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 forskningen ble støttet av en Hermes Fellowship Ola Negm og Tanya Monaghan og gjennom egen finansiering fra Nottingham Digestive sykdommer sentrum og NIHR Nottingham Digestive sykdommer biomedisinsk forskning.

1. klargjør en Microarray Plate
<ol><li>Fortynne C. difficile antigener med en utskrift buffer [PBS-Tween-trehalose (50 mM)] på optimal konsentrasjon (som var forhåndsdefinerte før pasienten sera): gift en (200 µg/mL) og gift B (100 µg/mL), pCDTb (200 µg/mL), og renset innfødt hele SLPs avledet fra ribotypes 001 og 002 027 (200 µg/mL). Merk: Gift B er Hentet fra en gift B-uttrykke belastning CCUG 20309 (90 µg/mL).<ol>
<li>Fortynne positiv kontrollene, lysates C. albicansog stivkrampe toxo...

<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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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. 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=An+oligonucleotide+microarray+for+multiplex+real-time+PCR+identification+of+HIV-1,+HBV,+and+HCV.">An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1, HBV, and HCV.</a> BioTechniques. 44 (2), 241-246 (2008).</li><li>Ryabinin, V. 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I denne protokollen, har vi vist at microarray er en passende plattform for definere humoral immunreaksjoner til C. difficile protein antigener i pasienten sera (tall 3 og 6) og kommersielle forberedelser av IVIg (figur 5). Vi har også vist at microarray teknikk utfører også sammenlignet med konvensjonelle ELISA (figur 2) og viser utmerket reproduserbarhet, intra - og inter - assay variabilities innenfor akseptable g...

Denne protokollen beskriver utvikling og validering av en roman og tilpassede protein microarray analysen for påvisning og semi kvantifisering av bakteriell belastning og isotype-spesifikke antistoffer Svar å C. difficile antigener. Vi kunne bruke våre C. difficile-spesifikke microarray analysen som et lovende nytt verktøy for den kompositoriske bioanalysis spesifikt antistoff innhold i pasienten sera1,2, forberedelser av IVIg3, og Identifikasjon av antistoff særegenheter som samsvarer med dårlige resultater i CDI4. Vi viser hvordan biobanked serumprøver og kommersielle preparater av IVIg kan analyseres på microarray lysbilder, slik at høy kvalitet reproduserbar profilering av C. difficile patogen-spesifikke antistoffer svar i denne analysen.
Mange friske barn og voksne har synlig serum IgG og IgA antistoffer mot C. difficile giftstoffer A og B5,6. Dette antas å oppstå etter forbigående eksponering under barndom og etter eksponering for C. difficile i voksen alder. Derfor polyklonale IVIg har vært brukt av etiketter til å behandle både tilbakevendende og fulminant CDI7,8,9. Sin definitive rolle og virkemåte er imidlertid uklart. Flere studier har vist at humoral immunrespons for C. difficile giftstoffer spiller en rolle i sykdom presentasjon og utfallet. Spesielt viser asymptomatisk pasienter en økt serum anti-gift A IgG konsentrasjon sammenlignet med pasienter som utvikler symptomatisk sykdom10. En påviselig forening er rapportert for median anti-gift A IgG titers og 30-dagers helt forårsake dødelighet11. Flere rapporter har også avdekket en tilknytning til beskyttelse mot regelmessighet og antistoff Svar å toksin A, B og flere ikke-toksin antigener (Cwp66, Cwp84, FliC, FliD og overflatelag proteiner (SLPs))12,13 , 14 , 15. disse observasjonene har påvirket utviklingen av første passiv immunterapi narkotika målretting C. difficile giften B (bezlotuxumab), som er nylig godkjent av US Food, Drug Administration og europeiske legemidler Kontoret for forebygging av tilbakevendende CDI16. Vaksinasjon strategies bruke deaktivert giftstoffer eller rekombinante gift fragmenter er også under utvikling17,18,19. Disse nye terapeutiske metoder vil utvilsomt stimulere behovet for evaluering av humoral immunreaksjoner mot flere antigener i store utvalgene.
I dag, er det en betydelig mangel av kommersielt tilgjengelig høy gjennomstrømming analyser kan samtidig vurdere bakterielle belastningen og isotype-spesifikke antistoffer Svar å C. difficile antigener. Det er et udekket behov for å utvikle slike analyser for å lette fremtidig forskningsinnsats og kliniske applikasjoner. Protein microarrays er en metode for å nakkens stort antall individuelt renset proteiner som en romlig organisert rekke flekker på en mikroskopisk lysbilde-baserte overflate ved hjelp av en robot-system, som kan være enten en kontakt20 eller en ikke-kontakt utskrift verktøyet21. Flekker kan representere komplekse blandinger som celle lysates, antistoffer, vev homogenates, endogen eller rekombinante proteiner eller peptider, kroppsvæsker eller narkotika22,23.
Protein microarray teknologi gir forskjellige fordeler over standard in-house ELISA teknikker, som har tradisjonelt blitt brukt til å vurdere anti -C. difficile antistoff svar. Disse inkluderer en økt kapasitet for å oppdage en rekke multi-isotype-spesifikke antistoffer mot et mer omfattende panel av protein mål, redusert volumkrav til antigener, eksempler og reagenser, og en forbedret evne til å inkludere en større antall tekniske gjentak, i tillegg til flere interne kvalitetskontroll (QC) måler1. Microarrays har derfor mer følsomme og nøyaktige reproduserbare og et større dynamisk område. Disse faktorene gjør microarrays en billigere og potensielt gunstig alternativ til ELISAs for store påvisning av kjente proteiner. Men ulempene microarray teknologi føre hovedsakelig fra store forhånd kostnadene forbundet med å etablere et panel av høyrenset antigener og sette opp den teknologisk plattformen.
Protein microarrays har blitt brukt de siste to tiårene som en diagnostisk og grunnleggende forskningsverktøy i kliniske applikasjoner. Bestemte programmer inkluderer protein uttrykk profilering, studiet av enzym-underlaget relasjoner, biomarkør screening, analyse av vert-mikrobe interaksjoner, og profilering antistoff spesifisitet23,24, 25,26,27,28. Mange nye patogen protein/antigen microarrays er etablert, inkludert malaria (Plasmodium)29, HIV-130, influensa31, alvorlig akutt åndedretts syndrom (SARS)32, viral hemoragisk feber33 , herpesviruses34og tuberkulose35.
Nåværende protokollen gjelder etableringen av en enkel drift C. difficile reaktive antigen microarray analysen, som muliggjør nøyaktig, presis og spesifikk kvantifisering av multi-isotype og stammen spesifikke antistoffer svar på C. difficile antigener i sera og polyklonale IVIg. Her, inkludere vi representant resultatene gjelder en akseptabel microarray analysen ytelse i forhold til ELISA samt analysen presisjon og reproduserbarhet profiler. Denne analysen kan utvikles profilen andre kliniske prøver og setter en ny standard for forskning i molekylær grunnlaget for 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>
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			<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 gir en detaljert oversikt over en roman høy gjennomstrømming protein microarray analysen for fastsettelse av anti -Clostridium difficile antistoff nivåer i menneskelig sera og i separate forberedelser av polyklonale intravenøs immunglobulin (IVIg). Protokollen beskriver metodologiske trinnene involvert i prøven forberedelse, utskrift av matriser, analysen prosedyre og dataanalyse. I tillegg kan denne protokollen bli ytterligere utviklet å innlemme forskjellige kliniske prøver inkludert plasma og celle kultur supernatants. Vi viser hvordan protein microarray kan brukes til å angi en kombinasjon av isotype (IgG, IgA, IgM), underklasse (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) og stammen spesifikke antistoffer mot svært renset hele C. difficile giftstoffer A og B (toxinotype 0, belastning VPI 10463, ribotype 087), gift B fra en C. difficile gift-B-bare uttrykke belastning (CCUG 20309), en forløper form for et B-fragment av binære gift, pCDTb, ribotype-spesifikke hele overflatelaget proteiner (SLPs; 001, 002, 027), og kontrollere proteiner (stivkrampe toxoid og Candida albicans). Under eksperimentet microarrays er analysert med sera fra personer med C. difficile infeksjon (CDI), personer med cystisk fibrose (CF) uten diaré, sunn kontroller (HC), og enkeltpersoner pre og post-IVIg terapi for den behandling av CDI kombinert immunsvikt lidelse og kronisk inflammatorisk demyeliniserende polyradiculopathy. Vi møter betydelige forskjeller i gift nøytralisering efficacies og multi-isotype spesifikt antistoff nivåer mellom pasient grupper, kommersielle preparater av IVIg, og sera før og etter IVIg administrasjon. Også er det en signifikant sammenheng mellom microarray og enzym knyttet immunosorbent analysen (ELISA) for Motgiften IgG nivåer i serumprøver. Disse resultatene tyder på at microarray kan bli et lovende verktøy for profilering antistoff Svar å C.difficile antigener i vaccinated eller infiserte mennesker. Med ytterligere raffinement av antigen paneler og en reduksjon i produksjonskostnadene forventer vi at microarray teknologi kan hjelpe optimalisere og velg de mest klinisk nyttig immunotherapies for C. difficile infeksjon i en pasient-spesifikk måte.

Figur 1 illustrerer et flytskjema som beskriver de viktigste trinnene i beskrevet protokollen. Figur 2 viser Spearman korrelasjon tester viser betydelig avtale mellom microarray og ELISA for IgG og IgA anti-gift A og B nivåer i pasienten test sera. Figur 3 viser differensial IgG og IgA antistoff-klassen spesifikt antistoff Svar å toksin A, gift B og binære gift (pCDTb) i pasienter med CF uten 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

Denne artikkelen beskriver en enkel protein microarray metode for profilering humoral immunreaksjoner til et 7-plex panel av svært renset Clostridium difficile antigener i menneskelig sera. Protokollen kan utvides for fastsettelse av spesifikt antistoff svar i forberedelsene til polyklonale intravenøs immunglobulin.

En Protein Microarray analysen for serologisk fastsettelse av Antigen-spesifikke antistoffer svar følgende Clostridium difficile infeksjon

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

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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

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

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

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 ...

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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

Dyrking og Vedlikehold<em&gt; Clostridium difficile</em&gt; I et anaerobt miljø

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-dyr Imaging og strømningscytometriske Teknikker for analyse av Antigen-spesifikke CD8 + T celle responser etter Nanopartikkel Vaksinasjon

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

Cefoperazone behandlet musemodell for klinisk relevante<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

Oppdagelse og berikelse av Rare antigen-spesifikke B-celler for analyse av fenotype og funksjon

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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

En protokoll for å karakterisere de morfologiske endringene av<em&gt; Clostridium difficile</em&gt; Som svar på antibiotisk behandling

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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I Vivo Analysen for påvisning av Antigen-spesifikke T-celle Cytolytic funksjon med en vaksinasjon 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 ...