Name: generation of recombinant human igg monoclonal antibodies from immortalized sorted b cells
Uploaded: 2015-06-05T13:00:00.000+00:00
Duration: 10 min 32 s
Description: Watch this Scientific Journal Video about Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells at JoVE.com

研究契約ミゲルServet（ISCIII CD14 / 00032）から（GN-G。）。 （CH）の科学研究「トランスレーショナル神経科学プログラム研究科 &quot;のオランダ機構（022005019）からフェローシップ。 プリンセスベアトリクスフォン（プロジェクトWAR08-12）と協会フランセーズcontreレミオパシーからの補助金（PM-M。）;ならびに科学研究費オランダ機構（916.10.148）のVeniフェローシップによるオランダの脳財団のフェローシップ（FS2008（1）-28）とプリンセスビアトリクスフォン（プロジェクトWAR08-12）（MLへ）。 我々は、フローサイトメトリーによる選別B細胞の彼女の助けのためJozienヤスパースに感謝します。

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+detection+in+automated+DNA+sequence+analysis.">Fluorescence detection in automated DNA sequence analysis.</a> Nature. 321 (6071), 674-679 (1986).</li></ol>

著者は、彼らが競合する金融利害関係がないことを宣言します。

本稿では、ヒトPBMCからのIgG抗体の生成のためのすべてのステップを詳細に示します。このプロトコルは、以前に公表された技術を超えるいくつかの利点が含まれています。利点の一つは、産生された抗体は、B細胞クローンの元の対に対応する重鎖および軽鎖を保つことです。 IgG抗体の同定は、ヒトドナーの任意のタイプで行うことができ、ワクチン接種5による免疫応答の悪化を必?...

この論文の目的は、具体的にヒト末梢血単核細胞（PBMC）から得られたヒトIgGモノクローナル抗体を作製し、特徴づけるための方法を記述することです。 ヒト抗体を研究するための関心は、研究のさまざまな分野に成長しました。特に、多くの研究グループは、自己抗体によって引き起こされる病理1-3に興味を持っています。我々は、クローン化され、1病原性の自己抗体を特徴としています。自己抗体の研究は、競合抗体4を使用して、 例えば 、それらの標的を同定し、治療戦略を開発するのに役立ちます。また、ヒト抗体の研究はまた、露出して特定の病原体6または研究するためにどのに耐性となった個体の抗体プロフィールを特徴付けるために、ワクチン接種後5免疫応答を評価するために、 すなわち 、研究の他の分野に関心のものであることができます抗体がです天然のレパートリー7,12。 いくつかの技術が、組換えヒトモノクローナル抗体8-12を生成するために開発されています。これらのほとんどは、ファージディスプレイおよびB細胞の不死化を使用します。ファージディスプレイの使用は、広範囲に新13抗体の検出に適用されています。しかし、となるヒト免疫グロブリンの重鎖および軽鎖対は、プロセス中に解離していること、すなわち、主要な欠点​​を有します。ヒトB細胞またはEBV形質転換とハイブリドーマの生産は、この欠点を克服します。 我々は、Toll様受容体9（TLR-9）、6,12を介して、ポリクローナルB細胞刺激と組み合わせたEBVで胸腺B細胞の感染を使用します。 本稿では、in vitroでの抗体生成にPBMC単離からのすべてのステップの完全な概要と、詳細に我々は、IgGヒト抗体の開発のために使用する技術が記載されています。このこのプロトコルは、ヒトIgGのプロファイルのいずれかのタイプの分析のために使用することができます。我々の研究室では、IgG抗体を産生するB細胞が正常にソートした後のPBMCの残りの部分から分離されました。五8は 、マルチウェルプレートに播種し、単一のB細胞のクローン性増殖のために、EBV及びTLR-9活性化によって不死化することができるB細胞を選別しました。フィーダー細胞としては、ヒト胎児肺組織からの線維芽細胞は、不死化B細胞の可視化を容易にする細胞系WI38が、使用されています。これらのB細胞から、免疫グロブリンの重鎖および軽鎖の配列を、PCRにより得ることができる、および抗体」遺伝子は、免疫グロブリンGを発現ベクター中にクローン化し、 インビトロで生成しました。この技術を用いて、ドナーに見られる全く同じ抗体配列を有する単一の抗体を試験することができます。

<table><tbody><tr><td>Histopaque-1077&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10771</td><td>solution containing polysucrose and sodium diatrizoate</td></tr><tr><td>FACSAria II cell sorter&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>96 U-bottom micro well plates&amp;nbsp;</td><td>Costar</td><td>3799</td><td></td></tr><tr><td>Advanced Roswell Park Memorial Institute (RPMI) 1640 medium&amp;nbsp;</td><td>Gibco, Life Technologies</td><td>12633-020</td><td></td></tr><tr><td>30% v/v EBV-containing supernatant of the B95-8 cell line&amp;nbsp;&amp;nbsp;</td><td>ATCC</td><td>CRL-1612</td><td>3.4 x 108 copies/ml</td></tr><tr><td>CpG2006&amp;nbsp;</td><td>Invivogen</td><td>ODN 7909</td><td></td></tr><tr><td>Wi38 cells&amp;nbsp;</td><td>Sigma-Aldrich</td><td>90020107</td><td></td></tr><tr><td>Interleukin-2</td><td>Roche&amp;nbsp;</td><td>10799068001</td><td></td></tr><tr><td>ELISA plates</td><td>Greiner Bio-One, Microlon</td><td>655092</td><td></td></tr><tr><td>AffiniPure F(ab&#039;)2 Fragment Goat Anti-Human IgG, Fc&amp;gamma; Fragment Specific (unconjugated)</td><td>Jackson ImmunoResearch</td><td>109-006-008</td><td></td></tr><tr><td>4% non-fat dry milk (Blotting Grade Blocker)&amp;nbsp;</td><td>Biorad</td><td>170-6404</td><td></td></tr><tr><td>Human IgG&amp;nbsp;</td><td>Sigma</td><td>I 2511</td><td>HUMAN IgG purified Immunoglobulin, 5.6 mg/ml</td></tr><tr><td>Goat F(ab)&lt;sub&gt;2&lt;/sub&gt; antihuman IgG Fc&amp;gamma; (conjugated with peroxidase (PO))</td><td>Jackson ImmunoResearch</td><td>109-036-008</td><td></td></tr><tr><td>ELISA reader (Perkin Elmer 2030)&amp;nbsp;</td><td>Perkin Elmer&amp;nbsp;</td><td>2030-0050</td><td></td></tr><tr><td>Peroxidase-conjugated AffiniPure Rabbit Anti-Human IgM, Fc&lt;sub&gt;5&amp;micro;&amp;nbsp;&lt;/sub&gt;</td><td>Jackson ImmunoResearch</td><td>309-035-095</td><td></td></tr><tr><td>SuperScript III Cells Direct cDNA Synthesis System&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>18080-200</td><td></td></tr><tr><td>Applied Biosystems (ABI) GeneAm PCR System 2700</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>High Pure RNA Isolation Kit&amp;nbsp;</td><td>Roche</td><td>11828665001</td><td></td></tr><tr><td>Reverse transcription system kit&amp;nbsp;</td><td>Promega</td><td>A3500</td><td></td></tr><tr><td>Recombinant Taq DNA Polymerase</td><td>TAKARA</td><td>R001A</td><td></td></tr><tr><td>Primers (2 &amp;mu;l)</td><td>Sigma</td><td></td><td></td></tr><tr><td>Ultrapure Agarose&amp;nbsp;</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>100 bp ladder</td><td>Invitrogen</td><td>15628-019</td><td></td></tr><tr><td>Quantity One 4.5.2 (Gel Doc 2000)</td><td>Biorad</td><td>170-8100</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>QIAGEN</td><td>28106</td><td></td></tr><tr><td>BigDye Terminator v3.1 cycle sequencing kit&amp;nbsp;</td><td>Applied Biosystems</td><td>4337455</td><td></td></tr><tr><td>0.1 ml reaction plate (MicroAMP Optical 96-well)</td><td>Applied Biosystems</td><td>4346906</td><td></td></tr><tr><td>Genetic analyser ABI300&amp;nbsp;</td><td>Applied Biosystems</td><td>4346906</td><td></td></tr><tr><td>DH5&amp;alpha; competent cells (E. coli)</td><td>Invitrogen</td><td>18263-012</td><td></td></tr><tr><td>pFUSEss-CHIg-hG1 (4,493 bp)</td><td>Invivogen</td><td>pfusess-hchg1</td><td></td></tr><tr><td>pFUSEss-CHIg-hG4 (4,484 bp)&amp;nbsp;</td><td>Invivogen</td><td>pfusess-hchg4</td><td></td></tr><tr><td>pFUSE2ss-CLIg-hk (3,875 bp)</td><td>Invivogen</td><td>pfuse2ss-hclk</td><td></td></tr><tr><td>pFUSE2ss-CLIg-hl2 (3,883 bp)&amp;nbsp;</td><td>Invivogen</td><td>pfuse2ss-hcll2</td><td></td></tr><tr><td>SOC medium</td><td>Invitrogen</td><td>15544-034</td><td></td></tr><tr><td>LB-based agar medium supplemented with Zeocin (Fast-Media Zeo Agar)</td><td>Invivogen</td><td>fas-zn-s</td><td></td></tr><tr><td>Terrific Broth (TB)-based liquid medium supplemented with Zeocin (Fast-Media Zeo TB)</td><td>Invivogen</td><td>fas-zn-l</td><td></td></tr><tr><td>DNA Miniprep kit&amp;nbsp;</td><td>Omega Bio Technology</td><td>D6942-02</td><td></td></tr><tr><td>Nanodrop (ND1000 Spectrophotometer)</td><td>Nanodrop</td><td></td><td></td></tr><tr><td>LB-based agar medium supplemented with Blasticidin (Fast-Media Blast Agar)</td><td>Invivogen</td><td>fas-bl-s</td><td></td></tr><tr><td>Terrific Broth (TB)-based liquid medium supplemented with Blasticidin (Fast-Media Blast TB)</td><td>Invivogen</td><td>fas-bl-l</td><td></td></tr><tr><td>EcoRI</td><td>New England Biolabs</td><td>R0101S</td><td>20,000 U/ml, in 10x NEBuffer EcoRI</td></tr><tr><td>NheI</td><td>New England Biolabs</td><td>R0131S</td><td>10,000 U/ml, in 10x NEBuffer 2.1</td></tr><tr><td>2-Log DNA ladder</td><td>New England Biolabs</td><td>N3200S</td><td>0.1-10.0 kb, 1,000 &amp;mu;g/ml</td></tr><tr><td>XmaI</td><td>New England Biolabs</td><td>R0180S</td><td>10,000 U/ml, in 10x CutSmart Buffer&amp;nbsp;</td></tr><tr><td>BsiWI</td><td>New England Biolabs</td><td>R0553S</td><td>10,000 U/ml, in 10x NEBuffer 3.1</td></tr><tr><td>AvrII</td><td>New England Biolabs</td><td>R0174S</td><td>5,000 U/ml, in 10x CutSmart Buffer&amp;nbsp;</td></tr><tr><td>FastAP Thermosensitive Alkaline Phosphatase</td><td>Thermo Scientific</td><td>EF0651</td><td>1 U/&amp;micro;l, in 10x FastAP Buffer</td></tr><tr><td>DH5&amp;alpha; competent cells&amp;nbsp;</td><td>Invitrogen</td><td>18263-012</td><td></td></tr><tr><td>PE Mouse Anti-Human IgG</td><td>BD Pharmingen</td><td>555787</td><td></td></tr><tr><td>anti-CD22, PerCP-Cy5.5, Clone: HIB22</td><td>Fisher scientific</td><td>BDB563942</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit&amp;nbsp;</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>BigDye Terminator v3.1</td><td>Applied Biosystems</td><td>4337455</td><td></td></tr></tbody></table>

generation of recombinant human igg monoclonal antibodies from immortalized sorted b cells

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.

CD22およびIgG陽性細胞を染色した後の選別ゲートは、 図1に示されているこの画像では二重陽性細胞の面積- 。IgG抗体を産生するB細胞が-別のチューブ内のすべてのこれらの細胞を選別するために選択されます。分析では、全PBMCの約1％が、この二重陽性集団に対応しています。得られた選別された細胞の数は、セクション1で得られた細胞の数に依存します。 ...

Watch this Scientific Journal Video about Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells at JoVE.com

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

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.

不死化ソートB細胞からの組換えヒトIgGモノクローナル抗体の作製

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

generation-recombinant-human-igg-monoclonal-antibodies-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

18.3K ビュー.  Maastricht University. 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.

ビデオ: Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

プラスミドDNAの組換えインフルエンザウイルスの生成

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

免疫・感染学

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

B細胞の組換えレトロウイルスの生産と感染症

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

タンパク質治療薬の製造細胞株の開発のためのハイスループット自動化されたプラットフォーム

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

医学

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

ハイブリドーマ技術によってマウスモノクローナル抗体の作製

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

まれな抗原特異的 B 細胞血液の循環から弁別に対するモノクローナル抗体の生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

濃縮ヒト記憶B細胞集団からの所望特異性を持つ抗体の選択と回収のための単細胞スクリーニング法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

抗体生成:ハイブリドーマを用いたモノクローナル抗体の産生

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

Education

JoVE Science Education

高等生物学

免疫学

Generating Recombinant Monoclonal Antibodies From Mammalian Cell Cultures

Source: Nogales-Gadea, G. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/52830">Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates a method of antibody generation from mammalian cells by introducing plasmid vectors with heavy and light chain genes. These vectors interact with polyethylenimine and enter the cells by forming polyplexes; later, they are translated into individual proteins and assembled into complete antibodies, which are secreted into the extracellular medium.

哺乳類細胞培養物からの組換えモノクローナル抗体の作製

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Peripheral Blood ...

JoVE Encyclopedia of Experiments

抗体ベースの技術

製造と精製

A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells

Source: Devilder, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56508">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood.</a> J. Vis. Exp. (2018).
The video demonstrates a technique for generating monoclonal antibodies from antigen-specific B cells. Eukaryotic expression vectors encoding the heavy and light chains of antibodies, cloned from isolated antigen-specific B cells, are introduced into mammalian cells using polyplexes. Once inside the cells, these vectors undergo translation, and the resulting heavy and light chain peptides assemble into antibodies, which are subsequently secreted by the cells.

抗原特異的B細胞からモノクローナル抗体を作製する技術

1. Production of mAbs

	The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 &#181;L of Dulbecco&#39;s ...

Hybridoma Technology

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges K&#246;hler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
Hybridoma Selection
Commonly used fusion techniques &#8212; electroporation, polyethylene glycol (PEG) mediated fusion, and the use of fusogenic viruses &#8212; generally produce hybridomas at a very low frequency. Fusion products thus have a high ratio of self-fused B-cells and myeloma cells, or unfused cells, with a low number of hybrid cells.
The hypoxanthine-aminopterin-thymidine (HAT) medium is used to select hybridomas by selectively allowing their growth. The aminopterin blocks the default nucleotide synthesis in cells. However, cells can utilize hypoxanthine and thymidine from the medium to synthesize nucleotides via the salvage pathway.
Myeloma cells deficient in the HGPRT enzyme cannot synthesize nucleotides via this pathway and thus, do not grow in HAT medium. Although B-cells produce functional HGPRT enzymes, they cannot divide indefinitely. Thus, only the hybrid cells with functional HGPT from the B-cells and immortality from the myeloma line can grow on the HAT selection medium. Such a culture of hybrid cells is called a hybridoma, which can be screened for monoclonal antibody production.
Hybridoma Screening
Hybridoma cultures from the HAT selection are plated onto a 96-well plate; each well containing only one cell. Using Enzyme-linked Immunosorbent Assay (ELISA), each cell is screened for the production of antibodies specific to the epitope of interest. Cells that test positive are then selected and grown in a larger culture vessel to establish hybridoma cell lines. These cell lines are a permanent source of unlimited monoclonal antibodies. Thus hybridoma technology is a convenient and cost-effective method for mass production of monoclonal antibodies.

ハイブリドーマテクノロジー

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or ...