Der Autor bedankt sich bei Frau Lora Matthews für die Durchführung der Technik und Herr Paul Shepherd für Kinematographie danken. Dieses Projekt wurde von der University of South Australia, Laboratoriumsmedizin Programm gefördert.

Vorbereitung von 5x Veronal Buffered Saline (VBS) <ol><li> Zur Vorbereitung der Veronal-gepufferte Kochsalzlösung (VBS), müssen drei separate Lösungen vorbereitet werden. </li><li> Bereiten Sie die Lösung 1 durch Auflösen 21.25gm von NaCl und 0.94gm von Sodium barbiturathaltiges Mittel in 350ml destilliertem Wasser. Die Endkonzentrationen der NaCl und Natrium barbiturathaltiges Mittel sind 1,02 M und 13mm bzw.. </li><li> Bereiten Sie die Lösung 2 durch Lösen 1.44gm von barbiturathaltiges Mittel in 125ml destilliertem Wasser heiß. Die Endkonzentration der barbiturathaltiges Mittel ist 62.5mm. </li><li> Bereiten Lösung 3 durch Auflösen 20.33gm von MgCl 2 und 4.41gm von CaCl 2 in 100 ml destilliertem Wasser. Die Endkonzentration von MgCl 2 und CaCl 2 ist 2,18 m und 440 jeweils. </li><li> Mix-Lösungen 1 und 2 und auf Raumtemperatur abkühlen. </li><li> Sobald die kombinierte Lösung abgekühlt ist, fügen Sie 1,25 ml Lösung 3 und pH-Wert auf 7,3-7,5 mit 1 M HCl. </li><li> Passen Sie das endgültige Volumen auf 500 ml mit destilliertem Wasser auf einem 5-fach Stammlösung. </li><li> So bereiten Sie eine 1x funktionierende Lösung, verdünnte die Aktie im Verhältnis 1:5 mit destilliertem Wasser. </li></ol> Eine Verwässerung des Revercells <ol><li> Vorsichtig resuspendieren Stammlösung Revercells durch Inversion, und entfernen Sie dann 1 ml Zellen und zentrifugieren Sie für 5 min bei 600g bei 4 ° C. </li><li> Nach der Zentrifugation vorsichtig den Überstand und resuspendieren Sie die Zellen in 3 ml 1x Veronal Kochsalzlösung. </li><li> Die Zellen können nun bei 4 ° C bis zur Verwendung gelagert werden. </li></ol> Verfahren <ol><li> Transfer 50ml 1x VBS in Zeilen A1-A12 von einer 96-well U Bodenplatte. </li><li> Um Well A1, fügen Sie 50 ul Anti-A Serum und gründlich mit Hilfe einer Pipette. </li><li> Mit einer frischen Pipettenspitze entfernen 50 ul von Flüssigkeit aus gut A1 und überträgt es auf gut A2. Gründlich mischen und wiederholen Sie diesen Vorgang, bis Sie auch A11 und erreichen dann verwerfen Sie die letzten 50 ul aus dieser Quelle. </li><li> Die Serumprobe wurde nun seriell 2-fach von 1:2 bis 1:2048 verdünnt. Kein Serum ist Well A12 aufgenommen, da dies die VBS negative Kontrolle. </li><li> Wenn mehr als eine Serumprobe zu beurteilen ist, wiederholen Sie die Schritte 4,1-4,4 für Zeile B etc. </li><li> Sobald alle Verdünnungen gemacht worden sind, fügen Sie 50 ul 1% Revercells in alle Vertiefungen. Hinweis: Die Revercells wird zufrieden über die Zeit und sollte vorsichtig suspendiert werden, bevor in die Vertiefungen verteilt. </li><li> Mischen Sie die Proben durch leichtes Antippen auf der Seite der Mikrotiterplatte Tray, Deckel und lassen Sie die Hämagglutination Reaktion für 1 Stunde bei 37 ° C. Ein Inkubator oder entsprechend beheizten Raum genutzt werden kann. </li><li> Nach der Inkubation vorsichtig das Tablett aus dem Inkubator und untersuchen Sie die Platte auf Hämagglutination. Die VBS-Steuerung und sollte die erste gut untersucht werden. Wenn die Reaktion negativ ist, dann sind die Ergebnisse gültig sind. Wenn das VBS Beispiel zeigt eine positive Hämagglutination Ergebnis, dann können die Ergebnisse nicht verwendet werden und der Prozess muss wiederholt werden. Eine positive Hämagglutination Reaktion wird ein breites Blatt, auf der Basis der gut erscheinen, während eine negative Reaktion wird als kleines konzentrierten Pellets von Zellen in der Mitte der gut erscheinen. Ein Zwischenergebnis wird eine Halo-positive Zellen mit einem zentralen Kern von pelletierten Zellen. Dieses Ergebnis kommt als das Serum hat einige Ab, dass reagiert werden kann, aber nicht hinreichende Mengen von Ab, eine vollständige Reaktion zu verursachen. </li><li> Die repräsentativen Ergebnisse Abschnitt hat zwei schematische Darstellungen von den erwarteten Ergebnissen. Ein Beispiel für eine ungültige und gültige Satz von Ergebnissen ist. Der Titer der Anti-Serum wird ebenfalls bestimmt werden. </li></ol> Repräsentative Ergebnisse: Das Video enthält ein Beispiel für repräsentative Ergebnisse. Unten (Abb. 1 und Abb. 2) sind zwei Beispiele in schematischer Weise dargestellt. Das erste Beispiel ist eine Reaktion, die nicht interpretiert werden können, wie das VBS Kontrolle positiv ist, während der zweite ist ein gültiger Test, der für die Titer bestimmt werden können. <img src="/files/ftp_upload/1752/1752fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Ein Beispiel für eine Hämagglutination, wo das positive Ergebnis ist fehlgeschlagen. In der schematischen Darstellung erwähnt, kann die heamagglutination Reaktion nicht akzeptiert werden. Das VBS Serum negative Kontrolle zeigt eine positive Hämagglutination Reaktion, wie durch die Blattbildung auf der Basis der gut belegt. Dies deutet darauf hin, dass ein Fehler aufgetreten ist und die Ergebnisse können nicht akzeptiert werden. In diesem Fall sollte der Vorgang wiederholt werden. <img src="/files/ftp_upload/1752/1752fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Ein Beispiel für eine Hämagglutination, wo das positive Ergebnis korrekt gearbeitet hat. In der schematischen Darstellung ist die heamagglutination Reaktion gültig. Das VBS negative Kontrolle zeigt eine negative Reaktion eine Hämagglutinations durch die Pellets, die auf der Basis der gut geformt hat nachgewiesen. Der letzte positive gut Zeile 7, was bedeutet, dass der Titer dieser Serumprobe 128 (dh der reziproke Wert 1 / 128) angibt. Dies wäre ein Beispiel für einen Antikörper, der in moderaten Mengen im Serum ist.

Die Hämagglutination-Assay ist eine sehr einfache Technik, die für eine große Anzahl von Serumproben, um innerhalb kurzer Zeit beurteilt werden können. Es hat auch den Vorteil, dass ohne die Notwendigkeit spezieller Ausrüstung zu lesen, mit dem Auge geeignet. Diese Technik kann auch für den Nachweis von humanem Chorion-Gonadotropin Hormon im Urin modifiziert werden, als Teil einer Hämaglutinationshemmungstest. In Bezug auf einen Standard Hämagglutinationstest, es gibt ein paar Bereiche, in denen darauf zu, um f?...

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<td>Revercells</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>02490201</td>
<td>Use at 1% final</td>
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<td>Anti-A serum</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>02611305</td>
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<td>96 well U bottom plate</td>
<td>&nbsp;</td>
<td>TRP</td>
<td>92097</td>
<td>Must be U-bottom</td>
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<td>Veronal buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use at 1x final</td>
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<td>37&deg;C room/incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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determining the reactivity and titre of serum using a haemagglutination assay

Hämagglutinations ist eine spezifische Form der Agglutination und wird verwendet, wenn Antikörper gegen rote Blutkörperchen, die als Partikel Antigen wirken zu binden. Rote Blutkörperchen sind besonders nützlich, Ziele, da sie leicht verfügbar sind und die Agglutination zu beobachten ist mit dem bloßen Auge. Diese Technik wird häufig verwendet, um den Titer eines Antikörpers (Ab), für die Blutgruppenbestimmung und virale Quantifizierung zu bestimmen. In diesem Video werden die Schritte bei der Vorbereitung und Durchführung einer Hämagglutination Test beteiligten demonstriert mit spezifischen Antikörpern gegen Blutgruppe A-Antigene hinzugefügt, um rote Blutkörperchen (Revercells). Das Antiserum wird seriell in eine 96-Well U-Boden Mikrotiterplatten Tablett verdünnt, an dem eine Aussetzung der Revercells aufgenommen. Die Proben werden gemischt und dann bei 37 ° C für 60 Minuten. Nach dieser Zeit können die Proben dann leicht ve, + ve und Zwischenprodukte (-/+) Hämagglutination Reaktionen erzielt werden. Dieser Ansatz ermöglicht die Reaktivität und die Titer von einer Serumprobe zu beurteilen mit einem schnellen und einfachen Technik. Das Video nimmt die Theorie hinter der Test, wie die Ergebnisse gelesen und interpretiert, wie der Titer bestimmt wird, wie der Test geändert werden können und alle Probleme mit dem Einsatz dieser Technik verbunden sind.

Watch this Scientific Journal Video about Determining the Reactivity and Titre of Serum using a Haemagglutination Assay at JoVE.com

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Hämagglutinations ist eine Form der Agglutination, wo Antikörper binden an roten Blutkörperchen. Rote Blutkörperchen sind beide verfügbar und die Ergebnisse sind leicht beobachtbaren mit dem bloßen Auge. Dieses Video zeigt die Schritte in einer Hämagglutination Test beteiligt sind, die Interpretation der Ergebnisse und die Bestimmung der Titer.

Die Bestimmung der Reaktivität und Titre von Serum mit einem Hämagglutinations 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 ...

determining-reactivity-titre-serum-using-haemagglutination

Research

JoVE Journal

Immunology and Infection

20.9K Aufrufe.  University of South Australia. Hämagglutinations ist eine Form der Agglutination, wo Antikörper binden an roten Blutkörperchen. Rote Blutkörperchen sind beide verfügbar und die Ergebnisse sind leicht beobachtbaren mit dem bloßen Auge. Dieses Video zeigt die Schritte in einer Hämagglutination Test beteiligt sind, die Interpretation der Ergebnisse und die Bestimmung der Titer.

Serie: Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

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

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

Die Messung der 50% Hämolytische Complement (CH 50) Aktivität von Serum

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

Forschung

Immunologie und Infektion

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Die Bestimmung der Phagozytoseaktivität of Clinical Antikörper Proben

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Bestimmung der optimalen zytotoxische Aktivität der Menschenrechte Her2neu spezifischen CD8 T-Zellen durch Vergleich der Cr51 Freisetzungsassay dem xCELLigence-System

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Paarweise Wachstum Wettbewerb Assay zur Bestimmung der Replication Fitness von Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Hochdurchsatz-quantitative Echtzeit-RT-PCR-Assay zur Bestimmung Expressionsprofile der Typen I und III Interferon-Formationsglieder

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

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.

Die Beurteilung Retinal Microglial Phagozytische Funktion<em&gt; In Vivo</em&gt; Mit einem Durchfluss-Zytometrie basierten Assay

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

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

Optimierung eines Assay Quantitative Mikroneutralisations

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Test zum Nachweis von Antigen-spezifischen T-Zell Zytolyse-Funktion mit einer Impfung-Modell

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Eine einfache Durchflusszytometrie basierten Test um festzustellen, In-vitro- Antikörper abhängige Verbesserung des Dengue-Virus mit Zika-Virus genesenden Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Schnelle, sichere und einfache manuelle am Krankenbett Nukleinsäure-Extraktion für den Nachweis des Virus in Vollblut-Proben

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...