CN104507965A - 作为血清和组织生化标志物的bag3 - Google Patents
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- CN104507965A CN104507965A CN201380032138.3A CN201380032138A CN104507965A CN 104507965 A CN104507965 A CN 104507965A CN 201380032138 A CN201380032138 A CN 201380032138A CN 104507965 A CN104507965 A CN 104507965A
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Abstract
本公开内容涉及诊断生物学标志物领域。具体地,本公开内容涉及用作诊断病理状态的生物学标志物的抗BAG3抗体。此外,本公开内容包括用于在生物样品中检测和评估抗BAG3抗体或BAG3/抗体复合物的特异性ELISA方法和试剂盒。
Description
说明
本发明涉及用作病理状态的诊断中的生化标志物的抗BAG3抗体。
现有技术
BAG3(RefSeq:NP_004272;基因ID 9531)为74kDa的细胞质蛋白,特别是集中在粗糙型内质网中。BAG3蛋白属于通过称为BAG域的结构域(110-124个氨基酸)与热激蛋白HSP70的ATPase域相互作用的共分子伴侣(co-chaperones)家族。除了BAG域,BAG3包括WW域以及可介导与其它蛋白结合的富含脯氨酸的重复(PXXP)。此外,两个保守的IPV(Ile-Pro-Val)基序位于WW和PXXP区域之间,并介导BAG3结合到分子伴侣HspB家族的成员HspB8。因此,由于它的多结构域结构的衔接性质,BAG3可与不同的伴侣蛋白相互作用。bag3基因表达在包括肌细胞的少数正常的细胞类型中和数个原发瘤或肿瘤细胞系中是组成性的。此外,它可被各种应激源诱导:事实上应激的刺激物激活热激转录因子(HSF)1,HSF1负责应激激活的基因包括bag3的表达(Rosati A,Graziano V,DeLaurenzi V,Pascale M,Turco MC.BAG3:a multifaceted protein that regulatesmajor cell pathways.Cell Death Dis.2011;2:e141)。证据表明,BAG3通过根据细胞的环境以依赖或不依赖Hsp70的方式调节细胞凋亡调节的蛋白质例如IKKγ、Bax或BRAF的水平或定位,具有维持细胞存活的功能。
BAG3蛋白质表现为在心肌成肌细胞分化期间表达,并且维持成肌素的表达。这些发现表明BAG3参与心脏的后期发育(De Marco M,TurcoMC,Rosati A,BAG3 protein is induced during cardiomyoblast differentiationand modulates myogenin expression.Cell Cycle.2011;10:850-852)。此外,在心肌细胞中,BAG3已经显示位于Z-盘,并且与肌动蛋白加帽蛋白CapZβ1相互作用,稳定肌原纤维结构,并且在机械应激过程中可能保持肌纤维的完整性。BAG3突变可损害Z-盘的组装,并提高对应激诱导的细胞凋亡的敏感性。与BAG3在心肌细胞并且通常在肌细胞中的存活和肌纤维的完整性中的作用一致,bag3基因的突变已经与一些形式的肌纤维肌病和扩张型心肌病相关联。
到目前为止,已经检测到了细胞质BAG3和可溶的串行形式的BAG3二者,并且发现它们与不同的病理,以及更广泛地与细胞存活相关联。
对允许快速鉴定这样的病理,而不具有以意外的特殊方式以及敏感的方式与创伤性诊断相关联的缺点,和/或可允许早期检测病理,监测治疗的效果,预测并发症的风险,进行信息追踪的生物学标志物的鉴定日益感到需求及重要性。
发明概述
本发明涉及用作病理状态的诊断中的生化标志物的抗BAG-3抗体。
优选地,所述抗BAG-3抗体结合到可溶的BAG3以形成免疫复合物。
根据本发明优选的实施方式,所述诊断是体外的(in vitro)或离体的(ex vivo)。
本发明的另一方面为,所述诊断的接受者为哺乳动物,优选人类。
如在本发明的详细说明中进一步描述的,本发明的抗BAG-3抗体的用途具有对选自由心脏病,癌症,糖尿病,皮肤、神经、骨、血管和结缔组织的炎症和炎症相关的疾病组成的组的病理状态特异性的优点。
根据本发明的实施方式,所述心脏病选自:心绞痛、梗塞前心绞痛、心肌梗塞、心力衰竭、局部缺血(ischemia)、急性冠心病(acute coronarydisease)、急性心力衰竭、慢性心力衰竭和医源性心脏病。
根据本发明的另一个实施方式,所述癌症选自:胰腺癌、膀胱癌和前列腺癌。
本发明的另一个实施方式为检测生物样品中抗BAG3抗体或结合到可溶的BAG3以形成免疫复合物的抗BAG3抗体的存在的方法,所述方法包括以下步骤:
a.获得生物样品,所述生物样品由血清、血浆、尿或唾液组成,
b.确定所述生物样品中抗BAG3抗体或BAG3缔合的抗体(BAG3associated antibodies)的存在。
根据优选的实施方式,本发明的方法还包括以下另外的步骤:
c.比较从所述生物样品获得的值与参考值或从健康供体获得的值。
在优选的实施方式中,所述确定步骤b通过ELISA试验进行。
根据本发明优选的实施方式,所述血清、血浆、尿或唾液来自哺乳动物,优选人类。
根据本发明的方法的优选的实施方式,所述抗BAG3抗体或所述免疫复合物的存在与病理状态相关。
优选地,所述病理状态选自由心脏病,癌症,糖尿病,皮肤、神经、骨、血管和结缔组织的炎症和炎症相关的疾病组成的组。
优选地,所述心脏病选自:心绞痛、梗塞前心绞痛、心肌梗塞、心力衰竭、局部缺血、急性冠心病、急性心力衰竭、慢性心力衰竭和医源性心脏病。
根据本发明优选的实施方式,所述癌症选自:胰腺癌、膀胱癌和前列腺癌。
本发明的另一个实施方式为ELISA试剂盒以及其用于检测生物样品中抗BAG3抗体或结合到可溶的BAG3以形成免疫复合物的抗BAG3抗体的用途,所述ELISA试剂盒包括用于捕获可溶的BAG3的BAG3重组蛋白或BAG3特异的小鼠单克隆抗体AC-1、AC-2、AC-3、AC-4、AC-5、AC-rb1a、AC-rb1b、AC-rb2a、AC-rb2b、AC-rb3a、AC-rb3b、AC-rb4a和AC-rb4b,以及能够识别人免疫球蛋白的抗体。
优选地,所述生物样品为血清、血浆、尿或唾液样品。
在本发明优选的实施方式中,所述血清、血浆、尿或唾液样品来自哺乳动物,优选人类。
本发明还进一步涉及用于检测生物样品中的BAG3蛋白的免疫组织化学(IHC)试剂盒,其中,所述生物样品优选为组织样品,所述试剂盒包括BAG3-特异的抗体和试剂,所述试剂包括染色需要的探针。
附图简述
本发明的特征和优点将从下面记录的详述、为了说明而非限制目的给出的实施例以及附图1-10中明显,其中:
图1.
图1A:在来自培养的心肌细胞的上清液中检测BAG3蛋白质。在5%的CO2气氛下于37℃使用或不使用10%的FBS培养80%融合的人(HCMa)和大鼠(H9c2)心肌细胞16个小时。在包含50mM NaCl和0.05%IGEPAL的缓冲液中透析所述上清液,冻干,并在1ml的RIPA缓冲液(50mM TrisHCl pH 7.6、150mM氯化钠、2mM原钒酸钠、4mM EDTA、10mM焦磷酸钠、1%NP-40、0.1%脱氧胆酸钠)中重悬,通过蛋白质印迹用抗BAG3抗体或抗GAPDH抗体分析。
图1B:在胞吐囊泡中检测BAG3蛋白质。从H9c2细胞获得的上清液(Surnatants)经连续的离心:(i)2’000×g 15分钟,以去除细胞;(ii)10’000×g30分钟,以去除细胞碎片;(iii)150’000×g 90分钟,以使胞吐囊泡成团块。在PBS中以150’000×g洗团块(pellet)90分钟一次,并通过蛋白质印迹用抗BAG3 TOS-2多克隆抗体分析,与全细胞裂解物比较。Rab-4作为胞吐囊泡的标志物被分析。细胞溶质蛋白GAPDH作为对照被分析。
图1C:在蛋白质印迹中用抗BAG3抗体TOS-2多克隆抗体分析来自两个健康供体和两个受慢性心力衰竭影响的患者的血清。
图1D:从凝胶切除在两个受慢性心力衰竭影响的患者中获得的条带,并使用程序MASCOT通过质谱分析它的身份。
图2.
图2A:在来自CHF患者的血清中检测BAG3蛋白质。通过蛋白质印迹用从心力衰竭患者获得的血清(1:40)分析BAG3重组蛋白和来自HCMa细胞的全细胞裂解物。进行来自健康供体的血清的分析作为阴性对照。
图2B:通过ELISA试验检测抗BAG3抗体。在特异性ELISA试验中比较来自50个CHF患者(具有<60%的射血分数)的血清与来自50个健康供体的血清中抗BAG3抗体的存在。结果以任意单位绘制。
图2C:ELISA试验结果的ROC分析。于0.083 A.U.的截断值导致74%的灵敏度和68%的特异性。
图3.
图3A:为了检测rBAG3-FITC结合到HCMa细胞(a、b、c)和J774A1细胞(d、e、f、g、h、i)而进行直接荧光的共聚焦显微分析。根据生厂商说明书使用购自SIGMA的FluoroTag FITC Conjugation Kit将BAG3重组蛋白和纯化的BSA(来自购自SIGMA的牛血清的白蛋白)共轭到FITC。根据生产商说明书计算的等量的rBAG3-FITC(b、e)和BSA-FITC(h)蛋白加到具有0.1%NaN3的HCM和J774 A1的培养介质中1个小时。β-整合素作为对照(a、d、g)被分析。通过Zeiss LSM共聚焦显微镜分析细胞。合并的图像在c、f和i中显示。
图3B:BAG3结合巨噬细胞。用不同浓度的Fitc-BAG3蛋白质(7、14和70nm)培养J774 A1巨噬细胞(1×106细胞/ml)。FITC-BSA(70nM)用作阴性对照(灰色)。通过流式细胞术分析细胞荧光。
图3C图a—分析用BAG3培养的J774 A1巨噬细胞中的Cox-2和iNOS水平。用对照培养基、BSA、LPS或rBAG3培养80%融合的J774 A1细胞20个小时。在所示处加入多粘菌素以证实,由大肠杆菌得到的rBAG3的效果独立于污染性内毒素的存在。通过蛋白质印迹分析细胞裂解物中的Cox-2和iNOS表达。
图3C图b—分析从与BAG3培养的J774 A1巨噬细胞释放的亚硝酸盐。用对照培养基、BSA、LPS或rBAG3培养80%融合的J774 A1细胞24个小时。用100μl的格里斯试剂培养来自每个样品的100μl上清液;用Beckman DU62分光光度计测量在550nm的光密度(OD550)。通过比较样品的OD550与亚硝酸钠的标准曲线来评估亚硝酸盐的浓度。
图3C图c—分析从与BAG3培养的J774 A1巨噬细胞释放的IL-6。用对照培养基、BSA、LPS或重组BAG3培养80%融合的J774 A1细胞5个小时。在所示处加入BAG3肽(肽1、肽2、肽3、肽4或乱序肽)625nM,以证实它们封闭BAG3活性的能力。使用ELISA试验测量细胞培养介质中的IL-6产生。通过比较样品的OD与重组IL-6的标准曲线而评价IL-6的浓度。
图4.
图4A:
在正常胰腺组织中使用单克隆抗BAG3抗体AC-1染色BAG3的代表图像。用苏木精复染色切片。染色揭示胰岛的中等阳性,而正常的胰管和胰腺腺泡细胞不具有BAG3的表达。
图4B:
使用以生物素化的二抗揭示的单克隆抗BAG3抗体染色的BAG3低阳性和BAG3高阳性肿瘤样品的代表图像。切片用苏木精复染色。以两个不同的放大率显示:100×(左图)和400×(右图)。我们通过使用400×的放大率在10个不重叠的视野的总癌细胞中计数阳性细胞,基于样品中阳性癌细胞的比例分配评分。如所述地计算的BAG3阳性细胞的中值百分比为40%,并且该值用作截断值使低阳性的样品和高阳性的样品分开。
图4C:做出了比较具有低的BAG3染色(≤40%的阳性细胞)的39个患者与27个具有高的BAG3染色(>40%的阳性细胞)的患者的存活曲线。所有分析的患者经历胰腺癌的R0切除。中位存活期从高阳性组中的12个月提高到低阳性组中的23个月。对数秩检验p值=0.0013。
图5.
来自数个类风湿性关节炎的滑膜组织中BAG3染色的代表图像。在滑膜成纤维细胞和炎性浸润物中观察到了BAG3阳性。用苏木精复染色切片。
图6.
BAG3染色在正常膀胱(normal urocystis)中产生阴性和在膀胱移行细胞癌中产生肿瘤细胞的细胞质中BAG3高阳性的代表图像。用苏木精复染色切片。
图7.
图7A:
在图中显示通过qRT-PCR评价的bag3 mRNA相对表达,其中,值报告为平均值+S.D。蓝色的线表示计算的中值。
图7B:
对qRT-PCR分析的所有患者做出存活分析。13个具有高的bag3表达(中位存活=19.0个月)的患者与12个具有低的bag3表达(中位存活=32.0个月)相比较具有较短的存活期。对数秩检验p值=0.0198。
图8.
图8A:
如图中表示,用不同浓度的吉西他滨处理胰腺癌细胞系(PSN1、Capan-1、AsPC-1、PANC-1和MIA PaCa-2)。在48个小时后,分析凋亡细胞的死亡。图描述了亚G0/G1细胞的平均百分比(±S.D.)。数据是3个独立的实验的代表。
图8B:
胰腺癌细胞系中BAG3的蛋白质印迹分析;GAPDH看家蛋白含量用于监测相等的装载条件。
图8C:
用2μM的吉西他滨(GEM)处理MIA PaCa-2和PANC-1细胞系显示的时间,通过蛋白质印迹监测BAG3蛋白表达水平。
图8D:
通过RT-PCR分析bag3 mRNA水平;图描述了相对的bag3 mRNA水平(±S.D.),并且数据是三个独立的实验的代表。
图8E:
用BAG3 siRNA或非靶向siRNA(NTsiRNA)转染MIA PaCa-2和PANC-1细胞系72个小时,然后用2μM吉西他滨(GEM)处理24个小时。通过蛋白质印迹分析BAG3水平,并检测GAPDH的水平,以监测相等的装载条件。
图8F:
如上述转染MIA PaCa-2和PANC-1细胞,并用2μM吉西他滨(GEM)处理24小时或48小时。如所述地分析凋亡性细胞死亡。图描述亚G0/G1细胞的平均百分比(±S.D.)。数据是3个独立的实验的代表。
图9.
图9A:通过ELISA试验检测BAG3特异的免疫复合物。来自55个胰腺癌患者的血清与来自51个健康供体的血清在特异性ELISA试验中比较BAG3特异的免疫复合物的存在。结果以任意单位+S.E.绘制。
图9B:ELISA试验结果的ROC分析。于0.183A.U.的截断值导致65%的灵敏度和78%的特异性。
图10.
在特异性ELISA试验中检测来自49个女性受试者在诊断为胰腺癌前0-1年的患者血清中的BAG3特异的抗体,与来自235个女性对照受试者的血清比较BAG3特异的抗体的存在。对照血清对于每个病例受试者为约5,并与各个病例年龄匹配。其它时间点包括:与251个对照相比诊断为PDAC前1-2年的53个受试者;与212个对照相比诊断为PDAC前2-3年的44个受试者;与187个对照相比诊断为PDAC前3-4年的42个受试者。
结果以任意单位±S.绘制。用student t检验计算P值。
发明详述
本发明涉及用作病理状态的诊断中的生化标志物的抗BAG-3抗体。
本发明中的术语“诊断”指医疗诊断(通常简称为诊断),指尝试确定或鉴别可能的疾病或紊乱的过程。本发明的诊断还包括早期诊断。
术语“早期诊断”指试验在特异或非特异的症状前辨别病理状态的能力。
现在在血清、血浆、尿或唾液中有利地检测到抗BAG3抗体。直到现在,还未在生理或病理状态的血清中发现这样的抗体。在血清中检测抗BAG3抗体作为鉴定和监测治疗的工具具有诊断、早期诊断和预后目的、风险分层使用的快速和无创技术的优点。
抗体的检测的另一个优点为需要非常少量的血清用于检测。实际上,可溶的BAG3蛋白还可在患一些病理的患者的血清中检测到,但是用于检测可溶蛋白所需要的血清量远高于检测抗体需要的量。此外,以下是可能的:可溶的BAG3蛋白的水平可以比抗体低很多,和/或相对于可溶的BAG3蛋白,抗体可以是在特定病理的早期阶段可检测的和/或可更有效地预测并发症的危险或监测治疗的效果。
本发明另一个实施方式为可在选自血清、血浆、尿或唾液的不同生物样品中有利地检测所述抗BAG3抗体。
在本发明中,血清意为既非血细胞也非凝血因子的血液的成分;它是去除纤维蛋白原的血浆。血清包括未用在凝血(凝固)中的所有蛋白质和所有电解质、抗体、抗原、激素和任何的外源物质。
在本发明中,血浆意为通常在悬浮液的全血中保留血细胞的血液的淡黄色/浅黄色液体组分。它包含凝血因子,例如纤维蛋白原。
在另外的实施方式中,本发明提供了抗BAG3抗体作为生化标志物的用途,其中,所述抗BAG3抗体结合到可溶的BAG3以形成免疫复合物。
现在已经在生物样品中有利地检测到游离的或结合到可溶的BAG3以形成免疫复合物的抗BAG3抗体,并可在病理状态的诊断中用作标志物。在生物样品中检测这样的抗体和/或免疫复合物还具有用于诊断和/或预后目的的快速和无创技术的优点。
根据本发明的优选的实施方式,所述诊断是体外的或离体的。
本发明的进一步的实施方式为所述诊断的接受者为哺乳动物,优选人类。
在本发明中,免疫复合物或蛋白/抗体复合物意为抗体对可溶抗原的整体结合,称为单一免疫复合物,结合的抗原作用为结合到抗体的特定的表位。
由于还需要非常少量的血清用于检测免疫复合物,这样的免疫复合物具有看作抗体的检测的相同的优点。检测可溶的BAG3需要的血清的量还比检测蛋白/抗体(免疫)复合物需要的高很多。可在特异病理的早期阶段检测免疫复合物及游离的抗体,并且可更有效地预测并发症的危险或监测治疗的效果。
本发明的再一个实施方式为抗BAG3抗体或所述免疫复合物(由抗BAG3抗体结合到可溶的BAG3形成)用作病理状态的生物学标志物的用途,其中,所述病理状态为心脏病,癌症,糖尿病,皮肤、神经、骨、血管和结缔组织的炎症和炎症相关的疾病。
优选地,所述心脏病选自以下组成的组:心绞痛、梗塞前心绞痛、局部缺血、心肌梗塞、心力衰竭、急性冠心病、急性心力衰竭、慢性心力衰竭和医源性心脏病。
根据本发明的优选的实施方式,所述癌症选自:胰腺癌、膀胱癌和前列腺癌。
本发明的另外的实施方式为检测生物样品中抗BAG3抗体或结合到可溶的BAG3以形成免疫复合物的抗BAG3抗体的存在的方法,所述方法包括以下步骤:
a.获得生物样品,所述生物样品由血清、血浆、尿或唾液组成,
b.确定所述生物样品中抗BAG3抗体或BAG3缔合的抗体的存在。
根据本发明的方法具有允许检测健康个体和被涉及BAG3的病理影响的患者之间的抗BAG3抗体和/或BAG3/抗体复合物之间的显著差别的优点。提出的检测方法允许心脏病患者的组与健康人群的组在统计上显著性地分离。还可在患者的亚组中以增加的危险(心力衰竭,HF)分层这样的心脏病患者。
本发明的再一个方面涉及在生物样品中检测抗BAG3抗体的存在或结合到可溶的BAG3以形成免疫复合物的抗BAG3抗体的方法,所述方法还包括以下步骤:
c.比较从所述生物样品获得的值与参考值或从健康供体获得的值。
在优选的方面,根据本发明的方法为其中所述确定步骤b通过ELISA试验进行的方法。
根据本发明的方法中的优选的实施方式,所述血清、血浆、尿或唾液来自哺乳动物,优选人类。
根据本发明的方法具有允许病理的、疾病和/或它们的并发症的危险评估、以及监测治疗的生物学标志物的快速和无创检测的优点。
根据另一个方面,本发明涉及检测方法,其中,所述抗BAG3抗体或结合到可溶的BAG3以形成免疫复合物的所述抗BAG3抗体的存在与病理状态相关。
在优选的实施方式中,所述病理状态选自由心脏病,癌症,糖尿病,皮肤、神经、骨、血管和结缔组织的炎症和炎症相关的疾病组成的组。具体地,所述心脏病选自由以下组成的组:心绞痛、梗塞前心绞痛、心肌梗塞、局部缺血、心力衰竭、急性冠心病、急性心力衰竭、慢性心力衰竭或医源性心脏病。
根据本发明优选的实施方式,所述癌症选自:胰腺癌、膀胱癌和前列腺癌。
本发明还涉及用于检测生物样品中抗BAG3抗体或结合到可溶的BAG3以形成免疫复合物的抗BAG3抗体的ELISA试剂盒。
根据本发明的ELISA试剂盒包括用于捕获抗BAG3抗体或BAG3特异的小鼠单克隆抗体AC-1、AC-2、AC-3、AC-4、AC-5、AC-rb1a、AC-rb1b、AC-rb2a、AC-rb2b、AC-rb3a、AC-rb3b、AC-rb4a和AC-rb4b、用于捕获可溶的BAG3的BAG3重组蛋白,以及能够识别人免疫球蛋白的抗体。
这样的抗体可为能够识别人免疫球蛋白的酶连接的抗体。
本发明还涉及用于在生物样品中检测BAG3相关的抗体的试剂盒,并且以用于捕获可溶的BAG3的BAG3特异的小鼠单克隆抗体AC-1、AC-2、AC-3、AC-4、AC-5、AC-rb1a、AC-rb1b、AC-rb2a、AC-rb2b、AC-rb3a、AC-rb3b、AC-rb4和AC-rb4b以及能够识别人免疫球蛋白的用于检测的酶连接的抗体通过ELISA进行。
优选地,所述生物样品为血清、血浆、尿或唾液样品。
在本发明优选的实施方式中,所述血清、血浆、尿或唾液样品来自哺乳动物,优选人类。
本发明还涉及用于检测生物样品中的BAG3蛋白的免疫组织化学(IHC)试剂盒,其中,所述生物样品优选为组织样品。这些样品可为活体组织切片、冷冻组织、石蜡包埋组织。
根据本发明的IHC试剂盒包括BAG3特异的抗体和试剂,所述试剂包括染色需要的探针。
所述BAG3特异的抗体可为小鼠单克隆抗体AC-1、AC-2和AC-3和/或能够识别小鼠免疫球蛋白的用于检测的酶连接的抗体。
尤其地,所述IHC试剂盒有利地允许在来自经受胰腺切除的患者的100%的胰腺癌组织样品中揭示BAG3蛋白,并在大部分膀胱癌样品中表达。此外,还可通过IHC在胰岛中的正常胰腺组织中用检测BAG3的试剂盒揭示BAG3蛋白,而其它正常组织结果为阴性(例如正常的膀胱)。还可在类风湿性关节炎的组织样品的滑膜成纤维细胞和炎性浸润中观察到BAG3阳性。
受PDAC影响的患者的长期生存非常差:仅约4%的患者在诊断后活5年。实际上,接受手术(surgical reception)目前是治愈的唯一选择,但是,仅约20%的患者诊断为可切除的疾病;此外,这样的患者的子集的大比例(约80%)中,在诊断时已经出现转移过程(metastatization process),并且实际的远端转移在手术切除后出现。因此,我们需要更好地理解胰腺癌发展的早期阶段,并且鉴定可允许检测它们的分子。同样,非常需要允许更好诊断和帮助治疗的选择的标志物。
有益地,BAG3 IHC试剂盒允许鉴定PDAC患者的预后。观察到,由IHC鉴定的BAG3表达的强度与患者的存活率相关。因此,它可用于预后及用于做出治疗的选择。
本发明的另一方面为用于在生物样品中检测BAG3基因表达的试剂盒,包括一组扩增引物。优选地,所述扩增引物适于通过实时定量RT-PCR检测。
下面描述并通过SEQ ID NO.1至SEQ ID NO.10识别的根据引物组1至5的特异性引物bag3引物允许通过实时定量PCR检测和定量BAG3的表达。
引物组1
正向:SEQ ID NO.1:AACGGTGACCGCGACCCTTT;
反向:SEQ ID NO.2:CCTTCCCTAGCAGGCGGCAG
引物组2
正向:SEQ ID NO.3:CCGGCTGGCCCTTCTTCGTG;
反向:SEQ ID NO.4:CAGCCTAGAGCCCTCCCGGG
引物组3
正向:SEQ ID NO.5:GTCACCTCTGCGGGGCATGC;
反向:SEQ ID NO.6:GGTGACTGCCCAGGCTGCTC
引物组4
正向:SEQ ID NO.7:CCAGCCTCCCACGGACCTGA;
反向:SEQ ID NO.8:CTGGTGACTGCCCAGGCTGC
引物组5
正向:SEQ ID NO.9:CAGGAGCAGCACGCCACTCC;
反向:SEQ ID NO.10:TGGTCCAACTGGGCCTGGCT。
用于在生物样品中检测bag3 mRNA的RT-PCR试剂盒允许使bag3基因表达水平与患者的存活关联,并可用于预后,以及用于治疗的选择。优选地,所述生物样品为组织样品。
本发明的再一个方面由能够阻断巨噬细胞活化,并且因此可用于炎症、涉及巨噬细胞活化的肿瘤或其它疾病的治疗的抗BAG3单克隆抗体、它们的片段、以及相应于BAG3蛋白质的特定氨基酸序列的肽代表。具体地,参见图3和表I。
本发明涉及BAG3特异的小鼠单克隆抗体AC-1、AC-2和AC-3或上述被修饰为F(ab)、F(ab’)2、F(ab)或人源化;或者包括如下序列的肽,作为能够结合和/或封闭可溶的BAG3效果的用途:
PEP1:DRDPLPPGWEIKIDPQ(SEQ ID NO.11)
PEP2:SSPKSVATEERAAPS(SEQ ID NO.12)
PEP3:DKGKKNAGNAEDPHT(SEQ ID NO.13)
PEP4:NPSSMTDTPGNPAAP(SEQ ID NO.14)。
实施例
实施例1.
在培养的人原代心肌细胞和大鼠心肌细胞系H9c2中剥夺血清引起的
应激
已知心肌细胞在提高针对胁迫物的响应中释放保护因子。由于应激诱导的蛋白,例如Hsp70、Hp27、Hsp90以及其它蛋白虽然发挥了细胞内活性,但是还可响应于应激而被分泌,在应激条件下分析了由心肌细胞释放的BAG3。为此目的,我们分析了在培养的人原代心肌细胞和大鼠心肌细胞系H9c2中剥夺血清引起的应激的影响。如图1中显示,我们可在暴露于血清剥夺16个小时的心肌细胞的上清液中检测到BAG3蛋白质(图1A)。由于细胞存活在那个时间点不受剥夺血清的影响(结果未显示),我们抛弃了BAG3由于细胞坏死而释放的假设。因此,我们证实了BAG3是否存在于胞吐囊泡中。实际上,通过由差速离心过程分离细胞外囊泡(16),我们发现它们包含BAG3蛋白质(图1B)。
为进一步证实可溶形式的BAG3的存在,我们研究了它在受慢性心力衰竭(CHF)影响的患者的两个血清中的存在。通过蛋白印迹分析,我们可鉴定由抗BAG3抗体识别的条带。我们切除所述条带,并使它经质谱,证实它的身份(图1C)。该证据证实,可检测到胞外形式的蛋白质。我们不能在来自健康供体的血清中检测到所述蛋白(图1C)。在整个BAG3蛋白序列上由质谱识别和匹配的肽由粗体表示:
MSAATHSPMM QVASGNGDRD PLPPGWEIkI DPQTGWPFFV
DHNSRTTTWN DPRVPSEGPK ETPSSANGPS REGSRLPPAR
FRTEAAAAA PQRSQSPLRQ CGQVAAAAAA
QPPASHGPER SQSPAASDCS SSSSSASLPS SGRSSLGSHQ
LPRGYISIPV IHEQNVTRPA AQPSFHQAQK
I QGDDWEPRPL RAASPFRSSV QGASSREGSP
ARSSTPLHSP SPIRVHTVVD RPQQPMTHRE TAPVSQPENK
PESKPGPVGP ELPPGHIPIQ VIRKEVDSKP VSQKPPPPSE
KVEVKVPPAP VPCPPPSPGP SAVPSSPKSV ATEERAAPST
APAEATPPKP GEAEAPPKHP GVLKVEAILE KVQGLEQAVD
NFEGKKTDKK YLMIEEYLTK ELLALDSVDP EGRADVRQAR
RDGVRKVQTI LEK
K
(SEQ ID NO:15).
人BAG 3蛋白质具有根据SEQ ID NO:15的氨基酸序列。
实施例2.
CHF患者血清中的抗BAG3抗体
我们发现,使用抗人IgG作为二抗,来自CHF患者的血清在蛋白印迹中识别BAG3蛋白(图2A中显示了表示来自3个不同患者的血清的实验的结果)。该结果表明抗BAG3抗体在CHF患者血清中的存在。为了证实该发现,我们在特定的ELISA实验中分析了来自50个CHF患者的血清(具有<60%的射血分数)与来自50个健康供体的血清相比,其中抗BAG3抗体的存在。如图2B中显示,我们检测到患者的血清中抗BAG3抗体的值显著地高于对照血清。ELISA试验结果的ROC分析,使用截断值0.083A.U.,74%的灵敏度和68%的特异性(图2C)。
实施例3.
BAG3结合到巨噬细胞
我们探讨了BAG3被心肌细胞释放的功能显著性。我们排除,该蛋白质可参与自分泌路径,因为它显然未结合到心肌细胞的表面,如我们在使用荧光异硫氰酸盐(FITC)缀合的BAG3的实验中评估的(图3A)。因此,我们研究了BAG3是否可与血细胞相互作用。实际上,我们发现,FITC-BAG3结合到细胞系J774的巨噬细胞(图3B)。BAG3结合到巨噬细胞尤其地被竞争性BAG3肽或被来自抗BAG3单克隆抗体的BAG3隔绝的F(ab’)2片段所破坏(表I)。尤其地,用14nM FITC-BAG3蛋白和用625nM的BAG3肽(肽1、肽2、肽3、肽4或乱序肽)或用420nM的来自抗BAG3单克隆和多克隆抗体(小鼠单克隆AC1、AC2和兔多克隆TOS2)的F(ab')2片段培养J774细胞。来自小鼠IgG的F(ab’)2片段或来自兔IgG的F(ab’)2片段用作阴性对照。
表I
为研究BAG3结合到巨噬细胞在功能上的结果,我们检测了重组BAG3对细胞中可诱导的一氧化氮合酶(iNOS)和环氧合酶(Cox)-2的表达的影响。如图3C的图a显示,这些酶的水平在BAG3处理的巨噬细胞中增强。此外,BAG3诱导的亚硝酸盐和白介素(IL)-6的释放(图3C的图b和图c)证实,巨噬细胞响应于它们结合到蛋白而被活化。
在来自CHF患者的血清中,我们可检测到显著量的抗BAG3抗体(图2A、B)。自身抗体的产生可能与通常胞内蛋白的胞外释放相关,例如在产生抗肌钙蛋白自身抗体的慢性局部缺血的患者中发生的。CHF患者血清中抗BAG3抗体的ELISA值显著高于在健康对照血清中的检测的值。
因此,ELISA检测的抗BAG3抗体的产生表现为慢性心力衰竭的生物标志物。它用于危险分层和治疗监控的用途值得研究。
由应激的心肌细胞的BAG3释放以及随后巨噬细胞的活化导致NO的局部释放,可构成心脏局部缺血的保护回路。实际上,血管舒张、新血管生成和重塑可被靶向。BAG3释放及其短暂或长期的效果值得研究,并可有助于我们对局部缺血和其它心脏应激状态的理解。
此外,BAG3特异的小鼠单克隆抗体AC-1、AC-2和AC-3和/或其它或其被修饰为F(ab)、F(ab’)2、F(ab)或人源化;或包括序列PEP 1至4的肽和/或其它肽为能够结合/或阻碍可溶性BAG3的效果的分子,所述分子可用于治疗炎症、肿瘤或涉及巨噬细胞活化的其它疾病。
实施例4.
通过免疫组织化学,BAG3在PDAC中的表达
我们开发了免疫组织化学(IHC)试剂盒,包括我们的抗BAG3单克隆抗体,并能够通过免疫组织化学(IHC)检测BAG3蛋白质。这个试剂盒在所有346(100%)个我们分析的PDAC活体组织切片中揭示BAG3的表达。BAG3染色显示胰岛中等阳性,而正常的胰管和胰腺腺泡细胞不具有BAG3表达。这在正常的胰腺和邻近肿瘤块的非赘生的胰腺组织中是真实的。主要在肿瘤细胞的细胞质中观察到了BAG3染色。BAG3染色的强度是可变的,如阳性癌细胞的肿瘤那样。此外,朗格汉斯细胞岛(Langerhansinsulae)为阳性,并构成IHC的良好的内标(图4A)。因此,我们的试剂盒允许通过IHC在PDAC中检测BAG3蛋白质。此外,它允许在其它肿瘤或正常组织中通过IHC检测BAG3蛋白质。
我们还研究了BAG3的表达与患者存活、及对治疗的响应的关联。我们分析了来自相同数目的患者的346个PDAC样品的群组(表II),该群组描述了通过免疫组织化学分析的所有肿瘤样品的数据和用存活数据分析的R0患者的亚组的数据;我们使用400×的放大率通过在10个不重叠的视野的总癌细胞中计数阳性细胞的数目,而基于样品中阳性癌细胞的比例分配评分。
表II
如所述地计算BAG3阳性细胞的中位百分比为40%,并且该值用作截断值以分开低阳性样品和高阳性样品。基于该分类,190个患者样品(55%)分类为低阳性(≤40%的阳性细胞),并且156(45%)分类为高阳性(>40%的阳性细胞)(图4,图B)。在检查的所有病变具有不含肿瘤细胞的切除边缘的66个患者的群组中进行存活分析(R0),并且仅3.7%显示转移到远端器官的转移的存在(表II)。获得的数据显示高表达的BAG3的患者(中位存活=12.0月)具有比低表达的BAG3的患者(中位存活=23.0月)显著短的存活(p=0.0013)(图4,图C)。基于Cox比例分析,高的BAG3表达与超过两倍的更高的死亡危险相关(表III)。
表III
实施例5.
响应于治疗的BAG3蛋白
用于治疗胰腺癌的一线化疗为吉西他滨。为了研究BAG3蛋白响应于治疗的功能,我们分析了在人PDAC细胞中下调BAG3的效果。我们用特异的靶向bag3 mRNA的siRNA或用非特异的(NT)siRNA转染细胞,并用吉西他滨处理细胞显示的时间。BAG3的沉默增强了响应于药物的细胞凋亡(图8)。
这些结果证明胰腺癌中BAG3蛋白和mRNA的过表达,以及高的表达水平与更高的死亡危险相关,使BAG3具有可用于预后和治疗选择的标志物的功能。此外,它们显示BAG3下调增强PDAC细胞中的凋亡。由于它在检测的所有损伤中广泛的表达,以及它参与维持胰腺癌细胞的存活,所以BAG3代表用于PDAC中创新性治疗的有价值的靶标。
实施例6.
胰腺癌患者的血清中的BAG3蛋白
因为在胰腺癌患者中广泛的表达,我们研究了BAG3是否存在于胰腺癌患者的血清中。我们发现确实可检测BAG3。同样虽然普遍与BAG3形成复合物,但是也可检测抗BAG3抗体。因此,我们开发了用于检测BAG3/抗体复合物的ELISA试验。我们分析了来自51个健康供体的血清和55个受PDAC影响的患者的血清(表IV)。
表IV
如图9A中显示,在来自患者的血清中的免疫复合物(以任意单位测量)显著高于来自健康供体的血清。此外,ELISA实验结果的ROC分析,使用截断值0.183 A.U.,65%的灵敏度和78%的特异性(图9B)。
实施例7.
实时定量PCR的BAG3表达
测量bag3 mRNA在25个PDAC组织样品中的水平还证实关于BAG3表达的免疫组织化学数据(表V)。
表V
具体地,25个患者中有16个存活者,而9个患者在分析时死于胰腺癌的发展。设定在分析的肿瘤中的bag3 mRNA的表达的中位数为0.0068(Q1=0.004;Q3=0.010)(图7,图A)。PDAC患者的所有考虑的人口统计资料和临床特征与bag3的mRNA水平不相关。因此,评估与存活的相关性,并且PDAC样品中bag3表达水平的中位数用作截断值,以分开具有低的bag3表达的患者与具有高的bag3表达的患者。因此,13个样品(52%)分类为高的bag3阳性,12个样品(48%)为低的bag3阳性。具有高的bag3表达的患者(生存中位数=19.0个月)具有比低的bag3表达的患者(生存中位数=32.0个月)短的存活,p值=0.0198(图7,图B)。基于Cox比例分析,高的bag3表达与超过六倍的更高死亡危险相关(单变量:HR=6.094;95%CI=1.105~33.597,p=0.038)。
通过实时定量RT-PCR检测BAG3表达的bag3引物。我们开发了RT-PCR试剂盒,包括用于bag3 mRNA检测和定量的特异的引物。
实施例8
我们评估了在源自UKCTOCS的预期的生物样本库中的病例对照研究集合中在诊断PDAC前的BAG3抗体水平(Menon等,BMJ,337:a2079,2008)。202,000个绝经后女性的实验群体在登记时提供单个血清,并且50,000个女性每年持续提供血清样品。
通过癌症登记数据和邮政跟踪调查问卷识别随机抽样后(post-randomisation)诊断为胰腺癌的女性。
对于诊断为PDAC的这些女性,我们获得了在PDAC诊断前不同的时间点的血清样品。从健康受试者获得对照血清,我们测量了与每个PDAC病例相比的约5个对照受试者中的BAG3抗体效价。此外,对照血清来自年龄与各个PDAC病例相匹配的受试者。对于每个时间点使用该对照血清匹配。
在特异性ELISA试验中,在来自诊断为胰腺癌前0~1年的49个女性受试者的患者血清中检测BAG3特异的抗体,与235个女性对照受试者的患者血清相比较BAG3特异的抗体的存在。
在特异性ELISA分析中检测BAG3抗体浓度。
如图10显示,与对照受试者相比,在诊断前至少3-4年的病例中BAG3抗体显著更高。
其它时间点包括:与251个对照相比诊断为PDAC前1-2年的53个受试者;与212个对照相比诊断为PDAC前2-3年的44个受试者;与187个对照相比诊断为PDAC前3-4年的42个受试者。
如图10中显示,相对于对照受试者,BAG3抗体在诊断前至少3-4年的病例中显著更高。
方法
细胞培养物
HCMa(人心肌细胞—成人)购自Sciencell Research Laboratories(SanDiego,CA),并在心肌细胞培养基(CMM,FBS 5%,心肌细胞生长补充剂1%、青霉素/链霉素溶液1%)(Sciencell Research Laboratories,San Diego,CA)中生长。在低传代细胞培养物上进行全部实验。大鼠胚胎成心肌细胞(H9c2系)购自美国模式培养物保藏中心(ATCC,Manassas,VA,USA),并在添加10%胎牛血清(FBS)、100U/mL的青霉素和100μg/mL的链霉素的密理博高糖培养基(Dulbecco's Modified Eagle's Medium,DMEM)中培养。J774A.1,鼠单核细胞巨噬细胞系(ATCC,Manassas,VA,USA)在添加10%的胎牛血清(FBS)、25mM HEPES、2mM谷氨酰胺、100u/mL青霉素和100μg/mL的链霉素的DMEM中生长。
从美国模式培养物保藏中心(ATCC,Manassas,VA,USA)细胞库获得胰腺癌细胞系(MIA PaCa-2、AsPC-1、PSN1、Capan-1和PANC-1)。在添加10%的FBS和2.5%的马血清的密理博高糖培养基(DMEM)中培养MIAPaCa-2细胞。AsPC-1和PSN1细胞在添加10%FBS的RPMI-1640培养基中培养。Capan-1在包含20%FBS的RPMI-1640中培养,而PANC-1在添加10%FBS的DMEM中培养。用于上述细胞系的所有培养基购自BioWhittaker-Lonza(Bergamo,Italy)MediaTech(Manassas,VA),并添加100单位的青霉素/mL和2μg的链霉素/mL(Sigma-Aldrich,St.Louis,MO)。在5%CO2的环境中于37℃培养细胞。用Eli Lilly(Sesto Fiorentino,Italy)提供的吉西他滨(2’,2’-二氟代脱氧胞苷,GEM,)以指示的浓度处理细胞。
分离人血清中的BAG3抗体
用分离缓冲液(具有1.5%BSA和0.2M的甘氨酸-乙酸盐的PBS,pH2.5)以1:40稀释血清至500μl的终体积,并室温培养20分钟。然后,吸取血清到Microcon离心过滤器,YM-100(100,000MW截断值;Millipore,Billerica,MA,USA)的样品容器,并在室温以14,100rpm离心20分钟。然后,然后通过倒转放入第二个管而从流分离样品容器,并在室温以5,000rpm离心3分钟。用1M Tris pH 9.0的缓冲液调节包含分离的抗体的收集的溶液至pH 7.0。用稀释缓冲液(具有1.5%的BSA和0.1%Tween-20的PBS)使保留物的体积重构为最初的体积(500μl)。14为了通过免疫印迹法检测BAG3,在4℃于包含5%的牛血清白蛋白的TBST中以1:200稀释分离的抗体。
蛋白质印迹分析
收集细胞,并在添加蛋白酶抑制剂混合物(1mM苯甲基磺酰氟、1mg/ml的胃酶肽素A、2mg/ml抑肽酶)的包含20mM HEPES(pH 7.5)、150mM NaCl、0.1%Triton(TNN缓冲液)的缓冲液中通过3个循环的冷冻和融化而裂解。在10,000g离心15分钟后,收集可溶的蛋白,并通过Bradford检验(Bio-Rad,Hercules,CA)确定它们的量。25μg的总蛋白和血清样品(PBS-T 0.05%中1:2)在8%或10%的SDS-PAGE凝胶上运行,并电泳转移到硝酸纤维素膜。用TBST缓冲液(20mM Tris-HCl pH 7.4,500mM NaCl和0.1%Tween 20)中10%的脱脂奶粉封闭硝酸纤维素的印迹,并在包含5%的牛血清白蛋白或5%的脱脂奶粉的TBST中用一抗在4℃培养过夜。用购自Pierce(Rockford,IL)的辣根过氧化物酶共轭的二抗和购自Amersham Life Sciences Inc.(Arlington Heights,IL,USA)的ECL检测试剂连续培养而检测免疫反应性。
用Image Scan(SnapScan 1212;Agfa-Gevaert NV)进行条带的扫描密度测定术。使用Gimp2软件确定关于每条带的曲线下的面积。从计算的值减去背景。
质谱分析
如Shevchenko方案所述,切除蛋白条带,然后用MilliQ水和乙腈洗凝胶块,并且原位消化蛋白。简略地,凝胶条在1,4-二硫苏糖醇(10mM)中还原,并用碘乙酰胺(50mM)烷基化,然后在冰上于胰蛋白酶(12ng/μL)中洗和再水化1个小时。在加入30μL的碳酸氢铵(10mM,pH 7.5)后,在25℃消化样品过夜。5μL的获得的肽混合物注入到nano Acquity LC系统(Waters Corp.Manchester,United Kingdom)。肽在1.7μm BEH C-18柱(Waters Corp.Manchester,United Kingdom)上以200nl/分钟的速度分离。梯度(溶液A:0.1%的甲酸,溶液B:0.1%的甲酸、100%的ACN)开始于5%的B,并在55分钟后终止于50%的B。使用Q-TOF Premier质谱仪(Waters Corp.,Micromass,Manchester,United Kingdom)获得MS和MS/MS数据。通过MassLynx软件自动选择带双重和三重电荷的肽离子,并且碎片化。自动处理MS数据,并且通过ProteinLynx软件产生用于通过数据库检索的蛋白质鉴定的峰列表(peaklist)。使用SwissProt蛋白数据库用MASCOT服务器进行数据库检索。检索SwissProt人数据库(405506个序列,146166984个残基),允许1个错误的裂缝,脲甲基(C)作为固定修饰。设定肽耐受性为60ppm,并且设定MS/MS耐受性为0.8Da。
通过差速超速离心法纯化胞吐囊泡
在4℃通过连续离心(2000×g 15分钟,10,000×g 30分钟)去除H9c2的无血清培养基的细胞和大的碎片。在前两次离心的每次后,弃除团块,保留上清液用于下一个步骤。然后,在4℃以150,000×g超速离心最终的上清液(用SW50.1转子,和Optima L-90K超高速离心机,Beckman Coulter)90分钟,以收集外泌体(Exosome)。在PBS中洗所述颗粒,以去除污染的蛋白质,并在4℃以150,000×g离心90分钟最后一次。16在清洗后,在20μl的PBS中重新悬浮团块(外泌体),并用抗BAG3 TOS-2多克隆抗体分析,通过蛋白质印迹与全细胞裂解物相比较。对于胞吐囊泡,Rab-4作为标志物被分析。
FACS分析
rBAG3结合—在冰上用PBS中2%FBS+0.1%NaN3封闭J774 A.1细胞15分钟,并在黑暗中4℃于包含2%FBS+0.1%NaN3的PBS中用不同浓度的FITC-rBAG3蛋白(7、14和70nm)或FITC-BSA(70nM)培养(2.5×105/100μl)30分钟。在用PBS洗后,在PBS+2%FBS+0.1%NaN3中重新悬浮细胞,并用FACScan(BD Biosciences)流式细胞分析仪分析。
竞争–在冰上于包含2%FBS+0.1%NaN3的PBS中用625nM的BAG3肽(肽1、肽2、肽3、肽4或乱序肽)或用420nM的来自抗BAG3单克隆和多克隆抗体(小鼠单克隆AC1、AC2和兔多克隆TOS2)的F(ab')2片段或来自小鼠IgG的F(ab’)2片段或来自兔IgG的F(ab’)2片段培养J774 A.1细胞(2.5×105/100μl)30分钟。在用PBS洗培养的细胞后,然后在黑暗中在4℃于包含2%FBS+0.1%NaN3的PBS中用FITC-rBAG3蛋白(14nM)培养细胞30分钟。在用PBS洗后,在PBS+2%FBS+0.1%NaN3中重悬细胞,并通过流式细胞计数器(BD Biosciences)分析。
通过ELISA检测IL6
在存在或不存在多粘菌素B硫酸盐(5μg/ml)下在用LPS(10ng/ml)或用rBAG3(14nM)或BSA(14nM)处理10或20个小时的J774 A.1细胞(5×104/在96孔微板)的上清液中测量IL6。在处理后,收集50μL的细胞培养基,并用小鼠IL6试剂盒(eBioscience)以一式三份分析。
荧光
于六孔板中在盖玻片上培养细胞至60-70%融合,并在具有0.1%的NaN3的HCMa和J774 A1的培养基中加入等量的rBAG3-FITC和BSA-FITC蛋白1个小时。在1×PBS中清洗盖玻片,并且室温在1×PBS中3.7%的甲醛中固定30分钟,然后用1×PBS 0.1M甘氨酸培养5分钟。在4℃以1:100稀释的抗β整合素单克隆抗体培养后,用1×PBS清洗盖玻片3次。在室温用1:500稀释的山羊抗小鼠IgG DyLight 594共轭的抗体(JacksonImmunoResearch,West Grove,PA,USA)培养45分钟后,再在1×PBS中清洗盖玻片3次。在室温用Hoechst 33342(Sigma Aldrich,2μg/ml)培养10分钟后,在PBS中再次清洗盖玻片3次,然后在蒸馏水中清洗盖玻片。然后,盖玻片装在具有含47%(v/v)甘油的间隙的载玻片。使用激光共聚焦扫描显微镜(德国Zeiss LSM共聚焦显微镜)分析样品。当比较实验材料和对照材料时,通过使用相同的采集参数(激光强度、增益光电倍增管、针孔光圈(pinhole aperture),物镜63×,变焦2)以连续的扫描模式采集图像。为了产生图像,通过小心地调节图像的亮度和对比度以为了最低的荧光强度特性的视觉评价而留下光细胞荧光背景并有助于不同的实验组之间的比较。使用Adobe Photoshop 7和Adobe Illustrator 10组合最终的图。Leica Q9 Confocal Software和ImageJ用于分析数据。
通过ELISA测量抗体的效价
在PBS,pH 7中用重组BAG3蛋白1μg/ml(50μl/孔)包被NUNCMaxisorp 96孔ELISA板,并在4℃过夜培养。用清洗缓冲液(PBS+0.05%Tween-20)洗板2次,然后用PBS中0.5%的鱼胶在室温封闭(150μl/孔)一个小时。封闭后,用清洗缓冲液清洗板2次,用清洗缓冲液中0.5%的鱼胶以1:70稀释血清,然后以一式三份应用(50μl/孔),并室温培养2个小时。然后,用清洗缓冲液洗板6次。用清洗缓冲液中0.5%的鱼胶以1:20,000稀释抗人IgG(H+L)抗体(Sigma Aldrich),以50μl/孔加入,并在4℃培养30分钟。培养后,洗板6次,用TMB(50μl/孔)(eBioscience)显色,用4.5M的硫酸终止反应(25μl/孔),并在450nm分光光度分析所述板。
NO
2
-
检验
在存在或不存在多粘菌素B硫酸盐(Sigma-Aldrich,St.Louis,MO,USA)5μg/ml下在用LPS(10ng/ml)或用rBAG3(7、14和28nM)或BSA(28nM)处理24个小时的J774 A.1细胞(5×104/在6孔微板)中测量在培养的上清液中由细胞释放的NO的稳定代谢物亚硝酸盐的含量(NO2 -)。通过格里斯反应测量NO2 -的量。简略地,100μL的细胞培养介质与100μL的格里斯试剂–相等体积的5%(v:v)的磷酸中的1%磺胺(w:v)以及0.1%(w:v)萘基乙二胺-HCl混合,并且在室温培养10分钟,然后在酶标仪Titertek(Dasit,Cornaredo,Milan,Italy)中于550nm测量吸光度。由亚硝酸钠标准曲线计算样品中NO2 -(以μM)的量。
通过ELISA测量BAG3/抗体免疫复合物
在PBS,pH 7中用抗BAG3单克隆抗体AC-1、AC-2或AC-3包被NUNC Maxisorp 96孔ELISA板,并在4℃培养过夜。用清洗缓冲液(PBS+0.05%Tween-20)洗板2次,然后用PBS中0.5%的鱼胶在室温封闭(150μl/孔)1小时。封闭后,用清洗缓冲液清洗板2次,并用清洗缓冲液中0.5%的鱼胶以1:70稀释血清,然后以一式三份应用(50μl/孔),并在室温培养2小时。然后用清洗缓冲液洗板6次。用清洗缓冲液中0.5%的鱼胶以1:20,000稀释抗人IgG(H+L)抗体(Sigma Aldrich),以50μl/孔加入,并在4℃培养30分钟。在培养后,洗板6次,用TMB(50μl/孔)(eBioscience)显色,用4.5M的硫酸(25μl/孔)终止反应,并在450nm分光光度分析所述板。
免疫组织化学
免疫组织化学方案包括:在二甲苯中脱蜡,通过减小醇的浓度直至纯水而再水合,在95℃在pH 6.0的柠檬酸盐缓冲液中非酶抗原修复30分钟,并在甲醇中用H2O2猝灭内源过氧化物酶20分钟。在用PBS漂洗后,用0.1%PBS/BSA中5%的标准马血清封闭样品。为检测BAG3,室温用3微克/ml浓度的BAG3单克隆抗体AC-1、AC-2或AC-3培养样品1个小时。在用PBS彻底洗涤后,用生物素化的二级抗小鼠IgG培养切片20分钟,然后漂洗,用亲合素-生物素复合物过氧化物酶(购自Novocastra-LeicaMicrosystems,Milano,IT)培养,并用二氨基联苯(Sigma-Aldrich,St.Louis,MO)显影。最终,用苏木精复染色切片,在醇中脱水,在二甲苯中清除,用Permount(Fisher Scientific,Milan,IT)贴片(mounted)。
实时定量RT-PCR
获得切除的胰腺癌的组织样本,立即在液氮中冷冻,并在﹣80℃储存直到提取RNA。通过酚提取的方式从冷冻的组织以及从胰腺癌细胞系中分离总RNA(TRIzol Reagent,Invitrogen Corporation,Carlsbad,CA,USA)。通过冷冻切片和大部分细胞区域的剥离来富集组织样品中的癌细胞结构。通过NanoDrop分光光度计验证RNA的浓度和纯度(A260:A280>2.0;A260/A230>1.8)(Thermo Fisher,Waltham,MA,USA)。根据制造商的说明书使用高容量的cDNA逆转录试剂盒(Applied Biosystems,Applera,FosterCity,CA,USA)逆转录1.0μg的总RNA。实时定量PCR检测用于评价肿瘤组织样品中BAG3的差异表达。通过Primm srl(Milano,Italy)合成用于人bag3基因的引物:(正向引物:(SEQ ID NO:16)CCT GTT AGC TGT GGTTG;反向引物:(SEQ ID NO:17)AAC ATA CAG ATA TTC CTA TGG C)。通过使用QuantiFast SYBR Green PCR试剂盒(QIAGEN,Hamburg,Germany)每个样品三个重复,以25-μl的终体积进行所有qPCR,根据下述条件在ABI7700 Sequence Detection System(AppliedBiosystems,Applera,Foster City,CA,USA)中运行:95℃5分钟,95℃10秒和在60℃30秒,40个循环。使用S.D.S软件v 2.1获得数据作为循环阈(Ct)值。在每个样品中,在对内源GAPDH的表达标准化后,使用对比方法获得bag3 mRNA的相对表达水平。
从上述描述和上面记录的实施例,根据本发明描述和获得的生物学标志物获得的益处是明显的。
Claims (18)
1.用于病理状态的诊断用途的抗BAG-3抗体。
2.根据权利要求1所述的用途的抗BAG-3抗体,其中,所述抗BAG-3抗体结合到可溶的BAG3以形成免疫复合物。
3.根据权利要求1或2的任一项所述的用途的抗BAG-3抗体,其中,所述诊断是体外的或离体的。
4.根据权利要求1或3的任一项所述的用途的抗BAG-3抗体,其中,所述诊断的接受者为哺乳动物,优选人类。
5.根据权利要求1或4的任一项所述的用途的抗BAG-3抗体,其中,所述病理状态选自:心脏病,癌症,糖尿病,皮肤、神经、骨、血管和结缔组织的炎症和炎症相关的疾病。
6.根据权利要求5所述的用途的抗BAG-3抗体,其中,所述心脏病选自:心绞痛、梗塞前心绞痛、心肌梗塞、心力衰竭、局部缺血、急性冠心病、急性心力衰竭、慢性心力衰竭和医源性心脏病。
7.根据权利要求5所述的用途的抗BAG-3抗体,其中,所述癌症选自:胰腺癌、膀胱癌和前列腺癌。
8.检测生物样品中抗BAG3抗体或结合到可溶的BAG3以形成免疫复合物的抗BAG3抗体的方法,包括以下步骤:
a.获得生物样品,所述生物样品由血清、血浆、尿或唾液组成,
b.确定所述生物样品中抗BAG3抗体或BAG3缔合的抗体的存在。
9.根据权利要求8所述的方法,还包括以下步骤:
c.比较从生物样品获得的值与参考值或从健康供体获得的值。
10.根据权利要求6或9的任一项所述的方法,其中,所述确定步骤b通过ELISA试验进行。
11.根据权利要求6至10的任一项所述的方法,其中,所述血清、血浆、尿或唾液来自哺乳动物,优选人类。
12.根据权利要求6至11的任一项所述的方法,其中,所述抗BAG3抗体或所述免疫复合物的存在与病理状态相关。
13.根据权利要求12所述的方法,其中,所述病理状态选自:心脏病,癌症,皮肤、神经、骨、血管和结缔组织的炎症和炎症相关的疾病。
14.根据权利要求13所述的方法,其中,所述心脏病选自:心绞痛、梗塞前心绞痛、心肌梗塞、心力衰竭、急性冠心病、局部缺血、急性心力衰竭、慢性心力衰竭或医源性心脏病。
15.根据权利要求13所述的方法,其中,所述癌症选自:胰腺癌、膀胱癌和前列腺癌。
16.ELISA试剂盒用于检测生物样品中抗BAG3抗体或结合到可溶的BAG3以形成免疫复合物的抗BAG3抗体的用途,所述ELISA试剂盒包括用于捕获可溶的BAG3的BAG3重组蛋白或BAG3特异的小鼠单克隆抗体AC-1、AC-2、AC-3、AC-4和AC-5以及能够识别人免疫球蛋白的抗体。
17.根据权利要求16所述的用途,其中,所述生物样品为血清、血浆、尿或唾液样品。
18.根据权利要求17所述的用途,其中,所述血清、血浆、尿或唾液样品来自哺乳动物,优选人类。
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EP12172531.1A EP2676966A1 (en) | 2012-06-19 | 2012-06-19 | BAG3 as biochemical serum and tissue marker |
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PCT/EP2013/061976 WO2013189778A1 (en) | 2012-06-19 | 2013-06-11 | Bag3 as biochemical serum and tissue marker |
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CN105021825A (zh) * | 2015-07-09 | 2015-11-04 | 陈勇 | 一种检测胰腺癌相关多肽dap44的elisa试剂盒 |
CN105769900A (zh) * | 2016-03-22 | 2016-07-20 | 山西大学 | Bag3基因在制备抗膀胱癌药物中的应用 |
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ITMI20130403A1 (it) * | 2013-03-18 | 2014-09-19 | Biouniversa Srl | Anticorpi anti-bag3 per uso terapeutico |
IT201600069391A1 (it) * | 2016-07-04 | 2016-10-04 | Univ Degli Studi Di Salerno | Uso della proteina bag3 e suoi frammenti peptidici per il controllo dell’omeostasi vascolare |
WO2021140173A1 (en) | 2020-01-10 | 2021-07-15 | Biouniversa S.R.L. | Methods and uses for treating fibrotic solid tumors with bags inhibitors |
US11940450B2 (en) | 2021-03-16 | 2024-03-26 | University Of Connecticut | Biomarker panel for non-invasive diagnosis of congenital renal dysfunction |
CN113862361B (zh) * | 2021-10-25 | 2023-08-15 | 中山大学孙逸仙纪念医院 | 一种诊断和治疗膀胱癌的分子标志物hsf1及其用途 |
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CA2876922A1 (en) | 2013-12-27 |
US20150132764A1 (en) | 2015-05-14 |
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AU2013279604A1 (en) | 2014-12-18 |
JP2015529633A (ja) | 2015-10-08 |
EP2861618B1 (en) | 2020-03-11 |
AU2013279604B2 (en) | 2018-03-29 |
JP6654432B2 (ja) | 2020-02-26 |
CN104507965B (zh) | 2019-04-26 |
WO2013189775A1 (en) | 2013-12-27 |
JP2015521475A (ja) | 2015-07-30 |
AU2013279601A1 (en) | 2015-01-15 |
KR102150903B1 (ko) | 2020-09-03 |
US20150147767A1 (en) | 2015-05-28 |
ES2795981T3 (es) | 2020-11-25 |
KR20150036111A (ko) | 2015-04-07 |
BR112014031958A2 (pt) | 2017-08-01 |
WO2013189778A1 (en) | 2013-12-27 |
CN104619861A (zh) | 2015-05-13 |
CA2876872A1 (en) | 2013-12-27 |
KR20150036102A (ko) | 2015-04-07 |
EP2861754A1 (en) | 2015-04-22 |
BR112014031957A2 (pt) | 2017-08-01 |
CA2876922C (en) | 2022-09-20 |
US10359433B2 (en) | 2019-07-23 |
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