禽病原性大肠杆菌Ⅰ型菌毛单克隆抗体的研制及其对分离株的检测  

Laboratory Preparation of Monoclonal Antibodies Specific to Type Ⅰ Pili of Avian Escherichia coli and Their Preliminary Application

作　　者：刘业兵[1] 高崧[1] 彭大新[1] 张如宽[1] 刘秀梵[1] 

机构地区：[1]扬州大学动物医学系,江苏扬州225009

出　　处：《中国兽医学报》2000年第2期141-151,共11页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目! ( 3 9770 5 62 )

摘　　要：从禽病原性大肠杆菌分离株 TK3 ( O1 )提取 型菌毛免疫 BALB/c小鼠 ,取其脾细胞与小鼠骨髓瘤细胞融合 ,获得 4株能稳定分泌针对 型菌毛的单克隆抗体杂交瘤细胞株 ,分别命名为 a B6 、b G5 、c F3和 c G3。单抗阻断甘露糖敏感血凝试验、免疫胶体金和 Western blot试验证明 ,单抗是 型菌毛特异的。这些单抗培养上清及腹水的 ELISA效价为分别为 1 0 - 2～ 1 0 - 3和 1 0 - 5～ 1 0 - 6 ;腹水的平板凝集价为 1∶ 1 2 8～ 1∶ 2 56。上述4株单抗对 77株带 型菌毛的禽病原性大肠杆菌优势血清型分离株进行了检测 :O1 8分离株阳性率为72 %,O78分离株阳性率为 1 7%,O2分离株阳性率为 3 3 %,O88分离株阳性率 89%,O1 1分离株阳性率为60 %,O2 6分离株阳性率为 3 3 %。表明研制的 型菌毛单抗能检出大多数 O1 8、O88、O1 1分离株的 型菌毛 ,而对 O78、O2、O2A set of four hybridoma cell lines, aB 6,bG 5,cF 3 and cG 3,secreting monoclonal antibodies specific to type Ⅰpili of avian E.coli, were obtained from two separate fusions between mouse myeloma cell line Sp2/0 Ag14 and spleen cells from mice immunized with purified type 1 pili antigens of avian E.coli isolate TK3. Their specificity was evaluated by the mannose sensitive hemagglutination blocking test,immunogold technigue and immunoblotting. The antibodies from culture medium and mouse ascites had the titers of 10 -2 10 -3 and 10 -5 10 -6 in the enzyme linked immunosorbent assay(ELISA) respectively and the mouse ascites had the titers of 1∶128~1∶256 in the direct agglutination test. The type Ⅰ pili of 78 avian Escherichia coli isolates were determined with these four monoclonal antibodies in the direct agglutination test.The positives for O18 isolates was 72%(13/18), and 17%, 33%, 89%, 60% and 33% for O78 , O2, O88 , O11 and O26 isolates respectively. These results suggested that most type Ⅰ pili were detectable for O18 , O88 and O11 serogroups with monoclonal antibodies specific to corresponding pili of TK3(O1) isolate but not for those of O78 ,O2,and O26 isolates.

关 键 词：大肠杆菌 I型菌毛 单克隆抗体 检测 

分 类 号：S852.612[农业科学—基础兽医学] S858.305.1[农业科学—兽医学]