CN110579610A - 用于检测t细胞活化的v域免疫抑制因子的试剂盒 - Google Patents

用于检测t细胞活化的v域免疫抑制因子的试剂盒 Download PDF

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CN110579610A
CN110579610A CN201910677351.9A CN201910677351A CN110579610A CN 110579610 A CN110579610 A CN 110579610A CN 201910677351 A CN201910677351 A CN 201910677351A CN 110579610 A CN110579610 A CN 110579610A
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Abstract

本发明提供了用于检测T细胞活化的V域免疫抑制因子(VISTA)的试剂盒，本发明提供的试剂盒中的人源化单克隆抗体能够高强度、特异性与VISTA抗原结合，具有高效检测VISTA表达相关疾病的辅助诊断作用。

Description

用于检测T细胞活化的V域免疫抑制因子的试剂盒
技术领域
本发明涉及体外检测领域，特别地涉及用于检测T细胞活化的V域免疫抑制因子的试剂盒。
背景技术
T细胞活化的V域免疫抑制因子(VISTA)是一种新型的抑制性免疫检查点蛋白。VISTA在肿瘤细胞上的表达及其相关调控机制尚不清楚。已有研究表明VISTA在人类卵巢癌和子宫内膜癌中高度表达。VISTA在子宫内膜癌中的上调与VISTA启动子甲基化状态有关。VISTA在肿瘤细胞中抑制T 细胞增殖和细胞因子的产生，并在体内减少肿瘤浸润的CD8+T细胞。抗 VISTA抗体延长荷瘤小鼠的存活时间。
近日，德克萨斯大学MD安德森癌症中心在PNAS期刊上发表论文“Comparison ofimmune infiltrates in melanoma and pancreatic cancer highlights VISTA as apotential target in pancreatic cancer”。研究小组发现，与黑色素瘤相比，VISTA优先在胰腺癌中高水平表达，研究人员的研究还提供了与黑色素瘤相比较的原发性和转移性胰腺癌免疫浸润的详细分析，这可能进一步有助于指导针对胰腺癌治疗的免疫治疗策略。另一研究表明， VISTA的表达与有无淋巴结转移、腺癌细胞分化程度、临床分期及预后相关(P<0.05)。
如此，需要开发针对动物模型或患者生物学样品中比现有ELISA灵敏度更高，更快捷的方法以辅助诊断和预后。
发明内容
为了解决上述技术问题，本发明提供用于检测T细胞活化的V域免疫抑制因子的试剂盒。
本发明是以如下技术方案实现的：
用于检测T细胞活化的V域免疫抑制因子的试剂盒，将VISTA抗体包被板制成固相抗体，所述VISTA抗体包括：
包含SEQ ID NO:01的氨基酸序列的CDR-L1，和/或
包含SEQ ID NO:02的氨基酸序列的CDR-L2，和/或
包含SEQ ID NO:03的氨基酸序列的CDR-L3，和/或
包含SEQ ID NO:04的氨基酸序列的CDR-H1，和/或
包含SEQ ID NO:05的氨基酸序列的CDR-H2，和/或
包含SEQ ID NO:06的氨基酸序列的CDR-H3。
进一步地，使用所述试剂盒的方法包括：将样品依次加入到抗体包被的微孔中，然后加入HRP标记的酶标液形成抗体-抗原-酶标记的复合物，彻底洗涤后加入TMB显色；TMB在HRP酶催化下呈蓝色，加入终止溶液后呈黄色，颜色深度与VISTA抗原含量呈正相关；测定溶液OD 450nm，根据标准曲线定量计算VISTA蛋白含量。
进一步地，所述抗体选自单克隆抗体，特异性结合人VISTA的抗体片段，包含特异性结合人VISTA抗体片段的融合蛋白，鼠源抗体，人源化抗体，鼠源单克隆抗体，优选地，所述抗体为人源化单克隆抗体。
进一步地，所述抗体包含抗体重链可变区氨基酸序列包括如SEQ ID NO:7所示氨基酸序列具有至少99％序列同一性的VH序列。
进一步地，所述抗体包含抗体轻链可变区氨基酸序列包括如SEQ ID NO:8所示氨基酸序列具有至少99％序列同一性的VL序列。
进一步地，所述抗体重链恒定区氨基酸序列如SEQ ID NO:7所示，所述抗体轻链恒定区氨基酸序列如SEQ ID NO:8所示。
进一步地，所述抗体的生产步骤包括在培养基及合适的培养条件下培养含有表达权利要求1-6所述抗体的核苷酸序列的宿主细胞，从所述培养基中和/或所述宿主细胞中通过常规方法回收并纯化抗体和/或抗原结合片段。
进一步地，所述试剂盒还包括酶标包被板、标品、标品缓冲剂、酶标液、样品稀释液、TMB显色剂、洗涤液、终止液。
进一步地，所述检测试剂盒辅助诊断肺癌胰腺癌、乳腺癌、卵巢癌、子宫内膜癌、黑色素瘤以及其他VISTA蛋白高表达的疾病类型。
具体实施方式
定义
本发明术语“抗体”以最广泛的含义使用，包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体、双特异性抗体和抗体片段，只要它们表现出特定的抗原结合活性，本发明术语“抗体片段”是指完整抗体以外的分子，其包含结合完整抗体所结合的抗原的完整抗体的一部分，抗体片段包括但不限于Fv、Fab、Fab'、双特异性抗体、线性抗体、单链抗体分子(例如scFv)和/或由抗体片段形成的多特异性抗体。
抗体的类别是指其重链所具有的恒定结构域或恒定区的类型。有五种主要类型的抗体：IgA、IgD、IgE、IgG和IgM，其中几种可进一步分成亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于免疫球蛋白的不同类别的重链恒定结构域分别称为α、δ、ε、γ和μ。
本发明术语“IgG”是免疫球蛋白G(Immuno globulin G)的缩写。本发明术语“人源化”是指包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在某些实施方案中，人源化抗体将包含至少一个、通常两个可变结构域的基本上全部，其中全部或基本上全部HVR对应于非人抗体的那些HVR，并且全部或基本上全部的FR对应于人抗体的那些FR。人源化抗体任选地可以包含源自人抗体的抗体恒定区的至少一部分。
抗体的结合特异性及亲合力均主要由CDR序列决定，根据成熟、公知的现有各项技术可轻易地将非CDR区域的氨基酸序列改变而获得具有相类似的生物活性的变体。本领域公知，抗原结合功能区是指可以与目标分子如抗原发生特异性相互作用的区域，其作用具有高度选择性，识别一种目标分子的序列通常不能识别其他分子序列。
本发明术语“结合”是指分子(例如抗体)的单个结合位点与其结合配偶体(例如抗原)之间的非共价相互作用。除非另有说明，本发明所述“结合力”是指结合对成员(例如抗体和抗原)之间1:1相互作用的内在结合亲和力。分子X对其配偶体Y的亲和力通常可以由解离常数(KD)表示，亲和力可以通过本领域已知的常用方法来测量。
本发明术语“同一性”、“相似度”为比对序列并引入间隙以实现最大百分比序列同一性后，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分比，不考虑任何保守取代作为序列同一性的一部分。可以以本领域技术范围内的各种方式实现为了确定百分比氨基酸序列同一性的目的比对。
术语“特异性地结合”是指，抗体或其抗原结合片段“特异性地结合”另一分子的区域，具体地，是指相对于另外的表位与该表位更加频繁地、更加快速地、以更长的持续时间和/或以更大的亲和力或亲合力反应或结合。在一些实施方案中，本发明的抗体或其抗原结合片段以至少10-7M的亲和力结合抗原，例如10-8M、10-9M、10-10M、10-11M或更高。优选地，抗体或其抗原结合片段在生理条件下(例如，在体内)结合。因此，特异性地结合到抗原是指该抗体或其抗原结合片段以上述特异性和/或在这样的条件下结合到抗原上的能力，适合于确定所述结合的方法是本领域已知的。
术语“结合”通常是指以对应于约10-6M或更小的KD的亲和力结合，该KD比该抗体对与除指定抗原或紧密相关抗原之外的非特异性抗原结合的亲和力低至少10倍，如低至少100倍、至少1,000倍。
如本文所用，术语“kd”(sec-1或1/s)是指特定抗体-抗原相互作用的解离速率常数，所述值又称为koff值。
如本文所用，术语“ka”(M-1x sec-1或1/Msec)是指特定抗体-抗原相互作用的结合速率常数，所述值又称为kon值。。
如本文所用，术语“KD”(M)是指特定抗体-抗原相互作用的解离平衡常数并且通过kd除以ka获得。
如本文所用，术语“KA”(M-1或1/M)是指特定抗体-抗原相互作用的结合平衡常数并且通过ka除以kd获得。
研究表明VISTA是肿瘤形成的关键因子。VISTA浓度肿瘤等疾病存在高度相关性。日前已有很多研究表明循环VISTA水平与肿瘤负荷相具有强关联性，并提示了VISTA水平为潜在的预后标志物显然，准确测量VISTA 对理解其在许多生物学过程中的具有潜在的重要作用。测量内源VISTA水平的能力依赖于灵敏且特异性测定法的可获得性。针对VISTA的基于比色法、化学发光、及荧光测定的酶联免疫吸附测定法(ELISA)已有报道。
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明作进一步地详细描述。
实施例1：
用6-8周龄雌性BALB/c小鼠(购自上海杰思捷实验动物有限公司)作为实验动物进行小鼠免疫试验。初次免疫使用50μg人VISTA蛋白(购自北京义翘神州)与完全弗氏佐剂混合形成乳剂，按1ml/只注射量注射小鼠腹腔，每隔2周用25μg人VISTA蛋白与不完全福氏佐剂充分混合形成乳液进行加强免疫，加强免疫三次，末次免疫1周后，收集小鼠静脉血并分离血清，通过ELISA方法测量抗体效价并选择具有高效价的小鼠细胞制备杂交瘤制备单脾细胞悬液。
收集对数生长的骨髓瘤细胞(SP2/0)制备免疫脾细胞悬液，将骨髓瘤细胞与脾细胞按1:5-1:10的比例混合在一起，用不完全培养液洗涤后离心8 分钟弃上清，将细胞沉淀置于40℃水浴中预热，向细胞沉淀中加入预热至 40℃的PEG-4000溶液1ml，出现颗粒后1min内向反应液中加入25ml预热至40℃的不完全培养基以终止反应。静置后加入2ml HAT培养基，轻吹沉淀细胞使其悬浮混匀，之后补充HAT培养基至离心管内的脾细胞浓度达到1.5×107/ml，将上述细胞悬浮液分装至96孔板培养并观察，待细胞表面积达到孔板面积1/2以上时，吸出上清液样品供抗体检测。
实施例2：
筛选杂交瘤培养上清液的抗人VISTA抗体。具体地，用缓冲液在96 孔高吸附酶标板上包被人VISTA(购自北京义翘神州)，包被量100μL每孔，之后用缓冲液洗涤3次后；用含1％BSA的缓冲液阻断并于25℃孵育1h，阻断量280μL/孔，孵育完成后缓冲液洗涤3次，分别向1-90号孔中加入75μL 上清液样品(S1-S85)以及阳性血清(对照，CK1-5)，25℃孵育1小时，缓冲液洗涤5次；每孔加入100μL以比例1/10000稀释于含1％BSA缓冲液里的抗小鼠IgG抗体，所述抗小鼠IgG抗体被辣根过氧化物酶标记，25℃孵育1小时，缓冲液洗涤5次；每孔加入100μL比色底物3,3',5,5'-四甲基联苯胺(TMB)，30℃显色10min后终止显色反应，在酶标仪上读取450nm 处的吸光度，根据OD450nm的强弱选取能够分泌人VISTA结合抗体的阳性克隆。
实施例3：
将筛选得到的同时具有抗原结合活性以及抗原中和活性的克隆，进行抗体DNA序列的测定。首先按照说明书使用RNA prep Pure试剂盒 (Tiangen)提取细胞mRNA。之后使用Quant Script RT试剂盒(Tiangen) 合成cDNA第一链。将逆转录产生的cDNA第一链用于后续PCR反应，将 PCR扩增得到的目的条带克隆到pGEM-T载体中，挑取单克隆由南京金斯瑞生物科技有限公司完成测序。
经过PCR扩增得到抗体轻链可变区和抗体重链可变区，排除骨架区序列后即可得到其互补决定区序列；其中轻链的三个互补决定区CDR-L1氨基酸序列如SEQ ID NO:1；CDR-L2氨基酸序列如SEQ ID NO:2、CDR-L3 的氨基酸序列如SEQ ID NO:3所示；重链的三个互补决定区CDR-H1氨基酸序列如SEQ ID NO:4、CDR-H2氨基酸序列如SEQ ID NO:5、CDR-H3氨基酸序列如SEQ ID NO:6所示；抗体轻链恒定区氨基酸序列来源自鼠源 IgVH4-21*07、抗体重链恒定区序列鼠源IgVH2-09*01，轻链全长序列为将抗体轻链可变区和轻链恒定区连接即得；重链全长序列为将抗体重链可变区和重链恒定区连接即得，将上述可变区序列及恒定区序列分别克隆到真核细胞表达载体TL10-11中(载体骨架pEGFP-N1，购自上海吉满生物)。将抗体轻链及抗体重链表达载体转染293F细胞系(购自上海纪宁生物科技)。转染前一天接种细胞，转染当天将细胞离心收集细胞，将细胞重悬于新鲜的表达培养基中，细胞密度为2×108细胞/mL。按照转染体积加入质粒，终浓度为45.2μg/mL，加入线性聚乙烯亚胺至终浓度为50μg/mL。将上述混合物放入细胞培养箱37℃培养1小时，之后向培养液中加入新鲜培养基至终体积为20倍转染体积，继续培养5～6天后收集上清。
实施例4：
检测实施例1获得的抗人VISTA鼠源单抗(以下简称OM-anti-VISTA) 与其抗原结合的动力学常数。利用仪器光学表面等离子体共振技术来检测偶联包被在生物芯片上的分子与待测分子之间的结合和解离，简要地，将 OM-anti-VISTA溶于的醋酸钠缓冲溶液(pH5.0)中并偶联到CM芯片上，用1M乙醇胺封闭，结合阶段将不同浓度的OM-anti-VISTA以25μL/min的速度注入3.0min，在解离阶段用PBS缓冲溶液以25μL/min的速度注入5.0 min，结合动力学常数和解离动力学常数通过Biacore 3000软件进行分析计算。OM-anti-VISTA的结合动力学常数为2.14E+05(1/Ms)、解离动力学常数为3.61E-05(1/s)、解离平衡常数为1.68E-09(M)。
实施例5：
人源化形式的抗人VISTA抗体参考Molecule Immunol的制备方法制备，在Germline数据库中选取与OM-anti-VISTA非CDR区匹配最好的人源化模板，其中重链可变区的模板为人源IgVH4-28*03，轻链可变区的模板为人源IGKV1-16*02，将鼠源抗体CDR区移植到选择的人源化模板上，替换得到人源化抗体重链可变区，氨基酸序列如SEQ ID NO:7所示，替换得到人源化抗体轻链可变区，氨基酸序列如SEQ ID NO:8所示。通过序列比对选择适合位点进行回复突变，获得的重链可变区氨基酸序列(VH)及轻链可变区氨基酸序列(VL)如表1所示。
表1.回复突变获得的氨基酸序列
VH SEQ ID NO
ORI SEQ ID NO:7
VisLC16-H SEQ ID NO:9
VisLC17-H SEQ ID NO:10
VisLC18-H SEQ ID NO:11
VL SEQ ID NO
ORI SEQ ID NO:8
VisLC16-L SEQ ID NO:12
VisLC17-L SEQ ID NO:13
VisLC18-L SEQ ID NO:14
将人源化的抗人VISTA单抗的重链可变区(SEQ ID NO：7-11)分别与人抗体IgG1重链恒定区(SEQ ID NO:15)连接，分别得到对应的重链全长序列。将人源化的抗人VISTA单抗的轻链可变区(SEQ ID NO：8-14) 分别与人抗体Kappa轻链的恒定区(SEQ ID NO:16)连接，分别得到对应的轻链全长序列，将上述所有重链全长序列与轻链全长序列组合得到人源化抗体全长序列，经酶切连接入TL10-11(载体骨架pEGFP-N1)载体中。
实施例6：
测定三种人源化VISTA单抗(ORI：SEQ ID NO:7和SEQ ID NO:8， VisLC16：SEQ IDNO:9和SEQ ID NO:12，VisLC17：SEQ ID NO:10和SEQ ID NO:13，VisLC18：SEQ ID NO:11和SEQ ID NO:14)与抗原VISTA蛋白的结合动力学常数，方法同实施例4，人源化VISTA单抗的结合动力学常数、解离动力学常数和解离平衡常数如表2所示。
表2.人源化VISTA单抗与其抗原结合的动力学常数
实施例7：
为了确定ORI、VisLC16、VisLC17、VisLC18单抗的热稳定性，将ORI、 VisLC16、VisLC17、VisLC18样品置于40℃高温条件下，在第2周、第3 周分别取样进行SE-HPLC检测以观察热稳定性，测试采用色谱法，流动相为磷酸盐缓冲液(0.1mol/L)，硫酸钠缓冲液(0.1mol/L，pH6.7)；流速为 0.6mL/min；色谱柱温度为27℃；样品池温度4℃；检测波长280nm；用缓冲液稀释样品至2mg/mL上样，体积20μL，用面积归一化法计算主峰比例处理数据，人源化VISTA单抗热稳定性测试结果如表3所示。
表3.人源化VISTA单抗热稳定性测试结果
热稳定性测试表明ORI单抗均显示出了优异的热稳定性。
实施例8：
ORI单抗用于制备检测试剂盒，具体方法为，将抗体包被板制成固相抗体，往包被抗体的微孔中依次加入样品，再加入HRP标记的酶标液，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色，TMB在 HRP酶的催化下转化成蓝色，并在终止液作用下转化为黄色，颜色深浅与 VISTA含量成正相关，利用酶标仪测定450nm波长下OD，通过标曲计算 VISTA含量，本试剂盒采用新型人源化VISTA单抗，与人VISTA结合能力更强，结合速率更高，可用于快速、准确的VISTA含量检测，为辅助诊断 VISTA相关疾病提供更灵敏、准确的数据，如表4所示，试剂盒具体地包括下表所述成分，保存条件为4℃。
表4.人VISTA高灵敏检测试剂盒
试剂盒组成 96孔
酶标包被板 1×96规格
TMB显色剂 5mL×2管
洗涤液 10mL×2管
终止液 5mL×2管
标品(500pg/mL) 1mL×2管
标品缓冲剂 10mL×1管
酶标液 1mL×2管
样品稀释液 10mL×1管
以上所揭露的仅为本发明较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。
序列表
<110> 郑权
<120> 用于检测T细胞活化的V域免疫抑制因子的试剂盒
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> CDR1-L
<400> 1
Ser Lys Asp Ser Lys Asp Glu His Asp
1 5
<210> 2
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> CDR2-L
<400> 2
Asn Tyr Lys Ser Cys His Trp
1 5
<210> 3
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> CDR3-L
<400> 3
Met Leu Thr Asp Arg Met Thr Tyr Ser
1 5
<210> 4
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> CDR1-H
<400> 4
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Ser Asp
1 5 10
<210> 5
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> CDR2-H
<400> 5
Asn Tyr Lys Ser Cys His Trp
1 5
<210> 6
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> CDR3-H
<400> 6
Lys Met Phe Pro Val Pro Val Ser Met
1 5
<210> 7
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> ORI VH
<400> 7
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Asn Cys His Arg Tyr
1 5 10 15
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
20 25 30
Ser Asp Gly Glu Asp Pro Trp Asp Lys His Glu Pro Met Ile Ile Pro
35 40 45
Lys Glu Ile Met Phe Asn Cys Gly Ala Gln Pro Ile Pro Glu Asn Tyr
50 55 60
Lys Ser Cys His Trp Trp Cys Gly Thr Met Cys Cys Trp Phe His Pro
65 70 75 80
Phe Cys Glu Asp Gly Ser Ser Gly Phe Ile Thr Asp His Asp Lys Met
85 90 95
Phe Pro Val Pro Val Ser Met Val Gly Ile Ala Asn Leu Cys Gln Gly
100 105 110
Thr Lys Val Glu Ile Lys Trp
115
<210> 8
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> ORI VL
<400> 8
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Tyr Val Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Lys Asp Glu His
20 25 30
Asp Asp Ile Thr Asp Tyr Asp Arg Asp Leu Phe Ile Gln Tyr Cys Gly
35 40 45
Ser Cys Lys Cys Met Gly Arg Val Arg Val Gln Asn Ser Ile Ser Gly
50 55 60
Phe Cys Cys Val His Cys Trp Asp Thr Val Leu Phe Asn His Thr Pro
65 70 75 80
Gln Leu Ala His Trp Phe Arg Gly Thr Tyr Met Leu Thr Asp Arg Met
85 90 95
Thr Tyr Ser Ser Ile Asn Ile Met Gly Val Thr His Gln Gly Leu Ser
100 105 110
Ser
<210> 9
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> VisLC16-H
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Asn Cys His Arg Asp
1 5 10 15
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
20 25 30
Ser Asp Gly Glu Asp Pro Ala Asp Lys His Glu Pro Met Ile Ile Pro
35 40 45
Lys Glu Ile Met Phe Asn Cys Gly Ala Gln Pro Ile Pro Glu Asn Tyr
50 55 60
Lys Ser Cys His Trp Trp Cys Gly Thr Met Cys Cys Trp Phe Gln Pro
65 70 75 80
Phe Cys Glu Asp Gly Arg Ser Gly Phe Ile Thr Asp His Asp Lys Met
85 90 95
Phe Pro Val Pro Val Ser Met Val Gly Ile Ala Asn Leu Cys Gln Gly
100 105 110
Thr Lys Val Glu Ile Lys Trp
115
<210> 10
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> VisLC17-H
<400> 10
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Asn Cys His Arg Tyr
1 5 10 15
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Arg Ser Asp Tyr Glu
20 25 30
Ser Asp Gly Glu Asp Pro Trp Asp Lys His Glu Pro Met Ile Ile Pro
35 40 45
Lys Glu Ile Met Phe Asn Cys Gly Ala Gln Pro Ile Pro Glu Asn Tyr
50 55 60
Lys Ser Cys His Trp Trp Cys Gly Thr Met Cys Cys Trp Phe His Pro
65 70 75 80
Phe Cys Glu Asp Gly Ser Ser Gly Phe Ile Thr Asp His Asp Lys Gln
85 90 95
Phe Pro Val Pro Val Ser Met Val Gly Ile Ala Asn Leu Cys Gln Gly
100 105 110
Thr Lys Val Glu Ile Lys Trp
115
<210> 11
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> VisLC18-H
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu His Cys His Arg Tyr
1 5 10 15
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
20 25 30
Ser Asp Gly Glu Asp Pro Gln Asp Lys His Glu Pro Met Ile Ile Pro
35 40 45
Lys Glu Ile Met Phe Asn Cys Lys Ala Gln Pro Ile Pro Glu Asn Tyr
50 55 60
Lys Ser Cys His Trp Trp Cys Gly Thr Met Cys Cys Trp Phe His Pro
65 70 75 80
Phe Cys Glu Asp Gly Ser Ser Gly Phe Ile Thr Asp His Asp Lys Met
85 90 95
Phe Pro Val Pro Val Ser Met Val Gly Ile Ala Asn Leu Cys Gln Ile
100 105 110
Thr Lys Val Glu Ile Lys Trp
115
<210> 12
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> VisLC16-L
<400> 12
Arg Thr Val Ala Ala Pro Ser Val Met Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Tyr Val Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Lys Asp Glu His
20 25 30
Asp Asp Ile Thr Asp Tyr Asp Arg Asp Leu Phe Ile Gln Tyr Cys Ala
35 40 45
Ser Cys Lys Cys Met Pro Arg Val Arg Val Gln Asn Ser Ile Ser Gly
50 55 60
Phe Cys Cys Val His Cys Trp Asp Thr Val Leu Phe Asn His Thr Pro
65 70 75 80
Gln Leu Ala His Phe Phe Arg Gly Thr Tyr Met Leu Thr Asp Arg Met
85 90 95
Thr Tyr Ser Ser Ile Asn Ile Met Gly Val Thr His Gln Gly Leu Ser
100 105 110
Ser
<210> 13
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> VisLC17-L
<400> 13
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Tyr Val Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Tyr Asp Glu His
20 25 30
Asp Asp Ile Thr Asp Tyr Asp Arg Asp Leu Phe Ile Gln Tyr Cys Gly
35 40 45
Ser Cys Lys Cys Met Gly Arg Val Arg Val Gln Asn Ser Ile Ser Gly
50 55 60
Phe Cys Cys Val His Cys Trp Asp Thr Val Leu Phe Asn His Thr Pro
65 70 75 80
Asp Leu Ala His Trp Phe Arg Gly Thr Tyr Met Leu Thr Asp Arg Met
85 90 95
Thr Tyr Ser Ser Ile Asn Ile Gln Gly Val Thr His Gln Gly Leu Ser
100 105 110
Ser
<210> 14
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> VisLC18-L
<400> 14
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Ala
1 5 10 15
Tyr Val Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Lys Asp Glu His
20 25 30
Asp Asp Ile Thr Asp Tyr Asp Arg Asp Leu Phe Ile Gln Tyr Tyr Gly
35 40 45
Ser Cys Lys Cys Met Gly Arg Val Arg Val Gln Asn Ser Ile Ser Gly
50 55 60
Phe Cys Cys Val His Cys Trp Asp Thr Asp Leu Phe Asn His Thr Pro
65 70 75 80
Gln Leu Ala His Trp Phe Arg Glu Thr Tyr Met Leu Thr Asp Arg Met
85 90 95
Thr Tyr Ser Ser Ile Asn Ile Met Gly Val Thr His Gln Gly Leu Ser
100 105 110
Ser
<210> 15
<211> 329
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> CH
<400> 15
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
1 5 10 15
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
20 25 30
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
35 40 45
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
50 55 60
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
65 70 75 80
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
85 90 95
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
100 105 110
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
115 120 125
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
130 135 140
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
145 150 155 160
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
165 170 175
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
180 185 190
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
195 200 205
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
210 215 220
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
225 230 235 240
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
245 250 255
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
260 265 270
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
275 280 285
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
290 295 300
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
305 310 315 320
Lys Ser Leu Ser Leu Ser Pro Gly Lys
325
<210> 16
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> CL
<400> 16
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
1 5 10 15
Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
20 25 30
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
35 40 45
Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
50 55 60
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
65 70 75 80
Asp Tyr Glu Lys His Lys Leu Tyr Ala Cys Glu Val Thr His Gln Gly
85 90 95
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105 110
<210> 17
<211> 450
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> TIM-3单抗重链
<400> 17
Asp Ile Leu Met Thr Gln Thr Pro Ser Leu Ile Tyr Gln Ala Arg His
1 5 10 15
Thr Ala Val Thr Gln Asp Phe Trp Pro Trp Gln Cys Thr Ala Met Met
20 25 30
Trp Thr Gly Thr Trp Tyr Leu Trp Cys Phe Leu Ala Ile Asn Cys Gln
35 40 45
Pro Glu His Gly Val Thr Arg Met Gln Tyr Asp Glu Pro His Ser Cys
50 55 60
Glu Arg Ala Pro Val Ser Ile Met Cys Cys His Gln His His Trp Thr
65 70 75 80
Asp Asn Cys Phe Asp Val Thr His Ile Ile Arg Asn Val Glu Trp Val
85 90 95
Asp Ala Cys Glu Ala Val Pro Lys Arg Trp Asn Thr Ala Lys Met Leu
100 105 110
Thr Pro Gly Asn Lys Leu Glu Trp Met Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 18
<211> 226
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<223> TIM-3单抗轻链
<400> 18
Asn Leu Met Gly Phe Lys Gln Cys Trp Asp Glu Ile Thr Leu Tyr Cys
1 5 10 15
Gly Asp Asp Asn Arg Met Phe Trp Glu Asn Glu Gln His Tyr Pro Met
20 25 30
Phe Gln Leu Met Glu Phe Glu Pro Ala Asp Asp Ala Trp Asn Asn His
35 40 45
Arg Ser Gln Asn Lys His Val Val Gln Phe Ser Ala Gln Pro Phe Trp
50 55 60
Leu Ala Gly Ser Cys Thr Trp Lys Asp Ile Arg Met Ile Ser Arg Lys
65 70 75 80
Cys Arg Cys Ser Ala Lys Ser His Gly Pro Met Glu Asp Arg Pro Cys
85 90 95
Asn Trp Phe Met Tyr Ala Thr Thr Gly Leu Ser Phe Arg Asp Gly Asn
100 105 110
Ile Tyr Phe Asn Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
115 120 125
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
130 135 140
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
145 150 155 160
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
165 170 175
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
180 185 190
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Leu Tyr Ala Cys Glu Val
195 200 205
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
210 215 220
Glu Cys
225

Claims (9)

1.用于检测T细胞活化的V域免疫抑制因子的试剂盒，其特征在于，将VISTA抗体包被板制成固相抗体，所述VISTA抗体包括：
包含SEQ ID NO:01的氨基酸序列的CDR-L1，和/或
包含SEQ ID NO:02的氨基酸序列的CDR-L2，和/或
包含SEQ ID NO:03的氨基酸序列的CDR-L3，和/或
包含SEQ ID NO:04的氨基酸序列的CDR-H1，和/或
包含SEQ ID NO:05的氨基酸序列的CDR-H2，和/或
包含SEQ ID NO:06的氨基酸序列的CDR-H3。
2.根据权利要求1所述的试剂盒，其特征在于，使用所述试剂盒的方法包括：将样品依次加入到抗体包被的微孔中，然后加入HRP标记的酶标液形成抗体-抗原-酶标记的复合物，彻底洗涤后加入TMB显色；TMB在HRP酶催化下呈蓝色，加入终止溶液后呈黄色，颜色深度与VISTA抗原含量呈正相关；测定溶液OD 450nm，根据标准曲线定量计算VISTA蛋白含量。
3.根据权利要求1所述的试剂盒，其特征在于，所述抗体选自单克隆抗体，特异性结合人VISTA的抗体片段，包含特异性结合人VISTA抗体片段的融合蛋白，鼠源抗体，人源化抗体，鼠源单克隆抗体，优选地，所述抗体为人源化单克隆抗体。
4.根据权利要求1所述的试剂盒，其特征在于，所述抗体包含抗体重链可变区氨基酸序列包括如SEQ ID NO:7所示氨基酸序列具有至少99％序列同一性的VH序列。
5.根据权利要求1所述的试剂盒，其特征在于，所述抗体包含抗体轻链可变区氨基酸序列包括如SEQ ID NO:8所示氨基酸序列具有至少99％序列同一性的VL序列。
6.根据权利要求1所述的试剂盒，其特征在于，所述抗体重链恒定区氨基酸序列如SEQID NO:7所示，所述抗体轻链恒定区氨基酸序列如SEQ ID NO:8所示。
7.根据权利要求1-6所述任一试剂盒，其特征在于，所述抗体的生产步骤包括在培养基及合适的培养条件下培养含有表达权利要求1-6所述抗体的核苷酸序列的宿主细胞，从所述培养基中和/或所述宿主细胞中通过常规方法回收并纯化抗体和/或抗原结合片段。
8.根据权利要求7所述试剂盒，其特征在于，所述试剂盒还包括酶标包被板、标品、标品缓冲剂、酶标液、样品稀释液、TMB显色剂、洗涤液、终止液。
9.根据权利要求1-8所述试剂盒，其特征在于，所述检测试剂盒辅助诊断肺癌胰腺癌、乳腺癌、卵巢癌、子宫内膜癌、黑色素瘤以及其他VISTA蛋白高表达的疾病类型。
CN201910677351.9A 2019-07-25 2019-07-25 用于检测t细胞活化的v域免疫抑制因子的试剂盒 Withdrawn CN110579610A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115677851A (zh) * 2021-07-29 2023-02-03 东莞市朋志生物科技有限公司 一种免疫阻断抗体或其抗原结合片段及其应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115677851A (zh) * 2021-07-29 2023-02-03 东莞市朋志生物科技有限公司 一种免疫阻断抗体或其抗原结合片段及其应用
CN115677851B (zh) * 2021-07-29 2024-03-22 东莞市朋志生物科技有限公司 一种免疫阻断抗体或其抗原结合片段及其应用

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