【作者】 温和心；

【导师】 陆芹章； 罗廷荣；

【作者基本信息】 广西大学 ， 预防兽医学， 2003， 硕士

【摘要】 本研究采用PK₁₅细胞培养猪瘟石门毒，经蔗糖密度梯度离心纯化后免疫小鼠，取免疫鼠脾细胞与SP_2/0-Ag₁₄在PEG4000作用下融合，经间接ELISA法筛选，有限稀释法克隆3次，最终获得4株稳定分泌抗CSFV-Shimen的McAb杂交瘤细胞株，分别命名为AC9、CF8、DG5和EC9，并接种小鼠腹腔生产单抗腹水，ELISA效价为1：2000至1：8000不等。四株单抗与基因工程CSFV E2蛋白反应结果表明AC9、CF8和EC9是抗CSFV E2蛋白的单抗。单抗AC9.DG5和EC9与SDS处理前后的CSFV反应无明显差异，而CF8不与SDS处理后的CSFV反应，CF8针对的抗原表位的抗原性与空间构象密切相关。IFA试验结果CF8和DG5与接种猪瘟石门毒的细胞反应产生较强的特异荧光，AC9和EC9反应较弱。 用AC9和CF8单抗对CSFV进行抗原捕获间接ELISA试验（ACI-ELISA），通过一元单抗和二元单抗CAI-ELISA试验的比较，结果表明AC9与CF8两种单抗协同作用可使ELISA反应值提高1倍。封闭对比试验表明封闭与否对试验结果无显著影响。通过方阵试验找出了McAb和血清多抗（PcAb）的最佳工作稀释度。用有限稀释法测定了试验的敏感度，并确定了病料的实际工作稀释度。通过取代试验肯定了ACI-ELISA试验的特异性。最后用ACI-ELISA与PCR对30份病料的检测结果比较，表明ACI-ELISA与PCR检测结果有很高的符合率。更多还原

【Abstract】 In this study four monoclonal antibodies designated as AC9, CF8, DG5 and EC9 against classical swine fever virus Shimen strain(CSFV Shimen strain) were generated by immunization of Balb/C mice with purified CSFV Shimen, which multiplied by PK15 cells culture. The hybridomas were inoculated in the mice bellies to produce McAbs. The ascites titer had been measured between 1:2000-1:8000. The McAbs except for DG5 could react with CSFV E2 protein in indirect ELISA, indicating that AC9, CF8 and EC9 are anti-CSFV E2 protein McAbs. AC9, DG5 and EC9 could still react with CSFV Shimen strain treated by SDS, while CF8 react negatively, indicating the structure is essential to the epitope antigenicity of CF8. CF8 and DG5 had strong reactivity CSFV Shimen strain prepared in cells PK15 slides in indirect immunofluorescent assay(IFA), while AC9 and EC9 react weakly in IFA.The comparison of antigen-capture indirect ELJSA(ACI-ELISA) experiments showed the corporation of AC9 and CF8 could doubly enhance reaction sensitivity than AC9 or CF8 alone. Throught chessboard test found out the most suitable dilution of AC9-CF8 and the CSFV postive standar serum respectively is 1:400 and 1:200. The average CSFV titer of samples is 1:32 by indirect ELISA, but 1:4 dilution is used in practice. The results of antigen substitution and antigen blocking experiments showed the ACI-ELISA has highly spectific reactivity with CSFV. At last, the ACI-ELISA was used to test 30 samples, it yielded nearly the same results as PCR.更多还原