Name: a semi-automated approach to preparing antibody cocktails for immunophenotypic analysis of human peripheral blood
Uploaded: 2016-02-08T15:00:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood at JoVE.com

We thank Hannah Puzas for assistance with system design and configuration, and Kevin Khovananth for technical advice. Funding for this work was provided by The Hearst Foundations and the Providence Portland Medical Foundation.

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YK，ILG，TLM，WLM，DPH和KSB:没有竞争的经济利益。 ANT是Beckman Coulter公司的员工，这个视频文章的出版公司是由Beckman Coulter，Inc赞助

外周血免疫是获得分析上市个别响应的免疫治疗至关重要。挑战在于标准化检测控制实验对实验变异1,14。变异性的一个主要来源在于样品的人的操纵。因此，可以想到，样本处理的部分或全部自动化将促进实验对实验变异性-1,14-显着减少。在这个协议中，我们报道了成功的努力通过导入装有二维条码阅读器，用于在全血免疫样品染色的自动化液体处理器，以尽量减少测定变异性。 在设计中，我们的方法是通用的，可以通过添加额外的"基地鸡尾酒"来定义他们监视其他免疫子集。这样的子集包括T辅助细胞，调节性T细胞，B细胞，NK细胞，树突细胞，和单核细胞（手稿中准备）18。我们通过引入额外的1标记适应财团的建议抗体面板。细胞群用"基地鸡尾酒"共轭荧光染料以最小排放到明亮的紫罗兰色421（BV421），藻红蛋白（PE）标识，别藻蓝蛋白（APC）的探测器，因为我们想保留这些渠道的检测诱导抗原。此功能不仅保证了最高灵敏度监测免疫细胞的激活状态，但也使我们在这三个荧光最常与抗诱导标志物的抗体结合的新的激活标记灵活的住宿。因此，我们的方法不仅适用于全血免疫鸡尾酒制剂，但也用于染色其它样品，包括从分列组织（例如，肿瘤）中回收外周血单核细胞和细胞是有用的。 成功的前奏我们的方法元币种需要实验室器皿准备特别注意步骤，编程和该方法的执行。二维条码管制备包括具有人可读的标签和抗体的转移到指定的管标签。为了防止错误的引入，我们建议两个人这样做。只要更改了电子表格，调查人员应验证该方法是否正常。编程过程中选择合适的移液的方法，确保成功的液体转移。 LLS使移液未经抗体管，为此，我们请使用P50或P200导电尖端的底部得到沉淀杂物。 LLS可能不是P1000提示一个不错的选择为LLS是P1000的提示更敏感，有时错误地气泡存在触发。在实验室器具定义的高度应为每个仪器可能发生作为次优的液体传输来调整，例如，如果没有提示的边缘之间有足够的空间和管子的底部。 如图 12所示，如果"基地鸡尾酒"和/或"激活鸡尾酒"都没有很好涡旋，它可能会导致次优的染色。 我们想过用冻干试剂细胞染色作为一种替代方法，以减少与试剂分装19相关的变异。然而，灿烂紫色染料的聚合物缀合物是已知的相互引起的非特异性信号进行交互。这样，加在冻干试剂两个以上聚合物缀合物（例如，BV421和BV650 等）可能会引起非特异性的信号（例如，在BV421 +群体 BV650背景信号增加）20。此外，冻干试剂缺乏包括新染色的灵活性。他们通常比较昂贵，并且需要大量订购。由于这些原因，我们选择使用搭载二维条码管自动化液体处理程序。虽然ŧ碟刹时间来建立较难，导致了前期投资购买仪器，从长远来看，这些因素将会由生产力的提高和检测的重复性进行补偿。事实上，几组先前报告的自动液体处理器的成功整合到他们的免疫或类似的应用程序21,22的工作流程。对于流式细胞仪分析的自动化解决方案也可从商业来源（FACS SPA三，自动鸡尾酒制备工作站，和FlowStainer）。这进一步表明有用于免疫自动鸡尾酒制剂极其需要。 掌握这一技术后，我们设想，全自动全血免疫的发展将进一步降低实验对实验变异，可能使全血免疫即使是在多中心临床试验设置23可行的。用裂解是我们已经开始^ h助手自动裂解和洗涤步骤。我们还设想，抗体的二维条码管的体积和试剂分的跟踪自动化决心将大大提高分别抗体的库存和在我们的方法中的质量控制。

的人末梢血免疫确定在免疫细胞亚群1的定量和定性的改变。它提供了洞察和行动性，预测性的生物标志物的发现艾滋病的机制，并促进联合免疫疗法的发展。因此，免疫的验证和标准化是相当感兴趣的学术研究人员，临床实验室，和工业的领域。 虽然外周血通过流式细胞方法免疫目前用于血液恶性肿瘤2,3和人类免疫缺陷病毒（HIV）感染4,5，免疫治疗需求的外周血可靠免疫在验证和标准化，因为它具体考虑的临床管理要求对免疫细胞亚群和激活/抑制性受体1,6-8覆盖范围更加广泛。帅客ssful免疫需要仔细标准化，以减少相关的仪器设置和细胞染色过程9,10实验对实验的可变性。虽然流式细胞仪的仪器设置标准化是公认的解决前者关注9,11,12，它如何在不限制免疫细胞亚群和他们的激活状态的覆盖范围最小化与细胞染色变化仍不清楚。 作为待检测的抗原的数目的增加，所以也没有用于误差和变异由于不理想的试剂分注和交叉污染的机会。成立于80年代末用于临床实验室检测为使用抗凝全血phenoptypic分析方法。这些相同的方法服务于科研实验室样品制备的需求。重要的是，在临床实验室中使用的抗体的鸡尾酒通常较不复杂和AVAilable预滴定和从制造商预混合。仅需要预混合抗体混合物的单个传输。在研究设置，10-16抗体的抗体鸡尾酒是典型的。每个鸡尾酒必须通过实验室验证的稳定性或制备的每个实验前新鲜。准备多种抗体鸡尾酒的样本可能带来50-80个人抗体的移液，既繁琐又容易出错的任务。 鸡尾酒制剂的自动化拥有手动鸡尾酒制剂几个优点，例如更少的错误，提高了移液的准确度，甚至可能降低试剂的浪费。这里，我们报告成功引入装有的二维（2D）条形码读取器到免疫的细胞染色过程中尽量减少样品制备相关的变异的自动液体处理器的。

<table><tbody><tr><td>BD LSRFortessa</td><td>BD Biosciences</td><td></td><td>Flow cytometer</td></tr><tr><td>NXp Span-8 Laboratory Automation Workstation&amp;nbsp;</td><td>Beckman Coulter</td><td>A31839</td><td>Laboratory Automation Workstation&amp;nbsp;</td></tr><tr><td>Insert, Tube, 11 mm, White, for 1.5 ml Microfuge Tubes (case of 25)</td><td>Beckman Coulter</td><td>373696</td><td>Accessary for 24-Position Tube Rack to accommodate microfuge tubes.&amp;nbsp;</td></tr><tr><td>LLS kit</td><td>Beckman Coulter</td><td>719262</td><td>Attach bottom of the deck in Laboratory Automation Workstation to enable liquid level sensing</td></tr><tr><td>24-Position Tube Rack</td><td>Beckman Coulter</td><td>373661</td><td>Tube rack to hold microfuge tubes for antibody cocktails or blood</td></tr><tr><td>BIOHAZARD AUTOCLAVE BAGS 12 x 24 INCHES RED</td><td>LabMart</td><td>M111416</td><td>Disposable autoclave bags for the Laboratory Automation Workstation</td></tr><tr><td>VisionMate High Speed 2D Barcode Reader</td><td>Thermo Scientific</td><td>AB-1850</td><td>2D barcode reader</td></tr><tr><td>8-Channel Handheld Screw Cap Capper/Decapper</td><td>Thermo Scientific</td><td>4105MAT</td><td>For de-capping and capping 2D barcode tubes&amp;nbsp;</td></tr><tr><td>Microcentrifuge tubes</td><td>Fisher Scientific</td><td>02-681-335</td><td>For making cocktail in or supplying blood speciment to the Laboratory Automation Workstation</td></tr><tr><td>CoolRack XT CFT24</td><td>biocision</td><td>BCS-534</td><td>To hold sample tubes.</td></tr><tr><td>1.0 ml Matrix ScrewTop Amber Tubes in Latch Racks</td><td>Thermo Scientific</td><td>3741AMB</td><td>2D barcode tubes to store fluorescent dye-cojugated antibodies</td></tr><tr><td>Biomek Span-8 P1000 Tips, Conductive, Pre-sterile with Barrier, 1,025 &amp;micro;l</td><td>Beckman Coulter</td><td>987925</td><td>Tips for Laboratory Automation Workstation&amp;nbsp;</td></tr><tr><td>Biomek Span-8 P50 Tips, Conductive, Pre-sterile with Barrier, 50 &amp;micro;l (Case of 10 racks)</td><td>Beckman Coulter</td><td>B01091</td><td>Tips for Laboratory Automation Workstation&amp;nbsp;</td></tr><tr><td>Biomek Span-8 P250 Tips, Conductive, Pre-sterile with Barrier (Case of 10 racks)</td><td>Beckman Coulter</td><td>394627</td><td>Tips for Laboratory Automation Workstation&amp;nbsp;</td></tr><tr><td>BD FACS Lysing Solution 10x Concentrate</td><td>BD Biosciences</td><td>349202</td><td>RBC lysis</td></tr><tr><td>Falcon Round-Bottom Polystyrene Tubes</td><td>Fisher Scientific</td><td>14-959-5</td><td>Sample tubes</td></tr><tr><td>Anti-CD45 FITC</td><td>BD Biosciences</td><td>347463</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CD3 Alexa Fluor 700</td><td>eBioscience</td><td>56-0038-42</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CD4 PerCPCy5.5</td><td>BD Biosciences</td><td>341654</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-TCRgd BV650</td><td>BD Biosciences</td><td>564156</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-TCRab BV786</td><td>BD Biosciences</td><td>563825</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CD8 APC-H7</td><td>BD Biosciences</td><td>560179</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CCR7 PE-CF594</td><td>BD Biosciences</td><td>562381</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CD45RA PE-Cy7</td><td>BD Biosciences</td><td>337167</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-HLA-DR BV421</td><td>BD Biosciences</td><td>562804</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD278(ICOS) PE</td><td>BD Biosciences</td><td>557802</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD38 APC</td><td>BD Biosciences</td><td>340439</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD279(PD1) BV421</td><td>BD Biosciences</td><td>562516</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD357(GITR) PE</td><td>eBioscience</td><td>12-5875-42</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD137(41BB) APC</td><td>BD Biosciences</td><td>550890</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD69 BV421</td><td>BD Biosciences</td><td>562884</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD314(NKG2D) PE</td><td>BD Biosciences</td><td>557940</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD134(OX40) APC</td><td>BioLegend</td><td>350008</td><td>Effector T cell panel (activation)</td></tr><tr><td>BD Vacutainer Plastic Blood Collection Tubes with Sodium Heparin: Conventional Stopper</td><td>Fisher Scientific</td><td>02-689-6</td><td>For collecting blood</td></tr><tr><td>Albumin, Bovine Serum, Fraction V, RIA and ELISA Grade</td><td>EMD Millipore</td><td>126593</td><td>For flow wash buffer</td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S8032</td><td>For flow wash buffer</td></tr><tr><td>Heparin, sodium salt</td><td>Affimetrix</td><td>16920</td><td>For flow wash buffer</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS), 1x, with Calcium, Magnesium, without Phenol Red</td><td>GE Healthcare Life Sciences</td><td>SH30588.02</td><td>For flow wash buffer</td></tr></tbody></table>

a semi-automated approach to preparing antibody cocktails for immunophenotypic analysis of human peripheral blood

外周血通过流式细胞免疫仪确定疾病和治疗过程中外周血白细胞的频率和激活状态的改变。它具有预测治疗功效，并确定新的治疗目标的潜力。全血染色使用未经处理的血液，最大限度地减少可样品制备过程中可能出现的文物。然而，全血染色还必须在新鲜采集的血液进行，以确保样品的完整性。此外，最好是在同一天准备抗体的鸡尾酒，以避免串联染料的潜在不稳定性，防止灿烂的紫色染料之间试剂反应。因此，全血染色需要仔细标准化来控制内和实验间的差异。 这里，我们报告装有的二维（2D）条形码读取器的自动液体处理器的调配成使得antibod的标准方法Ÿ鸡尾酒流式细胞仪。抗体被转移到设置在数据库中编译一个96孔格式及其内容的二维条形码的管中。然后，液体处理器可以通过在数据库中引用抗体名称查找源抗体的小瓶。我们的方法消除了繁琐的协调源抗体管的定位。它提供的多功能性允许用户容易地通过修改的数据库，而不在每个测定中软件的方法重写脚本更改任何数目的抗体分配工艺细节，如特异性抗体的使用，体积，和目的地。 概念实验的证明取得了突出的间和分子内精密度，由一个11色，17 - 抗体流式细胞仪检测的重复编写证明。这些方法增加的总吞吐量，流式细胞仪检测，并协助每天准备的详细苯氧所需的复杂抗体鸡尾酒新鲜收集抗凝外周血typic表征。

Watch this Scientific Journal Video about A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood at JoVE.com

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.7K 次观看.  Providence Cancer Center, Providence Portland Medical Center. Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

视频: A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

科研

遗传学

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

免疫与感染

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

Source: Tzeng, S. J. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody&#39;s isotype, which correlates with the B cells&#39; secretory activity.

使用 ELISpot 测定对分泌抗体的 B 细胞进行功能评估

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

Isolation and Purification of B Cells from Human Peripheral Blood

Source: Tzeng, S. J. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.