变异系数(CV%)分别为4.59%、8.42%、8.94%,平均7.32%；玉米各加标量的平均回收率分别为96.30%、98.67%、98.13%,变异系数(CV%)分别为5.74%、5.59%、9.56%,平均6.96%。用于检测小米中OA的ci-ELISA工作曲线线性方程为y=-0.3249x+1.079,相关系数r=0.9959,用于检测玉米中OA的ci-ELISA工作曲线线性方程为y=-0.2554x+0.9561,相关系数r=0.9971。小米、玉米OA检测下限均为0.04ng／ml,OA最低限出量均为0.2ng／g。采集的所有样品中,小米OA检出率为41%(41／100),玉米OA的检出率为38%(38／100),二者无显著性差异(P0.05),其中,集市小米OA的检出率为26%(13／50),集市玉米OA检出率为32%(16／50),二者无显著性差异(P0.05)；超市小米OA检出率为56%(28／50),超市玉米OA检出率为44%(22／50),二者无显著性差异(P0.05)；检测的所有小米样品中,超市小米OA检出率(56%)明显高于集市小米(26%)(P0.05)；检测的所有玉米样品中,超市玉米(44%)和集市玉米(32%)OA检出率无显著性差异(P0.05)。采集的所有样品中,小米(0.40-4.45ug/kg,中位数为1.73 ug/kg)和玉米(0.48-4.02ug/kg,中位数为1.61 ug/kg) OA含量无显著性差异(P0.05)。其中,集市小米(0.41-3.94ug／kg,中位数为1.63 ug／kg)和集市玉米(0.53-3.99ug/kg,中位数为1.52 ug/kg)、超市小米(0.40-4.45ug/kg,中位数为1.78ug/kg)和超市玉米(0.48-4.02ug/kg,中位数为1.74 ug/kg) OA含量均无显著性差异(P0.05)。检测的所有小米样品中,超市小米(中位数为1.78ug/kg)与集市小米(中位数为1.63ug/kg)OA含量无显著性差异(P0.05),检测的所有玉米样品中超市玉米(中位数为1.74ug/kg)和集市玉米(中位数为1.52ug/kg) OA含量均无显著性差异(P0.05)。本研究检测的所有样品的OA含量在0.40-4.45ug/kg之间,中位数为1.67ug/kg,根据《中国国家标准GB2715—2005(粮食卫生标准)》中对谷类、豆类的OA的限量≤5ug／kg的要求,所有样品中的OA含量均未超标。结论获得了分泌抗OA的McAb的杂交瘤细胞株。半抗原单抗的制备过程中,免疫抗原和检测抗原可以用同一种载体,从而使完全抗原的制备大大简化。在此抗OA-McAb基础上,建立了ci-ELISA方法,用于OA检测。该检测方法灵敏度较高,满足了《中国国家标准GB2715—2005(粮食卫生标准)》中对谷类、豆类的OA的限量≤5ug/kg的要求。以含有5%氯化钠的60%甲醇水溶液对小米、玉米中的OA的提取效果较为理想,方法重复性良好。济南市集市和超市出售的小米和玉米中均不同程度地存在OA污染情况；其中,超市小米的OA检出率高于集市小米OA的检出率。依照《中国国家标准GB2715—2005(粮食卫生标准)》中对谷类、豆类的OA的限量≤5ug／kg要求,所检测的样品中OA含量均未超标,中位数为1.67ug/kg。
[Abstract]:Objective to prepare a monoclonal antibody (monoclonal antibody, McAb) against ochratoxin A (ochratoxin A, OA) and to identify it preliminarily. On this basis, a competitive inhibition enzyme linked immunosorbent assay (competitive inhibition enzyme linked immunosorbent) was established to optimize the detection of millet and corn. Methods ci-ELISA was used to detect the content of OA in millet and corn samples sold in bazaar and supermarkets in five districts of Ji'nan, and preliminarily understand the pollution situation.
Methods OA- bovine serum albumin (bovine serum albumin, BSA) conjugate was used to immunize female BALB/c mice with OA-BSA. BSA as the antigen, and the antibody titer of the mice was detected by the enzyme linked immunosorbent assay (enzyme linked immunosorbent assay), and the intersections of the antibody were observed by the enzyme linked immunosorbent assay (enzyme linked immunosorbent assay). Reaction conditions. Using the method of cell fusion, using OA-BSA as the envelope antigen and BSA as the control, the McAb in the hybridoma cell culture supernatant was detected by ELISA method, and the hybridoma cell line secreting McAb against OA was obtained. The cross reaction between the McAb and BSA was studied to evaluate the specificity of McAb. The specific secretion of anti O by the finite dilution method was used. The hybridoma cells of A McAb were cloned, and the specificity of McAb was further determined by ci-ELISA method. McAb was prepared in a large number of ascites. The McAb in ascites was purified by octanoic acid saturated ammonium sulfate method. The titer of ascites before and after purification was detected by ELISA. At the same time, the cross reaction was studied with BSA as the anti original control. OA was the competitive antigen. The ci-ELISA method was established for the detection of OA. The solution of different concentration of methanol and sodium chloride was selected as the extraction solvent to extract the OA from the sample, and the recovery rate of the different extraction solvents was compared. The effects of methanol and sodium chloride concentration on the extraction effect of OA in millet and corn samples were observed, and the extraction method of OA was optimized. The Ji'nan city was collected. Millet and maize, millet and corn sold in five districts of almanac, millet and corn sold in five districts of the city, 50 millet and corn each sold in Ji'nan city supermarket. Using the standard addition method, the content of OA in the millet and corn samples was tested, the rate of detection was calculated, and according to the China national standard GB2715 - 2005 (food hygienic standard) The limit of OA for cereals and legumes is calculated, and the exceeding standard rate is calculated. All data are processed by SPSS software package.
Results the serum antibody titer of BALB/c mice immunized with OA-BSA was 1:512000, and there was a strong cross reaction with BSA. After the fusion, ELISA screened the hybridoma cell lines with positive antibody secretion, and the cross reaction rate of McAb and BSA against OA-BSA was only 3.50%. The cell lines secreting anti OA-BAS specific McAb were cloned in 3 rounds, and the positive rate of antibody secretion was reached. To 100%, 1 hybridoma cell lines that could stabilize the secretion of anti OA-BSAMcAb were established. Ci-ELISA further demonstrated that the antibody was specific to OA. A large number of McAb were prepared by the ascites induced method. The antibody titer of the purified ascites was 1:512000, and the.Ci-ELISA linear range of.Ci-ELISA was 0.04ng / ml-1250.OOng / ml, and the standard curve of BSA was no cross reaction to BSA. The linear equation y=-0.1368x+0.6796, the correlation coefficient r=0.9965, the detection limit is 0.04ng / ml. when the content of methanol in the extraction solvent is 60% and the extraction effect of 5% sodium chloride is ideal. OA plus the scalar is 50.OOng / g, 25.OOng / g, 12.50ng / g, the extraction solvent is confirmed by the extraction solvent, the average recovery rate of the millet is added to the scalar (%). The coefficient of variation (CV%) was 4.59%, 8.42%, 8.94%, and 7.32%, respectively, and the average recovery of each additive was 96.30%, 98.67% and 98.13%, respectively, and the coefficient of variation (CV%) was 5.74%, 5.59%, 9.56%, respectively, and the average 6.96%. used to detect the ci-ELISA working curve of OA in millet was y=-0.3249x+1.079, related lines, respectively. The linear equation of ci-ELISA working curve used to detect OA in maize was y=-0.2554x+0.9561, the correlation coefficient r=0.9971. millet was r=0.9971., the detection limit of maize OA was 0.04ng / ml, and the minimum output limit of OA was all 0.2ng / g. in all samples. The detection rate of millet OA was 41% (41 / 100), and the detection rate of maize was 38% (38 / 100) and two were not obvious. P0.05, among them, the detection rate of millet OA was 26% (13 / 50), the OA detection rate was 32% (16 / 50) and two (P0.05), the detection rate of OA in supermarket millet was 56% (28 / 50), the detection rate of OA in supermarket corn was 44% (22 / 50), and there was no significant difference (P0.05) in all millet samples, and the millet OA in all the samples of the millet was detected. The detection rate (56%) was significantly higher than that of Millet (26%) (P0.05), and there was no significant difference in the detection rate of corn (44%) and market corn (32%) OA in all corn samples (P0.05). Among all the samples, millet (0.40-4.45ug/kg, median of 1.73 ug/kg) and corn (0.48-4.02ug/kg, median of 1.61 ug/kg) had no significant OA content. Difference (P0.05). Among them, millet (0.41-3.94ug / kg, median 1.63 UG / kg) and market corn (0.53-3.99ug/kg, median of 1.52 ug/kg), supermarket millet (0.40-4.45ug/kg, median is 1.78ug/kg) and supermarket corn (0.48-4.02ug/kg, median of 1.74 ug/kg) no significant difference in OA content. All millet samples detected. There was no significant difference in the content of Millet (median 1.78ug/kg) and millet (median 1.63ug/kg) of market millet (median of 1.63ug/kg) in supermarkets (P0.05). There was no significant difference in OA content between supermarket corn (median is 1.74ug/kg) and market corn (median of 1.52ug/kg) in all corn samples (P0.05). The OA content of all the samples tested in this study was found to be no significant difference (P0.05). The median of 0.40-4.45ug/kg is 1.67ug/kg. According to the Chinese national standard GB2715 - 2005 (grain health standard), the limit of the OA of the beans is less than 5ug / kg, and the OA content in all the samples is not exceeding the standard.
Conclusion the hybridoma cell line that secretes anti OA McAb is obtained. During the preparation of the antigen monoclonal antibody, the immune antigen and detection antigen can be used the same carrier so that the preparation of the complete antigen is greatly simplified. On the basis of the anti OA-McAb, the ci-ELISA method is established and used for the test of OA. The sensitivity of this method is high and satisfies China. The standard GB2715 - 2005 (grain health standard) > the requirement for the limit of OA in cereals and legumes is less than 5ug/kg. The extraction effect of OA in millet and maize with 60% methanol water containing 5% sodium chloride is ideal, and the method repeatability is good. There are OA pollution in the millet and corn sold in Ji'nan market and supermarkets in different degrees. Among them, the OA detection rate of millet in supermarket is higher than that of millet OA in market market. According to China national standard GB2715 - 2005 (grain health standard), the limit of OA of the beans is less than 5ug / kg, and the content of OA in the samples is not exceeding the standard, the median is 1.67ug/kg.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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