首页>>学术检索

TEM1型β-内酰胺酶免疫胶体金层析检测方法的建立

标题: TEM1型β-内酰胺酶免疫胶体金层析检测方法的建立
英文标题: Establishment of Detection Methods for the Immune Colloidal Gold of TEM1 Type β-lactamase
作者: 张立静
出版时间: 2011-01-01
所在大学: 河北农业大学
关键词: TEM1型β-内酰胺酶,单克隆抗体,多克隆抗体,ELISA,胶体金
英文关键词: TEM1 typeβ-lactamase,monoclonal antibody,polyclonal antibody,ELISA,colloidal gold
论文级别: 硕士
学位: 学位论文
导师: 朱宝成%王建平
专业: 微生物与生化药学
提交时间: 2011
摘要: β-内酰胺酶是对β-内酰胺类抗生素耐药的细菌分泌的一种胞外酶,可选择性分解β-内酰胺类抗生素,是绝大多数致病菌对青霉素类药物产生耐药的主要原因.在牛奶中加入β-内酰胺酶后,能将β-内酰胺类抗生素全部降解,从而达到掩盖牛奶含有抗生素的目的.β-内酰胺酶在我国奶牛养殖和牛奶生产中很大程度上持续存在.因此,为确保牛奶及乳制品质量安全,保障消费者健康,建立牛奶中β-内酰胺酶的检测技术十分必要.牛奶中β-内酰胺酶的检测方法中微生物法灵敏度高,但检测时间较长;碘量法、高效液相色谱法等操作复杂,需要特殊的仪器设备.免疫胶体金层析法是一种快速、灵敏、特异和安全的检测技术,可以实现现场快速检测,为大规模筛选提供技术支持.本研究采用小鼠体内诱生法制备抗TEM1型β-内酰胺酶单克隆抗体,采用兔背部多点注射的方法制备抗TEM1型β-内酰胺酶的多克隆抗体.两种抗体经辛酸-硫酸铵法纯化后进行质量鉴定,单抗和多抗的蛋白质浓度分别为10mg/mL、6mg/mL.间接ELISA测得抗体效价分别为1:320 000和1:80 000.双抗夹心法结果表明两种抗体对TEM1型β-内酰胺酶的特异性较高,且对头孢菌素酶无交叉反应性.采用柠檬酸三钠还原法制备出质量高、直径20nm的胶体金颗粒,对多克隆抗体进行标记的最适蛋白量为72μg/mL,最适pH值以加入0.1mol/L K2CO3的体积衡量,为35μL.金标抗体经纯化后采用浸泡法吸附在Ahlstrom8964玻璃纤维素膜上.将抗TEM1型β-内酰胺酶的单抗包被在NC膜上作为检测线,羊抗兔二抗作为质控线,并对免疫层析试纸条的样品垫、NC膜、金标垫等材料筛选以及相关处理液进行了优化.分别经过处理干燥后与吸水纸、PVC板组装成试纸条.所制备出的试纸条对PBS基质和牛奶基质中的最小检出限为6U/mL和10U/mL,4℃可保存三个月.本研究对胶体金免疫层析检测TEM1型β-内酰胺酶进行了初步研究.采用双抗夹心法建立了TEM1型β-内酰胺酶的胶体金免疫层析方法,制备出快速检测试纸条,该试纸条灵敏度、特异性、稳定性均良好.
英文摘要: β-lactamase is an extracellular enzyme secreted by theβ-lactam antibiotic-resistant bacteria, with which to generate the resistance for penicillins and cephalosporins.β-lactamase can decompound all the antibiotics after a certain period of time when added into the milk, thus covered up the presence of antibiotic in milk. The illicit use of the enzyme continued to exist in dairy breeding and milk production. Therefore, in order to ensure milk quality and the health of consumer the development of detection technology for monitoring theβ-lactamase is necessary.Many methods for detection ofβ-lactamase in milk have been established by now. Microbiological method is sensitive but time-consuming; others such as iodimetry method, HPLC are not suitable for screening a large number of samples because these methods are cost and highly-skilled. Gold Immunochromatographic assay, as a rapid, special, sensitive and safe biological method, could then provide a technical support for large-scale screening purpose.In this study, the monoclonal antibody (McAb) and the polyclonal antibody (PcAb) against TEM1 typeβ-lactamase were prepared. The McAb and PcAb were collected, puried and identified. The concentration of the McAb was 10mg/mL, and the PcAb was 6mg/mL. The titer of the McAb and PcAb were 1:320 000 and 1:80 000, respectively. Two antibodies were specific for TEM1 and showed no cross reactivity to cephalosporinase. The colloidal gold particle was obtained by reducing the gold chloride with sodium citrate and the diameter was 20nm. Then the gold particle was labeled with PcAb. The optimum pH for labelling is about 35μL volume of 0.1mol/L K2CO3, and the amount of PcAb is 72μg/mL. The colloidal gold-labeled PcAb was sprayed on the Ahlstrom8964 glass-fiber as detection reagent. The McAb and the goat anti-mouse IgG were drawn on a nitrocellulose membrane (NC) of Millipore 135 as test line and control line, respectively.The immunochromatographic test strip of the sample pad, NC membrane, gold standard pad material and associated fluid were optimized and assembled to prepare the immuno chromatography strip. The lowest detection limits were 6U/mL and 10U/mL in PBS and milk. The test strip was stable for three months at 4℃.In this paper, a simple, rapid and sensitive colloidal gold strip for detection of TEM1 typeβ-lactamase was developped. The sensitive, specific and stable strip could be used to detect TEM1 in milk.