Este estudo foi financiado pelos Institutos Nacionais de Saúde concede R01HL105826 e K02HL114749 (Dr. Sadayappan) e American Heart Association Midwest Fellowships; 11PRE7240022 (Mr. Barefield) e 13POST14720024 (Dr. Govindan). Os autores agradecem o apoio de Dr. Jimmy Page, Jill Clampit e Dr. John Hudson, MSD, pelo excelente suporte técnico e literatura no desenvolvimento do ensaio.

1. Uniplex cMyBP-C Assay <ol><li> Um dia antes do experimento, casaco da placa padrão nu 96 poços MSD com anticorpo de captura. Para cMyBP-C, usar um volume il de 30 de rato anticorpo anti-cMyBP-C monoclonal (gelatina livre), a uma concentração de 5 ug / ml diluído em tampão fosfato salino (PBS). </li><li> Uma vez que o poço é hidrofóbico, pipetar a solução para o canto inferior do poço; toque os lados da placa para espalhar a solução ao longo de todo o poço. </li><li> A placa é coberta com um selante de placa e incubadas O / N a 4 ° C sem agitação. </li><li> Remover a solução de anticorpo de captura tocando a solução ao longo de um lavatório e, em seguida, sobre uma pilha de toalhas de papel. A ligação não específica para a placa é bloqueada por adição de 150 ul de uma de 5% (w / v) de bloqueador Uma solução (BSA alto grau) em PBS a cada poço. </li><li> Selar a placa e incubar durante 1 hora à temperatura ambiente, agitando a 700 rpm. </li><li> Durante o passo de bloqueio, preparar as normas e splos. Adicione a série padrão diluindo fragmento da proteína recombinante cMyBP-C (aminoácidos 1-271), para uma concentração de partida de 2,000 ng / ml em 1% (w / v) de bloqueador de A / PBS. Depois dilui-se sequencialmente por um factor de 5 em 1% (w / v) de bloqueador de A / PBS. Total de sete padrões + 1 em branco (1% (w / v) de bloqueador Um apenas PBS /) foram usadas. </li><li> Remover a solução de bloqueio e lavar a placa 3 vezes com 150 ul de 0,05% (v / v) Tween-20/PBS. Cada vez, remover a solução de lavagem através da inversão da placa de cima de uma pia. Após a terceira etapa de lavagem, vigorosamente apertar o prato sobre uma pia e seque a placa vigorosamente sobre uma camada de papel toalha até que esteja completamente seco. Este é um passo crucial, como volumes de incubação são pequenos, e qualquer solução de lavagem restante irá diluir significativamente a próxima incubação. </li><li> Pipetar 25 ul de padrões e das amostras nas cavidades. Selar a placa e incubar à temperatura ambiente, agitando a 700 rpm durante 1 hora. </li><li> Preparar a solução de anticorpo de detecção de 1 ug / ml em 1% bloqueador A / PBS. A cuestômagos feito anticorpo cMyBP-C (aminoácidos epitopo 2-14) com MSD sulfo-TAG rotulagem serve como anticorpo de detecção. Kits estão disponíveis para SULFO-TAG rotulagem, tornando-se um procedimento relativamente simples para rotular qualquer anticorpo. </li><li> Repetir o passo de lavagem, tal como descrito na etapa 1.4. </li><li> Adicionar 25 ul de solução de anticorpo de detecção para cada poço, selar a placa e incubar a temperatura ambiente durante 1 hora num agitador de placas definida a 700 rpm. </li><li> Durante o período de incubação, executar MSD da demo placa (placa com luzes LED) para garantir o funcionamento adequado do Imager Sector e software (consulte a seção de reagentes). Além disso, diluir o-tampão de leitura, que é fornecido pela MSD, por adição de 5 ml de 4x ler-tampão de 15 ml de água destilada. </li><li> Repetir o passo de lavagem, tal como descrito na etapa 1.4. </li><li> Adicionar 150 ul de leitura tampão em cada poço utilizando uma pipeta multicanal. Certifique-se de evitar bolhas de ar (pipetagem reversa é um método adequado). Após a adição da leitura tampão, verificar imediatamente a placa no sectorImager. </li></ol> 2. 3-plex cMyBP-C, CK-MB e cTnI Assay <ol><li> A placa de 96 poços 3-Plex e diluente soluções são deixadas a equilibrar à temperatura ambiente antes do uso. Esta placa é pré-revestido com anticorpos de captura para cMyBP-C, CK-MB e cTnI pela MSD. </li><li> Adicionar 25 ul de 1% (w / v) de bloqueador A / PBS a cada poço. Os lados da placa para assegurar que todo o poço é coberto pela solução. </li><li> Selar a placa com o selador de placa, coloque a placa em um shaker prato, e agitar a 700 rpm por 30 min. </li><li> No entanto, uma série de diluições de calibradores individuais é preparado. Porque cada poço tem três anticorpos de captura três calibradores necessidade de ser misturado e diluído em série antes da utilização. Diluir recombinante cMyBP-C, CK-MB (MSD), e TIc (MSD) em conjunto em 1% (w / v) de bloqueador de A / PBS, para criar uma amostra de 2,000 ng / ml cMyBP-C, com 100 ng / mL de CK- MB, e 25 ng / mL TIc (amostra A). </li><li> Um volume final de pelo menos 100 ul é preparado para cada padrão. Acompletar a série padrão, diluir em série das amostras são, por um factor de 5. Usar 25 ul da amostra A em 100 uL de 1% (w / v) de bloqueador A / PBS para criar amostra B. Em seguida, utilizar 25 ul de amostra B, em 100 ul de 1% (w / v) de bloqueador de A / PBS para criar Amostra C , e assim por diante. Amostras de soro humano de 16 pacientes com IAM e 16 indivíduos controle foram usados ​​para medir cMyBP-C, CK-MB, e os níveis de cTnI. Estas amostras foram diluídas 2x em 1% (w / v) de bloqueador de A / PBS. </li><li> Adicionam-se 25 ul dos padrões e das amostras diluídas aos 25 ul já presente nos poços da placa 3-Plex. É importante também incluir um espaço em branco (apenas solvente). Para estabelecer a técnica, as amostras são carregadas em triplicado técnicos. Caso contrário, as duplicatas são suficientes. </li><li> Selar a placa novamente (vedante de placa pode ser reutilizada) e incubar num agitador de placas (700 rpm) a temperatura ambiente durante 2 horas. </li><li> Imediatamente antes da incubação é terminada, a solução de anticorpo de detecção é preparado. O diluente utilizado é de 1% (w / v) de bloqueador A em PBS. Todos thrOs anticorpos de detecção de ee SULFO-marcadas são diluídos em conjunto a uma concentração final de 1 ug / ml cada. Anticorpos de detecção são normalmente fornecidos com uma concentração estoque de 50x. </li><li> Para uma placa de 96 poços completo, é necessário 2,700 ml de solução total diluído (com erro de pipetagem incluídas). Adicionar 54 ul de cada um dos anticorpos de detecção de 2538 ul de 1% (w / v) de bloqueador de A / PBS. </li><li> Após 2 horas, retire soluções sacudindo vigorosamente a placa acima de uma pia. Lavar os poços 3x com 150 mL de 0,05% de Tween-20 em PBS. Adicionar 25 ul de solução de anticorpo de detecção para cada poço utilizando uma pipeta multicanal, e os lados da placa são virados para garantir que todo o poço é coberto pela solução. </li><li> Selar a placa e incubar durante 1 hora, agitando a 700 rpm. </li><li> Durante o período de incubação, executar MSD da demo placa (placa com luzes LED) para garantir o funcionamento adequado do Imager Sector e software. Também diluir o-tampão de leitura, que é fornecido pela MSD, por adição de 5 ml de 4x leituraamortecer a 15 ml de água destilada. </li><li> Repetir o passo de lavagem descrito no passo 2.8. </li><li> Adicionar 150 ul de leitura tampão em cada poço utilizando uma pipeta multicanal. Evitando bolhas de ar é crítica (pipetagem reversa é um método adequado). Após a adição da leitura tampão, verificar imediatamente a placa na Imager sector. </li></ol>

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	<li>Thygesen, K., 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=Third+universal+definition+of+myocardial+infarction.">Third universal definition of myocardial infarction.</a> Eur. Heart J. 33, 2551-2567 (2012).</li><li>Leng, S. X., 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=ELISA+and+multiplex+technologies+for+cytokine+measurement+in+inflammation+and+aging+research.">ELISA and multiplex technologies for cytokine measurement in inflammation and aging research.</a> J. Gerontol. A. Biol. Sci. Med. Sci. 63, 879-884 (2008).</li><li>Thompson, I., 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=Competitive+electrochemiluminescence+wash+and+no-wash+immunoassays+for+detection+of+serum+antibodies+to+smooth+Brucella+strains.">Competitive electrochemiluminescence wash and no-wash immunoassays for detection of serum antibodies to smooth Brucella strains.</a> Clin. Vaccine Immunol. 16, 765-771 (2009).</li><li>Kingsmore, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+measurement:+technologies+and+applications+of+protein+and+antibody+arrays.">Multiplexed protein measurement: technologies and applications of protein and antibody arrays.</a> Nat. Rev. Drug. Discov. 5, 310-320 (2006).</li><li>Altan, Z. 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=A+long-acting+tumor+necrosis+factor+alpha-binding+protein+demonstrates+activity+in+both+in+vitro+and+in+vivo+models+of+endometriosis.">A long-acting tumor necrosis factor alpha-binding protein demonstrates activity in both in vitro and in vivo models of endometriosis.</a> J. Pharmacol. Exp. Ther. 334, 460-466 (2010).</li><li>Patel, K. 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=High+sensitivity+cytokine+detection+in+acute+coronary+syndrome+reveals+up-regulation+of+interferon+gamma+and+interleukin-10+post+myocardial+infarction.">High sensitivity cytokine detection in acute coronary syndrome reveals up-regulation of interferon gamma and interleukin-10 post myocardial infarction.</a> Clin. Immunol. 133, 251-256 (2009).</li><li>Fu, Q., Zhu, J., Van Eyk, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+multiplex+immunoassay+platforms.">Comparison of multiplex immunoassay platforms.</a> Clin. Chem. 56, 314-318 (2010).</li><li>Mahajan, V. S., Jarolim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+interpret+elevated+cardiac+troponin+levels.">How to interpret elevated cardiac troponin levels.</a> Circulation. 124, 2350-2354 (2011).</li><li>Govindan, S., 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=Cardiac+myosin+binding+protein-C+is+a+potential+diagnostic+biomarker+for+myocardial+infarction.">Cardiac myosin binding protein-C is a potential diagnostic biomarker for myocardial infarction.</a> J. Mol. Cell Cardiol. 52, 154-164 (2012).</li><li>Barefield, D., Sadayappan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+and+function+of+cardiac+myosin+binding+protein-C+in+health+and+disease.">Phosphorylation and function of cardiac myosin binding protein-C in health and disease.</a> J. Mol. Cell Cardiol. 48, 866-875 (2010).</li><li>Kuster, D. W., 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=Cardiac+myosin+binding+protein+C+phosphorylation+in+cardiac+disease.">Cardiac myosin binding protein C phosphorylation in cardiac disease.</a> J. Muscle Res. Cell Motil. 33, 43-52 (2012).</li><li>Sadayappan, S., de Tombe, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+myosin+binding+protein-C:+redefining+its+structure+and+function.">Cardiac myosin binding protein-C: redefining its structure and function.</a> Biophys. Rev. 4, 93-106 (2012).</li><li>Sadayappan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+myosin+binding+protein-C:+a+potential+early-stage,+cardiac-specific+biomarker+of+ischemia-reperfusion+injury.">Cardiac myosin binding protein-C: a potential early-stage, cardiac-specific biomarker of ischemia-reperfusion injury.</a> Biomark Med. 6, 69-72 (2012).</li><li>Fichorova, R. N., 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=Biological+and+technical+variables+affecting+immunoassay+recovery+of+cytokines+from+human+serum+and+simulated+vaginal+fluid:+a+multicenter+study.">Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.</a> Anal. Chem. 80, 4741-4751 (2008).</li><li>Malekzadeh, 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=Challenges+in+multi-plex+and+mono-plex+platforms+for+the+discovery+of+inflammatory+profiles+in+neurodegenerative+diseases.">Challenges in multi-plex and mono-plex platforms for the discovery of inflammatory profiles in neurodegenerative diseases.</a> Methods. 56, 508-513 (2012).</li></ol>

A plena aplicação da patente está pendente (Petição N º de série 13/464, 466, Pub. No. EUA 2012/0282618 A1 e Data:. 05/04/12) para determinar os fatores de risco associados com a degradação cMyBP-C e liberação no fluido corporal humano e quantificar o nível de cMyBP-C no fluido do corpo humano.

Neste estudo, mostra a aplicabilidade de um imunoensaio multiplex para a detecção de múltiplos marcadores cardíacos no soro de pacientes. Esta técnica tem inúmeras vantagens sobre ELISA tradicional. Primeiro, ECL no kit de ensaio é utilizado em vez de detecção colorimétrica. Em ECL, um sinal eléctrico estimula a produção local de propriedade medida (de luz), desacoplando assim o evento estimulação do sinal, o que reduz o fundo 14. Isto permite uma elevada sensibilidade, como pode ser visto na <...

A medição de baixas quantidades de proteína em amostras biológicas complexas, tal como o soro, é de crescente importância para a gestão clínica do paciente, assim como a ciência básica. Por exemplo, um aumento dos níveis séricos de marcadores cardíacos, tais como a troponina I, num ambiente clínico é consistente com enfarte agudo do miocárdio (MI) 1. Para detectar proteínas em amostras de soro, padrão de ensaio imunoenzimático (ELISA), é a técnica utilizada na maioria das vezes, uma vez que tem uma elevada sensibilidade e permite a quantificação absoluta de analito. No entanto, os testes ELISA tradicionais exigem uma quantidade relativamente grande de amostra (normalmente 100 l), tem alto sinal de fundo em alguns fluidos biológicos, e são restritas para a medição de um único analito por ELISA 2. Recentemente, uma nova técnica de imunoensaio foi introduzido que contorna muitos destes inconvenientes. Este ensaio modificado, desenvolvido pela MSD, utiliza electrochemiluminesccia (ECL) para a detecção de sinal, o que permite por um fundo muito baixo e uma sensibilidade aumentada, permitindo a utilização de pequenos volumes de amostra. Electroquimioluminescência é baseado na molécula repórter ruténio (II) trisbipyridal, que está ligado aos anticorpos de detecção. Esta molécula repórter emite luz a 620 nm após a estimulação eléctrica do fundo da placa de 96 poços, que tem eléctrodos de carbono integrados neles 3. Além disso, por meio de revestimento local, os anticorpos de captura múltiplas podem ser revestidos para um poço (até 10 numa placa de 96 poços), permitindo a quantificação simultânea de proteínas diferentes numa única amostra 4. Esta técnica tem sido recentemente usado para medir perfis de citoquinas pró-inflamatórias em soro 5,6. As placas de multiplex MSD comparam favoravelmente com outras plataformas de ensaio multiplex 7. Usando MSD como a plataforma de ensaio primário, desenvolvemos mais uma custom made placa 3-plex que cuma quantificar simultaneamente o nível de ligação às proteínas miosina cardíaca-C (cMyBP-C), creatina-quinase MB (CK-MB) e troponina I (cTnI), e os resultados foram comparados com detecção Monoplex de cMyBP-C. CK-MB e cTnI são biomarcadores bem estabelecidos para MI. No entanto, os aumentos destes biomarcadores pode ser causada por outras patologias que MI, por exemplo miocardite ou insuficiência renal 8. Isto argumenta a favor da adição de biomarcadores adicionais para aumentar a especificidade do diagnóstico de EM. Mostrámos recentemente que cMyBP-C é também um biomarcador potencial de MI 9. cMyBP-C é uma proteína associada filamento grosso que é expresso no coração, 10-12, mas não nos músculos esqueléticos ou lisa. Assim, o aumento do nível de cMyBP-C no sistema circulatório é um indicador específico de danos cardíacos 13. Neste estudo, comparou-se a detecção de Uniplex cMyBP-C com a utilização de um ensaio de 3-Plex personalizado para medir os níveis séricos decMyBP-C, CK-MB e cTnI no soro de pacientes com MI. No futuro, esta técnica de assinatura pode ser usado para diagnosticar MI em pacientes com dor torácica na sala de emergência. O conselho de revisão institucional (IRB), da Loyola University Chicago aprovou o estudo para o uso de amostras humanas deidentified eo uso do imunoensaio (LU # 20392).

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>MSD Read-Buffer T (4X) with surfactant</td>
			<td>Meso Scale Discovery</td>
			<td>R92TC-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Acros</td>
			<td>9005-64-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MSD Blocker A</td>
			<td>Meso Scale Discovery</td>
			<td>R93BA-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10X</td>
			<td>Fisher BioReagents</td>
			<td>BP399-4</td>
			<td>Filter before use</td>
		</tr>
		<tr>
			<td>Myosin binding protein C antibody, mouse monoclonal (E7), gelatin free</td>
			<td>Santa Cruz</td>
			<td>SC-137180L</td>
			<td>For coating, antibody has to be gelatin free</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Plates and Equipment</td>
		</tr>
		<tr>
			<td>Multi-Array 96-well standard plate</td>
			<td>Meso Scale Discovery</td>
			<td>L15XA-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A prototype 3-plex, four-spot, 96-well plate with cMyBP-C, cTnI and CK-MB</td>
			<td>Meso Scale Discovery</td>
			<td>N452A-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ThermaSeal Sealing Film</td>
			<td>Life Science Products Inc.</td>
			<td>ST-3098</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MSD SECTOR Imager 2400A</td>
			<td>Meso Scale Discovery</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MTS 2/4 digital microtiter shaker</td>
			<td>IKA</td>
			<td>3208001</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a sensitive and specific quantitation method for determination of serum cardiac myosin binding protein-c by electrochemiluminescence immunoassay

Biomarcadores são cada vez mais importante na tomada de decisão clínica, bem como a ciência básica. Diagnóstico de enfarte do miocárdio (MI) é amplamente determinado por detecção de proteínas cardíacas específicas no soro ou plasma do paciente, como um indicador de lesão do miocárdio. Tendo recentemente demonstrado que a proteína de miosina cardíaca-C (cMyBP-C) é detectável no soro após o IM, propusemos como um biomarcador potencial de MI. Os biomarcadores são tipicamente detectados por sandwich enzyme-linked immunosorbent tradicionais. No entanto, esta técnica requer um grande volume de amostra, tem uma pequena gama dinâmica e pode medir apenas uma proteína de cada vez. Aqui, mostramos um imunoensaio multiplex no qual três proteínas cardíacas pode ser medida simultaneamente com elevada sensibilidade. Medindo cMyBP-C em Uniplex ou em conjunto com creatina quinase MB e troponina I apresentaram sensibilidade comparáveis. Esta técnica utiliza a Meso Scale Descoberta(MSD) método de multiplexação de uma placa de 96 poços juntamente com electroquimioluminescência para a detecção. Enquanto apenas pequenos volumes de amostra são necessários, elevada sensibilidade e uma grande gama dinâmica são alcançados. Usando esta técnica, medimos cMyBP-C, MB da creatina quinase e os níveis de troponina I em amostras de soro de 16 pacientes com IAM e compararam os resultados com 16 indivíduos de controle. Fomos capazes de detectar todos os três marcadores nestas amostras e encontrou os três biomarcadores para ser aumentado após o IM. Esta técnica é, portanto, adequado para a detecção sensível de marcadores cardíacos em amostras de soro.

Os princípios básicos e fluxo de trabalho do ensaio 3-Plex são mostrados na Figura 1, e o fluxo de trabalho geral está descrito na Tabela 1. Ensaios UNIPLEX trabalhar ao longo do mesmo princípio, excepto que toda a parte inferior também é revestida com um anticorpo de captura. A detecção do sinal é feito por ECL em que um sinal eléctrico é aplicado à parte inferior do poço. Isto, por sua vez, inicia uma produção local de luz através de uma reacção química da leitura ...

Watch this Scientific Journal Video about A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay at JoVE.com

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarcadores de medição em amostras biológicas complexas é cada vez mais guiando tomada de decisão clínica. Nós descrevemos um método altamente sensível para medir simultaneamente miosina cardíaca binding protein-C, MB da creatina quinase e troponina I em amostras de soro de pacientes com infarto do miocárdio e controle de indivíduos saudáveis.

Um método de quantificação sensível e específico para Determinação do Serum miosina cardíaca Binding Protein-C por Imunoensaio electroquimioluminescência

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

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16.6K visualizações.  Loyola University Chicago. Biomarcadores de medição em amostras biológicas complexas é cada vez mais guiando tomada de decisão clínica. Nós descrevemos um método altamente sensível para medir simultaneamente miosina cardíaca binding protein-C, MB da creatina quinase e troponina I em amostras de soro de pacientes com infarto do miocárdio e controle de indivíduos saudáveis.

Vídeo: A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - um método para identificar em toda a Transcriptoma Sítios de Ligação de Proteínas RNA Binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biologia

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Um Método para Mouse ilhotas pancreáticas Isolamento e intracelular cAMP Determinação

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Método de purificação de proteína livre de Determinação Affinity Binding por termoforese Microscale

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Uma rápida e específica de microplaca de ensaio para a determinação de ascorbato intra-e extracelular em células cultivadas

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Uma estratégia para sensíveis, Large Scale quantitativos Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

qPCR é um método sensível e rápido para detecção de DNA por citomegalovírus em formalina-fixo, tecido da biópsia de parafina-embedded

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Ligadura altamente eficiente de pequeno RNA moléculas microRNA para quantificação por sequenciamento de alto rendimento

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

A quantificação da Adesividade celular endotelial<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

O isolamento de regiões específicas genômico e identificação de moléculas pela Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.