저자는 촬영에 대한 기술과 미스터 폴 목사는 수행 미스 로라 매튜스를 감사하고 싶습니다. 이 프로젝트는 사우스 오스트 레일 리아 대학, 연구소의 의학 프로그램에 의해 투자되었다.

5 배 Veronal 버퍼 살린 (VBS)의 준비 <ol><li> Veronal 버퍼 살린 (VBS)를 준비하려면, 별도의 세 가지 솔루션을 준비해야합니다. </li><li> 증류수의 350ml에 나트륨 Barbitone의 NaCl과 0.94gm의 21.25gm을 용해하여 용액 1을 준비합니다. NaCl과 나트륨 Barbitone의 최종 농도는 각각 1.02M과 13mM 있습니다. </li><li> 뜨거운 증류수의 125ml에 Barbitone의 1.44gm을 용해하여 용액 2를 준비합니다. Barbitone의 최종 농도는 62.5mM입니다. </li><li> 증류수의 100ml에 CaCl 2 MgCl 2 4.41gm의 20.33gm을 용해하여 용액 세를 준비합니다. MgCl 2, CaCl 2의 최종 농도는 각각 2.18M과 440mM입니다. </li><li> 믹스 솔루션 1, 2 룸 온도 냉각. </li><li> 통합 솔루션이 냉각되면, 솔루션 3 1.25 ML을 추가하고 1M HCL을 사용하여 7.3-7.5 수있는 산도를 조정합니다. </li><li> 배 재고 솔루션을 준비하는 증류수로 500ml로 최종 볼륨을 조정합니다. </li><li> 1X 작업 솔루션을 준비하려면, 증류수와 주식 1:5 희석. </li></ol> Revercells의 희석 <ol><li> 부드럽게 전도에 의해 Revercells의 주식 솔루션을 resuspend 후 4 600g에서 5 분 세포 원심 분리기의 1ml를 제거 ° C. </li><li> 원심 분리 후, 조심스럽게 뜨는을 제거하고 Veronal가 살린 버퍼 1X의 3ml에있는 세포를 resuspend. </li><li> 세포는 이제 4 ° C까지 사용할 수 저장할 수 있습니다. </li></ol> 절차 <ol><li> 96 잘 U 바닥 접시의 행 A1 - A12에 1X VBS의 50ml를 전송합니다. </li><li> 자 A1면, 안티 - A 혈청 50μl를 추가하고 피펫을 사용하여 철저히 섞는다. </li><li> 신선한 피펫 팁을 사용하여, 잘 A1에서 액체의 50μl을 제거하고 잘 A2로 전송할 수 있습니다. 철저하게 혼합하고 잘 A11에 도달하고이 우물에서 마지막 50μl를 삭제 때까지이 과정을 반복합니다. </li><li> 혈청 샘플은 현재 순차적으로 1시 2분에서 1:2048 2 배 희석되었습니다. 이 VBS 부정적인 컨트롤로 더 혈청 잘 A12에 추가되지 않습니다. </li><li> 하나 이상의 혈청 샘플은 평가되어야하는 경우, 반복 행의 B 등의 4.1-4.4 단계 </li><li> 일단 모든 dilutions가 있었고, 모든 우물 1 %의 Revercells의 50μl를 추가합니다. 참고 : Revercells 시간을 통해 해결하고 부드럽게 우물에 총동원되고 이전에 중단되어야합니다. </li><li> 부드럽게 microtitre 트레이의 측면을 두드리고하여 샘플을 믹스, 커버와 37 1H에 대한 진행 haemagglutination 반응 ° C.를 떠나 보육 또는 적절하게 가열 방을 사용할 수 있습니다. </li><li> 부화 다음, 조심스럽게 인큐베이터에서 트레이를 제거하고 haemagglutination에 대한 번호판을 검사합니다. VBS 컨트롤은 좋​​지 검사 가장 먼저해야합니다. 반응이 부정적이라면, 결과는 유효합니다. VBS 샘플은 긍정적인 haemagglutination 결과를 보여주는 경우, 결과는 사용할 수 없습니다와 과정은 반복되어야합니다. 부정적인 반응이 잘의 중앙에 세포의 작은 집중 펠렛으로 나타납니다 동안 긍정적인 haemagglutination 반응은 우물의 바닥에 넓은 시트로 표시됩니다. 중간 결과는 pelleted 세포의 중앙 코어와 함께 긍정적인 세포의 후광을 것입니다. 혈청 반응 수있는 AB를 가지고 본 결과가 발생하지만, AB의 부족한 금액은 전체 반응을 일으킬 수 있습니다. </li><li> 대표적인 결과 섹션 예상 결과 두 도표 표현이 있습니다. 결과의 무효와 유효 기간 설정의 예제는 제공됩니다. 백신 혈청 titre도 결정됩니다. </li></ol> 대표 결과 : 비디오는 대표적인 결과의 예를 포함하고 있습니다. 아래 (Fig1과 그림 2) 도표 방식에 표시된 두 예제입니다. 두 번째 결정하는 titre을 허용 유효한 분석하는 동안 첫 번째 예제는 VBS 컨트롤이 긍정적으로 해석될 수없는 반응입니다. <img src="/files/ftp_upload/1752/1752fig1.jpg" alt="그림 1" /> 그림 1. haemagglutination의 예를 들어 긍정적인 결과는 실패했습니다 어디에. 위의 도표 표현에서 heamagglutination 반응은 허용되지 않습니다. VBS 혈청 부정적인 컨트롤은 물론의 기본에있는 시트 형성에 의해 입증으로 긍정적인 haemagglutination 반응을 표시합니다. 이것은 오류가 발생하고 그 결과가 수락 수 없다는 것을 나타냅니다. 이 경우 프로세스는 반복해야합니다. <img src="/files/ftp_upload/1752/1752fig2.jpg" alt="그림 2" /> 그림 2. 긍정적인 결과가 올바르게 작동이 haemagglutination의 예. 도표 표현에서 heamagglutination 반응이 유효합니다. VBS 대조군은 부정적인 반응 haemagglutination에게 표시됩니다S는 우물의 바닥에 형성 하였다 펠렛에 의해 입증. 마지막으로 긍정적인 우물이 혈청 샘플에 대한 titre 128 (예 : 128분의 1의 상호)을 나타냅니다 행 7입니다. 이것은 혈청에서 적당한 금액에 존재하는 항체의 예입 것입니다.

haemagglutination 분석 시간이 짧은 기간 내에 평가하기 위해 혈청 샘플의 수많은 수있는 아주 간단한 방법입니다. 그것은 적합한되는 눈은, 전문 장비가 필요없이 읽을되는 장점도 있습니다. 이 기술은 또한 haemagglutination - 억제 테스트의 일환으로, 소변에있는 인간 chorionic gonadotrophin 호르몬의 검출을 위해 수정할 수 있습니다. 표준 haemagglutination 테스트와 관련하여, 관리가 제대로 수행할 수있는 분?...

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<td>Revercells</td>
<td>&nbsp;</td>
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<td>TRP</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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determining the reactivity and titre of serum using a haemagglutination assay

Haemagglutination는 응집의 구체적인 형태이며, 항체가 미립자 항원의 역할을 적혈구에 바인딩할 때 사용됩니다. 적혈구들이 쉽게 사용할 수 있으므로 특히 유용 타겟하고 응집이 육안을 사용하여 관찰할 수있다. 이 기술은 일반적으로 혈액 그룹화 및 바이러스에 대한 항체 부량의 titre (AB)를 결정하는 데 사용됩니다. 이 비디오에서는 haemagglutination 분석을 준비하고 수행에 관련된 단계는 혈액형 A - 항원은 적혈구 (Revercells)에 추가 특정 항체를 사용하여 증명합니다. 항혈청는 순차적으로 Revercells의 정지를 추가하는, U - 96 잘 하단 microtitre 트레이에 희석합니다. 샘플은 60 분 동안 혼합 후 ° C 37 incubated 수 있습니다. 이 시간 후, 샘플 그때 쉽게했습니다 + 적 및 중간 (-/+)의 haemagglutination 반응에 대한 채점 수 있습니다. 이 방법은 신속하고 간단한 방법을 사용하여 평가하는 혈청 샘플의 반응과 titre을 허용합니다. 비디오 분석을 수정할 수있는 방법을 titre를 결정하는 방법 결과를 읽고 해석하는 방법 분석 뒤에 이론, 그리고이 기술의 사용과 관련된 문제를 다룰 것입니다.

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

Haemagglutination는 항체가 적혈구에 바인딩 유착의 한 형태입니다. 적혈구가 모두 쉽게 사용할 수 있으며 그 결과는 즉시 육안을 사용하여 관찰할 수 있습니다. 이 비디오는 결과를 해석하고 titre을 결정, haemagglutination 분석에 관련된 단계를 설명합니다.

Haemagglutination 분석을 사용하여 혈청의 반응성 및 Titre를 결정

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 조회수.  University of South Australia. Haemagglutination는 항체가 적혈구에 바인딩 유착의 한 형태입니다. 적혈구가 모두 쉽게 사용할 수 있으며 그 결과는 즉시 육안을 사용하여 관찰할 수 있습니다. 이 비디오는 결과를 해석하고 titre을 결정, haemagglutination 분석에 관련된 단계를 설명합니다.

비디오: 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 % Haemolytic 보완 (CH 측정 50</sub혈청>) 활동

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

연구

면역학 및 감염병학

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.

임상 항체 샘플 Phagocytic 활동 결정

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 시스템으로 CR51 릴리스 분석을 비교하여 인간 Her2neu 특정한 CD8 T 세포의 최적 세포 독성 활동 결정

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.

인간 면역 결핍 바이러스의 복제 피트니스를 결정하기위한 인접 쌍 성장 경쟁 분석

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.

높은 처리량 정량 실시간 RT-PCR 분석 유형 I과 III 인터페론 하위 유형의 발현 양상을 결정하는

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.

망막 미세 아교 식세포의 기능을 평가<em&gt; 생체</em&gt;은 유동 세포 계측법 기반 분석을 사용하여

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.

정량적 마이크로 중화 분석의 최적화

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.

Vivo에서 예방 접종 모델을 사용 하는 항 원 특정 T-셀 Cytolytic 함수의 검색에 대 한 분석 결과

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.

간단한 Cytometry 결정 체 외에서 항 체 의존의 향상 뎅기열 바이러스 Zika 바이러스 미리 혈 청을 사용 하 여 분석 결과 기반으로

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).

전체 혈액 샘플에서 바이러스의 검출, 안전, 신속 하 고 간단한 수동 침대 핵 산 추출

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