Preparation And Preliminary Application Of Monoclonal Antibodies Against Various Important Proteins Of Influenza Virus

Influenza virus?IV?belongs to the Orthomyxoviridae family,which is an important zoonotic pathogen.It's responsible for the seasonal flu outbreak and periodic flu pandemics,bringing great harm to the development of livestock and poultry breeding and human public health safety.In this research,according to the sequence analysis of various proteins?PB2,PB1,PA and NP?and different subtypes of influenza virus,broad-spectrum and multi-application monoclonal antibodies were prepared and screened,which providing application support for the prevention,detection and diagnosis of influenza virus.In the study,two segment sequences were selected base on its sequence analysis as templates to immune mice,which are high homology and good immunogenicity.After cell fusion experiment,a total of 46 strains of positive hybridoma clones be screened by indirect ELISA and Western Blotting methods,and identify the monoclonal antibodies subclass.At last,nine broad-spectrum and high sensitivity monoclonal antibodies?PB2 MAbs:8A5,2C4,PB1 MAbs:7H11,1B7,PA MAbs:9A8,9E9,10E11,NP MAbs:10E9,3F3?were screened by western blot.The antigenic epitope of nine monoclonal antibodies were confirmed and analysed by using the amino acid site-specific mutation and pepscan methods.These results indicated the main epitope region of MAbs 8A5,2C4,7H11,1B7,9A8,9E9 and 10E11 were located respectively₃₇GRQEKNPALRMKWMM₅₁,₆₆₉GKDAGPLTEDPDEGTAGVESA₆₈₉,₂₂FPYTGDPPYSHGTGT₃₆,₆₈₇QMYQKCCNLFEKF₆₉₉,₁₉₆RGEETIEERF₂₀₅ and ₆₇₁ALRDNLEPGTFDLGGLYEAI₆₉₀.The recognition epitope of MAbs 10E9,3F3 were located respectively ₈₄SAGKDP₈₉ and ₃₂₀ENPAH₃₂₄.Besides,the application of these nine monoclonal antibodies were analyzed by Co-Immunoprecipitation and laser confocal assay.These results suggested that MAbs 8A5,2C4,7H11,1B7,9A8,9E9,10E11,10E9 and 3F3 could be all used in the Co-Immunoprecipitation.According to the results of laser confocal assay,these MAbs,8A5,2C4,7H11,10E9,can be applied in laser confocal assay,while MAbs 1B7,9A8,9E9,10E11,3F3 have non-specific phenomena.In order to establish efficient and rapid diagnostic technology to detect the influenza virus,the above-mentioned two NP monoclonal antibodies?10E9 and 3F3?,which were IgG2a subtypes,were purified in ascites.MAb 3F3 was labeled with colloidal gold particles of 20 nm,MAb 10E9 was dispensed as the test line and goat anti-mouse IgG was dispensed as the control line.Based on the principle of immunochromatography,a colloidal gold immunochromatographic strip for rapid detection of influenza virus was developed.