CN113748343A - pS396测定用于诊断TAU蛋白病的用途 - Google Patents

pS396测定用于诊断TAU蛋白病的用途 Download PDF

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CN113748343A
CN113748343A CN202080024099.2A CN202080024099A CN113748343A CN 113748343 A CN113748343 A CN 113748343A CN 202080024099 A CN202080024099 A CN 202080024099A CN 113748343 A CN113748343 A CN 113748343A
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J·T·彼得森
T·卡里卡里
K·霍格隆德
K·布莱诺
M·N·哈恩达尔
H·泽特伯格
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Abstract

本发明涉及用于测量样品中的磷酸化的tau的体外测定，所述测定包括2种抗体的使用，这两种抗体为i)对tau上的pS396特异的捕获抗体，和ii)在与该捕获抗体不同的表位上结合tau的检测抗体。

Description

pS396测定用于诊断TAU蛋白病的用途
本发明涉及抗tau抗体在区分不同tau病变中的tau种类的测定中的用途。根据本发明的测定可用于，即诊断患有如阿尔茨海默病(Alzheimer’s disease)、匹克氏病(Pick’s disease)、皮质基底节变性、进行性核上性麻痹和球状神经胶质tau蛋白病等tau蛋白病的患者。
背景技术
术语tau蛋白病定义一组特征为微管相关蛋白tau的沉积的病理性疾病。沉积的tau被异常磷酸化并作为细胞内的内含物而积聚。有许多特定的tau蛋白病，其中的每一种均随着含蛋白的内含物的分布和形态外观，以及影响神经元和神经元过程相对神经胶质和神经胶质过程的病理学的相对负担而变化(Dickson等人,2011)。最常见的tau蛋白病为进行性核上性麻痹(PSP)、皮质基底节变性(CBD)、匹克氏病和球状神经胶质tau蛋白病(GGT)以及慢性创伤性脑病变(CTE)。除了匹克氏病外，所有tau蛋白病通常都与运动障碍有关(Keith A.Josephs,第IX章，在Movement Disorders[运动障碍]中(第二版)，2015)。tau蛋白病还指额颞痴呆(FTD)、额颞叶变性(FTLD)。FTD的多数病例与MAPT(tau)或GRN(颗粒体蛋白)中的基因突变有关。FTD与tau蛋白的积聚有关。
为了对这些患者进行诊断并为他们找到正确疗法，重要的是具有一种可对这些患者进行诊断并区分每种疾病特有的tau病变的方法。本发明的发明人提供了可在这些疾病的诊断中有帮助的多种测定。
发明内容
本发明涉及用于测量样品中的磷酸化tau的体外测定，所述测定包括2种抗体的使用，这两种抗体为i)对tau上的磷酸化(p)丝氨酸(S)残基396(pS396)特异的捕获抗体，和ii)在与该捕获抗体不同的表位上结合tau的检测抗体。如本文进一步披露的，该检测抗体可结合tau上的非磷酸化残基。
本发明的第二方面涉及用于测量样品中磷酸化的tau的方法，该方法包括以下步骤
a.将附着于顺磁珠的对pS396特异的捕获抗体与生物素化的检测抗体以及样品混合，
b.孵育该混合物足够时间以使得这些抗体与该样品中的tau结合(例如1分钟、2分钟、3分钟或5分钟或更久)，
c.任选地，洗涤步骤b)中孵育后的混合物，
d.添加缀合有链霉亲和素的β-半乳糖苷酶，并允许所述链霉亲和素缀合物和该生物素化的检测抗体反应(例如1分钟、2分钟、3分钟或5分钟或更久)，
e.任选地，洗涤步骤d)中所获得的混合物，
f.向步骤d)或e)中的混合物中添加试卤灵β-D-半乳糖吡喃糖苷，并使试卤灵β-D-半乳糖吡喃糖苷水解(例如1分钟、2分钟、3分钟或5分钟或更久)，
g.读取荧光信号并将该信号与标准进行比较。
附图说明
图1pS396 tau测定的示意图。
(A)Tau12-pS396测定，也称为全长pS396(或FL pS396)测定，使用抗pS396抗体作为捕获抗体，并使用Tau12(表位在氨基酸6-18处)作为检测抗体。
(B)HT7-pS396(中央区pS396或MR pS396)测定测量从中央区(表位159-168)延伸的tau种类上的pS396。该测定使用pS396抗体作为捕获抗体，并使用HT7作为检测抗体。
(C)pS396-Tau46(C端pS396或CT pS396)测定对于含有极端C端区(氨基酸404-441)的pS396磷酸化的tau是特异的，该测定使用抗pS396抗体作为捕获抗体，并使用Tau46(表位404-441)作为检测抗体。
图2pS396磷酸化的tau种类区分具有tau病变的神经退行性疾病。通过FL、MR和CTpS396测定对分离自患有临床确定的tau蛋白病的个体的额灰质脑的TBS可溶级分进行测试。用每个pS396测定对五百倍稀释的等摩尔浓度(0.46mg/ml)的用测定稀释液制备的每个样本(n＝24总计)进行分析。此处示出了稀释校正数据(平均值±平均值的标准误差[SEM])。样品由阿尔茨海默病(AD，n＝5)、匹克氏病(PiD，n＝5)、皮质基底节变性(CBD，n＝5)、进行性核上性麻痹(PSP，n＝5)、球状神经胶质tau蛋白病(GGT，n＝2)、和健康对照(Ctrl，n＝2[对于MR测定n＝1])组成。
(A)tau蛋白病脑样品中的FL pS396的浓度。平均浓度±SEM：AD＝5769±621pg/ml、PiD＝4737±960pg/ml、CBD＝10716±2452pg/ml、PSP＝8888±1002pg/ml、GGT＝10122±3955pg/ml、Ctrl＝3326±73pg/ml。未记录组间统计学上的显著差异(Kruskal-Wallis检验后Dunn多重比较检验)。
(B)来自不同tau蛋白病的脑样本中的MR pS396浓度。平均水平±SEM：AD＝23432±4773pg/ml、PiD＝29949±6938pg/ml、CBD＝17434±9359pg/ml、PSP＝5563±1047pg/ml、GGT＝9830±3933pg/ml、Ctrl＝1862pg/ml。未记录组间统计学上的显著差异(单向方差分析[ANOVA])。
(C)与对照相比，来自五个不同tau蛋白病组的脑样品中CT pS396的水平。平均浓度±SEM：AD＝17692±2060pg/ml、PiD＝12549±1701pg/ml、CBD＝11203±5698pg/ml、PSP＝4766±706pg/ml、GGT＝7857±3097pg/ml、Ctrl＝397±54pg/ml。与PSP中(p<0.05)和Ctrl中的CT pS396浓度(p<0.01)相比，AD中的CT pS396浓度显著更高。类似地，PiD中的CTpS396水平显著高于对照(Ctrl)中的CT pS396水平(p<0.05；Kruskal-Wallis检验后Dunn多重比较检验)。
(D)MR与FL pS396水平的比率显著区分开不同的tau蛋白病。与PiD、CBD、PSP、GGT和对照中的MR/FL比率相比，AD的MR/FL比率明显不同(各自p<0.01)。此外，PiD患者中MR与FL的比率与CBD、PSP、GGT和对照中的MR与FL的比率明显不同(各自p＝0.0001；单向ANOVA后Dunnett多重比较检验)。
(E)疾病组中CT与FL pS396浓度的比率明显不同。与CBD、PSP、GGT和对照相比，AD中的CT/FL pS396水平明显不同(各自p<0.01)，并且与CBD、PSP、GGT和对照相比，PiD中的CT/FL pS396水平明显不同(各自p<0.01；单向ANOVA后Dunnett多重比较检验)。
所有三种测定均测量人tau蛋白病脑样品中的tau pS396。结果表明不同tau蛋白病中的pS396 tau群体关于所存在的tau群体而显著变化，这说明pS396和tau片段化的组合测量是区分这些疾病的有前途的方法。
图3阿尔茨海默病的rTg4510转基因tau小鼠模型中高水平的pS396-Tau46(CTpS396)。(A)在50或75倍稀释下，用CT pS396测定对来自不同动物的CSF样品进行分析。所测量的和稀释调节的浓度均有示出。(B)来自六种不同动物的10或20倍稀释的血浆样品的CTpS396浓度，包括CT pS396的CSF水平在(A)中示出的那些动物。测定检测限＝1.50pg/ml
CT pS396存在于rTg4510转基因tau小鼠动物的CSF和血浆中。CT pS396测定是用于研究该模型中CT pS396处理中的分子变化和用于药物疗效的临床前评价的重要工具。
图4人CSF中CT pS396的测量。通过旋转过滤(使用
Figure GDA0003335036700000041
设备；条件显示于表1中)，然后用尺寸排阻色谱(SEC)(具有S200 10/300GL柱，于50mM Tris pH 7.5，10％甘油流动的缓冲液中)来富集15ml人CSF中的CT pS396信号。用Simoa CT pS396直接分析洗脱级分。
(A)旋转过滤的人CSF的截留物的洗脱图，横轴表示洗脱体积，并且纵轴表示UV吸光度。网格线示出了如下分子量标记物的洗脱体积：蓝色葡聚糖(空隙容积，2000kDa；7.76ml)、白蛋白(66kDa；13.45ml)、碳酸酐酶(29kDa；16.28ml)、细胞色素(12.4kDa；20.39ml)、抑肽酶(6.5kDa；24.36ml)。
(B)旋转浓缩的人CSF的色谱图，横轴表示洗脱级分ID，并且纵轴表示UV吸光度。网格线是指在(A)中示出的相同分子量标记物。
(C)净(未处理的)CSF级分、旋转过滤的CSF(截留物)级分和洗脱的SEC级分中的CTpS396的浓度。在净CSF中未检测到CT pS396信号，这说明需要预处理以富集信号。过滤产物中也未检测到CT pS396。然而，在过滤产物的SEC筛分之后可以测量到CT pS396。在C1、C2和C3级分中发现最高浓度的CT pS396(该样品中分别为6.1、9.9和6.1pg/ml)，对应于洗脱体积12.0-13.5ml和分子量约66kDa。这些特性说明在12.0-13.5ml下洗脱的级分富集含有pS396和Tau46两种表位的tau单体。
在未处理的人CSF中不可测量到CT pS396。然而，通过旋转过滤和SEC进行的预分析处理富集了CT pS396信号，其中在12.0-13.5ml下洗脱达到最高浓度。通过改变CSF起始体积(表1)，以及通过改变每级分收集的SEC洗脱体积，使用具有不同容量和模型的旋转过滤器记录了一致结果(表1)。
通过旋转过滤对CSF样品进行顺序处理，然后通过SEC筛分对于CT pS396信号富集是必要的，因为省略任一步骤都会导致量大幅减少或检测不到的量。
具体实施方式
传统ELISA已持续多年用于分析样品中的不同种类分子。本发明涉及单分子阵列测定(Simoa)ELISA技术用于检测来自诊断患有如阿尔茨海默病、匹克氏病、皮质基底节变性、进行性核上性麻痹、球状神经胶质tau蛋白病和慢性创伤性脑病变等tau蛋白病的患者的样品中的tau种类的用途。
在本发明中，tau是以下序列的人tau，甲硫氨酸在第1位
Figure GDA0003335036700000061
在单分子免疫测定的第一步中捕获抗体附着于顺磁珠(约2.7um直径)的表面，将其用于浓缩样品中tau分子的稀释溶液。向该混合物中添加生物素化的检测抗体，并允许捕获抗体和检测抗体与样品中的tau反应。为了去除非特异性蛋白结合，可以对混合物进行洗涤。随后添加β-半乳糖苷酶标记的链霉亲和素，然后进行任选的洗涤步骤，并添加试卤灵β-D-半乳糖吡喃糖苷。使反应混合物反应并生成可在适当机器中读取和分析的荧光产物。
由本发明的发明人开发的测定基于两种抗体，即捕获抗体和生物素化的检测抗体。捕获抗体与如实例1所述的顺磁珠缀合。这些对于tau的磷酸化(p)S396是特定的。此类抗体的生成披露于例如WO 2017/009308中，并且在下表I中进一步描述了这些抗体。在本发明的实例中，所用的pS396特异性抗体是来自专利WO 2018/011073且在下表II中描述的指定为“C10-2人源化的”的抗体。
表I：WO 2017/009308中披露的pS396抗体
Figure GDA0003335036700000071
Figure GDA0003335036700000081
Figure GDA0003335036700000091
表II：WO 2018/011073中披露的pS396抗体
以下方案中的抗体均为指定为“C10-2人源化抗体”的抗体的工程化的版本。具体示出了与C10-2人源化抗体相比的差异，另外，表中的灰色方框旨在表示与C10-2人源化抗体相同的氨基酸。因此，例如，D55E与C10-2人源化抗体具有相同的轻链CDR 1-3和重链CDR1和3(灰色方框)，而重链的CDR2不同(已给出氨基酸残基)，并且由此VH与C10-2人源化抗体不同(已给出氨基酸残基)
Figure GDA0003335036700000101
Figure GDA0003335036700000111
Figure GDA0003335036700000121
Figure GDA0003335036700000131
Figure GDA0003335036700000141
Figure GDA0003335036700000151
Figure GDA0003335036700000161
Figure GDA0003335036700000171
所用的检测(detection/或detector，在本文可交替使用)抗体已如实例2所描述那样进行了生物素化，并且可在不同于pS396残基的位点处结合tau的C末端区、中央区或N末端区。检测抗体可结合非磷酸化的残基。具体而言，tau上的C端的表位为氨基酸1-20(如6-18)，tau的中央区为氨基酸140-170(如159-168)，并且tau的N端为400-441(如404-421)。在实例中使用以下抗体：
使用Tau12(#806502，百进生物科技公司(BioLegend))，并使其与tau的氨基酸6-18结合，检测抗体是HT7(#MN1000，英杰公司(Invitrogen))，其结合中央区159-168氨基酸，并且Tau46(#806601，百进生物科技公司)结合C末端区氨基酸404-441。
已开发了测量不同tau种类上pS396的三种测定(图1)；每种使用抗pS396捕获抗体，并且除了检测抗体，每种测定共用所有其他实验条件，这些实验条件已在下文描述。
1.全长pS396(FL测定)：该测定测量从N端区延伸的pS396磷酸化的tau种类。检测抗体(单克隆Tau12(#806502，百进生物科技公司))与tau的氨基酸6-18结合。
2.中央区pS396(MR测定)：该测定测量同时带有如下两种表位的tau形式：pS396磷酸化和中央区159-168氨基酸。检测抗体是HT7(#MN1000，英杰公司)。
3.C端pS396(CT测定)：该测定对于含有极端羧基端区(氨基酸404-441)的pS396磷酸化的tau是特定的。检测抗体是Tau46(#806601，百进生物科技公司)。
如实例4中描述的方法包括以下步骤：
a.将附着于顺磁珠的对pS396特异的捕获抗体与生物素化的检测抗体以及样品混合，
b.孵育该混合物足够时间以使得这些抗体与该样品中的tau结合(例如1分钟、2分钟、3分钟或5分钟或更久)，
c.任选地，洗涤步骤b)中孵育后的混合物，
d.添加缀合有链霉亲和素的β-半乳糖苷酶，并允许所述链霉亲和素缀合物和该生物素化的检测抗体反应(例如1分钟、2分钟、3分钟或5分钟或更久)，
e.任选地，洗涤步骤d)后所获得的混合物，
f.向步骤d)或e)中的混合物中添加试卤灵β-D-半乳糖吡喃糖苷，并使试卤灵β-D-半乳糖吡喃糖苷水解(例如1分钟、2分钟、3分钟或5分钟或更久)，
g.读取荧光信号并将该信号与标准进行比较。
使用的缀合有pS396的抗体珠的数量一般为至少1000颗珠，如至少10,000颗、100,000颗珠或更多。样品可以是来自哺乳动物的CSF、血浆或生物流体样品，例如来自患有如阿尔茨海默病、匹克氏病、皮质基底节变性、进行性核上性麻痹或球状神经胶质tau蛋白病等tau蛋白病的人的人CSF样品。例如通过使用实例3所示的旋转过滤柱和尺寸排阻色谱来浓缩关于tau的样品可能是有利的。
如实例4所示，独立使用或组合使用的测定结果可用于诊断或区分tau蛋白病，如阿尔茨海默病、匹克氏病、皮质基底节变性、进行性核上性麻痹和球状神经胶质tau蛋白病。例如，CT测定可用于诊断阿尔茨海默病和匹克氏病。通过比较MR/FL比率，其可用于将阿尔茨海默病与其他tau蛋白病和对照区分开，并且进一步地，匹克氏病明显不同于皮质基底节变性、进行性核上性麻痹、球状神经胶质tau蛋白病和对照。通过使用CT/FL比率，阿尔茨海默病可与其他tau蛋白病和对照区分开，且匹克氏病不同于皮质基底节变性、进行性核上性麻痹以及球状神经胶质tau蛋白病和对照。
实验细节
实例1：捕获抗体(pS396)与顺磁珠的缀合
使用Ultracel 50K旋转过滤柱(#UFC505096，艾美康恩有限公司(Amicon))将pS396抗体缓冲交换至珠缀合缓冲液(BCB；50mM MES pH 6.2)中。首先通过在14000xg、在室温(RT)下离心5min，用450ul BCB冲洗过滤器，并弃掉流出液(flow-through)。然后，通过在14000xg、在RT下离心5min，将1.6g/L抗体缓冲交换至BCB中。弃掉流出液，并将过滤器放回收集管。将BCB添加至截留物中使体积达到450ul，并在相同条件下再次离心。弃掉流出液之后重复该步骤一次。随后，用40ul BCB冲洗过滤器，转至新收集管中，并通过在1000xg、在RT下离心2min回收抗体。用Nanodrop Lite(赛默飞世尔科技公司(ThermoFisherScientific))估算抗体浓度，并4℃储存直至使用。
使用磁力分离器，用珠洗涤缓冲液(BWB；1x PBS+1％Tween 20)洗涤顺磁性羧基化单丛珠(singleplex bead)(#103207，Quanterix公司)三次，然后用BCB洗涤两次。通过添加0.3g/L 1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐(#A35391，赛默飞世尔科技公司)，并在4℃下孵育30min来活化浓度为1.4×106颗珠/μL的珠。然后，用冰冷的BCB洗涤活化的珠一次，并弃掉上清。将抗体(0.2g/L)添加至珠中，并且在RT下，震荡孵育混合物2h以使抗体与珠结合。一般在以下条件下(其中每步持续5秒)用HulaMixer样品混合器(#15920D，赛默飞世尔科技公司)进行震荡：转速＝5rpm，倒数＝90°，振动/暂停＝5°。然后，去除上清，并用BWB洗涤缀合有抗体的珠两次。在RT下伴随震荡用珠封闭缓冲液(1xPBS中1％BSA)来封闭反应1h。最后，用BWB洗涤珠两次，并且然后用珠稀释液(BD；50mM Tris pH 7.8、50mM NaCl、10mM EDTA、1％BSA、0.1％Tween20)洗涤一次。用磁力分离器去除上清后，在BD中重悬珠，并且在4℃储存直至使用。
实例2：生物素与检测抗体缀合
在Ultracel 50K旋转过滤柱(#UFC505096，艾美康恩有限公司)中将检测抗体缓冲交换至生物素化反应缓冲液中(BRB；100mM PBS pH 7.4)。通过在14000xg、在RT下离心450ul BRB持续5min清洗柱后，弃掉流出液并将抗体转移至过滤器。添加BRB使体积达到450ul，并在14000xg、在RT下离心5min。再重复缓冲交换两次，在每个阶段用BRB使得抗体体积达到450ul，并在14000xg、在RT下离心5min。用40ul BRB冲洗过滤器，转至新收集管中，并通过在1000xg、在RT下离心2min回收抗体。使用Nanodrop Lite估算抗体浓度。将四十倍过量的EZ-Link NHS-PEG4生物素(#21329，赛默飞世尔科技公司)添加至抗体中，并在RT下孵育30min。通过重复生物素标记之前进行的缓冲交换过程去除游离的生物素。在4℃下储存缀合有生物素的抗体直至使用。
实例3：样品和校准物的预分析处理
分析之前，通过用测定稀释液(Tau 2.0稀释液，#101556，Quanterix公司)稀释储备浓度来制备适当浓度的测定校准物(通过糖原合成酶激酶3β(#TO8-50FN，新格诺康公司(SignalChem)体外磷酸化的重组tau 441)。质量控制样品包括用测定稀释液稀释500倍和5000倍的TBS可溶性人阿尔茨海默病脑提取物。
用Tau 2.0稀释液将Tris缓冲盐水(TBS)可溶性人脑提取物、rTg4510转基因小鼠CSF和血浆样品稀释至图2和3及其图例中所示的期望浓度。
通过在旋转过滤柱中离心样品以及在运行于Ethan LC系统(通用电气医疗集团(GE Healthcare))的Superdex S200 10/300GL柱(#17-5175-01，通用电气医疗集团)上通过尺寸排阻色谱(SEC)筛分截留物来富集人CSF中pS396 tau的水平。运行缓冲液为50mMTris pH 7.5+10％甘油。使用Simoa pS396测定直接分析所收集的级分。已经使用具有不同容量和特性的旋转过滤柱验证了该方法(表1)。
表1.离心过滤器装置的细节和用于在基于尺寸的筛分之前浓缩人CSF以富集pS396信号的条件。所有装置购自默克密理博公司(Merck Millipore)。
Figure GDA0003335036700000211
*将来自所示数量的柱的截留物级分汇集用于进一步分析。
实例4：单分子阵列(Simoa)测定
每个pS396测定在Simoa HD-1仪器(Quanterix公司，美国马萨诸塞州列克星敦(Lexington，MA，USA))上使用两步方案。在该测定配置中，将100ul的珠混合物(由1000颗珠/ul，每个pS396抗体包被的珠和Helper珠(#103208，Quanterix公司)组成)吸入至反应容器中。然后，添加20ul生物素化检测抗体(2ug/ml)和100ul目标分析物，并孵育反应混合物47个节奏(1个节奏＝45秒)以使分析物与捕获抗体和检测抗体反应。随后洗涤珠，并添加100ul的450pM缀合有链霉亲和素的β-半乳糖苷酶(SBG；#100439，Quanterix公司)。在另外孵育7个节奏和随后的洗涤之后，添加25μl试卤灵β-D-半乳糖吡喃糖苷(RGP；#103159，Quanterix公司)。通过SBG催化RGP的水解，产生荧光产物试卤灵。将珠转移至200，000孔的盘上，每个孔的大小只够容纳一颗珠。除去额外的珠，并在成像之前将盘表面密封。将荧光信号转换成每珠酶均值(average enzyme per bead，AEB)，并用已知蛋白浓度生成的四参数逻辑校准曲线推算样品浓度。
测定设置
已开发了测量不同tau种类上pS396的三种测定(图1)；每种使用抗pS396捕获抗体，并且除了检测抗体，每种测定共用所有其他实验条件，这些实验条件已在下文描述。
1.全长pS396(FL测定)：该测定测量从N端区延伸的pS396磷酸化的tau种类。检测抗体(单克隆Tau12(#806502，百进生物科技公司))与tau的氨基酸6-18结合。
2.中央区pS396(MR测定)：该测定测量同时带有如下两种表位的tau形式：pS396磷酸化和中央区159-168氨基酸。检测抗体是HT7(#MN1000，英杰公司)。
3.C端pS396(CT测定)：该测定对于含有极端羧基端区(氨基酸404-441)的pS396磷酸化的tau是特定的。检测抗体是Tau46(#806601，百进生物科技公司)。
序列表
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1 5 10 15
Gly
<210> 16
<211> 6
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10.2 HC CDR3
<400> 16
Arg Gly Ala Met Asp Tyr
1 5
<210> 17
<211> 439
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10.2 VH
<400> 17
Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Asn Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val
130 135 140
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
180 185 190
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
195 200 205
Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
210 215 220
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
225 230 235 240
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
245 250 255
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
260 265 270
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
275 280 285
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
290 295 300
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
305 310 315 320
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
325 330 335
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
340 345 350
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
355 360 365
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
370 375 380
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
385 390 395 400
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
405 410 415
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
420 425 430
Leu Ser His Ser Pro Gly Lys
435
<210> 18
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C5.2 LC CDR1
<400> 18
Gln Ala Ser Gln Asp Thr Ser Ile Asn Leu Asn
1 5 10
<210> 19
<211> 7
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C5.2 LC CDR2
<400> 19
Gly Ala Ser Asn Leu Glu Asp
1 5
<210> 20
<211> 7
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C5.2 LC CDR3
<400> 20
Leu Gln His Thr Tyr Leu Pro
1 5
<210> 21
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C5.2 VL
<400> 21
Asp Val Gln Met Ile Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Ile Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Asn
20 25 30
Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Asp
65 70 75 80
Glu Asp Met Ala Thr Tyr Phe Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 22
<211> 13
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C5.2 HC CDR1
<400> 22
Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg Thr Ile His
1 5 10
<210> 23
<211> 17
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C5.2 HC CDR2
<400> 23
Tyr Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Asn Asp Asn Phe Lys
1 5 10 15
Gly
<210> 24
<211> 6
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C5.2 HC CDR3
<400> 24
Arg Gly Thr Met Asp Tyr
1 5
<210> 25
<211> 439
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C5.2 VH
<400> 25
Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Asn Asp Met Phe
50 55 60
Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Arg Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val
130 135 140
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
180 185 190
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
195 200 205
Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
210 215 220
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
225 230 235 240
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
245 250 255
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
260 265 270
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
275 280 285
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
290 295 300
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
305 310 315 320
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
325 330 335
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
340 345 350
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
355 360 365
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
370 375 380
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
385 390 395 400
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
405 410 415
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
420 425 430
Leu Ser His Ser Pro Gly Lys
435
<210> 26
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C8.3 LC CDR1
<400> 26
Gln Ala Ser Gln Gly Thr Ser Ile Asn Leu Asn
1 5 10
<210> 27
<211> 7
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C8.3 LC CDR2
<400> 27
Gly Ser Ser Asn Leu Glu Asp
1 5
<210> 28
<211> 7
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C8.3 LC CDR3
<400> 28
Leu Gln His Ser Tyr Leu Pro
1 5
<210> 29
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C8.3 VL
<400> 29
Asp Val Gln Met Ile Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Ile Val Thr Met Thr Cys Gln Ala Ser Gln Gly Thr Ser Ile Asn
20 25 30
Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ser Ser Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Asp
65 70 75 80
Glu Asp Met Ala Thr Tyr Phe Cys Leu Gln His Ser Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 30
<211> 13
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C8.3 HC CDR1
<400> 30
Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg Thr Ile His
1 5 10
<210> 31
<211> 17
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C8.3 HC CDR2
<400> 31
Tyr Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Asn Phe Lys
1 5 10 15
Gly
<210> 32
<211> 6
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C8.3 HC CDR3
<400> 32
Arg Gly Ala Met Asp Tyr
1 5
<210> 33
<211> 439
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C8.3 VH
<400> 33
Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Asn Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Asn Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val
130 135 140
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
180 185 190
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
195 200 205
Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
210 215 220
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
225 230 235 240
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
245 250 255
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
260 265 270
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
275 280 285
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
290 295 300
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
305 310 315 320
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
325 330 335
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
340 345 350
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
355 360 365
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
370 375 380
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
385 390 395 400
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
405 410 415
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
420 425 430
Leu Ser His Ser Pro Gly Lys
435
<210> 34
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10-2人源化的LC CDR1
<400> 34
Gln Ala Ser Gln Asp Thr Ser Ile Asn Leu Asn
1 5 10
<210> 35
<211> 7
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10-2人源化的LC CDR2
<400> 35
Gly Ala Ser Asn Leu Glu Thr
1 5
<210> 36
<211> 9
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10-2人源化的LC CDR3
<400> 36
Leu Gln His Thr Tyr Leu Pro Phe Thr
1 5
<210> 37
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10-2人源化的VL
<400> 37
Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Asn
20 25 30
Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 38
<211> 5
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10-2人源化的HC CDR1
<400> 38
Asp Arg Thr Ile His
1 5
<210> 39
<211> 17
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10-2人源化的HC CDR2
<400> 39
Tyr Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gln Lys Phe Gln
1 5 10 15
Gly
<210> 40
<211> 6
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10-2人源化的HC CDR3
<400> 40
Arg Gly Ala Met Asp Tyr
1 5
<210> 41
<211> 445
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> C10-2人源化的VH
<400> 41
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 42
<211> 18
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55E HC CDR2
<400> 42
Tyr Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ser Gln Lys Phe Gln
1 5 10 15
Gly Arg
<210> 43
<211> 444
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55E VH
<400> 43
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 44
<211> 18
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55Q HC CDR2
<400> 44
Tyr Ile Tyr Pro Gly Gln Gly Ser Thr Lys Tyr Ser Gln Lys Phe Gln
1 5 10 15
Gly Arg
<210> 45
<211> 444
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55Q VH
<400> 45
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Gln Gly Ser Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 46
<211> 18
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55S HC CDR2
<400> 46
Tyr Ile Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Ser Gln Lys Phe Gln
1 5 10 15
Gly Arg
<210> 47
<211> 444
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55S VH
<400> 47
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 48
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32S LC CDR1
<400> 48
Gln Ala Ser Gln Asp Thr Ser Ile Ser Leu Asn
1 5 10
<210> 49
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32S VL
<400> 49
Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Ser
20 25 30
Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 50
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32Q LC CDR1
<400> 50
Gln Ala Ser Gln Asp Thr Ser Ile Gln Leu Gln
1 5 10
<210> 51
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32Q VH
<400> 51
Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Gln
20 25 30
Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 52
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N34S LC CDR1
<400> 52
Gln Ala Ser Gln Asp Thr Ser Ile Asn Leu Ser
1 5 10
<210> 53
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N34S VL
<400> 53
Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Asn
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 54
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N34Q LC CDR1
<400> 54
Gln Ala Ser Gln Asp Thr Ser Ile Asn Leu Gln
1 5 10
<210> 55
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N34Q VL
<400> 55
Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Asn
20 25 30
Leu Gln Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 56
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32S,N34S LC CDR1
<400> 56
Gln Ala Ser Gln Asp Thr Ser Ile Ser Leu Ser
1 5 10
<210> 57
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32S,N34S VL
<400> 57
Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Ser
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 58
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32Q, N34S LC CDR1
<400> 58
Gln Ala Ser Gln Asp Thr Ser Ile Gln Leu Ser
1 5 10
<210> 59
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32Q, N34S VL
<400> 59
Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Gln
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 60
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32Q, N34Q LC CDR1
<400> 60
Gln Ala Ser Gln Asp Thr Ser Ile Gln Leu Gln
1 5 10
<210> 61
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32Q, N34Q VL
<400> 61
Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Gln
20 25 30
Leu Gln Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 62
<211> 11
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32S, N34Q LC CDR1
<400> 62
Gln Ala Ser Gln Asp Thr Ser Ile Ser Leu Gln
1 5 10
<210> 63
<211> 214
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> N32S, N34Q LC
<400> 63
Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Met Thr Cys Gln Ala Ser Gln Asp Thr Ser Ile Ser
20 25 30
Leu Gln Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln His Thr Tyr Leu Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 64
<211> 6
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> A101 HC CDR3
<400> 64
Arg Gly Thr Met Asp Tyr
1 5
<210> 65
<211> 444
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> A101 VH
<400> 65
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 66
<211> 18
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55E, A101T HC CDR2
<400> 66
Tyr Ile Tyr Pro Gly Gln Gly Ser Thr Lys Tyr Ser Gln Lys Phe Gln
1 5 10 15
Gly Arg
<210> 67
<211> 6
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55E, A101T HC CDR3
<400> 67
Arg Gly Thr Met Asp Tyr
1 5
<210> 68
<211> 444
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55E, A101T VH
<400> 68
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Glu Gly Ser Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 69
<211> 18
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55Q, A101T HC CDR2
<400> 69
Tyr Ile Tyr Pro Gly Gln Gly Ser Thr Lys Tyr Ser Gln Lys Phe Gln
1 5 10 15
Gly Arg
<210> 70
<211> 6
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55Q, A101T HC CDR3
<400> 70
Arg Gly Thr Met Asp Tyr
1 5
<210> 71
<211> 444
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55Q, A101T VH
<400> 71
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Gln Gly Ser Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 72
<211> 18
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55S, A101T HC CDR2
<400> 72
Tyr Ile Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Ser Gln Lys Phe Gln
1 5 10 15
Gly Arg
<210> 73
<211> 6
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55S, A101T HC CDR3
<400> 73
Arg Gly Thr Met Asp Tyr
1 5
<210> 74
<211> 444
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<223> D55S, A101T VH
<400> 74
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440

Claims (17)

1.一种用于测量样品中的磷酸化的tau的体外测定，所述测定包括2种抗体的使用，这两种抗体为i)对tau上的pS396特异的捕获抗体，和ii)在与该捕获抗体不同的表位上结合tau的检测抗体。
2.根据权利要求1所述的测定，其中所述检测抗体结合至tau上的氨基酸1-20(如6-18)、tau上的氨基酸140-170(如159-168)或tau上的氨基酸400-441(如404-421)内的表位。
3.根据权利要求1-2所述的测定，其中该检测抗体被生物素化。
4.根据前述权利要求中任一项所述的测定，其中该捕获抗体附着于顺磁珠的表面。
5.根据前述权利要求中任一项所述的测定，其中使用缀合有β-半乳糖苷酶的链霉亲和素来生成信号读数。
6.根据前述权利要求中任一项所述的测定，其中该样品是来自哺乳动物的CSF、血浆或其他生物流体样品。
7.根据前述权利要求中任一项所述的测定，其中该样品是来自患有如阿尔茨海默病、匹克氏病、皮质基底节变性、进行性核上性麻痹或球状神经胶质tau蛋白病等tau蛋白病的人的CSF或血浆样品。
8.根据前述权利要求中任一项所述的测定，其中该CSF样品已经例如通过使用旋转过滤柱和尺寸排阻色谱关于tau进行了浓缩。
9.根据前述权利要求中任一项所述的测定用于诊断如阿尔茨海默病、匹克氏病、皮质基底节变性、进行性核上性麻痹或球状神经胶质tau蛋白病等tau蛋白病的用途。
10.一种用于测量样品中的磷酸化的tau的方法，该方法包括以下步骤
a.将附着于顺磁珠的对pS396特异的捕获抗体与生物素化的检测抗体以及样品混合，
b.孵育该混合物足够时间以使得这些抗体与该样品中的tau结合(例如1分钟、2分钟、3分钟或5分钟或更久)，
c.任选地，洗涤步骤b)中孵育后的混合物，
d.添加缀合有链霉亲和素的β-半乳糖苷酶，并允许所述链霉亲和素缀合物和该生物素化的检测抗体反应(例如1分钟、2分钟、3分钟或5分钟或更久)，
e.任选地，洗涤步骤d)后所获得的混合物，
f.向步骤d)或e)中的混合物中添加试卤灵β-D-半乳糖吡喃糖苷，并使试卤灵β-D-半乳糖吡喃糖苷水解(例如1分钟、2分钟、3分钟或5分钟或更久)，
g.读取荧光信号并将该信号与标准进行比较。
11.根据权利要求10所述的方法，其中所述捕获抗体附着于顺磁珠，并且将至少1000颗所述顺磁珠添加至该样品中，如至少10000颗、100,000颗珠或更多。
12.根据权利要求10所述的方法，其中所述检测抗体结合至tau上的氨基酸1-20(如6-18)、tau上的氨基酸140-170(如159-168)或tau上的氨基酸400-441(如404-421)内的表位。
13.根据权利要求10-12所述的方法，其中该样品是来自哺乳动物的CSF、血浆或生物流体样品。
14.根据权利要求10-13所述的方法，其中该样品是来自患有如阿尔茨海默病、匹克氏病、皮质基底节变性、进行性核上性麻痹或球状神经胶质tau蛋白病等tau蛋白病的人的CSF或血浆样品。
15.根据权利要求10-14所述的方法，其中所述样品已经例如通过使用旋转过滤柱和尺寸排阻色谱关于tau进行了浓缩。
16.根据权利要求10-15所述的方法用于诊断如阿尔茨海默病、匹克氏病、皮质基底节变性、进行性核上性麻痹或球状神经胶质tau蛋白病等tau蛋白病的用途。
17.根据权利要求10-17所述的方法的用途，其中所述检测抗体结合tau上的氨基酸400-441(如404-421)内的表位以诊断阿尔茨海默病或匹克氏病。
CN202080024099.2A 2019-03-28 2020-03-24 pS396测定用于诊断TAU蛋白病的用途 Pending CN113748343A (zh)

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