CN111848799B - 特异性检测抗体及其在制备检测试剂盒中的用途 - Google Patents
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Abstract
本发明提供特异性检测抗体及其在制备检测试剂盒中的用途，包括将CD19抗体包被板制成固相抗体，所述CD19抗体包含SEQ ID NO:1的氨基酸序列的CDR‑L1，和/或包含SEQ ID NO:2的氨基酸序列的CDR‑L2，和/或包含SEQ ID NO:3的氨基酸序列的CDR‑L3，和/或包含SEQ ID NO:4的氨基酸序列的CDR‑H1，和/或包含SEQ ID NO:5的氨基酸序列的CDR‑H2，和/或包含SEQ ID NO:6的氨基酸序列的CDR‑H3。
Description
技术领域
本发明涉及试剂盒领域，特别地涉及特异性检测抗体及其在制备检测试剂盒中的用途。
背景技术
B淋巴细胞抗原CD19，也称为CD19分子(分化簇19)，B淋巴细胞表面抗原B4，T细胞表面抗原Leu-12和CVID3是一种跨膜蛋白，在人体中由CD19基因编码。在人类中，除了浆细胞和滤泡树突细胞外，CD19在所有B谱系细胞中均有表达。CD19在人B细胞中起两个主要作用。它充当衔接蛋白以将细胞质信号蛋白募集到膜上，并且它在CD19/CD21复合物内起作用以降低B细胞受体信号传导途径的阈值。由于其存在于所有B细胞中，它是B淋巴细胞发育，淋巴瘤诊断的生物标志物，并且可以用作白血病免疫疗法的靶标。
CAR-T细胞疗法或者CD19/CD3双特异性抗体药物在靶向治疗患有CD19淋巴瘤和白血病的患者中已经表现出显著的抗肿瘤活性。然而CD19表达阴性导致的免疫逃逸已经成为这类新型治疗中出现耐药性的主要模式。临床上并没有观察到CD19分子的完全缺失或是基因沉默，越来越多的观点认为免疫逃逸的主要机制很可能与CD19信号通路在B细胞恶性肿瘤中的关键作用有关。
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，本发明提供特异性检测抗体及其在制备检测试剂盒中的用途。
本发明是以如下技术方案实现的：
特异性检测抗体，将CD19抗体包被板制成固相抗体，所述CD19抗体包括：
包含SEQ ID NO:1的氨基酸序列的CDR-L1，和/或
包含SEQ ID NO:2的氨基酸序列的CDR-L2，和/或
包含SEQ ID NO:3的氨基酸序列的CDR-L3，和/或
包含SEQ ID NO:4的氨基酸序列的CDR-H1，和/或
包含SEQ ID NO:5的氨基酸序列的CDR-H2，和/或
包含SEQ ID NO:6的氨基酸序列的CDR-H3。
进一步地，所述试剂盒包括上述抗体、标品缓冲剂、酶标液、样品稀释液、TMB显色剂、洗涤液、终止液酶标、包被板、标品。
进一步地，使用所述试剂盒的方法包括：步骤一、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；步骤二、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CD19抗原含量呈正相关；步骤三、测定溶液OD450nm，根据标准曲线定量计算CD19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CD19的抗体片段，包含特异性结合人CD19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如SEQ ID NO:7所示或如SEQID NO:9所示的氨基酸序列或与SEQ ID NO:7或SEQ ID NO:9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如SEQ ID NO:8所示或如SEQID NO:10所示的氨基酸序列或与SEQ ID NO:8或SEQ ID NO:10具有至少99％序列同一性的VL序列。
进一步地，所述抗体重链可变区氨基酸序列如SEQ ID NO:9所示，所述抗体轻链可变区氨基酸序列如SEQ ID NO:10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上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，所述检测试剂盒辅助诊断CD19蛋白过表达的疾病。
CD19是参与B细胞活化与增殖的重要膜抗原之一，是所有B细胞共有的表面标志，B细胞活化后不消失，是最重要的B细胞标记因子，同时CD19也是B细胞表面的传达信号复合体的构成部分，CD19的细胞外部分同其他膜抗原结合进行信号传导。CD19阳性细胞升高见于B淋巴细胞系统的恶性肿瘤，如CD19在95％急性前B淋巴细胞白血病细胞和94％急性成熟B淋巴细胞白血病细胞表达，也见于慢性淋巴细胞性白血病和淋巴瘤等；CD19阳性细胞降低见于体液免疫缺陷病。
具体实施方式
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。
IgG型抗体包括许多不同的亚型，这些亚型的Fc区有轻微的差别，因此具有不同的功能。例如，IgG型抗体的一种亚型IgG3，可以较其他任何亚型更好地固定补体。相似地，IgG1亚型擅长于结合入侵者并调理它们以利于专职吞噬细胞的吞噬，这是因为在巨噬细胞和中性粒细胞表面有能与已结合入侵者的IgG1抗体Fc区结合的受体。
T细胞共刺激分子中研究最清楚的是表达于APC表面的B7蛋白(B7-1和B7-2)，B7蛋白可以通过插入T细胞表面的受体而对T细胞提供共刺激。迄今为止已分离出了这类受体中的两种—CD28和CTLA-4。大部分T细胞表达CD28蛋白，而CTLA-4分子只在激活的T细胞中表达。现在认为，APC上的B7蛋白通过与原初T细胞的CD28受体结合，而提供激活所必需的共刺激信号。随后，一旦细胞被激活，B7蛋白与CTLA-4受体结合就可促使关掉或“去活化”T细胞。获得性免疫应答行使完其功能后对其实施关闭是非常重要的。
代表性的抗原结合功能区包括：抗体的可变区、抗体可变区的结构变构体、受体的结合域、配体结合域或酶结合域。
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。
“可变区”是指在抗体间，可变区的某些区段的序列显著不同，所述高度可变区为各3-12个氨基酸，其主要采取β折叠构型，由三个高度可变区连接，这些高度可变区形成环，所述环连接β折叠结构及在一些情况下形成β折叠结构的一部分。各链中的高度可变区是由FR紧挨着结合在一起，而与其他链的高度可变区有助于形成抗体的抗原结合位点。
“恒定区”不直接涉及抗体对抗原的结合，但抗体恒定区涉及双特异性抗体的制备。
“抗体的抗原结合片段”指能够与表位结合的抗体的片段、部分、区域或结构域，因此术语“抗原结合”与“表位结合”以及“抗体的抗原结合片段”与“抗体的表位结合片段”是相同的。
“抗体的抗原结合片段”可以含有该类抗体的1、2、3、4、5或所有6个CDR域，并且尽管能够结合到所述表位，仍可以展现出不同的特异性、亲和力或选择性。
“抗体的抗原结合片段”可以是单条多肽链(例如，scFv)的一部分或包含单条多肽链，或者可以是两条或更多条多肽链(各自具有氨基末端和羧基末端，例如，双抗体、Fab片段、Fab2片段等)的一部分或包含两条或更多条多肽链。
术语“IgG”是免疫球蛋白G的缩写，人IgG有四个亚型：IgG1、IgG2、IgG3、IgG4，根据IgG分子中的r链区分，是血清中的主要抗体成分，。
一个或多个二硫键链间连接是指第一Fc链及第二Fc链通过一个或多个二硫键链间连接，形成异源二聚体片段。在本发明中，一个或多个二硫键的形成可以是第一Fc链及第二Fc链或者第一Fc链及第二Fc链及其相连接的抗原结合功能区在同一个细胞内合成时形成，也可以是第一Fc链及第二Fc链或者第一Fc链及第二Fc链及其相连接的抗原结合功能区在不同细胞内分别合成，之后通过体外还原氧化的方法形成。
实施例1：
小鼠免疫：选购6-8周龄雌性BALB/c小鼠(购自江苏省协同医药生物工程有限责任公司)作为实验动物。初次免疫使用45μg人CD19蛋白(购自南京双领生物科技有限公司)与完全弗氏佐剂混合形成乳剂，按0.5ml/只注射量注射小鼠，腹腔注射，每隔两周进行加强免疫，加强免疫方法为：将不完全福氏佐剂与22.5μg人CD19蛋白混合充分制成乳液，按0.5ml/只注射量注射小鼠，腹腔注射，一共加强免疫四次，末次免疫一周后，收集并分离血清ELISA方法检测抗体效价，方法参考说明书，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制备免疫脾细胞悬液，将骨髓瘤细胞与脾细胞按一定比例混合，用不完全培养液洗涤后，离心弃上清，将细胞沉淀和1ml PEG-4000分别置于40℃水浴中预热，之后混合为反应液并静置至出现颗粒，1min内向反应液中加入25ml预热至40℃的不完全培养基终止反应。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1×107/ml，将上述细胞悬浮液分装至96孔板培养，待细胞表面积达到孔板面积2/3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CD19抗体。在96孔高吸附酶标板上包被人CD19(购自南京双领生物科技有限公司)，包被量100μL每孔，之后冲洗3次；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上清液样品以及阳性血清作为对照，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CD19结合抗体的阳性克隆。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。使用RNA prep Pure试剂盒提取细胞mRNA，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。
实施例3：
经过PCR扩增得到鼠源抗CD19抗体的轻链可变区和重链可变区，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 IDNO: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)。将抗体轻链及抗体重链表达载体转染293F细胞系。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1.5×107细胞/mL。按照转染体积加入质粒，终浓度为39.1μg/mL，加入线性聚乙烯亚胺至终浓度为45μg/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CD19鼠源单抗(以下简称OM-anti-CD19)与其抗原结合的动力学常数。具体方法为：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-CD19溶于的醋酸钠缓冲溶液(pH 5.0)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-CD19以27μL/min的速度注入3min，在解离阶段用PBS缓冲溶液以27μL/min的速度注入8min，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-CD19的结合动力学常数为3.38E+02(1/Ms)、解离动力学常数为1.57E-02(1/s)、解离平衡常数为0.03(nM)。
实施例5：
人源化形式的抗人CD19抗体参考Molecule Immunol的制备方法制备，在Germline数据库中选取与OM-anti-CD19非CDR区匹配最好的人源化模板，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
| CD19抗体VH | 序列编号 |
| CD19-ORI-VH | SEQ ID NO:7 |
| AzzH-13-VH | SEQ ID NO:9 |
| CD19抗体VL | 序列编号 |
| CD19-ORI-VL | SEQ ID NO:8 |
| AzzL-13-VL | SEQ ID NO:10 |
实施例6:
将人源化的抗人CD19单抗的重链可变区(SEQ ID NO：9)与人抗体IgG1重链恒定区(SEQ ID NO:11)连接，得到对应的重链全长序列。将人源化的抗人CD19单抗的轻链可变区(SEQ ID NO：10)与人抗体Kappa轻链的恒定区(SEQ ID NO:12)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。分别构建抗人CD19的抗体的重链和轻链的TL10-11表达载体(重链氨基酸序列如SEQ ID NO:9所示，轻链氨基酸序列如SEQ ID NO:10所示)，根据常规方法，获得抗人CD19抗体重链和轻链的表达载体，分别用于在真核细胞中表达抗人CD19抗体的重链和轻链。
检测获得的抗体与抗原CD19的结合的动力学常数。方法为：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，将抗待测抗体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anti-CD19以25μL/min的速度注入3min，在解离阶段用PBS缓冲溶液以25μL/min的速度注入8min，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。anti-CD19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.双特异性抗体与其与其抗原结合的动力学常数
为了确定ORI、AzzH-13单抗的热稳定性，将ORI、AzzH-13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CD19单抗热稳定性测试结果如表3所示。
表3.人源化CD19单抗热稳定性测试结果
热稳定性测试表明ORI、AzzH-13单抗均显示出了很好且相当的稳定性。
实施例7：
上述2条氨基酸序列(ORI，AzzH-13)均可分别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CD19含量成正相关，利用酶标仪测定450nm波长下OD，通过标曲计算CD19含量，本试剂盒采用新型人源化CD19单抗，与人CD19结合能力更强，结合速率更高，可用于快速、准确的CD19含量检测，为辅助诊断CD19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CD19高灵敏检测试剂盒
| 试剂盒组成 | 96孔 |
| 标品(500pg/mL) | 1mL×3管 |
| 标品缓冲剂 | 10mL×3管 |
| 酶标液 | 1mL×3管 |
| 样品稀释液 | 10mL×3管 |
| 酶标包被板 | 1×96规格 |
| TMB显色剂 | 5mL×3管 |
| 洗涤液 | 10mL×3管 |
| 终止液 | 5mL×3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。
Claims (8)
1.特异性检测抗体，其特征在于，将CD19抗体包被板制成固相抗体，所述CD19抗体包括：氨基酸序列如SEQ ID NO:1所示的CDR-L1，氨基酸序列如SEQ ID NO:2所示的CDR-L2，氨基酸序列如SEQ ID NO:3所示的CDR-L3，氨基酸序列如SEQ ID NO:4所示的CDR-H1，氨基酸序列如SEQ ID NO:5所示的CDR-H2，和氨基酸序列如SEQ ID NO:6所示的CDR-H3。
2.特异性检测抗体在制备试剂盒中的用途，其特征在于，所述试剂盒包括权利要求1所述抗体、标品缓冲剂、酶标液、样品稀释液、TMB显色剂、洗涤液、终止液酶标、包被板、标品。
3.根据权利要求2所述的用途，其特征在于，所述抗体为人源化单克隆抗体。
4.根据权利要求2所述的用途，其特征在于，所述抗体的重链可变区氨基酸序列选自SEQ ID NO:7或SEQ ID NO:9。
5.根据权利要求2所述的用途，其特征在于，所述抗体的轻链可变区氨基酸序列选自SEQ ID NO:8或SEQ ID NO:10。
6.根据权利要求2所述的用途，其特征在于，所述抗体重链可变区氨基酸序列如SEQ IDNO:9所示，所述抗体轻链可变区氨基酸序列如SEQ ID NO:10所示。
7.根据权利要求6所述的用途，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所述的用途，其特征在于，所述用途为用检测试剂盒辅助诊断CD19蛋白过表达的疾病。
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| WO2019148656A1 (zh) * | 2018-02-05 | 2019-08-08 | 深圳市默赛尔生物医学科技发展有限公司 | 多信号嵌合抗原受体及其表达基因、其修饰的nk细胞及抗肿瘤的药物 |
| CN110105449A (zh) * | 2019-04-03 | 2019-08-09 | 浙江众意生物科技有限公司 | 一种特异性结合vegf的抗体及用途 |
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| WO2019148656A1 (zh) * | 2018-02-05 | 2019-08-08 | 深圳市默赛尔生物医学科技发展有限公司 | 多信号嵌合抗原受体及其表达基因、其修饰的nk细胞及抗肿瘤的药物 |
| CN110105449A (zh) * | 2019-04-03 | 2019-08-09 | 浙江众意生物科技有限公司 | 一种特异性结合vegf的抗体及用途 |
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