Ishiyamna S, Ono K, Rai S K, Uga S
Department of Medical Laboratory Sciences, Faculty of Health Sciences, Kobe Tokiwa University, 2-6-2 Otani-cho, Nagata-ku, Kobe, 653-0838, Japan.
Nepal Med Coll J. 2009 Mar;11(1):9-13.
Diagnosis of larval migrans (LM) is usually done by immunodiagnostic methods. These methods, however, simply show the presence or absence of antibody but not the active infection of the patients. Therefore, we aimed to establish a diagnostic method for detecting circulating Toxocara canis antigen using a sandwich, ELISA. Monoclonal antibodies (MAb) were produced against the excretory-secretory (ES) antigen of second-stage T canis larvae. Among the MAbs obtained, we selected one MAb (TCMAb12; molecular weight, 30-80 kDa, IgG) for use in the sandwich ELISA. The cross-reactivity of the sandwich-ELISA against thirteen different kinds of parasite antigens were examined. The results revealed that the antibody reacted with T canis ES antigen, T. canis female antigen, and T. canis second-stage larvae antigen, but did not react with any other antigens. From results obtained using an ES antigen concentration standard curve, we confirmed that the detection limit of the sandwich-ELISA was 5 ng/ml, which provides sufficient sensitivity for the diagnosis of toxocariasis (LM). We applied the method to suspected toxocariasis patients and examined the circulating antigen in their sera. We used nine serum samples collected from patients with suspected toxocariasis based on both their clinical symptoms and high antibody titers. Overall, five sera showed antigen-positive reactions, while the remaining four were negative. These results indicated that about 44.0% of the antibody-positive patients were antigen-negative, not ongoing active infection. The results obtained using this technique would provide us for understanding toxocariasis.
幼虫移行症（LM）的诊断通常采用免疫诊断方法。然而，这些方法仅能显示抗体的存在与否，而无法检测患者是否处于活动性感染状态。因此，我们旨在建立一种使用夹心酶联免疫吸附测定法（ELISA）检测循环中的犬弓首线虫抗原的诊断方法。我们制备了针对犬弓首线虫二期幼虫排泄分泌（ES）抗原的单克隆抗体（MAb）。在所获得的单克隆抗体中，我们选择了一种单克隆抗体（TCMAb12；分子量为30 - 80 kDa，IgG）用于夹心ELISA。我们检测了该夹心ELISA对13种不同寄生虫抗原的交叉反应性。结果显示，该抗体与犬弓首线虫ES抗原、犬弓首线虫雌虫抗原和犬弓首线虫二期幼虫抗原发生反应，但与其他任何抗原均无反应。根据ES抗原浓度标准曲线的结果，我们证实夹心ELISA的检测限为5 ng/ml，这为弓首线虫病（LM）的诊断提供了足够的灵敏度。我们将该方法应用于疑似弓首线虫病患者，并检测了他们血清中的循环抗原。我们使用了9份根据临床症状和高抗体滴度收集的疑似弓首线虫病患者的血清样本。总体而言，5份血清显示抗原阳性反应，其余4份为阴性。这些结果表明，约44.0%的抗体阳性患者抗原呈阴性，并非处于活动性感染状态。使用该技术获得的结果将有助于我们了解弓首线虫病。
