Nos gustaría reconocer el Fondo Común NIH - Proteína Programa de reactivos de captura para financiar el desarrollo de la selección y caracterización de anticuerpos tubería recombinante de anticuerpos de red y de la Fundación Canadiense para la Innovación para la compra de financiación de la plataforma de selección robótica.

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Los autores no tienen nada que revelar.

Al llevar a cabo en la selección de anticuerpos in vitro, los dos determinantes principales de éxito selección son 1) el aislamiento de dianas antigénicas bien plegadas para seleccionar sobre y 2) la disponibilidad de una biblioteca de anticuerpos de alta diversidad funcional. En muchos casos, la disponibilidad de cantidades suficientes de bien plegada, proteína de longitud completa puede ser limitante. Un enfoque para superar esta limitación es el uso de dominios identificados a partir del anál...

Con el inicio de la era post-genómica, la disponibilidad de reactivos de unión de alta calidad para caracterizar y modular proteínas es esencial para abrir nuevas investigaciones y vías terapéuticas. Los anticuerpos siguen siendo fundamentales para los investigadores académicos e industriales como la investigación básica y las herramientas de diagnóstico y terapéuticos potenciales. No en vano, se ha registrado un impresionante crecimiento de las empresas de desarrollo de anticuerpos contrato, la mayoría de los cuales se basan en tecnologías de hibridoma convencionales para generar anticuerpos personalizados. Sin embargo, la selección in vitro usando bibliotecas de anticuerpos en fagos se está convirtiendo en una tecnología poderosa alternativa que puede ofrecer ventajas únicas y éxito donde las tecnologías convencionales pueden enfrentar limitaciones 1, 2. A la luz de la considerable demanda de anticuerpos de alta calidad como herramientas de investigación, dos retos principales para la generación de anticuerpos renovables son 1) el rendimiento de la selección y 2) av antígenodimensiones pueden ser. Un número de grupos han descrito en las tuberías de selección in vitro destinadas a aumentar el rendimiento y la tasa de identificación de anticuerpos. Estas descripciones detalle una variedad de enfoques viables que incluyen la selección a ya sea de longitud completa se dirige a 3,4, o dominios 5,6,7 estructuralmente relacionado, utilizando ya sea 6,8 o placa de base 3,4 esquemas de inmovilización del antígeno basado en perlas. Además, la creciente adopción de las tecnologías de síntesis de genes 9 ha hecho la generación de antígeno sistemática, en particular de dominios aislados, razonablemente rentable y puede potencialmente aliviar la dificultad en la obtención de cantidades suficientes de antígeno purificado, de longitud completa. Mediante el uso de las dos tecnologías en tándem, un oleoducto autónomo y generación antígeno escalable y selección de anticuerpos se ideó que permita el aislamiento paralelo de anticuerpos para grandes conjuntos de dominios de antígenos expresados ​​y facilitar el desarrollo de reactivos para characterizing clases enteras de proteínas estructuralmente o funcionalmente relacionados. Con este objetivo, un gasoducto integrado que las parejas en la identificación in silico de dominios antígeno expresables, síntesis de genes, expresión bacteriana de alto rendimiento de antígenos y escalables fagos selecciones de anticuerpos se ha desarrollado. Este gasoducto requiere sólo la infraestructura básica disponible para la mayoría de los laboratorios de ciencias de la vida (incluidas las bibliotecas de anticuerpos que son cada vez más accesibles a través de un acuerdo de licencia o de transferencia de material), pero también es susceptible de automatización para el uso a escala industrial. El uso de este protocolo, es posible generar cientos de dominios antigénicos de afinidad de etiquetado, y aislar fragmentos de anticuerpos rutinariamente altamente específicos para muchos de estos antígenos. La tecnología de presentación en fagos ha demostrado demostrado la compatibilidad con una amplia variedad de formatos de reactivo de afinidad recombinantes incluyendo Fab, scFv, Fv y los dominios autónomos y unacreciente variedad de pequeños &quot;marcos alternativos&quot; (proteínas ankyrin repetir diseñados (DARPins), fibronectina (FN), dominios de lipocalina y más 10). Discusión se limita en este ejemplo para el aislamiento de fragmentos de anticuerpos Fab, aunque se presume que estos métodos pueden adaptarse a otros tipos de bibliotecas. Utilizando esta tecnología, los Fab con baja afinidad nanomolar a pequeña, etiquetados dominios de la proteína incluyendo dominios del factor de transcripción, dominios SH2, proteínas y otros ARN vinculante se han seleccionado con éxito, muchos de los que se unen para pedir una proteína y son funcionales en inmunoensayos, como la inmunofluorescencia , inmunoprecipitación e inmunohistoquímica. Es importante destacar que los clones de unión recombinantes son totalmente renovable y se puede volver a generar a partir de construcciones de expresión a través de la producción bacteriana oferta mayor coherencia, la reproducibilidad y la rentabilidad, lo que justifica el gasto de validación clon riguroso. En este protocol y video que lo acompaña, se demuestran los métodos básicos para la selección de anticuerpos de bibliotecas de fagos utilizando dominios de antígenos inmovilizados. Este método particular emplea dominios de la proteína marcada con GST inmovilizado por adsorción pasiva en placas de micropocillos, aunque otras etiquetas 11,12,13 y formatos de selección 13,14,2 también se han utilizado con éxito. Consideraciones críticas para la configuración y el comportamiento de las selecciones con control paralelo de parámetros de selección destinados a identificar y aislar anticuerpos monoclonales específicamente enriquecidos para la validación se detallan.

<table border="0" cellpadding="0" cellspacing="0" width="470">
	<tbody>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:471px;">MATERIALS</td>
		</tr>
		<tr height="43">
			<td colspan="2" height="43" style="height:43px;width:249px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:87px;">Catalog Number</td>
		</tr>
		<tr height="40">
			<td colspan="2" height="40" style="height:40px;width:249px;">New Brunswick Innova44 stackable incubator shaker</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:87px;">M1282-0004</td>
		</tr>
		<tr height="40">
			<td colspan="2" height="40" style="height:40px;width:249px;">Liquidator 96 channel manual benchtop pipetting system</td>
			<td style="width:135px;">Anachem Ltd</td>
			<td style="width:87px;">LIQ-96-200</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Microplate shaker</td>
			<td style="width:135px;">VWR</td>
			<td style="width:87px;">12620-926</td>
		</tr>
		<tr height="40">
			<td colspan="2" height="40" style="height:40px;width:249px;">ThermoScientific Sorvall ST-40 benchtop centrifuge</td>
			<td style="width:135px;">Thermo Product</td>
			<td style="width:87px;">75004525</td>
		</tr>
		<tr height="40">
			<td colspan="2" height="40" style="height:40px;width:249px;">ELx405 Select Deep Well Microplate Washer</td>
			<td style="width:135px;">Biotek</td>
			<td>ELX405USD</td>
		</tr>
		<tr height="41">
			<td colspan="2" height="41" style="height:41px;width:249px;">Custom High Throughput Automated Phage Selection Robot</td>
			<td style="width:135px;">S&#38;P Robotics</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Plastic conical Falcon tube: 50 mL</td>
			<td style="width:135px;">VWR</td>
			<td style="width:87px;">21008-178</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Plastic conical Falcon tube: 15 mL</td>
			<td style="width:135px;">VWR</td>
			<td style="width:87px;">89039-668</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Axygen 1.7 mL microcentrifuge tubes</td>
			<td style="width:135px;">Corning</td>
			<td style="width:87px;">MCT-175-L-C</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">96-well/384-well Maxisorp plate</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:87px;">M9410-1CS</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Corning 96-well V-bottom deep-well block</td>
			<td style="width:135px;">Corning</td>
			<td style="width:87px;">3960</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Axygen Mini-tube 96-well sterile microplate blue box</td>
			<td style="width:135px;">Corning</td>
			<td style="width:87px;">AXY-MTS-06-C-R-S</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Breathable adhesive plate sealing film</td>
			<td style="width:135px;">VWR</td>
			<td style="width:87px;">60941-084</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:471px;">REAGENTS</td>
		</tr>
		<tr height="43">
			<td colspan="2" height="43" style="height:43px;width:249px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:87px;">Catalog Number</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">polyethylene glycol</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">PEG800.1</td>
		</tr>
		<tr height="18">
			<td colspan="2" height="18" style="height:18px;">yeast extract</td>
			<td>Bioshop</td>
			<td>YEX401.1</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">bio-tryptone</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">TRP402.1</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">N-Z amine</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:87px;">C7290</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">glucose</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:87px;">G8270-1KG</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">lactose</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">LAC234</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">glycerol</td>
			<td style="width:135px;">Bioshop</td>
			<td>GLY001.500</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">carbenicillin&#160;</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">CAR544.10</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">kanamycin</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">KAN201.25</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">tetracycline&#160;</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">TET701.25</td>
		</tr>
		<tr height="43">
			<td colspan="2" height="43" style="height:43px;width:249px;">Tween 20</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">TWN510.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">monobasic potassium phophate</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">PPM302.1</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">dibasic sodium phosphate</td>
			<td style="width:135px;">Anachemia</td>
			<td style="width:87px;">84486-440</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">sodium chloride</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">SOD001.10</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">potassium chloride</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">POC308.1</td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;width:249px;">calcium chloride</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">CCL302.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">magnesium sulphate</td>
			<td style="width:135px;">EMD</td>
			<td style="width:87px;">MX0070-1</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">magnesium chloride</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">MAG510.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">NH4Cl</td>
			<td style="width:135px;">Amresco</td>
			<td style="width:87px;">0621-1KG</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Na2SO4</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">SOS513.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">agar</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">AGR001.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">SybrSafe DNA gel stain</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:87px;">S33102</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">phosphoric acid</td>
			<td style="width:135px;">Acros Organics</td>
			<td style="width:87px;">201140010</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">lysozyme</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">LYS702.25</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">benzonase</td>
			<td style="width:135px;">Novagen</td>
			<td style="width:87px;">71205</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:139px;">Triton X-100</td>
			<td style="width:111px;"></td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">TRX506.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">protease inhibitor cocktail tablets</td>
			<td style="width:135px;">Roche</td>
			<td style="width:87px;">11 836 170 001</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Ni-NTA resin&#160;</td>
			<td style="width:135px;">Qiagen</td>
			<td style="width:87px;">1018240</td>
		</tr>
		<tr height="42">
			<td colspan="2" height="42" style="height:43px;width:249px;">1000x helper phage (M13K07) stock (1013 phage per mL)</td>
			<td style="width:135px;">NEB</td>
			<td style="width:87px;">N0315S</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="39" rowspan="2" style="height:40px;width:249px;">HRP conjugated M13-specific antibody (anti-M13-HRP).</td>
			<td rowspan="2" style="width:135px;">GE Healthcare</td>
			<td rowspan="2" style="width:87px;">27-9241-01</td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="44">
			<td colspan="2" height="44" style="height:44px;width:249px;">TMB substrate: mix equal volumes of TMB and H2O2 peroxidase substrate&#160;</td>
			<td style="width:135px;">KPL</td>
			<td style="width:87px;">50-76-00</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">dNTPs</td>
			<td style="width:135px;">Biobasic</td>
			<td style="width:87px;">DD0056</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Taq polymerase</td>
			<td style="width:135px;">Genscript</td>
			<td style="width:87px;">E00007</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Exonuclease</td>
			<td style="width:135px;">GE Healthcare&#160;</td>
			<td style="width:87px;">EZ0073X-EZ</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Shrimp alkaline phosphatase</td>
			<td style="width:135px;">GE Healthcare</td>
			<td style="width:87px;">E70092Z-EZ</td>
		</tr>
	</tbody>
</table>

scalable high throughput selection from  phage-displayed synthetic antibody libraries

La demanda de los anticuerpos que se satisfagan las necesidades de las aplicaciones básicas y clínicas de investigación es alta y se incrementará dramáticamente en el futuro. Sin embargo, es evidente que las tecnologías tradicionales no son monoclonales solo hasta esta tarea. Esto ha llevado al desarrollo de métodos alternativos para satisfacer la demanda de alta calidad y reactivos de afinidad renovables a todos los elementos accesibles del proteoma. Con este fin, métodos de alto rendimiento para la realización de selecciones de bibliotecas de anticuerpos en fagos sintéticos se han diseñado para aplicaciones que implican diversos antígenos y optimizado para un rendimiento rápido y el éxito. En la presente memoria, un protocolo se describe en detalle que ilustra con la demostración de vídeo la selección paralela de clones Fab fagos a partir de bibliotecas de alta diversidad contra cientos de objetivos usando un manipulador de líquidos 96 manual de canales o el sistema de robótica automatizada. El uso de este protocolo, un solo usuario puede generar cientos de antígenos,seleccionar anticuerpos a ellos en paralelo y validar la unión de anticuerpos dentro de 6-8 semanas. Se destacan: i) un formato antígeno viable, ii) caracterización del antígeno preselección, iii) los pasos críticos que influyen en la selección de clones específicos y de alta afinidad, y iv) las formas de efectividad selección monitoreo y etapa temprana caracterización anticuerpo clon. Con este enfoque, hemos obtenido fragmentos de anticuerpos sintéticos (FABS) a muchas clases de objetivos, incluyendo los receptores de un solo paso de membrana, hormonas de proteínas secretadas y proteínas intracelulares multi-dominio. Estos fragmentos se convierten fácilmente a los anticuerpos de longitud completa y se han validado para exhibir una alta afinidad y especificidad. Además, se han demostrado para ser funcionales en una variedad de inmunoensayos estándar, incluyendo transferencia de Western, ELISA, inmunofluorescencia celular, inmunoprecipitación y ensayos relacionados. Esta metodología se acelerará el descubrimiento de anticuerpos y en última instancia, nos acercan a la realización del objetivo of generación de anticuerpos de alta calidad, renovables a la proteoma.

El protocolo descrito en el presente documento se ha utilizado para aislar fragmentos de anticuerpos a una variedad de ambos dominios de antígenos estructuralmente y funcionalmente relacionados con bibliotecas combinatorias de Fab presentados en fagos en paralelo. Cuestiones relacionadas con la disponibilidad de antígeno pueden eludirse, en muchos casos, por la identificación in silico de dominios antígeno expresables que son adecuados para la selección de anticuerpos 23. Mediante el acoplamiento de la e...

Watch this Scientific Journal Video about Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries at JoVE.com

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

Se describe un método con acompañamiento visual para conducir, las selecciones de rendimiento altos escalables de bibliotecas de anticuerpos sintéticos combinatorios fagos contra cientos de antígenos simultáneamente. El uso de este enfoque paralelo, hemos aislado fragmentos de anticuerpos que exhiben alta afinidad y especificidad para diversos antígenos que son funcionales en inmunoensayos estándar.

Escalable de alto rendimiento seleccion de bibliotecas de anticuerpos sintéticos fagos

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

scalable-high-throughput-selection-from-phage-displayed-synthetic

Research

JoVE Journal

Immunology and Infection

18.4K vistas.  The Recombinant Antibody Network.  Se describe un método con acompañamiento visual para conducir, las selecciones de rendimiento altos escalables de bibliotecas de anticuerpos sintéticos combinatorios fagos contra cientos de antígenos simultáneamente. El uso de este enfoque paralelo, hemos aislado fragmentos de anticuerpos que exhiben alta afinidad y especificidad para diversos antígenos que son funcionales en inmunoensayos estándar. 

Video: Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

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, ...

Investigación

Inmunología e Infección

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cuantitativo de alto rendimiento de una sola célula de ensayo de citotoxicidad de las células T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

Screening de alto rendimiento para los de amplio espectro químico Inhibidores de Virus ARN

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

ScanLag: De Alto Rendimiento La cuantificación del crecimiento de las colonias y Lag Tiempo

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Alto Rendimiento Purificación de<em&gt; Plasmodium falciparum</em&gt; Los merozoitos Para uso en opsonizante Antibody Ensayos

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

De Alto Rendimiento de genes de marcado en las<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Construcción de fagos sintético muestra fabulosa biblioteca con diversidad a medida

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

Un alto rendimiento, alto contenido líquido y basada en C. elegans patosistema

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un alto rendimiento Shigella-ensayo bactericida específico

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long &#945;-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Bloqueo de anticuerpos dirigido por un conjugado dual-funcional de péptido antigénico y méticos Fc-III (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...