Preparation Of Monoclonal Antibody Against Cyprinid Herpesvirus 3 And Preparation Of Colloidal Gold Immunoassay Test Strip

With the development of international trade,the import and export of ornamental fish has become an important part of China’s fishery development,and koi（Cyprinus Carpio haematopterus）is the main breed of ornamental fish in China.Since the outbreak of Koi herpesvirus disease（KHVD）in Germany in 1997,CyHV-3 is the main pathogen of this disease,which has been reported in more than 30 countries and regions,causing serious damage to koi breeding industry.Therefore,in order to prevent the outbreak of KHVD,it is necessary to establish effective and rapid detection methods.At present,there are many immunological detection methods for CyHV-3.The preparation of whole virus monoclonal antibody of CyHV-3 can provide multiple targets for the establishment of CyHV-3 immunological detection method,and provide technical support for the simple and rapid detection of CyHV-3.In addition,CyHV-2 can cause herpes virus necrosis of hematopoietic organs,bringing inestimable losses to crucian carp breeding industry.Since the homology of CyHV-3 and CyHV-2 reached 81.55%,the preparation of CyHV-3 and CyHV-2 cross monoclonal antibody,it can provide technical support for the establishment of immunological detection methods for CyHV-3 and CyHV-2.By screening monoclonal antibodies with high titer and using immunocolloidal gold technology,a preliminary test strip for CyHV-3 immunoassay was developed,which can detect CyHV-3 rapidly and sensitively.In this study,CyHV-3 virus particles were isolated and purified,CyHV-3 full virus monoclonal antibody and CyHV-3 and CyHV-2 cross monoclonal antibody were prepared and their functions were identified,and CyHV-3 colloidal gold immunoassay strip was preliminary developed.The research is mainly composed of the following three aspects:（1）CyHV-3-GZ1301 virus strain was used to infect the carp cell line（CCB）,and a large amount of virus infection fluid was collected.CyHV-3 virus was purified by sucrose density gradient super centrifugation.The 4-week-old female BALB/c mice were immunized with purified CyHV-3 for 4 times,and the spleen cells of the mice were fused with SP2/0 tumor cells.The positive cell lines were screened by dot-blot method,and the positive cell lines were verified by Western-blot.Ascites was prepared with the selected positive cell lines and the ascites titer was detected by dot-blot assay.CCB cells infected with CyHV-3 were detected with the selected monoclonal antibody.The results showed that CCB cells infected with CyHV-3 began to shrink and become round and vacuole on day 6,presenting a typical cytopathic effect.Many CYHV-3 viruses were collected under sucrose density gradients of 40%-50%and 50%-66%,and the purified CyHV-3 virus particles were observed by transmission electron microscopy.The positive cell lines screened by Dot-blot were subcloned and expanded by trans hole culture.Five positive cell lines that could specifically react with CyHV-3 were screened by Western-blot,which were 1G6,2A11,3D2,4B9 and 4E10,respectively.Their ascites titers were 2A11 and1:10000,respectively.The titer of 3D2 was 1:10000.The titer of 4B9 was 1:10000.The titer of 4E10 was 1:20000.Immunofluorescence detection of CCB cells infected with CyHV-3 showed that 1G6,2A11,3D2,4B9,4E10 antibody could detect CyHV-3 in CCB cells,presenting green fluorescence signal.In this study,monoclonal antibody against CyHV-3 was prepared to provide more antibody targets for the establishment of CyHV-3 immunological detection methods.（2）CyHV-2-YC01 strain stored in our laboratory was used to infect GiCF,a large amount of virus infection fluid was collected,and CyHV-2 virus was purified by sucrose density gradient super centrifugation.The fusion cells were droplet plates,and positive cell lines that could react with CyHV-3 and CyHV-2 were screened by dot-blot and Western-blot methods.Antibody subtypes were detected,ascites was prepared and ascites titer was detected.CCB cells infected with CyHV-3 and GiCF cells infected with cy HV-2 were detected by immunofluorescence assay.The results showed that a total of 6positive cell lines were screened,which could react with cy HV-2 and CyHV-3,respectively,3E11,Ig G₃,4E4 and Ig G_2a.4F4,Ig M;2B4 Ig G₁ type;2C8 Ig G₁ type;2D11Ig G₁ type.The ascites titer of 2B4 was 1:80,000 in CyHV-3 virus and 1:160,000 in CyHV-2 virus.The titer of 2C8 was 1:40,000 for CyHV-3 and 1:40,000 for CyHV-2.The titer of2D11 was 1:160000 in CyHV-3 and 1:40000 in CyHV-2.In cell immunofluorescence detection,3E11,4E4 and 4F4 could detect CCB cells infected with CyHV-3 and GiCF cells infected with CyHV-2.Since 2B4,2C8 and 2D11 were selected cell lines at a later stage,immunofluorescence detection has not been performed.The cross antibodies of CyHV-3 and CyHV-2 prepared in this study can provide more theoretical basis for the establishment of immunological detection methods of CyHV-3 and CyHV-2.（3）In order to prepare monoclonal antibody against CyHV-3 with a high titer,the fused cells were dropped again,and positive cell lines that could specifically react with CyHV-3 were screened by Dot-blot,Western-blot and immunofluorescence to prepare ascites and detect ascites titer.The monoclonal antibodies were paired by the principle of immunocolloidal gold to prepare CyHV-3 immunoassay strip.The results showed that four positive cell lines with high titer and strong specificity were screened by dot-blot and Western-blot,namely 1G11,2C9,3A6 and 3H4,which could recognize CCB cells infected with CyHV-3 in cell immunofluorescence.The ascites titers were detected by dot-blot,and the antibody titers were 1G11 strains 1:40000,2C9 strains 1:16000,3A6strains 1:80000,and 3H4 strains 1:40000.CyHV-3 colloidal gold immunoassay strip was preliminarily developed,which could detect purified CyHV-3.CyHV-3 colloidal gold immunoassay strip was preliminarily developed,which could detect purified CyHV-3.The immunoassay strip prepared with 3A6 antibody gold and 2C9 antibody film was better,which could specifically detect CyHV-3,had no cross reaction with CyHV-2,and could detect 20 times diluted CyHV-3 virus infection fluid.The sensitivity of the strip will be further optimized in the future.