發(fā)布時(shí)間：2019-05-10 17:03

【摘要】：隨著社會(huì)的進(jìn)步和生活水平的提高,人們對(duì)食品安全越來越重視,尤其關(guān)注食品中存在的對(duì)人體健康產(chǎn)生不利影響的獸藥殘留。作為七大獸藥殘留中的一大類,p-受體激動(dòng)劑因其顯著的營養(yǎng)再分配作用,常被非法用在畜牧業(yè)生產(chǎn)中。肉制品中殘留的p-受體激動(dòng)劑通過食物鏈可在人體內(nèi)累積,危害消費(fèi)者的身體健康。由p-受體激動(dòng)劑引發(fā)的各類中毒事件也是時(shí)有發(fā)生,造成了惡劣的社會(huì)影響。因此,急需建立方便快速的檢測(cè)方法加強(qiáng)對(duì)這類p-受體激動(dòng)劑的監(jiān)管。本文希望通過制備高靈敏度、高特異性的苯乙醇胺A和沙丁胺醇單克隆抗體,建立相應(yīng)的酶聯(lián)免疫吸附分析方法(ELISA),用來有效的監(jiān)測(cè)實(shí)際樣品中的β-受體激動(dòng)劑殘留。苯乙醇胺A (Phenylethanolamine A, PA)是近年來出現(xiàn)的一種新的β-受體激動(dòng)劑,目前作為促生長劑被非法用于畜牧業(yè)中。本研究成功制備了針對(duì)苯乙醇胺A的特異性單克隆抗體,在此基礎(chǔ)上建立了高靈敏度、高特異性的用于檢測(cè)動(dòng)物組織樣品中苯乙醇胺A的ELISA法。首先對(duì)苯乙醇胺A的分子結(jié)構(gòu)進(jìn)行修飾,使之成為帶氨基的半抗原衍生物,再與載體蛋白即牛血清白蛋白(Bovine serum albumin, BSA)或卵清白蛋白(Ovalbumin, OVA)偶聯(lián),形成免疫原以及包被原。其中以PA-BSA偶聯(lián)物為免疫原免疫小鼠,取免疫小鼠的脾細(xì)胞與小鼠的骨髓瘤細(xì)胞融合后獲得了一株靈敏度較高的單克隆抗體細(xì)胞株。以單克隆抗體為基礎(chǔ)建立了檢測(cè)苯乙醇胺A的間接競爭ELISA法,并對(duì)方法的靈敏度,特異性,精確度以及準(zhǔn)確性進(jìn)行了表征。在最優(yōu)條件下,基于同源性包被抗原/抗體測(cè)定苯乙醇胺A的ELISA法的IC50為0.16ng/mL,檢出限(LOD)為0.011 ng/mL,比已有文獻(xiàn)的靈敏度高1.3-17倍。與其它十四種β-受體激動(dòng)劑的交叉反應(yīng)低于0.59%,證明所建立的ELISA不僅靈敏度高,而且特異性強(qiáng)。選擇豬肉以及豬肝作為實(shí)際樣品進(jìn)行樣品加標(biāo)回收實(shí)驗(yàn),回收率在91.40-105.51%,批內(nèi)變異系數(shù)(Coefficients of variation, CV)為1.56-9.92%(n=3),批間CV為2.02-11.71%(n=3)。用高效液相色譜串聯(lián)質(zhì)譜(LC-MS/MS)對(duì)ELISA進(jìn)行驗(yàn)證,發(fā)現(xiàn)兩種方法結(jié)果的相關(guān)系數(shù)為0.98,相關(guān)性良好。本文所建立的ELISA法具有靈敏度高、特異性強(qiáng)、簡便、準(zhǔn)確可靠的特點(diǎn),可用于實(shí)際樣品中苯乙醇胺A的檢測(cè)。沙丁胺醇是繼克倫特羅之后另一種被普遍濫用的p-受體激動(dòng)劑。本研究通過丁二酸酐法合成沙丁胺醇衍生物,使之帶有活性基團(tuán)羧基,再與載體蛋白偶聯(lián),制成免疫原和包被原。對(duì)免疫小鼠抗血清進(jìn)行效價(jià)測(cè)定,發(fā)現(xiàn)血清滴度能達(dá)到104以上,基于抗血清建立的ELISA法的IC50為6.91 ng/mL, LOD值為0.19 ng/mL。希望在此良好的基礎(chǔ)上,后期能獲得穩(wěn)定分泌特異性單克隆抗體的細(xì)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，

本文編號(hào)：2473849