Cornelissen J B, Gaasenbeek C P, Boersma W, Borgsteede F H, van Milligen F J
DLO-Institute for Animal Science and Health, Department of Immunology, Pathobiology and Epidemiology, Lelystad, The Netherlands.
Int J Parasitol. 1999 May;29(5):685-96. doi: 10.1016/s0020-7519(99)00017-x.
A peptide-based indirect ELISA to detect cattle antibodies against Fasciola hepatica was developed and evaluated for its sensitivity and specificity. An immunogenic antigen released in vitro by F. hepatica was purified. After purification the sequence of the first 20 N-terminal aa of this protein showed considerable homology with cathepsin L-like proteinase. Based on its homology with cathepsin-L1, we further focused on this protein for diagnostic purpose. Predicted B-cell epitopes of cathepsin-L1 were synthesised as single synthetic peptides and tested with respect to their diagnostic potential. An indirect ELISA based on one of these peptides was (i) evaluated further and (ii) compared to the potential of an indirect ELISA with excretion/secretion antigens from adult F. hepatica, or (iii) purified cathepsin-L1. Specificity and sensitivity of the three ELISAs were assessed using sera from calves experimentally infected with pure isolates of Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Schistosoma mattheei, Ascaris suum, Taenia saginata or F. hepatica, respectively, and sera from parasite-naive calves. In addition, sera were analysed from calves naturally infected with F. hepatica. The sensitivities of all three ELISAs were also very high, 98.9% (i), 100% (ii) and 100% (iii). The specificity of the peptide ELISA was very high, 99.8%, whereas specificities of the ES antigens and cathepsin-L1 ELISAs were only 82.8% and 94.6%. In experimentally infected cattle, F. hepatica-specific antibodies were first detected between days 21 and 28 p.i. with all three ELISAs, and the antibody levels persisted in the peptide ELISA until day 183 p.i. All sera from naturally infected calves were positive in the peptide ELISA. These results demonstrate that the peptide-based F. hepatica ELISA is a useful method for detecting antibodies in the sera from cattle infected with F. hepatica. This type of immunodiagnostic will therefore contribute to more accurate diagnosis and to timely curative treatment of animals.
开发了一种基于肽的间接ELISA方法，用于检测牛抗肝片吸虫抗体，并对其敏感性和特异性进行了评估。纯化了肝片吸虫体外释放的一种免疫原性抗原。纯化后，该蛋白前20个N端氨基酸序列与组织蛋白酶L样蛋白酶具有相当高的同源性。基于其与组织蛋白酶L1的同源性，我们进一步将该蛋白用于诊断目的。合成组织蛋白酶L1的预测B细胞表位作为单个合成肽，并测试其诊断潜力。基于其中一种肽的间接ELISA进行了进一步评估，并与使用来自成年肝片吸虫的排泄/分泌抗原的间接ELISA或纯化的组织蛋白酶L1的潜力进行了比较。使用分别来自实验感染了胎生网尾线虫、奥斯特他线虫、牛古柏线虫、瑞士细颈线虫、马氏血吸虫、猪蛔虫、牛带绦虫或肝片吸虫纯分离株的犊牛血清以及未感染寄生虫的犊牛血清，评估了三种ELISA的特异性和敏感性。此外，还分析了自然感染肝片吸虫的犊牛血清。三种ELISA的敏感性也都非常高，分别为98.9%（i）、100%（ii）和100%（iii）。肽ELISA的特异性非常高，为99.8%，而ES抗原和组织蛋白酶L1 ELISA的特异性分别仅为82.8%和94.6%。在实验感染的牛中，所有三种ELISA在感染后21至28天首次检测到肝片吸虫特异性抗体，并且在肽ELISA中抗体水平一直持续到感染后183天。所有自然感染犊牛的血清在肽ELISA中均为阳性。这些结果表明，基于肽的肝片吸虫ELISA是检测感染肝片吸虫的牛血清中抗体的一种有用方法。因此，这种免疫诊断方法将有助于更准确的诊断和对动物的及时治疗。
