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 (R² = 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.

Keywords: Antibody; Competitive ELISA; NDV; Nanobody; Nanobody-HRP.

Scheme 1

Schematic representation of screening the nanobodies from the immunised camel (a), the platform for expressing nanobody-HRP fusion proteins (a), and designation of the developed cELISA (b)

Fig. 1

Expression, purification, and identification of the recombinant NDV-NP protein from the LaSota strain. a SDS-PAGE analysis, M, protein molecular markers; lane 1, pET-28a vector control; lane 2, bacterial lysates of the recombinant protein; lane 3, inclution body; lane 4, soluble protien; lane 5, purified protein. b Antigenic analysis of Western blotting, lane 1–5: same as a, reaction with the positive chicken sera for anti-NDV antibodies

Fig. 2

Screening the nanobodies against the NDV-NP protein. a Titres of antibodies against NDV-NP protein in the sera from the camel after the fifth immunisation. b Alignment of amino acid sequence of 9 screened nanobodies. Numbering and CDRs are according to the previous methods [4]. The residues at positions 37, 44, 45, and 47 are indicated by red arrows. c Specific reactions between the 9 screened nanobodies and NDV-NP protein. d Titration of the 9 screened nanobodies binding with the NDV-NP protein. e Analysis of the 9 screened nanobodies blocking the binding between the chicken sera and NDV-NP protein by blocking ELISA

Fig. 3

Construction of the platform for expressing the nanobody-HRP fusion protein. a Schematic presentation of the commercial vector pCMV-N1-EGFP changed into the vector to insert the main genes encoding vHRP. b Amino acid sequences encoded by the instering gene into the pCMV-N1-EGFP vector. Amino acids in the brown, yellow, blue, red, and green show the IgG signal peptide, HA tag, NDV-Nb5, HRP, and His tag, respectively

Fig. 4

Identification of NDV-Nb5-HRP fusion protein expression and secretion in the HEK293T cells. a Detection of NDV-Nb5-HRP expressed in the HEK293T cells with the anti-HA mAb as the first antibody by IFA. b Detection of the HRP activity in the NDV-Nb5-HRP fusions secreted into the culture medium of HEK293T cells. c Detection of the NDV-Nb5-HRP reaction with the NDV-NP protein using indirect ELISA

Fig. 5

Sensitivity of the cELISA for detecting anti-NDV antibodies using the NDV-Nb5-HRP fusion protein as a probe. a Distribution of the PI values from the cELISA for detecting the clinical positive chicken sera for anti-NDV antibodies. b Detection of antibodies against NDV in the serial sera from the challenged SPF chickens with NDV strain LaSota with the cELISA. c Determination of the largest dilution of positive chicken sera for anti-NDV antibodies

Fig. 6

Specificity the cELISA for detecting anti-NDV antibodies using the NDV-Nb5-HRP fusions as a probe. a Distribution of the PI values from the cELISA to detect the clinical negative chicken sera for anti-NDV antibodies. b Evaluation of the cELISA detecting the antibodies against other chicken disease viruses, including IBV, AIV, IBDV, and avian HEV

Fig. 7

Correlation between cELISA and the HI test and the commercial ELISA kit (IDEXX). a Serum titers for antibodies against NDV detected between the cELISA (PI) and HI test (log2). b Titres of antibodies against NDV in different dilution serum samples detected between the cELISA (PI) and commercial ELISA kit test (S/P)