Development Of Newcastle Disease Virus Sandwich Elisa Detection Assay Based On Fenobody And Ranbody Of Nanobody

Newcastle Disease(ND)is a highly contagious infectious disease caused by Newcastle Disease Virus(NDV)infection.NDV infections cause dyspnea,neurological disorders,bleeding in the mucous membranes,loose green stools,and high mortality rates,causing huge economic losses to the global poultry industry.Because the clinical symptoms of ND are easily confused with avian respiratory diseases such as avian influenza,infectious bronchitis,and infectious laryngotracheitis,the development of efficient and simple differential diagnosis technologies for respiratory diseases is imminent.At present,the ELISA is also applied to the detection of NDV.This method has the advantages of simple operation,rapid detection,and high specificity,but it cannot be widely used in early disease prevention and control due to the shortcomings of high commercial production costs,limited quantities,and difficulty in long-term storage.Nanobody is a special Ig G which is unique in camelidae and naturally lacks light chain and heavy chain first constant region.Nanobodies can be easily expressed in different systems through genetic recombination technology,and can be fused with multiple tags to provide an effective detection method for disease diagnosis.Recently,there have been studies designed to derive Fenobody(ferritin-fused Nanobody)and RANbody(Nanobody Fusion Reporter Gene)from Nanobodies for immunodiagnosis.However,there are no studies on the development of sandwich ELISAs using fenobody and RANbody as pairing reagents.Therefore,for the first time in this study,a sandwich ELISA method was established using fenobody as the capture antibody and RANbody as the detection antibody,laying a foundation for early detection of NDV and prevention and control of NDV.The main results of this study are as follows:1. The adult Bactrian camel was immunized with NDV La Sota inactivated vaccine as an antigen.After 5 immunizations,the titer of NDV La Sota-specific antibody in the serum can reach 1:10~6;Subsequently,a nanobody library with a storage capacity of 3.0×10~8 was successfully constructed by extracting total RNA from peripheral lymphocytes,nested RT-PCR,digestion,ligation and electrotransformation of TG1 cells;NDV La Sota virus particles were used as antigens,and 13 strains of specific nanobodies against NDV were screened;The affinity of the 13 strains nanobodies were analysed by indirect ELISA,5strains of Nb2,Nb4,Nb24,Nb30 and Nb49 were selected for subsequent expression to prepare fenobodies and RANbodies.2. Based on the characteristic that ferritin can self-assemble into a cage protein,5nanobodies(Nb2,Nb4,Nb24,Nb30 and Nb49)and ferritin fusion proteins(fenobodies)were constructed,expressed,and purified successfully by digestion,ligation,transformation and prokaryotic expression;the self-assembly of fenobody to form the 24 subunit structure were identified by TEM;identification of fenobodies that can specifically bind to NDV by indirect ELISA;In addition,compare the affinity and half-life of fenobody and nanobody to NDV.The affinity of fenobody to NDV is more than 12 times that of nanobody,and the half-life is also longer than 2.23 times that of nanobody.3. NDV-Nb-2,-4,-24,-30,-49 were successfully cloned into p EGFP-N1-HRP vectorby double digestion,ligation and transformation to construct RANbody-NDV-2,-4,-24,-30,-49;the successful expression of RANbody-NDV-2,-4,-24,-30,-49 were identified by IFA and Western blot;5 strains of RANbodies were identified to specifically bind to NDV by ELISA In addition,NDV structural proteins NDV-NP,-P,-M,-F,-HN were successfully constructed,and NDV-RANbodies-2,-4,-24,-30,-49 bind to the NDV-F protein were identified by Indirect immunofluorescence assay(IFA).4. The best pairing of Fenobody(NDV-fenobody-4,800 ng/well)and RANbodies(NDV-RANbody-49,1:10)was determined to establish a sandwich ELISA method for detecting NDV by checkerboard titration ELISA.The detection limit of this method is a virus suspension with a hemagglutination titer of 2~2 and 10 ng of purified NDV particles.Compared with two commercial detection methods,the sandwich ELISA method shows higher sensitivity and specificity.At the same time,it shows 98.7%consistency with HA detection,In summary,in this study,13 strains of nanobodies against NDV were screened.Then,for the first time,a sandwich ELISA method was developed using fenobody and RANbody derived from nanobodies to detect NDV in different samples.This method has high sensitivity,specificity,simple operation,and low commercial production cost.This study lays the foundation for the establishment of a new rapid detection method for NDV and the establishment of a differential diagnosis method for epidemic diseases.In addition,the sandwich ELISA platform established can be widely used for the establishment of different antigen sandwich ELISA detection methods.