A nanobody-based blocking enzyme-linked immunosorbent assay for detecting antibodies against pseudorabies virus glycoprotein E1

Pseudorabies (PR) is an acute infectious disease of pigs caused by the pseudorabies virus (PRV) and results in great economic losses to the pig industry worldwide. PRV glycoprotein E (gE)-based enzyme-linked immunosorbent assay (ELISA) has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries. Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent. However, there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies. In the present study, the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel. Then, two nanobodies against PRV-gE were screened from the immunized camel by phage display technique. Subsequently, two nanobody-HRP fusion proteins were expressed with HEK293T cells. The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA (bELISA) to detect anti-PRV-gE antibodies. Through optimizing the conditions of bELISA, the amount of coated antigen was 200 ng per well, and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5. The cut-off value of bELISA was 24.20%, and the sensitivity and specificity were 96.43% and 92.63%, respectively. By detecting 233 clinical pig sera with the developed bELISA and a commercial kit, the results showed that the coincidence rate of two assays was 93.99%. Additionallly, epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains. Simple, great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed. The bELISA could be used for monitoring and eradicating PR.