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

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1:12 800～1:102 400),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 Plasmodiumfalciporum.Methods The Plasmodiumfalciporum histidine-rich protein Ⅱ(HRP-Ⅱ) gene was cloned and expressed.Recombinant HRP-Ⅱ protein was used for immunizing mice to prepare monoclonal 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 Plasmodium 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 Plasmodiun falciparum 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 Plasmodium falciparum patients showed a sensitivity of 95.5% (85/89).Conclu sion Monoclonal antibodies specific to Plasmodium falciparum HRP-Ⅱ were established based on the re combinant 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.