抗癌胚抗原(CEA)纳米抗体的筛选鉴定及双纳米抗体夹心ELISA检测CEA

doi:10.13523/j.cb.2307014

中国生物工程杂志

2024, Vol. 44

Issue (2/3): 48-58 DOI: 10.13523/j.cb.2307014

技术与方法

抗癌胚抗原(CEA)纳米抗体的筛选鉴定及双纳米抗体夹心ELISA检测CEA

王新婷,胡倩倩,娄楚,杨天宁,李江伟^*(

)

新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室乌鲁木齐 830046

Screening and Identification of Anti-CEA Nanobody and Construction of a Double-nanobody ELISA for CEA Detection

WANG Xinting,HU Qianqian,LOU Chu,YANG Tianning,LI Jiangwei^*(

)

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China

全文: PDF(1028 KB) HTML

摘要：

目的: 筛选能与癌胚抗原(carcinoembryonic antigen, CEA)高亲和力结合的纳米抗体(nanobodies, Nbs),用于今后开发CEA的快速检测方法。方法: 使用人CEA蛋白免疫双峰驼,以淋巴细胞cDNA为模板,采用巢式PCR扩增重链抗体可变区(VHH)基因,连接到pMECS载体,电转化TG1大肠杆菌,构建VHH cDNA文库。经辅助噬菌体M13K07感染后生成噬菌体展示文库,采用ELISA固相亲和淘洗方法,富集与CEA结合的VHH克隆,通过胞间质可溶性ELISA(PE-ELISA)筛选与CEA高结合的纳米抗体,并在WK6中进行IPTG诱导表达和亲和纯化。ELISA检测纳米抗体与CEA的结合特异性,竞争ELISA筛选配对纳米抗体构建夹心ELISA。结果: 构建获得了库容为2.8×10⁸ cfu的VHH文库,菌液PCR表明VHH插入约为100%,随机挑取第2~3轮淘洗富集的90个克隆进行PE-ELISA检测,结果45个克隆为阳性,序列分析表明,它们编码7个纳米抗体序列。在WK6中,这些纳米抗体以可溶形式表达,经Ni亲和层析获得了纯度接近90%的重组纳米抗体。间接ELISA显示7株纳米抗体均特异结合CEA, 其中2株纳米抗体3G2和3F4与CEA具有较高亲和力且呈浓度依赖,即使在高浓度(5 μg/mL)下也不与无关蛋白溶菌酶结合,表现出较高特异性。竞争ELISA结果显示,3G2和3F4在结合CEA中没有竞争作用,表明3G2和3F4结合在CEA的不同表位。将3F4作为捕获抗体,3G2作为检测抗体,建立了双纳米抗体夹心ELISA方法(2Nbs-ELISA法),其对溶液中CEA的检测限(limit of detection, LOD)为1.8 ng/mL。结论: 采用获得的两株CEA特异纳米抗体建立了夹心ELISA检测方法,对CEA的检测具有更高敏感性和特异性。

关键词： CEA; 纳米抗体; 噬菌体展示; 表位归类; 双纳米抗体夹心ELISA

Abstract:

Objective: To screen nanobodies that bind to carcino-embryonic antigen (CEA) with high affinity for the development of rapid detection methods for CEA in the future. Methods: Using human CEA protein to immunize Bactrian camels, lymphocyte cDNA was used as a template, and nested PCR was used to amplify the variable domain of heavy chain of heavy-chain antibody (VHH) gene, ligated to pMECS vector, and electroporated into TG1 E. coli to construct a VHH cDNA library. After infection with helper phage M13K07, a phage display library was generated, and VHH clones bound to CEA were enriched by biopanning in ELISA, nanobodies with high binding to CEA were screened by periplasmic soluble ELISA (PE-ELISA), and IPTG-induced expression and affinity purification were performed in WK6. The binding specificity of the nanobodies to CEA was by measured by ELISA,and the epitope overlap was detected by competitive ELISA to screen paired nanobodies for the construction of sandwich ELISAs. Results: A VHH library with a size of 2.8×10⁸ cfu was constructed. Colony PCR showed that VHH insertion was about 100%. A total of 90 clones enriched in the 2nd-3rd round were randomly selected for PE-ELISA detection, and 45 clones were positive. Sequence analysis showed that they encoded 7 nanobody sequences. In WK6, these nanobodies are expressed in a soluble form, and recombinant nanobodies with a purity close to 90% are obtained by Ni affinity chromatography. Indirect ELISA showed that all 7 Nbs specifically bound CEA, and 2 nanobodies 3G2 and 3F4 had high affinity and concentration-dependent binding to CEA, and did not bind to the unrelated protein lysozyme even at high concentrations (5 μg/mL), showing high specificity. Competitive ELISA results showed that 3G2 and 3F4 did not compete in binding to CEA, indicating that 3G2 and 3F4 bind to different epitopes of CEA antigens. Using 3F4 as the capture antibody and 3G2 as the detection antibody, the double nanobody sandwich ELISA method (referred to as 2Nbs-ELISA) was established, and its limit of detection (LOD) for CEA in solution was 1.8 ng/mL. Conclusion: The two CEA-specific Nbs obtained in this study were used to establish a sandwich ELISA method with higher sensitivity and specificity for the detection of CEA.

Key words: CEA Nanobodies Phage display Epitope binning Double nanobody sandwich ELISA

收稿日期: 2023-07-11 出版日期: 2024-04-03

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通讯作者: *电子信箱:jwli67@sina.com

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引用本文:

王新婷, 胡倩倩, 娄楚, 杨天宁, 李江伟. 抗癌胚抗原(CEA)纳米抗体的筛选鉴定及双纳米抗体夹心ELISA检测CEA[J]. 中国生物工程杂志, 2024, 44(2/3): 48-58.

WANG Xinting, HU Qianqian, LOU Chu, YANG Tianning, LI Jiangwei. Screening and Identification of Anti-CEA Nanobody and Construction of a Double-nanobody ELISA for CEA Detection. China Biotechnology, 2024, 44(2/3): 48-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2307014 或 https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I2/3/48

表1 用于扩增VHH片段的巢式PCR引物

图1 ELISA检测骆驼免疫血清中CEA特异抗体滴度

图2 CEA免疫VHH文库的构建 A: PBMC cDNA中VH和VHH基因片段的PCR扩增。左:第一轮PCR;右:第二轮PCR B:通过在平板上生长的稀释后TG1-VHH克隆数量来确定VHH文库的大小 C:用集落PCR检测TG1-VHH文库中的VHH插入物。1~30列:随机选择30个TG1-VHH克隆;-:阴性对照

表2 亲和淘洗中的输入和输出

图3 各轮筛选中回收率及富集度的变化

图4 抗CEA纳米抗体的序列分析 A: CEA纳米抗体CDR氨基酸序列 B:7株非冗余CEA纳米抗体系统进化分析

图5 SDS-PAGE分析抗CEA纳米抗体的纯化 B:胞间质裂解液;F: 流川; E: 洗脱的纳米抗体

图6 ELISA分析纳米抗体与CEA的结合 A:ELISA分析纳米抗体与CEA的结合 B:7种纳米抗体在3种浓度下与CEA结合的检测 C: 纳米抗体3F4和3G2与CEA和溶菌酶的结合曲线

图7 竞争ELISA分析3G2和3F4结合CEA的表位重叠性 A:竞争性ELISA分析表位的示意图 B:3G2存在和不存在时M13-3F4的结合值

图8 2Nbs-ELISA 法标准曲线的建立 A:夹心ELISA示意图 B:2Nbs-ELISA 方法校准曲线 C:2Nbs-ELISA法标准曲线

[1]	Ek W E, Karlsson T, Höglund J, et al. Causal effects of inflammatory protein biomarkers on inflammatory diseases. Science Advances, 2021, 7(50): eabl4359.
[2]	Hall C, Clarke L, Pal A, et al. A review of the role of carcinoembryonic antigen in clinical practice. Annals of Coloproctology, 2019, 35(6): 294-305. doi: 10.3393/ac.2019.11.13 pmid: 31937069
[3]	Desai S, Guddati A K. Carcinoembryonic antigen, carbohydrate antigen 19-9, cancer antigen 125, prostate-specific antigen and other cancer markers: a primer on commonly used cancer markers. World Journal of Oncology, 2023, 14(1): 4-14. doi: 10.14740/wjon1425 pmid: 36895994
[4]	Ding Y F, Huang C X, Zhang Y M, et al. Magnetic microbead enzyme-linked immunoassay based on phage encoded protein RBP 41-mediated for rapid and sensitive detection of Salmonella in food matrices. Food Research International, 2023, 163: 112212. doi: 10.1016/j.foodres.2022.112212
[5]	Huang L J, Qiu S, Liu Z, et al. Proximity hybridization induced DNA assembly for label-free surface-enhanced Raman spectroscopic detection of carcinoembryonic antigen. Analytica Chimica Acta, 2022, 1191: 339314. doi: 10.1016/j.aca.2021.339314
[6]	Zhao S S, Zhao S N, Guo Y. Time-resolved fluorescence immunoassay for detection of CEA in saliva. Advances in Food Safety and Environmental Engineering. London: CRC Press, 2022: 107-115.
[7]	Singh S, Podder P S, Russo M, et al. Tailored point-of-care biosensors for liquid biopsy in the field of oncology. Lab on a Chip, 2023, 23(1): 44-61. doi: 10.1039/D2LC00666A
[8]	Jin C R, Wu Z Q, Molinski J H, et al. Plasmonic nanosensors for point-of-care biomarker detection. Materials Today Bio, 2022, 14: 100263. doi: 10.1016/j.mtbio.2022.100263
[9]	Kumar P, Sarkar N, Singh A, et al. Nanopaper biosensors at point of care. Bioconjugate Chemistry, 2022, 33(6): 1114-1130. doi: 10.1021/acs.bioconjchem.2c00213 pmid: 35658426
[10]	Wang Y D, Xianyu Y L. Nanobody and nanozyme-enabled immunoassays with enhanced specificity and sensitivity. Small Methods, 2022, 6(4): e2101576.
[11]	Bastos-Soares E A, Sousa R M O, Gómez A F, et al. Single domain antibodies in the development of immunosensors for diagnostics. International Journal of Biological Macromolecules, 2020, 165: 2244-2252. doi: 10.1016/j.ijbiomac.2020.10.031 pmid: 33058975
[12]	Salvador J P, Vilaplana L, Marco M P. Nanobody: outstanding features for diagnostic and therapeutic applications. Analytical and Bioanalytical Chemistry, 2019, 411(9): 1703-1713. doi: 10.1007/s00216-019-01633-4 pmid: 30734854
[13]	Yang E Y, Shah K. Nanobodies: next generation of cancer diagnostics and therapeutics. Frontiers in Oncology, 2020, 10: 1182. doi: 10.3389/fonc.2020.01182 pmid: 32793488
[14]	Koczula K M, Gallotta A. Lateral flow assays. Essays in Biochemistry, 2016, 60(1): 111-120. doi: 10.1042/EBC20150012 pmid: 27365041
[15]	Ibrahim J, Peeters M, Van Camp G, et al. Methylation biomarkers for early cancer detection and diagnosis: Current and future perspectives. Eur J Cancer, 2023,178:91-113.
[16]	Yang Y, Jiang X L, Liu Y, et al. Elevated tumor markers for monitoring tumor response to immunotherapy. EClinicalMedicine, 2022, 46: 101381. doi: 10.1016/j.eclinm.2022.101381
[17]	Tabatabaei M S, Ahmed M. Enzyme-linked immunosorbent assay (ELISA). Methods in Molecular Biology, 2022, 2508: 115-134. doi: 10.1007/978-1-0716-2376-3_10 pmid: 35737237
[18]	Jeong S, Park M J, Song W, et al. Current immunoassay methods and their applications to clinically used biomarkers of breast cancer. Clinical Biochemistry, 2020, 78: 43-57. doi: S0009-9120(19)31056-2 pmid: 32007438
[19]	Laraib U, Sargazi S, Rahdar A, et al. Nanotechnology-based approaches for effective detection of tumor markers: a comprehensive state-of-the-art review. International Journal of Biological Macromolecules, 2022, 195: 356-383. doi: 10.1016/j.ijbiomac.2021.12.052 pmid: 34920057
[20]	Zhang Y, Li M Y, Gao X M, et al. Nanotechnology in cancer diagnosis: progress, challenges and opportunities. Journal of Hematology & Oncology, 2019, 12(1): 137.
[21]	Suan Ng S, Ling Lee H, Bothi Raja P, et al. Recent advances in nanomaterial‐based optical biosensors as potential point‐of‐care testing (PoCT) probes in carcinoembryonic antigen detection. Chemistry:An Asian Journal, 2022, 17(14): e202200287.
[22]	Larkins M C, Thombare A. Point-of-care testing//StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing, 2024.

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