抗哈维氏弧菌单克隆抗体杂交瘤细胞系的建立及其特性鉴定

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187的细胞上清及腹水效价分别为1：3200和1：32000，交叉反应结果表明187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187腹水对GYC1108—1的敏感性达10^7／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。

Preparation and characterization of the mAb against Vibrio harveyi

AIM: To prepare the mAb against Vibrio harveyi and analyse their biological properties. METHODS: 8-week-old female BALB/c mice were immunized with pathogenic V.harveyi GYC1108-1, which was isolated from the diseased great yellow croaker in Zhejiang China in 2003. Spleen cells collected from the immunized mice after the fifth immunization were fused with Sp2/0-Ag-14 myeloma cells. ELISA was used to screen hybridoma cells and identify the specificity and sensitivity of mAbs. RESULTS: Limited dilution method was used to subclone the positive clones. After three cycles of subcloning, 5 mAbs against GYC1108-1 were obtained and were proved to have high titer of ELISA. The test of specificity suggested that 1B7 reacted on V.harveyi, V.alginolyticus and V.parahaemolyticus but did not react on V.mimicus, V.anguillarum, Aeromonas hydrophila and the other isolates among the 14 bacterium species tested. 1B7 was used to detect V.harveyi at the concentration of 10(4) CFU/mL by indirect ELISA. CONCLUSION: The obtained mAbs could be used to diagnosis and prevent of V.harveyi in aquaculture rapidly.