O autor gostaria de agradecer a senhorita Lora Matthews para executar a técnica eo Sr. Paul Pastor para a cinematografia. Este projeto foi financiado pela Universidade da Austrália do Sul, Laboratório curso de Medicina.

Preparação de Saline Veronal 5x Buffered (VBS) <ol><li> Para preparar a solução salina Veronal Buffered (VBS), três soluções distintas precisa estar preparado. </li><li> Prepare uma solução através da dissolução de NaCl e 21.25gm 0.94gm de Barbitone de sódio em 350ml de água destilada. A concentração final das Barbitone NaCl e de sódio são 1,02 e 13mm, respectivamente. </li><li> Prepare a solução 2, dissolvendo 1.44gm de Barbitone em 125ml de água destilada quente. A concentração final de Barbitone é 62,5 mm. </li><li> Prepare a solução 3 dissolvendo 20.33gm de MgCl 2 e 4.41gm de CaCl 2 em 100ml de água destilada. A concentração final de MgCl 2 e CaCl 2 é 2.18m e 440mm, respectivamente. </li><li> Mix de soluções 1 e 2 e arrefecer à temperatura ambiente. </li><li> Uma vez que a solução combinada esfriou, adicionar 1,25 ml de solução 3 e ajustar o pH a 7,3-7,5 usando HCl 1M. </li><li> Ajustar o volume final de 500ml com água destilada para preparar uma solução estoque de 5x. </li><li> Para preparar uma solução 1x de trabalho, diluir a 1:05 estoque com água destilada. </li></ol> Diluição de Revercells <ol><li> Delicadamente ressuspender a solução estoque de Revercells por inversão e remova 1ml de células e centrifugar por 5 minutos a 600g a 4 ° C. </li><li> Após a centrifugação, remover cuidadosamente o sobrenadante e ressuspender as células em 3 ml de 1x Veronal Buffered Saline. </li><li> As células podem agora ser armazenadas a 4 ° C até o uso. </li></ol> Procedimento <ol><li> Transferência de 50ml de 1x VBS em linhas A1-A12 de uma placa inferior 96 poços U. </li><li> Bem a A1, adicionar 50μl de soro anti-A e misture bem com uma pipeta. </li><li> Usando uma ponteira fresco, remover 50μl de líquido bem A1 e transferi-lo para o bem A2. Misturar bem e repetir este processo até chegar bem A11 e depois descartar a 50μl última deste bem. </li><li> A amostra de soro foi agora diluída em série de 2 vezes de 1:2 a 1:2048. Sem soro é adicionado ao Bem A12 como este é o controle VBS negativo. </li><li> Se mais de uma amostra de soro deve ser avaliada, repita os passos de 4,1-4,4 para a linha B etc </li><li> Uma vez que todas as diluições foram feitas, adicionar 50μl de Revercells 1% para todos os poços. Nota: O Revercells vai resolver com o tempo e deve ser suavemente suspenso antes de ser dispensado nos poços. </li><li> Misture as amostras batendo na lateral da bandeja de microtitulação, cubra e deixe a reação de hemaglutinação para prosseguir por 1h a 37 ° C. Uma sala de incubadora ou devidamente aquecida pode ser utilizada. </li><li> Após a incubação, remova cuidadosamente a bandeja da incubadora e examine a placa de hemaglutinação. O controle VBS bem deve ser o primeiro bem examinados. Se a reação for negativa, então os resultados são válidos. Se a amostra VBS mostra um resultado positivo hemaglutinação, então os resultados não podem ser utilizados eo processo deve ser repetido. A reação de hemaglutinação positiva aparecerá como uma folha larga na base do poço, enquanto que uma reação negativa aparecerá como uma pelota pequena concentração de células no centro do poço. Um resultado intermediário terá um halo de células positivas com um núcleo central de células peletizada. Este resultado ocorre como o soro tem algumas Ab que pode reagir, mas quantidades insuficientes de Ab para causar uma reação completa. </li><li> A seção de resultados representativos tem duas representações diagramáticas dos resultados esperados. Um exemplo de um conjunto inválido e válido de resultados é fornecido. O título do anti-soro também será determinada. </li></ol> Resultados representativos: O vídeo inclui um exemplo de resultados representativos. Abaixo (fig. 1 e Fig. 2) são dois exemplos mostrados de forma esquemática. O primeiro exemplo é de uma reação que não pode ser interpretada como o controle VBS é positivo, enquanto o segundo é um ensaio válido que permita o título a ser determinado. <img src="/files/ftp_upload/1752/1752fig1.jpg" alt="Figura 1" /> Figura 1. Um exemplo de um hemaglutinação, onde o resultado positivo falhou. Na representação esquemática acima, a reação heamagglutination não pode ser aceite. O VBS soro controle negativo exibe uma reação de hemaglutinação positiva como evidenciado pela formação da folha na base do poço. Isso indica que um erro ocorreu e os resultados não podem ser aceites. Neste caso, o processo deve ser repetido. <img src="/files/ftp_upload/1752/1752fig2.jpg" alt="Figura 2" /> Figura 2. Um exemplo de hemaglutinação, onde o resultado positivo tem funcionado corretamente. Na representação esquemática, a reação heamagglutination é válido. O controle negativo VBS exibe uma reação negativa hemaglutinação ums evidenciado pela pelota que se formou na base do poço. O bem é positivo última linha 7, que indica que o título para esta amostra de soro é de 128 (ou seja, o inverso de 1 / 128). Este seria um exemplo de um anticorpo que está presente em quantidades moderadas no soro.

O ensaio de hemaglutinação é uma técnica muito simples que permite um grande número de amostras de soro para ser avaliado dentro de um curto período de tempo. Ele também tem a vantagem de ser lido sem a necessidade de equipamentos especializados, com o olho que está sendo adequado. Esta técnica também pode ser modificada para a detecção do hormônio gonadotrofina coriônica humana presentes na urina, como parte de um teste de inibição da hemaglutinação. Em relação a um teste de hemaglutinação padrão...

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<td>Revercells</td>
<td>&nbsp;</td>
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<td>Use at 1% final</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>
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determining the reactivity and titre of serum using a haemagglutination assay

Hemaglutinação é uma forma específica de aglutinação e é usado quando os anticorpos se ligam a células vermelhas do sangue, que funcionam como um antígeno particulado. Os glóbulos vermelhos são alvos particularmente úteis como eles são facilmente disponíveis e de aglutinação é observável a olho nu. Esta técnica é comumente usado para determinar o título de um anticorpo (Ab), para tipagem de sangue e quantificação viral. Neste vídeo, as etapas envolvidas na preparação e realização de um ensaio de hemaglutinação é demonstrada utilizando anticorpos específicos para antígenos de grupo sanguíneo A-adicionados às células vermelhas do sangue (Revercells). O anti-soro é diluído em uma série de 96 poços de microtitulação bandeja de U-inferior, à qual é adicionada uma suspensão de Revercells. As amostras são misturados e, em seguida, incubados a 37 ° C por 60 minutos. Após este período, as amostras podem ser facilmente marcados para ve, ve + e reações de hemaglutinação intermediária (-/+). Esta abordagem permite a reatividade e título de uma amostra de soro a ser avaliados através de uma técnica rápida e simples. O vídeo irá cobrir a teoria por trás do ensaio, como os resultados são lidos e interpretados, como a determinação do título, como o ensaio pode ser modificado e problemas associados com o uso desta técnica.

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

Hemaglutinação é uma forma de aglutinação, onde anticorpos se ligam às células vermelhas do sangue. Os glóbulos vermelhos são facilmente disponíveis e os resultados são facilmente observáveis ​​a olho nu. Este vídeo mostra os passos envolvidos em um ensaio de hemaglutinação, interpretando os resultados e determinar o título.

Determinação da Reatividade e Titre de soro usando um ensaio de hemaglutinação

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 visualizações.  University of South Australia. Hemaglutinação é uma forma de aglutinação, onde anticorpos se ligam às células vermelhas do sangue. Os glóbulos vermelhos são facilmente disponíveis e os resultados são facilmente observáveis ​​a olho nu. Este vídeo mostra os passos envolvidos em um ensaio de hemaglutinação, interpretando os resultados e determinar o título.

Vídeo: 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.

Medir o Complemento de 50% hemolítica (CH 50) Atividade de soro

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

Imunologia e Infecção

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.

Determinação da atividade fagocítica de amostras de anticorpos Clínica

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.

Determinando a atividade citotóxica Optimal específicos de células CD8 Her2neu Humanos T, comparando a Cr51 Ensaio de Libertação do Sistema xCELLigence

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 ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Indução de enxerto-versus-hospedeiro de Doenças e<em&gt; Em Vivo</em&gt; Monitoramento celular T Usando um modelo murino de MHC de correspondência

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Investigando os efeitos dos probióticos sobre pneumocócica Colonização Usando um<em&gt; In Vitro</em&gt; Adesão Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

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.

Crescimento Pairwise competição ensaio para determinar a replicação de Fitness de Imunodeficiência vírus humanos

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.

De alto rendimento quantitativo em tempo real de RT-PCR Ensaio para Determinar Perfis de Expressão do tipo I e III do interferão subtipos

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.

Avaliando Retinal microglia fagocítica Função<em&gt; In Vivo</em&gt; Usando um ensaio baseado em citometria de fluxo

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.

Optimização de um Micro-quantitativa Ensaio de neutralização

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.

Na Vivo Ensaio para detecção de células T antígeno-específicas função citolítica usando um modelo de vacinação

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

Determinação da Reatividade e Titre de soro usando um ensaio de hemaglutinação

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