生物技术通报 ›› 2022, Vol. 38 ›› Issue (5): 248-256.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0862
收稿日期:
2021-07-04
出版日期:
2022-05-26
发布日期:
2022-06-10
作者简介:
王加利,女,博士,研究方向:噬菌体展示;E-mail: 基金资助:
WANG Jia-li(), HE Si-qi, KANG Zi-xi, WANG Jian-xun()
Received:
2021-07-04
Published:
2022-05-26
Online:
2022-06-10
摘要:
噬菌体抗体展示技术是第一个也是目前应用最广泛的体外抗体筛选技术,可用于发现治疗各种疾病的全人源抗体。虽然存在不同的抗体发现方法,但噬菌体抗体展示技术已成为发现和优化靶标特异性单克隆抗体不可或缺的工具。研究人员可通过在丝状噬菌体上创建组合抗体库,从而在几周内获得抗原特异性抗体。在当前冠状病毒病(COVID-19)大流行的情况下,对中和抗体的研究激励着研究人员寻找出抗SARS-CoV-2的候选治疗药物。通过对噬菌体抗体库的筛选,目前已有许多不同的SARS-CoV-2中和抗体被发现。因此,本文综述了噬菌体抗体展示技术的原理,抗体库的构建、分类及淘选流程,并对其优点及局限性进行了讨论。此外,还总结了利用该技术发现抗SARS-CoV-2中和抗体的最新进展。旨为今后该技术在抗体发现中的应用提供理论支持。
王加利, 和似琦, 康子茜, 王建勋. 噬菌体抗体展示技术及其在抗新冠病毒抗体发现中的应用[J]. 生物技术通报, 2022, 38(5): 248-256.
WANG Jia-li, HE Si-qi, KANG Zi-xi, WANG Jian-xun. Antibody Phage Display Technology and Its Application in the Discovery of Anti-SARS-CoV-2 Antibodies[J]. Biotechnology Bulletin, 2022, 38(5): 248-256.
抗体名称 Antibody description | 噬菌体抗体库 Phage antibody library | 靶抗原 Target antigen | 抗体类型 Antibody format | 中和活性 Neutralization activity | 参考文献 Reference |
---|---|---|---|---|---|
H014 | 免疫Fab文库 | RBD | 人源化Fab | 假病毒 3 nmol/L 活病毒 38 nmol/L | [ |
IgG1 ab1 | 天然Fab、scFv、VH文库 | RBD | IgG1 | 活病毒200 ng/mL | [ |
HB27 | 免疫Fab文库 | RBD | IgG1 | 活病毒0.22 nmol/L | [ |
II62 | 天然半合成scFv文库 | RBD | scFv、scFv- Fc、IgG1 | — | [ |
VHH-72 | 免疫VHH文库 | S | VHH-Fc | 假病毒 0.2 μg/mL | [ |
H11-D4、H11-H4 | 天然VHH文库 | RBD | VHH-Fc | 活病毒18 nmol/L(H11-D4)、4-6 nmol/L(H11-H4) | [ |
Ty1 | 免疫VHH文库 | RBD | VHH、VHH-Fc | 假病毒0.77 µg/mL(Ty1) 12 ng/mL(Ty1-Fc) | [ |
Nb11-59 | 免疫VHH文库 | RBD | VHH | 活病毒0.55 µg/mL | [ |
双特异性VHH | 天然和合成VHH文库 | S1 | VHH-Fc | — | [ |
VHH EEE | 免疫VHH文库 | RBD | 三价VHH | 假病毒 0.52 nmol/L | [ |
表1 噬菌体展示来源的靶向SARS-CoV-2刺突蛋白抗体的临床前研究
Table 1 Preclinical studies of phage display-derived antibodies targeting the spike protein of SARS-CoV-2
抗体名称 Antibody description | 噬菌体抗体库 Phage antibody library | 靶抗原 Target antigen | 抗体类型 Antibody format | 中和活性 Neutralization activity | 参考文献 Reference |
---|---|---|---|---|---|
H014 | 免疫Fab文库 | RBD | 人源化Fab | 假病毒 3 nmol/L 活病毒 38 nmol/L | [ |
IgG1 ab1 | 天然Fab、scFv、VH文库 | RBD | IgG1 | 活病毒200 ng/mL | [ |
HB27 | 免疫Fab文库 | RBD | IgG1 | 活病毒0.22 nmol/L | [ |
II62 | 天然半合成scFv文库 | RBD | scFv、scFv- Fc、IgG1 | — | [ |
VHH-72 | 免疫VHH文库 | S | VHH-Fc | 假病毒 0.2 μg/mL | [ |
H11-D4、H11-H4 | 天然VHH文库 | RBD | VHH-Fc | 活病毒18 nmol/L(H11-D4)、4-6 nmol/L(H11-H4) | [ |
Ty1 | 免疫VHH文库 | RBD | VHH、VHH-Fc | 假病毒0.77 µg/mL(Ty1) 12 ng/mL(Ty1-Fc) | [ |
Nb11-59 | 免疫VHH文库 | RBD | VHH | 活病毒0.55 µg/mL | [ |
双特异性VHH | 天然和合成VHH文库 | S1 | VHH-Fc | — | [ |
VHH EEE | 免疫VHH文库 | RBD | 三价VHH | 假病毒 0.52 nmol/L | [ |
[1] |
Winter G, Milstein C. Man-made antibodies[J]. Nature, 1991, 349(6307):293-299.
doi: 10.1038/349293a0 URL |
[2] |
Alfaleh MA, Alsaab HO, Mahmoud AB, et al. Phage display derived monoclonal antibodies:from bench to bedside[J]. Front Immunol, 2020, 11:1986.
doi: 10.3389/fimmu.2020.01986 URL |
[3] |
Throsby M, van den Brink E, Jongeneelen M, et al. Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells[J]. PLoS One, 2008, 3(12):e3942.
doi: 10.1371/journal.pone.0003942 URL |
[4] |
de Carvalho Nicacio C, Williamson RA, Parren PW, et al. Neutralizing human Fab fragments against measles virus recovered by phage display[J]. J Virol, 2002, 76(1):251-258.
doi: 10.1128/JVI.76.1.251-258.2002 URL |
[5] |
Kramer R, Marissen W, Goudsmit J, et al. The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries[J]. Eur J Immunol, 2005, 35(7):2131-2145.
pmid: 15971273 |
[6] |
Sokullu E, Soleymani Abyaneh H, Gauthier MA. Plant/bacterial virus-based drug discovery, drug delivery, and therapeutics[J]. Pharmaceutics, 2019, 11(5):211.
doi: 10.3390/pharmaceutics11050211 URL |
[7] |
Specthrie L, Bullitt E, Horiuchi K, et al. Construction of a microphage variant of filamentous bacteriophage[J]. J Mol Biol, 1992, 228(3):720-724.
pmid: 1469710 |
[8] |
van Wezenbeek PM, Hulsebos TJ, Schoenmakers JG. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome:comparison with phage fd[J]. Gene, 1980, 11(1/2):129-148.
doi: 10.1016/0378-1119(80)90093-1 URL |
[9] |
Kehoe JW, Kay BK. Filamentous phage display in the new millennium[J]. Chem Rev, 2005, 105(11):4056-4072.
pmid: 16277371 |
[10] |
Smith GP. Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface[J]. Science, 1985, 228(4705):1315-1317.
doi: 10.1126/science.4001944 pmid: 4001944 |
[11] |
Hoogenboom HR, Griffiths AD, Johnson KS, et al. Multi-subunit proteins on the surface of filamentous phage:methodologies for displaying antibody(Fab)heavy and light chains[J]. Nucleic Acids Res, 1991, 19(15):4133-4137.
pmid: 1908075 |
[12] |
Rakonjac J, Jovanovic G, Model P. Filamentous phage infection-mediated gene expression:construction and propagation of the gIII deletion mutant helper phage R408d3[J]. Gene, 1997, 198(1/2):99-103.
doi: 10.1016/S0378-1119(97)00298-9 URL |
[13] |
Ledsgaard L, Kilstrup M, Karatt-Vellatt A, et al. Basics of antibody phage display technology[J]. Toxins, 2018, 10(6):236.
doi: 10.3390/toxins10060236 URL |
[14] |
Rondot S, Koch J, Breitling F, et al. A helper phage to improve single-chain antibody presentation in phage display[J]. Nat Biotechnol, 2001, 19(1):75-78.
pmid: 11135557 |
[15] |
Lowman HB, Bass SH, Simpson N, et al. Selecting high-affinity binding proteins by monovalent phage display[J]. Biochemistry, 1991, 30(45):10832-10838.
pmid: 1932005 |
[16] |
Tohidkia MR, Barar J, Asadi F, et al. Molecular considerations for development of phage antibody libraries[J]. J Drug Target, 2012, 20(3):195-208.
doi: 10.3109/1061186X.2011.611517 URL |
[17] |
Dübel S, Stoevesandt O, Taussig MJ, et al. Generating recombinant antibodies to the complete human proteome[J]. Trends Biotechnol, 2010, 28(7):333-339.
doi: 10.1016/j.tibtech.2010.05.001 URL |
[18] |
Schirrmann T, Meyer T, Schütte M, et al. Phage display for the generation of antibodies for proteome research, diagnostics and therapy[J]. Molecules, 2011, 16(1):412-426.
doi: 10.3390/molecules16010412 pmid: 21221060 |
[19] | Kim S, Park I, Park SG, et al. Generation, diversity determination, and application to antibody selection of a human naïve fab library[J]. Mol Cells, 2017, 40(9):655-666. |
[20] |
Bradbury ARM, Marks JD. Antibodies from phage antibody libraries[J]. J Immunol Methods, 2004, 290(1/2):29-49.
doi: 10.1016/j.jim.2004.04.007 URL |
[21] | Tsuruta LR, Dos ML, Moro AM. Display technologies for the selection of monoclonal antibodies for clinical use[M]// Böldicke T. Antibody Engineering.London:intechopen, 2018:47-73. |
[22] |
Lai JY, Lim TS. Infectious disease antibodies for biomedical applications:a mini review of immune antibody phage library repertoire[J]. Int J Biol Macromol, 2020, 163:640-648.
doi: 10.1016/j.ijbiomac.2020.06.268 URL |
[23] |
Kessler C, Pardo A, et al. Novel PSCA targeting scFv-fusion proteins for diagnosis and immunotherapy of prostate cancer[J]. J Cancer Res Clin Oncol, 2017, 143(10):2025-2038.
doi: 10.1007/s00432-017-2472-9 URL |
[24] |
Barbas CF, Bain JD, Hoekstra DM, et al. Semisynthetic combinatorial antibody libraries:a chemical solution to the diversity problem[J]. PNAS, 1992, 89(10):4457-4461.
pmid: 1584777 |
[25] |
Griffiths AD, Williams SC, Hartley O, et al. Isolation of high affinity human antibodies directly from large synthetic repertoires[J]. EMBO J, 1994, 13(14):3245-3260.
doi: 10.1002/j.1460-2075.1994.tb06626.x pmid: 8045255 |
[26] |
Sheets MD, Amersdorfer P, Finnern R, et al. Efficient construction of a large nonimmune phage antibody library:the production of high-affinity human single-chain antibodies to protein antigens[J]. PNAS, 1998, 95(11):6157-6162.
pmid: 9600934 |
[27] |
Chan CE, Lim AP, et al. The role of phage display in therapeutic antibody discovery[J]. Int Immunol, 2014, 26(12):649-657.
doi: 10.1093/intimm/dxu082 URL |
[28] | Andris-Widhopf J, Steinberger P, Fuller R, et al. Generation of human scFv antibody libraries:PCR amplification and assembly of light- and heavy-chain coding sequences[J]. Cold Spring Harb Protoc, 2011, 2011(9):pdb.prot065573. |
[29] |
Chen W, Dimitrov DS. Human monoclonal antibodies and engineered antibody domains as HIV-1 entry inhibitors[J]. Curr Opin HIV AIDS, 2009, 4(2):112-117.
doi: 10.1097/COH.0b013e328322f95e URL |
[30] |
Beck A, Goetsch L, Dumontet C, et al. Strategies and challenges for the next generation of antibody-drug conjugates[J]. Nat Rev Drug Discov, 2017, 16(5):315-337.
doi: 10.1038/nrd.2016.268 URL |
[31] |
Mattes MJ. Radionuclide-antibody conjugates for single-cell cytotoxicity[J]. Cancer, 2002, 94(4 suppl):1215-1223.
pmid: 11877748 |
[32] |
Alewine C, Hassan R, Pastan I. Advances in anticancer immunotoxin therapy[J]. Oncologist, 2015, 20(2):176-185.
doi: 10.1634/theoncologist.2014-0358 URL |
[33] |
Bradbury AR, Sidhu S, Dübel S, et al. Beyond natural antibodies:the power of in vitro display technologies[J]. Nat Biotechnol, 2011, 29(3):245-254.
doi: 10.1038/nbt.1791 pmid: 21390033 |
[34] |
Dumoulin M, Conrath K, Van Meirhaeghe A, et al. Single-domain antibody fragments with high conformational stability[J]. Protein Sci, 2002, 11(3):500-515.
doi: 10.1110/ps.34602 URL |
[35] |
Zhao AZ, Tohidkia MR, et al. Phage antibody display libraries:a powerful antibody discovery platform for immunotherapy[J]. Crit Rev Biotechnol, 2016, 36(2):276-289.
doi: 10.3109/07388551.2014.958978 URL |
[36] |
Hoogenboom HR. Selecting and screening recombinant antibody libraries[J]. Nat Biotechnol, 2005, 23(9):1105-1116.
pmid: 16151404 |
[37] | Shen W, Li SQ, et al. Blocking agent optimization for nonspecific binding on phage based magnetoelastic biosensors[J]. J Electrochem Soc, 2012, 159(10):B818-B823. |
[38] |
Jones ML, Seldon T, Smede M, et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies[J]. J Immunol Methods, 2010, 354(1/2):85-90.
doi: 10.1016/j.jim.2010.02.001 URL |
[39] | Bazan J, Całkosiński I, Gamian A. Phage display—A powerful technique for immunotherapy[J]. Hum Vaccines Immunother, 2012, 8(12):1817-1828. |
[40] |
Smith GP, Petrenko VA. Phage display[J]. Chem Rev, 1997, 97(2):391-410.
doi: 10.1021/cr960065d URL |
[41] |
Zhou M, Meyer T, Koch S, et al. Identification of a new epitope for HIV-neutralizing antibodies in the gp41 membrane proximal external region by an Env-tailored phage display library[J]. Eur J Immunol, 2013, 43(2):499-509.
doi: 10.1002/eji.201242974 URL |
[42] |
Nizak C, Monier S, del Nery E, et al. Recombinant antibodies to the small GTPase Rab6 as conformation sensors[J]. Science, 2003, 300(5621):984-987.
doi: 10.1126/science.1083911 URL |
[43] |
Liang WC, Wu XM, et al. Cross-species vascular endothelial growth factor(VEGF)-blocking antibodies completely inhibit the growth of human tumor xenografts and measure the contribution of stromal VEGF[J]. J Biol Chem, 2006, 281(2):951-961.
doi: 10.1074/jbc.M508199200 URL |
[44] |
Frenzel A, Kügler J, et al. Designing human antibodies by phage display[J]. Transfus Med Hemother, 2017, 44(5):312-318.
doi: 10.1159/000479633 URL |
[45] |
Froude JW, Pelat T, et al. Generation and characterization of protective antibodies to Marburg virus[J]. MAbs, 2017, 9(4):696-703.
doi: 10.1080/19420862.2017.1299848 URL |
[46] |
Schofield DJ, Pope AR, Clementel V, et al. Application of phage display to high throughput antibody generation and characterization[J]. Genome Biol, 2007, 8(11):R254.
doi: 10.1186/gb-2007-8-11-r254 pmid: 18047641 |
[47] |
Löfblom J, Wernérus H, et al. Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display[J]. FEMS Microbiol Lett, 2005, 248(2):189-198.
pmid: 15964717 |
[48] |
Hoffmann M, Kleine-Weber H, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor[J]. Cell, 2020, 181(2):271-280. e8.
doi: S0092-8674(20)30229-4 pmid: 32142651 |
[49] |
Fan X, Cao D, Kong L, et al. Cryo-EM analysis of the post-fusion structure of the SARS-CoV spike glycoprotein[J]. Nat Commun, 2020, 11(1):3618.
doi: 10.1038/s41467-020-17371-6 URL |
[50] |
Lv Z, Deng YQ, Ye Q, et al. Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody[J]. Science, 2020, 369(6510):1505-1509.
doi: 10.1126/science.abc5881 URL |
[51] | Li W, Drelich A, Martinez DR, et al. Potent neutralization of SARS-CoV-2 in vitro and in an animal model by a human monoclonal antibody[J]. bioRxiv, 2020, 093088. |
[52] |
Zhu L, Deng YQ, Zhang RR, et al. Double lock of a potent human therapeutic monoclonal antibody against SARS-CoV-2[J]. Natl Sci Rev, 2021, 8(3):nwaa297.
doi: 10.1093/nsr/nwaa297 URL |
[53] |
Parray HA, Chiranjivi AK, Asthana S, et al. Identification of an anti-SARS-CoV-2 receptor-binding domain-directed human monoclonal antibody from a naïve semisynthetic library[J]. J Biol Chem, 2020, 295(36):12814-12821.
doi: 10.1074/jbc.AC120.014918 pmid: 32727845 |
[54] |
Detalle L, Stohr T, Palomo C, et al. Generation and characterization of ALX-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection[J]. Antimicrob Agents Chemother, 2016, 60(1):6-13.
doi: 10.1128/AAC.01802-15 URL |
[55] |
Stalin Raj V, Okba NMA, Gutierrez-Alvarez J, et al. Chimeric camel/human heavy-chain antibodies protect against MERS-CoV infection[J]. Sci Adv, 2018, 4(8):eaas9667.
doi: 10.1126/sciadv.aas9667 URL |
[56] |
Hufton SE, Risley P, Ball CR, et al. The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency[J]. PLoS One, 2014, 9(8):e103294.
doi: 10.1371/journal.pone.0103294 URL |
[57] |
Ibañez LI, De Filette M, Hultberg A, et al. Nanobodies with in vitro neutralizing activity protect mice against H5N1 influenza virus infection[J]. J Infect Dis, 2011, 203(8):1063-1072.
doi: 10.1093/infdis/jiq168 URL |
[58] |
Laursen NS, Friesen RHE, Zhu X, et al. Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin[J]. Science, 2018, 362(6414):598-602.
doi: 10.1126/science.aaq0620 pmid: 30385580 |
[59] |
Wrapp D, De Vlieger D, Corbett KS, et al. Structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies[J]. Cell, 2020, 181(5):1004-1015.e15.
doi: 10.1016/j.cell.2020.04.031 URL |
[60] |
Huo J, Le Bas A, Ruza RR, et al. Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2[J]. Nat Struct Mol Biol, 2020, 27(9):846-854.
doi: 10.1038/s41594-020-0469-6 URL |
[61] |
Hanke L, Vidakovics Perez L, Sheward DJ, et al. An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction[J]. Nat Commun, 2020, 11(1):4420.
doi: 10.1038/s41467-020-18174-5 pmid: 32887876 |
[62] | Gai J, Ma L, Li G, et al. A potent neutralizing nanobody against SARS-CoV-2 with inhaled delivery potential[J]. MedComm:Beijing, 2021, 2(1):101-113. |
[63] |
Dong J, Huang B, Jia Z, et al. Development of multi-specific humanized llama antibodies blocking SARS-CoV-2/ACE2 interaction with high affinity and avidity[J]. Emerg Microbes Infect, 2020, 9(1):1034-1036.
doi: 10.1080/22221751.2020.1768806 URL |
[64] |
Koenig PA, Das H, Liu H, et al. Structure-Guided Multivalent Nanobodies Block Sars-Cov-2 Infection and Suppress Mutational Escape[J]. Science, 2021, 371(6530):eabe6230.
doi: 10.1126/science.abe6230 URL |
[65] | Wilson PC, Andrews SF. Tools to therapeutically harness the human antibody response[J]. Nat Rev Immunol, 2012, 12(10):709-719. |
[66] | Sun Y, Ho M. Emerging antibody-based therapeutics against SARS-CoV-2 during the global pandemic[J]. Antib Ther, 2020, 3(4):246-256. |
[1] | 徐重新, 张霄, 刘媛, 仲建锋, 谢雅晶, 卢莉娜, 高美静, 刘贤金. 靶向模拟Bt Cry1C蛋白抗虫功能的人源化基因工程抗体筛选及鉴定[J]. 生物技术通报, 2022, 38(5): 191-200. |
[2] | 唐禄, 董丽平, 尹茉莉, 刘磊, 董媛, 王会岩. 成纤维细胞生长因子20单克隆抗体的制备及鉴定[J]. 生物技术通报, 2021, 37(10): 179-185. |
[3] | 许映雪, 尹焕才. MMP-9蛋白的抗原表位分析及单克隆抗体制备[J]. 生物技术通报, 2019, 35(7): 134-140. |
[4] | 叶尔那扎尔·努尔吐热, 邱丽芬, 张富春, 张茂祥. DNA 免疫技术在单克隆抗体开发中的研究进展[J]. 生物技术通报, 2019, 35(2): 204-211. |
[5] | 郎巧利, 吴梦, 黄楠, 何琦琳, 葛良鹏, 杨希. 伪狂犬病毒的gE蛋白胞外区真核表达以及单克隆抗体制备[J]. 生物技术通报, 2019, 35(11): 96-103. |
[6] | 孙静娟,邱景璇,曾海娟,丁承超,王广彬,李杰,王淑娟,刘箐. 单增李斯特菌CdaA的抗原表位分析及抗体的制备[J]. 生物技术通报, 2018, 34(5): 163-171. |
[7] | 王晨, 汪嘉琪, 赵亮, 范里, 刘旭平, 陈敏, 张理想, 谭文松. 二氧化碳分压和渗透压升高对CHO细胞维持期生长、代谢和产物表达的影响[J]. 生物技术通报, 2018, 34(3): 217-224. |
[8] | 乔玉玲, 黄铮, 秦海艳, 宋兰兰, 陈继军, 安晨, 叶星, 毛晓燕. 抗CD52单克隆抗体HPLC-肽图分析方法的建立[J]. 生物技术通报, 2018, 34(11): 216-222. |
[9] | 陈春野,刘剑,朱瑞,李姝璇,叶江辉,王玮,潘德全,徐飞海,程通,夏宁邵. 大肠杆菌可溶性表达人鳞状上皮细胞癌抗原的制备及应用[J]. 生物技术通报, 2017, 33(9): 252-258. |
[10] | 胡冬冬, 赵亮, 范里, 刘旭平, 邓献存, 缪仕伟, 谭文松. 酵母抽提物对CHO细胞生长及抗体表达的影响[J]. 生物技术通报, 2017, 33(6): 162-169. |
[11] | 张鑫涛,唐红萍,赵亮,范里,刘旭平,缪仕伟,谭文松. 金属离子对CHO细胞抗体表达及抗体电荷分布的影响[J]. 生物技术通报, 2016, 32(8): 233-241. |
[12] | 杨川,秦艺丹,胡潜,李强. 抗迟缓爱德华菌(Edwardsiella tarda)单克隆抗体的制备及双抗夹心ELISA检测方法的建立[J]. 生物技术通报, 2016, 32(7): 200-205. |
[13] | 刘鹏展, 黄绮玲, 何小维, 李文美, 张文琪, 张赛. NT-proBNP特异性单克隆抗体的制备及鉴定[J]. 生物技术通报, 2016, 32(6): 148-154. |
[14] | 陈坤,徐军,谢灿,周冬梅,杨彬,孙文正. 基于HUVEC细胞表面ELAM-1表达的抗TNF-α抗体活性检测方法[J]. 生物技术通报, 2015, 31(12): 70-74. |
[15] | 杨辉,杨彬,马旭通,孙文正,林小鹊,谭世杰. 两步串联层析法纯化抗TNF-α 单克隆抗体[J]. 生物技术通报, 2015, 31(12): 75-80. |
阅读次数 | ||||||
全文 |
|
|||||
摘要 |
|
|||||