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

Esta pesquisa foi apoiada por uma bolsa Hermes Ola Negm e Tanya Monaghan e através de financiamento separado do centro de doenças digestivas de Nottingham e a NIHR Nottingham digestivo doenças Biomedical Research Centre.

1. preparar um prato de Microarray
<ol><li>Diluir a antígenos de c. difficile com um buffer de impressão [PBS-Tween-trealose (50mm)] para a concentração ideal (que foi predefinida antes de executar o paciente sera): toxina um (200 µ g/mL) e toxina B (100 µ g/mL), pCDTb (200 µ g/mL) e purificado nativo SLPs todo derivado ribotypes 001, 002 e 027 (200 µ g/mL). Nota: Toxina B foi obtida uma toxina B-expressando estirpe CCUG 20309 (90 µ g/mL).<ol>
<li>Dilua os controles positivos, lysates de c. alb...

<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. 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Neste protocolo, mostramos que microarray é uma plataforma adequada para definir humorais respostas imunes aos antígenos de proteína c. difficile em soros de doentes (figuras 3 e 6) e preparações comerciais de IVIg (Figura 5). Nós também têm demonstrado que a técnica de microarray executa bem em comparação com o ELISA convencional (Figura 2) e mostra excelente reprodutibilidade, com intra e inter assay variab...

Este protocolo descreve o desenvolvimento e validação de um ensaio de microarray de proteína romance e personalizado para a deteção e a quantificação semi da estirpe bacteriana e respostas específicas do isotipo do anticorpo aos antígenos de c. difficile . Utilizamos com êxito o nosso c. difficile-ensaio de microarray específico como uma nova ferramenta promissora para a Bioanálise composicional de conteúdo de anticorpos específicos no soro dos doentes1,2, preparações de IVIg3, e identificação das especificidades de anticorpos que correlacionam com resultados pobres em CDI4. Demonstramos como amostras de soro biobanked e preparações comerciais de IVIg podem ser analisadas em slides de microarray, permitindo alta qualidade reprodutível caracterização das respostas de anticorpos específicos do patógeno c. difficile neste ensaio.
Muitas crianças saudáveis e adultos têm anticorpos IgG e IgA detectável soro c. difficile toxinas A e B5,6. Estes são pensados para surgir na sequência de exposição transitória durante a infância e após a exposição a c. difficile na idade adulta. Por esta razão, IVIg policlonal tem sido utilizado off-label para tratar recorrentes e fulminante CDI7,8,9. No entanto, seu papel definitivo e modo de ação ainda não está claro. Vários estudos têm mostrado que a resposta imune humoral a c. difficile toxinas desempenha um papel na apresentação da doença e dos resultados. Especificamente, pacientes assintomáticos mostram uma aumento anti-toxina IgG A concentração sérica comparada aos pacientes que desenvolvem doença sintomática10. Relatou-se uma associação demonstrável para títulos de IgG A anti-toxina medianos e mortalidade 30 dias11. Vários relatórios também revelaram uma associação com uma proteção contra a recorrência e anticorpo respostas a toxina A, B e vários antígenos não-toxina (Cwp66, Cwp84, FliC, FliD e proteínas da camada superficial (SLPs))12,13 , 14 , 15. estas observações têm estimulado o desenvolvimento da primeira imunoterapia passiva droga alvo c. difficile toxina B (bezlotuxumab), que recentemente foi aprovado pelo Food e Drug Administration e os medicamentos Europeu Agência para a prevenção da recorrente CDI16. Estratégias de vacinação usando toxinas inactivadas ou fragmentos de toxina recombinante também estão atualmente sob desenvolvimento17,18,19. Estas novas abordagens terapêuticas, sem dúvida, irão estimular a exigência de avaliação humorais respostas imunes aos antígenos múltiplos em tamanhos de amostra grande.
Hoje, há uma notável falta de ensaios comercialmente disponíveis de alta produtividade capaz de avaliar simultaneamente a estirpe bacteriana e respostas específicas do isotipo do anticorpo aos antígenos de c. difficile . Há uma necessidade não atendida de desenvolver tais ensaios para facilitar os esforços de investigação futuras e aplicações clínicas. Os microarrays da proteína são um método para imobilizar um grande número de proteínas purificadas individualmente como uma matriz espacialmente organizada de pontos sobre uma superfície com base em slide microscópica por meio de um sistema robótico, que pode ser um contato20 ou um contato não- 21. ferramenta da impressão. As manchas podem representar misturas complexas como lisados celulares, anticorpos, tecido homogenates, proteínas endógenas ou recombinantes ou peptídeos, fluidos corporais ou drogas22,23.
Tecnologia de microarray de proteína oferece vantagens distintas sobre ELISA in-house padrão técnicas, que tem sido tradicionalmente usadas para avaliar as respostas de anticorpos dec. difficile anti. Estes incluem um aumento da capacidade para a detecção de uma gama de multi-específicas do isotipo anticorpos contra um painel mais amplo de alvos de proteína, requisitos de volume reduzido de antígenos, amostras e reagentes e a capacidade avançada de incorporar uma maior número de repetições de técnicas, além de controle de qualidade interno múltiplo (QC) mede1. Microarrays são, portanto, mais sensíveis, precisos e reprodutíveis e têm uma maior gama dinâmica. Esses fatores tornam microarrays uma alternativa mais barata e potencialmente favorável para ELISA para a detecção em larga escala de proteínas conhecidas. No entanto, as desvantagens da tecnologia microarray resultam principalmente os grandes custos adiantados associados com o estabelecimento de um painel de antígenos altamente purificados e criação de plataforma tecnológica.
Os microarrays da proteína têm sido extensivamente usados nas últimas duas décadas como ferramenta de pesquisa básica e diagnóstico em aplicações clínicas. Aplicações específicas incluem a expressão da proteína de perfil, o estudo das relações de enzima-substrato, triagem de biomarcador, análise das interações do anfitrião-micróbio e criação de perfil de anticorpo especificidade23,24, 25de27,,26,28. Muitos novo patógeno/antígeno da proteína microarrays foram estabelecidos, incluindo malária (Plasmodium)29, HIV-130, gripe31, síndrome respiratória aguda grave (SARS)32, febre hemorrágica viral33 , herpesviruses,34e tuberculose35.
O presente protocolo refere-se ao estabelecimento de um fácil funcionamento C. difficile antígeno reativa microarray do ensaio, que permite a quantificação, precisa e específica de multi-isotype e respostas de anticorpos específicos de estirpe de C. difficile antígenos em soros e policlonal IVIg. Neste documento, que incluem resultados representativos referentes a um desempenho aceitável do microarray quando comparado com ELISA, bem como a precisão do ensaio e a reprodutibilidade perfis. Este ensaio poderia ser desenvolvido para o perfil de outras amostras clínicas e define um novo padrão para a investigação sobre a base molecular do 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

Nós fornecemos uma visão detalhada de um ensaio de microarray de romance da elevado-produção de proteínas para a determinação de antiníveis de anticorpos deClostridium difficile em soros humanos e em preparações separadas de polyclonal imunoglobulina intravenosa (IVIg). O protocolo descreve as etapas metodológicas envolvidas na preparação de amostra, impressão de matrizes, procedimento de ensaio e análise de dados. Além disso, este protocolo pode ser desenvolvido para incorporar diversas amostras clínicas incluindo plasma e sobrenadantes. Mostramos como proteína microarray pode ser usado para determinar uma combinação de isotipo (IgG, IgA, IgM), subclasse (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), e anticorpos específicos de estirpe altamente purificado toda c. difficile toxinas A e B (toxinotype-0, estirpe VPI ribotype 10463, 087), toxina B de uma c. difficile toxina B-somente expressando estirpe (CCUG 20309), uma forma de precursor de um fragmento B da toxina binária, pCDTb, proteínas de toda a superfície da camada de ribotype específicos (SLPs; 001, 002, 027) e controle de proteínas (tétano tetânico e Candida albicans). Durante o experimento, microarrays são analisados com soros de indivíduos com infecção por c. difficile (CDI), os indivíduos com fibrose cística (CF), sem diarreia, controles saudáveis (HC) e de indivíduos pré e post-IVIg terapia para o tratamento de transtorno de imunodeficiência combinada, CDI e polyradiculopathy desmielinizante inflamatória crônica. Encontramos diferenças significativas de eficácias de neutralização de toxina e níveis de anticorpos específicos de multi-isotipo entre grupos de pacientes, preparações comerciais de IVIg e sera antes e após a administração IVIg. Além disso, há uma correlação significativa entre microarray e ensaio imunoenzimático (ELISA) para os níveis de IgG antitoxina em amostras de soro. Estes resultados sugerem que microarray pode se tornar uma ferramenta promissora para o perfilamento respostas de anticorpos para antígenos de c nos seres humanos infectados ou vacinados. Com mais refinamento de painéis de antígeno e uma redução nos custos de produção, prevemos que tecnologia microarray pode ajudar a otimizar e selecione as imunoterapias mais clinicamente útil para c. difficile infecção de forma paciente específico.

A Figura 1 ilustra um fluxograma descrevendo as etapas principais no protocolo descrito. A Figura 2 mostra testes de correlação de Spearman demonstrando acordo significativo entre microarray e ELISA IgG e IgA antitoxina A e B os níveis no paciente teste sera. A Figura 3 mostra diferencial IgG e IgA de anticorpo-classe respostas de anticorpos específicos para A toxina, toxina B e toxina binária (...

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

Este artigo descreve um método de microarray de proteína simples para perfilação humorais respostas imunes a um painel de 7-plex de altamente purificado antígenos de Clostridium difficile em soros humanos. O protocolo pode ser estendido para a determinação de respostas de anticorpos específicos em preparações de imunoglobulina intravenosa policlonal.

Um ensaio de Microarray da proteína para determinação sorológica do antígeno-específicas anticorpo respostas seguintes Clostridium difficile infecção

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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Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

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