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

Denne forskning blev støttet af en Hermes Fellowship Ola Negm og Tanya Monaghan og gennem særskilte midler fra Nottingham fordøjelsessystemet sygdomme centrum og NIHR Nottingham fordøjelsessystemet sygdomme biomedicinsk forskning centrum.

1. udarbejde en Microarray plade
<ol><li>Fortynd C. difficile antigener med en udskrivning buffer [PBS-Tween-trehalose (50 mM)] i den optimale koncentration, (som var foruddefinerede før du kører de patient sera): toksin en (200 µg/mL) og toksin B (100 µg/mL), pCDTb (200 µg/mL), og renset indfødte hele SLPs stammer fra ribotypes 001, 002 og 027 (200 µg/mL). Bemærk: Toksin B blev indhentet fra et toksin B-udtrykker stamme CCUG 20309 (90 µg/mL).<ol>
<li>Fortynd de positive kontroller, lysates af ...

<ol>
	<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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I denne protokol, har vi vist, at microarray er en velegnet platform til at definere humorale immunrespons til C. difficile protein antigener i patient sera (tallene 3 og 6) og kommercielle præparater af IVIg (figur 5). Vi har også bevist at microarray teknik udfører godt i forhold til konventionelle ELISA (figur 2) og viser fremragende reproducerbarhed, med intra og Inter-Diesel assay variabilitet falder inden for a...

Denne protokol beskriver udvikling og validering af en roman og tilpassede protein microarray analyse til påvisning og semi-kvantificering af bakteriel stamme og isotypespecifikke antistof svar til C. difficile antigener. Vi kunne bruge vores C. difficile-specifikke microarray analyse som en lovende nye værktøj til den kompositoriske bioanalyse af specifikt antistof indhold i patientens sera1,2, præparater af IVIg3, og identifikation af antistof særtræk, der korrelerer med dårlige resultater i CDI4. Vi demonstrere, hvordan biobanked serumprøver og kommercielle præparater af IVIg kan analyseres på microarray dias, så høj kvalitet reproducerbare profilering af C. difficile patogen-specifikke antistof svar i denne analyse.
Mange raske børn og voksne har påviselige serum IgG og IgA antistoffer til C. difficile toksiner A og B5,6. Disse menes at opstå efter forbigående eksponering under vorden og efter udsættelse for C. difficile i voksenalderen. Derfor polyklonale IVIg har været brugt off-label til behandling af tilbagevendende og fulminant CDI7,8,9. Men dens endelige rolle og virkningsmekanisme er fortsat uklart. Flere undersøgelser har vist, at den LUN immun svar til C. difficile toksiner spiller en rolle i sygdom præsentation og resultatet. Specifikt, viser asymptomatiske patienter en øget serum anti-toksin A IgG koncentration sammenlignet med patienter, der udvikler symptomatisk sygdommen10. En påviselig tilknytning er blevet rapporteret til median anti-toksin A IgG titers og 30-dages all-dødelighed11. Flere rapporter har også afsløret en forening med en beskyttelse mod gentagelse og antistof svar til toksin A, B og flere ikke-toksin antigener (Cwp66, Cwp84, FliC, FliD og overflade lag proteiner (SLPs))12,13 , 14 , 15. disse observationer har ansporet udvikling af den første passiv immunterapi narkotika målretning C. difficile toksin B (bezlotuxumab), som er for nylig blevet godkendt af US Food and Drug Administration og det Europæiske Lægemiddelagentur Agentur for forebyggelse af tilbagevendende CDI16. Vaccinationsstrategier ved hjælp af inaktiverede toksiner eller rekombinant toksin fragmenter er også i øjeblikket under udvikling17,18,19. Disse nye terapeutiske tilgange vil utvivlsomt fremme kravet om evaluering LUN immun svar til flere antigener i store stikprøvestørrelser.
I dag, er der en bemærkelsesværdig mangel på kommercielt tilgængelige høj overførselshastighed assays i stand til samtidigt vurdere bakteriel stamme og isotypespecifikke antistof svar til C. difficile antigener. Der er et udækket behov for at udvikle sådanne assays for at lette fremtidig forskning og kliniske applikationer. Protein microarrays er en metode til at immobilisere stort antal individuelt renset proteiner som et rumligt organiseret vifte af steder på en mikroskopisk slide-baserede overflade ved hjælp af en robot system, som kan være enten en kontakt20 eller en ikke-kontakt udskrivning værktøj21. Steder kan repræsentere komplekse blandinger som cellelysater, antistoffer, væv homogeniseret, endogene eller rekombinante proteiner eller peptider, kropsvæsker eller narkotika22,23.
Protein microarray teknologi tilbyder forskellige fordele i forhold til standard in-house ELISA teknikker, som traditionelt er blevet brugt til at vurdere anti -C. difficile antistof reaktioner. Disse omfatter en øget kapacitet til at afsløre et udvalg af multi-isotypespecifikke antistoffer mod en mere omfattende panel af protein mål, reduceret volumen krav for antigener, prøver og reagenser, og en forbedret evne til at indarbejde en større antallet af tekniske replikater, foruden flere interne kvalitetskontrol (QC) foranstaltninger1. Microarrays er derfor mere følsomme, nøjagtige og reproducerbare og har et større dynamikområde. Disse faktorer gør microarrays en billigere og potentielt gunstige alternativ til ringprøver for storstilet påvisning af kendte proteiner. Imidlertid ulemper af microarray teknologi skyldes hovedsageligt store up-front udgifter i forbindelse med oprettelse af et panel af højtrenset antigener og oprette den teknologiske platform.
Protein microarrays har været flittigt brugt i de seneste to årtier som et diagnostisk og grundlæggende forskningsværktøj i kliniske applikationer. Specifikke anvendelser omfatter protein udtryk profilering, undersøgelse af enzym-substrat relationer, biomarkør screening, analyse af host-mikrobe interaktioner, og profilering antistof specificitet23,24, 25,26,27,28. Mange nye patogen protein/antigen microarrays er fastlagt, herunder malaria (Plasmodium)29, HIV-130, influenza31, alvorlig akut luftvejssyndrom (SARS)32, viral hæmoragisk feber33 , herpesvirus34, og tuberkulose35.
Denne protokol vedrører oprettelsen af en let drift C. difficile reaktive antigen microarray analyse, som muliggør præcis, præcis og specifik kvantificering af multi-isotypen og stamme-specifikke antistof svar til C. difficile antigener i sera og polyklonale IVIg. Heri, medtager vi repræsentative resultater vedrørende en acceptabel microarray analyse ydeevne i forhold til ELISA assay præcision og reproducerbarhed profiler. Denne analyse kunne udvikles yderligere for at profilere andre kliniske prøver og sætter en ny standard for forskning i den molekylære grundlag af 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>
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			<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

Vi giver en detaljeret oversigt over en roman høj overførselshastighed protein microarray analyse til bestemmelse af anti -Clostridium difficile antistof niveauer i menneskers sera og separat præparater af polyklonale intravenøs immunglobulin (IVIg). Protokollen beskriver de metodiske trin involveret i prøveforberedelse, udskrivning af arrays, analyseprocedure og dataanalyse. Derudover kunne denne protokol udvikles yderligere for at integrere forskellige kliniske prøver herunder plasma og celle kultur analysere. Vi viser, hvordan protein microarray kan bruges til at afgøre en kombination af isotype (IgG, IgA, IgM), underklasse (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), og stamme-specifikke antistoffer mod meget renset hele C. difficile toksiner A og B (toxinotype 0, stamme VPI 10463, ribotype 087), toksin B fra en C. difficile toksin B kun udtrykke stamme (CCUG 20309), en forløber form af et B fragment af binære toksin, pCDTb, ribotype-specifikke hele overflade lag proteiner (SLPs, 001, 002, 027), og kontrollere proteiner (stivkrampe toxoid og Candida albicans). Under eksperimentet, microarrays er aftestede med sera fra personer med C. difficile infektion (CDI), personer med cystisk fibrose (CF) uden diarré, raske kontrolpersoner (HC), og fra enkeltpersoner pre og post-IVIg behandling for den behandling af CDI, kombineret immundefekt sygdom og kronisk inflammatorisk demyeliniserende polyradiculopathy. Vi møder betydelige forskelle i toksin neutralisering efficacies og multi-isotypen specifikt antistof niveauer mellem patientgrupper, kommercielle præparater af IVIg, og sera før og følgende IVIg administration. Også, der er en markant sammenhæng mellem microarray og enzym-forbundet immunosorbent assay (ELISA) for antitoksin IgG niveauer i serumprøver. Disse resultater tyder på, at microarray kunne blive et lovende redskab for profilering antistof svar til C.difficile antigener på vaccineret eller inficerede mennesker. Med yderligere finjustering af antigen paneler og en reduktion af produktionsomkostningerne forventer vi, at microarray teknologi kan hjælpe med at optimere og vælge de mest klinisk nyttige immunoterapi til C. difficile infektion på en patient-specifikke måde.

Figur 1 illustrerer et rutediagram, der beskriver de vigtigste trin i den beskrevne protokol. Figur 2 viser Spearman korrelation tests viser betydelig aftale mellem microarray og ELISA for IgG og IgA anti-toksin A og B niveauer i patient testsera. Figur 3 viser differential IgG og IgA antistof-klasse specifikt antistof svar til toksin A, toksin B og binære toksin (pCDTb) i patienter med CF uden diar...

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

I denne artikel beskrives en simpel protein microarray metode til profilering LUN immun svar til en 7-plex panel af meget renset Clostridium difficile antigener i menneskelige sera. Protokollen kan udvides til bestemmelse af specifikt antistof reaktioner i forberedelserne af polyklonale intravenøs immunglobulin.

Et Protein Microarray analyse for serologisk bestemmelse af Antigen-specifikke antistof reaktioner følgende 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

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Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Dyrkning og vedligeholdelse<em&gt; Clostridium difficile</em&gt; I et anaerobt miljø

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

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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 behandlet musemodel af klinisk relevant<em&gt; Clostridium difficile</em&gt; Strain 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 og berigelse af Rare Antigen-specifikke B-Celler til analyse af fænotype og funktion

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

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