单克隆抗体间接免疫荧光检测猪瘟病毒方法的建立

Establishment of indirect immunofluorescence assay for detecting classical swine fever virus

以作者制备的抗猪瘟病毒单克隆抗体(anti-CSFV McAb)为一抗、荧光素标记羊抗小鼠IgG为二抗,通过反应条件的优化,建立检测猪瘟病毒抗原的间接免疫荧光(IFA)检测方法。确定IFA最佳工作条件:CSFV最佳接种浓度和培养条件为CSFV 10-3倍稀释后接种PK15细胞,37℃5%CO2恒温箱中培养36 h;McAb最适工作浓度为1∶1 000倍稀释;荧光素标记的羊抗小鼠IgG荧光抗体的最适工作浓度为1∶50倍稀释。特异性试验表明,用建立的IFA检测方法检测CSFV感染PK15细胞为阳性,而检测伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪2型圆环病毒(PCV-2)感染PK15细胞均为阴性。结果表明,建立的检测细胞培养中CSFV抗原的IFA检测方法具有敏感特异、简便快速等优点,可用于CSFV感染的实验室诊断及CSFV在感染细胞中的定位和动态分布研究。

An indirect immunofluorescence assay（IFA） test was developed to diagnosis CSFV using the anti-CSFV McAb as the primary antibody and the fluorescein isothiocyanated（FITC） labeled goat anti-mouse IgG as the second antibody.The optimum conditions of IFA was as follows：the optimum vaccinate concentration and culture condition of CSFV were that the CSFV was diluted by 10-3 to vaccinate PK15 cells and were cultivated for 36 h at temperator（37℃ 5% CO2）.The optimum working concentration of anti-CSFV McAb and FITC-labeled goat anti-mouse IgG were 1∶1 000 and 1∶50.IFA was adopted in detection of CSFV antigen in the PK15 cells.The results were positive in PK15 cells infected with CSFV and were negative in PK15 cells infected with PRV,PPV or PCV-2.It is evident that this IFA test is a sensitive,specific and rapid method for the detection of CSFV in PK15 cells.It is effective in studying the antigen locations of CSFV.IFA can be applied in diagnosing and studying the distribution of the CSFV in infected PK15 cells.