Preparation And Application Of Monoclonal Antibodies To Cyprinid Herpesvirus 2 And Development Of Colloidal Gold Immunochromatographic Test Strips

Carassius carassius is an important economic fish in China,and it accounts for a large proportion in freshwater aquaculture,but the frequent occurrence of herpesviral haematopoietic necrosis in recent years has brought serious economic losses to the healthy aquaculture of crucian carp.When the fish is infected with Cyprinid Herpesvirus2（CyHV-2）,the body surface and gills bleed,the eyes protrude,the internal organs are swollen with bleeding spots,and the swim bladder wall is spotted with bleeding,the virus is highly infectious and pathogenic,and the mortality rate of fish infected with the virus can be up to 80%or more.So far there are no effective measures to control CyHV-2infection,and the tests used to diagnose and identify CyHV-2 infection are usually based on molecular biology,In addition,some scholars use anti-CyHV-2 ORF25 and ORF90monoclonal antibodies to establish enzymelinked immunosorbent assay,and use anti-CyHV-2 ORF92,ORF72,ORF121,ORF66,ORF25 monoclonal antibodies to establish indirect Immunofluorescence Assay,but these methods are time-consuming and expensive,and are mostly limited to well-equipped laboratories,so it is important to develop a rapid,specific and convenient field detection method for the prevention and control of CyHV-2 infection.In this study,CyHV-2 was purified by sucrose density gradient ultracentrifugation and used as the antigen to immunize BALB/c mice.Ten monoclonal antibodies（MAbs）against CyHV-2 with good specificity were successfully prepared by cell fusion technique and Dot-blot screening,Among them,five MAbs belonged IgG₁ isotype,one MAbs belonged IgG_2a isotype,one MAbs belonged IgG₃ isotype and three MAbs belonged IgM isotype,named 3E11-H3,4E4-H9,3G9-B11,2E1-B10,1F5-A3,4C4-A7,3B4-G5,4F4-B7,3B8-C2 and 4F4-B3,respectively,and the possible proteins recognized by Western-blot detection combined with mass spectrometry identification were found to be ORF37R,ORF81,ORF88,ORF151R,ORF55R ORF115,ORF68 and ORF2A,and the temporal sequence of protein expression of each monoclonal antibody was analyzed.In vitro neutralization experiments showed that all ten Mabs can attenuate CPE by CyHV-2 in vitro among which 2E1-B10 had the best neutralization ability,and the neutralization index was determined to be 1:22 using the fixed virus dilution serum method.Hybridoma cells screened by intraperitoneal injection were used to prepare mouse ascites antibodies,which were purified using Protein A/G affinity chromatography,and antibody potency was detected by Dot-blot.These three MAbs were paired in pairs and colloidal gold immunoassay strips were prepared by double antibody sandwich method.The specificity results showed that the test strip could be used to detect not only CyHV-2 but also Koi herpesvirus（KHV）,but there was no cross-reactivity for other common freshwater fish viruses（e.g.GCRVⅠ,GCRVⅢ,SVCV,LYCIV）,indicating that the test strip has good specificity.In addition,the test strip has good stability and can be stored at room temperature for half a year and still detect CyHV-2.After homogenizing the tissues of CyHV-2 infected crucian carp and healthy crucian carp,the test strip was found to be positive for spleen and kidney tissues of CyHV-2 infected crucian carp and negative for spleen and kidney tissues of healthy crucian carp,which indicates that the test strip can be used for the clinical analysis of CyHV-2.The test strips were used for clinical rapid detection of CyHV-2.In conclusion,we established a colloidal gold immunochromatographic test strip for the detection of CyHV-2,which has good sensitivity,specificity and stability,and can detect CyHV-2 in samples within 10 min,which is faster and more convenient compared with conventional biomolecular and immunological detection methods,and can be applied to the initial diagnosis of CyHV-2 infection without the need for human intervention.In addition,2E1-B10 was found to have better neutralization abilities by in vitro neutralization assay,and four possible proteins were identified by mass spectrometry identification,and the eukaryotic plasmids pEGFP-N1-ORF84,pEGFP-N1-ORF88,pEGFP-N1-ORF90 and pEGFP-N1-ORF7 were constructed and transfected into HEK-293T cells.Western-blot results showed that they were successfully expressed in HEK-293T cells and the protein recognized by 2E1-B10 was ORF90.In addition,CyHV-2viral receptor with a molecular weight of about 130 KDa was identified by viral overlay protein binding assay（VOPBA）.This experiment will help to elucidate the function and mechanism of ORF90 protein in the process of CyHV-2 infestation of Gi CF cells,identify potential antiviral targets,and provide a theoretical basis for the development of novel antiviral strategies.