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>

<ol>
	<li>Milstein, C., Kohler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+variations+of+myelomatous+cells+(proceedings).">Clonal variations of myelomatous cells (proceedings).</a> Minerva Med. 68 (50), 3453 (1977).</li><li>Lai, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+neutralization+by+monoclonal+antibodies+against+yellow+grouper+nervous+necrosis+virus+(YGNNV)+and+immunolocalization+of+virus+infection+in+yellow+grouper+Epinephelus+awoara+(Temminck+&amp;+Schlegel).">In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper Epinephelus awoara (Temminck &amp; Schlegel).</a> J Fish Dis. 24 (4), 237-244 (2001).</li><li>Chen, C. W., Wu, M. S., Huang, Y. J., Cheng, C. A., Chang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+Linear+B-Cell+Epitope+of+Betanodavirus+Coat+Protein+by+RG-M18+Neutralizing+mAB+Inhibits+Giant+Grouper+Nervous+Necrosis+Virus+(GGNNV)+Infection.">Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection.</a> PLoS One. 10 (5), 0126121 (2015).</li><li>Lai, Y. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+yellow+grouper+nervous+necrosis+virus+(YGNNV)+in+a+new+nodavirus-susceptible+cell+line+from+yellow+grouper,+Epinephelus+awoara+(Temminck+&amp;+Schlegel),+brain+tissue.">Propagation of yellow grouper nervous necrosis virus (YGNNV) in a new nodavirus-susceptible cell line from yellow grouper, Epinephelus awoara (Temminck &amp; Schlegel), brain tissue.</a> J Fish Dis. 24 (5), 299-309 (2001).</li><li>Lougee, E., Morjaria, S., Shaw, O., Collins, R., Vaughan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+HLA+typing+designed+for+solid+organ+transplantation:+epityping+and+its+application+to+the+HLA-A+locus.">A new approach to HLA typing designed for solid organ transplantation: epityping and its application to the HLA-A locus.</a> Int J Immunogenet. 40 (6), 445-452 (2013).</li><li>Radulovich, N., Leung, L., Tsao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+gateway+system+for+double+shRNA+expression+and+Cre/lox+based+gene+expression.">Modified gateway system for double shRNA expression and Cre/lox based gene expression.</a> BMC Biotechnol. 11, 24 (2011).</li><li>Froger, A., Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+plasmid+DNA+into+E.+coli+using+the+heat+shock+method.">Transformation of plasmid DNA into E. coli using the heat shock method.</a> J Vis Exp. (6), e253 (2007).</li><li>Pronobis, M. I., Deuitch, N., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Miraprep:+A+Protocol+that+Uses+a+Miniprep+Kit+and+Provides+Maxiprep+Yields.">The Miraprep: A Protocol that Uses a Miniprep Kit and Provides Maxiprep Yields.</a> PLoS One. 11 (8), e0160509 (2016).</li><li>Metzker, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+in+DNA+sequencing.">Emerging technologies in DNA sequencing.</a> Genome Res. 15 (12), 1767-1776 (2005).</li><li>Chiu, C. C., John, J. A., Hseu, T. H., Chang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+ayu+(Plecoglossus+altivelis)+Pit-1+in+Escherichia+coli:+its+purification+and+immunohistochemical+detection+using+monoclonal+antibody.">Expression of ayu (Plecoglossus altivelis) Pit-1 in Escherichia coli: its purification and immunohistochemical detection using monoclonal antibody.</a> Protein Expr Purif. 24 (2), 292-301 (2002).</li><li>Atassi, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+structures+of+proteins.+Their+determination+has+revealed+important+aspects+of+immune+recognition+and+generated+strategies+for+synthetic+mimicking+of+protein+binding+sites.">Antigenic structures of proteins. Their determination has revealed important aspects of immune recognition and generated strategies for synthetic mimicking of protein binding sites.</a> Eur J Biochem. 145 (1), 1-20 (1984).</li><li>Opuni, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+epitope+mapping.">Mass spectrometric epitope mapping.</a> Mass Spectrom Rev. , (2016).</li><li>Chang, C. Y., Chiu, C. C., Christopher John, J. A., Liao, I. C., Lea&#241;o, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Aquaculture of Groupers. , 207-224 (2008).</li><li>Conti-Tronconi, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-bungarotoxin+and+the+competing+antibody+WF6+interact+with+different+amino+acids+within+the+same+cholinergic+subsite.">Alpha-bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite.</a> Biochemistry. 30 (10), 2575-2584 (1991).</li><li>Briggs, S., Price, M. R., Tendler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+specificity+of+antibody+recognition+of+carcinoma-associated+epithelial+mucins:+antibody+binding+to+synthetic+peptide+epitopes.">Fine specificity of antibody recognition of carcinoma-associated epithelial mucins: antibody binding to synthetic peptide epitopes.</a> Eur J Cancer. 29 (2), 230-237 (1993).</li><li>Chen, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structures+of+a+Piscine+Betanodavirus:+Mechanisms+of+Capsid+Assembly+and+Viral+Infection.">Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection.</a> PLoS Pathog. 11 (10), e1005203 (2015).</li><li>Badani, H., Garry, R. F., Wimley, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+entry+inhibitors+of+enveloped+viruses:+The+importance+of+interfacial+hydrophobicity.">Peptide entry inhibitors of enveloped viruses: The importance of interfacial hydrophobicity.</a> Biochim Biophys Acta. , (2014).</li><li>Qureshi, N. M., Coy, D. H., Garry, R. F., Henderson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+putative+cellular+receptor+for+HIV-1+transmembrane+glycoprotein+using+synthetic+peptides.">Characterization of a putative cellular receptor for HIV-1 transmembrane glycoprotein using synthetic peptides.</a> AIDS. 4 (6), 553-558 (1990).</li></ol>

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><tbody><tr><td>Hybrid-SFM medium</td><td>Gibco</td><td>12045-076</td><td></td></tr><tr><td>Dulbeccos&#039;s Phophate-Buffered Saline (PBS)</td><td>Gibco</td><td>21600-069</td><td></td></tr><tr><td>Pfu DNA Polymerase&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>EP0502&amp;nbsp;</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&amp;nbsp;</td><td>New England Biolabs</td><td>R0111S</td><td>Including buffers</td></tr><tr><td>XhoI&amp;nbsp;</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>&lt;em&gt;E. coli&lt;/em&gt; DH-5&amp;alpha; competent cell</td><td>RBC Bioscience</td><td>RH617</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; 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&amp;nbsp;</td><td>Invitrongen</td><td>12780-052</td><td></td></tr><tr><td>Isopropylthio-&amp;beta;-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>NaN&lt;sub&gt;3&lt;/sub&gt;</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

9.4K 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

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

科研

生物化学

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

个性化肽阵列在器官移植 HLA 抗体检测中的研究

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

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 ...

免疫与感染

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 ...

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 ...

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

<meta charset="utf8"></head><body>用于抗原特异性 B 细胞单步鉴定的 Tetrameric 荧光抗原阵列

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

用于抗原特异性 B 细胞单步鉴定的 Tetrameric 荧光抗原阵列

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

生物工程

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 ...

Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct functional subsets based on the cytokines they secreted: for example, IFN-&#947; for CD4 T helper 1 cells, IL-4 and IL-13 for CD4 T helper 2 cells, IL-21 for CD4 T follicular helper cells, and IL-17 for CD4 T helper 17 cells. Analysis of hepatitis B virus (HBV)-specific CD4 T cells based on cytokine secretion after HBV-derived peptides stimulation could provide information not only about the magnitude of HBV-specific CD4 T-cell response but also about the functional subsets of HBV-specific CD4 T cells. Novel approaches, such as transcriptomics and metabolomics analysis, could provide more detailed functional information about HBV-specific CD4 T cells. These approaches usually require isolation of viable HBV-specific CD4 T cells based on peptide-major histocompatibility complex-II multimers, while currently the information about HBV-specific CD4 T-cell epitopes is limited. Based on an HBV-derived peptide matrix, a method has been developed to evaluate HBV-specific CD4 T-cell responses and identify HBV-specific CD4 T-cell epitopes simultaneously using peripheral blood mononuclear cells samples from chronic HBV infection patients.

Based on a hepatitis B virus (HBV)-derived peptide matrix, HBV-specific CD4 T-cell responses could be evaluated in parallel with identification of HBV-specific CD4 T-cell epitopes.

基于肽矩阵的 HBV 特异性 CD4 T 细胞响应和 HLA-DR 受限 CD4 T 细胞表位的识别分析

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

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

摘要

探索更多视频

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

个性化肽阵列在器官移植 HLA 抗体检测中的研究

肽：MHC四聚体为基础的抗原表位特异性的T细胞的富集

重叠肽库在蛋白质 Qa-1 表位中的映射

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

用于抗原特异性 B 细胞单步鉴定的 Tetrameric 荧光抗原阵列

用于抗原特异性 B 细胞单步鉴定的 Tetrameric 荧光抗原阵列

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

基于肽矩阵的 HBV 特异性 CD4 T 细胞响应和 HLA-DR 受限 CD4 T 细胞表位的识别分析

高通量MHC II类分子结合分析抗原表位肽的定量分析