CN102482348B - 胶原新表位抗体 - Google Patents
胶原新表位抗体 Download PDFInfo
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- CN102482348B CN102482348B CN201080041396.4A CN201080041396A CN102482348B CN 102482348 B CN102482348 B CN 102482348B CN 201080041396 A CN201080041396 A CN 201080041396A CN 102482348 B CN102482348 B CN 102482348B
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Abstract
本发明提供了新的单克隆抗体和使用所述新单克隆抗体的免疫测定、测定方法、试剂盒等,所述单克隆抗体对胶原片段的C端新表位具有特异性,在所述新表位含有的脯氨酸是非羟基化形式的情况下的结合亲和力与羟基化形式情况下的结合亲和力实质上相同。不受新表位中有无脯氨酸羟基化形式的影响,可以定量生物样品中由于胶原酶消化产生的胶原片段。
Description
技术领域
本发明涉及可以识别胶原新表位的单克隆抗体,基于所述抗体的免疫测定、基于所述抗体的测定方法、基于所述抗体的筛选方法、基于所述抗体的患者鉴别方法和基于所述抗体的试剂盒。
背景技术
软骨降解是骨关节炎(OA)和慢性类风湿性关节炎(RA)等关节疾病的主要特征。监控此类疾病中软骨丢失的进展情况为管理疾病(包括预后、诊断和处置)提供有效的信息。目前,通过X射线检测关节腔变小来监控软骨丢失。但是,X射线诊断不能同时满足检测的灵敏度和准确度。而且,X射线只显示过去发生过软骨丢失,而不显示现在的软骨降解情况。因此,监控各种关节疾病中的软骨破坏的进展情况的替代方法是非常有价值的。
目前已知软骨中的胶原分解是通过被称为基质金属蛋白酶(MMP)的一组酶进行的。特别地,鉴于只有胶原酶(MMP-1、MMP-8和MMP-13)负责切断未变性的II型胶原的3重螺旋,认为胶原酶在975-976位的氨基酸残基之间的切断是之后由明胶酶等多种蛋白酶造成II型胶原从软骨基质脱离的关键事件。此外,此处的氨基酸残基参照GenBank数据库:COL2A1(登录号:NP001835)的全长序列。
降解后的胞外基质蛋白的片段从软骨释放到滑液中(SF),之后通过血液在全身循环。因而,虽然血清或尿中的软骨基质蛋白质片段的水平通常低于SF中的水平,但如果可以对其检测,则能够更简便地评估OA和RA中的软骨降解。
关于测定全身II型胶原片段存在2个主要问题。第一,关节软骨中的II型胶原的周转(turnover)通常非常低,因而血清或尿中的II型胶原片段的水平也非常低。即使OA软骨中II型胶原的转换增加,所述增加也不是明显容易检测的非常大的变化。因而需要灵敏度非常高的测定。
第二个问题是构成胶原的大部分脯氨酸和赖氨酸残基被羟基化修饰。特别地,由于脯氨酸是占胶原蛋白约3成的主要组成成分,在胶原酶切割位点附近也存在羟化脯氨酸,已知该羟化对结合亲和力具有很大影响。
包含在胶原中的脯氨酸的羟基化是由脯氨酰4-羟化酶以依赖于铁离子和维生素C的方式催化的,因此,羟基化程度易受胶原合成时的营养状态等的影响,不是恒定的。因而,期望的是用于测定II型胶原片段的抗体的结合亲和力不受脯氨酸有无羟基化的影响。
迄今为止,已经发明了若干测定由胶原酶产生的II型胶原片段的系统,但无一具有足够的灵敏度、准确度。例如在Robin Poole等人的应用单克隆抗体COL2-3/4C long(专利文献1)的竞争性ELISA方法中,对于胶原酶切割端结构的特异性低,且对于切割位点上游第5位(从N末端起第971位)的脯氨酸的非羟基化形式几乎没有反应性(非专利文献1)。此外,对于使用Otterness等人的单克隆抗体9A4(专利文献2)的夹心ELISA方法,对于胶原酶切割端结构的特异性高,但由于从切割端起上游第5个残基(从N末端起第971位)的脯氨酸的羟基化,结合亲和力降低至约1/90(非专利文献2)。因此,对于大部分为羟基化形式的II型胶原片段的检测灵敏度低。
由此可见,目前为止的任一种抗体都易于受胶原酶切割位点紧邻的脯氨酸羟基化修饰的影响,不能正确地测定羟基化形式和非羟基化形式混合存在的生物样品中的胶原片段的量。
现有技术文献
专利文献
专利文献1:特许第2999416号
专利文献2:特许第3258630号。
非专利文献
非专利文献1:J Immunol Methods 294 (2004) 第145-153页
非专利文献2:J Immunol Methods 247(2001)第25-34页。
发明内容
发明要解决的课题
本发明要解决的课题是正确地测定生物样品中由胶原酶消化产生的胶原片段(胶原新表位)的量。
用于解决课题的手段
本发明人深入研究了针对胶原新表位的单克隆抗体的制备,结果制备出新的单克隆抗体,即使新表位的脯氨酸变为羟基化形式,所述抗体的结合能力也不变化,从而完成了本发明。
换言之,本发明涉及:
(1)特异性结合胶原新表位片段的单克隆抗体,所述抗体结合序列号20所示氨基酸序列的962位至975位,其在包含于所述表位中的脯氨酸为非羟基化形式的情况下的结合亲和力与该脯氨酸为羟基化形式情况下的结合亲和力实质上相同;
(2)上述(1)记载的单克隆抗体,其中上述(1)记载的脯氨酸是序列号20所示氨基酸序列中的971位的脯氨酸;
(3)上述(1)记载的单克隆抗体,其中表位存在于序列号20所示氨基酸序列的957位至975位的区域中;
(4)上述(2)记载的抗体,在使由序列号14、17或18所示氨基酸序列组成的肽竞争以便抑制所述抗体与由序列号2所示氨基酸序列组成的肽的免疫反应的免疫测定中,对任一种肽的所述免疫反应的50%抑制浓度在0.04μM以下;
(5)单克隆抗体,其具有:
1)互补决定区中含有以下氨基酸序列的重链可变区:KYGIN(序列号5)、WINTYSGMTTYADDFKG(序列号6)和SLGYDYGGFAY(序列号7),以及
2)互补决定区中含有以下氨基酸序列的轻链可变区:RSGQTLVHDNENTYFH(序列号8)、KISNRFS(序列号9)和SQNTHVPFT(序列号10);
(6)单克隆抗体,其具有:
1)具有序列号3的氨基酸序列的重链可变区;和
2)具有序列号4的氨基酸序列的轻链可变区;
(7)上述(1)-(6)的任一项记载的单克隆抗体,其是被标记的;
(8)使用上述(1)-(6)的任一项记载的单克隆抗体的免疫测定;
(9)使用上述(1)-(6)的任一项记载的单克隆抗体测定胶原新表位片段含量的方法;
(10)测定胶原酶活性的方法,其以使用上述(1)-(6)的任一项记载的单克隆抗体测定的胶原新表位片段的含量为指标;
(11)筛选胶原酶抑制剂的方法,其以使用上述(1)-(6)的任一项记载的单克隆抗体测定的胶原新表位片段的含量为指标;
(12)鉴别胶原酶相关性疾病的患者的方法,包括使用上述(1)-(6)的任一项记载的单克隆抗体测定生物样品中含有的胶原新表位片段的含量的步骤;
(13)包含上述(1)-(6)的任一项记载的单克隆抗体的试剂盒;
(14)诊断胶原酶相关性疾病的方法,包括使用上述(1)-(6)的任一项记载的单克隆抗体测定生物样品中含有的胶原新表位片段的含量的步骤;和
(15)上述(1)-(6)的任一项记载的单克隆抗体,其用于诊断胶原酶相关性疾病。
发明的效果
本发明的单克隆抗体可以特异性识别新表位末端结构,不受脯氨酸羟基化修饰的影响,因而可以正确地检测、定量生物体中的胶原新表位片段量。
附图说明
图1:显示了胶原酶切割位点,其在三聚螺旋结构的纤维状胶原(I型、II型和III型)的示意图中的氨基末端起2/3的位置。下方显示了各种动物的II型胶原的胶原酶切割位点及其前后的氨基酸序列。从上至下是人、牛、狗、大鼠、小鼠的顺序。用星号显示了切割位点是975-976氨基酸残基之间,相当于人II型胶原的954-980位。
图2:显示了利用具有20A10的各种C末端结构的肽片段的竞争性免疫测定的结果。
图3:上部分的图显示了利用胶原酶消化和未消化的胶原对20A10的竞争性免疫测定的结果。下方的表显示胶原酶消化引起的特异性的改变以及I型、II型和III型胶原片段之间的交叉反应性。
图4:显示了利用与II性胶原特异性单克隆抗体组合的夹心免疫测定的结果。
图5:显示了在添加1.2 ng/孔MMP-13引起的II性胶原降解反应中,在添加各种浓度的MMP抑制剂的情况下,胶原新表位片段浓度的测定值。
图6:显示了在存在1 ng/ml白介素1的牛软骨外植体培养中,在添加各种浓度的MMP抑制剂的情况下,胶原新表位片段浓度的测定值。
图7:显示了20A10的可变区的氨基酸序列。上部分是重链的可变区,下部分是轻链的可变区。此外,分别附有下划线的序列表示互补决定区的位置。
具体实施方案
I.抗体
作为纤维状胶原的I型、II型和III型胶原是由3条肽链螺旋状卷曲在一起形成的,胶原酶(例如,MMP-1、MMP-8和MMP-13)在自N末端3:1的位置(975-976位氨基酸残基之间)切割未变性状态的这些纤维状胶原的三重螺旋。并且将新生成N末端四分之三片段的C末端、和C末端四分之一片段的N末端的末端结构称为“新表位”(图1)。此外,将切割生成的胶原酶片段称为“胶原新表位片段”。其中,已知人II型胶原(登录号NP_001835)C端的新表位部分(对应于969-975位;下文中也称为C末端新表位)的7个氨基酸残基Gly-Pro-Pro-Gly-Pro-Gln-Gly(序列号1)在从人到小鼠的动物物种之间是保守的。此外,胶原是以高羟脯氨酸含量为特征的蛋白质之一,其序列自C端第5位(或者自N端第971位)的脯氨酸残基在大部分情况下成为羟基化形式(下文中也称为羟化形式或羟脯氨酸),(序列号2:Gly-Pro-Hyp-Gly-Pro-Gln-Gly,Hyp是羟脯氨酸)其比例易受胶原合成时的健康状态和营养状态的影响,并非恒定,因而被认为阻碍胶原新表位片段的正确定量。因此,如果在免疫学测定胶原降解的情况下,检测新表位的抗体的特性固有地期望可以同等程度地免疫特异性识别羟基化形式和非羟基化形式(脯氨酸)。
本发明的单克隆抗体具有这样的特征,即使包含在C末端具有新表位的II型胶原新表位片段(序列号20)中的脯氨酸变为羟基化形式,所述单克隆抗体的结合亲和力也不发生改变。在此,“结合亲和力不发生改变”意指在构成新表位的氨基酸残基是脯氨酸(非羟基化形式)的情况下以及在羟脯氨酸(羟基化形式)的情况下,结合亲和力实质上相同。
“结合亲和力”一般指免疫球蛋白分子与该免疫球蛋白分子特异性的抗原之间产生的类型为非共价结合的相互作用的强度或亲和力,通常可以用解离常数(Kd)表示。
“实质上相同”具体意指羟基化形式(羟脯氨酸)的结合亲和力是在非羟基化形式的结合亲和力数值的80%-120%的范围内,优选在90%-110%的范围内,更优选在95%-105%的范围内。此外还意指在非羟基化形式(脯氨酸)的情况下和羟基化形式的情况下的交叉反应性是80%以上,优选90%以上,更优选95%以上。可以通过已知的方法,例如使用ELISA测定的Scatchard分析(例如,Campbell, 1991;Segel, 1976)确定抗体的结合亲和力。
此类单克隆抗体的代表性实例可列举20A10。图6显示了20A10的可变区的氨基酸序列。上部分表示重链(序列号3)的序列,下部分表示轻链(序列号4)的序列。此外,下划线部分表示互补决定区(CDR)(序列号5-10)。
用于制备本发明的单克隆抗体的免疫原可以是例如通过Antibodies:A Laboratory Manual(1989, Cold Spring Harbor Laboratory Press)等记载的方法制备的。
免疫方法可以通过常规方法进行,例如将免疫原通过静脉内、皮内、皮下、腹腔内注射等施用到哺乳动物体内。更具体的是,例如用含有生理盐水的磷酸盐缓冲液(PBS)、生理盐水等将免疫原稀释到恰当的浓度,按需要与常规的佐剂组合使用,以2-3周的时间间隔分若干次施用到受试动物体内。在使用小鼠的情况下,每次的施用量是一只50-100μg左右。在本文中,佐剂是指与抗原共同施用时,非特异性地增加对于抗原的免疫反应的物质。常用的佐剂可列举百日咳疫苗、弗氏佐剂等。通过在最后免疫后第3-10天对哺乳动物采血,可以获得抗血清。
可如下实施单克隆抗体的制备方法,制备用免疫原免疫的哺乳动物的浆细胞(免疫细胞)和哺乳动物的浆细胞瘤细胞(骨髓瘤细胞)的融合细胞(杂交瘤细胞),从杂交瘤细胞选择产生识别5-脱氧-5-甲硫腺苷的期望单克隆抗体的克隆,培养所述克隆。所述单克隆抗体的制备基本可以根据常规方法进行。
在所述方法中,考虑与细胞融合中使用的浆细胞瘤细胞的相容性来选择用免疫原免疫的哺乳动物是理想的,可使用小鼠、大鼠等。免疫方法与制备多克隆抗体的情况是相同的。但在最后免疫后第3-10天从免疫动物中采集脾细胞。
为了从所获得的免疫细胞获得杂交瘤,可以通过例如“分子细胞生物学基础实验方法”(南江堂 堀江武一等人,1994年出版)等记载的方法,以形成可以传代培养的细胞为目的,在存在例如仙台病毒或聚乙二醇的条件下,使浆细胞瘤细胞和产生抗体的免疫细胞融合,获得杂交瘤。在本文中使用的浆细胞瘤细胞期望使用同一恒温动物或同种恒温动物来源的浆细胞瘤细胞,例如,在与以小鼠为免疫动物获得的脾细胞融合的情况下,优选使用小鼠杂交瘤细胞。可以利用p3x63-Ag8.UI等公知的浆细胞瘤细胞。
可以如下获得杂交瘤,通过HAT培养基(补充了次黄嘌呤、氨喋呤、胸苷的培养基)选择,在确认有克隆的阶段,通过检查(筛选)分泌到培养基上清液中的抗体与抗原的结合,获得产生目标抗体的杂交瘤。
筛选方法可列举例如斑点法、凝聚反应法、Western印迹法、ELISA法等一般用于检测抗体的各种方法,优选例如下述实施例中详细描述的,对杂交瘤的培养上清液实施以与新表位肽的反应性为指标的ELISA方法。根据所述筛选,可以筛选出与新表位肽特异性反应的目标抗体生产株。克隆20A10是基于该过程获得的克隆的示例。
作为筛选结果所获得的可以生产目标抗体的株的克隆可以通过常规的有限稀释法、软琼脂法等来实施。必要时,可以用血清培养基或无血清培养基大量培养克隆的杂交瘤。通过所述培养,可以以培养上清液的方式获得较高纯度的所需抗体。此外,可以在与杂交瘤相容的哺乳动物例如小鼠等的腹腔中接种杂交瘤,以小鼠腹水的方式大量回收所需抗体。
含有产生本发明的单克隆抗体的杂交瘤的培养上清液和小鼠腹水可以不进行纯化或修饰地作为粗制抗体溶液使用。此外,可以通过使上述培养上清液或腹水接受饱和硫酸铵、离子交换层析(DEAE或DE52等)、抗免疫球蛋白柱或蛋白A柱等亲和柱层析等,对单克隆抗体进行分离、纯化。
此外,本发明的单克隆抗体可以使用重组抗体,所述重组抗体是通过克隆抗体基因、整合到合适的载体中、将所述载体导入宿主中,利用基因重组技术产生的(例如,Carl等人,THERAPEUTIC MONOCLONAL ANTIBODIES,1990年出版)。
具体而言,合成编码目标抗体(例如20A10)的可变区(例如,如果是20A10的话,是序列号3和4)的cDNA。cDNA的合成和扩增可使用5′-Ampli FINDER RACEKit(Clonetech生产)和利用PCR的5′-RACE方法(Frohman, M. A.等人,Proc. Natl. Acad. Sci. USA 1988,第85卷,第8998页等)。从获得的PCR产物中纯化目标DNA片段,与载体DNA连接。进一步如此制备重组载体,导入大肠杆菌等中挑选克隆,制备所需的重组载体。通过公知的方法例如双脱氧方法确定目标DNA的碱基序列。
一旦获得编码目标抗体的V区的DNA,就将其与编码所需的抗体恒定区(C区)的DNA连接,将连接产物整合到表达载体中。此外,也可以将编码抗体的V区的DNA整合到包含抗体C区的DNA的表达载体中。为了制备本发明中使用的抗体,将抗体基因整合到表达载体中以便在表达控制区例如增强子/启动子的控制下表达。之后,可以通过该表达载体转化宿主细胞来表达抗体。
对于抗体基因的表达,可以将抗体的重链(H链)或轻链(L链)分别整合到表达载体中并同时转化宿主,或者也可以将编码H链和L链的DNA整合到单个表达载体中,转化宿主(参考WO94/11523)。
使用抗体进行如下所述的免疫测定(免疫学测定方法)等时,为了可以检测抗体的行为,一般用各种物质标记抗体本身。本发明的单克隆抗体的优选形式,可列举标记的单克隆抗体。标记抗体可以通过使用例如“分子细胞生物学基础实验方法”(南江堂 堀江武一等人,1994年出版)等记载的常规方法来进行。各种物质可列举化学发光物质、酶、荧光物质、有色珠子、放射性同位素、元素、金属、生物素等。下文示例了具体实例,但不限于此。化学发光物质指例如鲁米诺和吖啶酯等。酶指例如β-半乳糖苷酶、碱性磷酸酶和过氧化物酶等。荧光物质指例如铕的穴状化合物(europium cryptate)、FITC和RITC等。着色珠子指例如蛋白A珠子、小麦胚芽凝聚素(WGA)珠子、链霉亲和素珠子等。放射性同位素指例如14C、125I和3H等。元素指例如铕等镧系元素。金属指例如铁蛋白和金胶粒等。
II.免疫测定
本发明的单克隆抗体可以特异性识别胶原新表位片段的C末端新表位结构。此外,该新表位是不依赖于胶原类型的。因此,例如,通过与能够识别各种胶原的特异性表位的抗体(制备方法参见上文)组合进行夹心测定,可以对任何胶原定量胶原新表位片段。对各类型胶原的特异性序列是本领域技术人员公知的。例如,可列举以下序列。
I型胶原特异性序列:Gly-Ser-Pro-Gly-Ala-Asp-Gly-Pro-Ala(序列号11)
II型胶原特异性序列:Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser(序列号12)
III型胶原特异性序列:Gly-Glu-Lys-Gly-Ser-Pro-Gly-Ala-Gln(序列号13)。
不论本发明的单克隆抗体是否被标记,其都可用于免疫测定(免疫学测定方法)中。使用本发明的单克隆抗体的免疫测定可以是竞争性的测定,也可以是非竞争性的测定。“在竞争性测定中,抗体的免疫反应的50%抑制浓度在0.04μM以下”意指,例如当添加了0.04μM的由序列号14、17或18所示氨基酸序列组成的肽时,由序列号2所示氨基酸序列组成的肽与抗体的结合的抑制率在50%以上(实施例2)。50%抑制浓度优选在0.04μM以下,更优选在0.022μM以下。此外,可以是均质测定法(由均质的体系测定),也可以是异质测定法(由非均质的体系测定)。具体而言,可列举例如酶免疫测定法(EIA)、酶联免疫吸附测定法(ELISA)、荧光免疫测定法(FIA)、放射性免疫测定法(RIA)、时间分辨荧光免疫测定(TR-FIA)、化学发光免疫测定法、免疫印迹法、Western印迹法、免疫染色法、SPA法、荧光偏振测定法(FP)、荧光共振能量转移(FRET)等。
本发明的免疫测定的优选形式可列举ELISA法。ELISA法是使用酶标记的抗体或抗原通过标记酶的活性定量抗体或抗原的量的方法。该方法使用用酶标记的抗原抗体复合物与游离的标记抗原,或用于分离抗体的固相化抗体或抗原。固相可利用琼脂、微量滴定板的内表面、乳胶粒子等。ELISA法具体可列举竞争性免疫测定或夹心免疫测定等。此外,标记的酶可列举辣根过氧化物酶(下文也称为HRP)、碱性磷酸酶等。
III.实用性
本发明的单克隆抗体和免疫测定可用于各种用途中。例如,本发明的单克隆抗体和免疫测定可用于测定MMP等胶原酶的活性中。已知三类胶原酶可以切割未变性状态下的作为纤维状胶原的I型、II型、III型胶原的三重螺旋链(Pendas AM等人,Genomics (1995) 26: 615-8;和Mitchell PG等人, J Clin Invest (1996) 97: 761-8)。本发明的单克隆抗体可以特异性识别由胶原酶切割产生的新表位片段,因此可以测定所述片段的量,可评估胶原酶的活性。
此外,本发明的单克隆抗体和免疫测定可用于以胶原酶新表位片段的含量为指标的筛选方法中。此类筛选是在存在测试物质时,将通过表达载体等制备的重组胶原酶(纯化或部分纯化的)维持在可以与所述酶的底物(胶原)结合的条件下(例如0.1 M磷酸缓冲溶液,pH 7.4,室温),研究测试物质是否抑制该酶与底物结合,换言之定量胶原新表位片段。此时,测试物质可以是肽、蛋白质、非肽类化合物、合成化合物(低分子化合物等)、发酵产物、细胞提取液、植物提取液、动物组织提取液等的任一种。此外也可以是含有上述物质的样品。
通过筛选,作为胶原酶抑制剂鉴别出的候选物质可以作为已知与胶原酶相关的疾病(例如骨关节炎、以癌症为首的增殖性疾病、骨质疏松、阿兹海默氏病、高血压)的预防、治疗剂。
此外,本发明的单克隆抗体和免疫测定可用于疾病患者的鉴别方法,所述方法包括测定包含在生物样品中的胶原新表位片段含量的步骤。例如,本发明的免疫测定可以测定来自患者的生物样品(在临床采样中一般被测试的任何生物学液体。例如血液、尿、唾液和汗等体液,且包括细胞和/或组织的提取物和上清液)中的胶原表位片段的含量。此外,生物样品是对患者没有危险、易于获得的,而且测定是简单且便宜的。因此,可以大量日常地诊断以胶原新表位片段的含量为进展指标的疾病(例如,骨质疏松、骨关节炎、慢性类风湿性关节炎,和其他产生骨的良性肿瘤和恶性肿瘤、软骨破坏的疾病)。进一步地,本发明的单克隆抗体和免疫测定能够用于确定患不同类型的骨关节炎或慢性类风湿性关节炎的患者中的进行性软骨胶原降解的程度。
此外,本发明的试剂盒的特征是含有本发明的单克隆抗体作为用于检测被测样品中的胶原新表位片段的结合剂。此类试剂盒更通常含有1个以上用于执行测定所需的构成要素。构成要素可以是标准品、试剂(稀释液、缓冲液等)、容器/或装置。例如,试剂盒内的1个容器可含有结合胶原类型特异性的序列(例如,序列号11-13)的单克隆抗体。此类抗体可以附着在本领域技术人员公知的任何支持材料(例如,微量滴定板中的孔、硝酸纤维素等合适的膜)上提供。进一步的,可以含有应该在测定中使用的构成要素(例如,试剂或缓冲液)。可选地,此类试剂盒还可以用适合直接检测或间接检测抗体结合的上述物质来标记。
在下文中,通过实施例具体说明本发明,但本发明并不限于下列实施例。另外,除非另外指定,则使用记载在Immunochemistry in Practice(Blackwell Scientific Publications)中的方法作为抗体制备方法。此外,除非另外指定,基因工程学技术使用记载在Molecular Cloning:A Laboratory Manual,第2版(Cold Spring Harbor Laboratory)中的方法。
实施例1
制备胶原新表位抗体
(1)抗原免疫:
合成含有Gly-Pro-Hyp-Gly-Pro-Gln-Gly(序列号:2)所示羟脯氨酸的新表位肽(Greiner Bio-one生产)。将10mg合成的新表位肽溶解在1ml含有5mM EDTA的0.1M磷酸盐缓冲液(pH 6.0)中,将10mg的巨匙孔血蓝蛋白(马来酰亚胺化KLH,PIERCE公司生产)溶解在1ml纯化水中,将上述溶液混合,室温下反应4小时,再在4℃反应过夜。用蒸馏水透析上述混合液后,冷冻干燥,获得13mg新表位肽-KLH复合物。
将0.1mg该肽-KLH复合物与弗氏完全佐剂一起施用到7只4周龄的A/J JmsSlc雌性小鼠的腹腔内,作为首次免疫。之后,在21天后、42天后和63天后,将0.1mg肽-KLH复合物与弗氏不完全佐剂一起加强免疫,再在71天后,将0.1mg该肽-KLH复合物悬浮在0.1ml生理盐水中的溶液施用到腹腔内,作为最终免疫。
(2)新表位肽的生物素标记:
将0.2mg合成的新表位肽溶解在0.4ml含有5mM EDTA的0.1M磷酸盐缓冲液(pH 6.0)中,将0.6mg的PEO-马来酰亚胺活化的生物素(PIERCE公司生产)溶解在0.1ml蒸馏水中,将上述溶液混合,室温下反应2小时后,用反相HPLC纯化生物素标记的新表位肽。
(3)制备杂交瘤:
在最终免疫后的第3天,摘出脾脏,回收脾细胞。用50%聚乙二醇4000将脾细胞和小鼠骨髓瘤细胞(p3×63-Ag8.U1,东京肿瘤研究所)融合,用含有次黄嘌呤、氨喋呤和胸苷的培养基选择。
(4)选择新表位抗体:
在细胞融合10天后,使用如下说明的ELISA筛选特异性抗体的生产细胞。在384孔微量滴定板(ヌンク公司生产)的各孔中,添加35μl含0.35μg抗小鼠IgG抗体(Shibayagi公司生产)的Tris缓冲液(50mM Tris-HCl,pH7.5),4℃下固定16小时。用90μl洗涤溶液(含0.01% Tween20的生理盐水)洗涤这些孔1次后,添加90μl Block Ace(大日本制药公司生产),在室温放置2小时,进行封闭(抗小鼠IgG抗体固相化平板)。用90μl洗涤溶液洗涤1次各孔后,将含有15μl杂交瘤培养上清液的10μl缓冲液A(含0.5% 牛血清白蛋白、0.01% Tween80、0.05% Proclin150、0.15M NaCl的50mM Tris缓冲液,pH7.4)和含有0.05ng生物素标记的新表位肽和2ng链霉亲和素-HRP(PIERCE公司生产)的10μl缓冲液A混合,在4℃下反应16小时。
之后,用90μl洗涤溶液洗涤3次各孔后,添加25μl的TMB-Substrate Chromogen(DAKO公司生产),室温下生色30分钟后,添加25μl的0.05M硫酸,终止反应,测量450nm下的吸光度。
根据筛选的结果,在与含羟脯氨酸的新表位肽反应的9个杂交瘤克隆中,选择3个与不含羟脯氨酸的新表位肽也表现出亲和力的杂交瘤克隆。在所获得的克隆中,选择1个克隆,所述克隆不论有无羟基化都对新表位肽表现出强亲和力,且胶原酶消化的II型胶原抑制它与上述新表位肽的结合,将其命名为20A10。使用小鼠单克隆抗体同种分型ELISA试剂盒(BD Biosciences公司生产)研究20A10同种型,结果是IgG1/κ。
实施例2
使用合成肽解析新表位抗体(20A10)的表位
使用具有以下氨基酸序列的新表位肽,通过以下方法进行对新表位抗体(20A10)的新表位识别特异性的研究。
Asp-Gly-Pro-Ser-Gly-Ala-Glu-Gly-Pro-HyP-Gly-Pro-Gln-Gly(对应于序列号20所示氨基酸序列的962-975位,序列号:14)
Gly-Pro-Gln-Gly(对应于972-975位,序列号:15)
Gly-Pro-Pro-Gly-Pro-Gln-Gly-Leu-Ala-Gly-Gln-Arg(对应于序列号20所示氨基酸序列的969-980位,序列号:16)
Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser-Gly-Ala-Glu-Gly-Pro-HyP-Gly-Pro-Gln-Gly(对应于序列号20所示氨基酸序列的957-975位,序列号:17)
Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser-Gly-Ala-Glu-Gly-Pro-Pro-Gly-Pro-Gln-Gly(对应于序列号20所示氨基酸序列的957-975位,序列号:18)。
在96孔微量滴定板(ヌンク公司生产)中添加150μl含有1.5μg抗小鼠IgG抗体(シバヤギ公司生产)的Tris缓冲液(50mM Tris-HCl,pH7.5),4℃下固定过夜。用0.3ml洗涤溶液(含0.01% Tween20的生理盐水)洗涤1次各孔后,添加0.3ml Block Ace(大日本制药公司生产),在室温放置2小时,进行封闭(抗小鼠IgG抗体固相化平板)。用0.3ml洗涤溶液洗涤1次各孔后,将含有0.001-125μM的上述各新表位肽的50μl缓冲液A,与含有0.1ng生物素标记的新表位肽(序列号2)和4ng链霉亲和素-HRP4的50μl缓冲液A,和含有0.5ng新表位抗体(20A10)的50μl缓冲液A分别混合,在4℃下反应16小时。之后,用0.3ml洗涤溶液洗涤3次各孔后,添加0.1ml的TMB-Substrate Chromogen,室温下生色30分钟后,添加0.1ml的0.05M硫酸,终止反应,测量450nm下的吸光度。
其结果是,新表位肽的975位的甘氨酸残基的羧基端是与抗新表位抗体20A10的结合必需的,以及从该末端起上游5个残基以上是必需的(图2的上部分的图和下部分的表格)。
此外,使用这样的肽按上述方法进行竞争性免疫测定,所述肽是在由19个残基组成的II型胶原的C端新表位部分的肽中第971位是羟脯氨酸的肽、和第971位是脯氨酸的肽。其结果是,非羟基化形式的交叉反应性为91%,确定几乎不受自末端起上游第5位的脯氨酸残基是否羟基化的影响(图2的中部的图和下部分的表格)。已报道,人软骨胶原中的第971位脯氨酸残基以81%的比例被羟基化修饰的例子。此外,已报道了被现有技术报道过的新表位抗体9A4对同一位置的脯氨酸羟基化形式的亲和力比对非羟基化形式的亲和力低90倍以上(Downs JT等人,Journal of Immunological methods, 247: 25-34 (2001))。与之相对,20A10可以以相等的亲和力结合非羟基化形式和羟基化形式中的任一种,因此对新表位片段的检测灵敏度高,即使羟基化修饰的比例发生改变也能够正确地定量。
实施例3
评估新表位抗体(20A10)对于胶原类型的特异性
在10μg/10μl的人I型、II型或III型胶原溶液(Chondrex公司生产)中,添加10μl的2 x酶反应缓冲液(含有0.3M NaCl、10mM CaCl2、0.005% Brij35的50mM Tris缓冲液,pH7.6)进行中和,添加0.2μg人活化MMP13(将人Pro-MMP13(Calbiochem公司生产)在1mM APMA中37℃下孵育2小时活化而成),37℃下反应过夜。之后,加入终止液(EDTA,终浓度5mM),形成天然型新表位溶液。
在384孔微量滴定板(ヌンク公司生产)中,添加35μl含0.35μg抗小鼠IgG-Fc抗体(Jackson Immuno Research公司生产)的Tris缓冲液(50mM Tris-HCl,pH7.5),4℃下固定过夜。用90μl洗涤溶液(含0.01% Tween20的生理盐水)洗涤1次各孔后,添加0.1ml Block Ace(大日本制药公司生产),在室温放置2小时,进行封闭(抗小鼠IgG抗体固相化平板)。
用90μl洗涤溶液洗涤1次各孔后,将含有6.4-250nM的天然型新表位溶液的10μl缓冲液A、和含有1ng/ml生物素标记的新表位肽(序列号2)和200ng/ml链霉亲和素-HRP的10μl缓冲液A、和含有15ng/ml的新表位抗体(20A10)的10μl缓冲液A分别混合,在4℃下反应16小时。
之后,用90μl洗涤溶液洗涤3次各孔后,添加25μl的TMB-Substrate Chromogen(DAKO公司生产),室温下生色30分钟后,添加25μl的0.05M硫酸,终止反应,测量450nm下的吸光度。
其结果是,确认抗新表位抗体20A10不与MMP13未消化的胶原反应,但与含有新表位末端的、MMP13消化的胶原特异性反应。此外,可确认20A10以几乎相同的亲和力与I型、II型或III型胶原的任一种的新表位结合(图3)。因此,即使所述抗体与大量未消化的胶原共存,也不产生背景,可以高灵敏度地检测胶原的消化片段,因而能够正确地定量胶原酶活性。此外,通过作为夹心抗体(捕获抗体)与能够识别I型或III型胶原中的特异性部位的抗体组合,也可以测定I型和III型胶原的分解。
实施例4
构建测定II型胶原特异性新表位的体系
为了测定II型胶原,以对应于C末端具有新表位的II型胶原新表位的一部分(对应于序列号20所示氨基酸序列的957-965位)的合成肽Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser(序列号12)作为免疫原,在氨基末端添加半胱氨酸接头,将羧基末端酰胺化。将2.1mg该合成肽溶解在1ml含5mM EDTA的0.1M磷酸盐缓冲液(pH6.0)中,将8mg的巨匙孔血蓝蛋白(马来酰亚胺化KLH,PIERCE公司生产)溶解在3ml含5mM EDTA的0.1M磷酸盐缓冲液(pH6.0)中,将上述溶液混合,室温下反应3小时。
用蒸馏水透析上述混合液后,冷冻干燥,获得8mg II型胶原特异性内部序列肽-KLH复合物。将0.004mg该肽-KLH复合物与弗氏完全佐剂一起施用到4周龄的A/J Jms Slc和Balb/c雌性小鼠各4只的腹腔内,作为首次免疫。
之后,在21天后、42天后和63天后,将0.1mg肽-KLH复合物与弗氏不完全佐剂一起加强免疫,再在71天后,将0.1mg该肽-KLH复合物悬浮在0.1ml生理盐水中的溶液施用到腹腔内,作为最终免疫。将0.2mg合成肽溶解在0.1ml含有5mM EDTA的0.1M磷酸盐缓冲液(pH 6.0)中,将0.25mg的HPDP-生物素(PIERCE公司生产)溶解在0.1ml二甲基甲酰胺中,将上述溶液混合,4℃下反应过夜后,用反相HPLC纯化生物素标记的肽。
在最终免疫后的第3天,摘出脾脏,回收脾细胞。
用50%聚乙二醇4000将脾细胞和小鼠骨髓瘤细胞(p3×63-Ag8.U1,东京肿瘤研究所)融合,用含有次黄嘌呤、氨喋呤和胸苷的培养基选择。在细胞融合10天后,使用如下说明的ELISA筛选特异性抗体的生产细胞。在384孔微量滴定板(ヌンク公司生产)的各孔中,添加35μl含0.35μg抗小鼠IgG抗体(シバヤギ公司生产)的Tris缓冲液(50mM Tris-HCl,pH7.5),4℃下固定16小时。用90μl洗涤溶液(含0.01% Tween20的生理盐水)洗涤1次这些孔后,添加90μl Block Ace(大日本制药公司生产),在室温放置2小时,进行封闭(抗小鼠IgG抗体固相化平板)。用90μl洗涤溶液洗涤1次各孔后,将含有15μl杂交瘤培养上清液的10μl缓冲液A(含0.5% 牛血清白蛋白、0.01% Tween80、0.05% Proclin150、0.15M NaCl的50mM Tris缓冲液,pH7.4)和含有0.05ng生物素标记的肽和2ng链霉亲和素-HRP(PIERCE公司生产)的10μl缓冲液A混合,在4℃下反应16小时。
之后,用90μl洗涤溶液洗涤3次各孔后,添加25μl的TMB-Substrate Chromogen(DAKO公司生产),室温下生色30分钟后,添加25μl的0.05M硫酸,终止反应,测量450nm下的吸光度。根据筛选的结果,选择4个与II型胶原的免疫原肽(序列号12)表现出强亲和力、但不与其他类型的胶原反应的杂交瘤克隆。在所获得的克隆中,选择1个与天然II型胶原反应,但不与I型、III型胶原反应的克隆,将其命名为6G4。6G4不与未变性的II型胶原反应,但与胶原酶消化后的变性II型胶原反应。
通过夹心ELISA方法构建定量测定II型胶原新表位的体系:
合成含有新表位和胶原类型特异性的内部序列的、由22个残基组成的合成肽Gly-Glu-Lys-Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser-Gly-Ala-Glu-Gly-Pro-Hyp-Gly-Pro-Gln-Gly(对应于954-975位,序列号19),作为校正标准肽。制备HRP标记的20A10。即,在0.5ml含有1mg 20A10的IgG级分和5mM EDTA的0.1M磷酸盐缓冲液(pH 6.0)中,添加0.1M巯基乙胺(mercaptoethylamine)水溶液0.05ml,37℃下反应1.5小时后,通过PD-10柱(GE Healthcare公司生产)进行凝胶过滤,分级分离还原型IgG级分。在0.2ml含有1mg过氧化物酶(辣根来源的,Roche公司生产,HRP)和5mM EDTA的0.1M磷酸盐缓冲液(pH 6.0)中,添加Sulfo-SMCC(PIERCE公司生产),室温下反应2小时后,通过PD-10柱(GE Healthcare公司生产)进行凝胶过滤,分级分离马来酰亚胺化的HRP级分。在HRP级分中加入上述20A10的还原型IgG级分,4℃下反应过夜,进行高速凝胶过滤(附带用含有5mM EDTA的0.1M磷酸盐缓冲液pH 6.0平衡的TSK-GEL G3000柱(東ソー公司生产)的LC-6A系统(岛津公司生产)),分级分离约0.5mg HRP标记的20A10级分。在96孔微量滴定板(ヌンク公司生产)的各孔中,添加150μl含有1.5μg的II型胶原内部序列特异性抗体6G4的Tris缓冲液(50mM Tris-HCl,pH7.5),4℃下固定16小时。用300μl洗涤溶液(含0.01% Tween20的生理盐水)洗涤1次这些孔后,添加150μl Block Ace(大日本制药公司生产),在室温放置2小时,进行封闭。用300μl洗涤溶液洗涤1次各孔后,将含有10-500pM标准肽或测试样品的50μl缓冲液A(含0.5% 牛血清白蛋白、0.01% Tween80、0.05% Proclin150、0.15M NaCl的50mM Tris缓冲液,pH7.4)与含有0.05ng HRP标记的新表位抗体20A10的100μl缓冲液A混合,在4℃下反应16小时。之后,用300μl洗涤溶液洗涤3次各孔后,添加100μl的TMB-Substrate Chromogen(DAKO公司生产),室温下生色30分钟后,添加100μl的0.05M硫酸,终止反应,测量450nm下的吸光度。
其结果是,抗体只与胶原酶消化的II型胶原反应,最低检测限度为10pM(图4)。与对上述羟基化形式亲和力低的新表位抗体9A4不同,所述抗体20A10对非羟基化形式和羟基化形式的任一种都以相同的亲和力结合,因而不需要以脯氨酸/羟脯氨酸比例换算测量值,而且能够不受羟基化修饰的影响正确地定量新表位浓度。
实施例5
体外胶原酶活性的测定体系
在96孔微量滴定板(NUNC公司生产)中添加5ng/ml的人II型胶原溶液(Chondrex公司生产),在4℃孵育过夜后,用洗涤缓冲液(0.05M Tris-HCl,pH7.6)洗涤2次,形成胶原包被的平板。在预孵育用的平板(Costar公司生产)中,加入酶反应缓冲液(含有0.3M NaCl、10mM CaCl2、0.005% Brij35的50mM Tris缓冲液,pH7.6)和作为一种人II型胶原酶的人活化MMP13和MMP抑制剂,室温下孵育30分钟后,按上述实施例4说明的,通过夹心ELISA系统测定酶反应溶液中的胶原新表位的量。
其结果是,新表位测量值依赖于所添加的MMP13的用量而增加,而MMP抑制剂用量依赖性地抑制通过该MMP13的新表位产生(图5)。
实施例6
在人软骨细胞培养体系中的胶原酶活性测定体系
在96孔培养平板(住友ベークライト公司生产)中添加10ng/ml的人II型胶原溶液,在4℃孵育过夜后,用培养基(含有0.1mg/ml BSA、ITS、50μM L-抗坏血酸的DMEM培养基)洗涤1次,形成胶原包被的平板。
在包被的平板中,每孔接种4 x 104个正常人来源的软骨细胞(Chondrex公司),用培养基在37℃、5%CO2的条件下培养。1天后,更换培养液,添加1ng/ml人白介素1β(Genzyme公司生产)和10ng/ml 制癌蛋白(Oncostatin) M(Sigma公司生产)以及各种浓度的作为测试对象的MMP抑制剂,再培养2天。4天后,在添加反应终止液(EDTA,终浓度5mM)后回收培养上清液,通过上述实施例4记载的夹心ELISA系统测定其中包含的胶原新表位浓度,从而测量MMP抑制剂的活性。
其结果确认了,通过IL-1β刺激,诱导培养的软骨细胞表达MMP13,促进II型胶原降解,MMP抑制剂用量依赖性地抑制软骨细胞的胶原降解(图6)。由此可知,本测定体系可用于测定检测对象的MMP抑制剂。
实施例7
20A10的氨基酸序列解析
从建系的杂交瘤细胞中,使用RNeasy Mini试剂盒(QIAGEN公司生产)进行RNA提取。使用用于快速扩增cDNA末端的5’RACE Syatem,2.0版(Invitrogen公司生产),从1μg提取的RNA中,扩增DNA片段。所扩增的片段用TOPO TA克隆试剂盒(Invitrogen公司生产)克隆,用Applied Biosystems 3130 Genetic Analyzer(Applied Biosystems公司生产)解析碱基序列。由此指明可变区的氨基酸序列(图7)。
工业实用性
根据本发明,可以按I型、II型、III型胶原的胶原类型分别正确地检测、定量新表位,不受随健康、营养状态而变化的脯氨酸羟基化修饰的影响。特别是在以胶原酶切割II型胶原作为软骨代谢指标的骨关节炎等软骨病变中,可用于评估病情进展或治疗效果的诊断方法或试剂盒。此外,胶原酶切割I型胶原作为全身的结缔组织胞外基质代谢的指标,可用于评估各种内脏的纤维化进展和抗纤维化治疗效果的诊断方法或试剂盒。
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Claims (6)
1.单克隆抗体,其具有:
1)具有序列号3的氨基酸序列的重链可变区;和
2)具有序列号4的氨基酸序列的轻链可变区。
2.使用权利要求1记载的单克隆抗体的非诊断免疫测定。
3.使用权利要求1记载的单克隆抗体测定胶原新表位片段含量的非诊断方法。
4.测定胶原酶活性的非诊断方法,其以使用权利要求1记载的单克隆抗体测定的胶原新表位片段的含量为指标。
5.权利要求1记载的单克隆抗体在制备用于鉴别胶原酶相关性疾病的患者的药物中的用途。
6.权利要求1记载的单克隆抗体在制备用于诊断胶原酶相关性疾病的药物中的用途。
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WO2002068675A2 (en) * | 2001-02-23 | 2002-09-06 | Colorado State University Research Foundation | Product, method and system for identifying collagenase-cleaved type ii collagen |
WO2006080396A1 (ja) * | 2005-01-28 | 2006-08-03 | Shionogi & Co., Ltd. | 組換え蛋白質の定量法 |
WO2006095654A1 (ja) * | 2005-03-07 | 2006-09-14 | Shionogi & Co., Ltd. | レポータ遺伝子アッセイ法 |
-
2010
- 2010-09-16 JP JP2011531960A patent/JPWO2011034128A1/ja not_active Ceased
- 2010-09-16 CN CN201080041396.4A patent/CN102482348B/zh not_active Expired - Fee Related
- 2010-09-16 KR KR1020127006740A patent/KR20120064072A/ko not_active Application Discontinuation
- 2010-09-16 WO PCT/JP2010/066029 patent/WO2011034128A1/ja active Application Filing
- 2010-09-16 US US13/496,483 patent/US20120237948A1/en not_active Abandoned
- 2010-09-16 EP EP10817241.2A patent/EP2479190A4/en not_active Withdrawn
Non-Patent Citations (4)
Title |
---|
A double epitope tag for quantification of recombinant protein using fluorescence resonance energy transfer;Koji Enomoto等;《Analytical Biochemistry》;20081231;第380卷;摘要、第252页左栏第4段 * |
Analysis of collagenase-cleavage of type II collagen using a neoepitope ELISA;James T. Downs等;《Journal of Immunological Methods》;20011231;第247卷;摘要 * |
James T. Downs等.Analysis of collagenase-cleavage of type II collagen using a neoepitope ELISA.《Journal of Immunological Methods》.2001,第247卷 |
Koji Enomoto等.A double epitope tag for quantification of recombinant protein using fluorescence resonance energy transfer.《Analytical Biochemistry》.2008,第380卷 |
Also Published As
Publication number | Publication date |
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CN102482348A (zh) | 2012-05-30 |
KR20120064072A (ko) | 2012-06-18 |
EP2479190A1 (en) | 2012-07-25 |
US20120237948A1 (en) | 2012-09-20 |
WO2011034128A1 (ja) | 2011-03-24 |
EP2479190A4 (en) | 2013-12-04 |
JPWO2011034128A1 (ja) | 2013-02-14 |
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