PDF(Pubmed)
Abstract：
Toxoplasmosis, while often asymptomatic and prevalent as a foodborne disease, poses a considerable mortality risk for immunocompromised individuals during pregnancy. Point-of-care serological tests that detect specific IgG and IgM in patient sera are critical for disease management under limited resources. Despite many efforts to replace the T. gondii total lysate antigens (TLAs) by recombinant antigens (rAgs) in commercial kits, while IgG detection provides significant specificity and sensitivity, IgM detection remains comparatively low in sensitivity. In this study, we attempted to identify novel antigens targeting IgM in early infection, thereby establishing an IgM on-site detection kit. Using two-dimensional gel electrophoresis (2DE) and mouse serum immunoblotting, three novel antigens, including EF1γ, PGKI, and GAP50, were indicated to target T. gondii IgM. However, rAg EF1γ was undetectable by IgM of mice sera in Western blotting verification experiments, and ELISA coated with PGKI did not eliminate cross-reactivity, in contrast to GAP50. Subsequently, the lateral flow reaction employing a strip coated with 0.3 mg/mL purified rAg GAP50 and exhibited remarkable sensitivity compared with the conventional ELISA based on tachyzoite TLA, which successfully identified IgM in mouse sera infected with tachyzoites, ranging from 103 to 104 at 5 dpi and 104 at 7 dpi, respectively. Furthermore, by using standard T. gondii-infected human sera from WHO, the limit of detection (LOD) for the rapid fluorescence immunochromatographic test (FICT) using GAP50 was observed at 0.65 IU (international unit). These findings underline the particular immunoreactivity of GAP50, suggesting its potential as a specific biomarker for increasing the sensitivity of the FICT in IgM detection.
摘要：
弓形虫病,虽然通常无症状且作为食源性疾病流行，对怀孕期间免疫功能低下的个体造成相当大的死亡风险。在有限的资源下检测患者血清中特异性IgG和IgM的即时血清学测试对于疾病管理至关重要。尽管在商业试剂盒中用重组抗原(rAgs)替换弓形虫总裂解物抗原(TLA)的许多努力,而IgG检测提供了显着的特异性和灵敏度，IgM检测的灵敏度仍然相对较低。在这项研究中,我们试图在早期感染中鉴定靶向IgM的新型抗原，从而建立IgM现场检测试剂盒。用双向凝胶电泳(2DE)和小鼠血清免疫印迹,三种新型抗原，包括EF1γ，PGKI,和GAP50，用于靶向弓形虫IgM。然而,在免疫印迹验证实验中，小鼠血清IgM检测不到rAgEF1γ，用PGKI包被的ELISA没有消除交叉反应性，与GAP50相比。随后，使用涂有0.3mg/mL纯化rAgGAP50的条带的侧流反应，与基于速殖子TLA的常规ELISA相比，具有显着的灵敏度，它成功地鉴定了感染了速殖子的小鼠血清中的IgM，在5dpi时从103到104，在7dpi时从104，分别。此外，通过使用世界卫生组织的标准弓形虫感染的人血清，使用GAP50的快速荧光免疫色谱检测(FICT)的检测限(LOD)为0.65IU(国际单位).这些发现强调了GAP50的特定免疫反应性，表明其作为特异性生物标志物的潜力，可提高FCT在IgM检测中的敏感性。
