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山东大学学报 (医学版) ›› 2018, Vol. 56 ›› Issue (10): 51-57.doi: 10.6040/j.issn.1671-7554.0.2018.1027

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CA50靶向抗体文库的构建筛选及应用评价

杜鲁涛1,2,赵巧辉3,田晓平3,吴学炜4,付光宇4,渠海4, 苗拥军4,王丽丽5,郑桂喜5,郭海洋6,李桂林3,王传新1,2   

  1. 1.山东大学第二医院检验医学中心, 山东 济南250033;2.山东省肿瘤标志物检测工程实验室, 山东 济南250033; 3.郑州伊美诺生物技术有限公司, 河南 郑州 450016;4.郑州安图生物工程股份有限公司, 河南 郑州 450016; 5.山东大学齐鲁医院检验医学中心, 山东 济南 250012;6.玛格丽特公主癌症中心/大学健康网络, 安大略 多伦多 M5G 1L7, 加拿大
  • 收稿日期:2018-09-04 出版日期:2018-10-10 发布日期:2022-09-27
  • 通讯作者: 王传新. E-mail:cxwang@sdu.edu.cn李桂林. E-mail:liguilin@autobio.com.cn
  • 基金资助:
    山东省重点研发(重点产业关键技术)(2016CYJS01A02)

Construction and screening of CA50 targeted antibody library and establishment of immunoassay system

DU Lutao1,2, ZHAO Qiaohui3, TIAN Xiaoping3, WU Xuewei4, FU Guangyu4, QU Hai4, MIAO Yongjun4, WANG Lili5, ZHENG Guixi5, GUO Haiyang6, LI Guilin3, WANG Chuanxin1,2   

  1. 1. Department of Clinical Laboratory, The Second Hospital of Shandong University, Jinan 250033, Shandong, China;
    2. Tumor Marker Detection Engineering Laboratory of Shandong Province, Jinan 250033, Shandong, China;
    3. Zhengzhou Immuno Bio-Tech Co., Ltd, Zhengzhou 450016, Henan, China;
    4. Autobio Diagnostics Co., Ltd, Zhengzhou 450016, Henan, China;
    5. Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China;
    6. Princess Margaret Cancer Centre/University Health Network, Toronto, M5G 1L7, Ontario, Canada
  • Received:2018-09-04 Online:2018-10-10 Published:2022-09-27

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摘要： 目的 获得针对CA50靶点的高亲和力、高特异性抗体,建立体外诊断CA50检测试剂盒,提高肿瘤诊断的特异性和灵敏度。 方法 PCR 技术获得单链抗体( ScFv)基因并克隆入pcomb3XSS噬粒载体,通过电转化获得ScFv噬菌体抗体库。用CA50特性识别表位LST a(唾液酸化-乳糖-N-四糖a)的抗原淘洗筛选,获得针对CA50靶点的特异性抗体基因序列。随机挑选克隆测序,验证ScFv抗体库的多样性。利用重叠PCR技术获得人鼠嵌合抗体基因,经过抗体表达纯化及配对筛选建立双抗体夹心法CA50检测试剂盒。同时收集临床诊断阳性样本54例和临床阴性对照样本146例,利用该研究建立的CA50检测试剂盒与罗氏CA50试剂盒进行平行对照,评价其检测相关性。 结果 建立噬菌体抗体库的容量为2.5×108,具有较好的多样性。24个PCR鉴定阳性克隆测序,结果为20个正确插入且插入序列均不相同。经过3轮淘洗筛选出5个阳性克隆,经过抗体配对筛选建立的CA50试剂盒临床样本检测结果与罗氏的临床相关性(r=0.98)。 结论 通过噬菌体展示抗体库技术成功筛选出了针对CA50的高特异性、高亲和力抗体,为CA50检测在相关肿瘤辅助诊断中的应用提供可能性。

关键词: CA50靶点, 诊断, 抗体库, 评价

Abstract: Objective To obtain antibodies against CA50 antigen with high affinity and specificity, and to establish a diagnostic kit so as to enhance the specificity and sensitivity of the diagnosis of tumor related diseases. Methods The mouse single chain antibody(ScFv)genes were obtained by PCR and cloned into pcomb3XSS phagemids. ScFv phage antibody library was obtained by electrotransformation. CA50 antigen containing epitope LST a(sialic acid lactose-N-four sugar a)was used for the elutriation screening. Random clone sequencing results showed that the antibody library 山 东 大 学 学 报 (医 学 版)56卷10期 -杜鲁涛,等. CA50靶向抗体文库的构建筛选及应用评价 \=-had good diversity. The human mouse chimeric antibody was constructed by overlap-PCR. The double antibody sandwich method CA50 kit was established after antibody expression purification and matching screening. Meanwhile, 54 clinical positive samples and 146 negative control samples were collected. The CA50 kit established was used for comparation with Roches CA50 kit to evaluate the detection correlation. Results The phage antibody library had a capacity of 2.5×108, and had a good diversity. The positive clones of 24 strains were identified by PCR. The sequencing results showed that 20 strains were correct and the sequences were different. After 3 rounds of panning, 5 positive clones were screened out, and the correlation between the results of CA50 kit and Roches CA50 kit was 0.98. Conclusion The antibodies with high specificity and affinity against CA50 were successfully screened by phage library technology, which made the application of CA50 possible in tumor diagnosis.

Key words: CA50 targets, Diagnosis, Antibody library, Evaluation

中图分类号: 

  • R392-33
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