Este estudio fue financiado por los Institutos Nacionales de Salud subvenciones R01HL105826 y K02HL114749 (Dr. Sadayappan), y la Asociación Americana del Corazón Midwest Becas; 11PRE7240022 (Mr. Barefield) y 13POST14720024 (Dr. Govindan). Los autores agradecen la asistencia del Dr. Jimmy Page, Jill Clampit y el Dr. John Hudson, MSD, por su excelente apoyo técnico y la literatura en el desarrollo del ensayo.

1. Uniplex cMyBP-C Ensayo <ol><li> El día antes del experimento, cubra la placa estándar de 96 pocillos desnuda MSD con el anticuerpo de captura. Para cMyBP-C, utilizar un volumen de 30 l de anticuerpo anti-ratón cMyBP-C monoclonal (gelatina libre) a una concentración de 5 g / ml diluido en solución salina tamponada con fosfato (PBS). </li><li> Dado que el pozo es hidrófobo, pipetear la solución en la esquina inferior del pozo; luego toque los lados de la placa para extender la solución sobre todo el bien. </li><li> La placa se cubre con un sellador de placas y se incubaron O / N a 4 ° C sin agitación. </li><li> Eliminar la solución de anticuerpo de captura pulsando la solución a lo largo de un sumidero y luego en una pila de toallas de papel. La unión no específica a la placa se bloqueó mediante la adición de 150 l de un 5% (w / v) de una solución bloqueador (BSA de alto grado) en PBS a cada pocillo. </li><li> Tapar la placa e incubar durante 1 hora a temperatura ambiente con agitación a 700 rpm. </li><li> Durante la etapa de bloqueo, se preparan las normas y sejemplos. Hacer la serie estándar mediante la dilución de fragmento de proteína recombinante cMyBP-C (aminoácidos 1-271) a una concentración de partida de 2000 ng / ml en 1% (w / v) Un bloqueador / PBS. A continuación, se diluye en serie en un factor de 5 en 1% (w / v) Un bloqueador / PBS. Total de los estándares se utilizaron 7 + 1 blanco (1% (w / v) Un bloqueador de PBS solo /). </li><li> Eliminar la solución de bloqueo y se lava la placa 3x con 150 0,05% Tween-20/PBS l (v / v). Cada vez, eliminar la solución de lavado invirtiendo la placa sobre un fregadero. Después de la tercera etapa de lavado, película vigorosamente la placa sobre un lavabo y golpea suavemente la placa vigorosamente sobre una capa de papel de cocina hasta que esté completamente seco. Este es un paso crucial, ya que los volúmenes de incubación son pequeñas, y cualquier solución de lavado restante se diluye significativamente la siguiente incubación. </li><li> Pipetear 25 l de estándares y muestras en los pocillos. Tapar la placa e incubar a temperatura ambiente, con agitación a 700 rpm durante 1 hora. </li><li> Preparar la solución de anticuerpo de detección a 1 g / ml en 1% bloqueador de A / PBS. A custom hizo anticuerpo cMyBP-C (aminoácidos epítopo 2-14) con MSD SULFO-TAG etiquetado sirve como anticuerpo de detección. Los kits están disponibles para sulfo-TAG etiquetado, por lo que es un procedimiento relativamente simple para etiquetar cualquier anticuerpo. </li><li> Repetir la etapa de lavado como se ha descrito en el paso 1.4. </li><li> Añadir 25 l de solución de anticuerpo de detección a cada pocillo, sellar la placa, y se incuba a TA durante 1 h en un agitador de placas ajustado a 700 rpm. </li><li> Durante el período de incubación, ejecute MSD de demostración-plate (placa con luces LED) para garantizar el correcto funcionamiento del Imager Sector y software (consulte la sección de reactivo). También, se diluye la lectura-tampón, que se proporciona por MSD, mediante la adición de 5 ml de tampón de lectura-4x a 15 ml de agua destilada. </li><li> Repetir la etapa de lavado como se ha descrito en el paso 1.4. </li><li> Añadir 150 l de buffer de lectura a cada pocillo utilizando una pipeta multicanal. Asegúrese de evitar burbujas de aire (pipeteo reverso es un método adecuado). Después de la adición de la lectura-buffer, escanear inmediatamente la placa en el SectorImager. </li></ol> 2. 3-plex cMyBP-C, CK-MB y cTnI Ensayo <ol><li> Una placa de 96 pocillos 3-plex y diluyente soluciones se dejaron equilibrar a la temperatura ambiente antes de su uso. Esta placa es pre-recubiertas con anticuerpos de captura para cMyBP-C, CK-MB y cTnI por MSD. </li><li> Añadir 25 l de 1% (w / v) Un bloqueador / PBS a cada pocillo. Pulse en los lados de la placa para asegurarse de que todo el pozo está cubierto por la solución. </li><li> Sellar la placa con un sellador de placas, coloque la placa en un agitador de placas, y agitar a 700 rpm durante 30 min. </li><li> Mientras tanto, se preparó una serie de dilución de los calibradores individuales. Debido a que cada pocillo tiene tres anticuerpos de captura tres calibradores deben ser mezclados y diluidos en serie antes de su uso. Diluir cMyBP-C recombinante, CK-MB (MSD), y cTnI (MSD) juntos en 1% (w / v) Un bloqueador / PBS para crear una muestra con 2000 ng / ml cMyBP-C, 100 ng / ml CK- MB, y 25 ng / ml de cTnI (muestra A). </li><li> Un volumen final de al menos 100 l se prepara para cada estándar. Acompletar la serie estándar, diluir en serie las muestras están en un factor de 5. Usa 25 l de muestra A en 100 l 1% (w / v) Un bloqueador / PBS para crear muestra B. A continuación, utilice 25 l de la muestra B en 100 l 1% (w / v) Un bloqueador / PBS para crear muestra C , y así sucesivamente. Muestras de suero humano de 16 pacientes con IM y 16 sujetos de control se utilizan para medir los niveles de cTnI cMyBP-C, CK-MB, y. Estas muestras se diluyeron 2 veces en 1% (w / v) Un bloqueador / PBS. </li><li> Añadir 25 l de los estándares y las muestras diluidas a la 25 l ya presente en los pocillos de la placa 3-plejo. Es importante también incluir un espacio en blanco (sólo diluyente). Para establecer la técnica, las muestras se cargan en triplicado técnicas. De lo contrario, los duplicados son suficientes. </li><li> Sellar la placa de nuevo (sellador de placas puede ser reutilizado) y se incuba en un agitador de placas (700 rpm) a temperatura ambiente durante 2 h. </li><li> Justo antes de que se terminó la incubación, se preparó la solución de anticuerpo de detección. El diluyente utilizado es 1% (w / v) en PBS Un bloqueador. Todos THRanticuerpos de detección ee sulfo-etiquetados se diluyen juntos en una concentración final de 1 mg / ml cada uno. Detección de anticuerpos se proporcionan normalmente a una concentración madre 50 veces. </li><li> Para una placa de 96 pocillos completa, se necesita 2.700 l de solución total diluido (con pipeteado de error incluido). Añadir 54 l de cada anticuerpo de detección a 2.538 l 1% (w / v) Un bloqueador / PBS. </li><li> Después de 2 horas, eliminar soluciones agitando vigorosamente por la placa por encima de un fregadero. Lavar los pocillos 3 veces con 150 l de 0,05% de Tween-20 en PBS. Añadir 25 l de solución de anticuerpo de detección a cada pocillo usando una pipeta multicanal, y los lados de la placa son parches para garantizar que todo el pozo está cubierto por la solución. </li><li> Tapar la placa e incubar durante 1 hora con agitación a 700 rpm. </li><li> Durante el período de incubación, ejecute MSD de demostración-plate (placa con luces LED) para garantizar el correcto funcionamiento del Imager Sector y software. También diluir la lectura-tampón, que se proporciona por MSD, mediante la adición de 5 ml de 4x lecturaamortiguar a 15 ml de agua destilada. </li><li> Repetir el paso de lavado descrito en el paso 2.8. </li><li> Añadir 150 l de tampón de lectura a cada pocillo usando una pipeta multicanal. Evitar las burbujas de aire es crítico (pipeteo inverso es un método adecuado). Después de la adición de la lectura-buffer, escanear inmediatamente la placa en el Imager Sector. </li></ol>

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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>

Una solicitud de patente completa está pendiente (solicitud n º de serie 13/464, 466, Pub. No. EE.UU. 2012/0282618 A1 y Fecha:. 05/04/12) para determinar los factores de riesgo asociados a la degradación cMyBP-C y la liberación en el fluido corporal humano y cuantificar el nivel de cMyBP-C en el fluido corporal humano.

En este estudio, mostramos la aplicabilidad de un inmunoensayo múltiplex para la detección de múltiples biomarcadores cardíacos en el suero de los pacientes. Esta técnica tiene numerosas ventajas sobre ELISA tradicional. En primer lugar, ECL en el kit de ensayo se utiliza en lugar de la detección colorimétrica. En ECL, una señal eléctrica estimula la producción local de la propiedad medida (la luz), desacoplando así el caso de la estimulación de la señal, lo que reduce el fondo 14. Esto permite u...

La medición de bajas cantidades de proteína en muestras biológicas complejas, tales como el suero, es de creciente importancia clínica para el tratamiento del paciente, así como la ciencia básica. Por ejemplo, un aumento en los niveles séricos de biomarcadores cardíacos, tales como la troponina I, en un entorno clínico es compatible con infarto agudo de miocardio (MI) 1. Para detectar las proteínas en muestras de suero, ensayo de inmunoabsorción ligado a enzima estándar (ELISA) es la técnica utilizada con mayor frecuencia, ya que tiene una alta sensibilidad y permite la cuantificación absoluta del analito. Sin embargo, las pruebas ELISA tradicionales requieren una cantidad relativamente grande de muestra (típicamente 100 l), tienen una alta señal de fondo en algunos fluidos biológicos, y están restringidos a la medición de un solo analito por ELISA 2. Recientemente, una nueva técnica de inmunoensayo se introdujo que evita muchos de estos inconvenientes. Este ensayo modificado, desarrollado por MSD, utiliza electrochemiluminesccia (ECL) para la detección de la señal, lo que permite un fondo muy bajo y un aumento de la sensibilidad, que permite el uso de volúmenes de muestra pequeños. Electroquimioluminiscencia se basa en el reportero molécula de rutenio (II) trisbipyridal, que se une a los anticuerpos de detección. Esta molécula indicadora emite luz a 620 nm de la estimulación eléctrica de la parte inferior de la placa de 96 pocillos que tiene electrodos de carbono integrados en ellos 3. Además, mediante el uso de recubrimiento lugar, múltiples anticuerpos de captura pueden ser recubiertas en un pocillo (hasta 10 en una placa de 96 pocillos), lo que permite la cuantificación simultánea de diferentes proteínas en una sola muestra 4. Esta técnica se ha utilizado recientemente para medir los perfiles de citoquinas proinflamatorias en suero de 5,6. Las placas multiplex de MSD se comparan favorablemente con otras plataformas de ensayo multiplex 7. Usando MSD como la plataforma de ensayo principal, desarrollamos además un encargo placa 3-plex que cuna vez cuantificar el nivel de miosina cardiaca proteína de unión-C (cMyBP-C), MB de creatina-cinasa (CK-MB) y troponina I (cTnI), y los resultados se compararon con la detección monoplex de cMyBP-C. CK-MB y cTnI son marcadores biológicos bien establecidos para MI. Sin embargo, los aumentos de estos biomarcadores pueden ser causadas por patologías distintas de MI, por ejemplo, miocarditis o insuficiencia renal 8. Esto aboga por la adición de biomarcadores adicionales para aumentar la especificidad del diagnóstico de MI. Recientemente hemos demostrado que cMyBP-C es también un biomarcador potencial para MI 9. cMyBP-C es una proteína asociada a filamento grueso que se expresa en el corazón, 10-12, pero no en los músculos esqueléticos o lisos. Por lo tanto, el aumento del nivel de cMyBP-C en el sistema circulatorio es un indicador específico de daño cardíaco 13. En este estudio, se comparó la detección de uniplex cMyBP-C con el uso de un ensayo de 3-complejo de encargo para medir los niveles séricos decMyBP-C, CK-MB y cTnI en el suero de los pacientes con infarto de miocardio. En el futuro, esta técnica de firma puede ser utilizada para diagnosticar infarto de miocardio en pacientes con dolor torácico en urgencias. La junta de revisión institucional (IRB) de la Universidad de Loyola de Chicago aprobó el estudio para el uso de muestras humanas deidentified y el uso del inmunoensayo (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

Los biomarcadores son cada vez más importante en la toma de decisiones clínicas, así como la ciencia básica. Diagnóstico de infarto de miocardio (MI) es impulsado en gran medida mediante la detección de proteínas cardiacos específicos en el suero o plasma de los pacientes como un indicador de daño miocárdico. Después de haber demostrado recientemente que la miosina cardiaca proteína de unión-C (cMyBP-C) es detectable en el suero después de MI, se ha propuesto como un biomarcador potencial de MI. Los biomarcadores se detectan generalmente mediante ensayos inmunoenzimáticos sándwich tradicionales. Sin embargo, esta técnica requiere un gran volumen de muestra, tiene un pequeño rango dinámico, y puede medir sólo una proteína a la vez. A continuación os mostramos un inmunoensayo múltiplex en el que tres proteínas cardíacas se pueden medir simultáneamente con una alta sensibilidad. Medición cMyBP-C en uniplex o junto con creatina quinasa MB y troponina I cardiaca mostró una sensibilidad comparable. Esta técnica utiliza la Meso Escala Descubrimiento(MSD) método de multiplexación en una placa de 96 pocillos junto con electroquimioluminiscencia para la detección. Aunque sólo se necesitan pequeños volúmenes de muestra, se logran una alta sensibilidad y un amplio rango dinámico. Usando esta técnica, que mide cMyBP-C, MB de creatina quinasa y troponina I cardiaca en muestras de suero de 16 pacientes con MI y se compararon los resultados con 16 sujetos control. Hemos sido capaces de detectar los tres marcadores en estas muestras y encontramos los tres biomarcadores que aumentaron después de un IM. Esta técnica es, por lo tanto, adecuado para la detección sensible de los biomarcadores cardíacos en muestras de suero.

Los principios básicos y de flujo de trabajo de ensayo de la 3-plex se muestran en la Figura 1, el flujo de trabajo y en general se describe en la Tabla 1. Ensayos de Uniplex trabajan a lo largo del mismo principio, excepto que toda la parte inferior así se recubre con un anticuerpo de captura. Detección de la señal se realiza mediante ECL en que se aplica una señal eléctrica a la parte inferior del pozo. Esto, a su vez, inicia una producción local de la luz a través de una reac...

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 medición en muestras biológicas complejas es cada vez guían la toma de decisiones clínicas. Se describe un método altamente sensible para medir simultáneamente la miosina cardíaca proteína de unión-C, creatina quinasa MB, troponina I cardiaca y en muestras de suero de pacientes con infarto de miocardio y los sujetos de control sanos.

Un método de cuantificación sensible y específica para la determinación de suero miosina cardíaca proteína de unión-C por inmunoensayo electroquimioluminiscencia

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 vistas.  Loyola University Chicago. Biomarcadores de medición en muestras biológicas complejas es cada vez guían la toma de decisiones clínicas. Se describe un método altamente sensible para medir simultáneamente la miosina cardíaca proteína de unión-C, creatina quinasa MB, troponina I cardiaca y en muestras de suero de pacientes con infarto de miocardio y los sujetos de control sanos.

Video: 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 - un método para identificar transcriptoma en todo los sitios de unión de las proteínas de unión a ARN

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

Investigación

Biología

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.

Un método para ratón islotes pancreáticos Aislamiento y cAMP intracelular Determinación

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.

Purificación de proteínas libre de Método de Determinación afinidad de unión por termoforesis 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.

Un ensayo rápido y específico de microplacas para la Determinación de ascorbato intra y extracelular en 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.

Una estrategia para sensibles, grande escala cuantitativa Metabolómica

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 es un método sensible y rápido para la detección de ADN de citomegalovirus en fija en formol, biopsia de tejido embebido en parafina

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 los Pequeños ARN moléculas de microARN cuantificación mediante secuenciación de alto rendimiento

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.

La cuantificación de la adhesividad de la célula 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.

El aislamiento de regiones genómicas específicas e identificación de moléculas asociadas por 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.