【作者】 马仲彬；

【导师】 王泽霖；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 将H9N2亚型禽流感病毒经差速离心和蔗糖密度梯度离心,纯化抗原免疫Balb/c小鼠,应用淋巴细胞杂交瘤技术,通过ELISA和HI筛选,建立了两株抗H9N2亚型禽流感病毒的杂交瘤细胞株IH6、3A4,其分泌抗体能力稳定,为针对HA蛋白的单抗,免疫球蛋白分别为IgG1和IgG2b。分泌的单克隆抗体与16株H9N2亚型AIV发生血凝抑制反应,而不与H5N1、H5N2亚型AIV及HIN1、H3N2 SIV发生反应,对鸡NDV、IBDV、EDSV及IBV也无交叉反应,表明该单抗具有AIV H9N2亚型特异性。IH6杂交瘤细胞培养上清和腹水HI效价分别为2⁸、2¹⁴;3A4杂交瘤细胞培养上清和腹水HI效价为2⁸、2¹⁶。这两株杂交瘤细胞分泌的抗体均有良好的热稳定性。腹水经辛酸硫酸铵纯化后,蛋白含量分别为19mg/mL、14.9mg/mL。此两种单克隆抗体的获得为H9N2亚型禽流感病毒的研究、流感快速诊断方法的建立奠定了基础。 利用所获得的单克隆抗体结合胶体金技术,在国内首次建立了用免疫层析（GICA）快速检测H9亚型禽流感病毒的试纸条检测方法。检测时,只需将试纸条插入待检样品,5min后观察结果。试纸条上检测区和质控区同时出现两条红线为阳性,只在质控区出现一条红线的为阴性。用此试纸条检测送检的16株H9N2AIV均为为阳性,对照的H5N1、H5N2AIV、SIV毒株及NDV、IBDV、EDSV、IBV均为阴性,试纸条对H9N2AIV纯化抗原最低检出量为23.37ng;含毒尿囊液的最低检出量为2个血凝单位。用此试纸条对H9N2AIV鸡胚繁殖动态进行检测,得知病毒在66-86h之间含量最高;用该试纸条对人工感染H9N2AIV SPF鸡的咽喉及泄殖腔进行检测,该试纸条呈阳性反应,而对照鸡为阴性,证明该试纸条具有很好的特异性、敏感性,该法操作过程简单、快速、方便、不需特殊仪器,5min内便可判定结果,大大缩短了常规实验室检测所需时间,尤其适用于基层实验室和现场应用。更多还原

【Abstract】 AIV H9N2 was grown in chicken embryonated eggs and purified by differential centrifugation and sucrose density centrifugation.Eight-week Balb/c mice were immunized according to Kazuhiro’s produre.Two hybridoma named 1H6、 3A3were generated by fusion of NSo myelomas and Splenocytes from immunized mice.The monoclonal antibody（McAb） produced was screened by ELISA and HI and proved to be IgG1、 IgG2b by AGP,mouse ascetic fluid(HI 2¹⁴、 2¹⁶) was purified（protein concentration : 19mg/mL、 14.9mg/mL） and proved to react with H9N2 AIV only in HI.The result of cross-reaction in HI demonstrated that two McAbs were subtype-sepecific.The purified rabbit anti-AIVIgG was labeled with colloidal gold particals of 10nm in diameter.Then it was immobilized to nitrocellulose membrane .The two kinds of antibody concentration was optimized by the sqaure matrix test .A McAb-mediated rapid diagnosis strip was developed for detection of H9N2AIV.When we want get the result ,put it into the sample ,wait for 5 minutes.The sample with obvious red line on the strip was judged as positive for H9N2AIV,others,negative.The detection limit of purified H9N2AIV allantioic fluid of H9N2AIV were 16.56ng and 2HAU respectively.The test strip was placed in 4°Cdesiccator,after 8 month its effect of detection and releasing still hold on the line.The test strip show is easy to be justified .It demonstrated that rapid diagnosis strip was a rapid, sensitive, simple and non-lable-intensive test for detection of H9N2 subtype AIV.These key techniques and the data will help us research and develop a series of GICA strip to detect the domestic animals and poultry.更多还原