SZ y DW reciben una subvención sin restricciones de Viropharma.

1. Preparación de los glóbulos rojos tratados con bromelina <ol><li> Glóbulos rojos Lave 0 mecanografiadas-3X con PBS 1x (centrifugación a 664 xg durante 2-5 min). </li><li> Incubar 1 volumen de glóbulos rojos empaquetados con dos volúmenes de una solución de bromelina 0,5% durante 10 min a 37 ° C. </li><li> Se lavan las células 3 veces con 1 × PBS. </li><li> Las células se pueden almacenar como una solución de 3% a 4 ° C en 1x PBS durante al menos una semana. </li></ol> 2. C3 y C4 deposición Análisis en glóbulos rojos por FACS <ol><li> Calor-inactivar la cantidad requerida de suero del paciente (u otra fuente de anticuerpo anti-RBC) por calentamiento de la muestra durante 30 min a 56 ° C. </li><li> Lavar los glóbulos rojos tratado con bromelaína tres veces en GVB - (5 mM de veronal, NaCl 150 mM, 0,05% de gelatina de pH 7,4) y resuspender en GVA + + (VBG - + 2 mM de MgCl 2 + CaCl2 10 mM) para dar una solución al 0,5%. </li><li> Pipetear los siguientes componentes en una placa de fondo redondo con una perla de vidrio (fo una mezcla adecuada): 25 mu l 0,5% glóbulos rojos, 37,5 l fresco suero AB (fuente de complemento) y 37,5 l anti-C5 200 g / ml. Anti-C5 es caro y podría ser difícil de obtener. Se podría considerar el uso de suero C5-/C6-/C7- o C8-deficiente en lugar de suero AB y anti-C5. </li><li> Añadir suero de paciente por pocillo a una concentración final de 0,1 a 33%. Corregir las diferencias en concentración de suero por pocillo debido utilizando suero AB inactivado por calor. Añadir GVA + + para obtener un volumen final de 150 l / pocillo. Cerrar la placa con papel de ELISA. </li><li> Incubar 1,5-2 horas a 37 ° C mientras se agita. </li><li> Lavar los eritrocitos tres veces con PBS + 0,5% de BSA (centrifugación a 664 xg durante 2 min). </li><li> Añadir marcadas fluorescentemente anti-C3 y anticuerpos monoclonales anti-C4 o sus fragmentos Fab (para reducir la aglutinación) en PBS + 0,5% de BSA a una concentración final de 1 g / ml. Aquí desarrollado de encargo anti-C3-Alexa Fluor 488 y se usan anti-C4-Alexa Fluor 647 anticuerpos monoclonales, la elección de los these etiquetas fluorescentes permite la detección simultánea de C3 y C4 por FACS. </li><li> Incubar 30-45 minutos a temperatura ambiente con agitación suave. </li><li> Lavar los hematíes 3 veces con PBS + 0,5% de BSA. </li><li> Resuspender los eritrocitos 150 l de PBS 0,5% de BSA y la pipeta en una nueva placa (para deshacerse de las perlas de vidrio antes del análisis FACS). </li><li> Realizar análisis FACS. Células individuales separadas de dobletes en una dispersión de conspirar FSC-A, establecer la puerta de los glóbulos rojos individuales; seleccionar el canal apropiado para la detección de las señales de fluorescencia (por ejemplo, FITC y APC). </li><li> Cuantificar los resultados utilizando la intensidad media de fluorescencia. </li></ol> Nota: Es posible utilizar un control de isotipo (por ejemplo, etiqueta fluorescente anti-IL-6 IgG1 de ratón). Sin embargo, se recomienda incluir un cierto control negativo, por ejemplo, mediante la incubación de los glóbulos rojos sin suero del paciente (sólo suero AB) o sin una fuente de complemento (suero AB inactivado por calor y suero del paciente inactivado por calor), unad entonces manchar estos con el mismo anti-C3-Alexa Fluor 488 y anti-C4-Alexa Fluor 647 anticuerpos. Esto da un control más válido que un control de isotipo en el caso de las células rojas de la sangre 12, aunque estos diferentes controles suelen dar la misma, resultado negativo. 3. Ensayo hemolítico cuantitativa <ol><li> Calor-inactivar la cantidad requerida de sueros de pacientes (u otra fuente de anticuerpo anti-RBC) por calentamiento de la muestra de 30 min a 56 ° C. </li><li> Lavar los hematíes 3 veces con VBG - y una vez con GVA + +. </li><li> Pipetear los siguientes componentes en una placa de fondo redondo con una perla de vidrio (para la mezcla adecuada): 35 l 3% RBC, 25 l fresco suero AB (fuente de complemento) y 1-50% de suero de paciente. Corregir las diferencias en concentración de suero utilizando suero AB inactivado por calor. Añadir GVA + + para obtener un volumen final de 150 l / pocillo. Cerrar la placa con papel de ELISA. </li><li> Incubar a 37 ° C durante 1,5-2 h con agitación. </li><li> Cplaca entrifuge a 664 xg durante 5 min con una reducción de deceleración (p. ej deceleración hasta la parada completa en 2-3 min). </li><li> Cuidadoso pipeteado 90 l sobrenadante en una nueva placa de microtitulación. Es importante en este paso para evitar las burbujas de aire y evitar de tomar a lo largo de las células intactas. </li><li> Mida A414/690 en un espectrofotómetro adecuado. </li><li> Expresar la lisis como un porcentaje de la lisis de los glóbulos rojos con una muestra de agua pura. </li></ol>

<ol>
	<li>Zeerleder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+haemolytic+anaemia+-+a+practical+guide+to+cope+with+a+diagnostic+and+therapeutic+challenge.">Autoimmune haemolytic anaemia - a practical guide to cope with a diagnostic and therapeutic challenge.</a> Neth. J. Med. 69 (4), 177-184 (2011).</li><li>Reardon, J. E., Marques, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+evaluation+and+transfusion+support+of+patients+with+autoimmune+hemolytic+anemia.">Laboratory evaluation and transfusion support of patients with autoimmune hemolytic anemia.</a> Am. J. Clin. Pathol. 125, 71-77 (2006).</li><li>Lai, M., Leone, G., Landolfi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+hemolytic+anemia+with+gel-based+immunohematology+tests.">Autoimmune hemolytic anemia with gel-based immunohematology tests.</a> Am. J. Clin. Pathol. 139 (4), 457-463 (2013).</li><li>Zantek, N. D., Koepsell, S. A., Tharp Jr, ., R, D., Cohn, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+direct+antiglobulin+test:+A+critical+step+in+the+evaluation+of+hemolysis.">The direct antiglobulin test: A critical step in the evaluation of hemolysis.</a> Am. J. Hematol. 87 (7), 707-709 (2012).</li><li>Natukunda, B., Brand, A., Schonewille, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell+alloimmunization+from+an+African+perspective.">Red blood cell alloimmunization from an African perspective.</a> Curr. Opin. Hematol. 17 (6), 565-570 (2010).</li><li>Packman, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolytic+anemia+due+to+warm+autoantibodies.">Hemolytic anemia due to warm autoantibodies.</a> Blood Rev. 22 (1), 17-31 (2008).</li><li>Berentsen, S., Tj&#248;nnfjord, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+cold+agglutinin+mediated+autoimmune+hemolytic+anemia.">Diagnosis and treatment of cold agglutinin mediated autoimmune hemolytic anemia.</a> Blood Rev. 26 (3), 107-115 (2012).</li><li>Petz, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+antibody+autoimmune+hemolytic+anemia.">Cold antibody autoimmune hemolytic anemia.</a> Blood Rev. 22 (1), 1-15 (2008).</li><li>Barros, M. M. O., Blajchman, M. A., Bordin, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warm+autoimmune+hemolytic+anemia:+recent+progress+in+understanding+the+immunobiology+and+the+treatment.">Warm autoimmune hemolytic anemia: recent progress in understanding the immunobiology and the treatment.</a> Transfus. Med. Rev. 24 (3), 195-210 (2010).</li><li>Endoh, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+prewarming+conditions+for+Rh+antibody+testing.">Optimal prewarming conditions for Rh antibody testing.</a> Transfusion. 46 (9), 1521-1525 (2006).</li><li>Wouters, 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=C1-esterase+inhibitor+concentrate+rescues+erythrocytes+from+complement-mediated+destruction+in+autoimmune+hemolytic+anemia.">C1-esterase inhibitor concentrate rescues erythrocytes from complement-mediated destruction in autoimmune hemolytic anemia.</a> Blood. 121 (7), 1242-1244 (2013).</li><li>Arndt, P. A., Garratty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Review+of+Published+Methods+for+Analysis+of+Red+Cell+Antigen-Antibody+Reactions+by+Flow+Cytometry,+and+Approaches+for+Resolving+Problems+with+Red+Cell+Agglutination.">A Critical Review of Published Methods for Analysis of Red Cell Antigen-Antibody Reactions by Flow Cytometry, and Approaches for Resolving Problems with Red Cell Agglutination.</a> Transfus. Med. Rev. 24 (3), 172-194 (2010).</li></ol>

Los autores no tienen nada que revelar.

Los ensayos anteriores son reproducibles y robusto. Ellos son fáciles de realizar y es posible llevar a cabo con muchas muestras al mismo tiempo (en una placa de 96 pocillos). Por lo tanto, este enfoque también sería adecuado para un sistema totalmente automatizado, por ejemplo, un sistema de robot de ELISA. En contraste con las técnicas utilizadas actualmente, estos ensayos son cuantitativos y esto ayudará por ejemplo, en el estudio del efecto de inhibidores del complemento. Por otra parte, la in...

Los anticuerpos contra los glóbulos rojos (GR) pueden ser inducidos por la transfusión de glóbulos rojos que expresan un antígeno que no está presente en los glóbulos rojos receptores. Estos aloanticuerpos pueden causar reacciones hemolíticas agudas graves de transfusión debido a la activación del complemento en la siguiente transfusión 5. En la anemia hemolítica autoinmune (AHA), los pacientes tienen autoanticuerpos contra sus propios glóbulos rojos. Esto conduce a acelerado de aprobación de las células a través de la interacción de IgG unida a los glóbulos rojos con Fc gamma-receptores en los fagocitos en el bazo y / o en caso de auto-anticuerpos capaces de activar el complemento a través de los receptores del complemento en el hígado 6,7. La activación del complemento fulminante resultando en hemólisis intravascular es raro pero a menudo mortal. Acelerado, complementar destrucción de glóbulos rojos mediada inducida por cualquiera de los resultados de alo-o autoanticuerpos en la anemia aguda y la hipoxia tisular, por tanto, potencialmente fatal. Los auto-anticuerpos en AIHA se clasifican en los anticuerpos fríos y calientes, depending de la temperatura óptima que se unen a los glóbulos rojos (37 ° C o inferior, respectivamente). Los anticuerpos calientes son por lo general de isotipo IgG y los anticuerpos fríos del isotipo IgM 8,9. AIHA puede ser secundaria a trastornos lymphoprolyferative por ejemplo, enfermedades del tejido conectivo, tumores sólidos, infecciones o medicamentos, pero en 50% de los casos AIHA es idiopática 9. La detección de gammaglobulinas (por ejemplo. IgG o IgM) y complementar unido a glóbulos rojos del paciente se realiza por medio de una prueba de antiglobulina directa semicuantitativa (Coombs) (DAT). En el DAT glóbulos rojos del paciente se incuban con anti-IgG o anti-C3d. Ocurrencia de RBC de aglutinación demuestra la presencia de componentes del complemento adjuntos o unión de IgG. La detección de alo-o autoanticuerpos en el suero del paciente se realiza a través de la prueba de antiglobulina indirecta (IAT). En el IAT, los RBCs de ensayo tratado con bromelaína se incuban con suero de paciente, se lavaron y después se incubaron con anti-hUman IgG. En el caso de los glóbulos rojos han sido sensibilizados con anti-IgG RBC presente en la aglutinación de suero del paciente se producirá. Anticuerpos IgM contra RBC conducirán directamente a la aglutinación tras la incubación de los eritrocitos de prueba bromelina tratados con suero del paciente. RBC aglutinación en la prueba de Coombs directa o en el IAT se evaluó visualmente, ya sea por el ojo en un tubo de ensayo o mediante la carga de la muestra en una columna de Sephadex pequeña separación de los glóbulos rojos aglutinados e individuales por tamaño 1. Otra técnica utilizada frecuentemente para medir la activación del complemento en los glóbulos rojos es el ensayo hemolítico 1, en ​​el que se evalúa la capacidad de suero del paciente para inducir (mediada por el complemento) hemólisis de los glóbulos rojos tratado con bromelaína 10. La prueba se considera como positiva cuando el sobrenadante después de la centrifugación se tiñe de color rojo debido a la hemoglobina liberada y considera negativa si permanece incoloro. Tanto la prueba de antiglobulina y el ensayo hemolítico son semicuantitativo, puesto que la más alta Serum dilución se denota en el que la prueba es todavía positiva. La prueba de Coombs y el ensayo hemolítico son ensayos robustos que se utilizan de forma rutinaria en el diagnóstico. Dado que estos ensayos son semicuantitativa y depende de la experiencia del técnico que realiza el ensayo no son adecuados para estudiar las diferencias sutiles de la activación del complemento en eritrocitos, según sea necesario cuando la evaluación de la eficacia de los inhibidores del complemento. Por lo tanto, hemos desarrollado dos ensayos cuantitativos para determinar la activación del complemento por la (auto)-anticuerpos contra los glóbulos rojos, que vamos a describir en este artículo. En primer lugar, hemos desarrollado un ensayo para medir la deposición de fragmentos de activación de complemento C3 y C4 en glóbulos rojos (Figura 1A). En este ensayo, humanos tratado con bromelaína tipo-0 glóbulos rojos se incuban con suero inactivado por calor paciente (fuente de anticuerpo anti-RBC), suero AB fresco (fuente de complemento) y anticuerpo monoclonal anti-C5 (eculizumab). Durante este Incubation, C3 y C4 deposición se producirá si el suero de paciente contiene complementar la activación de anticuerpos anti-RBC. Con el fin de evitar la lisis de glóbulos rojos por la activación del complemento aguas abajo se añade un anticuerpo monoclonal anti-C5 de bloqueo. Siguiente, C3 y C4 deposición sobre el RBC es detectada por FACS utilizando anticuerpos monoclonales marcados con fluorescencia o fragmentos Fab que reaccionan con C3 y C4, respectivamente. Supresión de un solo RBC es importante para garantizar la fiabilidad de los resultados. Ventajas de esta técnica incluyen que se requiere un pequeño volumen de material del paciente, la activación del complemento en una etapa temprana de la cascada es evaluada y el método es reproducible y cuantitativo. Una ventaja adicional de mirar tanto C3 y C4 es que una distinción se puede hacer entre la activación clásica y de lectina vía (tanto C3 y C4 deposición) y la activación de la vía alternativa (sólo la deposición de C3). El uso de glóbulos rojos tratado con bromelaína en lugar de los glóbulos rojos sin tratar aumenta la sensibilidad de la comodecir. El segundo ensayo se basa en el ensayo hemolítico utilizado actualmente (Figura 1B). Bromelina tratados Humanos de tipo 0 RBC se incuban con suero inactivado por calor paciente (fuente de anticuerpos anti-RBC) y suero AB fresca (fuente de complemento). Si el complemento se activa por anticuerpos anti-RBC del paciente, se producirá la lisis dependiente de la dosis de glóbulos rojos, lo que conduce a la liberación de hemoglobina. La cantidad de hemoglobina liberada se cuantificó midiendo su absorbancia a 414 nm en el sobrenadante después de centrifugar los glóbulos rojos intactos y fragmentados. La absorbancia se correlaciona con la cantidad de hemólisis producido. En contraste con el ensayo utilizado en la actualidad, este protocolo permite un objetivo, valor cuantitativo de hemólisis que es muy reproducible y no depende de la persona interpretar el ensayo. Una aplicación de estos métodos ha sido descrita en 11, en ​​el que el uso potencial de C1-inhibidor como inhibidor del complemento en la AIHA era studied.

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:195px;">Fresenius Kabi</td>
			<td style="width:119px;"></td>
			<td style="width:316px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">B-4287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Barbital</td>
			<td style="width:195px;">Fagron</td>
			<td style="width:119px;">0261</td>
			<td>This chemical is subject to drug regulation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Barbital</td>
			<td style="width:195px;">Fagron</td>
			<td style="width:119px;">0263</td>
			<td>This chemical is subject to drug regulation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelatin</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.0470.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.06404.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.05833.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.0238.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 488 amine reactive dye</td>
			<td style="width:195px;">Pierce</td>
			<td style="width:119px;">46402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 650 amine reactive dye</td>
			<td style="width:195px;">Pierce</td>
			<td style="width:119px;">62265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#945;C5 (Eculizumab)</td>
			<td style="width:195px;">Alexion Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS (Canto)</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;"></td>
			<td>Any FACS can be used that has the appropiate lasers.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectrophotometer (e.g. Multiskan spectrum)</td>
			<td style="width:195px;">Thermo Labsystems</td>
			<td style="width:119px;">1500-193</td>
			<td>Any spectrophotometer with the right wavelength range can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD FACSDiva software v 6.1.2</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;">643629</td>
			<td>Any compatible FACS analysis software can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bromelain</td>
			<td style="width:195px;">Sanquin</td>
			<td style="width:119px;">K1121</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for quantitative detection of antibody-induced complement activation on red blood cells

Los anticuerpos contra los glóbulos rojos (GR) pueden dar lugar a la activación del complemento que resulta en un procedimiento acelerado de aprobación a través de los receptores del complemento en el hígado (hemólisis extravascular) o bien dirigiéndose a la lisis intravascular de los glóbulos rojos. Aloanticuerpos (por ejemplo ABO) o los anticuerpos contra antígenos de glóbulos rojos (como se ve en la anemia hemolítica autoinmune, AHA) que conducen a la activación del complemento son potencialmente dañinas y pueden ser - especialmente cuando se conduce a la lisis intravascular - fatal 1. En la actualidad, la activación del complemento, debido a (auto)-anticuerpos en los glóbulos rojos se evalúa in vitro mediante el uso de la prueba de Coombs reflejando la deposición del complemento en eritrocitos o por un ensayo hemolítico no cuantitativo que refleja RBC lisis 1-4. Sin embargo, para evaluar la eficacia de los inhibidores del complemento, es obligatorio disponer de técnicas cuantitativas. Aquí se describen dos de estas técnicas. En primer lugar, un ensayo para detectar la deposición de C3 y C4 en las células rojas de la sangre que es inducida por anticuerpos en el suero del paciente es preSENTED. Para esto, el análisis FACS se utiliza con anticuerpos anti-C3 o anti-C4 marcados con fluorescencia. A continuación, se describe un ensayo hemolítico cuantitativa. Hemólisis mediada por el complemento En este ensayo, inducida por el suero del paciente se mide haciendo uso de la detección espectrofotométrica de la hemoglobina liberada. Ambos de estos ensayos son muy reproducible y cuantitativo, facilitar los estudios de activación del complemento inducida por anticuerpos.

La Figura 2A muestra un gráfico de dispersión representativo de glóbulos rojos. Gating Conveniente para los glóbulos rojos individuales se puede ver en de FSC-W - FSC-Una parcela (P1). Por lo general, alrededor de 95% de los glóbulos rojos caen dentro de esta puerta de P1 (células individuales), pero cuando se utiliza un alto porcentaje de suero del paciente, esto puede caer al 70-80%, especialmente cuando el anti-RBC IgM está presente en el suero del paciente. Los re...

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Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Aquí se describen dos ensayos para medir la activación del complemento inducida por anticuerpos contra las células rojas de la sangre. La principal ventaja sobre los ensayos actuales es su naturaleza cuantitativa y fácil de interpretar.

Los métodos para la detección cuantitativa de anticuerpos inducida por la activación del complemento en los glóbulos rojos

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

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Immunology and Infection

30.4K vistas.  University of Amsterdam.  Aquí se describen dos ensayos para medir la activación del complemento inducida por anticuerpos contra las células rojas de la sangre. La principal ventaja sobre los ensayos actuales es su naturaleza cuantitativa y fácil de interpretar. 

Video: Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

El agotamiento de las poblaciones de células específicas por el agotamiento del complemento

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Investigación

Inmunología e Infección

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Aislamiento de células de ratón cavidad peritoneal

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modelo murino de CD40-activación de las células B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Detección de actividad sialidasa Neu1 en la regulación de Toll-like receptor de activación

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Protocolo para las vacunas recombinantes para RBD basada en el SRAS: Preparación de la proteína, la vacunación de animales y la detección de neutralización

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

De alto rendimiento Método de detección de virus de la influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Antígenos funcional protegido glóbulos rojos por la membrana injerto de compactos Poligliceroles hiperramificados

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Los ensayos cualitativos y cuantitativos para la detección y caracterización de proteínas de antimicrobianos

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...