Ikeda T
Department of Medical Zoology, Kanazawa Medical University, Uchinada, Ishikawa, Japan.
Am J Trop Med Hyg. 1998 Aug;59(2):286-90. doi: 10.4269/ajtmh.1998.59.286.
An ELISA was developed using chicken cystatin as a capture agent for the immunodiagnosis of paragonimiasis and fascioliasis. The assay detected specific antibodies to fluke cysteine proteinases without the need for purified proteinases. An ELISA plate was sensitized with chicken egg white cystatin, incubated with excretory-secretory (ES) products of adult flukes, and standard ELISA procedures were then followed. The ELISA plates incubated with the ES products of Paragonimus westermani and Fasciola sp. showed high reactivity to sera from patients with paragonimiasis westermani and fascioliasis, respectively. The capture ELISA showed little cross-reactivity with sera from patients with paragonimiasis and fascioliasis, which showed immunodiagnostic cross-reactivity in a conventional ELISA using crude fluke antigens. Moreover, the capture ELISA showed little reactivity with sera from patients with five other helminth diseases and from healthy volunteers. Omitting either sensitization with cystatin or incubation with fluke ES products abolished high ELISA reactivity, as did a prior exposure of the ES products to cystatin. The addition of papain to an incubation solution of the ES products greatly reduced ELISA reactivity. Incubating the cystatin-sensitized plates with partially purified cysteine proteinases from flukes instead of the ES products also maintained a similar high ELISA reactivity. These results indicate that the cystatin capture ELISA elicits a cystatin and fluke cysteine proteinase antigen-mediated reaction and measures fluke cysteine proteinase-specific antibodies. Prior exposure to low molecular weight inhibitors of cysteine proteinases and other proteinases, such as E-64, leupeptin, aprotinin, and pepstatin, had no effect, suggesting that these inhibitors can be added to cysteine proteinase preparations to prevent autoproteolysis. This assay has good sensitivity and high specificity and is useful for the immunodiagnosis of paragonimiasis and fascioliasis.
开发了一种以鸡胱抑素作为捕获剂的酶联免疫吸附测定（ELISA），用于并殖吸虫病和片形吸虫病的免疫诊断。该检测方法可检测针对吸虫半胱氨酸蛋白酶的特异性抗体，无需纯化的蛋白酶。用鸡卵清白蛋白胱抑素致敏酶联免疫吸附测定板，与成虫的排泄-分泌（ES）产物孵育，然后遵循标准的酶联免疫吸附测定程序。用卫氏并殖吸虫和片形吸虫属的ES产物孵育的酶联免疫吸附测定板，分别对卫氏并殖吸虫病和片形吸虫病患者的血清显示出高反应性。捕获ELISA与并殖吸虫病和片形吸虫病患者的血清几乎没有交叉反应，而这些患者的血清在使用粗制吸虫抗原的传统ELISA中显示出免疫诊断交叉反应。此外，捕获ELISA与其他五种蠕虫病患者和健康志愿者的血清几乎没有反应性。省略胱抑素致敏或吸虫ES产物孵育会消除酶联免疫吸附测定的高反应性，吸虫ES产物预先与胱抑素接触也会如此。向ES产物的孵育溶液中添加木瓜蛋白酶会大大降低酶联免疫吸附测定的反应性。用吸虫部分纯化的半胱氨酸蛋白酶而非ES产物孵育胱抑素致敏板，也能保持类似的高酶联免疫吸附测定反应性。这些结果表明，胱抑素捕获ELISA引发了胱抑素和吸虫半胱氨酸蛋白酶抗原介导的反应，并检测吸虫半胱氨酸蛋白酶特异性抗体。预先接触半胱氨酸蛋白酶和其他蛋白酶的低分子量抑制剂，如E-64、亮抑酶肽、抑肽酶和胃蛋白酶抑制剂，没有影响，这表明这些抑制剂可添加到半胱氨酸蛋白酶制剂中以防止自身蛋白水解。该检测方法具有良好的敏感性和高特异性，可用于并殖吸虫病和片形吸虫病的免疫诊断。
