Name: generation of discriminative human monoclonal antibodies from rare antigen-specific b cells circulating in blood
Uploaded: 2018-02-06T14:00:00
Description: Watch this Scientific Journal Video about Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood at JoVE.com

専門的な技術支援ありがとうフローサイトメトリー施設"CytoCell"(SFR 健康、Biogenouest、ナント)。彼らのテクニカル サポートもすべてのスタッフが組換えタンパク質の生産 (P2R) とプラットフォームの影響 (INSERM 1232、SFR 健康、Biogenouest、ナント) にありがちましょう。原稿の建設的なコメント、エマニュエル Scotet とリチャード ・ ブレスナックに感謝します。この仕事財政的に、IHU セスタス プロジェクトで、フランス国立研究機関 (ANR) (ANR-10-IBHU-005) を管理する «Investissements d'Avenir» フランス政府プログラムによって資金を供給によって支えられました。IHU セスタス プロジェクトはナントとペイ ・ ド ・ ラ ・ ロワール地方でもサポートされます。この作品は、将来プログラム ANR-11-LABX-0016-01 の投資を通して国立研究機関によってサポートされて LabEX 囲碁プログラムのコンテキストで実現しました。

すべてのひと末梢血サンプルはローカル倫理委員会 (エタブリセモン Français デュを歌った、EFS、ナント、プロシージャ PLER NTS-2016-08) に基づき、インフォームド コンセント後匿名成人ドナーから得られました。
1. ひと末梢血単核細胞の分離
注: 開始材料合計ひと末梢血や樹形サンプルできます。サンプルは、8 h 以上べきではないと、抗凝固剤 (例?...

<ol>
	<li>Buss, N. A., Henderson, S. J., McFarlane, M., Shenton, J. M., de Haan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+therapeutics:+history+and+future.">Monoclonal antibody therapeutics: history and future.</a> Curr Opin Pharmacol. 12 (5), 615-622 (2012).</li><li>Park, J., Kwon, M., Shin, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+checkpoint+inhibitors+for+cancer+treatment.">Immune checkpoint inhibitors for cancer treatment.</a> Arch Pharm Res. 39 (11), 1577-1587 (2016).</li><li>Pendley, C., Schantz, A., Wagner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+therapeutic+monoclonal+antibodies.">Immunogenicity of therapeutic monoclonal antibodies.</a> Curr Opin Mol Ther. 5 (2), 172-179 (2003).</li><li>Kwakkenbos, M. J., 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=Generation+of+stable+monoclonal+antibody-producing+B+cell+receptor-positive+human+memory+B+cells+by+genetic+programming.">Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming.</a> Nat Med. 16 (1), 123-128 (2010).</li><li>Tiller, T., 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=Efficient+generation+of+monoclonal+antibodies+from+single+human+B+cells+by+single+cell+RT-PCR+and+expression+vector+cloning.">Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.</a> J Immunol Methods. 329 (1-2), 112-124 (2008).</li><li>Franz, B., May, K. F., Dranoff, G., Wucherpfennig, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+characterization+and+isolation+of+rare+memory+B+cells+with+antigen+tetramers.">Ex vivo characterization and isolation of rare memory B cells with antigen tetramers.</a> Blood. 118 (2), 348-357 (2011).</li><li>Morris, L., 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=Isolation+of+a+human+anti-HIV+gp41+membrane+proximal+region+neutralizing+antibody+by+antigen-specific+single+B+cell+sorting.">Isolation of a human anti-HIV gp41 membrane proximal region neutralizing antibody by antigen-specific single B cell sorting.</a> PLoS One. 6 (9), e23532 (2011).</li><li>Iizuka, A., 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=Identification+of+cytomegalovirus+(CMV)pp65+antigen-specific+human+monoclonal+antibodies+using+single+B+cell-based+antibody+gene+cloning+from+melanoma+patients.">Identification of cytomegalovirus (CMV)pp65 antigen-specific human monoclonal antibodies using single B cell-based antibody gene cloning from melanoma patients.</a> Immunol Lett. 135 (1-2), 64-73 (2011).</li><li>Ouisse, L. H., 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=Antigen-specific+single+B+cell+sorting+and+expression-cloning+from+immunoglobulin+humanized+rats:+a+rapid+and+versatile+method+for+the+generation+of+high+affinity+and+discriminative+human+monoclonal+antibodies.">Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies.</a> BMC Biotechnol. 17 (1), 3 (2017).</li><li>Kerkman, P. F., 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=Identification+and+characterisation+of+citrullinated+antigen-specific+B+cells+in+peripheral+blood+of+patients+with+rheumatoid+arthritis.">Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis.</a> Ann Rheum Dis. , (2015).</li><li>Hamilton, J. A., 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=General+Approach+for+Tetramer-Based+Identification+of+Autoantigen-Reactive+B+Cells:+Characterization+of+La-+and+snRNP-Reactive+B+Cells+in+Autoimmune+BXD2+Mice.">General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice.</a> J Immunol. 194 (10), 5022-5034 (2015).</li><li>Sanjuan Nandin, I., 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=Novel+in+vitro+booster+vaccination+to+rapidly+generate+antigen-specific+human+monoclonal+antibodies.">Novel in vitro booster vaccination to rapidly generate antigen-specific human monoclonal antibodies.</a> J Exp Med. , (2017).</li><li>Bowers, P. 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=Mammalian+cell+display+for+the+discovery+and+optimization+of+antibody+therapeutics.">Mammalian cell display for the discovery and optimization of antibody therapeutics.</a> Methods. 65 (1), 44-56 (2014).</li></ol>

提案プロトコルは血で循環 Ag 特定の B 細胞から直接人間の抗体の生成のための強力な方法です。それは 3 つの重要な側面を組み合わせた: (i)、四量体関連磁気濃縮、まれな Ag-結合 B 細胞の前のヴィヴォ分離ができるの使用(cytometry 流れによって、BCR に関する必要な Ag; を発現する B 細胞のみを分離する 3 つの異なる蛍光色素で標識 3 つの Ag テトラマー (2 つの関連するものと無関係な?...

An erratum was issued for: <a href="https://www.jove.com/video/56508/generation-discriminative-human-monoclonal-antibodies-from-rare">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood</a>.
The Affiliations section was corrected from:
Marie-Claire Devilder1,2,&#160;M&#233;linda Moyon1,2,&#160;Xavier Saulquin1, Laetitia Gautreau-Rolland1 
1Centre R&#233;gional de Recherche en Canc&#233;rologie et Immunologie Nantes/Angers, INSERM 1232, LabEx IGO, Universit&#233; de Nantes 
2CHU Nantes
to:
Marie-Claire Devilder1,2,3,&#160;M&#233;linda Moyon1,2,3,&#160;Xavier Saulquin1,2, Laetitia Gautreau-Rolland1,2&#160; 
1CRCINA, Inserm, CNRS, Universit&#233; d&#39;Angers, Universit&#233; de Nantes 
2LabEx IGO, &#34;Immunotherapy, Graft, Oncology&#34; 
3CHU, Nantes, France

An erratum was issued for: <a href="https://www.jove.com/video/56508/generation-discriminative-human-monoclonal-antibodies-from-rare">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood</a>

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

ここで説明する方法は、目的の抗原 (Ag) に対するモノクローナル抗体 (Mab) を完全に人間の迅速かつ汎用性の高い生産を可能します。Mab は、多くの基礎研究アプリケーションin vitroとin vivoで不可欠なツール: たとえばフローサイトメトリー、組織学、西部のしみを付けおよびブロックの実験の流れ。さらに、自己免疫疾患、がん、治療し、移植拒絶反応1を制御する Mab をより多くに医学で使用されています。たとえば、反 ctla の条項 4、抗 PD 1 (またはアンチ PD L1) Mab は最近がん治療2免疫チェックポイント阻害剤として使用されました。
免疫グロブリン (Ig) で作り出された最初のモノクローナル抗体-免疫マウスまたはラットの脾臓細胞から得られたハイブリドーマを分泌します。ただし、マウスまたはラット モノクローナル抗体と強い免疫反応は、その急速なクリアランスと過敏反応3のありそうな誘導のための人間の彼らの治療上の使用を妨げます。この問題に取り組むため、Mab の動物性タンパク質シーケンス部分的に取って代わられましたいわゆるキメラ マウス ヒトやヒト化抗体を生成する人間の物。ただし、この戦略は部分的にしかコストと生産時間規模の両方を大幅に増加させながら、免疫原性を減少します。よい解決策はヒト B 細胞から直接人間の抗体を生成する、このためのいくつかの戦略があります。それらの 1 つは、ファージや酵母のディスプレイの使用です。これは含まランダム人間 Ig 重のコンビナトリアル ライブラリから可変領域を表示し、光チェイン ファージや酵母と興味の特定の抗原を用いた選択のステップを実施します。この方法の主な欠点は、重く、軽鎖がランダムに関連付けられ、生成された抗体の多様性の非常に大きな増加につながることです。抗体は特定 Ag に対する自然免疫応答から生じるだろっているものに対応しています。また、人間のタンパク質の折りたたみおよびポスト翻訳の修正、複製は、体系的に原核生物や酵母。2 番目の人間 mAb 生産方法は、エプスタイン ‐ バーウイルス感染や抗アポトーシス因子 BCL 6 および BCL-XL4発現によって自然の人間の B 細胞の不死化です。ただし、このメソッドは、メモリー B 細胞にのみ適用され、多数 mAb 生産不死化 B 細胞が目的の抗原特異性と (もしあれば) いくつかの mAb のクローンを識別するためにスクリーニングを必要とする、効率的ではありません。メソッドはこのように高価な時間のかかるです。
新しいプロトコルは、隔離された単一 B 細胞5から人間の抗体の生産の最近記載されています。最適化された単一セル逆転写-ポリメラーゼの連鎖反応 (RT-PCR) の増幅の両方、重と光-鎖のセグメントを単一の並べ替えられた B 細胞からのエンコードを依存しています。真核生物発現系、完全に人間の mAb の再構築をできるように、クローニングと発現のこれらのセグメントのこの続きますです。このプロトコルは、ワクチン接種ドナーからの B 細胞から正常に使用されています。セルを目的の Ag に対して指示される B 細胞の高い周波数を取得し、6をスクリーニングするために必要な時間を制限するワクチン接種後数週間収穫されました。他の完全ひと抗体は、HIV+ (ひと免疫不全ウイルス) 感染患者7と黒色腫患者8から生産されています。これらの進歩にもかかわらずプロシージャがないまだ利用可能メモリ表現型や頻度の独立した Ag 固有の B 細胞の分離ができます。
手順ここで効率的な前のヴィヴォ完全ひと抗原特異的モノクローナル抗体低上映時間と高収率で生産が続く、BCR 特異性に基づいて人間の循環 B 細胞の分離につながる。メソッドは、メモリー B 細胞や抗体産生 B 細胞免疫反応を誘導する制限されていないが、人間ナイーブ B 細胞レパートリーにも適用することができます。Ag 特定 B 細胞は非常に低い周波数で存在からでも動作する、効率の良い指標です。法の原理は次のように: 末梢血単核細胞 (PBMC) は興味の抗原を提示 2 テトラマーと汚れる、それぞれ異なる蛍光色素 (例えば、フィコエ リスリン (PE)、アロフィコシアニン (APC))、というラベルの付いた密接に関連抗原提示第 3 四量体共役 3 蛍光色素 (例えば、ブリリアント バイオレット 421 (BV421))。抗原結合セルの豊かにする細胞は、抗 PE をコートしたビーズと孵化させると反 APC Abs、細胞分離カラムでソートします。PE+ APC+細胞画分が選択されます種類 PBMC 細胞 B 細胞の同定を許可するために特定の抗体の様々 なステンド グラスと受ける流れ cytometry 細胞選別します。PE+と APC+、鮮やかなバイオレット-、しかしである B 細胞は分離されています。この手順は、カウンター B 細胞ではないまたは tetramerized の抗原にはバインドしませんが、PE/APC (PE+ APC-または PE- APC+これらの細胞になる) または使用されるテトラマー (これらの非抗原部分にバインドを行うセルを選択しますセルになります BV421+)。B 細胞の高い関心のエピトープに固有ではないがこのステップでも反選択されます (これらの細胞に BV421 なるも+)。したがって、このメソッドは、表現する B 細胞受容体 (BCRs) 2 つの非常に密接に関連抗原の間で区別することができる非常に特定の B 細胞を浄化できます。チューブに収集され、単一の特定の B 細胞と、Ig の PCR 増幅 Cdna (相補的デオキシリボ核酸) を複製し、分泌された IgG 抗体としてのひと細胞ラインで表現。
概念の証拠としてこの調査は記述するペプチドが提示主要な組織適合性の複雑なクラスによって認識される人間の抗体の効率的な生成 (MHC-私) 分子とを区別することができますこのペプチドと同じで読み込まれる他のペプチドMHC-私の対立遺伝子。このメソッドは (i) に Ag 固有 Mab; の高収量回復を可能この Ag の複雑さのレベルは重要ですが、(ii) 構造的に 2 つを区別することができるモノクローナル抗体の生産は、Ags を閉じます。このアプローチは、プロトコルのままには、ワクチン接種や感染患者に拡張することができます、ヒト化ラット システム9にもすでに、正常に実装されています。したがって、本研究は基礎研究と免疫療法で使用することができます完全ひと抗体を生成する汎用性と効率的なアプローチについて説明します。

<table>
	<tbody>
		<tr>
			<td>HEK 293A cell line</td>
			<td>Thermo Fisher scientific</td>
			<td>R70507</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1X) Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco by life technologies</td>
			<td>21969-035</td>
			<td>(+) 4,5g/L D-Glucose 
			0,11g/L Sodium Pyruvate 
			(-) L-Glutmine</td>
		</tr>
		<tr>
			<td>RPMI medium1640 (1X)</td>
			<td>Gibco by life technologies</td>
			<td>31870-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine (BSA)</td>
			<td>PAA</td>
			<td>K45-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutridoma-SP</td>
			<td>Roche</td>
			<td>11011375001</td>
			<td>100X Conc</td>
		</tr>
		<tr>
			<td>PBS-Phosphate Buffered Saline (10X) pH 7,4</td>
			<td>Ambion</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (Ethylenediaminetetraacetic acid) 0,5M pH=8</td>
			<td>Invitrogen by Life Technologies</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine serum (FBS)</td>
			<td>Dominique Dutscher</td>
			<td>S1810-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll - lymphocytes separation medium</td>
			<td>EuroBio</td>
			<td>CMSMSL01-01</td>
			<td>density 1,0777+/-0,001</td>
		</tr>
		<tr>
			<td>streptavidin R-phycoerythrin conjugate</td>
			<td>Invitrogen by Life Technologies</td>
			<td>S21388</td>
			<td>premiun grade 1mg/ml contains 5mM sodium azide</td>
		</tr>
		<tr>
			<td>Streptavidin, allophycocyanin conjugate</td>
			<td>Invitrogen by thermoFisher scientific</td>
			<td>S32362</td>
			<td>1mg/ml 
			2mM azide premium grade</td>
		</tr>
		<tr>
			<td>Brilliant violet 421 streptavidin</td>
			<td>Biolegend</td>
			<td>405225</td>
			<td>conc : 0,5mg/ml</td>
		</tr>
		<tr>
			<td>Anti-PE conjugated magnetic MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-APC conjugated magnetic MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-855</td>
			<td></td>
		</tr>
		<tr>
			<td>MidiMACs or QuadroMACS separotor</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302/130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 BV510 BD horizon</td>
			<td>BD Pharmingen / BD Biosciences</td>
			<td>563109</td>
			<td>Used dilution 1:20</td>
		</tr>
		<tr>
			<td>CD19 FITC</td>
			<td>BD Pharmingen / BD Biosciences</td>
			<td>345788</td>
			<td>Used dilution 1:20</td>
		</tr>
		<tr>
			<td>CD14 PerCPCy5.5</td>
			<td>BD Pharmingen / BD Biosciences</td>
			<td>561116</td>
			<td>Used dilution 1:50</td>
		</tr>
		<tr>
			<td>CD16 PerCPCy5.5</td>
			<td>BD Pharmingen / BD Biosciences</td>
			<td>338440</td>
			<td>Used dilution 1:50</td>
		</tr>
		<tr>
			<td>7AAD</td>
			<td>BD Pharmingen / BD Biosciences</td>
			<td>51-68981E (559925)</td>
			<td>Used dilution 1:1000</td>
		</tr>
		<tr>
			<td>FACS ARIA III Cell Sorter Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-strip PCR tubes</td>
			<td>Axygen</td>
			<td>321-10-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Racks for 96 microtubes</td>
			<td>Dominique Dutscher</td>
			<td>45476</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAseOUT Ribonuclease Inhibitor (recombinant)</td>
			<td>Invitrogen by thermoFisher scientific</td>
			<td>10777-019</td>
			<td>qty:5000U (40U/ul)</td>
		</tr>
		<tr>
			<td>Distilled Water Dnase/Rnase Free</td>
			<td>Gibco</td>
			<td>10977-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligod(T)18 mRNA Primer</td>
			<td>New England BioLabs</td>
			<td>S1316S</td>
			<td>5.0 A260unit</td>
		</tr>
		<tr>
			<td>Random hexamers</td>
			<td>Invitrogen by thermoFisher scientific</td>
			<td>N8080127</td>
			<td>qty : 50uM, 5nmoles</td>
		</tr>
		<tr>
			<td>Superscript III Reverse transcriptase</td>
			<td>Invitrogen by thermoFisher scientific</td>
			<td>18080-044</td>
			<td>qty : 10000U (200U/ul)</td>
		</tr>
		<tr>
			<td>GoTaq G2 Flexi DNA polymerase</td>
			<td>Promega</td>
			<td>M7805</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Set, Molecular biology grade</td>
			<td>Thermo Scientific</td>
			<td>R0182</td>
			<td>4*100umol</td>
		</tr>
		<tr>
			<td>5LVH1</td>
			<td>Eurofins</td>
			<td>ACAGGTGCCCACT 
			CCCAGGTGCAG</td>
			<td>First round of PCR - Amplification of heavy chains - Outer primers - Forward Prmers</td>
		</tr>
		<tr>
			<td>5LVH3</td>
			<td>Eurofins</td>
			<td>AAGGTGTCCAGTG 
			TGARGTGCAG</td>
			<td>First round of PCR - Amplification of heavy chains - Outer primers - Forward Prmers</td>
		</tr>
		<tr>
			<td>5LVL4_6</td>
			<td>Eurofins</td>
			<td>CCCAGATGGGTCC 
			TGTCCCAGGTGCAG</td>
			<td>First round of PCR - Amplification of heavy chains - Outer primers - Forward Prmers</td>
		</tr>
		<tr>
			<td>5LVH5</td>
			<td>Eurofins</td>
			<td>CAAGGAGTCTGTT 
			CCGAGGTGCAG</td>
			<td>First round of PCR - Amplification of heavy chains - Outer primers - Forward Prmers</td>
		</tr>
		<tr>
			<td>3HuIgG_const_anti</td>
			<td>Eurofins</td>
			<td>TCTTGTCCACCTT 
			GGTGTTGCT</td>
			<td>First round of PCR - Amplification of heavy chains - Outer primers -Reverse primers for human Ig- Bacteria PCR screening</td>
		</tr>
		<tr>
			<td>3CuCH1</td>
			<td>Eurofins</td>
			<td>GGGAATTCTCACA 
			GGAGACGA</td>
			<td>First round of PCR - Amplification of heavy chains - Outer primers -Reverse primers for human Ig</td>
		</tr>
		<tr>
			<td>5AgeIVH1_5_7</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCC 
			GAGGTGCAGCTGGTGCAG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5AgeIVH3</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCT 
			GAGGTGCAGCTGGTGGAG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5AgeIVH3_23</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCT 
			GAGGTGCAGCTGTTGGAG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5AgeIVH4</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCC 
			CAGGTGCAGCTGCAGGAG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5AgeIVH4_34</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCC 
			CAGGTGCAGCTACAGCAGTG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5AgeIVH1_18</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCC 
			CAGGTTCAGCTGGTGCAG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5AgeIVH1_24</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCC 
			CAGGTCCAGCTGGTACAG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5AgeIVH3__9_30_33</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCT 
			GAAGTGCAGCTGGTGGAG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5AgeIVH6_1</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCC 
			CAGGTACAGCTGCAGCAG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>3SalIJH1_2_4_5</td>
			<td>Eurofins</td>
			<td>TGCGAAGTCGACG 
			CTGAGGAGACGGTGACCAG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Reverse primers</td>
		</tr>
		<tr>
			<td>3SalIJH3</td>
			<td>Eurofins</td>
			<td>TGCGAAGTCGACG 
			CTGAAGAGACGGTGACCATTG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Reverse primers</td>
		</tr>
		<tr>
			<td>3SalIJH6</td>
			<td>Eurofins</td>
			<td>TGCGAAGTCGACG 
			CTGAGGAGACGGTGACCGTG</td>
			<td>Second round of PCR - Amplification of heavy chains - Inner primers -Reverse primers</td>
		</tr>
		<tr>
			<td>5&#39;LVk1_2</td>
			<td>Eurofins</td>
			<td>ATGAGGSTCCCYG 
			CTCAGCTGCTGG</td>
			<td>First round of PCR - Amplification of light chains k - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;LVk3</td>
			<td>Eurofins</td>
			<td>CTCTTCCTCCTGC 
			TACTCTGGCTCCCAG</td>
			<td>First round of PCR - Amplification of light chains k - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;LVk4</td>
			<td>Eurofins</td>
			<td>ATTTCTCTGTTGC 
			TCTGGATCTCTG</td>
			<td>First round of PCR - Amplification of light chains k - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>3&#39;Ck543_566</td>
			<td>Eurofins</td>
			<td>GTTTCTCGTAGTC 
			TGCTTTGCTCA</td>
			<td>First round of PCR - Amplification of light chains k - Outer primers -Reverse primers- Bacteria PCR screening</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVk1</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCT 
			GACATCCAGATGACCCAGTC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVk1_9_1&#8211;13</td>
			<td>Eurofins</td>
			<td>TTGTGCTGCAACCGGTGTAC 
			ATTCAGACATCCAGTTGACCCAGTCT</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVk1D_43_1_8</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTGT 
			GCCATCCGGATGACCCAGTC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVk2</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATGGG 
			GATATTGTGATGACCCAGAC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVk2_28_2_30</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATGGG 
			GATATTGTGATGACTCAGTC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeVk3_11_3D_11</td>
			<td>Eurofins</td>
			<td>TTGTGCTGCAACCGGTGTAC 
			ATTCAGAAATTGTGTTGACACAGTC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeVk3_15_3D_15</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCA 
			GAAATAGTGATGACGCAGTC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeVk3_20_3D_20</td>
			<td>Eurofins</td>
			<td>TTGTGCTGCAACCGGTGTAC 
			ATTCAGAAATTGTGTTGACGCAGTCT</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeVk4_1</td>
			<td>Eurofins</td>
			<td>CTGCAACCGGTGTACATTCG 
			GACATCGTGATGACCCAGTC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>3&#39;BsiWIJk1_2_4</td>
			<td>Eurofins</td>
			<td>GCCACCGTACGTT 
			TGATYTCCACCTTGGTC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>3&#39;BsiWIJk3</td>
			<td>Eurofins</td>
			<td>GCCACCGTACGTT 
			TGATATCCACTTTGGTC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>3&#39;BsiWIJk5</td>
			<td>Eurofins</td>
			<td>GCCACCGTACGTT 
			TAATCTCCAGTCGTGTC</td>
			<td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;LVl1</td>
			<td>Eurofins</td>
			<td>GGTCCTGGGCCCA 
			GTCTGTGCTG</td>
			<td>First round of PCR - Amplification of light chains &#955; - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;LVl2</td>
			<td>Eurofins</td>
			<td>GGTCCTGGGCCCA 
			GTCTGCCCTG</td>
			<td>First round of PCR - Amplification of light chains &#955; - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;LVl3</td>
			<td>Eurofins</td>
			<td>GCTCTGTGACCTC 
			CTATGAGCTG</td>
			<td>First round of PCR - Amplification of light chains &#955; - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;LVl4_5</td>
			<td>Eurofins</td>
			<td>GGTCTCTCTCSCA 
			GCYTGTGCTG</td>
			<td>First round of PCR - Amplification of light chains &#955; - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;LVl6</td>
			<td>Eurofins</td>
			<td>GTTCTTGGGCCAA 
			TTTTATGCTG</td>
			<td>First round of PCR - Amplification of light chains &#955; - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;LVl7</td>
			<td>Eurofins</td>
			<td>GGTCCAATTCYCA 
			GGCTGTGGTG</td>
			<td>First round of PCR - Amplification of light chains &#955; - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>5LVl8</td>
			<td>Eurofins</td>
			<td>GAGTGGATTCTCA 
			GACTGTGGTG</td>
			<td>First round of PCR - Amplification of light chains &#955; - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>3&#39;Cl</td>
			<td>Eurofins</td>
			<td>CACCAGTGTGGCC 
			TTGTTGGCTTG</td>
			<td>First round of PCR - Amplification of light chains &#955; - Outer primers -Forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVl1</td>
			<td>Eurofins</td>
			<td>CTGCTACCGGTTCCTGGGCC 
			CAGTCTGTGCTGACKCAG</td>
			<td>Second round of PCR - Amplification of light chains &#955; - Inner primers -forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVl2</td>
			<td>Eurofins</td>
			<td>CTGCTACCGGTTCCTGGGCC 
			CAGTCTGCCCTGACTCAG</td>
			<td>Second round of PCR - Amplification of light chains &#955; - Inner primers -forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVl3</td>
			<td>Eurofins</td>
			<td>CTGCTACCGGTTCTGTGACC 
			TCCTATGAGCTGACWCAG</td>
			<td>Second round of PCR - Amplification of light chains &#955; - Inner primers -forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVl4_5</td>
			<td>Eurofins</td>
			<td>CTGCTACCGGTTCTCTCTCS 
			CAGCYTGTGCTGACTCA</td>
			<td>Second round of PCR - Amplification of light chains &#955; - Inner primers -forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVl6</td>
			<td>Eurofins</td>
			<td>CTGCTACCGGTTCTTGGGCC 
			AATTTTATGCTGACTCAG</td>
			<td>Second round of PCR - Amplification of light chains &#955; - Inner primers -forward primers</td>
		</tr>
		<tr>
			<td>5&#39;AgeIVl8</td>
			<td>Eurofins</td>
			<td>CTGCTACCGGTTCCAATTCY 
			CAGRCTGTGGTGACYCAG</td>
			<td>Second round of PCR - Amplification of light chains &#955; - Inner primers -forward primers</td>
		</tr>
		<tr>
			<td>3&#39;XhoICl</td>
			<td>Eurofins</td>
			<td>CTCCTCACTCGAG 
			GGYGGGAACAGAGTG</td>
			<td>Second round of PCR - Amplification of light chains &#955; - Inner primers -Reverse primers - Bacteria PCR screening</td>
		</tr>
		<tr>
			<td>Ab-vec-sense</td>
			<td>Eurofins</td>
			<td>GCTTCGTTAGAAC 
			GCGGCTAC</td>
			<td>Bacteria PCR screening</td>
		</tr>
		<tr>
			<td>QA Agarose-TM, Molecular Biology Grade</td>
			<td>MP Bio</td>
			<td>AGAH0500</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoFast 96 PCR Plate</td>
			<td>Macherey Nagel</td>
			<td>743.100.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme Age I HF</td>
			<td>New England Biolabs</td>
			<td>R3552L</td>
			<td>20000U/ml</td>
		</tr>
		<tr>
			<td>Enzyme SalI HF</td>
			<td>New England Biolabs</td>
			<td>R3138L</td>
			<td>20000U/ml</td>
		</tr>
		<tr>
			<td>Enzyme Xho I</td>
			<td>New England Biolabs</td>
			<td>R0146L</td>
			<td>20000U/ml</td>
		</tr>
		<tr>
			<td>Enzyme BSIWI</td>
			<td>New England Biolabs</td>
			<td>R0553L</td>
			<td>10000U/ml</td>
		</tr>
		<tr>
			<td>HCg1 (Genbank accession number FJ475055)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LCk (Genbank accession number FJ475056 )</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LCl (Genbank accession number FJ517647)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Invitrogen by thermoFisher scientific</td>
			<td>15224.017</td>
			<td>100U (1U/ul)</td>
		</tr>
		<tr>
			<td>2X YT medium</td>
			<td>Sigma Aldrich</td>
			<td>Y1003-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>10835242001</td>
			<td></td>
		</tr>
		<tr>
			<td>LB (Luria Bertani) Broth (Lennox)</td>
			<td>Sigma Aldrich</td>
			<td>L3022-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Plasmid DNA, RNA and protein purification</td>
			<td>Macherey Nagel</td>
			<td>740588.250</td>
			<td></td>
		</tr>
		<tr>
			<td>Jet PEI DNA transfection reagent</td>
			<td>PolyPlus</td>
			<td>101-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat bottom96-well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>V-bottom 96-well plate</td>
			<td>Nunc/Thermofisher</td>
			<td>055142</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc easy 175 cm2 flasks</td>
			<td>Nunc/Thermofisher</td>
			<td>12-562-000</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA/ELISPOT coating buffer</td>
			<td>eBiosciences</td>
			<td>00-0044-59</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc maxisorp flat bottom 96 well ELISA plates</td>
			<td>Nunc/Thermofisher</td>
			<td>44-2404-21</td>
			<td>high protein binding</td>
		</tr>
		<tr>
			<td>Anti-human IgG Ab conjugated to horseradish peroxidase (HRP)</td>
			<td>BD Pharmingen / BD Biosciences</td>
			<td>55788</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB substrate</td>
			<td>BD Biosciences</td>
			<td>555214</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Sigma</td>
			<td>S0677</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL-HiTrap protein A HP column</td>
			<td>GE Healthcare</td>
			<td>17-0402-01</td>
			<td></td>
		</tr>
		<tr>
			<td>&#196;KTA FPLC</td>
			<td>GE Healthcare</td>
			<td>18190026</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 10/300 GL column</td>
			<td>GE Healthcare</td>
			<td>17517501</td>
			<td></td>
		</tr>
		<tr>
			<td>NGC Quest 10 Plus Chromatography System</td>
			<td>BioRad</td>
			<td>7880003</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of discriminative human monoclonal antibodies from rare antigen-specific b cells circulating in blood

モノクローナル抗体 (Mab) は、両方の基礎研究および生体医に役立つ強力なツールです。抗体は (例えば、正常な蛋白質およびその変異バージョン) の関連性の高い構造を区別する必要がある場合、高い特異性が必要不可欠です。このような差別の Mab を生成する現在の方法には高価な時間のかかる複数の Ab-産生 B 細胞のスクリーニングが含まれます。提案する迅速かつコスト効果の高い方法の識別、生成のため B リンパ球を循環血液から始まって完全ひと抗体.この戦略の独創性は、容易に利用可能な末梢血単核細胞 (PBMC) を使用して、他のすべてのセルのカウンターの選択を組み合わせる特異抗原結合 B セルの選択によるものです。単一のセルを逆転写ポリメラーゼの連鎖反応 (RT-PCR) 技術を使用して人間で表されるその後に対応する mAb のコーディング (相補的デオキシリボ核酸) cDNA シーケンスを取得特定の B 細胞が分離されたら、セルです。わずか 1 ヶ月で、内自然 B 細胞レパートリーによって検出されたあらゆる目的の抗原に対して指示される高い識別人間 Mab のミリグラムを生成することが可能です。

このプロジェクトでは、ペプチド Pp65495を認識する人間の Mab の世代に健康なドナーから PBMC から始まって主要な組織適合性の複雑なクラスによって (Pp65、ヒトサイトメガロから) 提示私 (MHC-私) 分子 HLA A * 0201 (HLA-A2)。これらの Mab を区別このコンプレックスと同じ MHC に読み込まれる他のペプチドを表す複合体-私の分子。
<p class="jove_content" fo:keep-together.withi...

Watch this Scientific Journal Video about Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood at JoVE.com

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

希少な B 細胞人間の血液循環から始まって、興味の抗原のために特定の抗体の生産のための手法について述べる。これらの自然抗体の生成は効率的かつ迅速、得られた抗体関連性の高い抗原の間で区別することができます。

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 ...

The overall goal of this procedure is to produce fully human monoclonal antibodies against a desired antigen starting from human B lymphocytes circulating in the blood. This protocol allows the generation of monoclonal antibodies that can discriminate between highly related antigens. For some applications such as immunotherapy, precise targeting of an antigen can be indispensable.The advantages of this technique are that this can be performed starting from the blood of healthy or immunized donors and allows the isolation of even rare antigen-binding B cells. While this method is described starting from human samples, it can also be applied to animals that can be pre-immunized. Visual demonstration of this method is critical as the procedure really is under quite complex multiple analytic gating strategy of discriminated B lymphocytes.The procedure will be performed by Melinda. She is an assistant engineer from our laboratory. First pipette 35 milliliters of diluted cell suspension on 15 milliliters of density gradient in a 50 milliliter conical tube.Then place the conical tube in a swing bucket rotor with brakes off and centrifuge at 1290 times gravity for 25 minutes at room temperature. Next, at the interface of the density gradient medium and plasma layer, carefully aspirate the mononuclear cell layer and transfer it to a new 50 milliliter conical tube. Add the cell suspension to a 15 milliliter conical tube and centrifuge at 460 times gravity for five minutes.Then, resuspend the pellet in 200 microliters of phosphate buffered saline with 2%fetal bovine serum. Add conjugated tetramers to a final concentration of 10 micrograms per milliliter. Mix well and then incubate at room temperature for 30 minutes in the Laminar flow hood, protected from the light.Then add 10 milliliters of ice cold cell separation buffer to the cell suspension. Centrifuge the cell suspension at 460 times gravity for five minutes. To the cell pellet obtained, add 500 microliters of ice cold cell separation buffer and 50 microliters of two variants of magnetic microbeads and incubate the cell suspension for 20 minutes.Next, resuspend the cell pellet in ice cold cell separation buffer. Carefully transfer the cell suspension on the top of an equilibrated column and let it drain completely. Then rinse the tube with three milliliters of cell separation buffer and transfer the buffer to the column.After rinsing the column three times, remove it from the magnet and place on a 25 milliliter conical collecting tube. After adding five milliliters of ice cold separation buffer on top of the column, quickly flush the cells from the column with the help of a plunger. Resuspend the cell pellet with a cocktail of anti-human antibodies and 7AAD in a total volume of 100 microliters of phosphate buffered saline with 2%fetal bovine serum.Then, incubate the cells at four degrees Celsius for 30 minutes. Next, use a cell sorter cytometer to sort specific B cells at the single cell level not exceeding beyond on 1, 000 events per second. Then, collect the single sorted B cells into 8-strip PCR tubes previously filled with 10 microliters of 1X phosphate buffered saline and 10 units of RNAse inhibitor and place on a rack for 96 microtubes.Quickly freeze the tubes at negative 80 degrees Celsius. Heat the frozen samples in a dry bath maintained at 70 degrees Celsius for five minutes to lyse the cells. Then place the strip on ice and start constituting the reverse transcription master-mix.Once the reverse transcription master-mix is prepared, program instruct thermocycler. At the end of the reverse transcription, perform nested PCRs to amplify immunoglobulin genes. Identify the positive clones by visualizing on a 1.5%agarose gel the migration of a 500 base pair PCR product for variable heavy chain and coding segments and 350 base pair PCR product for variable light chain encoding segments.Add one microliter of the cloning product to 45 microliters of electrocompetent TOP10 E.coli cells.Electroporate. Then quickly add 500 microliters of 2X YT medium to the cells and incubate in a water bath at 37 degrees Celsius for 30 minutes. Spread the transformed bacteria on 2X YT ampicillin plate.Then, incubate the plates overnight at 37 degrees Celsius. Next, setup a reaction premix and add 25 microliters of it in per 8-strip PCR tube kept on ice. Then, use a sterile toothpick to pick colonies and streak the colonies on the 2X YT ampicillin plate.Then, dip the toothpicks in the PCR reaction tube. Identify the positive clones by visualizing on a 1.5%agarose gel the migration of an 850 base pair PCR product for the heavy chain vector and a 600 base pair PCR product for the light chain vector. In two milliliters of LB medium, inoculate four positive colonies.Incubate the inoculate at 37 degrees Celsius overnight at 200 revolutions per minute shaking. Seed 15, 000 human embryonic kidney cells in 200 microliters of Dulbecco's Modified Eagle Medium supplemented with 10%fetal bovine serum in each well of a flat bottom 96 well plate a day before the transfection. Then incubate the plate at 37 degrees Celsius overnight at 5%carbon dioxide.Cotransfect the cells using a linear polyethylenimine derivative transfection reagent. Dilute 0.5 microliters of DNA transfection reagent with 10 microliters of 150 millimolar of sodium chloride. Next, dilute 0.125 microgram of each variable heavy and light chain expressing vectors in 10 microliters of 150 millimolar sodium chloride.Vortex all the dilutions for 10 seconds each. Then, mix the previously diluted DNA transfection reagent with 10 microliters of DNA solution. Quickly vortex the DNA transfection mixture for 15 seconds and incubate for 15 minutes at room temperature.Add the DNA transfection mixture drop-wise on the cells and then gently swirl the plate. Replace the medium 16 hours after transfection and culture the cells for five days in serum-free medium at 37 degrees Celsius. Next, use a multichannel pipette, to aspirate the supernatant, and transfer it into the v-bottom 96 well plate.Coat each well in a 96 well ELISA plate with relevant antigen at four degrees Celsius for overnight in 100 microliters of reconstituted ELISA coating buffer at a final concentration of two micrograms per milliliter. Next add 50 microliters of a transfected cell supernatant to the wells of a 96 well ELISA plate and incubate the plate for two hours at room temperature. Then add 100 microliters of antihuman immunoglobulin G antibody conjugated to horseradish peroxidase enzyme at one microgram per milliliter concentration and incubate the well at room temperature for one hour.Finally, add 50 microliters of chromogenic substrate to the well and incubate for another 20 minutes. At the end of incubation, read the optical density at 450 nanometers on a spectrophotometer. To isolate the specific B cells a gating strategy was used that discriminated the B cells expressing receptors against the HLA-A2/Pp65 complex.Initially, the cells were CD19 positive, CD3 negative gated. Finally, cells were stained with both HLA-A2/Pp65PE and HLA-A2/Pp65APC, but not HLA-A2/MelABV421 tetramers, were gated to obtain the highly specific B cells. Using this gating strategy, selective B cells can be isolated, which can differentiate between HLA-A2/Pp65 and other related HLA-A2 peptide complex.Next, to study the presence of PC1.02 antibody against HLA-A2/Pp65 tetramer an ELISA assay was performed. In the bar plot obtained, the y-axis corresponds to the optimal density readings and the x-axis correspond to the HLA-A2 peptides. From the assay, it shows that a monoclonal antibody binds with HLA-A2/Pp65 tetramer in a peptide and major histocompatibility complex dependent manner.Next, surface plasmin residence was used to study the binding affinity of the PC1.02 antibody. Then, a tabular data was obtained to analyze the HLA-A2/Pp65 specific B cells. The impact of the exclusion strategy of unspecific B cells, the yields of immunoglobulin gene amplification, and monoclonal antibody production from HLA-A2/Pp65 specific B cells have been shown.Once mastered, the technique ranging from isolation of antigens specific B cells from human blood to monoclonal antibody production can be done in as little as one month. When performing this procedure, remember go straight from sorting of B cells to RT-PCR without pausing.

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

抗原特異的インビボでの</emCFSE標識した標的細胞を用いた>キリングアッセイ

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

免疫・感染学

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

臍帯血から前駆B細胞サブセットの分離

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

効率的なためキラー人工抗原提示細胞（KaAPC）<em&gt;体外</em&gt;ヒト抗原特異的T細胞の枯渇

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

末梢血からのヒトアロ抗原特異的T細胞の生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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

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, ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

単離、分化、および血液からヒト抗体を分泌するB細胞の定量化：体液性免疫の機能読み出しなどのELISpot

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

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 Human Monocyte-derived Dendritic Cells from Whole Blood

全血からのヒト単球由来樹状細胞の生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

表現型および機能解析のためのレア抗原特異的B細胞の検出および濃縮

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...