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
• • 上一篇
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
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
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。
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[1] Lourdes, Roque-Navarro, Krittalak, et al. Anti-ganglioside antibody-induced tumor cell death byloss of membrane integrity [J]. Mol Cancer Ther, 2008, 7(7): 2033-2041. [2] Wu JH, Hao PP, Chen YG, et al. Intracoronary glycoprotein IIb/IIIa inhibitors improve short-Term mortality and reinfarction in east asian patients with sT-Segment elevation myocardial infarction after thrombus aspiration: a meta-analysis [J]. Evid Based Complement Alternat Med, 2018, 2018: 5174714. doi: 10.1155/2018/5174714. [3] Grainger JD, Thachil JandWill AM. How we treat the platelet glycoprotein defects; Glanzmann thrombasthenia and Bernard Soulier syndrome in children and adults [J]. Br J Haematol, 2018, 182(5): 621-632. [4] Reilly SW, Puentes LN, Hsieh CJ, et al. Altering Nitrogen Heterocycles of AZD2461 Affords High Affinity Poly(ADP-ribose)Polymerase-1 Inhibitors with Decreased P-Glycoprotein Interactions [J]. ACS Omega, 2018, 3(8): 9997-10001. [5] Llop E, P EG, Duran A, et al. Glycoprotein biomarkers for the detection of pancreatic ductal adenocarcinoma [J]. World J Gastroenterol, 2018, 24(24): 2537-2554. [6] Nishibori M, Wake HandMorimatsu H. Histidine-rich glycoprotein as an excellent biomarker for sepsis and beyond [J]. Critical care, 2018, 22(1): 209. [7] Shah AK, Hartel G, Brown I, et al. Evaluation of serum glycoprotein biomarker candidates for detection of esophageal adenocarcinoma and surveillance of Barrett's esophagus [J]. Mol Cell Proteomics, 2018. pii: mcp. RA118.000734.doi:10.1074/mcp.RA118.000734. [8] Wang X, Lin JM, Ying X. Evaluation of carbohydrate antigen 50 in human serum using magnetic particle-based chemiluminescence enzyme immunoassay [J]. Anal Chim Acta, 2007, 598(2): 261-267. [9] Woldike PM, Charib K,Siboni A. [Use of tumor marker CA50] [J]. Ugeskr Laeger, 2008, 170(38): 2985. [10] Liao Q, Zhao YP, Yang YC, et al. Combined detection of serum tumor markers for differential diagnosis of solid lesions located at the pancreatic head [J]. Hepatobiliary Pancreat Dis Int, 2007, 6(6): 641-645. [11] 段泽星, 罗俊卿, 李伟强, 等. 血清CEA、CA242、CA50、CA19-9对结直肠癌诊断和生物学特性评估的意义[J]. 胃肠病学, 2014, 19(4): 217-220. DUAN Zexing, LUO Junqing, LI Weiqiang, et al. Significance of serum CEA, CA242, CA50, and ca19-9 in diagnosis and evaluation of biological characteristics colorectal cancer [J]. Chinese Journal of Gastroenterology, 2014, 19(4): 217-220. [12] 陶晓军, 冯晓鸿, 孙业富. CA72-4、CA19-9、CA50、CA242联合检测在胃癌诊断中的应用[J]. 实验与检验医学, 2012, 30(2): 169-171. [13] 尹莉莉, 杜叶平, 武春梅. 血清CA50与CA199联合检测在胰腺癌诊断中的意义[J]. 解放军医药杂志, 2013, 25(1): 31-32. YIN Lili,DU Yeping,WU Chunmei. Significance of the Detection of Serum CA50 Combined with CA199 in Diagnosis of Pancreatic Cancer[J]. Medical & Pharmaceutical Journal of Chinese Peoples Liberation Army, 2013, 25(1): 31-32. [14] 朱剑峰, 李志辉, 朱红静, 等. CEA、CA199、CA724、CA242、CA125、CA50对消化道肿瘤的诊断价值[J]. 实用癌症杂志, 2014, 29(5): 501-502. ZHU Jianfeng, LI Zhihui, ZHU Hongjing, et al. Diagnostic value of CEA、CA199、CA724、CA242、CA125 and CA50 in gastrointestinal tumors[J]. The Practical Journal of Cancer, 2014, 29(5): 501-502. [15] Lindholm L, Holmgren J, Svennerholm L, et al. Monoclonal antibodies against gastrointestinal tumour-associated antigens isolated as monosialogangliosides[J]. In Archi Allergy Appl Immunol, 2009, 71(2):178-181. [16] Weiner GJ. Building better monoclonal antibody-based therapeutics [J]. Nat Rev Cancer, 2015, 15(6): 361-370. [17] Lei XF, Jia SZ, Ye J, et al. Application values of detection of serum CA199, CA242 and CA50 in the diagnosis of pancreatic cancer [J]. J Biol Regul Homeost Agents, 2017, 31(2): 383-388. [18] Watanabe M, Chigusa M, Takahashi H, et al. High level of CA19-9, CA50, and CEA-producible human cholangiocarcinoma cell line changes in the secretion ratios in vitro or in vivo [J]. In Vitro Cell Dev Biol Anim, 2000, 36(2): 104-109. [19] Xie M, Huang H, Hang J, et al. Evaluation of the analytical and clinical performances of time-resolved fluoroimmunoassay for detecting carcinoma antigen 50 [J]. J Immunoassay Immunochem, 2015, 36(3): 265-283. [20] Wei H, Hu J, Wang L, et al. Rapid gene splicing and multi-sited mutagenesis by one-step overlap extension polymerase chain reaction [J]. Anal Biochem, 2012, 429(1): 76-78. [21] Edenberg HJ, Moss LGandRutter WJ. Convenient vectors for cloning and sequencing EcoRI and HindIII fragments [J]. Gene, 1987, 58(2-3): 297-298. [22] Xu HX, Li S, Wu CT, et al. Postoperative serum CA19-9, CEA and CA125 predicts the response to adjuvant chemoradiotherapy following radical resection in pancreatic adenocarcinoma [J]. Pancreatology, 2018, 18(6): 671-677. [23] Sauzay C, Petit A, Bourgeois AM, et al. Alpha-foetoprotein(AFP): A multi-purpose marker in hepatocellular carcinoma [J]. Clin Chim Acta, 2016, 463: 39-44. doi: 10.1016/j.cca.2016.10.006. [24] Liu L, Xu HX, Wang WQ, et al. Serum CA125 is a novel predictive marker for pancreatic cancer metastasis and correlates with the metastasis-associated burden [J]. Oncotarget, 2016, 7(5): 5943-5956. [25] Peracaula R, Tabares G, Lopez-Ferrer A, et al. Role of sialyltransferases involved in the biosynthesis of Lewis antigens in human pancreatic tumour cells [J]. Glycoconj J, 2005, 22(3): 135-144. [26] Romao E, Poignavent V, Vincke C, et al. Construction of high-quality camel immune antibody libraries [J]. Methods Mol Biol, 2018, 1701: 169-187. doi: 10.1007/978-1-4939-7447-4_9. [27] Tullila A and Nevanen TK. Utilization of multi-immunization and multiple selection strategies for isolation of hapten-specific antibodies from recombinant antibody phage display libraries [J]. Int J Mol Sci, 2017, 18(6). pii: E1169. doi: 10.3390/ijms18061169. |
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