基于蛋白质芯片技术疟疾血清学监测方法的建立  

Establishment of a method for malaria sero-surveillance based on protein chip technology

作　　者：崔延雯 黄芳 尹建海 夏志贵 CUI Yan-wen;HUANG Fang;YIN Jian-hai;XIA Zhi-gui(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention(Chinese Center for Tropical Diseases Research),NHC Key Laboratory of Parasite and Vector Biology,WHO Collaborating Centref or Tropical Diseases,National Center for International Research on Tropical Diseases,Shanghai 200025,China)

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心)国家卫生健康委员会寄生虫病原与媒介生物学重点实验室世界卫生组织热带病合作中心国家级热带病国际联合研究中心,上海200025

出　　处：《中国病原生物学杂志》2022年第6期635-642,共8页Journal of Pathogen Biology

基　　金：上海市第五轮公共卫生体系建设三年行动计划重点学科项目(No.GWV-10.1-XK13)。

摘　　要：目的建立基于蛋白质芯片技术的疟疾血清学监测方法方法将间日疟原虫(Plasmodium vivax)裂殖子表面蛋白1_(19)(merozoite surface protein-1_(19),MSP-1_(19))、环子孢子蛋白(circumsporozoite protein,CSP)和恶性疟原虫(Plasmodium falciparum)的MSP-1_(19)作为特异性抗原包被在蛋白质芯片上,通过倍比稀释法筛选抗原并确定滤纸血洗脱液稀释倍数和筛选抗原的最佳包被浓度,建立基于蛋白质芯片技术的疟疾血清学监测方法。与间接ELISA法共同检测间日疟和恶性疟现症患者、健康者和高风险地区人群的滤纸血样本,比较两种方法的敏感性和检出效率,并对新建方法的特异性和质控品板间相关性进行验证。结果PvMSP-1_(19)(R01601)和PfMSP-1_(19)(R01604)两种抗原与阳性质控品间的决定系数R^(2)分别为0.9849和0.9317。滤纸血洗脱液的最佳稀释度为1∶4,筛选抗原最佳包被浓度为0.2μg/μl。新建方法和间接ELISA法对间日疟和恶性疟现症患者的检出率差异无统计学意义(P>0.05);检测间日疟现症患者的ROC曲线下面积分别为0.9167和0.9358,检测恶性疟现症患者的ROC曲线下面积分别为0.9167和1;对高风险地区人群的检出率分别为75.9%(22/29)和31.0%(9/29)(P<0.05),对其中19例有既往感染史人群的检出率分别为68.4%(13/19)和26.3%(5/19)(P<0.05)。新建方法中PvMSP-1_(19)(R01601)抗原对恶性疟患者和间日疟患者的检出率分别为28.6%(2/7)和83.3%(20/24)(P<0.05),PfMSP-1_(19)(R01604)抗原对间日疟患者和恶性疟患者检出率分别为29.2%(7/24)和85.7%(6/7)(P<0.05)。阳性质控品板间相关系数为0.9-1,阴性质控品板间相关系数为0.965-1。结论基于蛋白质芯片技术的疟疾血清学监测方法对疟疾现症患者和高风险人群的检出率高,具有较好的敏感性和特异性,芯片板间结果的一致性好,可作为疟疾消除和消除后阶段高效的血清学监测工具。Objective To establish a malaria sero-surveillance method based on the protein chip technology.Methods The merozoite surface protein-1_(19)(MSP-1_(19))and circumsporozoite protein(CSP)of Plasmodium vivax and the MSP-1_(19)of Plasmodium falciparum have been coated on the protein chip as specific antigens.The optimal antigens,the dilution of serum eluted from filter paper and the optimal coating concentration of screening antigens is determined by multiple dilution method,so as to establish a malaria sero-surveillance method based on the protein chip technology.Filter paper blood samples from patients with P.vivax and P.falciparum,healthy persons and negative persons in high-risk areas have been tested together with the indirect ELISA and the new method to compare the sensitivity,detection efficiency.Meanwhile,the specificity of the new method and the inter-board correlation of quality control serum have been verified.Results PvMSP-1_(19)(R01601)and PfMSP-1_(19)(R01604)have strong correlation with the positive quality control serum,the square of determination coefficient are 0.9849 and 0.9317 respectively.The optimal dilution of serum eluted from filter paper is 1∶4,and the optimal concentration of coated antigens is 0.2μg/μl.Two methods have no statistically significant difference in the detection rate of patients with P.vivax and P.falciparum(P>0.05).The area under ROC curve of these two methods are 0.9167 and 0.9358 for P.vivax patients,and 0.9167 and 1 for P.falciparum patients.The detection rate of the two methods in high-risk negative population are 75.9%(22/29)and 31.0%(9/29)respectively(P<0.05),and in 19 patients with previous infection history are 68.4%(13/19)and 26.3%(5/19)(P<0.05).In the new method,the detection rate of PvMSP-1_(19)(R01601)in patients with P.falciparum and P.vivax are 28.6%(2/7)and 83.3%(20/24)(P<0.05).The detection rate of PfMSP-1_(19)(R01601)in patients with P.vivax and P.falciparum are 29.2%(7/24)and 85.7%(6/7)(P<0.05).The correlation coefficient of inter-board positive quality co

关 键 词：疟疾 血清学方法 监测 蛋白质芯片技术 

分 类 号：R531.3[医药卫生—内科学]