双抗体夹心酶联免疫方法检测丁香假单胞杆菌斑点致病变种病菌

Sandwich Immunoassy Against Pseudomonas syringae pv. Maculicola

摘要:

丁香假单胞杆菌斑点致病变种病菌可以引起十字花科细菌性黑斑病，是侵染萝卜、白菜、花椰菜等农作物的重要病害之一。以灭活的丁香假单胞杆菌斑点致病变种病菌（NCPPB1820）为免疫原免疫小鼠，通过杂交瘤细胞技术，筛选最终获得6株产生特异性单克隆抗体的杂交瘤细胞。将辣根过氧化物酶（horseradish peroxidase，HRP）与抗体偶联，作为检测探针，分别将6种单克隆抗体作为包被抗体，进行两两配对筛选。结果表明，所制备的抗体特异性较好，对水稻细菌性谷枯病菌、水稻细菌性条斑病菌、玉米细菌性枯萎病菌、丁香假单胞菌丁香致病变种均没有交叉反应。以6号抗体作为检测抗体（6-HRP），以1号抗体作为包被抗体，建立酶联免疫分析法获得了最佳的敏感度。检测丁香假单胞杆菌斑点致病变种的最低检测限为 1.5×105 cfu/mL，定量限为4.12×105 cfu/mL。基于双抗体夹心的酶免疫方法特异性好，便捷，可以实现对丁香假单胞杆菌斑点致病变种病菌高通量测定。

Abstract:

A sandwich immnuoassay based on monoclonal antibody for detection of Pseudomonas syringae pv. Maculicola was developed. Killed Pseudomonas syringae pv. Maculicola cell was used as immunogen to immunize BALB/c mice. The antisera was detected and the mice of highest antisera titer was selected for cell fusion. With hybirdoma technology，six cell lines secreting specific antibody to Pseudomonas syringae pv. Maculicola were obtained. Monoclonal antibodies （mAb） coded mAb1 and mAb6 were selected used as coating antibody and detection antibody based on pair-matching screening results. The sandiwich measure based on double antibodies were found to be high specific to targeted bacteria and no cross reactions to other bacteria including Burkholderia glumae，Xanthomonas oryzae pv. Oryzicola，Pantoea stewartii subsp.stewartii and Pseudomonas syringae pv.syringae. Under optimized conditions，the limit of detection was 1.5×105 cfu/mL and quantitative limit was 4.12×105 cfu/mL. This immunoassy was highly specific to Pseudomonas syringae pv. Maculicola and suitable for in-field detction，which provide a convient and fast tool in plant bacteria analysis.