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

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.