抗禽流感病毒H7亚型血凝素特异性单克隆抗体的研制

目的:研制禽流感病毒H7亚型血凝素特异性单克隆抗体(mAb)。方法:以H7亚型禽流感诊断抗原为免疫原免疫6～8周雌性BALB/c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14进行融合。通过HA和HI试验筛选阳性克隆。应用HI试验和Western blot试验测定mAb的反应性和特异性。结果:共获得4株分泌抗AIVH7亚型HAmAbs的杂交瘤细胞株,分别命名为2E2、2A4、5F5、7G5。这些mAb的腹水HI效价在5×27～5×211之间,其中2E2属于IgM亚类,2A4属于IgG1亚类,5F5、7G5属于IgG2a亚类。Western blot分析结果显示,4株AIVH7亚型HAmAb能与AIVH7蛋白在Mr75000处反应,但不与新城疫病毒(NDV)蛋白发生反应,表明这些mAb能特异性识别AIVH7亚型HA。mAbHI反应性测定结果表明:4株mAb中,2E2、5F5、7G5只与H7亚型AIV发生特异性HI反应,而不与其他亚型AIV以及NDV、传染性支气管炎病毒(IBV)反应,显示出良好的特异性;而2A4除了与H7亚型AIV反应外,还与H15N8标准株发生低水平交叉反应。结论:这些mAb不仅为H7亚型AIV的HA结构分析提供了工具,而且为建立快速廉价的H7亚型禽流感诊断方法提供了核心试剂。

Development and characterization of monoclonal antibodies against H7 hemagglutinin of avian influenza virus

AIM: To prepare monoclonal antibodies (mAbs) against the hemagglutinin protein of H7 subtype of avian influenza virus (AIV). METHODS: (6-8 weeks old) BALB/c mice of were immunized endermicly with H7 subtype of AIV. The splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cells after the last immunization. Hybridoma cells were screened by hemagglutination (HA) and hemagglutination inhibition (HI) tests. The reactivity and specificity of mAbs were evaluated by HI test and Western blot assay. RESULTS: Four hybridoma cell lines secreting specific mAbs named 2E2, 2A4, 5F5 and 7G5 were developed. The HI titers of these mAbs were 5 x 2(7)-5 x 2(11), and the immunoglobulin subclass of 2E2 was IgM, that of 2A4 was IgG1, and that of 5F5 and 7G5 was IgG2a. Western blot analysis confirmed that the mAbs only reacted with M(r) 75 000 HA protein of H7 subtype of AIV but did not react with the proteins of Newcastle disease virus (NDV). The results of HI reactivity assay suggested that 2E2, 5F5 and 7G5 only reacted with H7 subtype of AIV but did not react with other subtypes of AIV, NDV and infectious bronchitis virus (IBV). However 2A4 reacted not only with H7 subtype of AIV but also with H15N8 reference strain of AIV at low HI level. CONCLUSION: These mAbs can be used as a useful tool to analyze the HA structure of AIV. They also provide the effective reagents for the rapid detection of H7 subtype of AIV.