食品中链霉素残留快速检测技术研究

发布时间：2018-04-26 06:23

  本文选题：链霉素 + 快速检测　； 参考：《中国计量学院》2012年硕士论文

【摘要】：链霉素（Streptomycin，SM）具有性质稳定、抗菌谱广、生产工艺简单、疗效好等特点，尤其对结核分枝杆菌有强大抗菌作用，是一种广泛应用于动物疾病治疗、预防的氨基糖苷类抗生素。但链霉素具有严重的耳毒性和肾毒性，它在动物源性食品中的残留已引起国内外的普遍关注。开发快速、准确、经济的检测链霉素类抗生素残留的方法在生产和生活中具有重要意义。 本文采取当前普遍应用的免疫检测法——酶联免疫检测法（ELISA）和一种新型的检测方法——基于核酸适配子快速检测法，对食品中链霉素残留检测技术进行研究，本研究获得以下结果： 1. SM人工抗原的制备与鉴定：采用碳二亚胺法（EDC）和戊二醛法（GA）将分别与牛血清白蛋白（BSA）、卵清白蛋白（OVA）偶联，制得免疫抗原（SM-cBSA）和包被抗原（SM-OVA）。经紫外扫描和SDS-PAGE电泳鉴定，SM成功偶联到蛋白质载体上，偶联比分别为7.6:1和17.7:1。 2. SM多克隆抗体和单克隆抗体的制备与鉴定：将SM-cBSA分别免疫新西兰大白兔6只和Balb/C小鼠3只，用间接ELISA和间接竞争ELISA测定抗血清效价和半数抑制浓度，并在小鼠多抗的基础上筛选最佳融合小鼠，制备单克隆抗体。 经8次免疫新西兰大白兔，，获得了兔多抗（rSM pAb），经鉴定，其效价可达为1:8000，阻断ELISA测定半数抑制浓度（IC50）为3.32ng/mL，与链霉素和双氢链霉素的交叉反应率分别为100%与120.78%，与其它化合物的交叉反应率（CR%）＜0.1%。经5次免疫Balb/C小鼠，获得效价可达1:80000，IC50为3.44ng/mL的鼠多抗（mSM pAb）；根据间接ELISA结果，选择2号小鼠细胞做细胞融合，筛得一株敏感特异的杂交瘤细胞1G1-E3，用体内诱生腹水法制备鼠单抗（mSM mAb）；经间接ELISA鉴定，细胞培养上清和腹水的效价分别为1:1280和1:1×106，阻断ELISA测定其IC50为2.91ng/mL，与其他化合物的CR%均＜0.01%。本实验制备多抗和单抗均可用于SM残留检测的免疫学试验，但mSM mAb的性能更好。 3.应用mSM mAb研制SM残留快速检测阻断ELISA试剂盒（SM-Kit）。SM-Kit的标准曲线呈S型，线性检测范围为0.5~40.5ng/mL，IC50为3.60ng/mL；牛奶、蜂蜜样品的平均添加回收率均在80%~120%之间；批内和批间变异系数均小于15%；除与双氢链霉素有交叉反应，与其他化合物的交叉反应率均小于0.01%，不同生物基质对SM-Kit的检测结果影响小；稳定性试验表明SM-Kit在4℃可保存6个月以上。 4.建立了基于两种基于适配子的SM残留快速检测方案，两种方案的检测IC50分别为160.83ng/mL和168.16ng/mL，初步证明方案的可行性，并为进一步研究奠定良好基础。
[Abstract]:Streptomycinia Streptomycinae (SMSM) is a kind of aminoglycoside antibiotic which is widely used in animal disease treatment and prevention because of its stable properties, wide antibacterial spectrum, simple production process and good curative effect, especially to Mycobacterium tuberculosis. However, streptomycin has serious ototoxicity and nephrotoxicity, and its residues in animal-derived foods have attracted widespread attention at home and abroad. It is of great significance to develop a rapid, accurate and economical method for detecting streptomycin residues in production and life. In this paper, the method of detection of streptomycin residues in food was studied by using Elisa (enzyme linked immunosorbent assay) and a new method of rapid detection based on aptamer of nucleic acid. The results of this study are as follows: 1. Preparation and identification of SM artificial antigen: the immune antigen (SM-cBSAA) and coated antigen (SM-OVA) were prepared by coupling bovine serum albumin (BSA) and ovalbumin (OVA) with carbodiimide (EDCA) and glutaraldehyde (GA) respectively. The results of UV scanning and SDS-PAGE electrophoresis showed that SM was successfully coupled to the protein vector, and the coupling ratios were 7.6: 1 and 17.7: 1, respectively. 2. Preparation and identification of SM polyclonal antibody and monoclonal antibody: 6 New Zealand white rabbits and 3 Balb/C mice were immunized with SM-cBSA. The antiserum titers and half inhibitory concentrations were determined by indirect ELISA and indirect competitive ELISA. The best fusion mouse was selected on the basis of mouse polyclonal antibody to prepare monoclonal antibody. New Zealand white rabbits were immunized for 8 times. Its titer was 1: 8 000, IC50 was 3.32 ng / mL, the cross reaction rates with streptomycin and dihydrostreptomycin were 100% and 120.78, respectively, and the cross reaction rate with other compounds was less than 0.1%. After 5 times Balb/C mice were immunized, the titer of mouse polyclonal antibody mSM pAbhe was 1: 80000 and IC50 was 3.44ng/mL. According to the results of indirect ELISA, mouse cell line 2 was selected for cell fusion. A sensitive and specific hybridoma cell line 1G1-E3 was screened, and mouse mSM mAb1 was prepared by in vivo induced ascites method, the titers of the supernatant and ascites were 1: 1280 and 1:1 脳 106, respectively, and the IC50 of blocking ELISA was 2.91 ng / mL, and the CR% of the other compounds was less than 0.01%. In this experiment, polyclonal antibodies and monoclonal antibodies can be used for the detection of SM residues in immunological tests, but the performance of mSM mAb is better. 3. The standard curve of SM-Kitt SM-Kit kit developed by mSM mAb was S-shaped, the linear detection range was 0.5 ~ 40.5ng / mL ~ (-1) IC50 was 3.60 ng / mL, the average recovery of milk and honey samples was between 80 ~ 120%. The coefficient of variation within and between batches was less than 15. The cross reaction rate with other compounds was less than 0.01 except for the cross reaction with dihydrostreptomycin. Different biological substrates had little effect on the detection results of SM-Kit. The stability test shows that SM-Kit can be stored for more than 6 months at 4 鈩

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