Preparation And Protective Efficacy Of Monoclonal Antibody Against Goose Parvovirus

Gosling plague caused by goose parvovirus(GPV),is an acute or subacute sepsis within one month-old goslings.The disease spreads rapidly and has a high fatality rate.Infected gosling characterized with septicemia,enteritis,and substantial heart,liver,kidney injuries.Vaccine is widely used to laying goose and expect the maternal antibody protect gosling avoid GPV infection.Actually,gosling plague has not been completely controlled in some area.Recently,the passive injection of viral neutralizing m Ab has been proved to be a fast,specific and effective method to prevent virus infection and will be a promising therapeutic agent.In this study,a GPV named DWY isolated from birds with gosling plague was identified after PCR and sequencing.Then,DWY was cultured and amplified on goose embryo fibroblasts(GEF).After being concentrated and purified,DWY was mixed with Freshman's adjuvant to emulsify and immunize six weeks female BALB/c mice,and myeloma cells(SP2/0)were fused with spleen cells of immunized mice in the presence of PEG,The hybridoma cells were screened by indirect ELISA method and subclone was performed in the highest pore of OD_450nm for three times,The hybridioma cell culture supernatant was used as the primary antibody for indirect immunofluorescence test,and the SP2/0 cell culture supernatant was used as the control.The obtained cell lines and mice were used to prepare ascites.After purifying the harvested ascites,antibody subtypes were determined and microcell neutralization test was conducted to determine whether the monoclonal antibody had neutralization activity.One-day-old goslings were infected at the dose of 100LD₅₀,and intramuscular injection of 5H3at the dose of 10 mg/kg body weight was performed at 24 h and 48 h after post infection.On the 5th day after infection,small intestines of goslings in the control group and experimental group were taken and slices were made for immunohistochemical test.Results indicate that the subtypes of m Abs were identified as Ig G,among which the heavy chains of 1A6,3F5,and 4D8 were Ig G1,and 2C5,5H3,and 6B9 were Ig G2b.All light chains were?and six hybridoma cell lines(1A6,2C5,3F5,5H3,6B9,and 4D8)were obtained,which could secrete monoclonal antibody(m Ab)against GPV steadily.Indirect immunofluorescence(IFA)assay showed that six m Abs could react with GEF cells infected with goose parvovirus and positive cells showed specific green fluorescence in the nucleus.The neutralization test showed that only the m Ab secreted by5H3 cell line had neutralization activity and could react specifically.The neutralization titer of its ascites against the virus in the microcell neutralization test was1:10⁴.The protective test showed that birds could be 100%protected with 5H3 m Ab injection at 10 mg/kg body weight within 48 hours post infection.Immunohistochemistry assay showed that the number of goose parvovirus in small intestine of infected birds was largely eliminated after treatment with m Ab 5H3.All these results indicate that m Ab 5H3 have potential therapeutic effect for GPV infection in goslings.The protective test showed that birds could be 100%protected with 5H3 m Ab injection at 10mg/kg body weight within 48 hours post infection.5H3 not only provides a new method and idea for the prevention and control of goose parvovirus disease,but also lays a foundation for the development of the gene engineering antibody against GPV.