免疫磁珠的制备及其富集、分离单增李斯特菌的研究

发布时间：2018-07-26 18:08

【摘要】：目的：探讨磁珠与抗体偶联的最佳条件,制备抗单增李斯特菌免疫磁珠；将免疫磁珠与显色培养法结合,初步建立起利用免疫磁珠分离技术对单增李斯特菌进行分离鉴定的检测方法。从而为食品中单增李斯特菌的检测提供一种快速、高效和灵敏的检测新方法。 方法：以单增李斯特菌体作为免疫抗原,免疫新西兰兔,获得兔抗单增李斯特菌多克隆抗体,鉴定多抗活性以及与单增李斯特菌体的结合能力；用激光粒度仪和透射电镜分析不同活性基团修饰磁珠的粒径大小和分散性,选择性质稳定的磁珠用以制备免疫磁珠；设计正交试验,摸索pH、温度、时间对氨基修饰磁珠与多抗偶联的影响,选择最优条件制备免疫磁珠并用于捕获单增李斯特菌；设计不同的偶联缓冲溶液、偶联时间、偶联温度,通过比较羧基修饰磁珠与抗体偶联后上清中剩余的抗体量来确定最佳偶联条件,运用制备的免疫磁珠对样品中的单增李斯特菌进行捕获,并与显色平板结合试验,鉴定制备的免疫磁珠捕获单增李斯特菌的能力。 结果：制备的兔抗单增李斯特菌多克隆抗体的效价为1.3×105,该多抗与单增李斯特菌菌体有较好的结合；通过激光粒度仪和透射电镜分析,平均粒径为743nm的EM2-100/40氨基修饰磁珠和粒径为180nm的PM3-020羧基修饰磁珠具有较好的分散性；正交试验结果显示,氨基修饰磁珠与抗体偶联的最佳条件组合为pH9.6、37℃偶联1h,在该条件下,0.5 mg EM2-100/40氨基修饰磁珠可偶联抗体量达68μg,偶联率为45.3%,以最优条件制备的1 mg/mL免疫磁珠用于捕菌的最佳工作体积为50μL,其捕菌率为35%；抗体与1 mg PM3-020羧基修饰磁珠在pH6.0的0.01 mol/L一水吗啉乙磺酸缓冲溶液中,37℃偶联2h,偶联抗体的量为160μg,偶联率约为32%；制得的免疫磁珠对单增李斯特菌的捕获率可达77.0%。 结论：成功制备了对单增李斯特菌体具有较好亲和力的兔多克隆抗体；获得磁珠与抗体偶联的最佳条件,成功制备了免疫磁珠,并应用于样品中单增李斯特菌检测的前处理,表现出良好的捕菌能力,较之常规平皿增菌培养显色法,检测时间至少缩短20h。为食品中单增李斯特菌的检测建立更为灵敏、快速的检测新方法奠定了基础。
[Abstract]:Objective: to study the optimal conditions for coupling magnetic beads with antibodies, to prepare immune beads against Listeria monocytogenes, and to combine immunomagnetic beads with chromogenic culture. A method for isolation and identification of Listeria monocytogenes by immunomagnetic beads was established. It provides a rapid, efficient and sensitive method for the detection of Listeria monocytogenes in food. Methods: rabbit polyclonal antibody against Listeria monocytogenes was obtained by immunizing New Zealand rabbits with single Listeria monocytogenes as immune antigen, and the activity of polyclonal antibody and its binding ability to Listeria monocytogenes were identified. The particle size and dispersity of magnetic beads modified by different active groups were analyzed by laser particle size analyzer and transmission electron microscope. The immunomagnetic beads with stable properties were selected, and the orthogonal test was designed to explore the pH and temperature. The effect of time on the coupling of amino-modified magnetic beads with polyresistance was studied. The immune beads were prepared under optimum conditions and used to capture Listeria monocytogenes. Different coupling buffer solution, coupling time, coupling temperature, The optimum coupling conditions were determined by comparing the amount of antibodies remaining in the supernatant of carboxyl modified magnetic beads coupled with antibodies. The prepared immunomagnetic beads were used to capture Listeria monocytogenes in the sample and to combine with the chromogenic plate. To identify the ability of the prepared immunomagnetic beads to capture Listeria monocytogenes. Results: the titer of rabbit polyclonal antibody against Listeria monocytogenes was 1.3 脳 10 5, and the antibody was well combined with Listeria monocytogenes. The EM2-100/40 amino modified magnetic beads with average diameter of 743nm and the PM3-020 carboxyl modified magnetic beads with average diameter of 180nm have good dispersion. The optimum conditions of coupling of amino modified magnetic beads with antibodies were pH9.6 ~ (37) 鈩，

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