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

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.

Novel P22-monoclonal antibody based blocking ELISA for the detection of African swine fever virus antibodies in serum

African swine fever(ASF)is a highly infectious,transboundary viral disease of domestic and wild pigs,and is currently the most serious threat to world swine production,resulting in significant economic loss.In the absence of vaccines and treatments,the control of the disease entirely depends on accurate and early diagnosis accompanied by the culling of infected pigs.Thus,a highly specific and sensitive diagnostic assay is required during an outbreak and surveillance of the disease.In this study,a highly sensitive,specific,rapid and repeatable P22-monoclonal antibody-based blocking enzyme-linked immunosorbent assay(bELISA)assay was developed for the detection of antibodies against genotype I and II African swine fever viruses(ASFVs).A total of 806 pig serum samples were tested to evaluate the performance of the diagnostic assay.To determine the PI(percent Inhibition)cut-off value,receiver-operating characteristic(ROC)analysis was applied.According to the ROC analysis of the data,98.10%specificity and 100%sensitivity were recorded when the threshold cut-off value of PI was established at 47%.In addition,the assay was able to detect ASFV antibodies as early as 9 days post-infection when serum samples from experimentally infected pigs were used.Taking all together,the results of the present study indicated that the P22-mAb based bELISA assay can be used for rapid and accurate detection of antibodies against ASFV,which could play a valuable role in the containment and prevention of ASFV as an alternative to other serological diagnostic methods.Also,this study will assist researchers to further investigate the immunogenic importance of P22 protein in ASFV infection.