| Chicken Newcastle disease is an acute,highly contagious and infectious disease of poultry caused by Newcastle disease virus(NDV),which has brought serious economic losses to the world’s chicken industry.At present,virus isolation and identification,RT-PCR,HA test,HI test and gene sequencing are often used in the diagnosis of Newcastle disease.However,due to the widespread use of live vaccines,NDV isolated from the field or NDV antigens detected by various methods may also be vaccine viruses,which often couldn’t be diagnosed in time due to the lack of a method to quickly distinguish the virulence of NDV.Based on the monoclonal antibody 1E3,this study established a double-antibody sandwich ELISA method,which could quickly distinguish between velogen NDV strains and lentogen vaccine strains,and provide an important tool for rapid diagnosis and epidemic prevention of Newcastle disease in chickens.This study mainly includes the following aspects;1.Preparation of monoclonal antibodies against chicken Newcastle disease virus La Sota strain.In this study,the NDV La Sota strain virus was inoculated into the chicken embryo allantoic cavity.The chicken embryo allantoic fluid was collected under aseptic conditions,the virus was concentrated and purified,and the virus was inactivated by formaldehyde,mixed with adjuvants and emulsified to achieve the effect of water-in-oil.The SPF BALB/c mice aged 6-8 weeks were inoculated subcutaneously in the neck,and the fusion requirements were met after three immunizations.After cell fusion and subcloning,five hybridoma cells were screened and named 1E3,5E1,7A8,8E2,8F11.2.Identify the reactivity of the above monoclonal antibodies with different virulence NDV.Preliminary identification of the five hybridoma cells showed that all monoclonal antibodies had good stability;they showed good specific reaction with La Sota strain,but negative with avian influenza virus(AIV),avian infectious bursal disease virus(IBDV)and serum 4 fowl adenovirus(FAdV-4);Antibody isotyping test showed that 1E3,5E1,8E2,and 8F11 were IgG1 subtypes,7A8 was IgM subtype,and their light chains were κ chains;7A8 had certain neutralizing activity against La Sota strain,and had important clinical therapeutic potential;1E3 had significantly different binding ability with the velogen and lentogen NDV strains,and occurred with the vaccine lentogen La Sota strain Strong binding reaction,and the reaction with NDV HN09-68 strain and NDV F4RE9 strain was negative.The results of SDS-PAGE,Western blot and secondary mass spectrometry showed that 1E3 was the monoclonal antibody corresponding to the NP protein of NDV La Sota strain.It could be seen that the anti-NDV NP protein monoclonal antibody 1E3 had important potential application value in distinguishing between the velogen and lentogen strains of NDV.3.The monoclonal antibody 5E1,which had extensive binding activity to NDV,was selected as the capture antibody,and 1E3 was the enzyme-labeled antibody.The optimal coating antibody concentration,the optimal enzyme-labeled antibody concentration,and the optimal reaction time were determined.A double-antibody sandwich ELISA method was established to detect NDV.The method had strong specificity,high sensitivity and good stability.After preliminary application,it could effectively distinguish NDV with different virulence,providing a reliable and convenient means for epidemiological research and rapid diagnosis of NDV. |
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