BACKGROUND Antibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV. RESULTS In the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni-NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests. CONCLUSIONS For the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed immunoassays eliminate the use of commercial secondary antibodies and shorten detection time. Meanwhile, both assays display great developmental prospect for further commercial production and application.

中文翻译：

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纳米抗体-辣根过氧化物酶和-EGFP融合蛋白可在免疫测定中检测猪细小病毒。

背景技术抗体是确定各种疾病的诊断免疫测定的特异性和准确性的重要试剂。然而，由于传统抗体的丰度有限，难以永久存储以及需要使用第二抗体，因此具有若干缺点。源自单链骆驼科动物抗体的纳米抗体可以规避许多这些限制，因此似乎是一个有前途的替代品。在本研究中，具有高灵敏度，特异性好，操作简便的夹心ELISA样免疫测定法和直接荧光测定法是首次开发用于检测猪细小病毒（PPV）的方法。筛选PPV病毒颗粒2（VP2）特异性纳米抗体后，辣根过氧化物酶（HRP）和增强型绿色荧光蛋白（EGFP）融合物是通过重组技术从纳米抗体衍生而来的。最后，使用纳米抗体-HRP和-EGFP融合体作为探针，已开发的免疫检测方法可特异性，灵敏且快速检测PPV。结果在该研究中，从细菌双峰驼中筛选出的五个PPV-VP2特异性纳米抗体已成功通过细菌系统表达，并使用Ni-NTA柱进行了纯化。在reporter-nanobody平台的基础上，通过转染HEK293T细胞分别产生HRP和EGFP融合蛋白。首先以PPV-VP2-Nb19为捕获抗体，以PPV-VP2-Nb56-HRP融合蛋白为检测抗体，开发了一种用于检测样品中PPV的三明治式ELISA法。测定显示92。1％与实时PCR一致，可普遍用于监测猪群中的PPV感染。另外，开发了使用PPV-VP2-Nb12-EGFP融合蛋白作为探针的直接荧光测定法，以检测ST细胞中的PPV。该测定表明与实时PCR的一致性为81.5％，可用于实验室测试。结论首次生产了五种PPV-VP2特异性纳米抗体-HRP和-EGFP融合体作为开发免疫测定的试剂。以PPV-VP2-Nb19为捕获抗体，以PPV-VP2-Nb56-HRP融合蛋白为检测抗体的三明治式ELISA法是首次开发用于检测不同样品中PPV的方法。结果表明，该免疫检测方法可普遍用于猪群PPV感染的监测。还开发了使用PPV-VP2-Nb12-EGFP作为探针的直接荧光测定法，以检测ST细胞中的PPV。两种开发的免疫测定法消除了商业二抗的使用，并缩短了检测时间。同时，这两种测定法都显示出巨大的发展前景，可进一步用于商业生产和应用。