纳米抗体-辣根过氧化物酶融合蛋白作为一种超灵敏探针，用于免疫分析中检测抗新城疫病毒的抗体。

Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.

机构信息

Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.

Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling, 712100, Shaanxi, China.

出版信息

J Nanobiotechnology. 2019 Mar 1;17(1):35. doi: 10.1186/s12951-019-0468-0.

DOI:10.1186/s12951-019-0468-0

PMID:30823927

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6396497/

Abstract

BACKGROUND

Sensitive and specific antibodies can be used as essential probes to develop competitive enzyme-linked immunosorbent assay (cELISA). However, traditional antibodies are difficult to produce, only available in limited quantities, and ineffective as enzymatic labels. Nanobodies, which are single-domain antibodies (sdAbs), offer an alternative, more promising tool to circumvent these limitations. In the present work, a cELISA using nanobody-horseradish peroxidase (HRP) fusion protein firstly designed as a probe was developed for detecting anti-Newcastle disease virus (NDV) antibodies in chicken sera.

RESULTS

In the study, a platform for the rapid and simple production of nanobody-HRP fusion protein was constructed. First, a total of 9 anti-NDV-NP protein nanobodies were screened from a immunised Bactrian camel. Then, the Nb5-HRP fusions were produced with the platform and used for the first time as sensitive reagents for developing cELISA to detect anti-NDV antibodies. The cut-off value of the cELISA was 18%, and the sensitivity and specificity were respectively 100% and 98.6%. The HI test and commercial ELISA kit (IDEXX) separately agreed 97.83% and 98.1% with cELISA when testing clinical chicken sera and both agreed 100% when testing egg yolks. However, for detecting anti-NDV antibodies in the sequential sera from the challenged chickens, cELISA demonstrated to be more sensitive than the HI test and commercial ELISA kit. Moreover, a close correlation (R = 0.914) was found between the percent competitive inhibition values of cELISA and HI titers.

CONCLUSIONS

A platform was successfully designed to easily and rapidly produce the nanobody-HRP fusion protein, which was the first time to be used as reagents for establishing cELISA. Results suggest that the platform supports the development of a cELISA with high sensitivity, simplicity, and rapid detection of anti-NDV antibodies. Overall, we believe that the platform based on nanobody-HRP fusions can be widely used for future investigations and treatment other diseases and viruses.

摘要

背景

敏感和特异的抗体可用作开发竞争性酶联免疫吸附试验（cELISA）的基本探针。然而，传统抗体难以生产，数量有限，且作为酶标记物效果不佳。纳米抗体是单域抗体（sdAb），提供了一种替代方法，更有希望克服这些限制。本研究中，首先设计了一种纳米抗体-辣根过氧化物酶（HRP）融合蛋白作为探针，用于检测鸡血清中的抗新城疫病毒（NDV）抗体。

结果

在本研究中，构建了一个快速简便生产纳米抗体-HRP 融合蛋白的平台。首先，从免疫的双峰驼中筛选出了 9 种抗 NDV-NP 蛋白纳米抗体。然后，利用该平台生产了 Nb5-HRP 融合蛋白，并首次将其用作开发 cELISA 检测抗 NDV 抗体的灵敏试剂。cELISA 的截断值为 18%，灵敏度和特异性分别为 100%和 98.6%。HI 试验和商业 ELISA 试剂盒（IDEXX）分别与 cELISA 检测临床鸡血清时的一致性为 97.83%和 98.1%，与 cELISA 检测蛋黄时的一致性为 100%。然而，在检测攻毒鸡的连续血清中的抗 NDV 抗体时，cELISA 比 HI 试验和商业 ELISA 试剂盒更灵敏。此外，cELISA 的竞争抑制百分率与 HI 滴度之间存在密切相关性（R=0.914）。