著者は、映画撮影のためのテクニックと氏ポールシェパードを実行するためのミスローラマシューズに感謝したいと思います。このプロジェクトは、南オーストラリア大学、研究室医学のプログラムによって資金を供給された。

5倍のベロナール緩衝生理食塩水の調製（VBS） <ol><li>ベロナール緩衝生理食塩水（VBS）を準備するには、3つの別々のソリューションが用意する必要があります。 </li><li>蒸留水の350ミリリットルでナトリウムBarbitoneのNaClおよび0.94gmの21.25gmを溶解して溶液1を準備します。 NaClおよびナトリウムBarbitoneの最終濃度は、それぞれ1.02Mと13mmです。 </li><li>ホット蒸留水の125​​ミリリットルでBarbitoneの1.44gmを溶解して溶液2を準備します。 Barbitoneの最終濃度が62.5mMである。 </li><li>蒸留水の100mlにCaCl 2のMgCl 2と4.41gmの20.33gmを溶解して溶液3を準備。 MgCl 2とCaCl 2の最終濃度は2.18M、それぞれ440mMである。 </li><li>ミックスのソリューション1と2と室温まで冷却。 </li><li>組み合わせた溶液を冷却した後、溶液3の1.25 mlを追加し、1M HClを用いて7.3から7.5にpHを調整する。 </li><li> 5倍のストック溶液を調製するために蒸留水で500ミリリットルに、最終的な音量を調整します。 </li><li> 1Xワーキング溶液を調製するために、蒸留水で株式1:5に希釈する。 </li></ol> Revercellsの希釈<ol><li>静かに転倒混和してRevercellsのストック溶液を再懸濁し、その後4℃で600グラムで5分間細胞および遠心の1mlを取り除く℃に</li><li>遠心後、上清を慎重に除去し、ベロナール緩衝生理食塩液1Xの3ミリリットルで細胞を懸濁します。 </li><li>細胞は現在、4℃で使用時まで保存することができます。 </li></ol>手順<ol><li>行96ウェルU底プレートのA1 - A12に1X VBS 50mlのを転送する。 </li><li>まあA1に、抗血清50μlのを追加し、ピペットを用いてよく混ぜる。 </li><li>新鮮なピペットチップを使用して、よくA1から液体の50μlのを削除してもA2に転送します。完全に混和して、よくA11に到達し、この井戸から最後の50μlのを破棄するまで、このプロセスを繰り返します。 </li><li>血清サンプルは、現在シリアル1:2から1:2048に2倍に希釈されています。これはVBSネガティブコントロールであるため、血清はよくA12に追加されません。 </li><li>複数の血清サンプルを評価する場合、Bなどの行のためのステップ4.1から4.4を繰り返します。 </li><li>一度全ての希釈が行われている、すべてのウェルに1％のRevercells50μlの追加。注：Revercellsは時間をかけて解決し、穏やかにウェルに分注される前に中断されるべきである。 </li><li>軽くマイクロタイタートレイの側面にタップすることによってサンプルを混合、37℃で1時間進行さ血球凝集反応をカバーし、残す℃のインキュベーターまたは適切に加熱室を使用することができます。 </li><li>インキュベーション後、慎重にインキュベーターからトレイを取り外して、赤血球凝集のためのプレートを調べます。 VBSのコントロールは、よくよく調べ最初にしてください。反応が負の場合、結果は有効です。 VBSのサンプルが陽性赤血球凝集の結果が表示されている場合、結果は使用することができるとプロセスを繰り返す必要があります。否定的な反応がウェルの中央のセルの小さな濃厚ペレットとして表示されますが、正赤血球凝集反応は、よくの基盤上に広いシートとして表示されます。中間結果は、ペレット化した細胞の中核と陽性細胞のハローを持つことになります。血清が反応することができますが、抗体の不十分な量が全反応を引き起こすために、いくつかの抗体を持たないので、この結果が発生します。 </li><li>代表的な結果のセクションでは、期待される結果の2つの図式表現を持っています。結果の無効と有効なセットの例が提供されています。抗血清の力価も決定されます。 </li></ol>代表的な結果： ビデオでは、代表的な結果の例が含まれています。 （Fig1および図2）の下には、図式的に示すように、2つの例です。秒が決定される力価を可能にする有効な検定のときに最初の例では、VBSの制御が正であると解釈することができる反応である。 <img src="/files/ftp_upload/1752/1752fig1.jpg" alt="図1" /> 陽性の結果が失敗した赤血球凝集の1例を図 。上記の図式表現では、heamagglutination反応が受け付けられません。 VBS血清陰性コントロールは井戸の底部にシートを形成することによって証明されるように正の赤血球凝集反応を表示します。これは、エラーが発生し、結果を受け入れることができないことを示します。このケースでは、プロセスが繰り返される。 <img src="/files/ftp_upload/1752/1752fig2.jpg" alt="図2" /> 図2。陽性の結果が正しく動作していた赤血球凝集の例。図式表現では、heamagglutination反応は有効です。 VBS陰性コントロールは陰性赤血球凝集反応が表示されますsは、井戸の基部に形成されたペレットからも明らか。最後の肯定的な井戸はこの血清サンプルの力価は128（すなわち1 / 128の逆数）であることを示す行7、です。これは、血清中の適度な量で存在する抗体の例になるであろう。

赤血球凝集アッセイは、時間の短い期間内に評価される血清サンプルの多数を可能にするとても簡単なテクニックです。それが適している目で、特殊な装置を必要とせずに読み取られるという利点もあります。この手法はまた、赤血球凝集阻害試験の一環として、尿中のヒト絨毛性ゴナドトロピンホルモンの存在の検出のため変更することができます。標準血球凝集試験に関連して、ケアが?...

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<td>Revercells</td>
<td>&nbsp;</td>
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<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>
<td>&nbsp;</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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<tr>
<td>Veronal buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use at 1x final</td>
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<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

赤血球凝集は凝集の特定の形式であり、抗体が粒子状抗原として​​機能する赤血球、にバインドするときに使用されます。赤血球は、容易に入手できるように特に有用な標的であると凝集は肉眼を用いて観測される。この手法は、一般的に血液型判定およびウイルス定量のために、抗体（Ab）の力価を決定するために使用されます。このビデオでは、赤血球凝集アッセイの準備と実行に必要なステップは、血液型抗原は、赤血球（Revercells）に追加するに特異的な抗体を用いて示されています。抗血清は、シリアルRevercellsの懸濁液を添加された96ウェルU底マイクロタイタートレイ、中に希釈される。サンプルを混合し、その後60分間37℃でインキュベートする。この時間後、次いで、試料を容易にVE、+ VEと中間(-/+)の赤血球凝集反応のために得点することができます。このアプローチは、迅速かつ簡単な手法を用いて評価される血清サンプルの反応性と力価が可能になります。ビデオは、アッセイを変更する方法、力価が決定される方法、結果を読み、どのように解釈されるか、分析の背後にある理論をカバーし、この技術の使用に関連するあらゆる問題になります。

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

赤血球凝集は、抗体が赤血球に結合凝集の形式です。赤血球は、両方とも容易に入手可能であり、結果は容易に肉眼を使って観測している。このビデオでは、結果を解釈し、力価を決定する、赤血球凝集アッセイに必要な手順を示しています。

赤血球凝集アッセイを用いて血清の反応性と力価の決定

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 ビュー.  University of South Australia. 赤血球凝集は、抗体が赤血球に結合凝集の形式です。赤血球は、両方とも容易に入手可能であり、結果は容易に肉眼を使って観測している。このビデオでは、結果を解釈し、力価を決定する、赤血球凝集アッセイに必要な手順を示しています。

ビデオ: 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.

50パーセント溶血補体（CHの測定 50</sub血清中>）活動

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

免疫・感染学

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.

臨床抗体サンプルの貪食活性を決定する

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システムにCR51リリースアッセイを比較することによって、人間Her2neu特異的CD8 T細胞の最適な細胞傷害活性を決定する

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.

ヒト免疫不全ウイルスの複製フィットネスを決定するためのペアごとの成長競合アッセイ

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.

ハイスループット定量的リアルタイムRT-PCRアッセイのタイプIおよびIIIインターフェロンサブタイプの発現プロファイルを決定するための

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.

網膜ミクログリアの貪食機能を評価します<em&gt;インビボ</em&gt;フローサイトメトリーベースのアッセイを使用しました

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.

定量的マイクロ中和アッセイの最適化

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.

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

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.

単純なフローサイトメトリーによるジカ回復期血清を用いたデング ウイルスのIn Vitro抗体依存強化測定法

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).

迅速・安全・簡易マニュアル ベッドサイド核酸抽出の全血試料中のウイルス検出

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