烟草普通花叶病毒抗血清的吸附纯化及应用

Adsorption Purification of TMV Antiserum and Detection Application

为了检测烟草普通花叶病毒(TMV),使用提纯病毒免疫小白鼠,制备TMV抗血清,对抗血清进行了吸附纯化,并建立了基于自制TMV抗血清的ELISA检测体系。蛋白电泳研究结果表明,提取的TMV纯度和浓度都很高;经过烟草总蛋白吸附纯化后,有效去除了非特异性反应;通过效价测定,确定TMV抗血清的效价高达1∶40960;正交连续稀释法确定的ELISA检测最佳试剂浓度为:酶标二抗稀释度1∶20000,TMV抗血清稀释度1∶2000,病叶样品的稀释度为1∶20。

For the detection of TMV in tobacco, antiserum was prepared by injecting purified TMV virion into mouse and the titer of antiserum was determined after adsorption purification, an ELISA analysis system was established basing on the in-house TMV antiserum. The results of PAGE indicated the extracted TMV virion was of high purity and concentration; non-specific antibody could be effectively diminished by adsorption with tobacco total protein; the titer of antiserum was as high as 1：40960; the concentrations of reagents in ELISA detection optimized by orthogonal continuous dilution method were 1：20000 in dilution for secondary antibody, 1：2000 for TMV antiserum and 1：20 for sample supernatant.