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目的:研制抗哈维氏弧菌特异性单克隆抗体(mAb)并进行免疫学特性分析。方法:以哈维氏弧菌GYC1108-1颗粒性抗原免疫8周龄雌性BALB/c小鼠,5次免疫后取其脾细胞与Sp2/0-Ag-14骨髓瘤细胞融合。用间接酶联免疫吸附试验(ELISA)对杂交瘤进行筛选,并鉴定mAb特异性及敏感性。结果:各株阳性克隆经3次亚克隆后,共获得5株抗GYC1108-1的mAb,其中1B7的细胞上清及腹水效价分别为1∶3 200和1∶32 000,交叉反应结果表明1B7除了与测试的多株哈维氏弧菌发生反应外,还与溶藻弧菌、麦氏弧菌和副溶血弧菌发生免疫交叉反应,但不与拟态弧菌、鳗弧菌、嗜水气单胞菌等发生交叉反应。ELISA检测1B7腹水对GYC1108-1的敏感性达107/L。结论:制备的抗哈维氏弧菌mAb可为哈维氏弧菌的检测及防治等提供有用的试剂。
Objective: To develop a specific monoclonal antibody against Vibrio harveyi (mAb) and analyze its immunological characteristics. Methods: 8-week-old female BALB / c mice were immunized with Vibrio harveyi GYC1108-1 granulocyte antigen. After 5 immunizations, the spleen cells were fused with Sp2 / 0-Ag-14 myeloma cells. The hybridoma was screened by indirect enzyme-linked immunosorbent assay (ELISA) and the specificity and sensitivity of mAb were identified. Results: Five positive clones against GYC1108-1 were obtained after three times of subcloning of the positive clones. The supernatant and ascites titer of 1B7 were 1: 300 and 1: 32 000, respectively. The cross-reactivity results showed that 1B7 in addition to the test with a number of strains of Vibrio harveyi reacts with Vibrio alginolyticus, Vibrio alginolyticus and Vibrio parahaemolyticus immune cross-reaction occurs, but not with Vibrio mimicus, Vibrio anguillarum, Aeromonas and other cross-reactions occur. The sensitivity of 1B7c ascites to GYC1108-1 by ELISA was 107 / L. Conclusion: The prepared anti-Vibrio harveyi mAb can provide useful reagents for the detection and prevention of Vibrio harveyi.