抗头孢氨苄单克隆抗体的制备与初步应用

doi:10.16431/j.cnki.1671-7236.2017.04.031

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (4): 1159-1167.doi: 10.16431/j.cnki.1671-7236.2017.04.031

抗头孢氨苄单克隆抗体的制备与初步应用

张伟¹, 王兴², 刘金萍¹, 徐声乐¹, 秦海艳¹, 刘若璇¹, 蔡云虹¹, 汝晓飞¹, 侯珂¹, 王捍东¹

1. 扬州大学兽医学院, 扬州 225009;
2. 张浦镇动物防疫站, 昆山 215300

收稿日期:2016-09-30 出版日期:2017-04-20 发布日期:2017-05-03
通讯作者: 王捍东 E-mail:ydwhd@sina.com
作者简介:张伟(1991-),男,安徽淮南人,硕士生,研究方向:动物性食品卫生安全,E-mail:zhangw0216@126.com
基金资助:
江苏高校优势学科建设工程；江苏省动物重要疫病与人兽共患病防控协同创新中心；江苏省高校重点实验室开放课题

Preparation and Preliminary Application of Anti-cephalexin Monoclonal Antibody

ZHANG Wei¹, WANG Xing², LIU Jin-ping¹, XU Sheng-le¹, QIN Hai-yan¹, LIU Ruo-xuan¹, CAI Yun-hong¹, RU Xiao-fei¹, HOU Ke¹, WANG Han-dong¹

1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. Animal Epidemic Prevention Station of Zhangpu Town, Kunshan 215300, China

Received:2016-09-30 Online:2017-04-20 Published:2017-05-03

摘要/Abstract

摘要：

为制备抗头孢氨苄（cephalexin，CEX）的单克隆抗体并建立鲜奶中CEX残留检测的ELISA方法，本试验采用戊二醛两步法分别将CEX与牛血清白蛋白（BSA）、卵清白蛋白（OVA）偶联，用合成的CEX-BSA作为免疫原免疫BALB/c雌鼠；5次免疫后，应用杂交瘤细胞融合技术建立并筛选出两株（2G4、5B3）分泌抗CEX单克隆抗体（monoclonal antibodies，McAb）的杂交瘤细胞株，将2G4细胞株注入BALB/c小鼠腹腔制备腹水，并对腹水进行纯化及鉴定。结果显示，2G4腹水效价大于1∶1.28×10⁵，其抗体亚型为IgG2b（κ），亲和力常数K=1.51×10⁹ L/mol；交叉试验结果显示，该单克隆抗体除了与头孢拉定有52.55%的交叉反应率外，与头孢噻呋、头孢曲松、头孢噻肟、头孢噻吩、青霉素、链霉素、四环素均无明显交叉反应；建立了测定鲜奶样品中CEX的ELISA方法，线性范围为20~1 000 ng/mL，线性方程y=0.4674x-0.5359（R²=0.9909），检测下限为19.68 ng/mL，平均回收率为91.31%，平均变异系数低于15%，低于中国及欧盟规定的头孢氨苄最高残留限量，具有一定的应用前景。

关键词: 单克隆抗体; 头孢氨苄; ELISA; 人工抗原

Abstract:

The aim of the study was to prepare a monoclonal antibody (McAb) against cephalexin(CEX),and preliminarily establish an ELISA method to detect residues of CEX in milk. The CEX was conjugated to carrier bovine serum albumin (BSA) and ovalbumin (OVA) by two-step glutaraldehyde method. BALB/c mice were immunized with the prepared CEX-BSA conjugate. After five times immunization, we built and screened two strains of hybridoma cells (2G4 and 5B3) which secreted McAb against CEX by hybridoma technology.The 2G4 cell lines were injected into BALB/c mouse's abdominal cavity to produce ascites. Finally the ascites were purified and identified. The results showed that antibody titer of the McAb 2G4 was above 1∶1.28×10⁵,its antibody subtype was IgG2b(κ) and affinity constant K was 1.51×10⁹ L/mol. The cross experiment result evidenced that the cross reaction rate of cefradine was 52.55%, ceftiofur, ceftriaxone, cefotaxime, cephalothin, penicillin, streptomycin and tetracycline had no cross reaction. We established an ELISA method to detect CEX residues in milk and its linear range was 20 to 1 000 ng/mL, linear equation y=0.4674x-0.5359(R²=0.9909), its limit of detection (LOD) was 19.68 ng/mL and the average recovery rate was 91.31%, the average coefficient of variation was below 15%. The detection limit was lower than the maximum residue limit of cefalexin in China and European Union. So this detection method might have good application foreground.

Key words: monoclonal antibody; cephalexin; ELISA; artificial antigen

中图分类号:

S859.79⁺6

张伟, 王兴, 刘金萍, 徐声乐, 秦海艳, 刘若璇, 蔡云虹, 汝晓飞, 侯珂, 王捍东. 抗头孢氨苄单克隆抗体的制备与初步应用[J]. 《中国畜牧兽医》, 2017, 44(4): 1159-1167.

ZHANG Wei, WANG Xing, LIU Jin-ping, XU Sheng-le, QIN Hai-yan, LIU Ruo-xuan, CAI Yun-hong, RU Xiao-fei, HOU Ke, WANG Han-dong. Preparation and Preliminary Application of Anti-cephalexin Monoclonal Antibody[J]. , 2017, 44(4): 1159-1167.

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抗头孢氨苄单克隆抗体的制备与初步应用

Preparation and Preliminary Application of Anti-cephalexin Monoclonal Antibody

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