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

作　　者：Huanhuan Lü Pinpin Ji Siyu Liu Ziwei Zhang Lei Wang Yani Sun Baoyuan Liu Lizhen Wang Qin Zhao 

机构地区：[1]Department of Preventive Veterinary Medicine,College of Veterinary Medicine,Northwest A&F University/Shaanxi Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology,Ministry of Agriculture and Rural Affairs,Yangling 712100,China

出　　处：《Journal of Integrative Agriculture》2024年第4期1354-1368,共15页农业科学学报（英文版）

基　　金：supported by the National Natural Science Foundation of China(32273041);the Key R&D Program of Shaanxi Province,China(2022NY-104);the Natural Science Foundation of Shaanxi Province,China(2022JC-12)。

摘　　要：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.

关 键 词：NANOBODY nanobody-HRP blocking ELISA PRV-gE ANTIBODY 

分 类 号：S858.28[农业科学—临床兽医学]