Preparation And Application Of Monoclonal Antibodies Against Avian Infectious Bronchitis Virus GX-YL5 Strain And Its S1 And N Proteins

Avian infectious bronchitis（IB）is an acute,highly contagious and viral respiratory tract disease of chickens,caused by infectious bronchitis virus（IBV）.IB clinical symptoms are characterized by trachea rales,cough and sneeze,which can lead to decrease of weight gain,feed efficiency,egg production and egg quality,and cause serious economic losses to the poultry industry.IBV contains four structural proteins of S,E,M and N proteins.The S protein can be cleaved into S1 and S2proteins by protease.The S1 protein can stimulate the body to produce hemagglutination inhibition antibody,neutralizing antibody and cell-mediated immune response,and is closely related to pathogenicity and tissue tropism.The N protein is highly conserved and has immunologically recognized associated antigen sites,which can activate the body’s cytotoxic T lymphocytes and helper T cells,and enhance the ability of lymphocytes to produce antibodies.IBV genome is prone to mutation,which leads to complicated serotypes and incomplete cross protection between different serotypes,which brings great difficulties for the diagnosis and control of IB.Therefore,the establishment of accurate,rapid and efficient detection method is of great practical significance for IB clinical diagnosis and prevention and control.In view of this,in this study,monoclonal antibodies against Guangxi IBV dominant serotype representative strain GX-YL5 and its S1 and N proteins were prepared,and were further applied to the immunohistochemical detection and establishment of double sandwich antibody ELISA methods.The details are as follows:1.Preparation of IBV monoclonal antibody immunogenThe IBV GX-YL5 allantoic fluid stored by the research group was concentrated by low temperature gradient centrifugation and ultracentrifugation and quantified by q PCR.Identification of eukaryotic expression S1 protein was carried out and the concentration was determined.The positive plasmid p GEX-4T-N containning the N gene of IBV GX-YL5 strain constructed by the research group was transformed and induced to express a large amount of N protein.The N protein was purified by GST high affinity purification column,and further identified and determined the concertration by SDS-PAGE,Western blot and BCA Protein Assay Kit.The results showed that IBV GX-YL5 allantoic fluid was concentrated 107.04 times.The corresponding target proteins were detected by Western blot.The S1 protein concentration was 2.35 mg/m L,and the N protein concentration was 142.205 mg/m L.2.Preparation and identification of monoclonal antibodies against IBV GX-YL5 strain and its S1 and N proteinsIBV GX-YL5 strain and its S1 and N proteins were used as immunogen to immunize 6-week-old female BALB/c mice.After cell fusion,indirect ELISA method screening and three rounds of limited dilution method cloning,hybridoma cell lines which can stably secrete monoclonal was obtained for each immunogen,named Y2D1,S1D1 and N2D5,respectively.A large number of monoclonal antibodies were prepared by ascites induction in mice.Western blot results showed that monoclonal antibody Y2D1 reacted specifically with IBV GX-YL5 whole virus and the S1 protein,S1D1 with the S1 protein,and N2D5 with the N protein.IFA verification results showed that all three monoclonal antibodies showed specific fluorescent signals in CEK cells infected with GX-YL5 strain.The antibody titer test results showed that the titers of Y2D1,S1D1 and N2D5 were≥2¹⁶,≥2¹⁷and≥2¹⁷,respectively.The antibody subtype identification results showed that Y2D1,S1D1 and N2D5 were Ig G2b,Ig G1 and Ig G2b,respectively.The results of specific tests showed that the three monoclonal antibodies only reacted specifically with IBV,but did not reacted with 7 viruses including avian influenza virus（AIV）.The cross-reactivity test was carried out with IBV reference strains Beaudette and M41,vaccine strains H120,4/91 and LDT3 and field isolates including LDT3-type,4/91-type,Mass-type,LX4-type and Taiwan-type.The results showed that the Y2D1monoclonal antibody only had a positive reaction with the vaccine strain H120 and LDT3-type isolates;the S1D1 monoclonal antibody only had a negative reaction with the reference strain Beaudette and LX4-type isolates;and the N2D5 monoclonal antibody cross-reacted with all tested strains.The neutralization test results showed that the neutralizing titers of Y2D1,S1D1 and N2D5 monoclonal antibodies to IBV GX-YL5 were1:32,1:128 and 1:256,respectively.3.Application of monoclonal antibodies in immunohistochemical detectionImmunohistochemical detection can be used for antigen localization,qualitative and relative quantification analyses.In this study,the prepared Y2D1,S1D1 and N2D5 monoclonal antibodies were used as primary antibodies,and immunohistochemical techniques were used to dectect the IBVs in trachea and kidney of SPF chickens in 5 days post infection（dpi）,7 dpi,11 dpi,14 dpi after artificial infection with IBV isolates GX-NN130048,GX-NN160421,GX-QZ170728 and GX-QZ171023.The results showed that the virus signals could be detected in trachea and kidney tissues in 5 dpi,7 dpi,11 dpi,14 dpi.More virus signals were detected in trachea tissue of infection with GX-NN130048 and GX-NN160421 strains.The highest viral levels were dected in kidney tissue of infection with GX-NN130048 strain at 5 dpi.A large number of virus signals were detected in kidney tissue of infection with GX-QZ171023 strain at 5 dpi,7dpi,11dpi.4.Establishment of double antibody sandwich ELISA detection methodDouble antibody sandwich ELISA is an efficient and rapid method for antigen detection.In this study,the S1D1 and N2D5 monoclonal antibodies against the S1 and N proteins of GX-YL5 strain were used to establish double antibody sandwich ELISA.The rabbit polyclonal serum against the S1-N protein of IBV strain GX-YL5 was used as coating antibody and monoclonal antibodies S1D1 and N2D5 were used as enzyme-labeled antibodies,namely S1D1（HRP-S1）and N2D5（HRP-N）.The optimal reaction conditions for double antibody sandwich ELISA based on S1D1 were determined after optimizing the conditions.The coating concentration of rabbit S1-N polyclonal antibody was 20μg/m L,and the detection concentration of HRP-S1 was 1μg/m L.The blocking time was 60 min;the sample incubation time was 30 min;the action time of enzyme-labeled antibody HRP-S1 was 90 min;the action time of substrate chromogenic solution was 15 min.And the minimum detection virus amount was 10^3.025 EID₅₀/m L.The optimal reaction conditions for double antibody sandwich ELISA based on N2D5 were determined after optimizing the conditions.The coating concentration of rabbit S1-N polyclonal antibody was 5μg/m L;the detection concentration of HRP-N was 2.5μg/m L.The blocking time was 60 min;the sample incubation time time was 90 min;the action time of enzyme-labeled antibody HRP-N was 2 h;the action time of the substrate chromogenic solution was 20 min.And the minimum detection virus amount was 10^1.025EID₅₀/m L.There was no cross reaction when negative allantoic fluid and AIV,NDV,ILTV,a MPV,IBDV,ALV,MDV were detected by the two double antibody sandwich ELISA methods.The coefficients of variation within and between batches were≤10%.62 clinical samples were detected by the two double antibody sandwich ELISA methods and RT-PCR.The results showed that the coincidence rate between the double antibody sandwich ELISA based on monoclonal antibody S1D1 and RT-PCR was 69%,and that between the double antibody sandwich ELISA based on monoclonal antibody N2D5 and RT-PCR was 87%.The results showed that double antibody sandwich established in this study ELISA is specific,sensitive and convenient,and is suitable for the detection of large number of clinical samples.In summary,specific and highly efficient monoclonal antibodies Y2D1,S1D1,N2D5 against IBV GX-YL5 strain and its S1 and N proteins have been successfully prepared in the present study.All the three monoclonal antibodies can be used to detect the distribution of virus by immunohistochemistry.The two double-antibody sandwich ELISA methods based on S1D1 and N2D5 have good specificity,sensitivity and repeatability.This study laid a foundation for the mass,rapid and accurate diagnosis of IBV clinical samples,and also provided a convenient and simple choice for the diagnosis of IBV.