【作者】 薛琳；

【导师】 夏咸柱；

【作者基本信息】 吉林大学 ， 预防兽医学， 2007， 硕士

【摘要】 荧光抗体实验是狂犬病毒(Rabies virus,RV)实验室诊断的一种最精确、快速、可靠的方法。本实验旨在通过制备RV荧光抗体,结合直接免疫荧光实验(dFAT)和荧光抗体病毒中和实验(FAVN)建立RV抗原、抗体检测方法,并应用该方法进行了RV疫苗毒力的测定和其免疫原性的检测。将狂犬病病毒ERA株纯化后免疫雌性BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经克隆和间接ELISA筛选,获得3株稳定分泌抗狂犬病病毒单克隆抗体的杂交瘤细胞株,分别命名为C7、C4和H3。经过鉴定:其腹水效价分别为1 : 1×106、1 : 5×105和1 : 1×105 ;且与犬瘟热病毒(CDV),犬细小病毒(CPV)、犬腺病毒(CAV)不发生交叉反应;3株单抗均为IgG类型;Western-blot分析表明,3株单抗均为抗狂犬病毒核蛋白单抗。采用G蛋白亲和层析柱对腹水进行纯化,将纯化的狂犬病毒核蛋白单抗用异硫氰酸荧光素(FITC)标记制备荧光抗体,并对该荧光抗体进行质量鉴定,结果表明,所制备的荧光抗体具有较强的特异性强和敏感性。用所制备的荧光抗体检测RV弱毒疫苗的毒价及其免疫原性,试验结果表明,所制备的荧光抗体可初步用于RV的实验室检测。更多还原

【Abstract】 Rabies, caused by rabies virus(RV)is an acute and fatal zoonotic diseases. The fatality rate is almost 100%. All warm-blooded animals are susceptible to infection by the virus. It is considered to be a serious public health problem. The control and eradication of rabies depends on the development of safe, effective and economical vaccine that might be used in preexposure vaccination. However, checking virus dilution or effectiveness of rabies vaccines depends on the rapid,accurate and safe diagnostic method. Monoclonal antibody against rabies virus nucleoprotein was developed by means of the conventional protocol. The IgG was purified by affinity chromatography and conjugated with FITC. Virus titer and immunogenicity of rabies vaccines were detected by direct fluorescent antibody test (dFAT) and fluorescent antibody virus neutralization test (FAVN).After immunization of BALB/c mice with purified rabies virus(RV), three hybridoma cell strain against RV named C7、C4 and H3, respectively, were developed after fusion between SP2/0 myeloma cell and the stimulated splenocytes. The indirect ELISA results showed that the McAbs ascites titer were 1:1×106、1:5×105and 1:1×105; No cross reaction was found when McAbs reacted with CDV, CPV and CAV; Identification of subclass showed that C7 belonged to IgG2b, C4 and H3 belonged to IgG1 respectively; The result of western-blotting with purified RV is positive, which indicates thesemonoclonal antibodies all aimed directly at rabies virus nucleoprotein.The three McAbs were purified by protein G and labelled with FITC. The results of absorption test and blocking test showed that the fluorescent antibody (FA) has highly specificity and sensitivity , and has no interaction with CPV, CDV and CCV. Its optimal working concentration was 1:128.The FA to rabies nucleoprotein that was characterized correctly was used to detect virus titers of Rabies Vaccine with FAT and 30 serum samples from dogs with FAVN assay. The result of fluorescein stain showed that the TCID50 of RV ERA strain had been increased from 10 - 5.64 , 50μL before rejuvenescence to 10-8.16, 50μL after rejuvenescence; ED50 of the 30 examined serum samples were all > ED50 of the reference serum, then the titre was > 0.5 IU/mL, that is to say the titer of the antibody to RV had reached a protective level.FA diagnostic method that was established provided a convenient, fast and reliable technic to the antigen detection and epidemiologic survey of RV as well as immunologic surveillance and the detection of vaccine effectiveness. It has laid a foundation for prevention and cure of RV.更多还原