单增李斯特菌多克隆抗体制备及其在乳胶凝集试验中的应用  

Preparation of anti-Listeria monocytogenes polyclonal antibody and its application in latex agglutination test

作　　者：鞠文[1] 宋秀玲[1] 原野[1] 张士尧[1] 方珍[2] 徐坤[1] 李娟[1] 孟日增[1,3] 

机构地区：[1]吉林大学公共卫生学院卫生检验教研室,吉林长春130021 [2]吉林大学军需科技学院食品质量与安全教研室,吉林长春130012 [3]吉林出入境检验检疫局检验检疫技术中心,吉林长春130062

出　　处：《吉林大学学报（医学版）》2012年第5期821-826,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(81072337);国家质检总局科技基金资助课题(2010IK018);吉林省科技厅科研基金资助课题(YYZX201123-2);"2012年吉林大学研究生创新研究计划"资助课题(20121120)

摘　　要：目的:制备高效价高纯度兔抗单增李斯特菌(LM)多克隆抗体,并初步探讨其在乳胶凝集试验(LAT)中的应用。方法:复苏并扩大培养LM标准菌株,制备灭活疫苗免疫家兔,采用ELISA法测定耳缘静脉血抗体效价。4次免疫后,颈动脉采血分离血清,采用辛酸-饱和硫酸铵法粗分离,经蛋白亲和层析柱HiTrap Protein GHP进行IgG纯化,采用SDS-PAGE电泳、紫外分光光度法、间接ELISA法分别测定蛋白纯度、蛋白含量及抗体效价。得到的高效价提纯IgG与乳胶微球进行共价偶联,并选择偶联乳胶的IgG含量及偶联时间等条件,建立快速检测模拟样品中LM的方法。结果:制备并纯化的兔抗LM IgG蛋白浓度为23.364g.L-1,效价为1∶12 800～1∶25 600,与霍乱弧菌、沙门氏菌、志贺菌等多种菌均无交叉反应,与斯氏李斯特菌有弱交叉反应;致敏时IgG与乳胶微球偶联比率为1∶40;利用LAT方法检测246份样品,阳性检出率为87.5%,最低检出抗原含量为0.039 8g.L-1。结论:制备了高效价、高纯度的兔抗LM多克隆抗体;建立的LAT方法特异性强,敏感性较高,重复性好,便于基层推广使用,为LM的现场检测提供了一种新手段。Objective To prepare the anti-Listeria monocytogenes（LM） polyclonal antibody derived from rabbit and to study the application of LM in latex agglutination test（LAT）.Methods Standard LM strain were cultured and multiplied using selective agents and enrichment procedures.Freund＇s complete inactivated vaccine and Freund＇s incomplete inactivated vaccine were made for immunizing rabbits.Blood samples were collected from the marginal ear vein and the antibody titers were measured by ELISA.Four times after immunization,blood serum was separated from the carotid artery and crude IgG was purified by using the method of caprylic acid-sulfide saturated soution precipitation.IgG molecules were purified by using the HiTrap Protein G chromatography column on AKTA Protein purification device and identified using the methods of SDS-PAGE electrophoresis,UV spectrophotometry,and indirect ELISA.Purified IgG were covalent conjuncted with latex microspheres and the content of latex and interaction time were optimized.The method was established for rapid detection of LM in simulated samples.Results The protein content of the purified anti-LM IgG was 23.364 g·L-1 and its antibody titer was 1∶12 800-1∶256 00,no cross-reaction occured with a variety of bacteria such as Vibrio cholerae,Salmonella,Shigella,but a weak cross-reaction with Steinmann Listeria monocytogenes.The covalent conjuncted ratio of IgG on the latex microspheres was 1∶40 and the positive rate was 87.5% for detetion in 246 samples by the method of LAT.The minimum detectable content of the antigen was 0.039 8 g·L-1.Conclusion LAT is a promising method to facilitate the grassroots level for field testing LM with its specificity,high sensitivity and good reproducibility.

关 键 词：单增李斯特菌 多克隆抗体 乳胶凝集试验 

分 类 号：R155[医药卫生—营养与食品卫生学]