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

Denna forskning stöddes av en Hermes-stipendium till Ola Negm och Tanya Monaghan och genom separat finansiering från Nottingham mag sjukdomar centrum och de NIHR Nottingham mag sjukdomar Biomedical Research Centre.

1. förbereda en Microarray tallrik
<ol><li>Späd C. difficile antigener med en utskrift buffert [PBS-Tween-trehalos (50 mM)] på den optimala koncentrationen (som var fördefinierade innan du kör patientsera): toxin en (200 µg/mL) och toxin B (100 µg/mL), pCDTb (200 µg/mL), och renad native hela SLPs härrör från ribotypes 001, 002 och 027 (200 µg/mL). Obs: Toxin B erhölls från ett toxin B-uttryckande stam CCUG 20309 (90 µg/mL).<ol>
<li>Späd de positiva kontrollerna, lysates av C. albicans</...

<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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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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S., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+protein+microarrays.">Overview of protein microarrays.</a> Current Protocols in Protein Science. , 21 (2013).</li><li>Huang, Y., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+array-based+approaches+for+biomarker+discovery+in+cancer.">Protein array-based approaches for biomarker discovery in cancer.</a> Genomics, Proteomics &amp; Bioinformatics. 15 (2), 73-81 (2017).</li><li>Vigil, A., Davies, D. H., Felgner, P. 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Z., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+in+high-content+functional+protein+microarrays.">Applications in high-content functional protein microarrays.</a> Current Opinion in Chemical Biology. 30, 21-27 (2016).</li><li>Crompton, P. 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=A+prospective+analysis+of+the+Ab+response+to+Plasmodium+falciparum+before+and+after+a+malaria+season+by+protein+microarray.">A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (15), 6958-6963 (2010).</li><li>Khodakov, D. 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I detta protokoll, har vi visat att microarray är en lämplig plattform för att definiera humorala immunsvar mot C. difficile proteinantigener i patientsera (figur 3 och 6) och kommersiella preparat av IVIg (figur 5). Vi har också visat att microarray tekniken presterar väl jämfört med konventionella ELISA (figur 2) och visar utmärkt reproducerbarhet, med intra - och inter - analysen variabilitet som faller inom ...

Det här protokollet beskriver utveckling och validering av en roman och anpassade protein microarray analys för detektion och semi kvantifiering av bakteriell stam och isotyp-specifika antikroppssvaret mot C. difficile antigener. Vi använder framgångsrikt våra C. difficile-specifika microarray analys som ett lovande nytt verktyg för den kompositionella bioanalys av specifika antikroppen innehåll i patientsera1,2, beredningar av IVIg3, och identifiering av antikropp särdrag som korrelerar med dålig utfall i CDI4. Vi visar hur biobanked serumprover och kommersiella preparat av IVIg kan analyseras på microarray diabilder, möjliggör hög kvalitet reproducerbara profilering av C. difficile patogen-specifika antikroppen svaren i denna analys.
Många friska barn och vuxna har detekterbara serum IgG och IgA antikroppar mot C. difficile toxiner A och B5,6. Dessa tros uppstå efter övergående exponering under spädbarnstiden och efter exponering för C. difficile i vuxen ålder. Av denna anledning polyklonala IVIg har varit används off-label för att behandla både återkommande och fulminant CDI7,8,9. Dock oklar sin slutgiltiga roll och verkningsmekanismen. Flera studier har visat att det humorala immunsvaret för C. difficile toxiner spelar en roll i sjukdomen presentation och resultatet. Specifikt, uppvisar asymtomatiska patienter en ökad anti toxin A IgG koncentration i serum jämfört med patienter som utvecklar symtomatisk sjukdom10. En påvisbar association har rapporterats för median anti toxin A IgG titrar och 30-dagars totalmortalitet11. Flera rapporter har också visat en association med ett skydd mot återfall och antikroppen Svaren till toxin A, B och flera icke-toxin antigener (Cwp66, Cwp84, FliC, FliD och ytskiktet proteiner (SLPs))12,13 , 14 , 15. dessa observationer har sporrat utvecklingen av den första passiva immunterapi drog inriktning C. difficile toxinen B (bezlotuxumab), som nyligen har godkänts av den amerikanska Food and Drug Administration och Europeiska läkemedelsmyndighetens Byrån för förebyggande av återkommande CDI16. Vaccination strategier med inaktiverade toxiner eller rekombinant toxin fragment är också för närvarande under utveckling17,18,19. Dessa nya terapeutiska strategier kommer utan tvekan att stimulera krav på utvärdering humorala immunsvar mot flera antigener i stort urvalsstorlekar.
Idag finns det en anmärkningsvärd brist på kommersiellt tillgängliga hög genomströmning analyser kan samtidigt bedöma bakteriestam och isotyp-specifika antikroppssvaret mot C. difficile antigener. I området i närheten finns det ett icke tillgodosett behov att utveckla sådana analyser för att underlätta framtida forskningsinsatser och kliniska tillämpningar. Protein microarrays är en metod för att immobilisera stort antal individuellt-renat protein som en rumsligt organiserade matris av fläckar på en mikroskopisk bild-baserade yta med hjälp av en robotarm-system, som kan vara antingen en kontakt20 eller en icke-kontakt skriva ut verktyg21. Fläckarna kan representera komplexa blandningar såsom cell lysates, antikroppar, vävnad homogenates, endogen eller rekombinanta proteiner eller peptider, kroppsvätskor eller droger22,23.
Protein Mikroarrayteknik erbjuder tydliga fördelar jämfört med standard in-house ELISA teknik, som har traditionellt använts för att bedöma anti -C. difficile antikroppssvar. Dessa inkluderar en ökad kapacitet för att upptäcka ett utbud av multi-isotyp-specifika antikroppar mot en mer omfattande panel av protein mål, minskad volymkrav för antigener, prover och reagenser, och en ökad förmåga att införliva en större antal tekniska replikat, förutom flera interna kvalitetskontroll (QC) mäter1. Microarrays är därför mer känsliga, exakt och reproducerbar och har en större dynamiskt omfång. Dessa faktorer gör microarrays ett billigare och potentiellt gynnsamma alternativ till ELISAs för storskaliga detektion av kända proteiner. Men nackdelarna med Mikroarrayteknik leda främst från stora initiala kostnader i samband med att upprätta en panel av högrenade antigener och inrätta en teknisk plattform.
Protein microarrays har använts flitigt under de senaste två decennierna som ett verktyg för diagnostik och grundläggande forskning i kliniska tillämpningar. Specifika tillämpningar inkluderar proteinuttryck profilering, studiet av enzym-substrat relationer, biomarkör screening, analys av värd-mikrobinteraktioner, och profilering antikropp specificitet23,24, 25,26,27,28. Många nya patogen protein/antigen microarrays har fastställts, inklusive malaria (Plasmodium)29, HIV-130, influensa31, svår akut respiratorisk sjukdom (SARS)32, viral hemorragisk feber33 , herpesviruses34, och tuberkulos35.
Detta protokoll gäller inrättandet av en lätt operativa C. difficile reaktiva antigen microarray analys, som möjliggör korrekt, exakt och specifika kvantifiering av multi-isotyp och stamspecifika antikroppssvaret mot C. difficile antigener i sera och polyklonala IVIg. Häri, inkluderar vi representativa resultat som hänför sig till en godtagbar microarray analys prestanda i jämförelse med ELISA samt Analysens precision och reproducerbarhet profiler. Denna analys kan utvecklas ytterligare för att profilera andra kliniska prover och sätter en ny standard för forskning i den molekylära basen för CDI.

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		<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>
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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 ger en detaljerad översikt över en roman hög genomströmning protein microarray analys för bestämning av anti -Clostridium difficile antikroppsnivåer i Humansera och i separata beredningar av polyklonala intravenöst immunglobulin (IVIg). Protokollet beskrivs de metodologiska steg som ingår i provberedning, utskrift av matriser, analysförfarande och dataanalys. Detta protokoll kan dessutom utvecklas ytterligare för att införliva olika kliniska prover inklusive plasma och cell cellkulturer. Vi visar hur protein microarray kan användas för att bestämma en kombination av isotyp (IgG, IgA, IgM), underklass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) och stam-specifika antikroppar mot höggradigt renat hela C. difficile toxiner A och B (toxinotype 0, stam VPI 10463, ribotyp 087), toxin B från en C. difficile toxin-B-only uttrycker stam (CCUG 20309), en föregångare form av ett B fragment av binära toxin, pCDTb, ribotyp-specifika hela ytskiktet proteiner (SLPs; 001, 002, 027), och styra proteiner (stelkramp tetanustoxoid och Candida albicans). Under experimentet, microarrays är utforskad med sera från individer med C. difficile -infektion (CDI), personer med cystisk fibros (CF) utan diarré, friska kontroller (HC), och från enskilda pre- och post-IVIg-terapi för den behandling av CDI, kombinerad immunbrist sjukdom och kronisk inflammatorisk demyeliniserande polyradiculopathy. Vi möter betydande skillnader i toxin neutralisering efficacies och multi-isotyp specifika antikroppsnivåer mellan patientgrupper, kommersiella beredningar av IVIg, och sera före och efter IVIg-administrering. Dessutom finns det en signifikant korrelation mellan microarray och enzyme-linked immunosorbent assay (ELISA) för antitoxin IgG-nivåer i serumprover. Dessa resultat tyder på att microarray skulle kunna bli ett lovande verktyg för profilering antikroppssvaret mot C.difficile antigener hos vaccinerade eller infekterade människor. Med ytterligare förfining av antigen paneler och en minskning av produktionskostnaderna, räknar vi med att Mikroarrayteknik kan hjälpa optimera, och välj de mest kliniskt användbar immunterapier för C. difficile infektion i ett patient-specifika sätt.

Figur 1 visar ett flödesschema som beskriver de stora stegen i protokollet beskrivs. Figur 2 visar Spearman korrelation tester visar betydande avtal mellan microarray och ELISA IgG och IgA anti toxin A och B nivåer i patientens testserumen. Figur 3 visar differentiell IgG och IgA antikroppar-klass specifika antikroppen Svaren till toxin A, toxin B och binära toxin (pCDTb) hos patienter med CF utan...

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

Denna artikel beskriver en enkel protein microarray metod för profilering humorala immunsvar mot en 7-plex panel av mycket renad Clostridium difficile antigener i humant serum. Protokollet kan förlängas för bestämning av specifika antikroppen svaren i förberedelserna av polyklonala intravenöst immunglobulin.

En Protein Microarray analys för serologisk bestämning av Antigen-specifika antikroppen Svaren följande 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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Trans-vivo Fördröjd överkänslighet analys för Antigenspecifikt förordning

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Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Odling och underhålla<em&gt; Clostridium difficile</em&gt; I en anaerob miljö

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

Hel-djur Imaging och Flödescytometrisk Tekniker för analys av antigen-specifika CD8 + T-cellssvar efter Nanoparticle Vaccination

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

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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

Detection och anrikning av sällsynta antigenspecifika B-celler för analys av Fenotyp och funktion

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

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

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In Vivo Test för påvisande av Antigen-specifika T-cell cytolytisk funktion använder en Vaccination 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 ...