禽白血病病毒衣壳蛋白单克隆抗体研制及其双夹心ELISA的建立  

Development of Monoclonal Antibodies Against p27 of Avian Leukosis Virus and the Establishemnt of a Sandwich ELISA for Detection

作　　者：尹丽萍[1,2] 秦爱建[1,2] 钱琨[1,2] 邵红霞[1,2] 金文杰[1,2] 史荣华[1,2] 梁有志[1,2] 杭柏林[1,2] 孙薇薇[1,2] 

机构地区：[1]扬州大学江苏省动物预防医学重点实验室,江苏扬州225009 [2]扬州大学教育部禽类预防医学重点实验室,江苏扬州225009

出　　处：《中国家禽》2013年第3期15-19,共5页China Poultry

基　　金：联合国家基金重点项目(U0831002);教育部创新团队项目(IRT0978);江苏省优势学科项目

摘　　要：为制备禽白血病病毒(ALV)核衣壳蛋白p27的单克隆抗体,本研究以原核表达的融合蛋白GST-ALV p27免疫小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,通过间接免疫荧光(IFA)和间接ELISA进行筛选,制备了5株能够稳定传代并分泌抗p27单抗的杂交瘤细胞株,分别命名为5D3、4F12、5B10、1C5和3C6,亚类鉴定3C6为IgG2b,其余为IgG1。通过IFA、Western-blot及竞争抑制ELISA,表明该5株单抗与J亚群禽白血病病毒(ALV-J)呈阳性反应,而与其他常见禽源病毒无交叉反应。利用5D3和4F12单抗建立了双抗体夹心ELISA方法,经663份临床样本验证,该方法与商品化试剂盒的符合率达到95.17%,证明所研制的针对ALV p27蛋白的单抗及建立的双抗体ELISA方法具有较高的应用价值。A panel of monoclonal antibodies （MAbs） against avian leukemia virus （ALV） nucleocapsid protein p27 were developed by fusions between SP2/0 and spleen cells from Balb/c mice immunized with purified fusion protein GST-p27 expressed in prokaryotic expression system. 5 of hybridomas cell strains stably secreted MAbs against ALVp27 were named as ALVp27-5D3, ALVp27-4F12, ALVp27-SB10, ALVp27-1C5 and ALVp27-3C6. These Mabs showed high titer and specific to ALV p27 in indirect immunofluorescence assay, indirect ELISA assay and Western blot. The subtypes of immunoglubin of MABs were IgG2b with kappa light chain for MAb ALVp27-3C6 and IgG1 with kappa light chain for the others. A sandwich ELISA kit to detect ALV p27 was established with 5D3 and 4F12. Compared with commercial kits to test 663 clinical samples,our kit showed 95.17% of coincidence rate. This kit will be very useful in eradication of ALV in pultry industry.

关 键 词：禽白血病病毒 P27 单克隆抗体 双抗体夹心ELISA 

分 类 号：S852.65[农业科学—基础兽医学]