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

Diese Forschung wurde durch eine Hermes-Fellowship Ola Negm und Tanya Monaghan und durch gesonderte Finanzierung von Nottingham verdauungsfördernden Krankheiten Zentrum und die NIHR Nottingham verdauungsfördernden Krankheiten Biomedical Research Centre unterstützt.

1. Vorbereitung einer Microarray-Platte
<ol><li>C. Difficile Antigene mit einem Drucken Puffer [PBS-Tween-Trehalose (50 mM)] bei der optimalen Konzentration (das vordefiniert wurde vor dem Ausführen der Patientenseren) verdünnen: Toxin ein (200 µg/mL) und Toxin B (100 µg/mL), pCDTb (200 µg/mL) und gereinigte Native ganze SLPs abgeleitet ribotypen 001 und 002 027 (200 µg/mL). Hinweis: Toxin B war ein Toxin B exprimierenden Stamm CCUG 20309 (90 µg/mL) entnommen.<ol>
<li>Die Positivkontrollen Lysates vo...

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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. 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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=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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In diesem Protokoll haben wir gezeigt, dass diese Microarray eine geeignete Plattform für die Humorale Immunantwort auf C. Difficile -Protein-Antigene in Patientenseren (Abbildungen 3 und 6) und kommerzielle Vorbereitung der IVIg (Abbildung 5) Definition ist. Wir haben auch gezeigt, dass die Microarray-Technik führt auch im Vergleich zu konventionellen ELISA (Abbildung 2) und zeigt hervorragende Reproduzierbarkeit mit...

Dieses Protokoll beschreibt die Entwicklung und Validierung eines neuartigen und kundenspezifische Protein Microarray Assays für die Erkennung und semi-Quantifizierung der Bakterienstamm und Isotype-spezifischen Antikörper-Reaktionen auf C. Difficile -Antigene. Wir nutzen erfolgreich unsere C. Difficile-spezifische Microarray-Assay als viel versprechende neue Werkzeug für die kompositorische Bioanalytik der spezifischen Antikörper Inhalte in Patientenseren1,2, Vorbereitungen von IVIg3, und Kennung der Antikörper-Besonderheiten, die mit schlechten Ergebnissen im CDI4korrelieren. Wir zeigen wie Biobanked Serumproben und kommerzielle Vorbereitung der IVIg auf Microarray Folien, analysiert werden können, ermöglicht qualitativ hochwertige reproduzierbare profiling von C. Difficile Erreger-spezifischen Antikörper-Reaktionen in diesem Assay.
Viele gesunde Kinder und Erwachsene haben nachweisbar Serum IgG und IgA Antikörper gegen C. Difficile Toxine A und B5,6. Diese werden gedacht, um nach vorübergehender Exposition in der Kindheit und nach Exposition gegenüber C. Difficile im Erwachsenenalter auftreten. Aus diesem Grund wurde polyklonale IVIg off-Label-zur Behandlung von wiederkehrenden und fulminanten CDI7,8,9verwendet. Seine endgültige Rolle und Wirkungsweise bleibt jedoch unklar. Mehrere Studien haben gezeigt, dass die Humorale Immunantwort auf C. Difficile Toxine in Krankheit Präsentation und das Ergebnis eine Rolle spielt. Insbesondere zeigen asymptomatische Patienten eine erhöhte Serum Anti-Toxin A IgG Konzentration im Vergleich zu Patienten, die Symptomen der Krankheit10zu entwickeln. Eine nachweisbare Verbindung wurde für mittlere Anti-Toxin-A IgG-Titer und 30-Tage Mortalität aller Ursachen11berichtet. Mehrere Berichte haben auch gezeigt, dass eine Vereinigung mit einem Schutz gegen Rezidive und Antikörper Reaktionen auf Toxin A, B und mehrere nicht-Toxin-Antigene (Cwp66, Cwp84, FliC, FliD und Oberflächenschicht Proteine (SLPs))12,13 , 14 , 15. diese Beobachtungen haben die Entwicklung der ersten passiven Immuntherapie Drug targeting C. Difficile Toxin B (Bezlotuxumab), die vor kurzem von der US Food and Drug Administration und die Europäische Arzneimittel-Agentur genehmigt wurde angespornt Agentur für die Prävention von wiederkehrenden CDI16. Impfstrategien mit inaktivierten Toxinen oder rekombinantes Toxin Fragmente sind auch derzeit in der Entwicklung17,18,19. Diese neue Therapieansätze werden zweifellos die Voraussetzung für die Bewertung der humoralen Immunantwort auf mehrere Antigene in große Stichprobenumfänge stimulieren.
Heute gibt es ein bemerkenswerten Mangel an handelsüblichen Hochdurchsatz-Assays in der Lage, gleichzeitig Bakterienstamm und Isotype-spezifischen Antikörper-Reaktionen auf C. Difficile -Antigene zu beurteilen. Es besteht ein ungedeckter Bedarf, solche Tests Erleichterung künftiger Forschung und klinische Anwendungen zu entwickeln. Protein Microarrays sind eine Methode, um große Anzahl von einzeln-gereinigte Proteine als räumlich organisierten Array von Flecken auf einer mikroskopischen folienbasierten Oberfläche zu immobilisieren, mit einem Robot-System, die entweder ein Kontakt20 oder ein berührungsloses sein kann Drucken Tool21. Die Spots können komplexe Mischungen wie Zelle Lysates, Antikörper, Gewebe Homogenates, endogenen oder rekombinante Proteine oder Peptide, Körperflüssigkeiten oder Drogen22,23darstellen.
Protein-Microarray-Technologie bietet deutliche Vorteile gegenüber standard im Haus ELISA-Techniken, die traditionell verwendet wurden, um Anti -C. Difficile Antikörperantworten zu beurteilen. Dazu gehören eine erhöhte Kapazität für eine Reihe von Multi-Isotype-spezifische Antikörper gegen eine umfangreichere Gruppe von Protein-Targets, geringerer Anforderungen für Antigene, Proben und Reagenzien, Erkennung und eine verbesserte Fähigkeit, eine größere integrieren Anzahl der technischen Replikationen, zusätzlich mehrere interne Qualitätskontrolle (QC) misst1. Microarrays sind daher sensibel, präzise und reproduzierbare und haben einen größeren Dynamikbereich. Diese Faktoren machen Microarrays eine billigere und potenziell günstige Alternative zu ELISAs für die groß angelegte Erkennung von bekannten Proteinen. Jedoch Nachteile der Microarray-Technologie resultieren im Wesentlichen aus der große Vorabkosten zugeordnet zur Gründung einer Gruppe von hochgereinigten Antigenen und die technologische Plattform einrichten.
Protein Microarrays haben in den letzten zwei Jahrzehnten als Diagnose- und grundlegende Recherche-Tool in der klinischen Anwendung intensiv genutzt. Spezielle Anwendungen sind Protein-Expression profiling, das Studium der Enzym-Substrat-Beziehungen, Biomarker Screening, Analyse von Host-Mikroben-Interaktionen und profiling Antikörper Spezifität23,24, 25,26,27,28. Viele neue Erreger Protein/Antigen Microarrays entstanden unter anderem Malaria (Plasmodium)29, HIV-130, Grippe31, schwere akute respiratorische Syndrom (SARS)32, virale hämorrhagische Fieber33, , Herpesviren34und Tuberkulose35.
Dieses Protokoll bezieht sich auf die Einrichtung eine einfach operative C. Difficile reaktive Antigen Microarray Assay, ermöglicht präzise, genaue und spezifische Quantifizierung der Multi-Isotype und Stamm-spezifischen Antikörper-Reaktionen auf C. Difficile Antigene in Sera und polyklonalen IVIg. Hier zählen wir repräsentative Ergebnisse in Bezug auf eine akzeptable Microarray Testleistung im Vergleich zu ELISA sowie Assay-Präzision und Reproduzierbarkeit Profile. Dieser Test könnte weiter entwickelt werden, um andere klinikmustern Profil und setzt einen neuen Standard für die Forschung in der molekularen Grundlagen des CDI.

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			<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

Wir bieten eine detaillierte Übersicht über ein neuartiges Hochdurchsatz-Protein-Microarray-Assay für die Bestimmung der Anti -Clostridium Difficile -Antikörpern im menschlichen Serum und in separaten Vorbereitungen von polyklonalen intravenöses Immunglobulin (IVIg). Das Protokoll beschreibt die methodischen Schritte zur Probenvorbereitung, Druck von Arrays, Testverfahren und Datenanalyse. Darüber hinaus könnte dieses Protokolls weiter ausgebaut werden, um vielfältigen klinischen Proben einschließlich Plasma und Zell-Kultur Überstände. Wir zeigen, wie Protein Microarray verwendet werden kann, um festzustellen, eine Kombination aus Isotype (IgG, IgA, IgM), Unterklasse (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) und Stamm-spezifische Antikörper gegen hoch gereinigt ganze C. Difficile Toxin A und B (Toxinotype 0, Stamm VPI 10463, ribotyp 087), Toxin B aus einem C. Difficile Toxin-B-nur mit dem Ausdruck ihrer Stamm (CCUG 20309), eine Vorstufe-Form eines B-Fragments des binären Toxins, pCDTb, ribotyp-spezifische ganze Oberflächenschicht Proteine (SLPs, 001, 002, 027), und Steuern Proteine (Tetanus Toxoid und Candida Albicans). Während des Experiments sind Microarrays sondiert mit Sera von Einzelpersonen mit C. Difficile -Infektion (CDI), Einzelpersonen mit Cystischer Fibrose (CF) ohne Durchfall, gesunden Kontrollpersonen (HC), und von Einzelpersonen Pre- und Post-IVIg Therapie für die Behandlung von CDI, kombinierte Immundefizienz Unordnung und chronisch entzündliche demyelinisierende polyradikulopathie. Wir begegnen erhebliche Unterschiede in Toxin Neutralisation Wirksamkeiten und Multi-Isotype spezifischen Antikörpertiter Patientengruppen, kommerzielle Vorbereitung der IVIg, und Sera vor und nach IVIg Verabreichung. Außerdem gibt es eine signifikante Korrelation zwischen Microarray und Enzym-linked Immunosorbentprobe Assay (ELISA) für Antitoxin-IgG-Werte in Serumproben. Diese Ergebnisse deuten darauf hin, dass diese Microarray ein viel versprechendes Instrument für die Profilerstellung Antikörperantworten auf C.difficile Antigene in geimpften oder infizierten Menschen werden könnte. Mit der Verfeinerung der Antigen-Panels und eine Reduzierung der Produktionskosten rechnen wir Microarray-Technologie optimieren und wählen Sie die klinisch nützlichen Immuntherapien für C. Difficile Infektion in einer Patienten-spezifischen Weise helfen kann.

Abbildung 1 zeigt ein Flussdiagramm, beschreibt die wichtigsten Schritte in der beschriebenen Protokoll. Abbildung 2 zeigt Spearman Korrelationstests zeigen bedeutende Abkommen zwischen Microarray und ELISA für IgG und IgA Anti-Toxin A und B im Patiententest Sera Ebenen. Abbildung 3 zeigt differenzierte IgG und IgA Antikörper-Klasse spezifische antikörperreaktionen auf Toxin A und Toxin B binäres...

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

Dieser Artikel beschreibt eine einfache Protein Microarray Methode für Clostridium Difficile Antigene im menschlichen Serum Profilerstellung humorale Immunantworten auf einer 7-Plex Jury hoch gereinigt werden. Das Protokoll kann zur Bestimmung der spezifischen Antikörperantworten in die Vorbereitungen der polyklonalen intravenöses Immunglobulin erweitert werden.

Ein Protein-Microarray-Assay für die serologische Bestimmung der Antigen-spezifische Antikörper Antworten folgende Clostridium Difficile Infektion

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

Antigen-spezifische In Vivo Killing-Assay mit CFSE markierten Zielzellen

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

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Immunologie und Infektion

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

Mit einem Pan-Viral Microarray-Assay (Virochip) zur klinischen Proben, die für virale Erreger Bildschirm

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

Darmkrebs Zelloberflächenprotein Profiling mit einem Antikörper Microarray und Fluoreszenz Multiplexing

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

Trans-vivo Überempfindlichkeit vom verzögerten Typ Assay für Antigen-spezifische Verordnung

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

Kultivierung und Pflege<em&gt; Clostridium difficile</em&gt; In einer anaeroben Umgebung

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

Whole-Tier Imaging und Fließzytometrieverfahren zur Analyse von Antigen-spezifischen CD8 + T-Zellantworten nach der Nanopartikel-Impfung

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

Cefoperazon behandelten Maus-Modell der klinisch relevanten<em&gt; Clostridium difficile</em&gt; Dehnungs 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

Detektion und Anreicherung von Rare Antigen-spezifische B-Zellen für die Analyse von Phänotypen und Funktion

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

Ein Protokoll zur Charakterisierung der morphologischen Veränderungen von<em&gt; Clostridium difficile</em&gt; In Antwort auf Antibiotika-Behandlung

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

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In Vivo Test zum Nachweis von Antigen-spezifischen T-Zell Zytolyse-Funktion mit einer Impfung-Modell

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