Summary

从排序的永生B细胞产生的重组人IgG单克隆抗体

Published: June 05, 2015
doi:

Summary

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Abstract

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Introduction

本文的目的是详细描述的方法来产生和表征从人外周血单核细胞(PBMC)中得到的人IgG单克隆抗体。

研究人类抗体的兴趣增加在研究许多不同的领域。特别是,许多研究小组有兴趣引起自身抗体1-3的病理。我们已经克隆和表征致病的自身抗体1。自身抗体的研究能够帮助确定他们的目标,并制定治疗策略, 例如 ,采用竞争性抗体4。另外,人抗体的研究也可以是在研究等领域, 兴趣,以评估疫苗接种5后的免疫反应,以表征该被暴露,并成为抗特定病原体的6或研究个体的抗体分布曲线抗体是在自然剧目7,12。

几种技术已经开发了产生重组人单克隆抗体8-12;大多数的这些使用噬菌体展示和B细胞永生化。使用噬菌体展示已被广泛应用于新抗体13的发现。然而,它具有一个主要的缺点,即重链和轻链对人免疫球蛋白成为离解的过程中。生产的杂交瘤与人类B细胞或EBV转化的克服了这一缺点。

我们通过Toll样受体9(TLR-9)6,12使用胸腺B细胞的感染与EBV与多克隆B细胞刺激的组合。

在本文中,我们详细描述了我们使用的抗体人源抗体的发展,从PBMC隔离的所有步骤, 体外抗体生成一个完整的概述的技术。这个协议可用于任何类型的人IgG轮廓的分析。在我们的实验室中,B细胞产生IgG抗体已成功地从PBMC中的其余部分分选后分离。五十排序B细胞8然后可以镀在多孔板和由EB病毒和TLR-9的激活永久化,对于单一B细胞的克隆扩增。作为饲养细胞,从人胚胎肺组织的成纤维细胞已被使用,细胞系WI38,这有利于该永生化B细胞的可视化。从这些B细胞,免疫球蛋白的重和轻链的序列可以通过PCR来获得,并且该抗体“基因克隆中免疫球蛋白G的表达载体和体外生产。使用这种技术,单个的抗体与正好在供体中发现的相同的抗体序列可以研究。

Protocol

知情同意从研究的参与者获得的。这项研究是由伦理委员会。 1.分离外周血单个核细胞(PBMC的) 离心机25毫升的参与者的肝素化血液,在900×g离心15分钟后,尽快在血液抽取之后。如果有血少,相应地缩减试剂。执行罩所有的下一步骤。 血清转移到干净的管中。它可用于在后面的步骤为冷冻的PBMC或在自身免疫患者,以测试自身抗体。 稀释血液用10ml的RPM…

Representative Results

染色CD22和IgG阳性细胞之后的分选门控示于图1在该图像中的双阳性细胞的面积。 -产生IgG抗体的B细胞-被选择为所有这些细胞在一个单独的试管中进行排序。在分析中,总的PBMC约1%的对应于这种双重阳性群体。得到的分选的细胞的数目将取决于在第1获得的细胞的数量。 5周EB病毒永生化和CpG(ODN2006)刺激后的不同结果示于图2中的生长克隆的检测容易?…

Discussion

在这个手稿,所有步骤从人PBMC的IgG抗体的产生,详细介绍。该协议包括一些优势较以前的发布技术。的优点之一是,产生的抗体保持对应于原始对中的B细胞克隆的重链和轻链。 IgG抗体的鉴定可以在任何类型的人供体的工作要做,而且也没有必要对由于疫苗接种5免疫应答的恶化。使用成纤维细胞系WI38作为饲养细胞的,允许更快速检测的生长克隆的,因为它们在形态上不同,很容易区分,…

Declarações

The authors have nothing to disclose.

Acknowledgements

研究合同米格尔SERVET(ISCIII CD14 / 00032)至(GN-G。)。奖学金由荷兰科学研究组织“转化神经科学计划的研究生院”(022005019)至(CH)。

赠款从Prinses贝娅特丽克丝全宗(项目WAR08-12)和法语协会驳LES肌病为(PM-M。);以及由荷兰科学研究组织的VENI奖学金(916.10.148)荷兰脑基金会的奖学金(FS2008(1)-28)和Prinses贝娅特丽克丝全宗(项目WAR08-12)(以ML )。

我们感谢Jozien雅斯贝尔斯为她在B细胞通过流式细胞分选的帮助。

Materials

Histopaque-1077  Sigma-Aldrich 10771 solution containing polysucrose and sodium diatrizoate
FACSAria II cell sorter  BD Biosciences 
96 U-bottom micro well plates  Costar 3799
Advanced Roswell Park Memorial Institute (RPMI) 1640 medium  Gibco, Life Technologies 12633-020
30% v/v EBV-containing supernatant of the B95-8 cell line   ATCC CRL-1612 3.4 x 108 copies/ml
CpG2006  Invivogen ODN 7909
Wi38 cells  Sigma-Aldrich 90020107
Interleukin-2 Roche  10799068001
ELISA plates Greiner Bio-One, Microlon 655092
AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (unconjugated) Jackson ImmunoResearch 109-006-008
4% non-fat dry milk (Blotting Grade Blocker)  Biorad 170-6404
Human IgG  Sigma I 2511 HUMAN IgG purified Immunoglobulin, 5.6 mg/ml
Goat F(ab)2 antihuman IgG Fcγ (conjugated with peroxidase (PO)) Jackson ImmunoResearch 109-036-008
ELISA reader (Perkin Elmer 2030)  Perkin Elmer  2030-0050
Peroxidase-conjugated AffiniPure Rabbit Anti-Human IgM, Fc5µ  Jackson ImmunoResearch 309-035-095
SuperScript III Cells Direct cDNA Synthesis System  Invitrogen  18080-200
Applied Biosystems (ABI) GeneAm PCR System 2700 Applied Biosystems
High Pure RNA Isolation Kit  Roche 11828665001
Reverse transcription system kit  Promega A3500
Recombinant Taq DNA Polymerase TAKARA R001A
Primers (2μl) Sigma
Ultrapure Agarose  Invitrogen 16500-500
100 bp ladder Invitrogen 15628-019
Quantity One 4.5.2 (Gel Doc 2000) Biorad 170-8100
QIAquick PCR purification kit QIAGEN 28106
BigDye Terminator v3.1 cycle sequencing kit  Applied Biosystems 4337455
0.1 ml reaction plate (MicroAMP Optical 96-well) Applied Biosystems 4346906
Genetic analyser ABI300  Applied Biosystems 4346906
DH5α competent cells (E. coli) Invitrogen 18263-012
pFUSEss-CHIg-hG1 (4493 bp) Invivogen pfusess-hchg1
pFUSEss-CHIg-hG4 (4484 bp)  Invivogen pfusess-hchg4
pFUSE2ss-CLIg-hk (3875 bp) Invivogen pfuse2ss-hclk
pFUSE2ss-CLIg-hl2 (3883 bp)  Invivogen pfuse2ss-hcll2
SOC medium Invitrogen 15544-034
LB-based agar medium supplemented with Zeocin (Fast-Media Zeo Agar) Invivogen fas-zn-s
Terrific Broth (TB)-based liquid medium supplemented with Zeocin (Fast-Media Zeo TB) Invivogen fas-zn-l
DNA Miniprep kit  Omega Bio Technology D6942-02
Nanodrop (ND1000 Spectrophotometer) Nanodrop
LB-based agar medium supplemented with Blasticidin (Fast-Media Blast Agar) Invivogen fas-bl-s
Terrific Broth (TB)-based liquid medium supplemented with Blasticidin (Fast-Media Blast TB) Invivogen fas-bl-l
EcoRI New England Biolabs R0101S 20,000 U/ml, in 10x NEBuffer EcoRI
NheI New England Biolabs R0131S 10,000 U/ml, in 10x NEBuffer 2.1
2-Log DNA ladder New England Biolabs N3200S 0.1-10.0 kb, 1,000 μg/ml
XmaI New England Biolabs R0180S 10,000 U/ml, in 10x CutSmart Buffer 
BsiWI New England Biolabs R0553S 10,000 U/ml, in 10x NEBuffer 3.1
AvrII New England Biolabs R0174S 5,000 U/ml, in 10x CutSmart Buffer 
FastAP Thermosensitive Alkaline Phosphatase Thermo Scientific EF0651 1 U/µL, in 10x FastAP Buffer
DH5α competent cells  Invitrogen 18263-012
PE Mouse Anti-Human IgG BD Pharmingen 555787
anti-CD22, PerCP-Cy5.5, Clone: HIB22 Fisher scientific BDB563942
QIAprep Spin Miniprep Kit  QIAGEN 27106
BigDye Terminator v3.1 Applied Biosystems 4337455

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Nogales-Gadea, G., Saxena, A., Hoffmann, C., Hounjet, J., Coenen, D., Molenaar, P., Losen, M., Martinez-Martinez, P. Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells. J. Vis. Exp. (100), e52830, doi:10.3791/52830 (2015).

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