The authors thank Miss Ching-Chun Lin and Miss Diana Lin of the Core Facility of the Institute of Cellular and Organismic Biology (ICOB) of Academia Sinica for offering their expertise on peptide synthesis and DNA sequencing, respectively. This study was supported by Academia Sinica.

1. Preparación del anticuerpo monoclonal <ol><li> Cultivar las células de hibridoma monoclonales de ratón RG-M56 2 en medio libre de suero en matraces 175T a 37 ºC con 5% de CO 2 suplemento. Recoger el sobrenadante cuando el color del medio se vuelve amarillo después de cinco días de incubación. NOTA: Las células de hibridoma se cultivaron en medio libre de suero para evitar la contaminación de anticuerpos de suero bovino fetal. </li><li> Centrifugar el sobrenadante a 4.500 xg durante 30 min a 4 ºC y desechar el sedimento de restos celulares. </li><li> Añadir 2 ml de agarosa de proteína G (suministrado como una suspensión 50%) a una columna de 5 ml y se equilibre con 10 volúmenes de resina (10 ml) de PBS enfriado en hielo. </li><li> Carga de 200 ml del sobrenadante de anticuerpo (paso 1.2) en la columna y deseche el paso a través. </li><li> Añadir 10 ml de helado de PBS a la columna para lavarla. Repetir dos veces. </li><li> Añadir 10 ml de glicina 50 mM, pH 2,7 a la columna para eluir el anticuerpo asociado a la proteína G. Columnalect 900 fracciones mu l en un tubo de microcentrífuga que contiene 100 l de 10x tampón de neutralización (1 M Tris, 1,5 M NaCl, y EDTA 1 mM, pH 8,0). </li><li> Almacenar el anticuerpo purificado en 50% de glicerol con 0,03% NaN 3 a -20 ºC. </li></ol> 2. Construcción y expresión de proteínas recombinantes en serie truncadas <ol><li> Preparar una mezcla de reacción PCR: 5 l de 10x tampón Pfu, 0,2 mM de cada dNTP, 0,2 mM de cebador directo 3, 0,2 M cebador inverso 3, 2 mM MgSO 4, 1 ng de pET20b-1A59 DNA 3 plásmido, y 2,5 U ( unidad) de ADN polimerasa Pfu; añadir ddH 2 O a un volumen final de 50 mL. <ol><li> Las muestras se ejecutan en un termociclador automático utilizando los siguientes parámetros: Ciclo 1 (94 ºC durante 5 min); 2-36 ciclos (94 ° C durante 30 s, 63 ° C durante 30 s, y 72 ° C durante 60 s); y el ciclo 37 (72 ºC durante 7 minutos). </li></ol></li><li> Se extrae la reacción en cadena de la polimerasa (PCR) los productos mediante el uso de un kit de purificación de PCR 5 para facilitar la continuación de la digestión con enzimas de restricción. </li><li> Digerir los fragmentos de ADN amplificados por PCR con enzimas de restricción Nde I y Xho I y se liga a cada uno de estos fragmentos de ADN en Xho I enzima escindido pET-20b (+) Nde I y vector. Transformar las construcciones en las células DH-5a-competente de Escherichia coli 6. <ol><li> Preparar la mezcla de digestión en tampón de digestión (20 mM Tris-acetato, Mg 10 mM (CH 3 COO) 2, KCH 50 mM 3 COO, y DTT 1 mM, pH 7,9) con 1 g de ADN amplificado por PCR o pET-20b (+) DNA vector, y 2 U de Nde I y Xho I restricción enzimas en un volumen final de 20 mL. </li><li> Mezclar la mezcla de digestión suave y rápidamente girar hacia abajo. Incubar a 37 ºC durante 2 h en un baño seco para asegurar el corte completo de la restricción sentarseES. </li><li> Extraer la enzima de fragmentos de ADN digerido de restricción usando un kit de purificación de PCR 5 para facilitar la siguiente construcción de plásmido. </li><li> Preparar la mezcla de ligación en tampón de ligación (Tris 66 mM, 5 mM MgCl 2 mM, ATP 1, y DTT 5 mM, pH 7,5) con 100 ng de ADN predigerido, amplificado por PCR, 10 ng de predigerido pET-20b (+) ADN vector, y 5 U de la ligasa de T4 ADN en un volumen final de 10 mL. </li><li> Mezclar la mezcla de unión suave y rápidamente girar hacia abajo. Se incuba a 16 ° C durante 18 h en un baño de agua. </li><li> Ponga 10 l de las muestras de ligación en 100 l de las células DH-5α-competentes y mezclar suavemente antes de colocar el tubo de microcentrífuga en hielo durante 30 min. Poner el tubo de microcentrífuga en un baño seco a 42 ºC durante 90 s para inducir choque térmico 7. transferir inmediatamente el tubo en hielo durante 2 min. </li><li> Añadir 900 l de caldo de Luria-Bertani (LB) (1% de Bacto triptona, 0,5% Bacto EXTRACTION levadurat, y 0,5% de NaCl, pH 7,0) al tubo. Incubar a 37 ºC con 150 rpm agitación durante 45 min. Sedimentar las células por centrifugación a 4000 x g durante 10 min y descartar el sobrenadante. </li><li> Resuspender el precipitado con 50 l de caldo LB y se extendió cada transformación en placas LB precalentado que contienen 100 mg / ml de ampicilina. Incubar las placas a 37 ° C durante 16 h. </li><li> Recoger una sola colonia usando una punta de 200 l y colocarlo en un caldo de 3 ml de LB que contiene 100 mg / ml de ampicilina en un tubo sin cubiertas 15 ml. Incubar a 37 ºC con 150 rpm agitación durante 12 h. </li><li> Extraer el ADN plasmídico de cada cultivo 8 y la secuencia usando el promotor T7 y cebadores del terminador T7 para confirmar la secuencia 9. </li></ol></li><li> Después de la confirmación de secuencia, transformar 10 ng de ADN de estos pET-20b (+) plásmidos con diferentes longitudes de gen de la proteína capa YGNNV en la cepa BL-21 (DE3) de E. coli por nosotrosing el método de choque térmico-7. Siga los pasos 2.3.6-2.3.8 para llevar a cabo la transformación. </li><li> Transferir una sola colonia de cada E. coli transformado BL-21 de células en caldo de 3 ml de LB que contiene 100 mg / ml de ampicilina en un tubo sin cubiertas 15 mL. Incubar el cultivo a 37 ºC con agitación 150 rpm. </li><li> Se enfría la cultura a 25 ºC cuando la OD 600 del cultivo es aproximadamente 0,6 y añadir IPTG a una concentración final de 0,4 mM para inducir la expresión de la proteína recombinante. Incubar el cultivo de un extra de 4 horas a 25 ° C con 200 rpm de agitación. </li><li> Transferir 1 ml del cultivo en un tubo de microcentrífuga. Sedimentar las células por centrifugación a 12.000 xg durante 1 min y descartar el sobrenadante. </li><li> Resuspender el sedimento celular en 100 l de tampón de desnaturalización (8 M urea, fosfato de sodio 20 mM, y 0,5 M NaCl, pH 7,4) pipeteando arriba y abajo con una micropipeta. Mezclar mediante agitación vigorosa. NOTA: El sol de la muestraluciones ahora deben ser semi-transparente, un poco pegajoso, y listo para el ensayo de hibridación dot-blot. </li></ol> 3. Diseño y síntesis de péptidos superpuestos <ol><li> Diseño y síntesis de péptidos 3 de 20 unidades de serie que cada superposición con su sucesor por 10 aa residuos de la región de 195 a 338 aa de la proteína de la cubierta YGNNV para delimitar la región epítopo de RG-M56 mAb mediante transferencia de puntos. </li><li> Diseñar y sintetizar péptidos 3 tres 8-mer (195 VNVSVLCR 202, 197 VSVLCRWS 204 y 199 VLCRWSVR 206) con un solapamiento de 6 aa residuos a la siguiente péptido sintético para limitar la región de epítopo 195-206 aa mediante transferencia de puntos. </li><li> Diseño y síntesis de 3 7-mer (196 NVSVLCR 202 y 195 VNVSVLC 201), 6-mer (195 VNVSVL 200, 196 NVSVLC 201, y 197 VSVLCR 202), y 5-mer péptidos (195 VNVSV 199, 196 NVSVL 200, 197 VSVLC 201, y 198 SVLCR 202) con una superposición de 6, 5, y 4 residuos de aa, respectivamente, en sus péptidos vecinos para reducir al mínimo la región epítopo mediante transferencia de puntos. </li></ol> 4. La hibridación dot-blot <ol><li> Disolver cada péptido sintetizado en sulfóxido de dimetilo (DMSO) a una concentración final de 10 mg / mL. NOTA: Para superar la variada solubilidad de péptidos sintéticos, todos los péptidos sintéticos debe disolverse en DMSO. DMSO es un buen disolvente para disolver completamente los péptidos hidrófobos o hidrófilos. </li><li> Remojar la membrana de fluoruro de polivinilideno (PVDF) con metanol durante 2 min. NOTA: membrana de PVDF es de hasta resistente a DMSO 100%; otros pueden no serlo. </li><li> Equilibrar la membrana de PVDF con tampón modificado Towbin (Tris 25 mM, glicina 192 mM, y 0,1% de SDS, pH 8,3) para2 minutos. NOTA: 10-20% (v / v) de metanol se puede añadir a un tampón de Towbin modificado para mejorar los resultados de la transferencia. </li><li> Enjuague un pedazo de papel de cromatografía con el tampón de Towbin modificado. Colocar la membrana de PVDF en el papel de cromatografía. Espere hasta que el tampón Towbin modificado ha desaparecido de la superficie de la membrana de PVDF antes de continuar con el siguiente paso. </li><li> Añadir 2 l de cada muestra de péptido a la membrana con una punta de 10 l. El aire seco de la membrana de PVDF en el papel de cromatografía durante 10 minutos. Añadir cada muestra péptido lenta y gradualmente sobre la membrana para evitar demasiada difusión. </li><li> Bloquear la membrana en tampón de TBST (0,05% (v / v) pH Tween-20, Tris 20 mM, y NaCl 150 mM, 7,4) con 5% de leche sin grasa para 30 min a temperatura ambiente con agitación suave. </li><li> Añadir RG-M56 mAb a una dilución final de 1: 1000 en tampón TBST con 5% de leche sin grasa a la membrana. Incubar la membrana a 37 ° C durante 1 h con agitación suave. </li><li> Retire el anticuerpo asílución. Lavar la membrana en tampón de TBST durante 5 min con agitación suave. Repetir dos veces. </li><li> Añadir el anticuerpo secundario (de cabra anti-IgG de ratón, Fc, conjugado con fosfatasa alcalina) en una dilución final de 1: 5000 en tampón TBST con 5% de leche sin grasa a la membrana. Incubar la membrana a 37 ° C durante 1 h con agitación suave. </li><li> Descartar la solución de anticuerpo. Lavar la membrana en tampón de TBST durante 5 min con agitación suave. Repita el paso de lavado dos veces. </li><li> Desarrollar la membrana con solución de sustrato BCIP / NBT a temperatura ambiente durante 15 min en la oscuridad. Detener el desarrollo lavando la membrana con ddH2O cuando aparezca la señal. </li><li> El aire seco de la membrana y capturar la imagen de transferencia puntual usando un sistema de imagen. </li><li> Medir la intensidad de cada transferencia de puntos utilizando el software de análisis de imagen 3. </li></ol> 5. barrido de alanina y sustitución <ol><li> Diseñar y sintetizar 3 alanina y methipéptidos de sustitución onine. Reemplazar cada residuo a bis con alanina para el péptido 8-mero 195 VNVSVLCR 202 y sintetizar péptidos 195 ANVSVLCR 202, 195 VAVSVLCR 202, 195 VNASVLCR 202, 195 VNVAVLCR 202, 195 VNVSALCR 202, 195 VNVSVACR 202, 195 VNVSVLAR 202 y 195 VNVSVLCA 202 . Reemplazar leucina residuos aa 200 a metionina para crear el genotipo SJNNV epítopo péptido 195 202 VNVSVMCR. </li><li> Siga la sección 4 para realizar el rastreo de alanina y mutagénesis de sustitución transferencia de puntos. </li></ol>

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The authors have no conflicts of interest related to this report.

Este protocolo ofrece una técnica rápida y simple para identificar un epítopo lineal mAb-reconocido. Teniendo en cuenta el coste de la síntesis de péptidos y la eficiencia de la producción de péptidos que sintetizan, la región antigénica de la proteína de cubierta del virus se redujo mediante la expresión de proteínas recombinantes en serie truncados antes del análisis de barrido de péptidos. Como tal, se utilizó el sistema de expresión de E. coli pET confiable y eficiente para producir estas pr...

En el sistema inmune, la recombinación de los segmentos V, D y J permite anticuerpos para crear enormes variaciones de las regiones determinantes de la complementariedad (CDR) para la unión a diversos antígenos para proteger al huésped de la infección patógena. La defensa de neutralización de anticuerpos contra los antígenos depende de la complementariedad espacial entre las CDR de los anticuerpos y los epítopos de los antígenos. Por lo tanto, la comprensión de esta interacción molecular ayudará diseño vacuna profiláctica y desarrollo de fármacos péptido terapéutico. Sin embargo, esta interacción de neutralización puede estar influenciada tanto por múltiples dominios antigénicos de un antígeno único y de múltiples CDR de anticuerpos, que en consecuencia hacen que el proceso de determinación de epítopo más complejo. Afortunadamente, el desarrollo de la tecnología de hibridoma, que se fusiona células productoras de anticuerpos individuales con células de mieloma, permite para un lote dividen constantemente de las células para secrete un anticuerpo específico, conocido como un anticuerpo monoclonal (mAb) 1. Las células de hibridoma producen estos mAbs, puros de alta afinidad para unirse a un único dominio antigénico de un antígeno específico. Con la relación de la antígeno-anticuerpo establecido, varios enfoques, incluyendo la exploración de péptidos, se puede utilizar para determinar el epítopo de un antígeno utilizando su mAb correspondiente. Los acontecimientos recientes en la tecnología de péptidos sintéticos han hecho que la técnica de exploración de péptidos más accesible y más conveniente para llevar a cabo. En pocas palabras, un conjunto de la superposición de péptidos sintéticos se producen según una secuencia de antígeno objetivo y están asociados a una membrana en un soporte sólido para el mAb hibridación. de barrido de péptidos no sólo ofrece una forma simple de un mapa de la región de unión a anticuerpo, sino que también facilita de aminoácidos (aa) mutagénesis de barrido a través de residuo o sustitución para evaluar la interacción de unión entre cada residuo aa del péptido epítopo y las CDR del anticuerpo. <p class = &quot;jove_content&quot;&gt; Aquí, el presente estudio describe un protocolo para la identificación eficiente del epítopo lineal de la proteína de la cubierta amarilla mero virus de la necrosis nerviosa (YGNNV) utilizando un anticuerpo monoclonal neutralizante 2, 3, 4. El protocolo incluye mAb preparación, construcción y expresión de proteínas recombinantes en serie truncadas, diseño de péptidos solapantes sintético, la hibridación dot-blot, barrido de alanina, y mutagénesis de sustitución. Teniendo en cuenta el alto costo de la síntesis de péptidos, el paso de truncar en serie las proteínas recombinantes de una proteína diana deseada se modificó, y la región antigénica se redujo a alrededor de 100 a 200 residuos de aa antes de que se realizó el análisis dot-blot array péptido sintético.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hybrid-SFM medium</td>
			<td>Gibco</td>
			<td>12045-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeccos&#39;s Phophate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu DNA Polymerase&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>EP0502&#160;</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Roche</td>
			<td>10799009001</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>NdeI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>XhoI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>pET-20b(+) vector</td>
			<td>Novagen, Merck Millipore</td>
			<td>69739</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli DH-5&#945; competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH617</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli BL-21(DE3) competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH217</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Amresco</td>
			<td>0339-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth&#160;</td>
			<td>Invitrongen</td>
			<td>12780-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropylthio-&#946;-D-thiogalactoside (IPTG)</td>
			<td>MDBio, Inc.</td>
			<td>101-367-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T.Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Amresco</td>
			<td>0167-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Affymetrix, USB</td>
			<td>75825</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Amresco</td>
			<td>0241-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Amresco</td>
			<td>0105-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Amresco</td>
			<td>0854-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>PerkinElmer</td>
			<td>NEL937001PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG, Fc fragment antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-055-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P (Polyvinylidene fluoride, PVDF)</td>
			<td>Merck Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Agarose Fast Flow</td>
			<td>Merck Millipore</td>
			<td>16-266</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP BioSpectrum 600 Image System</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>VisionWorks LS Analysis Software Ver 6.8</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>MyCycler thermal cycler</td>
			<td>BioRad</td>
			<td>1709713</td>
			<td></td>
		</tr>
	</tbody>
</table>

peptide scanning-assisted identification of a monoclonal antibody-recognized linear b-cell epitope

La identificación de un epítopo antigénico por el sistema inmune permite la comprensión del mecanismo de protección de anticuerpos neutralizantes que pueden facilitar el desarrollo de vacunas y fármacos peptídicos. de barrido de péptidos es un método simple y eficiente que los mapas de forma directa el epítopo lineal reconocido por un anticuerpo monoclonal (mAb). A continuación, los autores presentan una metodología de determinación epítopo participación de las proteínas truncadas recombinantes en serie, el diseño de péptidos sintéticos, y la hibridación dot-blot para el reconocimiento antigénico de la proteína de cubierta del virus de la necrosis nerviosa utilizando un anticuerpo monoclonal neutralizante. Esta técnica se basa en la hibridación dot-blot de péptidos y mAbs sintéticos sobre una membrana de fluoruro de polivinilideno (PVDF). La región antigénica mínima de una proteína de la cubierta viral reconocido por el RG-M56 mAb puede ser reducido a paso a paso recortado mapeo de péptidos en un péptido epítopo 6-mer. Además, barrido de alanina y mutagénesis residuo subinstitución puede llevar a cabo para caracterizar el significado unión de cada residuo de aminoácido que compone el epítopo. Se encontró que los residuos que flanquean el sitio epítopo para jugar un papel crítico en la regulación del péptido conformación. El péptido epítopo identificado puede ser utilizado para formar cristales de complejos de péptido-anticuerpo de epítopo para un estudio de difracción de rayos X y la competencia funcional, o para la terapéutica.

El objetivo de este experimento fue identificar un epítopo a través de transferencia de puntos usando mAb. Para obtener rápida y eficientemente por la región antigénica reconocida por mAb, de longitud completa y proteínas de la cubierta recombinante YGNNV en serie truncado con un marcador de fusión 6xHis en el extremo C-terminal se expresaron a partir de un sistema de expresión de E. coli PET 10 (Figura 1A). Las proteínas recombi...

Watch this Scientific Journal Video about Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope at JoVE.com

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

La identificación de un epítopo lineal de células B reconocido por anticuerpos monoclonales péptido Escaneo-asistida

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

peptide-scanning-assisted-identification-monoclonal-antibody

Research

JoVE Journal

Immunology and Infection

9.3K vistas.  Academia Sinica. Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Video: Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Péptido: MHC tetrámero basado en el enriquecimiento de células T específicos para el epítopo

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Investigación

Inmunología e Infección

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

La identificación basada en péptidos de motivos funcionales y sus parejas de unión

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Aislamiento de los precursores de células B subconjuntos de Sangre del Cordón Umbilical

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

El aislamiento, la identificación y purificación de células epiteliales del timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Generación de anticuerpos monoclonales murino por el hibridoma Tecnología

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generación de variantes de Escape de neutralizar anticuerpos Monoclonal del Virus de la Influenza

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Purificación de ADN Viral para la identificación de las proteínas virales y celulares asociadas

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

Biblioteca de péptido superpuestas a epitopos Qa-1 en una proteína

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika Virus específico diagnóstico del epítopo descubrimiento

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

Generación de anticuerpos monoclonales contra productos naturales

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...