恶性疟与间日疟双标记时间分辨免疫荧光检测试剂盒的研制  

Development of a dual-label time-resolved fluoroimmunoassay for the detection of Plasmodium falciparum and Plasmodium vivax

作　　者：孟庆东[1] 王燕[2] 梁焕坤 王珊霞 孙文俏[4] 任守雷[4] 信波[4] 朱鹏飞[4] 张立成[4] 

机构地区：[1]山东省医学科学院山东省寄生虫病防治研究所山东省医学科学院第三附属医院,山东济宁370800 [2]山东医药技师学院,山东泰安271016 [3]广州优迪生物科技有限公司,广东广州510663 [4]解放军第88医院,山东泰安271000

出　　处：《中国病原生物学杂志》2017年第3期258-261,共4页Journal of Pathogen Biology

基　　金：广东省科技型中小企业技术创新专项资金项目(No.2016A010119067)

摘　　要：目的采用双标记时间分辨荧光免疫分析(TRFIA)技术研制一种同时检测恶性疟原虫、间日疟原虫及混合感染试剂盒,并评价试剂盒各项性能指标。方法用抗恶性疟原虫/间日疟原虫乳酸脱氢酶(PLDH)抗体1H12作为捕获抗体包被96孔板,以Sm3+标记抗恶性疟原虫乳酸脱氢酶(PfLDH)抗体2A5和Eu3+标记抗间日疟原虫乳酸脱氢酶(PvLDH)抗体4F6作为检测抗体,建立恶性疟与间日疟双标记时间分辨荧光分析法。结果试剂盒检测恶性疟原虫的灵敏度为0.15ng/ml,线性范围为0.5500ng/ml,平均稀释回收率为102.2%,批内与批间变异系数分别为6.48%和6.63%;检测间日疟原虫的灵敏度为0.25ng/ml,线性范围为0.5500ng/ml,平均稀释回收率为100.78%,批内与批间变异系数分别为5.39%和6.16%,恶性疟与间日疟检测不相互干扰。用自制试剂盒检测212份疟疾患者血标本,阳性率为95.28%,高于镜检法的92.45%和Optimal法的51.89%。结论 PfLDH/PvLDH-TRFIA试剂盒能同时检测恶性疟、间日疟及二者混合感染,且灵敏度高,可测范围宽,稳定性好,具有良好应用前景。Objective The aim of this study was to create a kit for the detection of P. falciparum, P. vivaw, and mixed infections using a dual-label time-resolved fluoroimmunoassay （TRFIA）. Methods To establish a PfI.DH/Pv LDH dual-label TRFIA, anti-PLDH McAb （1H12） served as capture antibodies and were coated onto 96-well microplates. Sm^3＋ labeled anti-PfLDH McAb （2A5） and Eu^3＋ -labeled anti-PvLDH McAb （4F6） served as tracers. Results For the created kit, the sensitivity of PfLDH detection was 0.15 ng/ml, with a measurement range of 0.5-500 ng/ml. The average recovery ratio was 102.2%, and intra- and inter-assay CV were 6.48% and 6.63%, respectively. The sensitivity of PvLDH detection was 0.25 ng/ml, with a measurement range of 0. 5-500 ng/ml. The average recovery was 100.78%, and the intra and intevassay CV were 5.39%/00 and 6.12%/00, respectively. The created kit was used to test 212 blood samples collected from patients with malaria in comparison to microscopy and the OptiMAL malaria test kit. Sam ples tested positive at a higher rate with the created kit （95.28%） than with microscopy （92.45%） or the OptiMAL malaria test kit （51.89%）. Conclusion The PfLDH/ PvLDH-TRFIA had a high sensitivity, wide working range, and good stability, so it was able to detect P. falciparum, P. vivax, and mixed infections and it was useful in detecting malaria.

关 键 词：恶性疟 间日疟 乳酸脱氢酶 时间分辨荧光检测分析 

分 类 号：R382.31[医药卫生—医学寄生虫学]