School of Bioscience and Bioengineering, South China University of Technology, University City, Panyu District, Guangzhou 510006, China.
Malar J. 2013 Jun 12;12:199. doi: 10.1186/1475-2875-12-199.
Most rapid diagnostic tests (RDTs) currently used for malaria diagnosis cannot distinguish the various Plasmodium infections. The development of a Plasmodium vivax specific RDTs with high sensitivity to sufficiently differentiate the two most common Plasmodium infections would be very crucial for disease treatment and control.
Plasmodium vivax aldolase gene (PvALDO) was amplified from the extracted genomic DNA and constructed into pET30a vector. Plasmodium vivax aldolase protein was successfully expressed in Escherichia coli in soluble form and the overall purity was over 95% after one-step affinity chromatography purification. The purified products were used for the immunization of mice and rabbits. Rabbit polyclonal antibodies generated were deployed to develop a novel antibody-capture ELISA for hybridoma screening.
Three PvALDO specific mAbs (14C7, 15F1 and 5H7) with high affinities were selected and used in immunochromatographic test strips. Clinical blood samples (n=190) collected from Yunnan (China) were used for evaluation and the RDT's sensitivity for P. vivax was 98.33% (95% Confidence Interval (CI): 91.03% to 99.72%) compared with microscopic examination. There was specificity of 99.23% (95% CI: 95.77% to 99.87%) for P. vivax. Only one Plasmodium falciparum sample was detected among the P. falciparum samples (n=20). All Plasmodium malariae samples (n=2) as well as healthy uninfected samples (n=108) were negative. Overall performance of this RDT was excellent with positive predictive value (PPV) and negative predictive value (NPV) of 98.33% and 99.23%, respectively, at 95% CI and a very good correlation with microscopic observations (kappa value, K=0.9757). Test strips show high sensitivity even at 6.25 ng/ml of recombinant P. vivax aldolase (rPvALDO).
This study further elucidates the possibility of developing aldolase-specific RDTs which can differentiate the different Plasmodium infections and improve accurate diagnosis of malaria. This RDT could adequately differentiate between P. vivax and P. falciparum infections. The novel mAb screening method developed here could find application in the screening of highly specific antibodies against other antigens.
目前用于疟疾诊断的大多数快速诊断检测（RDT）无法区分各种疟原虫感染。开发一种对两种最常见的疟原虫感染具有高灵敏度的间日疟原虫特异性 RDT 对于疾病的治疗和控制将非常关键。
从提取的基因组 DNA 中扩增间日疟原虫醛缩酶基因（PvALDO），并构建到 pET30a 载体中。间日疟原虫醛缩酶蛋白在大肠杆菌中以可溶性形式成功表达，经一步亲和层析纯化后总纯度超过 95%。纯化产物用于免疫小鼠和兔子。用纯化产物免疫兔子产生的多克隆抗体被用于开发一种新的抗体捕获 ELISA 用于杂交瘤筛选。
从杂交瘤中筛选出三种具有高亲和力的 PvALDO 特异性单克隆抗体（14C7、15F1 和 5H7）。用来自云南（中国）的 190 份临床血液样本进行评估，该 RDT 对间日疟原虫的灵敏度为 98.33%（95%置信区间（CI）：91.03%至 99.72%），与显微镜检查相比。对间日疟原虫的特异性为 99.23%（95%CI：95.77%至 99.87%）。在 20 份恶性疟原虫样本中仅检测到 1 份恶性疟原虫样本。所有间日疟原虫样本（n=2）和健康未感染样本（n=108）均为阴性。该 RDT 的整体性能非常出色，阳性预测值（PPV）和阴性预测值（NPV）分别为 98.33%和 99.23%，在 95%置信区间内，与显微镜观察具有很好的相关性（kappa 值，K=0.9757）。即使在 6.25ng/ml 的重组间日疟原虫醛缩酶（rPvALDO）下，检测条也具有很高的灵敏度。
本研究进一步阐明了开发间日疟原虫特异性 RDT 的可能性，该 RDT 可以区分不同的疟原虫感染，提高疟疾的准确诊断。该 RDT 可以充分区分间日疟原虫和恶性疟原虫感染。这里开发的新型单克隆抗体筛选方法可用于筛选针对其他抗原的高度特异性抗体。
