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

Bu araştırma bir Hermes bursu Ola Negm ve Tanya Monaghan ve ayrı Nottingham Sindirim Hastalıkları Merkezi ve NIHR Nottingham sindirim hastalıkları Biyomedikal Araştırma Merkezi fon aracılığıyla tarafından desteklenmiştir.

1. hazırlanması Mikroarray plaka
<ol><li>C. difficile antijenleri Yazdırma arabelleği [PBS-ara-trehalose (50 mM)] ile (ki hasta sera çalıştırmadan önce önceden tanımlanmış) en iyi konsantrasyon, seyreltik: toksin bir (200 µg/mL) ve toksin B (100 µg/mL), pCDTb (200 µg/mL) ve arıtılmış yerli Tüm SLPs elde edilen ribotypes 001, 002 ve 027 (200 µg/mL). Not: Toksin B bir toksin B ifade zorlanma CCUG 20309 (90 µg/mL) elde edildi.<ol>
<li>Pozitif denetimleri, lysates C. albicansve t...

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Bu protokol için o Mikroarray C. difficile protein antijenleri (rakamlar 3 ve 6) hasta sera ve IVIG (Şekil 5) ticari hazırlıkları için humoral bağışıklık yanıtı tanımlamak için uygun bir platform olduğunu göstermiştir. Biz de Mikroarray tekniği de geleneksel ELISA (Şekil 2) karşılaştırıldığında gerçekleştirir ve hassas ( kabul edilebilir sınırlar içinde düşen içi ve arası ass...

Bu iletişim kuralı geliştirme ve tespiti için bir roman ve özelleştirilmiş protein mikroarray testin geçerliliği ve bakteriyel zorlanma ve C. difficile antijenleri izotip özgü antikor yanıt yarı miktar açıklanmaktadır. Başarılı bir şekilde bizim C. difficilekullanmak-belirli Mikroarray tahlil Kompozisyon bioanalysis hasta sera1,2, hazırlıklar IVIG3, spesifik antikor içerik için umut verici yeni bir araç olarak ve CDI4yoksul sonuçları ile ilişkili antikor özelliklerine tanımlaması. Biobanked serum numuneleri ve IVIG ticari hazırlıkları Mikroarray slaytlarda nasıl çözümlenebilir C. difficile patojen özgü antikor yanıt bu tahlil tekrarlanabilir yüksek kaliteli profilleme izin göstermektedir.
Birçok sağlıklı çocuk ve yetişkin tespit serum IgG ve IgA antikorları C. difficile toksinler A ve B5,6var. Bunlar bebeklik ve C. difficile maruz yetişkinlikte takip sırasında geçici pozlama ardından ortaya çıkan düşünülmektedir. Bu nedenle, poliklonal IVIG olmuştur dışı tekrarlayan ve fulminan CDI7,8,9tedavi etmek için kullanılır. Ancak, onun kesin rolünü ve eylem kalır belirsiz. C. difficile toksinler humoral bağışıklık yanıtı hastalığı sunum ve sonucu bir rol oynar çeşitli çalışmalar göstermiştir. Özellikle, asemptomatik Hastalar semptomatik hastalık10geliştirmek hastalara göre bir artan serum Anti-toksin A IgG konsantrasyon göster. Kanıtlanabilir bir dernek medyan Anti-toksin A IgG titreleri ve 30 günlük all-neden ölüm11için rapor edildi. Çeşitli raporlar da yineleme ve antikor yanıt toksin A, B ve birkaç sigara toksin antijenleri (Cwp66, Cwp84, FliC, hızlı ve yüzey katman proteinleri (SLPs)) karşı bir koruma ile bir ilişki ortaya koymuştur12,13 , 14 , 15. bu gözlemler son zamanlarda ABD Gıda ve ilaç idaresi ve Avrupa İlaç tarafından onaylanıp gelişme ilk pasif immünoterapi uyuşturucu hedefleme C. difficile toksini B (bezlotuxumab), mahmuzlu Tekrarlayan CDI16önlenmesi için Ajansı. Inaktif toksinler, uyuşturucu ve rekombinant toksin parçaları kullanarak aşılama stratejileri de vardır şu anda altında geliştirme17,18,19. Bu yeni tedavi yaklaşımları şüphesiz örneklerin boyutu büyük içinde birden çok antijenleri humoral bağışıklık yanıtlarını değerlendirmek için gereksinimin teşvik edecek.
Bugün, aynı anda bakteriyel zorlanma ve C. difficile antijenleri izotip özgü antikor yanıt değerlendirirken yetenekli ticari olarak mevcut yüksek üretilen iş deneyleri önemli bir eksikliği var. Gelecekteki araştırma çabalarını ve klinik uygulamaları kolaylaştırmak için bu tür deneyleri geliştirmek için bir karşılanmamış ihtiyaç vardır. Protein microarrays kişi20 veya olmayan bir kişi olabilir bir robotik sistem kullanarak çok sayıda tek tek saf protein lekelerin mikroskobik bir slayt tabanlı yüzeye dağınık şekilde organize bir dizi olarak hareketsiz için bir yöntemdir baskı aracı21. Noktalar hücre lysates, antikor, doku homogenates, endojen veya rekombinant proteinler veya peptidler, vücut sıvıları veya uyuşturucu22,23gibi karmaşık karışımlar temsil edebilir.
Protein mikroarray teknolojisi standart kurum içi ELISA ayrı avantaj geleneksel anti -C. difficile antikor yanıt değerlendirmek için kullanılan teknikleri sunar. Bunlar çok izotip özel antikorlar hedeflerinden protein, düşük hacim gereksinimleri antijenleri, örnekleri ve reaktifler, daha geniş bir panel karşı bir dizi tespit için artan bir kapasite dahil ve bir daha büyük dahil etmek için geliştirilmiş bir yetenek birden çok iç kalite kontrol (QC) yanı sıra teknik çoğaltır sayısı1ölçer. Microarrays bu nedenle daha doğru duyarlı ve tekrarlanabilir ve büyük bir dinamik alana sahip. Bu faktörler microarrays bilinen proteinler büyük ölçekli tespiti için ELISAs için potansiyel olarak uygun ve ucuz bir alternatif olun. Ancak, esas olarak bir panel yüksek oranda saflaştırılmış antijenleri kurulması ve teknolojik platformu kurma ile ilgili büyük ön maliyetleri Mikroarray teknoloji dezavantajları sonucu.
Protein microarrays klinik uygulamalarında tanılama ve temel araştırma aracı olarak son yirmi yılda yoğun olarak kullanılmaktadır. Belirli uygulamaları, enzim-substrat ilişkileri, biyomarker tarama, ana bilgisayar-mikrop etkileşimlerin analizi çalışma profil oluşturma ve antikor özgüllük23,24, profil oluşturma protein ifade 25,26,27,28. Birçok yeni patojen protein/antijen microarrays, sıtma (Plasmodium)29, HIV-130, grip31, şiddetli akut solunum Sendromu (SARS)32, viral kanamalı ateşi33 de dahil olmak üzere kurulmuş olan , herpesviruses34ve tüberküloz35.
Mevcut Protokolü doru, hassas ve belirli miktar multi-izotip ve C. zorlanma özgü antikor yanıt sağlayan bir kolay işletim C. difficile reaktif antijen Mikroarray yöntemi, kurulması ile ilgilidir difficile sera ve poliklonal IVIG antijenleri. Burada, ELISA yanı sıra tahlil hassasiyet ve tekrarlanabilirlik için profilleri karşılaştırıldığında bir kabul edilebilir Mikroarray tahlil performans için ilgili temsilcisi sonuçları içerir. Bu tahlil diğer klinik örnekler profil için daha fazla geliştirilebilir ve CDI moleküler temeli araştırma için yeni bir standart ayarlar.

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

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

Clostridium difficile antikor düzeyleri insan sera ve poliklonal intravenöz immünglobulin (IVIG) ayrı hazırlıklar belirlenmesi anti - için bir roman yüksek üretilen iş protein mikroarray tahlil ayrıntılı bir özetini sağlar. Protokol numune hazırlama, diziler, tahlil yordam ve veri analizi yazdırma ilgili metodolojik adımları açıklar. Ayrıca, bu iletişim kuralı plazma dahil olmak üzere çeşitli klinik örnekler dahil ve kültür supernatants hücre için daha fazla geliştirilebilir. Protein mikroarray izotip (IgA, IgG, IgM), alt sınıf (Igg1, Igg2, Igg3, Igg4, Iga1, Iga2) bir arada belirlemek için nasıl kullanılabileceğini göstermektedir ve zorlanma özel antikorlar için son derece saf tüm C. difficile toksinler A ve B (toxinotype 0, zorlanma VPI 10463, ribotype 087), toksin B bir C. difficile toksin B yalnızca ifade zorlanma (CCUG 20309), ikili toksin, pCDTb, ribotype özgü tüm yüzey katman protein B parçası habercisi şeklinde (SLPs; 001 002, 027) ve kontrol proteinler (tetanoz toksoidi ve Candida albicans). Deneme sırasında microarrays sera C. difficile enfeksiyon (CDI), kistik fibrozis (CF) ishal, sağlıklı kontrol (HC), olmadan kişilerle olan bireyler ve bireyler öncesi ve sonrası-IVIG tedavisi ile probed tedavi CDI, kombine immün yetmezlik hastalığı ve kronik inflamatuvar demiyelinizan polyradiculopathy. Hasta grupları, IVIG, ve sera önce ticari hazırlıkları ve aşağıdaki IVIG yönetim arasında önemli farklılıklar toksini nötralize efficacies ve multi-izotip spesifik antikor düzeyleri karşılaşıyoruz. Ayrıca, serum örneklerinde antitoksin IgG düzeyleri için Mikroarray ve enzim bağlı immunosorbent assay (ELISA) arasında önemli bir ilişki yoktur. Bu sonuçlar bu Mikroarray antikor yanıt C.difficile antijenleri aşı veya enfekte insanlar için profil oluşturma için umut verici bir araç haline gelebilir öneririz. İle arıtma antijen panelleri ve üretim maliyetleri azaltma biz Mikroarray teknoloji optimize etmek ve C. difficile enfeksiyon için en klinik olarak yararlı immunotherapies bir hastaya özgü şekilde seçin yardımcı olabilir tahmin.

Açıklanan protokolünde önemli adımları açıklayan bir akış çizelgesi Şekil 1 gösterir. Şekil 2 Spearman korelasyon testleri anti-toksin A ve B hasta test sera seviyeleri önemli arasındaki anlaşma Mikroarray ve ELISA IgG ve IgA gösteren gösterir. Şekil 3 fark IgG ve IgA antikor-sınıf CF olmadan ishal olan hastalar, ishal olan CDI hastalar ve HC spesifik antikor yanıt toksin A, to...

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

Bir 7-plex panel humoral bağışıklık yanıtlarını yüksek profil oluşturma saf insan sera içinde Clostridium difficile antijenleri için bu makalede basit protein mikroarray yöntemi açıklanır. Protokol spesifik antikor yanıt müstahzarları poliklonal intravenöz immünglobulin belirlenmesi için genişletilebilir.

Bir Protein Mikroarray tahlil antijen spesifik antikor yanıt aşağıdaki Clostridium difficile enfeksiyon serolojik belirlenmesi için

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

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

Research

JoVE Journal

Immunology and Infection

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Antijen Özgül In Vivo</emKAKE Etiketli Hedef Hücreler> Killing Testi

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

İmmünoloji ve Enfeksiyon

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

Viral Patojenler Klinik Örnekler Ekranına bir Pan-Viral Mikroarray Testi (Virochip)

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

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

Kolorektal Kanser Hücre Yüzey Protein Profil Antikor Mikroarray ve Floresans Multiplexing

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

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Antijen Özgül Düzenleme için Trans-vivo Gecikmeli Tip Aşırı Duyarlılık Testi

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

Kültürleme ve bakımı<em&gt; Clostridium difficile</em&gt; Bir Anaerobik Çevre

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

Nanopartikül Aşılama sonrası analizi antijene özgü CD8 + T hücresi tepkileri için tüm hayvan Görüntüleme ve Akış Sitometrisi Teknikleri

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

ve Cefoperazone-tedavi edilmiş fare modeli klinik olarak ilgili<em&gt; Clostridium difficile</em&gt; Gerilme 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

Fenotip ve Fonksiyon Analizi için algılama ve Nadir zenginleştirilmesi antijene özgü B hücreleri

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

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

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

Morfolojik Değişiklikleri Karakterize Eden Bir Protokol<em&gt; Clostridium difficile</em&gt; Antibiyotik Tedavisine Yanıt Vermek

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

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

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

Vivo Tahlil antijen spesifik T-hücre Cytolytic işlevi bir aşı modeli kullanılarak tespiti için

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