CN104487847A - 与阿尔茨海默氏病和轻度认知障碍相关的原肌球蛋白同种型 - Google Patents
与阿尔茨海默氏病和轻度认知障碍相关的原肌球蛋白同种型 Download PDFInfo
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Abstract
本发明提供了有助于阿尔茨海默氏病(AD)或轻度认知障碍(MCI)诊断的体外方法，包括测定患者样品中对应于P09493-3和/或P09493-1的一种或多种原肌球蛋白同种型的表达水平。还提供了包含结合原肌球蛋白同种型P09493-3和P09493-1的探针的试剂盒。
Description
发明背景
由于寿命的不断增加，基于痴呆的疾病逐渐成为了全球与健康相关的医疗预算的主要负担，并且被认为是受影响护理者/家庭的压力源。阿尔茨海默氏病(AD)表现出最普遍的神经退行性病理，并且一直在寻找用于患者治疗和筛选的治疗和改进的诊断方法的鉴定。虽然心理计量测试如简易精神状态检查通常是AD的良好预报器，尸体解剖仍然是阿尔茨海默氏病诊断的唯一明确的方法。记忆力衰退在老年人中是常见的现象。在严重的记忆力衰退为主要症状的事件中，该病症被称为轻度认知障碍(MCI)，并且常常被看作是AD的初期阶段。在该阶段，治疗可能比后期更为有效。据记载，MCI患者在3年内转化为临床型AD的比率为50％(Karas et al.2008)。诊断医学中蛋白生物标志物的应用在逐渐增加。AD的蛋白生物标志物，尤其是存在于易获取的生物液体如血液和尿液中的那些蛋白生物标志物的鉴定，代表了当前诊断方法的合适的有效的替代选择。原肌球蛋白是由两个α-螺旋组成的纤维分子。其广泛分布于所有细胞类型中，在那里其调节肌丝肌动蛋白和肌球蛋白的收缩。在哺乳动物中，4种高度保守的基因的不同剪接可产生多于40种同种型(Schevzovet al.2005)。此外，每种同种型可接受不同程度的翻译后修饰，包括磷酸化和糖基化。用于蛋白分离和鉴定的两种最常见的分析技术为二维凝胶电泳(2-D DIGE)和2D蛋白质印迹。通过采用2-D DIGE确定血小板样品中AD可能的生物标志物，EP2293075强调了两种原肌球蛋白同种型，指定为Swissprot/Uniprot登录号P07951以及NCBI/Genbank登录号BAB14554/AK023385。
发明概述
本发明提供了用于检测MCI和AD相关的包含外显子1a以及任选地外显子9d的原肌球蛋白同种型的方法和试剂盒。
根据第一个方面，本发明提供了有助于阿尔茨海默氏病(AD)或轻度认知障碍(MCI)诊断的体外方法，包括测定患者样品中对应于P09493-3和/或P09493-1的一种或多种原肌球蛋白同种型的表达水平。
根据第二个方面，本发明提供了试剂盒，其包含结合原肌球蛋白同种型P09493-3和P09493-1的探针，特征在于结合P09493-3和P09493-1的不包含外显子1a的共同表位的第一探针，和特异性结合外显子1a的第二探针。
根据第三个方面，本发明提供了改变一种或多种原肌球蛋白同种型P09493-3和P09493-1的浓度水平的试剂，其被用于治疗AD或MCI。
附图说明
图1显示了由外显子1a和9d激发的抗体的钌染色；
图2显示了针对外显子1a的抗体的2D蛋白质印迹评估；并且
图3显示了针对外显子9d的抗体的2D蛋白质印迹评估。
发明详述
除非另有规定，提及外显子1a和9d是指如下所示的氨基酸序列：
外显子1a：-Leu-Asp-Lys-Glu-Asn-Ala-Leu-Asp-Arg-Ala-Glu-Gln-Ala-Glu-Ala-Asp-Lys-Lys-Ala-Ala-(SEQ ID NO.1)
外显子9d：-Glu-Lys-Val-Ala-His-Ala-Lys-Glu-Glu-Asn-Leu-Ser-Met-His-Gln-Met-Leu-Asp-Gln-Thr-Leu-Leu-Glu-Leu-Asn-Asn-Met-(SEQ ID NO.2)
术语“患者”和“个体”在本文中可互换使用，并且指任何动物(例如，哺乳动物)，包括但不限于人、非人的灵长类、犬类、猫科动物、啮齿类等，其将成为诊断的受试者。优选地，个体或患者为人。
术语“药物”和“试剂”在本文中可互换使用，并且指对生物系统产生影响的化学或生物物质。
如本文所用，术语“原肌球蛋白同种型”包括所有基于原肌球蛋白的蛋白质，包括来源于差异化遗传剪接和翻译后修饰形式的那些。对应于图1、2和3的斑点2、3和4的3种原肌球蛋白同种型基于2D-WB和质谱数据被指定为UniprotKB(UniprotKB；www.uniprot.org/uniprot)登录号P09493-3；因此，在本文中提及P09493-3是指斑点2、3和4，除非另有说明。如本文所述，提及P09493是指P09493-3(斑点2、3和4)和P09493-1；本文中提及的P09493-1是如UniprotKB中所述，并且对应于斑点1。
根据第一个方面，本发明提供了有助于轻度认知障碍(MCI)和/或阿尔茨海默氏病(AD)诊断的方法，包括检测和测量体外患者样品中P09493和/或其翻译后类似物的一种或多种的表达水平。在优选的实施方案中，待检测和测量的原肌球蛋白同种型对应于P09493-3(斑点2、3和4)的一种或多种。如本文所用，“表达水平(level of expression/expression level)”是指所指蛋白或编码该蛋白的mRNA的量。P09493可与MCI和AD的其他生物标志物一起使用以提高AD或MCI检测的诊断能力。蛋白生物标志物并非单独使用，并且技术人员认可在使用P09493诊断MCI或AD过程中，诊断还将包括使用身体和认识测试如MMSE进行患者的临床评估，以助于将患者分类为AD或MCI。基于连同P09493使用的生物标志物的诊断能力(例如，通过ROC曲线分析及灵敏度和特异性评估)，有可能的是，生化生物标志物检测可为用于诊断AD或MCI的主要诊断工具。待检测的原肌球蛋白同种型包含外显子1a以及任选地外显子9d；原肌球蛋白同种型P09493-3包含外显子1a和9d，而原肌球蛋白同种型P09493-1包含外显子1a，但没有外显子9d。待检测和测量的原肌球蛋白同种型优选来源于获自患者血液样品的血小板。在MCI和AD患者中，已表明P09493浓度相比对照值显示显著的增加。
优选地，对照值是来源于获自健康个体的样品优选血小板样品的P09493的浓度。可选地，对照可为参照值。预期AD患者和健康对照中的待测量血小板优选使用EP 1891445中描述的方法获得，其描述了从血液样品中提取未活化血小板的方法。对照数据还可为来自在MCI/AD发作前取自MCI/AD患者本身的样品的值。此类生物标志物诊断方法中的通常做法是使用先前得到和存档的对照数据集。在优选的实施方案中，一种或多种原肌球蛋白同种型表达水平相比对照值的增加指示AD或MCI。本发明的方法任选地还可包括以能改变P09493浓度水平的试剂治疗被诊断为患有AD或MCI的患者(或本发明方法指示可能会患AD或MCI的患者)。
在第二方面，本发明提供了用于MCI和AD诊断的试剂盒，其包含特异性识别外显子1a的第一探针和特异性识别P09493-3和P09493-1的不包含外显子1a的共同表位的第二探针。为了诊断AD或MCI，第二探针可为外显子9d特异性的。具体而言，指在测试的检测范围内，探针实质上仅检测P09493，即探针与除P09493外的蛋白的任何结合的水平足够低，使得该结合不影响诊断结果的完整性。探针优选为抗体。
如本文所用，术语“抗体”是指特异性识别靶标上的表位的免疫球蛋白，如通过免疫球蛋白重链和轻链的可变域(VHS和VLS)，更具体而言互补决定区(CDR)的结合特征所确定的。许多可能的抗体形式为本领域所已知，其可包括但不限于多种完整的单克隆抗体或包含完整的单克隆抗体的多克隆混合物、抗体片段(例如，Fab、Fab'和Fr片段、线性抗体单链抗体和包含抗体片段的多特异性抗体)、单链可变片段(scFvS)、多特异性抗体、嵌合抗体、人源化抗体和包含识别靶标上给定表位所必需的结构域的融合蛋白。优选地，在本发明上下文中提及抗体是指单克隆抗体。抗体还可与多种报告部分缀合用于产生诊断作用，包括但不限于放射性核素、荧光素或染料。提及抗体还包括“完整”抗体的短链可变片段和其他亚结构变体。技术人员认可的是对于可用于本发明方法和试剂盒的抗体，其需要识别外显子1a以及任选地9d。此类识别可包括识别完整外显子氨基酸序列或外显子序列亚集的每种外显子特异性的抗体。例如，抗体识别的外显子1a的亚集可仅包含7个氨基酸，但仍然为外显子1a特异性的。其他适合用于本发明的探针为能够识别P09493的分子，如适体、分子印迹的聚合物等。
本发明的第三个方面涉及改变P09493的浓度水平的试剂的用途；此类用途意图作为减轻或抑制MCI和AD症状的治疗。
本发明还提供了治疗MCI和/或AD的方法，包括：(i)确定患者样品中对应于P09493-3和/或P09493-1的一种或多种原肌球蛋白同种型表达水平相比对照是否增加；以及(ii)给予能改变P09493的浓度水平的试剂。
本文所引用的所有出版物的内容通过引用并入本文。
本发明参照附图，通过以下非限制性实施例进行描述。
实施例
方法
研究人群
阿尔茨海默氏病患者：47名AD患者参与该研究。在取血当天进行了CERAD(阿尔茨海默病联合注册表)的神经心理学成套测试。患者均未以AD相关的药物如乙酰胆碱酯酶抑制剂或美金刚进行治疗。此外，痴呆患者未接受长期的慢性抗精神病药物治疗，这可能已影响了Mao-B表达。为了排除认知障碍的其他起因(例如，中风或肿瘤)，除了有两名患者接受了计算机断层扫描(幽闭恐惧症、金属植入物)外，所有患者均接受了脑部的结构成像扫描(磁共振成像)。基于CERAD标准(http://cerad.mc.duke.edu/Assesment.htm/)，例如，病史、脑成像和神经心理学CERAD成套测试，进行了以下诊断：47名AD(9名尸检确认)。总共在13名患者中进行了神经病理学的尸体解剖。这些患者在取血后10-18个月死亡。在患有临床疑似AD的47名患者的9名中，临床诊断得到神经病理学确认，并且13名血管性痴呆(VD)病例中的4名满足混合型痴呆(AD和VD)的神经病理学标准。神经病理学检查遵循标准方案，包括tau蛋白(抗体AT-8,Innogenetics,Ghent,Belgium)、β-淀粉体(克隆4G8,SignetLabs,Dedham,MA)和α-突触核蛋白(单克隆抗体和多克隆抗体,Chemicon,Temecula,CA)的苏木精/曙红染色、改良皮尔苏斯基氏浸渍(Bielschowskyimpregnation)和免疫组织化学。对于AD，根据确立的尸检一致标准进行神经病理学诊断，包括CERAD评分。AD病例显示指示CERAD C和Braak阶段V/VI的神经病理学，从而根据NIA里根研究所“痴呆”标准满足较高的AD可能性。对照：无神经退行性疾病或认知障碍征兆的49名AD个体的年龄和性别匹配的对照组。面见了所有个体，并由经验丰富的心理学家进行神经心理学检查(MMSE)。包括在我们研究中的所有个体均为不吸烟者。根据Petersen et al(1999&2004)确定轻度认知障碍。
研究设计的伦理方面
方案得到当地伦理委员会的批准，并且根据2000年修订的赫尔基辛宣言的原则进行研究。在解释研究的目的和方案后从每名个体或律师获得了知情同意书。
样品准备
不停顿从肘前静脉将血液抽入含有0.129mol/L柠檬酸钠作为抗凝剂(与血液的混合比为1：9)的真空管(Greiner Bio-One GmbH,Kremsmunster,Austria)中。丢弃抽吸的第一管，以避免来自静脉穿刺的任何组织污染。在MicroDiff 18血液分析仪(Coulter Electronics,Miami,FL,USA)上进行差别血像检查。按先前的描述进行血小板分离程序和蛋白沉淀[31]。简而言之，通过在4℃震荡过夜将沉淀再溶解于变性2-D样品缓冲液中，其含有7M尿素、2M硫脲、4％CHAPS、20mM Tris-HCl(pH 8.5)。每100x106个血小板使用70微升的样品缓冲液。使用考马斯亮蓝蛋白检测试剂盒以BSA作为标准蛋白(Pierce Biotechnology,Rockford,IL,USA)，以一式三份测定再溶解的样品中的蛋白浓度。利用合适的稀释，2-D样品缓冲液组分不干扰蛋白检测。因此，使用PBS以1:20稀释样品，并且将5％的2-D样品缓冲液加入至BSA标准液中。通过将每个研究样品的相同量的总蛋白合并来组成2D DIGE分析的内部标准。将等份的内部标准和研究个体样品储存在-80℃。在电泳前，根据厂商说明书并经少量修改以荧光花青染料(CyDyes,GE Healthcare,Uppsala,Sweden)标记蛋白。将CyDye与蛋白的比值降低至每μg蛋白5pmol的染料。以Cy2标记内部标准，并可选地用Cy3或Cy5来标记样品。对于实时定量逆转录PCR(qRT-PCR)，按先前所述从凝胶过滤的血小板提取总RNA。
2D DIGE电泳和凝胶图像分析
按先前所述进行电泳和凝胶图像处理。简而言之，在含有染料标记的样品的改良的再水合溶液(7M尿素、2M硫脲、4％CHAPS、70mM DTT和0.5％两性电解质pH 4-7)中，使24cm、pH 4-7的9个IPG-Drystrip被动地再水合。其后，通过被动再水合施加每一染料标记蛋白25μg。进行等电点聚焦直至达到30kVh。在第二维度中进行11.5％SDS-PAGE(10℃下35V 1h,50V 1.5h和最终110V 16.5h)，并使用Typhoon 9410成像仪(GE Healthcare,Uppsala,Sweden)在100μm分辨率下扫描凝胶。使用DeCyder软件模块凝胶内差异分析(version 6.00.28；GE Healthcare,Uppsala,Sweden)，将靶标斑点数设定为2500，在凝胶图像上进行斑点检测。将每个凝胶添加至合适的工作区和组，并使用DeCyder模块生物差异分析(版本6.01.02)针对主凝胶进行匹配。凝胶成像分析程序在先前有更为详细的描述。
蛋白鉴定
通过质谱分析鉴定蛋白。按先前所述进行后者的胰蛋白酶蛋白水解物的制备。将肽加载至Zorbax 300SB-C8(5μm,0.3mm x 5mm)柱上，并使用0.2％甲酸和3％乙腈至0.2％甲酸和45％乙腈的梯度，以250nl/min的流速在12分钟内，利用Zorbax 300SB-C18(5μm,75μm x 150mm)柱，通过纳流液相色谱(1100Series LC system,Agilent,Palo Alto,CA)进行分离。利用装配垂直纳喷雾离子源的离子肼质谱仪(XCT-Plus,Agilent)，通过串联质谱(MS/MS)碎片分析完成肽鉴定。通过Spectrum Mill MS蛋白质组工作台软件(Version A.03.03,Agilent)解读MS/MS数据，并针对人蛋白的SwissProt数据库(版本14.3，含有20,328个条目)进行搜索，所述数据库允许1.5Da的前体质量偏差、0.7Da的产物质量公差和70％的最小匹配峰值强度(％SPI)，以及一个切割的丢失。由于先前的化学修饰，半胱氨酸的脲甲基化被设置为固定的修饰。肽评分高于13的错误发现率始终小于1％，使得产生了对于确定具有两个或更多个高于13的肽评分的蛋白的好于99.9％的确定性。
抗体产生
按月以对应于H-Cys-Leu-Asp-Lys-Glu-Asn-Ala-Leu-Asp-Arg-Ala-Glu-Gln-Ala-Glu-Ala-Asp-Lys-Lys-Ala-Ala-NH2和H-Cys-Glu-Lys-Val-Ala-His-Ala-Lys-Glu-Glu-Asn-Leu-Ser-Met-His-Gln-Met-Leu-Asp-Gln-Thr-Leu-Leu-Glu-Leu-Asn-Asn-Met-OH的肽序列免疫绵羊，该肽序列通过N端半胱氨酸残基与牛血清白蛋白(BSA)缀合。按月将产生的免疫原给予成年绵羊，以产生多克隆响应。然后收获淋巴细胞并与异源骨髓瘤细胞融合。采用基于ELISA的检测，从产生的杂交瘤的上清液筛选外显子特异性抗体的存在，其中微量滴定板包被有全长蛋白。将阳性杂交瘤克隆至稳定。纯化通过所产生的单克隆杂交瘤生成的抗体，通过1维和2维蛋白质印迹(1D和2D WB)进行表征，并用于建立生物芯片三明治免疫检测。将检测施加于Evidence Investigator分析仪，其利用了基于ELISA免疫检测原理的生物芯片阵列技术(Fitzgerald et al 2005)。
2维蛋白质印迹分析
对于2D蛋白质印迹分析，通过24cm、pH 4-7的IPG胶带(GEHealthcare)上的IEF，在第一维中分离30μg的TCA沉淀的和尿素/硫脲/CHAPS再溶解的血小板蛋白。通过SDS PAGE在13x 16cm凝胶上进行第二维，并将分离的蛋白转移至硝酸纤维素膜(Pall,East Hills,NY)。使用基于钌-(II)-三-(红菲绕啉二磺酸盐)(RuBPS)的荧光染料对膜上的总蛋白进行染色。其后，使用于含有0.3％Tween-20的PBS(PBS-T)中的5％脱脂干燥奶粉将膜封闭2小时。通过孵育膜检测AD相关的原肌球蛋白同种型：a)对于抗1a外显子检测，HRP标记的抗1a外显子抗体(1/1000)，孵育1h，使用Luminol PLUS/过氧化物检测，暴露10sec；b)对于抗9d外显子检测，HRP标记的抗9d外显子抗体(1/1000)，孵育1h，使用LuminolPLUS/过氧化物检测，暴露30sec-1min。
结果
选择外显子9d特异性的捕获抗体固定于生物芯片表面上，并将外显子1a特异性的检测抗体与辣根过氧化物酶缀合以产生检测示踪物。采用这些免疫试剂开发的生物芯片免疫检测展现对4种AD和MCI相关的P09493原肌球蛋白同种型中的3种的特异性。1D和2D蛋白质印迹确定，产生的针对外显子1a和9d的抗体结合至斑点2、3和4(图1)。还发现AD和MCI样品中每种上调的原肌球蛋白同种型都包含外显子1a。通过质谱指定的4种其他的原肌球蛋白同种型对应于斑点5、6、7和8(图1)，其均不包含外显子1a，且在AD和MCI样品中均未上调。检测灵敏度为<10ng/ml(测量范围0-700ng/ml)，且对于标准水平浓度，批内精确度为<10％。在所用的实验条件下，斑点1具有pI＝4.5，且MW＝～37kDa，斑点2具有pI＝4.5，且MW＝～37kDa，斑点3具有pI＝4.6，且MW＝～35kDa，并且斑点4具有pI＝4.56，且MW＝～33kDa。在分离的血液血小板中，检测到了多达12μg/ml的水平。表1(％变化行)证实，相比对照，对应于斑点1、2、3和4的P09493原肌球蛋白同种型，单独地或组合地，在AD和MCI患者中均上调。t-检验强调了相比对照，斑点2和所有4个斑点的组合在AD和MCI患者中均显著上调(P<0.05)。该研究的结果表明，疑似患有AD或MCI的患者的体外样品中上调的P09493-3和P09493-1原肌球蛋白同种型的检测和测量预示AD和MCI。
参考文献
Karas et al(2008).Am.J.Neuroradiol,29(5):944-949
Schevzov G.et al(2005).J.Histochem.Cytochem.,53(5):557-570
Fitzgerald et al.(2005).Clin.Chem.,51(7):1165-1176
Petersen et al(1999).Arch.Neurol,56(3):303-308
Petersen et al(2004).J.Intern.Med.256(3):183-194.
Claims (7)
1.有助于阿尔茨海默氏病(AD)或轻度认知障碍(MCI)的诊断的体外方法，包括测定患者样品中对应于P09493-3和/或P09493-1的一种或多种原肌球蛋白同种型的表达水平。
2.如权利要求1所述的方法，其中所述原肌球蛋白同种型为P09493-3。
3.如权利要求1或2所述的方法，其中相比对照值，一种或多种原肌球蛋白同种型的表达水平的增加指示AD或MCI。
4.如前述权利要求中任一项所述的方法，其中所述患者样品为血液、血清、血浆或血小板样品。
5.试剂盒，其包含结合原肌球蛋白同种型P09493-3和P09493-1的探针，特征在于结合P09493-3和P09493-1的不包含外显子1a的共同表位的第一探针，和特异性结合外显子1a的第二探针。
6.如权利要求5所述的试剂盒，其中所述探针为抗体。
7.改变一种或多种原肌球蛋白同种型P09493-3和P09493-1的浓度水平的试剂，其被用于治疗AD或MCI。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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| GBGB1212084.6A GB201212084D0 (en) | 2012-07-06 | 2012-07-06 | Tropomyosin isoforms related to alzheimers disease and mild cognitive impairment |
| GB1212084.6 | 2012-07-06 | ||
| PCT/EP2013/064397 WO2014006224A1 (en) | 2012-07-06 | 2013-07-08 | Tropomyosin isoforms related to alzheimers disease and mild cognitive impairment |
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| CN104487847A true CN104487847A (zh) | 2015-04-01 |
| CN104487847B CN104487847B (zh) | 2016-08-17 |
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| US (1) | US9927429B2 (zh) |
| EP (2) | EP2870481B1 (zh) |
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| CN (1) | CN104487847B (zh) |
| AU (1) | AU2013285362B2 (zh) |
| BR (1) | BR112014033124A2 (zh) |
| CA (1) | CA2878418A1 (zh) |
| GB (1) | GB201212084D0 (zh) |
| RU (1) | RU2661022C2 (zh) |
| WO (1) | WO2014006224A1 (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111323597A (zh) * | 2018-12-14 | 2020-06-23 | 陈志成 | 用于检测受试者中mci和/或ad的方法、试剂盒及筛选化合物的方法 |
| CN112858697A (zh) * | 2021-03-29 | 2021-05-28 | 鲁东大学 | ALG-2-interacting protein X在制备分子标志物中的应用 |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6927212B2 (ja) * | 2016-07-08 | 2021-08-25 | 味の素株式会社 | 軽度認知障害又はアルツハイマー型認知症の評価方法 |
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| JP5695075B2 (ja) * | 2010-10-20 | 2015-04-01 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | アルツハイマー病又は軽度認知症のバイオマーカー |
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2012
- 2012-07-06 GB GBGB1212084.6A patent/GB201212084D0/en not_active Ceased
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2013
- 2013-07-08 RU RU2014152876A patent/RU2661022C2/ru not_active IP Right Cessation
- 2013-07-08 CA CA2878418A patent/CA2878418A1/en not_active Abandoned
- 2013-07-08 JP JP2015519246A patent/JP6262727B2/ja not_active Expired - Fee Related
- 2013-07-08 US US14/411,496 patent/US9927429B2/en active Active
- 2013-07-08 CN CN201380032698.9A patent/CN104487847B/zh not_active Expired - Fee Related
- 2013-07-08 WO PCT/EP2013/064397 patent/WO2014006224A1/en active Application Filing
- 2013-07-08 BR BR112014033124A patent/BR112014033124A2/pt not_active Application Discontinuation
- 2013-07-08 EP EP13734774.6A patent/EP2870481B1/en active Active
- 2013-07-08 EP EP17189117.9A patent/EP3290923B1/en active Active
- 2013-07-08 AU AU2013285362A patent/AU2013285362B2/en not_active Ceased
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| CN111323597A (zh) * | 2018-12-14 | 2020-06-23 | 陈志成 | 用于检测受试者中mci和/或ad的方法、试剂盒及筛选化合物的方法 |
| CN112858697A (zh) * | 2021-03-29 | 2021-05-28 | 鲁东大学 | ALG-2-interacting protein X在制备分子标志物中的应用 |
| CN112858697B (zh) * | 2021-03-29 | 2024-03-01 | 鲁东大学 | ALG-2-interacting protein X在制备分子标志物中的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2013285362A1 (en) | 2015-01-22 |
| AU2013285362B2 (en) | 2016-12-01 |
| US20150198590A1 (en) | 2015-07-16 |
| BR112014033124A2 (pt) | 2017-06-27 |
| EP2870481B1 (en) | 2017-09-06 |
| CA2878418A1 (en) | 2014-01-09 |
| EP3290923A1 (en) | 2018-03-07 |
| JP2015524551A (ja) | 2015-08-24 |
| US9927429B2 (en) | 2018-03-27 |
| GB201212084D0 (en) | 2012-08-22 |
| CN104487847B (zh) | 2016-08-17 |
| RU2661022C2 (ru) | 2018-07-11 |
| EP2870481A1 (en) | 2015-05-13 |
| JP6262727B2 (ja) | 2018-01-17 |
| WO2014006224A1 (en) | 2014-01-09 |
| EP3290923B1 (en) | 2021-04-28 |
| RU2014152876A (ru) | 2016-08-27 |
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