玉米内州萎蔫病菌免疫学检测方法的建立

Development of an Immunosorbant Assay for Seedborne Clavibacter michiganensis subsp. nebraskense in Corn Seeds

摘要:

以玉米内州萎蔫病菌单抗(4H4和4G12)为基础, 纯化抗体后, 进行亚类鉴定、效价及特异性测定。比较间接ELISA和双单抗夹心ELISA (DAS-ELISA)的检测灵敏度, 并应用于玉米种子中萎蔫病菌的检测。结果表明, 两株单克隆抗体(0.4 g/L)效价均可达1 : 256000, 亚类鉴定结果分别为IgG2a和IgG2b, 轻链均为K链。与供试的16株非目标细菌均无交叉反应。DAS-ELISA对萎蔫病菌种子悬浮液的检测灵敏度为1.0 × 109 CFU/L, 在此基础上建立了灵敏、特异的玉米内州萎蔫病菌双单抗DAS-ELISA检测方法。

Abstract:

We purified the McAbs and then determined the subclasses, activity and specificity of the Clavibacter michiganensis subsp. nebraskense McAbs (4H4 and 4G12). We compared the detection sensitivity between indirect ELISA and double antibody sandwich ELISA (DAS-ELISA) and utilized both of them on the detection of the corn bacteria. Titers of both McAbs in 0.4 g/L were 1 : 256000, subclasses of 4H4 belongs to IgG2a while 4G12 belongs to IgG2b. No cross reactivity was found within 16 bacterial strains tested from corn and other plants by ELISA. The minimun detection limit of the bacteria in the seed suspension with DAS-ELISA was 1.0 × 109 CFU/L. A specific and sensitive DAS-ELISA for detection of the C. michiganensis subsp. nebraskense was developed.