Yazar Miss Lora Matthews sinematografi teknikleri ve Bay Paul Shepherd gerçekleştirmek için teşekkür etmek istiyorum. Bu proje, Güney Avustralya Üniversitesi, Tıp Laboratuarı programı tarafından finanse edildi.

5x veronal Tamponlu Salin (VBS) hazırlanması <ol><li> Veronal Tamponlu Salin (VBS) hazırlamak için, üç ayrı çözüm hazırlıklı olması gerekiyor. </li><li> Distile su 350ml Sodyum barbital NaCl ve 0.94gm 21.25gm çözme çözüm 1 hazırlayın. NaCl ve Sodyum barbital son konsantrasyonları sırasıyla 1.02m ve 13mm. </li><li> Sıcak distile su 125ml barbital, 1.44gm eriterek çözüm 2 hazırlayın. Barbital son konsantrasyon 62.5mm. </li><li> Distile su 100 ml CaCl 2 MgCl 2 ve 4.41gm 20.33gm eriterek çözüm 3 hazırlayın. MgCl 2 ve CaCl 2 final konsantrasyon sırasıyla 2,18 m ve 440mm. </li><li> Mix çözüm 1 ve 2 ve oda sıcaklığına kadar soğumasını. </li><li> Kombine çözüm soğuduktan sonra, 1.25 ml çözüm 3 ve pH 7,3-7,5 1M HCl kullanarak ayarlayabilirsiniz. </li><li> 5x bir stok solüsyonu hazırlamak için 500ml için son hacim distile su ile ayarlayın. </li><li> 1x çalışma çözeltisi hazırlamak için, saf su ile seyreltilmiş hisse 01:05. </li></ol> Revercells, seyreltilmesi <ol><li> Yavaşça tersine çevirerek Revercells stok solüsyonu tekrar süspansiyon haline getirin ve sonra 4 600g az 5 dakika boyunca santrifüj hücre ve 1ml kaldırmak ° C </li><li> Santrifüj sonra dikkatle süpernatant kaldırmak ve veronal Tuzlu Tamponlu 1x 3ml hücrelerin tekrar süspansiyon haline getirin. </li><li> Hücreler 4 ° C kullanana kadar saklanabilir. </li></ol> Prosedür <ol><li> 50ml 1x VBS 96 U alt plakası satır A1-A12 içine aktarın. </li><li> Eh, A1, anti-serum, 50μl ekleyin ve bir pipet kullanarak iyice karıştırın. </li><li> Taze bir pipet kullanarak, A1 kuyusunda sıvı 50μl kaldırmak ve iyi A2 transfer. İyice karıştırın ve iyi A11 ulaşmak ve daha sonra bu kuyudan son 50μl atmak kadar bu işlemi tekrarlayın. </li><li> Şimdi serum örneği seri 01:02 dan 1:2048 2 kat sulandırılmış oldu. Bu VBS negatif kontrol olarak serum Peki A12 eklenir. </li><li> Eğer birden fazla serum örneği değerlendirilecek, tekrar satır B vb 4,1-4,4 adımları </li><li> Bir kez tüm dilüsyonları yapılmış, tüm kuyulara 1% Revercells 50μl ekleyin. Not: Revercells zaman içinde çözecek ve kuyuların içine yavaşça reçete edilmeden önce ara verilmelidir. </li><li> Mikrotitre tepsinin yan hafifçe dokunarak örnekleri karıştırın, kapak ve 37 1s için devam etmek hemaglutinasyon reaksiyonu ° C bırakın Bir inkübatör veya uygun ısıtılmış bir oda kullanılabilir. </li><li> Inkübasyon sonrasında, dikkatli bir şekilde inkübatör tepsiyi kaldırın ve hemaglutinasyon plaka inceleyin. VBS kontrolü de iyi incelendiğinde ilk olmalıdır. Reaksiyonun negatif ise, daha sonra sonuçları geçerlidir. VBS örnek olumlu bir hemaglutinasyonun sonuç gösteriyorsa, o zaman sonuç ve süreç tekrar edilmelidir. Olumsuz bir tepki kuyunun merkezinde küçük bir hücre konsantre pelet olarak görünecektir olumlu bir hemaglutinasyon reaksiyonu, iyi tabanı geniş bir sayfa olarak görünecektir. Bir ara sonuç pelet hücrelerinin merkezi bir çekirdek ile pozitif hücrelerin bir hale olacaktır. Bu sonuç, serum tepki verebilir bazı Ab olarak oluşur, ancak tam bir reaksiyona neden yetersiz miktarda Ab. </li><li> Temsilcisi sonuç bölümünde iki şematik gösterimleri beklenen sonuçlar vardır. Sonuçların geçerli ve geçersiz bir set bir örnek verilir. Aynı zamanda anti-serum titresi tespit edilecektir. </li></ol> Temsilcisi Sonuçlar: Video temsilcisi sonuçlar bir örnek içerir. Aşağıda şematik bir biçimde gösterildiği gibi iki örnek k (fiekil 1 ve Şekil 2). İlk örnek, VBS kontrolü pozitif olarak yorumlanır olamaz bir reaksiyon ikinci belirlenecek titresi sağlar geçerli bir testtir. <img src="/files/ftp_upload/1752/1752fig1.jpg" alt="Şekil 1" /> Şekil 1 hemaglutinasyonun bir örnek pozitif sonuç başarısız oldu. Yukarıdaki şematik temsili, heamagglutination tepki kabul edilemez. VBS serum negatif kontrol kuyunun dibinde tabaka oluşumu ile kanıtlandığı gibi olumlu bir hemaglutinasyon reaksiyonu gösterir. Bu bir hata oluştu ve sonuç kabul edilemez olduğunu gösterir. Bu durumda, işlem tekrarlanmalıdır. <img src="/files/ftp_upload/1752/1752fig2.jpg" alt="Şekil 2" /> Şekil 2 pozitif sonuç doğru çalıştı hemaglutinasyonun bir örnek. Şematik temsili, heamagglutination reaksiyon geçerlidir. VBS negatif kontrol olumsuz hemaglutinasyon reaksiyonu gösterir.kuyunun dibinde oluşan pelet ile kanıtlanır. Son olumlu yanı, bu serum örneği için titresi 128 (yani karşılıklı 1 / 128) olduğunu gösterir satır 7,. Bu, orta miktarda serum bulunan bir antikor bir örnek olacaktır.

Hemaglutinasyon testi zaman kısa bir süre içinde değerlendirilmesi gereken çok sayıda serum örneklerinin sağlayan çok basit bir teknik. Uygun olması göz, özel ekipman ihtiyacı olmadan okumak olmanın avantajı da vardır. Bu teknik de bir hemaglutinasyon-inhibisyon testi parçası olarak, idrarda insan koryonik gonadotropin hormonu tespiti için değiştirilmiş olabilir. Standart bir hemaglutinasyon testi ile ilgili olarak, bakım doğru yapılacak tahlil için alınması gereken birkaç alanları vardır....

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<td>Revercells</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>02490201</td>
<td>Use at 1% final</td>
</tr>
<tr>
<td>Anti-A serum</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>02611305</td>
<td>&nbsp;</td>
<tr>
<td>96 well U bottom plate</td>
<td>&nbsp;</td>
<td>TRP</td>
<td>92097</td>
<td>Must be U-bottom</td>
</tr>
<tr>
<td>Veronal buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use at 1x final</td>
</tr>
<tr>
<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

Hemaglütinasyon aglütinasyon belirli bir biçimi ve antikorlar, bir parçacık antijen olarak hareket kırmızı kan hücreleri, bağladığınızda kullanılır. Kırmızı kan hücreleri, bunlar da hazır olarak özellikle yararlı hedefler ve aglütinasyon çıplak gözle kullanarak gözlemlenebilir. Bu teknik genellikle kan gruplama ve viral kantifikasyon bir antikor titresi (Ab) belirlemek için kullanılır. Bu video, hemaglutinasyon tahlil hazırlanması ve sahne alan adımları spesifik antikorlar kan grubu A-antijenleri, kırmızı kan hücreleri (Revercells) gösterilmiştir. Antiserum, seri Revercells bir süspansiyon eklendi hangi, iyi bir 96 U-alt mikrotitre tepsi seyreltilir. Örnekleri 60 dakika karıştırılır ve daha sonra 37 ° C&#39;de inkübe. Bu süreden sonra, örnekleri daha sonra kolayca Ve + ve orta (-/+) hemaglutinasyon reaksiyonlar için gol olabilir. Bu yaklaşım, basit ve hızlı bir tekniği kullanılarak değerlendirilmiştir bir serum örnek reaktivite ve titresi sağlar. Video testin nasıl değiştirilebilir titre nasıl belirlenir sonuçlar okunur ve nasıl yorumlanır Testin ardındaki teori, ve bu tekniğin kullanımı ile ilişkili herhangi bir sorun kapsayacaktır.

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

Hemaglütinasyon antikorlar kırmızı kan hücrelerinin bağlamak aglütinasyon şeklidir. Kırmızı kan hücreleri de hazır ve sonuçları çıplak gözle kolayca gözlemlenebilir. Bu video sonuçları yorumlama ve titre belirlenmesi, hemaglutinasyon tayininde yer alan adımları göstermektedir.

Hemaglütinasyon Testi kullanarak Reaktivite ve Serum Titre Belirlenmesi

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 Görüntüleme.  University of South Australia. Hemaglütinasyon antikorlar kırmızı kan hücrelerinin bağlamak aglütinasyon şeklidir. Kırmızı kan hücreleri de hazır ve sonuçları çıplak gözle kolayca gözlemlenebilir. Bu video sonuçları yorumlama ve titre belirlenmesi, hemaglutinasyon tayininde yer alan adımları göstermektedir.

Video: 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.

Ölçüm% 50 Hemolitik Kompleman (CH 50</subSerum) Faaliyet

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

İmmünoloji ve Enfeksiyon

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.

Klinik Antikor Örnekleri fagositik Aktivitesi Belirlenmesi

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.

XCELLigence Sistemi Cr51 Yayın Assay Karşılaştırılması tarafından İnsan Her2neu Özgül CD8 T hücrelerinin Optimal Sitotoksik Aktivite Belirlenmesi

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.

İnsan İmmün Yetmezlik Virüs çoğaltma Fitness belirlenmesi için İkili Büyüme Rekabet Analizi

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.

Yüksek verim Kantitatif Gerçek zamanlı RT-PCR Testi Türleri I ve III İnterferon Alttipleriyle İfadesi Profilleri belirlenmesi için

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.

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi<em&gt; İn Vivo</em&gt; A Akış Sitometri bazlı analiz kullamlarak

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.

Bir Kantitatif Mikro-nötralizasyon Testi optimizasyonu

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.

Vivo Tahlil antijen spesifik T-hücre Cytolytic işlevi bir aşı modeli kullanılarak tespiti için

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.

Basit Akış Sitometresi Vitro antikor bağımlı Enhancement-in dang virüs Zika virüs nekahat Serum kullanarak belirlemek için tahlil dayalı

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).

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