Himananto Orawan, Thummabenjapone Petcharat, Luxananil Plearnpis, Kumpoosiri Mallika, Hongprayoon Ratchanee, Kositratana Wichai, Gajanandana Oraprapai
Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand; Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok 10900, Thailand; and National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand.
Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE); and Agricultural Biotechnology Research Center for Sustainable Economy, Khon Kaen University, Khon Kaen 40002, Thailand.
Plant Dis. 2011 Sep;95(9):1172-1178. doi: 10.1094/PDIS-12-10-0889.
A novel monoclonal antibody (MAb) specific to the seedborne bacterium Acidovorax citrulli was produced. MAb 11E5 reacted specifically with 19 strains of A. citrulli but not with three closely related bacteria in the family Comamonadaceae (i.e., A. facilis, Comamonas acidovorans, and C. testosteroni) and another seven phytopathogenic bacteria. Moreover, this MAb detected a strain of A. citrulli that was not detected by a commercial enzyme-linked immunosorbent assay (ELISA)-based kit and a commercial immunochromatographic strip test. In Western blot analysis, MAb 11E5 reacted with an A. citrulli protein of a molecular mass >170 kDa. MAb 11E5 was employed to develop two sandwich ELISA systems: MAb captured-sandwich ELISA (MC-sELISA) and polyclonal antibody captured-sandwich ELISA (PC-sELISA). MC-sELISA was 10 times more sensitive than PC-sELISA for detection of A. citrulli in cucurbit leaf and seed extracts. The detection limit of the MC-sELISA was 5 × 10 CFU/ml. Detection of A. citrulli in naturally infected cucurbit leaves, fruit, and seed was also feasible using MC-sELISA. The newly established MCsELISA provides another alternative for specific detection of A. citrulli in cucurbits and can be applied for routine field inspection.
制备了一种针对种传细菌西瓜嗜酸菌的新型单克隆抗体（MAb）。单克隆抗体11E5与19株西瓜嗜酸菌发生特异性反应，但与丛毛单胞菌科的三种密切相关细菌（即易变嗜酸菌、食酸丛毛单胞菌和睾丸酮丛毛单胞菌）以及另外七种植物病原菌不发生反应。此外，该单克隆抗体检测到了一株西瓜嗜酸菌，而基于商业酶联免疫吸附测定（ELISA）的试剂盒和商业免疫层析试纸条检测未检测到该菌株。在蛋白质印迹分析中，单克隆抗体11E5与一种分子量>170 kDa的西瓜嗜酸菌蛋白发生反应。单克隆抗体11E5被用于开发两种夹心ELISA系统：单克隆抗体捕获夹心ELISA（MC-sELISA）和多克隆抗体捕获夹心ELISA（PC-sELISA）。在检测葫芦叶片和种子提取物中的西瓜嗜酸菌时，MC-sELISA的灵敏度比PC-sELISA高10倍。MC-sELISA的检测限为5×10 CFU/ml。使用MC-sELISA检测自然感染的葫芦叶片、果实和种子中的西瓜嗜酸菌也是可行的。新建立的MCsELISA为葫芦中西瓜嗜酸菌的特异性检测提供了另一种选择，可用于常规田间检测。
