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.
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.
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, 即兴趣,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。
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,…
The authors have nothing to disclose.
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。
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 |