This study reports development of a novel point of care assay, namely an enhanced immuno-dot blot assay, for discrimination of anti-Toxoplasma IgG and anti-Toxoplasma IgM antibodies. This method has been designed based on formation of a sandwich complex between a gold nanoprobe (chitosan gold nanoparticle-anti-human IgG or anti-IgM) and anti- Toxoplasma lysate antigen (TLA) which holds anti-TLA antibodies, either IgG or IgM. Briefly, anti-human IgG or anti-IgM antibody was conjugated to chitosan gold nanoparticles via glutaraldehyde chemistry. Then, lysate antigen was immobilized on the surface of nitrocellulose membrane, which followed by addition of the sera sample and gold nanoprobes. The positive signals were readily detectable via observation with naked eye. This positive color change was further intensified via gold enhancement chemistry. The intensity of biosensor signal was proportional to the concentration of active antibodies on the surface of nanoparticles, titer of T. gondii antibodies in the sera samples and concentration of Toxoplasma lysate antigen coated on the nitrocellulose membrane. A minimum concentration to use the antibodies for conjugation, to detect titer of Toxoplasma IgG and IgM antibodies, and the concentration of TLA coated in nitrocellulose membrane were 0.5 mg/mL, 2 IU/mL, 10 IU/mL, and 20 μg/mL, respectively. This enhanced immuno-dot blot assay offers a simple diagnostic technique without expensive equipment requirement for distinguishing of anti- T. gondii IgM and IgG antibodies in field conditions, pregnant women, and immunocompromised patients.

本研究报告开发了一种新型即时检测，即增强型免疫斑点印迹检测，用于区分抗弓形虫IgG 和抗弓形虫IgM 抗体。该方法是基于在金纳米探针（壳聚糖金纳米颗粒-抗人 IgG 或抗 IgM）和抗弓形虫之间形成夹心复合物而设计的裂解抗原 (TLA)，它含有抗 TLA 抗体，IgG 或 IgM。简而言之，抗人 IgG 或抗 IgM 抗体通过戊二醛化学与壳聚糖金纳米颗粒结合。然后，将裂解物抗原固定在硝酸纤维素膜表面，然后加入血清样品和金纳米探针。通过肉眼观察很容易检测到阳性信号。这种积极的颜色变化通过金增强化学进一步加强。生物传感器信号的强度成比例的活性的抗体的纳米颗粒的表面上的浓度，滴度的刚地弓形虫的血清样品中的抗体和浓度弓形虫裂解液抗原包被在硝酸纤维素膜上。使用抗体进行偶联、检测弓形虫IgG和IgM抗体滴度的最低浓度以及包被硝酸纤维素膜的TLA浓度为0.5 mg/mL、2 IU/mL、10 IU/mL和20 μg/mL ， 分别。这种增强的免疫斑点印迹分析提供了一种简单的诊断技术，无需昂贵的设备即可在野外条件、孕妇和免疫功能低下的患者中区分抗弓形虫IgM 和 IgG 抗体。