H9亚型禽流感病毒血凝素单克隆抗体制备及鉴定

Preparation and identification of monoclonal antibodies against hemagglutinin of H9 subtype avian influenza virus

【目的】制备H9亚型禽流感病毒(AIV)血凝素(HA)单克隆抗体,为检测诊断AIV提供技术支持。【方法】以灭活H9亚型AIV广西分离株为免疫原,免疫6~8周BALB/c雌性小鼠,然后取其脾细胞与Sp2/0骨髓瘤细胞进行细胞融合,经血凝抑制试验(HI)及间接免疫荧光试验(IFA)筛选H9亚型AIV血凝素单克隆抗体的杂交瘤细胞。【结果】经过4次亚克隆,获得3株稳定的H9单克隆抗体杂交瘤细胞株,分别命名为8A4、1B11和7F1细胞株,其细胞株培养上清液HI效价为2~7~2^(10),腹水HI效价为2^(16)~2^(19);单克隆抗体亚类鉴定结果表明,3株H9单克隆抗体均属于Ig G1亚类,其轻链均为K链。3株H9单克隆抗体只与H9亚型AIV发生特异性HI反应,而与其他HA亚型AIV及新城疫病毒(NDV)、减蛋综合症病毒(EDS)、传染性支气管炎病毒(IBV)无交叉反应。3株H9单克隆抗体细胞株的抗体分泌能力稳定性良好。【结论】用灭活H9亚型AIV为免疫原能成功制备针对H9亚型AIV血凝素的单克隆抗体,且具有良好的亚型广谱性和特异性。

[ Objective ]The monoclonal antibodies （MAbs） against hemagglutinin （HA） of H9 subtype avian influenza virus （AIV） were prepared, in order to provide technical support for detection and diagnosis of AIV. [Method]Six to eight- week-old BALB/c female mice were immunized with the immunogen of inactivated H9 subtype AW isolated from Guangxi. Splenocytes from immunized mice were fused with Sp2/0 myeloma cells. The hybridoma cells were screened by Hemag- glutinin inhibition （HI） test and indirect immunofluorescence assay （IFA）. [ Result ]After subcloning 4 cycles, three stable hybridoma cell lines secreting specific MAbs against H9 subtype were obtained, and named 8A4,1 B11 and 7F1, respec- tively. And the HI titers of their culture supernatants ranged from 27 to 2I~, and the HI titers of ascites ranged from 216 to 219. The identification results of immuno-globulin type showed that, MAbs of three hybirdomal cell lines were belong to IgG1 subtype, with K chain. Furthermore, the HI assay results showed that, three obtained McAbs only reacted with H9 subtype of AIV but did not react with other subtypes of AIV, newcastle disease virus （NDV）, egg drop syndrome virus （EDSV） and infectious bronchitis virus （IBV）. The three hybridoma cell lines had good stability for ability of secreting specific MAbs. [Conclusion]H9 subtype AIV hemagglutinin specific MAbs are successfully prepared with inactivated H9 subtype AW as immunogen, and have good subtype broad-spectrum and specificity.