Development And Primary Application Of Hybridoma Cell Strains Respectively Secreting Monoclonal Antibodies Against H9N2 Subtype Avian Influenza Virus And Newcastle Disease Virus

Several hybridoma cell strains were developed by fusion of SP2/0 mouse myeloma cells with splenocytes isolated from a Balb/c mouse immunized with the mix immunity antigen constituted by the inactive purified H9N2 subtype Avian Influenza virus(AIV),Newcastle Disease virus(NDV)LaSota strain and immunity intensifier Bursin,and the technique which fused together,filtrated separately was adopted.Two indirect enzyme-linked immunosorbent assay(ELISA)methods were established to respectively detect monoclonal antibodies(McAbs)against H9N2 subtype AIV and NDV secreted by hybridoma cell strains.Two hybridoma cell strains secreting the monoclonal antibodies against H9N2 subtype AIV were named C5,D5 and four hybridoma cell strains secreting the monoclonal antibodies against NDV were named 4B4,4B8,4C4,4E8.Three hybridoma cell strains secreting McAbs against H9N2 subtype AIV, named C8,D7,E8 were obtained after fusion with the single immunity antigen H9N2 subtype AIV,two hybridoma cell strains secreting McAbs against NDV,named 7D2, 8C10 were obtained after fusion with the single immunity antigen NDV by traditional monoclonal antibody technique.The maximal ELISA titers of the McAbs against H9N2 subtype AIV in cell supernatants and ascites were 1:1024 and 1:102400,respectively.The maximal ELISA titers of the McAbs against NDV in cell supernatants and ascites were 1:512 and 1:51200,respectively.All McAbs were categorized into IgG subtype.Average chromosome number of each hybridoma cell strain was between 96 and 106.These hybridoma cell strains can stably secrete McAbs after twenty-five serial passages and freezing and anabiosis three times in six months.The hybridoma cell strains C8,7D2,4B4 showed good stability to acid,alkali and heat.The result of cross-reaction in HI demonstrated that the McAbs against NDV were subtype-specific.The result of the hybridoma cell strain C8 and 7D2 by interdicted ELISA indicated that the two McAbs had good specificity to relevant viruses.The McAbs against H9N2 subtype AIV had high HI titer to H9N2 subtype AIV,but failed to neutralize the H9N2 subtype AIV in chicken egg embryo.These conflicting results of HI cross-reaction and neutralization test may be concluded that the HA epitopes is not same as the sites of neutralization in AIVs.Antigen capture enzyme-linked immunosorbent assay(AC-ELISA)was developed by the McAbs in ascite of the cell strain C8.The optimum conditions in some critical steps of the ELISA were set as follows:the optimal concentrations of the coating antigen and the McAbs against H9N2 subtype AIV were 1:1600 and 1:1600. The limit of quantitative detection in H9N2 subtype AIV was 843.75ng/mL.The AC-ELISA established in the present study should be further consummated to suit producing rapid diagnostic slip by combining gold-labeled chromatography to be applied in practice.