基于IgY的免疫磁珠ELISA法检测中国患者血清中华支睾吸虫循环抗原
Detection of Clonorchis sinensis circulating antigen in sera from Chinese patients by immunomagnetic bead ELISA based on IgY.
作者信息
Nie Ge, Wang Ting, Lu Shengjun, Liu Wenqi, Li Yonglong, Lei Jiahui
机构信息
Department of Parasitology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
出版信息
PLoS One. 2014 Dec 4;9(12):e113208. doi: 10.1371/journal.pone.0113208. eCollection 2014.
BACKGROUND
Clonorchiasis, caused by Clonorchis sinensis, is widely distributed in Southeast Asia including China. Clonorchiasis is included in control programs of neglected tropical diseases by World Health Organization (WHO) because it is one of the major health problems in most endemic areas. Diagnosis of clonorchiasis plays a key role in the control programs. However, so far, there is no satisfactory method for clonorchiasis because of low sensitivity, poor practicality and high false positivity of available diagnostic tools.
METHODOLOGY/PRINCIPAL FINDINGS: We developed an immunomagnetic bead enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) against cysteine proteinase of C. sinensis for detection of circulating antigen in serum samples of patients infected with C. sinensis. The polyclonal IgY, coated with magnetic beads, was used as a capture antibody and a monoclonal IgG labeled with horseradish peroxidase as a detection antibody in the IgY-based immunomagnetic bead ELISA system (IgY-IMB-ELISA). The results showed that the sensitivity of IgY-IMB-ELISA was 93.3% (14 of 15) in cases of heavy infection (5000 to 9999 eggs per gram feces, i.e, EPG 5000-9999), 86.7% (13 of 15) in cases of moderate infection (EPG 1000-4999) and 75.0% (9 of 12) in cases of light infection (EPG <1000) of clonorchiasis. Together 36 of total 42 (85.7%) serum samples of human clonorchiasis gave a positive reaction. There was a significant correlation between ELISA optical density and egg counts (EPG) with a correlation coefficient of 0.83 in total 42 patients. There were no positive results in patients with trichinosis (n = 10) or cysticercosis (n = 10). Cross-reactivity was 6.7% (2 of 30) with schistosomiasis japonica and 10.0% (3 of 30) with paragonimiasis, respectively. No positive reaction was found in 20 healthy persons.
CONCLUSIONS
Our findings suggest that IgY-IMB-ELISA appears to be a sensitive and specific assay for detection of circulating antigen in human clonorchiasis.
背景
华支睾吸虫病由华支睾吸虫引起，在中国等东南亚地区广泛流行。华支睾吸虫病被世界卫生组织（WHO）纳入被忽视热带病控制项目，因为它是大多数流行地区的主要健康问题之一。华支睾吸虫病的诊断在控制项目中起着关键作用。然而，由于现有诊断工具灵敏度低、实用性差和假阳性率高，目前尚无令人满意的华支睾吸虫病诊断方法。
方法/主要发现：我们开发了一种基于抗华支睾吸虫半胱氨酸蛋白酶的卵黄免疫球蛋白（IgY）的免疫磁珠酶联免疫吸附测定（ELISA），用于检测华支睾吸虫感染患者血清样本中的循环抗原。在基于IgY的免疫磁珠ELISA系统（IgY-IMB-ELISA）中，包被有磁珠的多克隆IgY用作捕获抗体，辣根过氧化物酶标记的单克隆IgG用作检测抗体。结果显示，在华支睾吸虫重度感染（每克粪便虫卵数5000至9999个，即EPG 5000 - 9999）病例中，IgY-IMB-ELISA的灵敏度为93.3%（15例中的14例）；中度感染（EPG 1000 - 4999）病例中为86.7%（15例中的13例）；轻度感染（EPG <1000）病例中为75.0%（12例中的9例）。42例人类华支睾吸虫病血清样本中共有36例（85.7%）呈阳性反应。在42例患者中，ELISA光密度与虫卵计数（EPG）之间存在显著相关性，相关系数为0.83。旋毛虫病患者（n = 10）或囊尾蚴病患者（n = 10）均未出现阳性结果。与日本血吸虫病的交叉反应率为6.7%（30例中的2例），与并殖吸虫病的交叉反应率分别为10.0%（30例中的3例）。20名健康人未出现阳性反应。
结论
我们的研究结果表明，IgY-IMB-ELISA似乎是一种检测人类华支睾吸虫病循环抗原的灵敏且特异的检测方法。
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