H9N2亚型禽流感病毒中和单克隆抗体的制备与应用

doi:10.16431/j.cnki.1671-7236.2021.10.028

Abstract

Abstract: In order to establish a rapid immunochromatographic assay for H9N2 subtype Avian influenza virus (AIV), BALB/c mice were immunized with H9N2 subtype AIV purified by differential centrifugation. The splenocytes of immunized mice were fused with myeloma cell SP2/0 and selectively cultured with HAT. MDCK cells were infected with H9N2 subtype AIV to establish a monoclonal antibody detection method of heterologous immunoperoxidase monolayer assay (IPMA). The neutralizing monoclonal antibody against H9N2 subtype AIV was screened and identified by IPMA screening and continuous cloning of hybridoma cells. Using colloidal gold labeled HA monoclonal antibody, pairing HA monoclonal antibody and sheep anti mouse IgG as detection line and quality control line, a rapid detection strip for H9N2 subtype AIV was prepared, and its specificity and sensitivity were determined. The results showed that 11 hybridoma cells stably secreting monoclonal antibody against H9N2 subtype AIV were obtained, and the titers of monoclonal antibodies ascites IPMA were 1.28×10^-4 to 2.56×10^-5. Monoclonal antibodies 3A2, 5H6, 6B8, 7E10 and 9G12 showed hemagglutination inhibitory activity in hemagglutination inhibition test (HI), and their HI titers ranged from 6log2 to 9log2. Monoclonal antibodies 3A2, 6B8 and 9G12 had significant virus neutralizing activity against H9N2 subtype AIV in virus neutralization test. The neutralizing titers were 1:6 400, 1:25 600 and 1:25 600 respectively. Western blotting showed that the neutralizing monoclonal antibody recognized the linear epitope of HA protein. The titer of H9N2 subtype AIV allantoic fluid detected by paired monoclonal antibodies 3A2 and 9G12 was 9log2, the sensitivity was equivalent to that of classical hemagglutination test (HA), and there was no cross reaction with other subtypes of AIV (H1, H3, H5, H7), Newcastle disease virus and chicken infectious bursal disease virus. In this study, the monoclonal antibody against H9N2 subtype AIV with virus neutralizing activity was prepared, and the H9N2 subtype AIV detection strip was preliminarily developed, which laid a good research foundation for the new vaccine development and rapid detection of H9N2 subtype AIV.

Key words: H9N2 subtype Avian influenza virus; monoclonal antibodies; hemagglutination inhibition; virus neutralization; immunochromatographic strip

CLC Number:

S852.65⁺7

LI Qingmei, GUO Junqing, MENG Zekun, LI Yanhua, LIU Xiao, SHI Jianzhou, LI Ge, CHAI Shujun, LUO Jun, DENG Ruiguang, ZHANG Gaiping. Preparation and Application of Neutralizing Monoclonal Antibodies Against H9N2 Subtype Avian Influenza Virus[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3761-3769.

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Preparation and Application of Neutralizing Monoclonal Antibodies Against H9N2 Subtype Avian Influenza Virus

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