The authors thank Miss Ching-Chun Lin and Miss Diana Lin of the Core Facility of the Institute of Cellular and Organismic Biology (ICOB) of Academia Sinica for offering their expertise on peptide synthesis and DNA sequencing, respectively. This study was supported by Academia Sinica.

1.单克隆抗体的制备<ol><li>培养的RG-M56小鼠单克隆杂交瘤细胞2在175T瓶中的无血清培养基在37ºC用5％的CO 2补充。收集上清液当介质的颜色变成后孵育五天黄色。 注:杂交瘤细胞细胞在无血清培养基中培养，以避免从胎牛血清抗体污染。 </li><li>离心上清在4ºC4500 xg离心30分钟，弃去细胞碎片沉淀。 </li><li>加入2毫升的G蛋白琼脂糖（作为50％的浆液提供）到5毫升柱，并用冰冷的PBS的10树脂体积（10毫升）中平衡。 </li><li>负载200毫升的抗体上清液（步骤1.2）到柱上并丢弃直通。 </li><li>添加10毫升冰冷的PBS到柱中来清洗。重复两次。 </li><li>加入10 mL的50mM甘氨酸，pH值2.7的到柱中来洗脱G蛋白相关的抗体。关口LECT 900微升馏分中含有100微升10X中和缓冲液（1摩尔Tris，1.5M NaCl和1mM EDTA的，pH值8.0）微量离心管中。 </li><li>在-20 C含0.03％NaN 3的存储在50％甘油的纯化的抗体。 </li></ol> （二）建设和串行截断重组蛋白的表达<ol><li>制备PCR反应混合物:5微升10X 的Pfu缓冲液，0.2mM的每种dNTP，0.2μM的正向引物3，0.2μM反向引物3，2毫硫酸镁 ，pET20b-1A59 3质粒DNA 1ng的，和2.5 U（单元） 的Pfu DNA聚合酶;添加的DDH 2 O至50微升的最终体积。 <ol><li>使用下列参数中的自动热循环运行的样品:循环1（94ºC5分钟）;周期2-36（94ºC30秒，63ºC30秒，72ºC60秒）;和循环37（72ºC7分钟）。 </li></ol></li><li>提取聚合酶链反应（PCR）产物通过使用PCR纯化试剂盒5，以方便下面的限制性内切酶消化。 </li><li>消化用NdeI I和Xho I限制性内切酶的PCR扩增的DNA片段和每个这些DNA片段的结扎成的Nde I和Xho I酶裂解的pET-20b中（+）载体。变换构建到大肠杆菌 DH-5α感受态细胞6。 <ol><li>准备在消化缓冲液中消化混合物（20毫摩尔Tris乙酸盐，10毫镁（CH 3 COO）2，50毫KCH 3 COO，和1mM DTT，pH值7.9）与1 PCR扩增的DNA或PET-20b的微克的Nde I和Xho I限制性的（+）载体DNA和2 U酶在20微升的最终体积。 </li><li>轻轻地混合消化混合物并迅速降速。在37ºC2小时，在干燥浴，以确保限制的完全切割坐上课。 </li><li>提取使用PCR纯化试剂盒5，以方便下面的质粒构建有关的限制性酶消化的DNA片段。 </li><li>准备在连接缓冲液中连接混合物（66毫摩尔Tris，5mM的MgCl 2的，1毫摩尔ATP，和5mM DTT，pH值7.5）用100ng简化的，PCR扩增的DNA的，预消化的pET-20b的10毫微克（+）载体DNA，并在10微升的最终体积5单位的T4 DNA连接酶。 </li><li>轻轻混合连接混合物并迅速降速。孵育16ºC用于在水浴18小时。 </li><li>把结扎样品入100微升在DH-5α感受态细胞的10微升和放置微量离心管在冰上进行30分钟，然后轻轻地混合。把离心管在干浴在42ºC为90秒诱导热休克7。紧接在管转移到冰上2分钟。 </li><li>添加900μL的Luria-BERTANI（LB）肉汤（1％Bacto胰蛋白胨，0.5％细菌酵母为Extract和0.5％的NaCl，pH 7.0）中到管。在37ºC孵育150 rpm振摇45分钟。沉淀通过离心将细胞在10分钟4000×g下，弃上清。 </li><li>重悬的LB肉汤的50微升沉淀和每个变换涂布在含有100μg/ mL氨苄青霉素预热LB平板上。在37ºC16小时孵育所述板。 </li><li>用200微升尖端拿起单个菌落并将其放入含有一个松散皑皑的15毫升管100微克/毫升氨苄青霉素3毫升的LB肉汤。在37ºC孵育150 rpm振摇12小时。 </li><li>提取来自每个培养物的质粒DNA 8和使用T7启动子和T7终止子引物，以确认序列9测序它。 </li></ol></li><li>序列确认后，将10纳克这些PET-20B的DNA（+）与美国YGNNV外壳蛋白基因的长度变化到大肠杆菌的BL-21（DE3）菌株质粒ING热休克法7。按照步骤2.3.6-2.3.8执行转换。 </li><li>从每个转化的大肠杆菌 BL-21细胞传输单个菌落到含有在一个松松地盖上瓶盖15毫升管100微克/毫升氨苄青霉素的3毫升的LB肉汤。孵育文化在37ºC以150rpm下振荡。 </li><li>冷却该培养至25ºC当培养物的OD 600为约0.6，并添加IPTG至0.4mM的最终浓度来诱导重组蛋白的表达。孵育一个额外的4小时培养在25ºC以200rpm振荡。 </li><li>传送1毫升培养成一个离心管中。沉淀通过离心将细胞在12,000×g离心1分钟，弃上清。 </li><li>通过上下吹打用微量重悬在100微升变性缓冲液（8M脲，20mM磷酸钠，和0.5 M氯化钠，pH 7.4）中的细胞沉淀。剧烈震荡混合。 注:该样品溶胶以及英现在应该是半透明的，有点粘，并准备好了斑点杂交检测。 </li></ol>重叠肽的3设计与合成<ol><li>设计并合成3串行20聚体的肽，每个具有其后继通过从YGNNV外壳蛋白的195-338氨基酸区域10氨基酸残基通过斑点印迹法来缩小的RG-M56单克隆抗体的表位区重叠。 </li><li>设计并合成3三个8聚体肽（195 VNVSVLCR 202，197 VSVLCRWS 204和199 VLCRWSVR 206）与6节AA残留到下一个合成肽重叠斑点杂交缩小195-206 AA的表位区。 </li><li>设计并合成3 7聚体（196 NVSVLCR 202和195 VNVSVLC 201），6聚体（195 VNVSVL 200，196 NVSVLC 201，和1个97 VSVLCR 202），和5-mer肽（195 VNVSV 199，196 NVSVL 200，197 VSVLC 201，和198 SVLCR 202）分别与6，5重叠，和4个氨基酸残基，到它们相邻的肽，以尽量减少斑点杂交表位区。 </li></ol> 4.斑点杂交<ol><li>溶解在二甲基亚砜（DMSO）各合成肽的10毫克/毫升的最终浓度。 注意:要克服的合成肽的不同的溶解度，所有合成肽应溶解在DMSO中。 DMSO是良好的溶剂，以完全溶解疏水性或亲水性的肽。 </li><li>泡用甲醇聚偏二氟乙烯（PVDF）膜2分钟。 注:PVDF膜是高达100％的DMSO抗性;其他人可能并非如此。 </li><li>改性Towbin缓冲液（25mM的Tris，192毫甘氨酸，和0.1％SDS，pH值8.3），用于平衡的PVDF膜2分钟。 注:10〜20％（V / V）的甲醇可以加入到改性Towbin缓冲改善传输结果。 </li><li>冲洗修改Towbin缓冲一张色谱纸。 PVDF膜放置到层析纸上。等到改性Towbin缓冲已经从PVDF膜表面在继续下一步骤之前消失。 </li><li>加入2微升每种肽样品在膜上，用10微升小费。风干的色谱纸PVDF膜10分钟。缓慢和逐渐加入各肽样品在膜上，以避免过多的扩散。 </li><li>在室温下温和振荡30分钟，阻断在TBST缓冲液中的5％脱脂牛奶的膜（0.05％（体积/体积）吐温20，20毫摩尔Tris，150 mM氯化钠，pH 7.4）中。 </li><li>添加RG-M56单克隆抗体以1:1的最终稀释度:1,000在TBST缓冲液，用5％脱脂牛奶的膜。在37ºC膜上轻轻摇动1小时。 </li><li>取出抗体等等（分辨率）。洗在TBST缓冲液将膜5分钟，轻轻摇动。重复两次。 </li><li> 5000在TBST缓冲液中的5％脱脂牛奶的膜:以1:1的最终稀释度加入二级抗体（山羊抗小鼠IgG，Fc的，共轭的碱性磷酸酶）。在37ºC膜上轻轻摇动1小时。 </li><li>弃去抗体溶液。洗在TBST缓冲液将膜5分钟，轻轻摇动。重复洗涤步骤两次。 </li><li>开发在室温BCIP / NBT底物溶液在黑暗中15分钟的膜。通过与DDH 2 O的洗膜出现信号时，停止发展。 </li><li>风干的膜和使用图像系统捕获斑点印迹图像。 </li><li>测量通过图像分析软件3每个斑点的强度。 </li></ol> 5.丙氨酸扫描和替代<ol><li>设计并合成3丙氨酸和methionine替代肽。用丙氨酸替换每个氨基酸残基的8聚体肽195 VNVSVLCR 202和合成肽195 ANVSVLCR 202，195 VAVSVLCR 202，195 VNASVLCR 202，195 VNVAVLCR 202，195 VNVSALCR 202，195 VNVSVACR 202，195 VNVSVLAR 202和195 VNVSVLCA 202 。更换AA残留亮氨酸200蛋氨酸创建SJNNV基因型表位肽195 VNVSVMCR 202。 </li><li>按照第4节进行丙氨酸扫描和替代诱变斑点印迹。 </li></ol>

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The authors have no conflicts of interest related to this report.

该协议提供了一个快速，简单的技术来识别单抗公认的线性表位。考虑到肽合成和合成肽的生产效率的成本，病毒外壳蛋白的抗原区由前肽扫描分析表达串联截短重组蛋白减少。因此，使用可靠的和有效的大肠杆菌 pET表达系统产生这些串联截短的重组蛋白，与50 10之间的分子量的重组蛋白质kDa的可以容易地通过该系统中表达。在这种方式中，表位可以容易地缩小到一个更容易管理的100到200?...

在免疫系统中，V，D和J片段的重组允许抗体结合至各种抗原，以保护从病原体感染的主机创建互补决定区（CDR）的巨大变化。针对抗原的抗体的中和防御依赖于抗体的CDR和抗原的抗原决定簇之间的空间互补性。因此，这种分子相互作用的理解将有助于预防性疫苗的设计和治疗肽的药物开发。然而，这种中和的相互作用可以都通过从一个单一的抗原多抗原结构域和由抗体的多个的CDR，其结果使表位判定处理更复杂的影响。幸运的是，杂交瘤技术的发展，该熔断器个体抗体产生细胞与骨髓瘤细胞，允许细胞不断地分裂批次秒RETE一个特定抗体称为单克隆抗体（mAb）1。杂交瘤细胞生产这些纯，高亲和力的单克隆抗体绑定到特定抗原的单一抗原域。既定的抗原 - 抗体，几种方法，包括肽扫描的关系，可用于确定使用其相应的单克隆抗体的抗原的表位。在合成肽技术的最新发展使得肽扫描技术更容易，更方便进行。简单地说，一组重叠的合成肽的根据靶抗原序列产生和关联到用于单抗杂交的固体支持膜。肽扫描不仅提供了一种简单的方式来映射抗体结合区，但也有利于氨基酸（AA）的诱变通过残扫描或取代来评估表位肽的每个氨基酸残基和抗体的CDR之间的结合相互作用。 2，3，4中的黄色石斑鱼神经坏死病毒（YGNNV）外壳蛋白的线性表位的有效识别的协议。该协议包括单克隆抗体制备，结构和串联截短重组蛋白的表达，合成的重叠肽的设计，斑点杂交，丙氨酸扫描，和​​替代诱变。考虑肽合成的高成本，的串行截断所需靶蛋白的重组蛋白的步骤被修改，进行合成肽阵列斑点印迹分析前的抗原区被缩小到约100至200个氨基酸残基。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hybrid-SFM medium</td>
			<td>Gibco</td>
			<td>12045-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeccos&#39;s Phophate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu DNA Polymerase&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>EP0502&#160;</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Roche</td>
			<td>10799009001</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>NdeI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>XhoI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>pET-20b(+) vector</td>
			<td>Novagen, Merck Millipore</td>
			<td>69739</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli DH-5&#945; competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH617</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli BL-21(DE3) competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH217</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Amresco</td>
			<td>0339-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth&#160;</td>
			<td>Invitrongen</td>
			<td>12780-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropylthio-&#946;-D-thiogalactoside (IPTG)</td>
			<td>MDBio, Inc.</td>
			<td>101-367-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T.Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Amresco</td>
			<td>0167-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Affymetrix, USB</td>
			<td>75825</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Amresco</td>
			<td>0241-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Amresco</td>
			<td>0105-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Amresco</td>
			<td>0854-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>PerkinElmer</td>
			<td>NEL937001PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG, Fc fragment antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-055-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P (Polyvinylidene fluoride, PVDF)</td>
			<td>Merck Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Agarose Fast Flow</td>
			<td>Merck Millipore</td>
			<td>16-266</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP BioSpectrum 600 Image System</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>VisionWorks LS Analysis Software Ver 6.8</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>MyCycler thermal cycler</td>
			<td>BioRad</td>
			<td>1709713</td>
			<td></td>
		</tr>
	</tbody>
</table>

peptide scanning-assisted identification of a monoclonal antibody-recognized linear b-cell epitope

抗原表位的免疫系统的识别允许用于中和可便于疫苗和肽类药物的发展抗体的保护机制的理解。肽扫描是直截了当映射由单克隆抗体（mAb）识别的线性表位的简单而有效的方法。在这里，作者提出了涉及连续截断重组蛋白，合成肽设计和斑点杂交使用中和单克隆抗体神经坏死病毒外壳蛋白的抗原识别表位确定方法。此技术依赖于合成肽和单克隆抗体上的聚偏二氟乙烯（PVDF）膜的斑点杂交。由RG-M56单克隆抗体识别的病毒外壳蛋白的最小抗原区可以通过一步一步的收窄修整肽作图到6聚体的肽表位。此外，丙氨酸扫描诱变和残余物分可以进行stitution来表征每个氨基酸残基组成的表位的结合的意义。发现侧翼表位的现场残留发挥肽结构调控关键作用。所识别的表位肽可被用来形成用于X射线衍射研究和功能的竞争，或用于治疗的表位肽 - 抗体复合物的晶体。

该实验的目的是通过点来识别的表位使用单克隆抗体印迹。以快速和有效地缩小由单克隆抗体识别的抗原区，全长和串联截断YGNNV重组外壳蛋白与在C末端带有6xHis融合标记是从大肠杆菌 pET表达系统10（ 图1A）来表示。所得重组蛋白点样到用的RG-M56单克隆抗体和抗的6xHis抗体进行斑点杂交的PVDF膜。在斑点印迹显示抗1-338氨基酸（全长），5...

Watch this Scientific Journal Video about Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope at JoVE.com

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

peptide-scanning-assisted-identification-monoclonal-antibody

Research

JoVE Journal

Immunology and Infection

9.3K 次观看.  Academia Sinica. Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

视频: Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

肽:MHC四聚体为基础的抗原表位特异性的T细胞的富集

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

科研

免疫与感染

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

肽为基础的识别功能图案和具有约束力的合作伙伴

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

鼠单克隆抗体的产生杂交瘤技术

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

重叠肽库在蛋白质 Qa-1 表位中的映射

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

针对天然产物的单克隆抗体的产生

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...