酶联免疫吸附反应对猕猴桃溃疡病菌效应子Hopz5多克隆抗体的特性分析

Characterization of Polyclonal Antibody Against Pseudomonas syringae pv. actinidiae Effector Hopz5 by Enzyme Linked Immunoabsorbent Assay

为了建立一种猕猴桃溃疡病菌(Pseudomonas syringae pv.actinidiae,PSA)的血清学检测方法,我们利用致病性极强的PSA3类群特异效应子Hopz5制备的多克隆抗体(PAb-Hopz5),通过酶联免疫吸附反应对样品类型、特异性、灵敏度及田间样品进行检测,全面分析了该抗体的特性。结果表明,PAb-Hopz5不仅能够检测发病组织,还能检测人工培养的PSA;即可利用发病组织蛋白,也可利用发病组织浸泡液进行检测。此外,PAb-Hopz5的检测不受PSA侵染时期的影响。PAb-Hopz5与其他供试菌包括P.syringae pv.tomato、P.fluorescens和P.putida等无交叉反应,具有较好的特异性。ELSIA检测菌悬液的最低浓度为3×105CFU/m L,明显低于PCR检测灵敏度,但对田间发病组织样品的检测效果优于PCR。研究表明,PAb-Hopz5可作为猕猴桃溃疡病菌PSA3类群检测用试剂,进而为猕猴桃溃疡病菌提供新的检测工具。

In order to establish a serological detection method for Pseudomonas syringae pv. actinidiae, we used highly pathogenic PSA3 group specific effector Hopz5 to prepare the polyclonal antibody （ PAb-Hopz5 ） . The characteristics of PAb-Hopz5 was comprehensively analyzed by detecting sample type, specificity, sensitivity and field samples via enzyme linked immunoabsorbent assay . The results showed that PAb-Hopz5 could be used for detecting not only the kiwifruit tissues, but also the PSA of artificial culture ; and not only the protein of disease tissue but also the soak solution of tissues could be used for the detection. Besides, the samples of PSA different infection periods could be detected by PAb-HopzS. No cross-reaction were observed with other testing bacteria, including P. syringae pv. tomato, P. fluorescens and P. putida, etc, indicating that the specificity of PAb-Hopz5 was solid. The minimum detectable concentration of bacterial suspension by ELISA was 3 x 10s CFU/mL, which was significantly lower than that by PCR, but for the detection of disease samples in the field it was better than that by PCR. Overall, the results showed that PAb-Hopz5 could be used as the test reagent for the PSA3 group detection of P. syringae pv. actinidiae, thus it provided a new detection tool for P. syringae pv. actinidiae.