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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 To develop a kit for the simultaneous detection of Plasmodium falciparum, Plasmodium vivax and mixed infection by dual-labeled time-resolved fluorescence immunoassay (TRFIA) and to evaluate the performance of the kit. Methods The anti-Plasmodium falciparum / Plasmodium vivax lactate dehydrogenase (PLDH) antibody 1H12 was used as the capture antibody to coat the 96-well plate. The anti-Plasmodium falciparum lactate dehydrogenase (PfLDH) antibody 2A5 and Eu3 + Plasmodium vivax lactate dehydrogenase (PvLDH) antibody 4F6 was used as the detection antibody to establish a dual-labeled time-resolved fluorescence analysis method for P. falciparum and P. vivax. Results The sensitivity of the kit to detect Plasmodium falciparum was 0.15ng / ml, the linear range was 0.5500ng / ml, the average dilution recovery was 102.2%, the coefficient of variation within the batch and the batch were 6.48% and 6.63%, respectively. The sensitivity of protozoa to 0.25ng / ml, the linear range of 0.5500ng / ml, the average dilution recovery was 100.78%, intra-and inter-assay coefficient of variation were 5.39% and 6.16%, respectively, and did not interfere with Plasmodium falciparum and Plasmodium vivax . The blood samples of 212 malaria patients were detected by homemade kit. The positive rate was 95.28%, which was higher than 92.45% of microscopy and 51.89% of Optimal method. Conclusion The PfLDH / PvLDH-TRFIA kit can detect both Plasmodium falciparum and Plasmodium vivax and their combined infections with high sensitivity, wide measurable range, good stability and good application prospect.