Martínez-Sernández Victoria, Orbegozo-Medina Ricardo A, González-Warleta Marta, Mezo Mercedes, Ubeira Florencio M
Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.
Laboratorio de Parasitología, Centro de Investigaciones Agrarias de Mabegondo, INGACAL, Abegondo, A Coruña, Spain.
PLoS Negl Trop Dis. 2016 Jul 20;10(7):e0004872. doi: 10.1371/journal.pntd.0004872. eCollection 2016 Jul.
ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1-10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis.
基于酶联免疫吸附测定（ELISA）的粪便中肝片吸虫组织蛋白酶检测方法是诊断肝片吸虫和巨片吸虫感染的有力技术。在过去十年中，内部研发的MM3 - 粪抗原ELISA及其商业版本BIO K 201（比利时BIO X Diagnostics公司）已被公认为是检测此类吸虫早期感染以及监测人和动物驱虫治疗效果的有用工具，因为它们比传统的粪便虫卵计数具有一些优势。然而，MM3 - 粪抗原ELISA的灵敏度有时会受到肝片吸虫整个生物周期（主要在牛体内）粪便样本中组织蛋白酶浓度高度变异性以及过氧化物酶标记的抗小鼠IgG多克隆抗体批次间性能差异的影响。为防止此类问题，我们研究了加入商业链霉亲和素 - 聚合辣根过氧化物酶缀合物以揭示结合的生物素化单克隆抗体MM3是否能提高MM3 - 粪抗原ELISA的灵敏度。我们观察到加入该试剂后，该检测方法先前的检测限从0.6 ng/mL降至150 pg/mL，并且改良后的检测方法能够识别仅感染一条吸虫的奶牛。此外，我们证明在室温下振荡孵育（每个步骤30分钟）而非标准孵育能够获得最大光密度值，这显著加快了诊断过程。最后，我们发现牛粪便中抗原浓度与寄生虫负荷之间没有显著相关性，这可能是由于本研究中所用动物的寄生虫负荷较低（1 - 10条成虫）。由于经典的MM3 - 粪抗原检测在检测动物和人类感染方面的有效性已经得到证实，预计本研究报告的改进将为肝片吸虫病的诊断和控制提供新的见解。
