国际医学寄生虫病杂志
國際醫學寄生蟲病雜誌
국제의학기생충병잡지
INTERNATIONAL JOURNAL OF MEDICAL PARASITIC DISEASES
2013年
2期
61-66
,共6页
杨玥涛%高春花%汪俊云%石锋%周晓农
楊玥濤%高春花%汪俊雲%石鋒%週曉農
양모도%고춘화%왕준운%석봉%주효농
恶性疟原虫%富组蛋白Ⅱ%单克隆抗体%免疫层析试条
噁性瘧原蟲%富組蛋白Ⅱ%單剋隆抗體%免疫層析試條
악성학원충%부조단백Ⅱ%단극륭항체%면역층석시조
Plasmodium falciparum%Histidine-rich protein Ⅱ%Monoclonal antibodies%Immunochromatographic strip
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.
目的 建立一種快速、簡便的診斷噁性瘧的膠體金免疫層析試條方法,併對其進行評價.方法 剋隆、錶達噁性瘧原蟲富組蛋白Ⅱ基因,以錶達的重組蛋白免疫BALB/c小鼠,製備單剋隆抗體,篩選噁性瘧原蟲富組蛋白Ⅱ特異的單剋隆抗體,分彆作為包被抗體和標記抗體,製備免疫層析試條.用該試條檢測流行區非瘧疾髮熱患者血樣80份、內髒利什曼病患者血樣20份和確診的間日瘧患者血樣75份以評價其特異性；檢測確診的噁性瘧患者血樣89份,以評價其敏感性. 結果 成功剋隆併錶達瞭噁性瘧原蟲富組蛋白Ⅱ,用重組蛋白免疫小鼠製備單剋隆抗體,採用雜交瘤技術共篩選齣5株能高效分泌特異抗體的細胞株(效價為1:12 800～1:102 400),抗體亞類均為IgG1.所有抗體均能唯一識彆噁性瘧原蟲蟲源蛋白組分,而與疫區非瘧疾髮熱患者的紅細胞組分無交扠反應.以篩選到的單剋隆抗體製備的免疫層析試條檢測疫區非瘧疾髮熱患者、內髒利什曼病患者和間日瘧患者血樣的特異度為97.7％(171/175),其中20份內髒利什曼病患者血樣全部為陰性.檢測噁性瘧患者血樣敏感度為95.5％(85/89). 結論 製備瞭能識彆天然噁性瘧原蟲富組蛋白Ⅱ的特異性單剋隆抗體,以此單抗為基礎研製齣的快速診斷噁性瘧的膠體金免疫層析試條敏感度、特異度均較高.
목적 건립일충쾌속、간편적진단악성학적효체금면역층석시조방법,병대기진행평개.방법 극륭、표체악성학원충부조단백Ⅱ기인,이표체적중조단백면역BALB/c소서,제비단극륭항체,사선악성학원충부조단백Ⅱ특이적단극륭항체,분별작위포피항체화표기항체,제비면역층석시조.용해시조검측류행구비학질발열환자혈양80빈、내장리십만병환자혈양20빈화학진적간일학환자혈양75빈이평개기특이성；검측학진적악성학환자혈양89빈,이평개기민감성. 결과 성공극륭병표체료악성학원충부조단백Ⅱ,용중조단백면역소서제비단극륭항체,채용잡교류기술공사선출5주능고효분비특이항체적세포주(효개위1:12 800～1:102 400),항체아류균위IgG1.소유항체균능유일식별악성학원충충원단백조분,이여역구비학질발열환자적홍세포조분무교차반응.이사선도적단극륭항체제비적면역층석시조검측역구비학질발열환자、내장리십만병환자화간일학환자혈양적특이도위97.7％(171/175),기중20빈내장리십만병환자혈양전부위음성.검측악성학환자혈양민감도위95.5％(85/89). 결론 제비료능식별천연악성학원충부조단백Ⅱ적특이성단극륭항체,이차단항위기출연제출적쾌속진단악성학적효체금면역층석시조민감도、특이도균교고.
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.
