Estamos muy agradecidos a K. Nakagaki, el Dr. H. Ishii, el Dr. K. Suzuki, Yamagata A., Oofusa K. por su valiosa contribución.

1. Producción y purificación de sGMRα <ol><li> Amplificar cDNA de sGMRα de placenta humana biblioteca de cDNA por reacción en cadena de polimerasa (PCR). </li><li> Añadir 50 bases 5&#39;-UTR secuencia de final de baculovirus polihedrina y 27 RGS base de codificación de su etiqueta en el extremo 3 &#39;a la amplificación de PCR por otro PCR. </li><li> Inserte la amplificación de PCR en un pMSG1.1MG plásmido. </li><li> Inyectar el psGMR/M1.1MG construir con el vector de ayuda pHA3PIG en los huevos de gusanos de seda PND-w1 cepa. </li><li> Trasera del cascarón G0 larvas de las polillas a 25 ° C. Seleccionar embriones G1 obtenido en el apareamiento entre hermanos o con PND-w1 para la expresión MGFP en los ojos para obtener los gusanos de seda transgénicos que llevan el gen sGMRα. </li><li> Extracto recombinante sGMRα de los capullos de gusanos de seda transgénicos con tampón fosfato que contiene NaCl 500 mm y se agita a 4 ° C durante 24 horas. </li><li> Centrifugar la muestra a 20000 g durante 15 minutos para eliminar un precipitado. </li><li> Aplicar sobrenadante a una columna de afinidad de níquel. </li><li> Lavar la columna con 10 mM imidazol, NaCl 500 mm y fosfato de sodio 20 mM, pH6.3, y eluir con un gradiente lineal de imidazol de 10 mm a 250 mm. Piscina de las fracciones que contiene el sGMRα purificada y diálisis frente a tampón fosfato salino (pH 7,4) (PBS). </li></ol> 2. Biotinilación de GM-CSF recombinante <ol><li> Retire la preparación de sorbitol por diálisis contra PBS. </li><li> Añadir 1 ml de 20 mM de sodio frío meta-peryodato solución a 4 ° C a la solución en hielo. </li><li> Incubar la muestra durante 30 minutos sobre el hielo en la oscuridad. </li><li> Agregar a la solución de glicerol a una concentración final de 15 mm. </li><li> La solución de diálisis frente a tampón acetato de sodio 100 mM (pH 5,5) </li><li> Añadir biotina hidrazida a la solución a una concentración final de 5 mM y continuamente agitar la solución durante 2 horas a temperatura ambiente. </li><li> Retire el material que no ha reaccionado por diálisis contra PBS, pH 7,4. </li></ol> 3. Una célula sin GM-CSF receptor-ligando vinculante ensayo <ol><li> Un abrigo de placas de microtitulación con 50μl de anticuerpo monoclonal anti-anticuerpos polihistidina la noche a 4 ° C. </li><li> Lavar cinco veces con 500μl de PBST. </li><li> Agregar 100μl de una solución de bloqueo y se incuba durante 3 horas a 4 ° C. </li><li> Lavar cinco veces con 500μl de PBST. </li><li> Añadir 50μl de sGMRα en la solución de bloqueo y se incuba durante la noche a 4 ° C. </li><li> Lavar cinco veces con 500μl de PBST. </li><li> Añadir 25μl de las muestras que contienen GM-CSF autoanticuerpos y 25μl de biotina GM-CSF. Incubar durante 1h a 4 ° C para formar sGMRα-GM-CSF complejo. </li><li> Lavar cinco veces con 500μl de PBST. </li><li> Añadir 50μl de 0,4 mm de fosfatasa alcalina estreptavidina durante 1 hora a 4 ° C para detectar la biotina GM-CSF obligado sGMRα. </li><li> Lavar cinco veces con 500μl de PBST. </li><li> Añadir 50μl de un sustrato quimioluminiscente para la solución y se incubó durante 1 hora a temperatura ambiente. </li><li> El uso de un lector de placas de quimioluminiscencia, detectar la actividad de quimioluminiscencia. </li></ol> 4. Los resultados representativos: El primer paso en la vía de transducción de GM-CSF receptor de señal es la unión de GM-CSF a la alfa del receptor de GM-CSF en la superficie celular. Debido a que GM-CSF autoanticuerpos se une específicamente al GM-CSF y bloquear su unión al receptor in vitro 16,17, la hipótesis de que los anticuerpos inhiben esta primera reacción uniéndose directamente a GM-CSF. Como se describió previamente (Ref. 9-15), se aplicó la tecnología de transgénicos del gusano de seda para obtener una gran cantidad de sGMRα recombinante de alta pureza. Cuando el gusano de seda de origen recombinante sGMRα fue cargada en el SDS-PAGE en condiciones no reductoras, el sGMRα mostró tanto monomérica (45 kDa) y diméricas (90 kDa), las formas, mientras que sólo la forma monomérica se detectó en condiciones reductoras (fig. 1B) , lo que indica que la sGMRα recombinante era una mezcla de monómeros y reguladores disulfuro vinculados 18. Utilizando el sistema libre de células (Fig. 2A), se evaluó la inhibición de la GM-CSF unión a sGMRα por GM-CSF autoanticuerpos (Fig. 2B y 2C fig.). Estos métodos se describe en la referencia 19 por Urano et al. La inhibición del crecimiento se correlaciona estrechamente con la inhibición de la unión (r = 0,988, p = 0,002) en diversas concentraciones de los autoanticuerpos GM-CSF (Fig. 3) 19. Del mismo modo, la inhibición de la unión se correlacionó significativamente con la inhibición del crecimiento. La inhibición de la unión aumenta de manera dosis-dependiente de GM-CSF autoanticuerpos (Fig. 3B) 19. Estos dos parámetros para el suero de las fracciones IgG de los distintos pacientes se correlacionaron entre sí (r = 0,589 p = 0,006, fig. 3C). Ni inhibición de la unión ni la inhibición del crecimiento correlacionado con los valores de Kd, y por lo tanto, ambos parámetros no se vieron afectados por la afinidad de unión 19. Por lo tanto la reproducibilidad de los datos entre la unión y la grecimiento inhibición obtenidos a través de tres experimentos independientes en tres diferentes muestras se evaluó. El coeficiente de variación indica que el sistema libre de células fue más excelente que el de bioensayo (tabla 1). <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="Figura 1" /> Figura 1. Diagrama de flujo del procedimiento para la producción de transgénicos sGMRα con gusanos de seda. A) Las estructuras de las 09.11 vectores de transformación. B) SDS-PAGE y Coomassie Brilliant Blue-tinción de la sGMRα purificada virtud de la reducción y las condiciones no reductoras. sGMRα, soluble GM-CSF receptor alfa; MGFP, el monstruo de la proteína verde fluorescente, BmNPVpol5&#39;-UTR, 5 &#39;no traducida secuencia de la región de Bombyx mori poliedrosis nuclear virus polihedrina; SV40 poliA, SV40 secuencia señal poliA; P 3xP3, 3xP3 promotor; P SER1, SER1 promotor; FiBL poliA, fibroína L-secuencia de la cadena de señal poliA, HR3, Bombyx mori poliedrosis nuclear virus HR3 potenciador. <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="Figura 2" /> Figura 2. Esquema del sistema de ensayo libre de células. A) Un ensayo de unión competitiva con sGMRα producida por gusanos de seda. B) Efecto de los anticuerpos neutralizantes y no neutralizantes en la inhibición de la unión de células sin sistema. C) La diferencia de la inhibición de la unión entre las diferentes concentraciones de anticuerpos neutralizantes y los anticuerpos no neutralizantes. GM-CSF, granulocitos y macrófagos factor estimulante de colonias; sGMRα, soluble GM-CSF receptor alfa, Su, RGS-Su-etiqueta; AP, la fosfatasa alcalina. <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="Figura 3" /> Figura 3. GM-CSF inhibición de la unión de sGMRα por efecto de GM-CSF anticuerpos policlonales o el suero de las fracciones IgG de los pacientes con PAP autoinmunes. A) Relación entre la inhibición de la unión y la inhibición del crecimiento de GM-CSF autoanticuerpos. B) inhibición de la unión a varias concentraciones de GM-CSF autoanticuerpos. C) La relación entre IC 50 por ciento de inhibición de unión y la inhibición del crecimiento por ciento en el suero de las fracciones IgG 19. IC 50, 50% de la concentración inhibitoria; GM-CSF, granulocitos y macrófagos factor estimulante de colonias. <table border="1"><tbody><tr><td> Muestra </td><td> A </td><td> B </td><td> C </td></tr><tr><td colspan="4"> Inter-ensayo </td></tr><tr><td> Número de determinaciones </td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td> El valor medio (% inhibición de la unión) </td><td> 61.6 </td><td> 63.8 </td><td> 69.7 </td></tr><tr><td> El valor medio (% de inhibición del crecimiento) </td><td> 21.0 </td><td> 73.4 </td><td> 82.8 </td></tr><tr><td> Coeficiente de variación (%) (% inhibición de la unión) </td><td> 6.0 </td><td> 5.6 </td><td> 8.3 </td></tr><tr><td> Coeficiente de variación (%) (% de inhibición del crecimiento) </td><td> 64.4 </td><td> 18.3 </td><td> 10.4 </td></tr></tbody></table> Ambos ensayos se realizaron a 5 ng / ml de GM-CSF y una concentración igual a GM-CSF autoanticuerpos. Tabla 1. Comparación de coeficiente de variación entre el enlace por ciento y las inhibiciones del crecimiento obtenida a través de tres experimentos independientes.

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El ensayo libre de células calcula la capacidad de neutralización de GM-CSF autoanticuerpos con una excelente reproducibilidad y rapidez. La inhibición de la unión de GM-CSF o fracciones de autoanticuerpos en suero de los pacientes IgG se evaluó en este ensayo. Los datos mostraron una correlación entre la inhibición de la unión de la prueba libre de células y la inhibición del crecimiento de un bioensayo con células TF-1, respectivamente. El bioensayo ha sido ampliamente utilizado, pero albergaba las dificult...

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

ANTECEDENTES: Anteriormente, hemos demostrado que la capacidad de neutralizar, pero no la concentración de GM-CSF autoanticuerpos se correlaciona con la severidad de la enfermedad en pacientes con proteinosis alveolar pulmonar autoinmune (PAP) 1-3. Como la derogación de GM-CSF en la bioactividad de pulmón es la causa probable de PAP 4,5 autoinmune, es prometedor para medir la capacidad de neutralización de anticuerpos GM-CSF para evaluar la gravedad de la enfermedad en cada paciente con PAP. Hasta ahora, la capacidad de neutralización de anticuerpos GM-CSF ha sido evaluado mediante la evaluación de la inhibición del crecimiento de las células de la médula ósea o la TF-1 las células estimuladas con GM-CSF 6-8. En el sistema de bioensayo, sin embargo, es a menudo problemático obtener datos fiables, así como para comparar los datos de diferentes laboratorios, debido a las dificultades técnicas en el mantenimiento de las células en un estado constante. OBJETIVO: Para imitar GM-CSF unión a receptores de GM-CSF en la superficie celular utilizando células libres de unión al receptor-ensayo. Métodos transgénicos gusano de seda se ha aplicado la tecnología para la obtención de una gran cantidad de soluble recombinante alfa del receptor de GM-CSF (sGMRα) de alta pureza 13.09. El sGMRα recombinante estaba contenido en las capas sericina hidrofílico de hilos de seda sin ser fusionadas a las proteínas de la seda, y por lo tanto, es fácil extraer de los capullos de buena pureza con soluciones acuosas neutras 14,15. Afortunadamente, las estructuras de oligosacáridos, que son críticos para la unión con GM-CSF, son más similares a las estructuras de sGMRα humanos que las producidas por otros insectos o levaduras. Resultados: El sistema de ensayo libre de células con sGMRα dado los datos con alta plasticidad y la fiabilidad. Unión a sGMRα GM-CSF fue dosis-dependiente inhibido por policlonal GM-CSF autoanticuerpos en una forma similar a los bioensayos con la TF-1 en las células, lo que indica que nuestra libre de células nuevo sistema de ensayo utilizando sGMRα es más útil para la medición de la actividad neutralizante de GM-CSF autoanticuerpos que el sistema de bioensayo con TF-1 de células o células humanas de médula ósea. Conclusiones: Se estableció un ensayo libre de células cuantificar la capacidad de neutralización de GM-CSF autoanticuerpos.

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Se diseñó un ensayo de receptor de la célula-unión libre con el fin de estimar la unión de granulocitos y macrófagos factor estimulante de colonias (GM-CSF) a los receptores. Que nos permite evaluar la inhibición competitiva de biotina GM-CSF unión a los receptores alfa soluble GM-CSF por GM-CSF autoanticuerpos con una excelente reproducibilidad.

Un sistema de células de ensayo gratis Estimación de la capacidad de neutralización de GM-CSF recombinante soluble de anticuerpos con GM-CSF receptor

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

a-cell-free-assay-system-estimating-neutralizing-capacity-gm-csf

Research

JoVE Journal

Immunology and Infection

12.6K vistas.  Niigata University Medical and Dental Hospital. Se diseñó un ensayo de receptor de la célula-unión libre con el fin de estimar la unión de granulocitos y macrófagos factor estimulante de colonias (GM-CSF) a los receptores. Que nos permite evaluar la inhibición competitiva de biotina GM-CSF unión a los receptores alfa soluble GM-CSF por GM-CSF autoanticuerpos con una excelente reproducibilidad.

Video: A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergética y los Oxidativo ráfaga: Los protocolos para el aislamiento y la Evaluación de leucocitos humanos y plaquetas

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Investigación

Inmunología e Infección

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

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. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generación e identificación de GM-CSF Derivado alveolares-como los macrófagos y las células dendríticas A partir de médula ósea de ratones

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

La evaluación de la función retiniana Microglial fagocítica<em&gt; En Vivo</em&gt; El uso de un ensayo basado en citometría de flujo

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murina de linfocitos Etiquetado por<sup&gt; 64</sup&gt; Cu-Anticuerpo objetivo de receptores de<em&gt; En Vivo</em&gt; Tráfico de células mediante PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

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 ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Medición de Influenza neutralizando las respuestas del anticuerpo al virus a (H3N2) en suero humano por microneutralización ensayos utilizando células de MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un novedoso sistema de alimentador-libre para la producción masiva de células de asesino naturales murinos In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

In Vitro Diferenciación del Factor del Granulocyte-macrófago-estimulante de colonias (GM-CSF) de ratón-producción de linfocitos de T (THGM)

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.