检测恶性疟原虫富组蛋白Ⅱ的疟疾诊断免疫层析试条的研制与评价  

Establishment and evaluation of colloid gold labeled strip test for rapid diagnosis of falciparum malaria based on capturing antigen histidine.rich protein Ⅱ of Plasmodiun falciparum

作　　者：杨玥涛[1] 高春花[1] 汪俊云[1] 石锋[1] 周晓农[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,世界卫生组织疟疾、血吸虫病和丝虫病合作中心,上海200025

出　　处：《国际医学寄生虫病杂志》2013年第2期61-66,共6页International JOurnal of Medical Parasitic Diseases

基　　金：基金项目：国家科技部重大专项（2012ZX10004220）;卫生行业科研专项经费资助项目（201202019）

摘　　要：目的建立一种快速、简便的诊断恶性疟的胶体金免疫层析试条方法，并对其进行评价。方法克隆、表达恶性疟原虫富组蛋白Ⅱ基因，以表达的重组蛋白免疫BALB/c小鼠，制备单克隆抗体，筛选恶性疟原虫富组蛋白Ⅱ特异的单克隆抗体．分别作为包被抗体和标记抗体．制备免疫层析试条。用该试条检测流行区非疟疾发热患者血样80份、内脏利什曼病患者血样20份和确诊的间日疟患者血样75份以评价其特异性：检测确诊的恶性疟患者血样89份．以评价其敏感性。结果成功克隆并表达了恶性疟原虫富组蛋白Ⅱ．用重组蛋白免疫小鼠制备单克隆抗体．采用杂交瘤技术共筛选出5株能高效分泌特异抗体的细胞株（效价为1：12800～1：102400），抗体亚类均为IgG1。所有抗体均能唯一识别恶性疟原虫虫源蛋白组分．而与疫区非疟疾发热患者的红细胞组分无交叉反应。以筛选到的单克隆抗体制备的免疫层析试条检测疫区非疟疾发热患者、内脏利什曼病患者和间日疟患者血样的特异度为97．7％（171／175），其中20份内脏利什曼病患者血样全部为阴性。检测恶性疟患者血样敏感度为95．5％（85／89）。结论制备了能识别天然恶性疟原虫富组蛋白Ⅱ的特异性单克隆抗体，以此单抗为基础研制出的快速诊断恶性疟的胶体金免疫层析试条敏感度、特异度均较高。Objective To establish and evaluate a gold immunochromatographic strip test for rapid diagnosis of malaria infected by Plasmodium falciporum. Methods The Plasmodium falciporum histidine-rich protein Ⅱ （HRP- Ⅱ ） gene was cloned and expressed. Recombinant HRP- Ⅱ protein was used for immunizing mice to prepare monoelonal antibodies （McAbs）. Monoclonal antibodies were screened, then conjugated with colloid gold as detecting reagent and immobilized on nitrocellulose in proper position. Blood samples from 80 febrile patients in endemic area of malaria, 20 patients with visceral leishmaniasis and 75 patients infected with P/asmodium vivax were used for evaluating the specificity. Eighty-nine blood samples offalciparum malaria patients were used for evaluating the sensitivity. Results The HRP-Ⅱ gene was cloned and expressed successfully. Five cell lines of McAbs with high titer against HRP-Ⅱ were obtained using the recombinant HRP-Ⅱ as immunogen. Western blotting analysis showed that these McAbs recognized native Plasmodiunfalciparum protein without cross-reaction with constituents of red blood cell of febrile patients from endemic area of malaria. Two samples out of 80 febrile patients and 2 patients with Plasmodium vivax showed false positive reaction with a specificity of 97.7%（171/175）, all the 20 samples from patients with visceral leishmaniasis were negative.Eighty-nine blood samples of Plasmodiumfalciparum patients showed a sensitivity of 95.5% （85/89）. Conclu sion Monoclonal antibodies specific to Plasmodiumfalciparum HRP-Ⅱ were established based on the recombinant HRP-Ⅱ. The immunochromatographic strip test based on the recombinant HRP-Ⅱ protein is a sensitive, specific, simple and rapid assay for falciparum malaria diagnosis.

关 键 词：恶性疟原虫 富组蛋白Ⅱ 单克隆抗体 免疫层析试条 

分 类 号：R392.33[医药卫生—免疫学]