柑桔黄龙病病原类细菌单克隆抗体的研制及诊断

Preparation and Diagnosis of Monoclonal Antibodies Against Citrus Yellow Shoot Disease

在柑桔黄龙病病原类细菌（BLO）建立免疫血清ELISA法诊断的基础上，利用人工培养柑桔黄龙病病原BLO制备单克隆抗体，能有效提高诊断黄龙病的准确性及灵敏度。利用人工培养的柑桔黄龙病病原BLO免疫的Balb／c小鼠脾细胞与SP2／0骨髓瘤细胞融合，用ELISA间接法筛选阳性抗体，经有限稀释法克隆，筛选出2株可持续分泌抗柑桔黄龙病BLO单克隆抗体（MCAb）的杂交瘤细胞株2F11、1E10，该2杂交瘤细胞腹水抗体效价以间接ELISA法检测滴度均超过1．7×105。对柑桔黄龙病的病株、健株检测，其结果显示带病植株显阳性，健株显阴性；将腹水McAb与辣根过氧化物酶（HRP）进行标记（McAb－HRP），制成酶标抗体，用酶标定位法检测柑桔病株，病症明显或不明显的均呈阳性反应，健株则显阴性反应。

Bacterial -like organism (BLO) of cztrus yellow shoot disease (CYSD) determined with ELISA diagnosis was cultured and isolated,and monoclonal antibodies against the BLO were prepared, facilitating accurate and sensitive diagnosis of CYSD. Splenic cells from immunizcd Balb/c mice were fused with SP2/0 murine myeloma cells. Screened by indirect ELISA and cloned by limiting dilution methods,two hybirdoma clones (2F11, 1E10)were obtained secreting continuously specific antibodies against the BLO. Antibody titers of ascitic fluids were both over 1. 7×105 when assayed by indirect ELISA. The application on several specimens showed that the symptom citrus plants were detected as positive and the healthy ones as negative.The monoclonal antibodies were labeled with the horse radish peroxidase (HRP) to prepare immunoenzyme localized monoclonal antibodies. It is proved that the affected citrus plants showed positive reaction while the healthy ones negative.