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目的建立一种简单、灵敏的疟疾诊断方法,并能对疟原虫感染进行定量分析。方法运用双抗体夹心免疫层析法,胶体碳标记间日疟原虫乳酸脱氢酶(Pv-LDH)和恶性疟原虫富组氨酸蛋白Ⅱ(Pf-HRP2)特异抗体,与疟原虫相应抗原结合,胶体碳抗原抗体复合物被NC膜上包被的疟原虫特异性抗体捕获并显色。曲线拟合疟原虫抗原浓度与对应的检测带灰度值,选取相关系数最高的拟合方程式。检测不同贮存条件的试纸条灵敏度稳定性,确定最佳保存条件。检测疟疾病人及健康人全血,判断试纸条与镜检法相比较的特异性、敏感性、符合率。结果成功制作了间日疟原虫和恶性疟原虫胶体碳标记特异抗体试纸条,灵敏度分别为5、2.5 ng/ml;检测线性范围分别为5~500 ng/ml、2.5~500 ng/ml。检测带灰度值与疟原虫抗原浓度呈正相关,可对疟原虫感染定量分析。4℃保存6个月后,灵敏度基本不变。与镜检法比较,试纸条的敏感性、特异性、符合率在95%以上。结论疟原虫胶体碳试条操作简便、快速,敏感性和特异性高,适合疟疾的快速定量诊断。
Objective To establish a simple and sensitive method for the diagnosis of malaria and to quantitatively analyze the infection of Plasmodium. Methods Double antibody sandwich immunoassay was used to detect the expression of Plasmodium falciparum lactic dehydrogenase (Pv-LDH) and Plasmodium falciparum-rich histone Ⅱ (Pf-HRP2) specific antibody, The colloidal carbon antigen antibody complex was captured and developed by the Plasmodium specific antibody coated on the NC membrane. Curve fitted to the concentration of Plasmodium antigen and the corresponding detection zone gray value, select the highest correlation coefficient fitting equation. Test strips of different storage conditions sensitivity stability, to determine the best storage conditions. Detection of malaria patients and healthy people whole blood, to determine the test strip compared with the specificity of microscopic examination, sensitivity, coincidence rate. Results The strips of colloidal carbon labeled special antibody against Plasmodium vivax and Plasmodium falciparum were successfully prepared. The sensitivity was 5 and 2.5 ng / ml, respectively. The linear range was 5 ~ 500 ng / ml and 2.5 ~ 500 ng / ml, respectively. The gray value of detection zone was positively correlated with the concentration of antigen of Plasmodium, which could be used for quantitative analysis of Plasmodium infection. After 4 months of preservation at 6 months, the sensitivity remained essentially unchanged. Compared with the microscopic examination, the test strip sensitivity, specificity, in line with the rate of 95%. Conclusion The malaria colloidal carbon test strip is simple, rapid, sensitive and specific and suitable for the rapid and quantitative diagnosis of malaria.