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

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 2 = 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. Electronic supplementary material The online version of this article (10.1186/s12951-019-0468-0) contains supplementary material, which is available to authorized users.

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…A nanobody-peroxidase fusion would be directly suitable for colorimetric or electrochemical diagnostics. The expression in mammalian cells of such a construct allowed obtaining a chimera in which both moieties were functional and that was successfully used in competitive ELISA [45]. Recently, the search for the production of equivalent immunoreagents in bacteria moved to two promising alternatives.…”

The cysteine‐free single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications

“…Heme peroxidases are very useful enzymes for biological applications owing to their great activity and broad substrate specificity . For instance, one of the most widely utilized heme peroxidases, horseradish peroxidase (HRP), is routinely applied as the reporters of chemiluminescence‐based western blot, immunohistochemistry, and enzyme‐linked immunosorbent assays (ELISA) . Furthermore, HRP can activate various tyramide substrates, such as biotin or fluorophore derivatives, into short‐lived radical forms, which in turn covalently conjugate to neighboring proteins within 10–20 nm.…”

“…heme peroxidases, horseradish peroxidase (HRP), is routinely applied as the reporters of chemiluminescence-based western blot, 2 immunohistochemistry, 3 and enzyme-linked immunosorbent assays (ELISA). 4 Furthermore, HRP can activate various tyramide substrates, such as biotin 5 or fluorophore derivatives, 6 into short-lived radical forms, which in turn covalently conjugate to neighboring proteins within 10-20 nm. This technique, known as proximity labeling, has been widely adopted in various applications, including tyramide signal amplification, 7 electron microscopy tag, 8 and extracellular proteomic mapping.…”

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

“…For example, one study designed a reporter-nanobody fusion (RANbody) platform, in which RANbody was used in immunohistochemical detection [9]. Other works have reported the application of nanobody-HRP, EGFP, or nano-luciferase fusions derived from nanobodies to develop detection assays, label cells and tissues, and for other purposes [10][11][12][13].…”

“…The pMECS vector was used to construct phage display library. The pCMV-N1-HRP vector described in a previous study was utilised to produce nanobody-HRP fusions [11]. The pEGFP-N1 vector (Clontech, Japan) was employed as a backbone to construct the platform for nanobody-EGFP fusions.…”