免疫磁分离及免疫比浊分析对福氏志贺菌的自动快速定量检测  

Establishment of nephelometric immunoassay combined with immunomagnetic separation for the rapid automatic quantitative detection of Shigella flexneri

作　　者：肖敏[1,2] 易勇[2] 毛旭虎[1] 罗萍[1] 聂棱 贾莉萍[2] 魏平[2] 王惠[2] 敬华[2] 邹全明[1] 

机构地区：[1]第三军医大学检验系临床微生物学及免疫学教研室 [2]解放军第306医院检验科 [3]北京金纳信生物科技有限公司

出　　处：《免疫学杂志》2009年第3期341-345,共5页Immunological Journal

摘　　要：目的以SPA包被的磁珠和富含SPA的金黄色葡萄球菌分别与福氏志贺菌的多抗血清结合,制备福氏志贺菌特异的多抗磁珠和抗体致敏的金黄色葡萄球菌作为协同凝集试剂与免疫比浊试剂,利用免疫比浊分析探索其在福氏志贺菌自动快速定量检测中的初步应用。方法福氏志贺菌F1a免疫日本大耳白兔制备多抗血清,磁珠包被SPA后再与多抗偶联,制成多抗免疫磁珠。以该磁珠捕获模拟标本的福氏志贺菌,接种MH平板37℃培养18～24h,菌落计数后计算免疫磁珠的特异富集作用。富含SPA的金黄色葡萄球菌(No1800株)经甲醛灭活后,与福氏志贺菌的多抗血清结合,制备特异抗体致敏的金黄色葡萄球菌,作为协同凝集试剂和自动定量检测的免疫比浊试剂,在日立7060全自动生化分析仪编制程序进行自动检测并进行初步的方法学评价。结果福氏志贺菌F1a免疫日本大耳白兔获得了高质量的多抗血清,特异性好,凝集效价达1∶320;制备多抗免疫磁珠对福氏志贺菌具有一定的富集作用,菌落计数显示磁珠处理的细菌捕获效率约为30%;抗体致敏的金黄色葡萄球菌,作为协同凝集试剂和自动定量检测的免疫比浊试剂,在玻片上能与待测福氏志贺菌产生明显的凝集现象,在日立7060全自动生化分析仪上定量检测灵敏、特异、线性良好。结论以福氏志贺菌的多抗血清分别致敏SPA包被的磁珠和富含SPA的金黄色葡萄球菌,制备特异的多抗磁珠对福氏志贺菌有一定的特异捕获与富集作用,抗体致敏的金黄色葡萄球菌作为协同凝集试剂与免疫比浊试剂对福氏志贺菌的自动、快速、定量检测具有较好的效果,为病原菌的快速诊断和自动定量分析提供了新的思路。Objective To establish a nephelometric immunoassay for the rapid automatic quantitative detection of Shigella flexneri. Methods The specific antiserum was prepared by rabbits immunized with Shigella flexneri 1a,while the magnetic beads coated with SPA binding to the specific IgG from the antiserum were used to capture the S. flexneri from the sample. SPA-rich Staphylococcus aureus killed by formalin was also combined with the specific IgG from the antiserum,and then used as nephelometric immunoassay reagent. The rapid automatic quantitative detection of Shigella flexneri was performed on the Hitachi 7060 biochemistry analyzer. Results In the present study,the high quality antiserum with agglutination titer of 1:320 and high specificity of Shigella flexneri 1a was obtained from the immunized rabbits. Immunomagnetic beads had shown some enrichment effect of Shigella flexneri 1a,while the colony counting of plate culture after enriched by immunomagnetic beads indicated that the capture efficiency was about 30%. The rapid automatic quantitative detection method of Shigella flexneri was established on the Hitachi 7060 analyzer with the SPA-rich Staphylococcus aureus combined with the specific IgG used as nephelometric immunoassay reagent. This immunoassay had a detection limit of 1000 cfu and relatively satisfied specificity and linear range. Conclusion With the immunomagnetic beads and the SPA-rich Staphylococcus aureus combined with the specific IgG of the antiserum,the rapid automatic quantitative immunoassay for the detection of Shigella flexneri is established,which provides a new way for the rapid diagnosis and automatic quantitative analysis of pathogenic bacteria.

关 键 词：福氏志贺菌 免疫磁珠 免疫比浊 SPA 病原菌快速定量分析 

分 类 号：R446.6[医药卫生—诊断学]