Name: a protein microarray assay for serological determination of antigen-specific antibody responses following clostridium difficile infection
Uploaded: 2018-06-15T14:00:00
Description: Watch this Scientific Journal Video about A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection at JoVE.com

この研究は、エルメス交わりオラ Negm とターニャ モナハンとノッティンガム消化器病センターと NIHR ノッティンガム消化器疾患医療研究センターから個別の資金によって支えられました。

1. マイクロ プレートの準備
<ol><li>(患者の血清を実行する前に事前に定義された) を最適な濃度で印刷バッファー [PBS-トゥイーン-トレハロース (50 mM)] c. ディフィシル抗原を希釈: 毒素 (200 μ g/ミリリットル) と毒素 B (100 μ g/mL)、pCDTb (200 μ G/ml) と精製ネイティブ全体の Slp は、ribotypes 001、002、027 (200 μ g/mL) から派生します。 注: 毒素 B 毒素 B 表現するひずみ CCUG 20309 (90 μ g/mL) から得?...

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	<li>Negm, O. 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=Profiling+humoral+immune+responses+to+Clostridium+difficile.-specific+antigens+by+protein+microarray+analysis.">Profiling humoral immune responses to Clostridium difficile.-specific antigens by protein microarray analysis.</a> Clinical and Vaccine Immunology. 22 (9), 1033-1039 (2015).</li><li>Monaghan, T. 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=High+prevalence+of+subclass-specific+binding+and+neutralizing+antibodies+against+Clostridium+difficile.+toxins+in+adult+cystic+fibrosis+sera:+possible+mode+of+immunoprotection+against+symptomatic+C.+difficile+infection.">High prevalence of subclass-specific binding and neutralizing antibodies against Clostridium difficile. toxins in adult cystic fibrosis sera: possible mode of immunoprotection against symptomatic C. difficile infection.</a> Clinical and Experimental Gastroenterology. 10, 169-175 (2017).</li><li>Negm, O. 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=Protective+antibodies+against+Clostridium+difficile.+are+present+in+intravenous+immunoglobulin+and+are+retained+in+humans+following+its+administration.">Protective antibodies against Clostridium difficile. are present in intravenous immunoglobulin and are retained in humans following its administration.</a> Clinical &amp; Experimental Immunology. 188 (3), 437-443 (2017).</li><li>Monaghan, 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=OC-058+A+protein+microarray+assay+for+predicting+severe+outcomes+in+Clostridium+difficile+infection.">OC-058 A protein microarray assay for predicting severe outcomes in Clostridium difficile infection.</a> Gut. 66, 31 (2017).</li><li>Bacon, A. 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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=Serum+anti-toxin+B+antibody+correlates+with+protection+from+recurrent+Clostridium+difficile+infection+(CDI).">Serum anti-toxin B antibody correlates with protection from recurrent Clostridium difficile infection (CDI).</a> Vaccine. 28 (4), 965-969 (2010).</li><li>Drudy, D., 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=Human+antibody+response+to+surface+layer+proteins+in+Clostridium+difficile+infection.">Human antibody response to surface layer proteins in Clostridium difficile infection.</a> FEMS Immunology and Medical Microbiology. 41 (3), 237-242 (2004).</li><li>Bauer, M. P., Nibbering, P. H., Poxton, I. R., Kuijper, E. J., van Dissel, J. 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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=Bezlotoxumab+for+prevention+of+recurrent+Clostridium+difficile+infection.">Bezlotoxumab for prevention of recurrent Clostridium difficile infection.</a> The New England Journal of Medicine. 376 (4), 305-317 (2017).</li><li>Leuzzi, R., Adamo, R., Scarselli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccines+against+Clostridium+difficile.">Vaccines against Clostridium difficile.</a> Human Vaccines &amp; Immunotherapeutics. 10 (6), 1466-1477 (2014).</li><li>Ghose, C., Kelly, C. 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このプロトコルでは、マイクロ アレイは (図 3および6) 患者血清および IVIg (図 5) の商業準備のdifficile C.タンパク質抗原に対する液性免疫応答を定義するための適切なプラットフォームを示しました。マイクロ アレイ技術が従来の elisa 法 (図 2) と比較した場合にも実行して測定内および測定間変動 (精度の許?...

このプロトコルでは、開発、検出のための新規およびカスタマイズされたタンパク質マイクロ アレイ アッセイの検証と菌株のアイソタイプ特異抗体のc. ディフィシル抗原半定量をについて説明します。我々 は正常に私たちのdifficile C.を使用して-患者血清1、2IVIg3の準備で特異抗体コンテンツの組成の生化学分析のための有望な新しいツールとして特定のマイクロ アレイ分析とCDI4の転帰不良と相関する抗体の特異性の id。Difficile C.病原体特異抗体のこのアッセイでの質の高い再現性のあるプロファイリングできるように biobanked 血清と市販の IVIg をマイクロ アレイ スライドに分析する方法を紹介します。
多くの健康な子供および大人検出血清 IgG, IgA 抗体c. ディフィシル毒素 A と B5,6があります。これらは幼児期と成人期のdifficile C.への暴露を次時に一時的な露出の後で発生すると考えられています。このため、ポリクローナル IVIg がずっと再発と劇症 CDI7,8,9を治療するためにオフにラベルを使用します。しかし、その決定的な役割と作用は不明のまま。いくつかの研究は、 C. difficile毒素に体液性免疫反応が病気の発表や結果に役割を果たすことを示しています。具体的には、無症候性の患者は、増加血清抗毒素 A IgG 濃度10症候性疾患を発症した患者と比較して表示します。明白な協会中央抗毒素 A IgG 抗体と 30 日間全ての原因の死亡率11報告されています。いくつかのレポートはまた毒素 A、B、およびいくつかの非毒素抗原 (Cwp66、Cwp84、フリック、ありますと表層タンパク質 (Slp)) 再発・抗体反応に対する保護との関連を明らかにした12,13,14,15. これらの観察が誕生して最初受動免疫療法薬ターゲットc. ディフィシル毒素 B の (bezlotuxumab)、最近、米国食品医薬品局や欧州の医薬品が承認されています。再発の CDI16防災機関。不活化毒素または組換え毒素断片を使用して予防接種戦略もまた中開発17,18,19。これらの新しい治療上のアプローチは間違いなく大きいサンプルの大きさの複数の抗原に対する液性免疫応答を評価するための要件を刺激します。
今日、菌株、アイソタイプ特異抗体のc. ディフィシル抗原を同時に評価することができる市販の高スループット試金の著しい欠乏があります。今後の研究活動や臨床応用を容易にするためにこのようなアッセイを開発するニーズがあります。蛋白質のマイクロ アレイ、接触20または非接触にすることができますロボット システムを用いた顕微鏡スライド ベース表面にスポットの空間的組織化配列として個別に浄化された蛋白質の多数を固定する方法印刷ツール21。スポットのセル lysates、抗体、組織ホモジネート、内因性または組換え蛋白質やペプチド、体液薬22,23など複雑な混合物があります。
蛋白質のマイクロ アレイの技術では、標準的な社内の elisa 法にはなかった利点伝統的反difficile C.の抗体の応答を評価するために使用されている技術を提供しています。マルチ アイソタイプ特異抗体抗原、サンプル、および試薬の量を削減要件標的蛋白質のより広範なパネルの範囲を検出するために増加した能力が含まれますとより大きいを組み込むための強化された機能複数の内部品質管理 (QC) に加えて、技術的な複製の数は、1を測定します。マイクロ アレイは従ってより敏感、正確かつ再現性のある、大きなダイナミック レンジを持っています。これらの要因はマイクロ アレイに知られている蛋白質の大規模検出 Elisa に安く、潜在的に有利な代わりを作る。ただし、マイクロ アレイ技術の不利な点は高度精製抗原のパネルを確立し、技術的なプラットフォームの設定に関連付けられている大規模なアップ フロント コストから主に発生します。
蛋白質のマイクロ アレイは、臨床応用の診断および基本的な研究ツールとして過去 20 年間で広く使用されています。特定のアプリケーションは、タンパク質発現プロファイリング、酵素-基質の関係、バイオ マーカーのスクリーニング、宿主微生物間相互作用の解析に関する研究とプロファイリング抗体特異性23,24, 25,26,,2728。マラリア (原虫)29, HIV 130, インフルエンザ31, 重症急性呼吸器症候群 (SARS)32、ウイルス性出血熱33 を含む多くの新しい病原体の抗原/マイクロ アレイが確立されています。、ヘルペス ウイルスの34、および結核35。
簡単営業C. difficile反応性抗原マイクロ アレイ分析、マルチ アイソタイプとC. 株特異抗体の正確な正確なおよび特定の定量化を可能にするのに係る現在のプロトコルdifficileポリクローナル IVIg および血清中の抗原。ここで、elisa 法と同様、測定精度と再現性プロファイルと比較されたとき許容可能なマイクロ アレイ分析性能に係る代表の結果が含まれています。この試金は他の臨床サンプルをプロファイルにさらに開発できるし、CDI の分子基盤研究のための新しい標準を設定します。

<table border="0" cellpadding="0" cellspacing="0" style="width:1827px;" width="1826">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BioRobotics MicroGrid II arrayer</td>
			<td style="width:296px;">Digilab, Malborough, MA, USA</td>
			<td style="width:296px;">N/A</td>
			<td style="width:836px;">Contact arrayer used to automated spotting of the antigens onto the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanner InnoScan 710.</td>
			<td>Innopsys</td>
			<td>N/A</td>
			<td>A fluorescent microarray slide scanner with a red (Cy5) laser to read the reaction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAPIX software version 7.2.0</td>
			<td>Innopsys</td>
			<td>N/A</td>
			<td>Measure signal intensities of the spots.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon contact pin</td>
			<td>Parallel Synthesis Technologies</td>
			<td>SMT-P75</td>
			<td>Print the samples onto the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific Nalgene Desiccator</td>
			<td>Thermo Scientific</td>
			<td>41102426</td>
			<td>To store the new and printed slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384-well plate</td>
			<td>Genetix</td>
			<td>X7022UN</td>
			<td>To prepare the antigens.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate cover</td>
			<td>Sigma Aldrich, UK</td>
			<td>CLS6570-100EA</td>
			<td>To reduce evaporation of the samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminosilane slides</td>
			<td>Schott,&#160; Germany</td>
			<td>1064875</td>
			<td>The slide of choice for printing the antigens.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide holders</td>
			<td>GraceBio Labs, USA</td>
			<td>204862</td>
			<td>Divide the slides into identical 16 subarrays. These holders are re-usable, removable, leak-proof wells .&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Candida albicans lysate</td>
			<td>NIBSC</td>
			<td>PR-BA117-S</td>
			<td>Positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetanus Toxoid&#160;</td>
			<td>Athens Research and Technology</td>
			<td>04/150</td>
			<td>Positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G (IgG), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707</td>
			<td>Purified Native Human Immunoglobulin G IgG, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G1 (IgG1), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-1</td>
			<td>Purified Native Human Immunoglobulin G1 IgG1, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G2 (IgG2), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-2</td>
			<td>Purified Native Human Immunoglobulin G2 IgG2, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G3 (IgG3), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-3</td>
			<td>Purified Native Human Immunoglobulin G3 IgG3, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G4 (IgG4), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-4</td>
			<td>Purified Native Human Immunoglobulin G4 IgG4, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A (IgA), Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701</td>
			<td>Purified Native Human Immunoglobulin A (IgA), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A1 (IgA1), Human Myeloma Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701-1M</td>
			<td>Purified Native Human Immunoglobulin A1 (IgA1), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A2 (IgA2), Human Myeloma Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701-2M</td>
			<td>Purified Native Human Immunoglobulin A2 (IgA2), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin M (IgM), Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090713</td>
			<td>Purified Native Human Immunoglobulin M (IgM), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated Goat Anti-Human IgG Antibody, gamma chain specific</td>
			<td>Vector Labs</td>
			<td>BA-3080</td>
			<td>Goat anti- human IgG (&#947;-chain specific)-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG1 Hinge-BIOT</td>
			<td>Southern Biotec</td>
			<td>9052-08&#160;</td>
			<td>Goat anti- human IgG1 biotin antibody reacts specifically with human IgG1 but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG2 Fc-BIOT</td>
			<td>Southern Biotec</td>
			<td>9060-08&#160;&#160;</td>
			<td>Goat anti- human IgG2 -biotin antibody reacts specifically with human IgG 2but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG3 Hinge-BIOT</td>
			<td>Southern Biotec</td>
			<td>9210-08&#160;&#160;&#160;</td>
			<td>Goat anti- human IgG3-biotin antibody reacts specifically with human IgG3 but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG4 pFc&#39;-BIOT</td>
			<td>Southern Biotec</td>
			<td>9190-08&#160;&#160;</td>
			<td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Human IgA, alpha chain specific, made in goat - Biotinylated</td>
			<td>Vector Labs</td>
			<td>BA-3030</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgA1-BIOT</td>
			<td>Southern Biotec</td>
			<td>9130-08</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgA2-BIOT</td>
			<td>Southern Biotec</td>
			<td>9140-08&#160;&#160;</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgM-BIOT</td>
			<td>Southern Biotec</td>
			<td>9020-08&#160;</td>
			<td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 mm syringe filter</td>
			<td>Thermo scientific</td>
			<td>723-2520</td>
			<td>Filter the 5% BSA.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich, UK</td>
			<td>A7284</td>
			<td>Use 5% BSA for blocking the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody diluent</td>
			<td>Dako, UK</td>
			<td>S3022</td>
			<td>To dilute the serum and the secondary antibody.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Cy5</td>
			<td>eBioscience</td>
			<td>SA1011</td>
			<td>Detection of the immune reaction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087)</td>
			<td>Toxins Group, Public Health England</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified whole C. difficile toxin B (CCUG 20309 toxin B only expressing strain)</td>
			<td>Toxins Group, Public Health England</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precursor form of B fragment of binary toxin, pCDTb</td>
			<td>University of Bath&#160;</td>
			<td>NA</td>
			<td>Produced in E. Coli from wholly synthetic recombinant gene construct. Amino acid sequence based on published sequence from 027 ribotype (http:www.uniprot.org/uniprot/A8DS70)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified native whole ribotype-specific (001, 002, 027) surface layer proteins</td>
			<td>Dublin City University&#160;</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vigam (IVIg preparation 1)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Privigen (IVIg preparation 2)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Intratect (IVIg preparation 3)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protein microarray assay for serological determination of antigen-specific antibody responses following clostridium difficile infection

クロストリジウム ・ ディフィシル抗体ポリクローナルの静脈内の免疫グロブリン (IVIg) の別の準備、ひと血清中で反による高スループット蛋白質マイクロ アレイ分析の詳細な概要をいたします。プロトコルでは、サンプル作製、印刷配列、試金プロシージャ、およびデータ分析の方法論的手順について説明します。さらに、このプロトコルは、プラズマを含む多様な臨床サンプルを取り込んで細胞培養上清にさらに開発できます。蛋白質のマイクロ アレイを使用して、アイソタイプ (IgG、IgA、IgM), サブクラス (IgG1、IgG2, IgG3, IgG4、IgA1、iga2 は) の組み合わせを決定する方法を示す、株特異抗体高純度全体c. ディフィシル毒素 A と B (toxinotype 0、ひずみ VPI10463、ribotype 087) 毒素 B、 c. ディフィシル毒素 B だけ表現するひずみ (CCUG 20309) から、バイナリ毒素、pCDTb、ribotype 全体の表層抗原 B フラグメントの前駆体フォーム (Slp; 001、002、027)、制御タンパク質 (破傷風、トキソイドとカンジダ)。血清c. ディフィシル感染症 (CDI)、健常者 (HC)、下痢症嚢胞性線維症 (CF) を持つ個人に個人個人の事前のポスト IVIg 療法からもプローブ、マイクロ アレイ実験中に、CDI、複合型免疫不全疾患、慢性炎症性脱髄性 polyradiculopathy の治療。我々 は、IVIg と前に血清の商業準備の患者グループと次の IVIg 投与間毒素の中和効果とマルチ アイソタイプ特異抗体レベル差が発生します。また、あるマイクロ アレイと酵素免疫測定法 (ELISA) 相関抗毒素 igg の血清中。そのマイクロ アレイがC.difficileワクチン接種や感染したヒトの抗原抗体反応をプロファイリングするための有望なツールになる可能性が示唆されました。さらに洗練された抗原パネルの生産コストの削減、最適化し、患者固有の方法でc. ディフィシル感染症の最も臨床的に有用な免疫療法を選択マイクロ アレイ技術が役立つことを期待しています。

記述されていたプロトコルの主な手順を示すフローチャートを図 1に示します。図 2は、スピアマンの相関関係テスト テスト患者血清中の抗毒素 A と B レベル IgG および IgA のマイクロ アレイと ELISA との重要な契約を示しています。図 3は、下痢なし CF 患者、下痢、CDI 患者と HC 毒素は、毒素 B、およ?...

Watch this Scientific Journal Video about A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection at JoVE.com

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

この記事では、ひと血清中におけるクロストリジウム ・ ディフィシル抗原を精製の 7 プレックス パネルに対する体液性免疫応答の高プロファイルの単純なタンパク質マイクロ アレイ法について説明します。特定抗体ポリクローナル免疫グロブリン製剤の定量プロトコルを拡張することができます。

蛋白質のマイクロ アレイの試金抗原特異的抗体の応答次のクロストリジウム ・ ディフィシル感染症の血清学的定量

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

This method may help with optimizing and selecting clinically useful immunotherapies for Clostridium difficile infection in a patient-specific manner, and elucidating human immune responses to Clostridium difficile. The main advantage of this technique is that it enables accurate and simultaneous quantification of multi isotype and strain-specific antibody responses to C.difficile antigens. Generally, individuals that are new to this method will struggle because expertise is required in establishing and optimizing a panel of highly purified antigens, and setting up the technological platform.Visual demonstration of this method is critical, as methodological list types involved in preparing the microwave plate, arrays printing, a safe procedure, and the data analysis require attention to the details. Demonstrating some of the procedure will be by Sam Greenwood, a technician from my laboratory. To begin, dilute Clostridium difficile antigens and printing buffer to optimum concentration.Next, transfer 10 microliters of antigen solution to a 384 well plate. Cover the plate with a plate seal, and centrifuge at 300 times gravity for five minutes at room temperature. For microarray printing, use a hand-held gas burner to heat the silicon PIN three times for two seconds each.Then, rinse the silicon PIN in clean water three times for three seconds each. Place the microarray plate in the loading cartridge of an arrayer. Eject the tray, and then place the amino silane slides in the slide tray and press the OK button to turn the vacuum on.Insure that all of the slides are secured, and then click OK.Return the tray to the origin, and close the lid. Next, launch the appropriate program and go to File, then Microarraying Parameters, then Open. Select Clostridium difficile file for printing all antigens in quadruplicate.Under this window, open the Source tab to indicate sample storage, and Target tab to input microarray details. Then, open the Options tab to select the washing tool. Under the General tab of Run Preferences, on the TAS applications suite toolbar, select Wash at start of run, and Wash at end of run.Open the Climate tab, and set the humidity level between 55 to 60%Now start the run, and periodically check the arrayer to insure proper functioning. After printing, carefully place the printed slides in a slide holder with 16 multi-well chambers. Then add 100 microliters of 5%BSAT to each block.Cover the slides and incubate for one hour at room temperature with shaking. Use 120 microliters of PBST to wash each block five times for one minute each with shaking. Next, thaw out the serum samples obtained from C difficile infected patients and healthy controls on ice for 20 minutes.Add commercially available antibody diluent to the master block, and then add samples to the master block and mix. Discard the washing buffer and add 100 microliters of each diluted sample to the experimental blocks. For the negative control block, add 100 microliters of only the antibody diluent.Then, incubate the slides for one hour at room temperature with gentle shaking. Next, use 120 microliters of PBST to wash each block five times for three minutes each with shaking. Then add 100 microliters of biotinylated goat anti-Human antibody diluted in antibody diluent to each block.Cover the slides and incubate for one hour at room temperature with gentle shaking. Use 120 microliters of PBST to wash each block five times for three minutes each with shaking. Next, add 100 microliters of streptavidin SY5 conjugate diluted in 5%BSAT, in a ratio of one to two thousand to each block.Cover the slides with foil and incubate for 15 minutes at room temperature with gentle shaking. Wash the slides first with 120 microliters of PBST five times for one minute each, and then wash them two more times for one minute each with PBS. To dry the slides, spin them for five minutes at 300 times gravity.Turn the laser scanner on 30 minutes prior to scanning, to allow it to warm up. Then place the slide in the scanner with the printed surface facing up until the plate light turns solid green. Next, press the Settings button.On the first tab, insure that the Automatic scrolling and Barcode are unchecked. Then, set the Pixel size to 10, Scan Mode to Medium, Acquisition to 635 only, Acquisition Mode to Manual, gain to 20, and power to low. On the second tab, insure that the slide type is unlabeled.On the third tab, set the Focus setting to Auto focus. Press the import grid button to select the appropriate array list associated with the scanned image. Then, press the autofind button to align the grid to the spots on the slide.Finally, press the Quantification Process button to measure the fluorescence intensity of each spot. Then, save the results in an appropriate format for further analysis. In this protocol, a protein microarray is compared with ELISA to assess the reproducibility between these two techniques.The Spearman correlation coefficient analysis shows significant agreement between the microarray and ELISA for detecting IgG Anti-toxin A, and Anti-toxin B levels in serum samples. Intro assay and inter assay reproducibility of the protein micro array are calculated using the serum samples of seven patients. The samples analyzed on two different array slides against Toxin A, Toxin B, SLP001, SLP002, SLP027, Toxin B from Toxin B only producing strain CCUG, and pCDTb antigens reveal good reproducibility.A protein microarray is performed to detect an isotype-specific antibody response against Clostridium difficile toxins. The serum samples of patients and healthy individuals are used to detect the levels of IgG and IgA against Toxin A, Toxin B, Toxin B from a Clostridium difficile Toxin B only expressing strain, and precursor form of a B fragment of binary toxin. Once mastered, this technique can be done in seven hours.The quality control experiment, different parameters such as selection of appropriate surface chemistry, printing buffers, blocking buffers, and dilutions of serum samples and secondary antibodies should be evaluated before running samples. Printing a high-quality array requires a good understanding of the array or software to adjust the printing of parameters according to the experimental protocol. It is crucial to maintain optimum performance of the arrayer, and a dust-free environment throughout the experiment.This technique will pave the way for researchers in the field of immunology and molecular biology, in terms of characterizing humoral immune responses to infection, and discovering serodiagnostic antigens. Don't forget that working with infected blood samples and bacterial toxins can be hazardous. In general, in laboratory, safety and standard precautions should always be taken into consideration while performing this procedure.

Research

JoVE Journal

Immunology and Infection

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile 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

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

ウイルス性病原体のために臨床サンプルをスクリーニングするためにパンウイルスマイクロアレイアッセイ（Virochip）を使用して、

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

抗体マイクロアレイと蛍光多重を用いた大腸癌の細胞表面タンパク質のプロファイリング

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

抗原特異的規制用のトランス生体内遅延型過敏アッセイ

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

培養およびメンテナンス<em&gt;クロストリジウム·ディフィシル</em&gt;嫌気性環境での

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

ナノ粒子ワクチン接種後の抗原特異的CD8 + T細胞応答の分析のための全動物イメージングとフローサイトメトリー技術

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

臨床的に関連のセフォペラゾン処理されたマウスモデル<em&gt;クロストリジウム・ディフィシル</em&gt;ひずみR20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

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 ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

形態変化を特徴付けるプロトコル<em&gt;クロストリジウム・ディフィシル</em&gt;抗生物質治療に対する反応

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

蛋白質のマイクロ アレイの試金抗原特異的抗体の応答次のクロストリジウム ・ ディフィシル感染症の血清学的定量

蛋白質のマイクロアレイの試金抗原特異的抗体の応答次のクロストリジウム・ディフィシル感染症の血清学的定量