Establishment Of Sandwich ELISA For Differential Diagnosis Of Class Ⅰ And Class Ⅱ Newcastle Disease Viruses

Newcastle disease(ND)is a highly contagious and highly contagious disease caused by the Newcastle disease virus(NDV).The disease can lead to nervous system,respiratory system and digestive system disorders of diseased birds.Its high incidence rate,rapid transmission and high fatality rate have brought great economic losses to poultry industry worldwide.NDVs can be divided into two categories:Class I and Class II.The vast majority of Class I NDV are either weakly or non-toxic strains,while Class II NDVs include the majority of highly virulent and some weakly virulent strains currently isolated.Class II NDV is mainly prevalent,and Class I NDV is also prevalent in certain regions in poultry farming industry of China.Studies have shown that Class I NDV can enhance its virulence through continuous passage through airbags or chicken embryos,so there is a risk of increased virulence in Class I NDV.The differential diagnosis of Class I and Class II NDV is of great significance for ND prevention and control.At present,the main methods used for the differential diagnosis of Class I and Class II NDV are molecular biology detection methods such as RT-PCR or fluorescence quantitative PCR.Although these methods have high sensitivity,they have disadvantages such as high cost,complex operation,long cycle,and difficulty in large-scale detection.ELISA has the advantages of simple operation and short detection cycle,but currently there is no commercial ELISA kit for differential diagnosis of Class I and Class II NDV.Nanobodies are a new type of genetically engineered antibodies,and some scholars have used their derivatives Fenobody and RANbody to establish sandwich ELISA for detecting pathogens.This study screened specific and shared antigenic epitopes of nanobodies against Class I and Class II NDV,and established a sandwich ELISA for rapid and accurate differential diagnosis of Class I and Class II NDV using Fenobody and RANbody platforms.It provides technical support for rapid differential diagnosis and precise prevention and control of NDV.The main results of this study are as follows:1.18 strains of nanobodies against NDV were screened and obtainedCamels were immunized with Class II gene type VII NDV inactivated virus particles as immunogens.After the final immunization,the titer of anti NDV antibodies in the serum reached 1×10~7.After isolation of peripheral blood lymphocytes,RNA extraction,nested PCR amplification,digestion,connection and transformation,the reservoir with a capacity of 1.2×10~9 was successfully constructed.Positive rate of NDV phage nanobody display library is94.79%.After three rounds of screening,18 specific nanobodies against NDV were successfully screened(NDV-VII-7,-9,-13,-19,-28,-38,-41,-43,-50,-54,-71,-75,-79,-80,-82,-92,-94,-95),laying a good foundation for the subsequent screening of specific and shared epitope nanobodies against Class I and Class II NDV.2.Five nanobodies that bind with high affinity to epitopes common to class I and class II NDV and one nanobody that binds to an epitope unique to class II were obtained from the screening18 NDV nanobodies were fused with HRP to prepare anti NDV RANbodies using RANbody technology(named NDV-VII-RANbody-7,-9,-13,-19,-28,-38,-41,-43,-50,-54,-71,-75,-79,-80,-82,-92,-94,-95).Five RANbodies(NDV-VII-RANbody-7,-13,-41,-71,-92)with high affinity for the common epitopes of Class I and Class II NDVs and one RANbody(NDV-RANbody-95)that only binds to the specific epitopes of Class II NDVs were identified through ELISA and IFA screening.Constructed the six Fenobodies using Fenobody technology.Western blot and SDS-PAGE analysis showed that the six Fenobodies(Fenobody7,-13,-41,-71,-92,-95)were successfully constructed,expressed,and purified.Transmission electron microscopy observation showed that all Fenobodies self-assembled into 24aggregates.Indirect ELISA and sandwich ELISA were used to verify the binding ability of six Fenobodies with NDV.3.A sandwich ELISA common to class I and class II and a sandwich ELISA specific to class II were establishedFenobody-92 and Fenobody-95 were determined by chessboard titration as capture antibodies for Class I and Class II universal sandwich ELISA and Class II specific sandwich ELISA,respectively,and RANbody-7 is a detection antibody for two types of ELISA.The optimal encapsulation amount for the capture antibody Fenobody-92 in the universal sandwich ELISA was determined to be 200 ng/well,and Fenobody-95 in the Class II specific sandwich ELISA was determined to be 400 ng/well by optimizing the detection conditions of two types of ELISA.The optimal dilution ratio for detecting antibody RANbody-7 using two methods is 1:10.The Cut-off value of the universal ELISA is 0.158 and Class II specific sandwich ELISA is 0.218.When the NDV HA titer in the sample is higher than 2~1 or the copy number is greater than 2.9×10~6copies/can be detected by two ELISA methods.Ten poultry pathogens including AIV,FAd V,Mg were tested with the two established ELISA methods with negative results.The results of animal experiments indicate that the two sandwich ELISA methods established can detect NDV in different tissue samples.Compared with the commercial NDV sandwich ELISA kit and HA,the both sandwich ELISA methods showed higher sensitivity.In summary,this study successfully screened 18 specific nanobodies against Class II gene type VII NDV and expressed and prepared their fusion protein(RANbody)with HRP.Five nanobodies that can bind to the common antigenic epitopes of Class I and Class II NDV were screened using IFA and ELISA,as well as one nanobody that only binds to the specific antigenic epitope of Class II NDV.Subsequently,the six nanobodies were expressed to prepare their fusion protein with ferritin(Fenobodies).Fenobody-92 and Fenobody-95 were used as capture antibodies for Class I and Class II NDV universal sandwich ELISA and Class II NDV specific sandwich ELISA.RANbody-7 was used as the detection antibody for two sandwich ELISA methods,and a universal sandwich ELISA method that can detect Class I and Class II NDV and a Class II NDV specific sandwich ELISA method that can only detect Class II NDV were established.The combination of two sandwich ELISA methods established can quickly and accurately differentiate and diagnose Class I or Class II NDV infections.Therefore,the two sandwich ELISA methods established can be applied to monitor Class I or Class II NDV infection in poultry or wild bird populations,providing technical support for the prevention and control of ND.