The authors thank Miss Ching-Chun Lin and Miss Diana Lin of the Core Facility of the Institute of Cellular and Organismic Biology (ICOB) of Academia Sinica for offering their expertise on peptide synthesis and DNA sequencing, respectively. This study was supported by Academia Sinica.

モノクローナル抗体の作製<ol><li>培養物を5％CO 2補充37ºCで175Tフラスコ中の無血清培地中のRG-M56マウスモノクローナルハイブリドーマ細胞2。媒体の色は、インキュベーションの5日後に黄色になるときに、上清を収集します。 注：ハイブリドーマ細胞は、ウシ胎児血清からの抗体の混入を避けるために、無血清培地で培養しました。 </li><li> 4ºCで30分間、4500×gで上清を遠心し、細胞破片ペレットを捨てます。 </li><li> 5 mLのカラム（50％スラリーとして供給される）プロテインGアガロースの2 mLを加え、氷冷PBSの10樹脂体積（10mL）で平衡化。 </li><li>抗体上清の負荷200 mLをカラムに（ステップ1.2）とパススルーを捨てます。 </li><li>それを洗浄するために列に氷冷PBSの10 mLを加え。二回繰り返します。 </li><li>プロテインG-関連抗体を溶出するカラムに50 mMグリシン、pHが2.7の10ミリリットルを追加します。コル10倍の中和緩衝液（1Mトリス、1.5 MのNaCl、および1mM EDTA、pH8.0）を100μLを含むマイクロチューブに900μLの分画をLECT。 </li><li> -20ºCで0.03％のNaN 3で50％グリセロール中で精製された抗体を保管してください。 </li></ol> 2.構築およびシリアル切り捨てられた組換えタンパク質の発現<ol><li> （10倍のPfu緩衝液の5μL、各dNTP 0.2mMの、0.2μMのフォワードプライマー3、0.2μMリバースプライマー3、2 mMのMgSO 4を、のpET20b-1A59 3プラスミドDNA 1ngの、および2.5 U：PCR反応混合物を準備しますPfu DNAポリメラーゼの単位）。 50μLの最終容量までのddH 2 Oを追加します。 <ol><li>自動サーマルサイクラー中で実行したサンプル次のパラメータを使用して：サイクル1（5分94ºC）。サイクル2-36（30秒94ºC、30秒間63ºC、60秒72ºC）。サイクル37（7分72ºC）。 </li></ol></li><li>以下の制限酵素消化を容易にするために、PCR精製キット5を用いて、ポリメラーゼ連鎖反応（PCR）産物を抽出します。 </li><li> んで IおよびXho I制限酵素でPCR増幅DNA断片を消化し、NdeIおよびのXhoI酵素切断PET-20B（+）ベクターにこれらのDNA断片の各々を連結。 6 大腸菌 DH-5α-コンピテント細胞にコンストラクトを変換します。 <ol><li> PCR増幅DNAまたはPET-20bの1μgの消化緩衝液（20 mMトリス酢酸、10mMのMgの（CH 3 COO）2、50mMのKCH 3 COO、および1mM DTT、pHは7.9）での消化混合物を準備しますNdeIおよびXhoI制限の（+）ベクターDNA、および2 Uは、20μLの最終容量で酵素。 </li><li>優しく消化混合物を混合し、すぐにスピンダウン。制限SITの完全な切断を確実にするために、乾燥槽中で2時間、37ºCでインキュベートしますエス。 </li><li>以下のプラスミドの構築を容易にするために、PCR精製キット5を用いて、制限酵素消化DNA断片を抽出します。 </li><li>予め消化、PCR増幅DNAの100 ngの、予め消化のpET-20bの10 ngの（+）とライゲーションバッファー（66 mMトリス、5mMのMgCl 2、1mMのATP、および5mM DTT、pH7.5）を内ライゲーション混合物を準備しますベクターDNA、および10μLの最終容量で5 UのT4 DNAリガーゼ。 </li><li>優しく連結混合物を混合し、すぐにスピンダウン。水浴中で18時間、16ºCでインキュベートします。 </li><li> DH-5α-コンピテント細胞の100μLにライゲーションサンプルの10μLを入れ、30分間氷上でマイクロ遠心チューブを配置する前に、穏やかに混合します。ヒートショック7を誘導するために90秒間42ºCで乾燥浴中にマイクロチューブを入れてください。すぐに2分間氷上にチューブを移します。 </li><li>ルリア - ベルターニ（LB）ブロス（1％バクトトリプトン、0.5％バクト酵母extracの900μLを追加管にT、および0.5％のNaCl、pH7.0）に。 150 rpmで45分間振盪しながら37ºCでインキュベートします。 10分間4000×gでの遠心分離により細胞をペレット化し、上清を捨てます。 </li><li> LBブロスの50μLでペレットを再懸濁し、100μg/ mLのアンピシリンを含む予め温めたLBプレート上に各変換を広げます。 16時間37ºCでプレートをインキュベートします。 </li><li> 200μLチップを用いて、単一のコロニーをピックアップし、ゆるくキャップした15 mLチューブに100μg/ mLのアンピシリンを含むLBブロス3mLの中にそれを配置します。 150rpmで12時間振盪しながら37ºCでインキュベートします。 </li><li>各培養物からプラスミドDNA 8を抽出し、シーケンス9を確認するために、T7プロモーターとT7ターミネータープライマーを用いて配列決定します。 </li></ol></li><li>配列確認後、当社による大腸菌のBL-21（DE3）株にYGNNVコートタンパク質遺伝子の長さを変化させて、これらのpET-20bは（+）プラスミドからDNA 10ngの変換ヒートショック法7る。手順に従ってください2.3.6-2.3.8変換を実行します。 </li><li>ゆるくキャップした15 mLチューブに100μg/ mLのアンピシリンを含む3mLのLBブロスに各形質転換した大腸菌 BL-21細胞からの単一コロニーを転送します。 150 rpmで振盪しながら37ºCで培養物をインキュベートします。 </li><li>文化のOD 600が約0.6のとき25ºCに文化を冷却し、組換えタンパク質の発現を誘導するために最終濃度0.4mMになるようにIPTGを追加します。 200 rpmで振盪しながら25ºCで余分4時間培養をインキュベートします。 </li><li>マイクロ遠心チューブに培養液を1mLを転送します。 1分間12,000×gの遠心分離により細胞をペレット化し、上清を捨てます。 </li><li>マイクロピペットでピペッティングすることによって、およびダウン変性緩衝液100μL（8M尿素、20mMリン酸ナトリウム、及び0.5MのNaCl、pH7.4）中で細胞ペレットを再懸濁します。活発ボルテックスで混和します。 注：サンプルゾルutionsは今、半透明の少し粘着性、およびドットブロットハイブリダイゼーションアッセイのために準備する必要があります。 </li></ol>重複ペプチドの3設計と合成<ol><li>設計と各ドットブロット法によりRG-M56 mAbのエピトープ領域を絞り込むためにYGNNVコートタンパク質の195から338のaa領域からの10アミノ酸残基によって、その後継と重なる3シリアル20量体ペプチドを合成します。 </li><li>ドットブロット法により195から206アミノ酸のエピトープ領域を絞り込むために、次の合成ペプチドに6アミノ酸残基のオーバーラップで3 3 8マーペプチド（195 VNVSVLCR 202、197 VSVLCRWS 204、および199 VLCRWSVR 206）を設計し、合成します。 </li><li>設計と3 7マーを合成（196 NVSVLCR 202と195 VNVSVLC 201）、6量体（195 VNVSVL 200、196 NVSVLC 201、および1その隣接するペプチド上に97 VSVLCR 202）、および5量体ペプチド（195 VNVSV 199、196 NVSVL 200、それぞれ197 VSVLC 201、及び198 SVLCR 202）6のオーバーラップを有する、5、4アミノ酸残基は、最小限にしますドットブロットによるエピトープ領域。 </li></ol> 4.ドットブロットハイブリダイゼーション<ol><li> 10mg / mLの最終濃度になるようにジメチルスルホキシド（DMSO）中の各合成ペプチドを溶解させます。 注：合成ペプチドの多様な溶解性を克服するために、すべての合成ペプチドは、DMSO中に溶解されるべきです。 DMSOは完全に疎水性又は親水性ペプチドを溶解する良溶媒です。 </li><li> 2分間メタノールでポリフッ化ビニリデン（PVDF）膜を浸します。 注：PVDF膜は、DMSOに耐性の100％までです。他の人はそうではないかもしれません。 </li><li>ために修飾Towbin緩衝液（25 mMトリス、192 mMグリシン、0.1％SDS、pHが8.3）でPVDFメンブレンを平衡化2分。 注：10〜20％（v / v）のメタノールの転送結果を改善するように改変Towbin緩衝液に添加することができます。 </li><li>修正されたTowbin緩衝液でクロマトグラフィー紙を洗浄します。クロマトグラフィーペーパー上にPVDF膜を配置します。修正されたTowbinバッファが次のステップに進む前に、PVDF膜表面から消失するまで待ちます。 </li><li> 10μLチップでメンブレンに各ペプチドサンプルの2μLを追加します。 10分間クロマトグラフィーの紙にPVDF膜に空気が乾燥します。あまりにも多くの拡散を避けるために、膜上にゆっくりと徐々に各ペプチドサンプルを追加します。 </li><li>穏やかに振盪しながら室温で30分間、5％脱脂乳でTBST緩衝液（0.05％（v / v）のTween-20を、20mMのトリス、および150mMのNaCl、pH7.4）中で膜をブロックします。 </li><li>膜への5％脱脂乳でTBST緩衝液1,000：1の最終希釈でRG-M56モノクローナル抗体を追加します。穏やかに振盪しながら1時間、37ºCで膜をインキュベートします。 </li><li>そう抗体を除去リューション。穏やかに振盪しながら5分間、TBST緩衝液中で膜を洗浄します。二回繰り返します。 </li><li>膜を5％脱脂乳を有するTBST緩衝液で5,000：1の最終希釈で二次抗体（ヤギ抗マウスIgG、Fcを、アルカリホスファターゼ結合）を加えます。穏やかに振盪しながら1時間、37ºCで膜をインキュベートします。 </li><li>抗体溶液を廃棄してください。穏やかに振盪しながら5分間、TBST緩衝液中で膜を洗浄します。二回洗浄ステップを繰り返します。 </li><li>暗所で15分間、室温でBCIP / NBT基質溶液を有する膜を開発。信号が表示されたときのddH 2 Oで膜を洗浄することにより現像を停止します。 </li><li>膜を空気乾燥し、画像システムを用いたドットブロットの画像を取り込みます。 </li><li>画像解析ソフト3を使用して、各ドットブロットの強度を測定します。 </li></ol> 5.アラニンスキャニングと交代<ol><li> 3アラニンおよびメティを設計・合成onine置換ペプチド。 VAVSVLCR 202、195 VNASVLCR 202、195 VNVAVLCR 202、195 VNVSALCR 202、195 VNVSVACR 202、195 VNVSVLAR 202 8-merペプチド195 VNVSVLCR 202のために、アラニンで各アミノ酸残基を交換し、合成ペプチド195 ANVSVLCR 202、195、および195 VNVSVLCA 202 。 VNVSVMCR 202 SJNNV遺伝子型エピトープペプチド195を作成するために、メチオニンへのaa残基のロイシン200を交換してください。 </li><li>アラニンスキャニングおよび置換変異誘発は、ドットブロッティング実行するために第4節に従ってください。 </li></ol>

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The authors have no conflicts of interest related to this report.

このプロトコルは、mAb-認識線形エピトープを同定するための迅速かつ簡単な手法を提供しています。考慮ペプチド合成およびペプチド合成の生産効率のコストを考えると、ウイルスコートタンパク質の抗原性領域は、ペプチドスキャン分析の前に直列に切り捨て組換えタンパク質を発現させることによって減少しました。 10 kDaの50の間の分子量を有する組換えタンパク質は、容易にこのシス...

免疫系では、V、D、およびJセグメントの再結合は、抗体が、病原性感染からホストを保護するために、種々の抗原に結合するための相補性決定領域（CDR）の多大な変化を作成することを可能にします。抗原に対する抗体の中和防衛は、抗体のCDRと抗原のエピトープとの間の空間的な相補性に依存します。したがって、この分子間相互作用の理解は、予防ワクチンの設計および治療用ペプチド薬剤の開発を支援します。しかしながら、この中和相互作用は、単一の抗原からの複数の抗原性ドメインにより、その結果エピトープ決意プロセスをより複雑にする抗体の複数のCDR、の両方によって影響され得ます。幸いなことに、骨髄腫細胞と個々の抗体産生細胞を融合ハイブリドーマ技術の開発は、秒細胞の恒常分割バッチを可能にしますモノクローナル抗体（mAb）として知られている網一つの特定の抗体、1。ハイブリドーマ細胞は、特定抗原の単一の抗原性ドメインに結合するために、これらの純粋な、高親和性モノクローナル抗体を産生します。確立された抗原 - 抗体、ペプチドスキャンを含むいくつかの方法の関係で、その対応するモノクローナル抗体を用いて抗原のエピトープを決定するために用いることができます。合成ペプチド技術における最近の開発は、ペプチドスキャン技術をよりアクセスし、実行することがより便利になりました。簡単に述べると、重複する合成ペプチドのセットは、標的抗原の配列に従って製造されたmAbのハイブリダイゼーションのための固体支持膜に関連しています。ペプチドスキャンは、抗体結合領域をマッピングするための簡単な方法を提供していますが、また、エピトープペプチドと抗体のCDRの各アミノ酸残基との間の結合相互作用を評価するために、残留物をスキャンまたは置換によりアミノ酸（AA）突然変異誘発を促進するだけでなく。 2、3、4を使用して、黄色のハタ神経壊死症ウイルス（YGNNV）コートタンパク質の線状エピトープの効率的な同定のためのプロトコルについて説明します。プロトコルは、mAb製剤、連続切断型組換えタンパク質の構築および発現、合成重複ペプチドの設計、ドットブロットハイブリダイゼーション、アラニンスキャニング、および置換突然変異を含みます。ペプチド合成の高いコストを考えると、直列に所望の標的タンパク質の組換えタンパク質を切り捨てるステップは、変更された、および合成ペプチドアレイドット - ブロット分析を行った前の抗原性領域は、約100〜200アミノ酸残基に絞りました。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hybrid-SFM medium</td>
			<td>Gibco</td>
			<td>12045-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeccos&#39;s Phophate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu DNA Polymerase&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>EP0502&#160;</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Roche</td>
			<td>10799009001</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>NdeI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>XhoI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>pET-20b(+) vector</td>
			<td>Novagen, Merck Millipore</td>
			<td>69739</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli DH-5&#945; competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH617</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli BL-21(DE3) competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH217</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Amresco</td>
			<td>0339-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth&#160;</td>
			<td>Invitrongen</td>
			<td>12780-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropylthio-&#946;-D-thiogalactoside (IPTG)</td>
			<td>MDBio, Inc.</td>
			<td>101-367-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T.Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Amresco</td>
			<td>0167-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Affymetrix, USB</td>
			<td>75825</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Amresco</td>
			<td>0241-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Amresco</td>
			<td>0105-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Amresco</td>
			<td>0854-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>PerkinElmer</td>
			<td>NEL937001PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG, Fc fragment antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-055-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P (Polyvinylidene fluoride, PVDF)</td>
			<td>Merck Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Agarose Fast Flow</td>
			<td>Merck Millipore</td>
			<td>16-266</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP BioSpectrum 600 Image System</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>VisionWorks LS Analysis Software Ver 6.8</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>MyCycler thermal cycler</td>
			<td>BioRad</td>
			<td>1709713</td>
			<td></td>
		</tr>
	</tbody>
</table>

peptide scanning-assisted identification of a monoclonal antibody-recognized linear b-cell epitope

免疫系による抗原エピトープの同定は、ワクチン及びペプチド薬物の開発を容易にすることができる中和抗体の防御機構の理解が可能になります。ペプチドスキャンは直接的モノクローナル抗体（mAb）によって認識される線状エピトープをマッピングする簡単かつ効率的な方法です。ここで、著者らは、中和モノクローナル抗体を使用して神経壊死症ウイルスコートタンパク質の抗原認識のために直列に切り捨て組換えタンパク質、合成ペプチドの設計、およびドットブロットハイブリダイゼーションを含むエピトープ決意方法を提示します。この技術は、ポリフッ化ビニリデン（PVDF）膜上に合成ペプチドとモノクローナル抗体のドットブロットハイブリダイゼーションに依存します。 RG-M56モノクローナル抗体により認識されるウイルスコートタンパク質の最小の抗原性領域は、6量体ペプチドエピトープにペプチドマッピングをトリミングステップ・バイ・ステップによって絞り込むことができます。また、アラニンスキャニング変異誘発、残渣サブstitutionエピトープを構成する各アミノ酸残基の結合重要性を特徴付けるために実施することができます。エピトープ部位に隣接する残基は、ペプチドのコンホメーションの調節において重要な役割を果たすことが見出されました。同定されたエピトープペプチドは、X線回折研究および機能競合のために、または治療のためのエピトープペプチド - 抗体複合体の結晶を形成するために使用することができます。

この実験の目的は、mAbを用いて、ドットブロッティングを通じてエピトープを同定することでした。迅速かつ効率的にモノクローナル抗体によって認識される抗原性領域を絞り込むために、C末端での6×His融合タグを有する完全長およびシリアル切り捨てYGNNV組換えコートタンパク質は、 大腸菌 pET発現システム10（ 図1A）から発現?...

Watch this Scientific Journal Video about Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope at JoVE.com

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

モノクローナル抗体認識リニアB細胞エピトープのペプチドスキャンアシスト識別

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

peptide-scanning-assisted-identification-monoclonal-antibody

Research

JoVE Journal

Immunology and Infection

9.3K ビュー.  Academia Sinica. Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

ビデオ: Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

ペプチドエピトープ特異的T細胞のMHCテトラマーベースの濃縮

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

免疫・感染学

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

機能的モチーフとその結合パートナーのペプチドベース識別

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

単離、同定、およびマウス胸腺上皮細胞の精製

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

エスケープ中和抗体でインフルエンザ ウイルスの亜種の世代

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

蛋白質の Qa 1 エピトープをマップするペプチド ライブラリを重複

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

ジカ特定診断エピトープの探索

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

天然物に対するモノクローナル抗体の作製

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...