Pseudorabies (PR) is an acute infectious disease of pigs caused by the PR 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.
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