苯乙醇胺A和沙丁胺醇单克隆抗体的制备及其酶联免疫吸附分析方法的建立
发布时间：2019-05-10 17:03
【摘要】：随着社会的进步和生活水平的提高,人们对食品安全越来越重视,尤其关注食品中存在的对人体健康产生不利影响的兽药残留。作为七大兽药残留中的一大类,p-受体激动剂因其显著的营养再分配作用,常被非法用在畜牧业生产中。肉制品中残留的p-受体激动剂通过食物链可在人体内累积,危害消费者的身体健康。由p-受体激动剂引发的各类中毒事件也是时有发生,造成了恶劣的社会影响。因此,急需建立方便快速的检测方法加强对这类p-受体激动剂的监管。本文希望通过制备高灵敏度、高特异性的苯乙醇胺A和沙丁胺醇单克隆抗体,建立相应的酶联免疫吸附分析方法(ELISA),用来有效的监测实际样品中的β-受体激动剂残留。苯乙醇胺A (Phenylethanolamine A, PA)是近年来出现的一种新的β-受体激动剂,目前作为促生长剂被非法用于畜牧业中。本研究成功制备了针对苯乙醇胺A的特异性单克隆抗体,在此基础上建立了高灵敏度、高特异性的用于检测动物组织样品中苯乙醇胺A的ELISA法。首先对苯乙醇胺A的分子结构进行修饰,使之成为带氨基的半抗原衍生物,再与载体蛋白即牛血清白蛋白(Bovine serum albumin, BSA)或卵清白蛋白(Ovalbumin, OVA)偶联,形成免疫原以及包被原。其中以PA-BSA偶联物为免疫原免疫小鼠,取免疫小鼠的脾细胞与小鼠的骨髓瘤细胞融合后获得了一株灵敏度较高的单克隆抗体细胞株。以单克隆抗体为基础建立了检测苯乙醇胺A的间接竞争ELISA法,并对方法的灵敏度,特异性,精确度以及准确性进行了表征。在最优条件下,基于同源性包被抗原/抗体测定苯乙醇胺A的ELISA法的IC50为0.16ng/mL,检出限(LOD)为0.011 ng/mL,比已有文献的灵敏度高1.3-17倍。与其它十四种β-受体激动剂的交叉反应低于0.59%,证明所建立的ELISA不仅灵敏度高,而且特异性强。选择猪肉以及猪肝作为实际样品进行样品加标回收实验,回收率在91.40-105.51%,批内变异系数(Coefficients of variation, CV)为1.56-9.92%(n=3),批间CV为2.02-11.71%(n=3)。用高效液相色谱串联质谱(LC-MS/MS)对ELISA进行验证,发现两种方法结果的相关系数为0.98,相关性良好。本文所建立的ELISA法具有灵敏度高、特异性强、简便、准确可靠的特点,可用于实际样品中苯乙醇胺A的检测。沙丁胺醇是继克伦特罗之后另一种被普遍滥用的p-受体激动剂。本研究通过丁二酸酐法合成沙丁胺醇衍生物,使之带有活性基团羧基,再与载体蛋白偶联,制成免疫原和包被原。对免疫小鼠抗血清进行效价测定,发现血清滴度能达到104以上,基于抗血清建立的ELISA法的IC50为6.91 ng/mL, LOD值为0.19 ng/mL。希望在此良好的基础上,后期能获得稳定分泌特异性单克隆抗体的细胞株。
[Abstract]:With the progress of society and the improvement of living standard, people pay more and more attention to food safety, especially the residues of veterinary drugs which have adverse effects on human health. As one of the major residues of seven veterinary drugs, p-receptor agonists have been illegally used in animal husbandry because of their significant nutritional redistribution. Residual p-receptor agonists in meat products can accumulate in the human body through the food chain, endangering the health of consumers. All kinds of poisoning events caused by p-receptor agonists also occur from time to time, resulting in bad social effects. Therefore, it is urgent to establish convenient and rapid detection methods to strengthen the supervision of this kind of p-receptor agonists. In this paper, we hope to establish an enzyme-linked immunosorbent assay (ELISA),) for the effective monitoring of 尾-receptor agonist residues in practical samples by preparing highly sensitive and specific monoclonal antibodies against phenylethanolamine A and salbutamol. Phenylethanolamine A (Phenylethanolamine A, PA) is a new 尾-receptor agonist, which has been illegally used in animal husbandry as a growth-promoting agent in recent years. In this study, a specific monoclonal antibody against phenylethanolamine A was successfully prepared. On the basis of this, a highly sensitive and specific ELISA method for the determination of phenylethanolamine A in animal tissue samples was established. The molecular structure of phenylethanolamine A was modified to make it a semi-antigen derivative with amino group. Then it was coupled with carrier protein, bovine serum albumin (Bovine serum albumin, BSA) or ovalbumin (Ovalbumin, OVA). The formation of immunogen and envelope progen. A highly sensitive monoclonal antibody cell line was obtained by fusion of spleen cells of immunized mice with myeloma cells of mice using PA-BSA coupling as immunogen. An indirect competitive ELISA method for the detection of phenylethanolamine A was developed based on monoclonal antibody. The sensitivity, specificity, accuracy and accuracy of the method were characterized. Under the optimum conditions, the IC50 of ELISA method based on homology coating antigen / antibody was 0.16 ng 路mL, and the detection limit (LOD) was 0.011 ng/mL,. The sensitivity of the method was 1.3 鈮，
本文编号：2473849
[Abstract]:With the progress of society and the improvement of living standard, people pay more and more attention to food safety, especially the residues of veterinary drugs which have adverse effects on human health. As one of the major residues of seven veterinary drugs, p-receptor agonists have been illegally used in animal husbandry because of their significant nutritional redistribution. Residual p-receptor agonists in meat products can accumulate in the human body through the food chain, endangering the health of consumers. All kinds of poisoning events caused by p-receptor agonists also occur from time to time, resulting in bad social effects. Therefore, it is urgent to establish convenient and rapid detection methods to strengthen the supervision of this kind of p-receptor agonists. In this paper, we hope to establish an enzyme-linked immunosorbent assay (ELISA),) for the effective monitoring of 尾-receptor agonist residues in practical samples by preparing highly sensitive and specific monoclonal antibodies against phenylethanolamine A and salbutamol. Phenylethanolamine A (Phenylethanolamine A, PA) is a new 尾-receptor agonist, which has been illegally used in animal husbandry as a growth-promoting agent in recent years. In this study, a specific monoclonal antibody against phenylethanolamine A was successfully prepared. On the basis of this, a highly sensitive and specific ELISA method for the determination of phenylethanolamine A in animal tissue samples was established. The molecular structure of phenylethanolamine A was modified to make it a semi-antigen derivative with amino group. Then it was coupled with carrier protein, bovine serum albumin (Bovine serum albumin, BSA) or ovalbumin (Ovalbumin, OVA). The formation of immunogen and envelope progen. A highly sensitive monoclonal antibody cell line was obtained by fusion of spleen cells of immunized mice with myeloma cells of mice using PA-BSA coupling as immunogen. An indirect competitive ELISA method for the detection of phenylethanolamine A was developed based on monoclonal antibody. The sensitivity, specificity, accuracy and accuracy of the method were characterized. Under the optimum conditions, the IC50 of ELISA method based on homology coating antigen / antibody was 0.16 ng 路mL, and the detection limit (LOD) was 0.011 ng/mL,. The sensitivity of the method was 1.3 鈮，
本文编号：2473849
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