H9N2亚型禽流感病毒中和单克隆抗体的制备与应用

为建立H9N2亚型禽流感病毒（Avian influenza virus，AIV）免疫层析快速检测技术，本研究以差速离心法纯化H9N2亚型AIV免疫BALB/c小鼠，将免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合和HAT选择性培养；以H9N2亚型AIV感染MDCK细胞建立异源免疫过氧化物酶单层细胞试验（IPMA）的单克隆抗体检测方法，通过对杂交瘤细胞的IPMA筛选和连续克隆化筛选鉴定抗H9N2亚型AIV中和性单克隆抗体；以胶体金标记HA单克隆抗体，配对HA单克隆抗体和羊抗小鼠IgG为检测线和质控线，制备H9N2亚型AIV快速检测试纸条，测定其特异性和敏感性。结果显示，获得了11株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞，其单克隆抗体腹水IPMA效价在1.28×10^-4至2.56×10^-5之间。单克隆抗体3A2、5H6、6B8、7E10和9G12血凝抑制试验（HI）显示血凝抑制活性，其（HI）效价在6log2~9log2之间。单克隆抗体3A2、6B8和9G12在病毒中和试验中对H9N2亚型AIV有显著病毒中和活性，中和效价分别1∶6 400、1∶25 600和1∶25 600。Western blotting结果提示，该中和单克隆抗体识别HA蛋白线性抗原表位。利用配对单克隆抗体3A2和9G12研制的H9N2亚型AIV检测试纸条检测H9N2亚型AIV尿囊液的效价为9log2，灵敏度与经典血凝试验（HA）相当，与其他亚型AIV （H1、H3、H5、H7），以及新城疫病毒和鸡传染性法氏囊病病毒等相关病毒均无交叉反应。本研究制备了具有病毒中和活性的抗H9N2亚型AIV单克隆抗体，并初步研制了H9N2亚型AIV检测试纸条，为H9N2亚型AIV新型疫苗研制和快速检测奠定良好的研究基础。

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

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.