El autor desea agradecer a la Srta. Lora Matthews para la realización de la técnica y el Sr. Pablo Pastor de la cinematografía. Este proyecto fue financiado por la Universidad de Australia del Sur, el programa de Medicina de Laboratorio.

Preparación de 5x buffer salino veronal (VBS) <ol><li> Para preparar la solución salina tamponada con veronal (VBS), tres soluciones distintas que estar preparados. </li><li> Prepare una solución de 1 por disolución de NaCl y 21.25gm 0.94gm de barbital sódico en 350 ml de agua destilada. La concentración final de NaCl y de sodio son barbitona 1.02m y 13 mm respectivamente. </li><li> Prepare una solución de 2 por disolución de 1.44gm barbitona en 125 ml de agua destilada caliente. La concentración final de barbitona es 62,5 mm. </li><li> Prepare una solución de 3 por disolución 20.33gm de MgCl 2 y 4.41gm de CaCl2 en 100 ml de agua destilada. La concentración final de MgCl2 y CaCl2 es 2,18 m y 440 mm respectivamente. </li><li> Mezclar las soluciones 1 y 2 y enfriar a temperatura ambiente. </li><li> Una vez que la solución combinada se ha enfriado, agregue 1.25ml de la solución 3 y ajustar el pH a 7,3-7,5 con HCl 1M. </li><li> Ajustar el volumen final de 500 ml con agua destilada para preparar una solución de 5x. </li><li> Para preparar una solución de trabajo 1x, diluir la 1:05 madre con agua destilada. </li></ol> Sensibilización de los glóbulos rojos de oveja con hemolisina <ol><li> Prepare la hemolisina por un lado se diluye 1:50 con VBS </li><li> A 4 ml de SRBC añadir 6 ml de VBS y mezcle suavemente por inversión </li><li> Centrifugar a 600 g x 5 minutos </li><li> Descartar el sobrenadante y lavar las células otro 2 veces </li><li> Después del último lavado, centrifugación de las células en 900g x 5 minutos para empacar las células </li><li> Eliminar el sobrenadante y resuspender las células en cantidad suficiente VBS para preparar una solución al 10% es decir, 0,5 ml de concentrado de hematíes se resuspenden en 5 ml de solución tampón </li><li> Gota a gota, añadir un volumen igual de hemolisina (conejo anti-oveja de anticuerpos de glóbulos rojos) a las células mientras agita continuamente </li><li> Se incuba a 30 ° C durante 30 minutos en un baño de agua </li><li> Mezclar suavemente las células cada 15 minutos </li><li> Sensibilizados SRBC se pueden almacenar durante la noche a 4 ° C </li></ol> CH 50 ensayo <ol><li> Etiqueta de una serie de tubos por duplicado, con 1:08, 1:16, 1:32, 1:64 y 1:128. </li><li> Prepare una serie de diluciones dobles de serie de control y suero problema en VBS cada una por duplicado </li><li> Comenzará a las 01:04 (100 ml de suero + 300 ml de VBS) y la transferencia de 200 ml de muestra en el tubo con la etiqueta siguiente. </li><li> Mezclar bien las diluciones entre 200 ml y la transferencia a la siguiente dilución con una punta de pipeta. </li><li> Repita hasta que los cinco diluciones. </li><li> Deseche 200ml de la final de 1:128 de dilución. </li><li> Añadir 200 ml de suspensión SRBC sensibilizados a todos los tubos. </li><li> Etiqueta de dos tubos separados como en blanco y añadir 200 ml de SRBC sensibilizados VBS + 200ml. Estos tubos se medirá lisis espontánea del SRBC en el VBS. </li><li> Etiqueta de otros dos tubos separados como lisis total y añadir 200 ml de SRBC sensibilizados + 200 ml de agua destilada. </li><li> Mezclar con cuidado todos los tubos. </li><li> Se incuba a 37 ° C durante 30 minutos en un baño de agua de mezcla después de 15 minutos. </li><li> Centrifugar las muestras a 1500 g durante 5 minutos para sedimentar los glóbulos rojos. </li><li> Transferencia de 100 ml de sobrenadante de cada tubo de un pozo en una placa de fondo plano de 96 pocillos. </li><li> Añadir 100 ml de agua destilada a cada pocillo. </li><li> Leer la absorbancia de las muestras a 540 nm utilizando un espectrofotómetro de placas. </li></ol> Cálculos <ol><li> Calcular la absorbancia media para cada muestra </li><li> Restar la absorbancia del blanco (lisis espontánea) de todas las muestras </li><li> Calcular el% de lisis de cada dilución utilizando la siguiente fórmula: <img src="/files/ftp_upload/1923/1923eq1.jpg" alt="Ecuación" /></li><li> Trazar el porcentaje de lisis (eje vertical) frente a la dilución del suero en el eje horizontal. </li><li> Calcular la dilución requerida para el 50% de hemólisis para el control y la prueba de suero (Fig. 2). </li></ol> Los resultados representativos: El video incluye un ejemplo de resultados representativos. En la práctica, un suero de control también se ejecuta en el momento mismo que el suero problema y se trata de la misma manera. A continuación (Tabla 1) se muestra de datos de una muestra de suero analizadas. Los datos se manipulan de acuerdo con la ecuación presentada en 5.3. <table border="1"><tbody><tr><td> Muestra </td><td> OD 540 </td><td> Media </td><td></td><td></td></tr><tr><td> En blanco </td><td> 0,042, 0,044 </td><td> 0.043 </td><td></td><td></td></tr><tr><td> Lisis total </td><td> 0.183, 0.183 </td><td> 0.183 </td><td></td><td></td></tr><tr><td colspan="5"></td></tr><tr><td> Dilución </td><td> OD 540 </td><td> Media </td><td> Significa-en blanco </td><td> % De lisis </td></tr><tr><td> 01:08 </td><td> 0.200, 0.219 </td><td> 0.210 </td><td> 0.168 </td><td> 116 </td></tr><tr><td> 01:16 </td><td> 0,179, 0,173 </td><td> 0.176 </td><td> 0.134 </td><td> 93 </td></tr><tr><td> 01:32 </td><td> 0.134, 0.110 </td><td> 0.122 </td><td> 0.08 </td><td> 55 </td></tr><tr><td> 1:64 </td><td> 0.053, 0.066 </td><td> 0.059 </td><td> 0.017 </td><td> 12 </td></tr><tr><td> 1:128 </td><td> 0,044, 0,045 </td><td> 0.045 </td><td> 0.003 </td><td> 2 </td></tr></tbody></table><img src="https://www.jove.com/files/ftp_upload/1923/1923fig1.jpg" alt="Figura 1" /> Figura 1. La activación de la vía clásica del complemento. La vía clásica se activa por la unión de inmunoglobulina M (IgM) o inmunoglobulina G (IgG) en la superficie de una célula diana. La porción Fc del Ab se une a C1q, C1r se activa y esta a su vez activa otra molécula de C1r que en conjunto activar dos moléculas de C1s. C1s ahora se unirá C4 que expone el sitio de unión de C2 que también se escinde. La unión de C4b y C2a conduce a la formación de un complejo conocido como una C3 convertasa. Este complejo se unirá ahora la formación de C3 C3a y C3b, algunos de los cuales se combinan con la C3 convertasa C5 convertasa formar una. Este complejo, actúa hoy en el C5, con la que resulta vinculante para C5b C6 iniciar la formación del complejo de ataque a la membrana (MAC). C5b6 actúa en C7, que a su vez actúan en C8 y C9 en última instancia resulta en la formación de la final de MAC. (Goldsby et al, 2003). <img src="https://www.jove.com/files/ftp_upload/1923/1923fig2.jpg" alt="Figura 2" /> Figura 2. Imprimir los registros de la muestra y el cálculo de la CH50 para un control y muestra de suero. (A), el porcentaje calculado (%) lisis de que tanto el control (•) y test () muestras de suero se trazan contra el factor de dilución. (B) Para el cálculo de la lisis del 50%, se dibuja una línea desde el valor del porcentaje del 50% hasta que se cruza con la línea gráfica y una línea vertical que se dibuja hacia abajo a la dilución. En este ejemplo, una dilución de 35 veces del control y la dilución de 21,6 veces del suero problema se requieren para alcanzar el 50% de lisis. Estos datos indican que hay menos complemento presente en el suero de prueba en comparación con el control, ya que requiere una menor dilución antes de los 50% de lisis se alcanzó. La muestra de control también muestra&gt; 100% de lisis en la dilución 1:08. Esto es más probable debido a la lisis incompleta de los SRBC por el agua destilada y más eficiente la lisis por los componentes del complemento.

<ol>
	<li>Goldsby, R. A., Kindt, T. J., Osborne, B. A., Kuby, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunology. , (2003).</li></ol>

El CH 50 ensayo está sujeto a muchas interferencias. Algunos SRBC son más frágiles que otros, resultando en hemólisis espontánea que no está relacionada con la actividad del complemento. La afinidad de los anticuerpos de conejo varía de un lote a otro y de un fabricante a otro, lo que afecta a la cantidad de anticuerpo que se une a los SRBC. Además, el proceso de sensibilización de SRBC con resultados de anticuerpos en las células con diferentes cantidades de anticuerpos del recubrimiento SRBC. Reco...

<table><tbody><tr><td>Sheep red blood cells</td><td>CSL</td><td>2490201</td><td>Use at 1% final</td></tr><tr><td>Serum</td><td></td><td></td><td>Human serum</td></tr><tr><td>Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated</td><td>AbD Serotec</td><td>C12HSB</td><td></td></tr><tr><td>96 well flat bottom plate</td><td>Sarstedt Ltd</td><td>83.1839</td><td></td></tr><tr><td>Veronal buffered saline</td><td></td><td></td><td>Use at 1x final</td></tr><tr><td>Waterbath</td><td></td><td></td><td></td></tr><tr><td>Plate reader</td><td></td><td></td><td></td></tr><tr><td>37&amp;#x00B0;C room/incubator</td><td></td><td></td><td></td></tr></tbody></table>

measuring the 50% haemolytic complement (ch50) activity of serum

El sistema del complemento es un grupo de proteínas que cuando se activa conducir a la lisis celular de destino y facilita la fagocitosis por opsonización. Los componentes individuales del complemento pueden ser cuantificados sin embargo, esto no proporciona ninguna información en cuanto a la actividad de la vía. El CH<sub&gt; 50</sub&gt; Es un ensayo de cribado para la activación de la vía clásica del complemento (Fig. 1) y es sensible a la reducción, la ausencia y / o inactividad de cualquiera de los componentes de la vía. El CH<sub&gt; 50</sub&gt; Pruebas de la capacidad funcional de los componentes del complemento del suero de la vía clásica de lisar glóbulos rojos de oveja (SRBC) pre-recubiertos con conejo anti-oveja de anticuerpos de glóbulos rojos (hemolisina). Cuando recubiertas de anticuerpos SRBC se incuban con suero, la vía clásica del complemento se activa y los resultados de hemólisis. Si un componente del complemento está ausente, el CH<sub&gt; 50</sub&gt; El nivel va a ser cero, si uno o más componentes de la vía clásica se reducen, el CH<sub&gt; 50</sub&gt; Se reducirá. Un volumen fijo de SRBC sensibilizados de forma óptima se añade a cada dilución de suero. Después de la incubación, se centrifuga la mezcla y el grado de hemólisis se cuantifica mediante la medición de la absorción de la hemoglobina liberada en el sobrenadante a 540 nm. La cantidad de la actividad del complemento se determina al examinar la capacidad de las diferentes diluciones del suero para lisar SRBC recubiertas de anticuerpos. Este video muestra los pasos experimentales que participan en el análisis del nivel de actividad del complemento de la vía clásica del complemento.

Watch this Scientific Journal Video about Measuring the 50% Haemolytic Complement CH50 Activity of Serum at JoVE.com

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

La vía clásica es activada por anticuerpos y que culmina en la lisis de las células diana. El CH<sub&gt; 50</subEnsayo&gt; proporciona una medida de la actividad del complemento de una muestra de suero. Este video muestra los pasos a seguir para determinar el CH<sub&gt; 50</sub&gt; De una muestra de suero, el cálculo y la interpretación de los resultados.

La medición del complemento hemolítico 50% (CH 50) La actividad de suero

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

measuring-the-50-haemolytic-complement-ch50-activity-of-serum

Research

JoVE Journal

Immunology and Infection

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

37.6K vistas.  University of South Australia. La vía clásica es activada por anticuerpos y que culmina en la lisis de las células diana. El CH 50 proporciona una medida de la actividad del complemento de una muestra de suero. Este video muestra los pasos a seguir para determinar el CH 50 De una muestra de suero, el cálculo y la interpretación de los resultados.

Video: Measuring the 50% Haemolytic Complement CH50 Activity of Serum

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

La determinación de la reactividad y el título de suero utilizando un ensayo de hemaglutinación

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

Investigación

Inmunología e Infección

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

Medición de actividad de factor V en el plasma humano usando un ensayo de coagulación de microplacas

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

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

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

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

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

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Aislamiento de exosomas del plasma de VIH-1 en individuos positivos

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

Un ensayo de hemaglutinación optimizado inhibición (HI) para cuantificar los títulos de anticuerpos específicos de la gripe

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Medicina

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Pruebas de hemocompatibilidad de implantes que contactan con la sangre en un modelo de bucle de flujo que imita el flujo sanguíneo humano

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen

Source: Kaufmann, L. et al., <a href="https://app.jove.com/v/55833">An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers.</a> J. Vis. Exp. (2017)
The video demonstrates a hemagglutination inhibition assay for detecting serum antibodies against specific antigens. Confirmation of hemagglutination inhibition in the test well and hemagglutination in the control well is achieved through distinct visual patterns. This assay plays a crucial role in assessing immune response, vaccine effectiveness, and studying viral infections in virology and immunology.

Ensayo de inhibición de la hemaglutinación para detectar anticuerpos séricos contra un antígeno diana

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

Source: Gross, F. L. et al., <a href="https://app.jove.com/v/56448">Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates Microneutralization assays to measure neutralizing antibodies using Madin-Darby Canine Kidney cells overexpressing &#945;2,6-linked sialic acid in human sera. This assay is often used for influenza human serology

Un ensayo de microneutralización para medir los títulos de anticuerpos neutralizantes en el suero humano

All influenza viruses should be handled according to appropriate biosafety level requirements (BSL-2 or higher) as defined in the Biosafety on ...

Assessing Functional SARS-CoV-2 Neutralizing Antibodies

This video showcases a neutralization assay measuring serum efficacy in neutralizing SARS-CoV-2 viruses. It involves pseudotyped viruses, serum dilution, and luminescence-based quantification, revealing the neutralization titer.

Evaluación de anticuerpos neutralizantes funcionales contra el SARS-CoV-2

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Pseudotype-based neutralization ...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

To assess the ability of specific antibodies of interest to target the hemagglutinin-expressing cells, replace the supernatant of the virally infected cell cultures with 25 microliters of antibody-dependent cell-mediated cytotoxicity, or ADCC, assay medium.
Next, in a separate 96-well plate, perform 4-fold dilutions of the antibodies of interest in triplicate and fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic, and taking care to include the background and no antibody controls.
When all of the antibody has been diluted, transfer 25 microliters of the diluted antibody to the appropriate corresponding wells in the experimental cell plate and place the plate in the cell culture incubator. After 15 minutes, add 7.5 times 10 to the 4th modified effector Jurkat cells, expressing the appropriate Fc receptor per well in 25 microliters of ADCC medium for a final volume of 75 microliters of ADCC medium per well.
Return the plate to the cell culture incubator for 6 hours, thawing luciferase substrate buffer in a 37-degree Celsius water bath during the last hour of the incubation. When the buffer is ready, add 75 microliters of luciferase substrate buffer to all of the wells except the background control well, and read the plate on a luminometer. The fold induction of antibody-dependent cell-mediated cytotoxicity in the transfected or infected cell cultures can then be calculated.

La medición del complemento hemolítico 50% (CH₅₀) La actividad de suero

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