CN1138146C - 体外检测体液中是否存在抗-组织转谷氨酰胺酶抗体的方法 - Google Patents
体外检测体液中是否存在抗-组织转谷氨酰胺酶抗体的方法 Download PDFInfo
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- CN1138146C CN1138146C CNB971965269A CN97196526A CN1138146C CN 1138146 C CN1138146 C CN 1138146C CN B971965269 A CNB971965269 A CN B971965269A CN 97196526 A CN97196526 A CN 97196526A CN 1138146 C CN1138146 C CN 1138146C
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Abstract
本发明涉及一种通过与组织转谷氨酰胺酶tTG,含tTG的复合物,及其抗原结构物、免疫活性序列或类似物的免疫反应,来检测体液抗体的方法。该方法可用于诊断和治疗控制那些与针对tTG,含tTG的复合物,及其抗原结构物、免疫活性序列或类似物的免疫反应相关的疾病。本发明还涉及将tTG和这些物质用于诊断和治疗控制,较佳地用于诊断和治疗控制慢性炎性疾病或自身免疫疾病,更佳地用于诊断和治疗控制口炎性腹泻或腹腔病。
Description
本发明涉及一种检测体液中抗体的方法,它通过与组织转谷氨酰胺酶(tissuetransglutaminase,简称为“tTG”),tTG的抗原结构物、其免疫活性序列或类似物之间,以及通过与含有tTG的复合物(tTG-containing compound),该复合物的抗原结构物(structure)、其免疫活性序列或类似物之间的免疫反应而进行检测。该方法可用于诊断和治疗控制与针对tTG,含tTG的复合物及其抗原结构物、免疫活性序列或类似物的免疫反应相关的疾病。因此,本发明还涉及tTG和上述物质在诊断和治疗控制中的用途,较佳地在诊断和治疗控制慢性炎性疾病或自身免疫疾病中,更佳地在诊断和治疗控制口炎性腹泻(sprue)和腹腔病(coeliac disease)中的用途。本发明还涉及一种口服药剂,它含有tTG,含tTG的复合物及其抗原结构物、免疫活性序列或类似物作为活性成分。该药剂可用于治疗伴有针对这些物质的免疫反应的疾病,因为口服施用上述化合物可造成免疫耐受性。
本发明基于一个发现,即组织转谷氨酰胺酶(tTG,EC 2.3.2.13)是口炎性腹泻(sprue)和腹腔病的自身抗原。
基于上述发现,开发出了本发明的检测针对tTG和含tTG的复合物的抗体的免疫学方法。
腹腔病是一种小肠粘膜疾病,初次症状表现主要发生在婴儿后期和刚学步的儿童期。如果相应的临床病症没有在成人期之前发生,就被称为“非热带性口炎性腹泻”。因此,这两种术语描述的是同一种疾病。口炎性腹泻伴有粘膜的炎性改变并导致综合性吸收不良。在大多数病例中,用不含谷蛋白的饮食进行治疗会产生形态上和临床上的反应。
众所周知作为病因的是小麦、大麦、黑麦以及某种程度上燕麦中的谷蛋白,而来自种系发生关系程度较低的其他植物品种(如玉米、水稻和大豆)的谷蛋白却是非致病的。在这些谷蛋白中,致病因子的作用被归咎于醇溶谷蛋白,尤其是α-麦醇溶蛋白(α-gliadin)。
出于该原因,口炎性腹泻主要发生在以小麦为主食的国家中(欧洲、美国、澳大利亚),例如其发病率在丹麦为0.14/1000,在西班牙为0.7/1000,在意大利为1/1000,在德国为0.45/1000,在瑞典为2.42/1000。
然而,最近的研究表明,一种亚临床形式,即粘膜发生形态变化而没有严重症状的情况,比以前据信的更为广泛。因此,在1994年意大利进行的研究揭示,在学龄儿童中的发病率为3.28/1000。在双亲患口炎性腹泻的家系的下一代中,患隐性口炎性腹泻的危险可高达50%。
隐性口炎性腹泻主要是经常伴有多形性皮肤病即疱疹样皮炎,其中可观察到特征性的表皮下小泡,并且在皮肤乳头处有颗粒状IgA沉着。对小肠进行活组织检查会显示不规则的、或重或轻的粘膜损伤。
另一种已了解清楚的相关性可在口炎性腹泻和胰岛素依赖性糖尿病(Diabetes mellitus)、甲状腺疾病和选择性IgA缺陷症之间观察到。
口炎性腹泻除了众多的临床并发症状(例如贫血(它被归咎于维生素B12吸收不良)和维生素K不足(这是导致出血倾向增加的原因))之外,其对肠胃道恶性肿瘤的危险大幅增加起着特殊作用。在高达15%的口炎性腹泻病人(大多数年龄在50岁以上)中,会发展成肿瘤疾病,其中约50%涉及肠T细胞淋巴瘤,约25%涉及食道、口咽和小肠的肿瘤。
口炎性腹泻的治疗包括严格遵守终身无谷蛋白的饮食,其中不仅必须排除由小麦制成的含谷蛋白的产品,而且还必须排除由黑麦、大麦和燕麦制成的产品。对于病人,这意味着在饮食习惯和社交活动两方面受到重大限制。
如果口炎性腹泻的诊断和治疗及时,那么将预后良好。然而,一旦发生并发症,通常不能完全逆转。相反,如果疾病仍然未被认识到并且也没有被治疗,那么吸收不良将导致严重症状。最后,患肠淋巴瘤和其他肠胃道肿瘤的危险将上升。
目前,小肠的活组织检查代表了诊断口炎性腹泻以及食用无谷蛋白饮食后随访标准的最高水平,但是以免疫标记为基础的非侵害性(non-invasive)诊断方法变得越来越重要。因为IgA和IgG类抗体存在于口炎性腹泻病人的血清中,这些抗体一方面针对麦醇溶蛋白,另一方面也针对肌内膜(肌内膜是一种特殊的结缔组织,它含有胶原I、III和V,以及弹性纤维、非胶原蛋白如纤连蛋白和蛋白多糖)的自身抗原,因此可以用ELISA法测试血清中针对麦醇溶蛋白的IgG和IgA抗体,并且用间接免疫荧光法测试针对肌内膜的IgG和IgA抗体。尽管针对麦醇溶蛋白的抗体对口炎性腹泻还没有足够的特异性,但已报道对于针对肌内膜的IgA抗体有高灵敏度和高特异性(97-100%)。然而,需要灵长动物的食道切片来进行免疫荧光检测。目前,还尝试在脐带材料上检测肌内膜抗体。
在1984年,Maury和Teppo(柳叶刀(Lancet)1984;20.892-894)描述了一种90kDa的富含甘露糖的糖蛋白(糖含量20%),它是正常皮肤和小肠粘膜的一种成分。在被检查的20位腹腔病患者中的10位病人和12位患疱疹皮炎(该疾病被认为是腹腔病的表皮症状)中的7个病例中,他们鉴别出在循环系统中能识别该蛋白质的IgG免疫复合物。所述的90kDa糖蛋白被确定为含有20%甘露糖,而tTG是非糖基化的,尽管存在6个糖基化(glycosilation)位点(Ichinose等人,生物化学杂志(J.Biol.Chem.),1990,265:13411-13414)。
在1986年,Maury等人(参见Gut,1986,27:147-152)用腹腔病患者的血清和对照血清对该抗体进行了ELISA测试。腹腔病患者表现出抗体水平明显高于(p<0.001)对照血清的抗体水平。在进一步用无谷蛋白饮食疗法治疗后,该水平降至正常水平。没有证明与抗网硬蛋白的抗体之间的相关性。
通过及时诊断和严格遵守无谷蛋白的饮食,该疾病可以被缓解,因此病人升高的恶性肿瘤患病危险也可被降至正常水平。因此,很有兴趣来开发一种合适的检测口炎性腹泻的分析法。因为患隐性口炎性腹泻的人群仍然属于高危群体,因此所有的有关个体(尤其是家系中的下一代),而且最终所有的学龄儿童(正如目前在意大利已被纳入考虑范围),都应当用灵敏、特异、易操作和低成本的方法进行检查。
然而由于下列原因,目前还不能进行大规模的过筛计划:
-对无症状个体所进行的侵害性十二指肠活组织检查不合理而且过于昂贵。
-基于抗麦醇溶蛋白抗体的ELISA检测法几乎不实用,因为其特异性太差。
-作为通用过筛方法,基于灵长动物食道的,检测IgA类肌内膜抗体的免疫荧光检测法过于昂贵。此外,评估有主观性,而且不能鉴别出IgA不足的口炎性腹泻病人(2%病人)。
因此目前为止,对于口炎性腹泻/腹腔病还没有非侵害性的、特异的、定量的、快速的、简便的、价廉和可行的检测方法和治疗控制手段。
这一问题被本发明所解决。基于惊人的发现,即组织转谷氨酰胺酶(tTG,EC 2.3.2.13)是口炎性腹泻的自身抗原,开发出了权利要求1-6所述的免疫方法,它们检测体液(尤其血清)中抗tTG和含tTG的复合物的抗体。该方法不仅可诊断口炎性腹泻或腹腔病,而且可检测所有伴有针对tTG、含tTG的复合物、及其抗原结构物、免疫活性序列或类似物的免疫反应的疾病。
组织转谷氨酰胺酶属于转谷氨酰胺酶(TG)类。TG(EC 2.3.2.13)是依赖于Ca2+的、催化酰基转移的酶,其中肽键相连的谷氨酰胺残基上的γ-氨甲酰(carboxamide)作为酰基供体。主要地,与蛋白质相连的赖氨酸残基作为酰基受体,因此转移形成了ε-(γ-谷氨酰基)赖氨酸键。TG对于酰基供体的底物特异性非常高(取决于氨基酸序列),但却有异常广谱的受体可供利用(Folk J.E.Annu.Rev.Biochem.,1980;49:517-531)。形成的共价肽键很稳定并且耐蛋白酶,这导致交联的蛋白质对化学、酶或物理作用有更高的抗性。
而且,在各种器官、组织、血浆和间隙体液中广泛存在各种TG,这与被转谷氨酰胺酶修饰的蛋白质存在于血块、细胞膜、表皮的角质层、毛发、指甲、和细胞外基质是相关的(Greenberg C.S.等人,FASEB J.,1991;5:3071-3077)。
所述的转谷氨酰胺酶可以根据它们的物理性质、它们在身体中的位置和它们的一级结构而加以区分。
组织TG(tTG)还被称为细胞的、红血球的、内皮的、胞浆的、肝脏的TG、或II型TG,而且它是分子量为75-85KDa的单体。
包括687个残基的完整氨基酸序列是来自于cDNA。在蛋白质水平,人的酶与来自小鼠巨噬细胞的酶之间有84%同源度,而人和豚鼠的酶之间有81%同源度。通常,在这些物种之间核苷酸的交换对氨基酸序列没有影响。活性中心是高度保守的,在3种物种之间有显著的蛋白质同源性(51个残基中有49个是相同的),而且与XIII因子的a-亚基有高度的蛋白质同源度(75%)(参见Gentile V.等人,生物化学杂志(J.Biol.Chem.),1991;266:478-483;Greenber C.S.等人,FASEB J.,1991;5:3071-3077)。
既没有信号肽,也没有糖基化,而且尽管有多个半胱氨酸残基,但是显然没有二硫键桥连。通过荧光杂交法,人组织转谷氨酰胺酶的基因被定位于染色体20q12(Gentile V.等人,Genomics,1994;20:295-297)。尽管酶的释放机制还不清楚,但是已有明确的证据表明,tTG在细胞内的普遍存在使得它在胞外基质(extracellular matrix,ECM)中具有重要功能。此外,tTG活性所需的胞内Ca2+浓度不可能在生理条件下达到,而在胞外区域是存在足够高的Ca2+浓度的(Gentile V.等人,细胞生物学杂志(J.Cell.Biol.),1992;119:463-474)。
若干研究建立起了tTG与纤连蛋白ECM蛋白之间的联系。除了纤连蛋白之外,ECM分子巢蛋白(nidogen)、N-端前胶原III肽、胶原V和XI、骨连蛋白(osteonectin)(它是一种与微纤丝缔合的糖蛋白)、高分子量的硫酸皮肤素蛋白聚糖(proteoglycan)、和galectin 3外源凝集素都可被鉴定为tTG的特异底物。
通过对培养的WI38细胞(肺胚胎成纤维细胞)的免疫荧光研究,也获得了tTG在伤口愈合中起重要作用的暗示:WI38细胞在正常条件下不表现胞外tTG活性,但是一旦产生人工伤口就在胞外发生此酶的沉积。此酶可能被动地从受损的细胞中释放出之后,首先是非共价地结合于ECM,尤其是结合于纤连蛋白和纤维胶原蛋白,此时酶具有数小时的催化活性(Upchurch,H.F.等人,J.Cell.Physiol.,1991;149:375-382)。使用大鼠模型,同样在人工产生伤口之后检测到5天tTG活性上升(Bowness,J.M.等人,Biochem.Biophys.Acta;1988;967:234-240)。同样,将人红细胞裂解液与血浆进行温育时,释放的tTG与纤连蛋白表现出强亲和力(Loraud,L.等人,美国科学院院报(Proc.Natl.Acad.Sci USA),1988;85:1057-1059)。所有这些发现都表明,结合于ECM的tTG在伤口愈合早期起中心作用,而且它与因子XIII一起使血纤维蛋白稳定化,从而通过胞外蛋白的交联而形成围绕受伤细胞的保护层和稳定的粘性物质。目前,在脊椎动物中还没有检测到一种酶能够酶切tTG所催化形成的极其稳定的蛋白质间的交联键。
基于tTG是口炎性腹泻的自身抗原这一发现,本发明还涉及tTG、含tTG的复合物、及其抗原结构物、免疫活性序列或类似物的用途,它们可用于诊断和治疗控制伴有针对这些物质的免疫反应的有关疾病。具体地,可以用这种方法诊断急性炎性疾病如肺炎、肾小球性肾炎、病毒性肝炎,或慢性炎性疾病如克罗恩氏病(Morbus Crohn)、溃疡性结肠炎(colitis ulcerosa),或自身免疫疾病如自身免疫性肝炎、Sjoegren氏综合症、Wegener氏肉芽肿病、类风湿性关节炎、自发性器官纤维化(如肺纤维化)。特别合适的是用于诊断和治疗控制口炎性腹泻的检测方法。因为该方法可以快速且低成本地进行,因此,它可有效地过筛检查群体是否有tTG抗体。
用于本发明的tTG可以源自动物、合成的或重组的,对于含tTG的复合物(它还额外地含有合并成分,例如与合成肽相连的动物tTG)同样如此。在本发明中,含tTG的复合物应理解为tTG与蛋白质形成的化学复合物,或其类似物。在本发明中,tTG类似物或这些含tTG的复合物的类似物意指所有能与抗tTG或含tTG的复合物(如合成肽)的抗体发生免疫反应的抗原结构物。免疫活性序列意指通过蛋白水解、合成或遗传工程而产生的tTG或含tTG的复合物的片段,以及通过氨基酸替换而获得的各种变异体。
本发明的免疫检测可用公知的方法进行。因此,任何直接(例如使用传感器芯片)或间接程序都可用于检测病人的抗体。
在直接程序中,待检测抗体与抗原的结合是通过化学或物理性质的改变而测定的,因此不需要用标记的结合物进行检测的后续步骤。
根据本发明,宜用免疫分析法,较佳地固相免疫分析法来检测tTG抗体,其中将反应物直接或间接地偶连于易检测的标记物。更佳地,检测可以用ELISA、RIA、或免疫荧光分析法进行。这些检测方法的程序是本领域技术人员众所周知的。
例如在ELISA中,抗原(在本例子中例如是tTG)被直接或间接地连于载体物质(如聚苯乙烯)上。在与待检测的抗体(例如来自病人血清的抗体)一起温育之后,用与酶偶连的物质来直接或间接地检测结合于抗原的抗体。这些物质可以是抗体、抗体片段、或高亲和力的配体,例如可结合于生物素标记物的亲和素。例如,过氧化物酶、碱性磷酸酯酶、β-半乳糖苷酶、脲酶或葡萄糖氧化酶都可以作为酶。例如,可以通过进入生色底物来定量检测结合的酶,并从而定量检测结合的tTG抗体。
在放射性免疫分析中,抗原(例如tTG)也可直接或间接地结合于载体物质如聚苯乙烯。在与待检测的抗体(例如来自病人血清的抗体)一起温育之后,用携带放射性标记如125I的物质来检测结合于抗原的抗体。这些物质可以是抗体、抗体片段、或高亲和力的配体,例如可结合于生物素标记物的亲和素。用合适的测量仪器来定量测定结合放射性。
在免疫荧光分析中,可根据相同的原理,使用携带荧光标记(如异硫氰酸荧光素(FITC))的物质来检测结合于抗原的抗体。这些物质可以是抗体、抗体片段、或高亲和力的配体,例如可结合于生物素标记物的亲和素。然后用合适的测量仪器来定量测定荧光染料的结合量。
根据本发明,还可以在凝集分析或凝胶扩散分析中检测病人的抗体。这些检测分析法也是本领域技术人员所熟悉的。因此,例如在凝胶扩散分析中,将抗原或抗体溶液分别置于琼脂或琼脂糖平板上紧紧毗邻的孔中。这种情况下,抗原溶液可以是例如tTG溶液,而抗体溶液可以是例如血液的血清。当物质扩散出所在孔时,会产生从孔开始的浓度梯度。如果在凝胶中重迭的抗原和抗体浓度在具体的比率之内,而且抗体溶液中含有针对抗原的抗体,那么就会在凝胶中形成看得见的沉淀。
在凝集分析中,将携带抗原(如tTG)的颗粒(如乳胶或聚苯乙烯颗粒)与例如来自血清的抗体进行交联。然后可用例如浊度测定法来检测产生的凝集物。
根据本发明,特别优选的是用IgA-特异性或IgG-特异性ELISA来检测口炎性腹泻病人的血清。已发现,新开发的基于tTG的、检测口炎性腹泻病人血清中IgA抗体的ELISA检测法极其适合诊断和治疗控制口炎性腹泻,因为其灵敏度和特异性高。这在受治疗的病人的随访中也很明显(在治疗中滴度下降)。将本发明的ELISA数据与第三者的免疫荧光评估(检测抗肌内膜的IgA)进行比较,呈现出良好的一致性。不一致的情况主要发生在抗体滴度低的时候,然而这是由于间接免疫荧光法被认为是目前顶尖水平的标准所造成的。这主要是由于主观的评估以及这种已有技术方法同时存在非特异性反应所造成的,
基于其他类别的抗体(例如以IgG抗体为例)的相应检测方法,适用于鉴别IgA缺乏的口炎性腹泻病人,还适用于检测伴有抗tTG的免疫反应的其他疾病。
当在测试体系中使用纯化的来自豚鼠的tTG、人tTG,通过蛋白水解或遗传工程而获得的序列或类似物,以及合成的免疫原性tTG肽时,可进一步改善这种检测方法。在实施例3.3中描述了一种诊断和随访伴有针对tTG的免疫反应的其他疾病的ELISA法。
本发明还涉及权利要求10所述的口服药剂,它用于治疗伴有针对tTG、含tTG的复合物及其抗原结构物、免疫活性序列或类似物的免疫反应的疾病。较佳地,该口服给药形式是片剂或胶囊,其中通过施用tTG、含tTG的复合物及其抗原结构物、免疫活性序列或类似物而产生口服耐受性。一方面,该口服耐受性是通过经口供给自身抗原而实现的。另一方面,有所谓的“旁观者效应”:如果诱导该疾病的自身抗原是未知的,那么在靶器官中接触免疫系统的其他抗原可在某些情况下用于口服治疗,该抗原能够局部刺激抗原特异性的抑制性T细胞,从而抑制全身性免疫应答。只有在较高的抗原剂量时,才会诱发自身反应性T细胞的无反应性。
口服耐受性是治疗各种自身免疫疾病的实用方法。
本发明的药剂宜用于治疗口炎性腹泻,但是也可用于治疗其他慢性炎性肠道疾病和自身免疫性肝炎。
根据本发明,tTG,含tTG的复合物及其抗原结构物、免疫活性序列或类似物以0.01-100毫克/千克体重的剂量进行施用。
本发明的药剂可任选地含有药学上可容忍的佐剂,例如在GALENISM中常用的填料、润滑剂、崩解剂、粘合剂,或释放剂。药物佐剂的比例可根据选定的活性成分种类在大范围内变化,在各种情况下为0.1-20%(重量)。
具体地,本发明的优点可以在口炎性腹泻的检测分析法和治疗控制中体现出来,该分析法是非侵害性、高特异性、和直接针对疾病相关因素的。此外,开发出的该分析法的重要优点是在实用性方面快速、简便和成本低,并且可以在不同的实验室之间实现标准化。由此,该分析法可以有效地过筛人群是否有针对tTG的抗体。
而且,因为分析数据是客观的,所以与涉及主观色彩的免疫荧光评估法相比,这种定量评估法的潜力占优。此外,免疫荧光评估法(尤其在低滴度时)受到同时发生的非特异性反应的阻碍。通过在测试系统中使用特异性自身抗原,可以最大限度地在免疫荧光中消除灵长动物食管材料或脐带上的非特异性反应。
因为该分析法可用于IgA类抗体以及其他类别的抗体,所以IgA缺乏的口炎性腹泻病人也能被鉴别。基于抗tTG抗体的检测法还适用于鉴别、检查和治疗控制其他伴有针对tTG的免疫反应的疾病。
同样,因为tTG被鉴定为口炎性腹泻的自身抗原,因此在口服治疗口炎性腹泻或其他伴有针对tTG的免疫应答的疾病时,可以使用完整的tTG或其免疫活性表位(通过蛋白水解或遗传工程而产生的序列、类似物或合成肽)。
实施例
实施例1自身抗原的分离和特性分析1.1.免疫荧光法,APAAP染色
对在-20℃,于100%甲醇中固定2分钟的各种细胞系进行染色。
在免疫荧光检测中,将制品分别与口炎性腹泻和对照血清一起温育,然后洗涤,再用TRITC标记的兔抗-人IgA抗体进行检测(Schuppan等人,J.Biol.Chem.1990;265,8823-32)。
在细胞与口炎性腹泻血清一起温育、洗涤之后进行APAAP标记,随后用APAAP复合物进行检测(Cordell J.L.,等人,J.Histochem.Cytochem.1984;32,219-229)。
此处,HT1080(人纤维肉瘤细胞)、WI38(人肺胚胎成纤维细胞)、Hepl和HepG2(肝癌细胞)都明确地显示出与病人血清反应的阳性胞质信号,而正常血清或用人IgA预处理后都没有显示出标记。人包皮成纤维细胞、人横纹肌肉瘤(RD)/大鼠Ito/大鼠Morris肝细胞瘤、和狗MDCK细胞表现出的反应仅为非常弱至阴性。1.2代谢细胞标记和自身抗原的免疫沉淀
对自身抗原的定性和分离是用HT1080细胞进行。
用L-丙氨酰基-L-谷氨酰胺,10%胎牛血清(FCS,Gibco),100U/毫升青霉素,和100微克/毫升链霉素(Seromed),在Dulbecco改进的Eagle培养基(DMEM,Gibco)中于37℃和8%二氧化碳下培养细胞。对于代谢标记,将细胞转入直径5厘米的培养皿中。一旦达到约90%汇合度,就将其保持在不含甲硫氨酸和FCS的培养基中,之后该培养基被3毫升无FCS但含35S-甲硫氨酸(0.2mCi,Expre35S35S,NEN-Dupont)的培养基替换。在温育16-20小时后,除去上清液。用磷酸盐缓冲液(PBS,Seromed)洗涤细胞,然后用3毫升裂解缓冲液(50mM Tris-HCl,150mMNaCl,0.5%Triton X-100,0.5%IGEPAL CA-630非离子型洗涤剂[Sigma],Complete蛋白酶抑制剂[Boehringer],pH7.5)进行裂解。随后,使用CNBr-活化的Sepharose 4B(Pharmacia),用培养基和细胞裂解液两者进行免疫沉淀。
活化和结合于Sepherose都根据制造商的说明进行。在膨胀和用1mM HCl(pH2.5)洗涤之后,将CNBr-活化的Sepharose 4B与针对人IgA的兔抗体(Dianova,2.4毫克抗体/毫升Sepharose)在0.1M碳酸氢钠,0.5M氯化钠,pH 8.3,于4℃一起温育过夜。未结合的抗体通过偶联缓冲液的洗涤而除去,然后在室温下加入1M乙醇胺(pH 9.0,2小时)而使未被占据的结合位点饱和。随后,用0.1M乙酸钠、0.5M氯化钠(pH 4.0)和0.1M Tris-HCl,0.5M氯化钠(pH 8.0)交替地洗涤Sepharose 3次(每次10×体积)。再将Sepharose分别与口炎性腹泻病人和健康人的血清(0.5毫升血清/毫升Sepharose),在偶联缓冲液(50mM Tris-HCl,150mM氯化钠,lmM氯化钙,1mM氯化镁,pH 8.0)中4℃温育过夜。过量的血清抗体通过偶联缓冲液洗涤3次而去除。
每次,将1毫升HT1080的培养基或代谢标记细胞的细胞裂解液(约5×104细胞)与50微升C14B Sepharose(Pharmacia),在室温下预温育30分钟,以去除非特异性结合的蛋白。在离心(10,000×g,4℃,5分钟)之后,将各上清液与50微升Sepharose在4℃温育搅拌过夜,其中该Sepharose早已分别结合有病人或对照个体的IgA。然后,Sepharose沉积物每次用3×1毫升洗涤缓冲液(10mM Tris-HCl,1%IGEPAL CA-630[Sigma],0.5%脱氧胆酸钠,0.1%月桂基磺酸钠,Complete蛋白酶抑制剂[Boehringer],pH 8.0)洗涤,再用1毫升10mM Tris-HCl(pH 8.0)洗涤。接着,沉淀物被置于SDS分析缓冲液中,于还原或非还原条件下在95℃孵育5分钟,在10-12.5%SDS聚丙烯酰胺凝胶上进行分离(Lammli,U.K.,自然(Nature)1970;227,680-685),然后用放射自显影进行检测(图1)。
在进一步检查中,发现结合的来自培养基的高分子量蛋白是纤连蛋白,它可非特异地结合于Sepharose。
然而,一种与细胞结合的85kDa蛋白质,在这种方式下可被全部30种所用的口炎性腹泻血清所沉淀,而用15种对照血清(其中包括正常血清、溃疡性结肠炎和Sjoegren氏综合症病人的血清)却没有沉淀。从这一事实得出结论:该蛋白质是口炎性腹泻的主要自身抗原。
用硝酸银对凝胶进行蛋白质染色(Henkeshoven,J.等人,Electtrophoresis 1985;6,103-112),在放射性自显影时可见的85kDa条带处有一明显的蛋白质条带。
1.3.85kDa自身抗原的分离和纯化
为了分离数量更多的自身抗原,总计培养了65个培养皿(每个175平方厘米)的HT1080细胞(约109细胞)。在得到汇合前不久,用无FCS的培养基替换原培养基,再在二氧化碳孵育箱中孵育16-20小时。如上分别进行裂解和免疫沉淀。Sepharose沉积物在总共4.5毫升含2%DL-二硫苏糖醇(Sigma)的SDS分析液中,于95℃温育5分钟,以脱去结合的蛋白质,随后在分析性SDS聚丙烯酰胺凝胶中进行仔细检查。
为了进一步纯化自身抗原,使用Prep Cell(491型,BIO-RAD),如下通过洗脱电泳分离免疫沉淀物:将4.5毫升蛋白质混合物置于圆凝胶(外径:3厘米)的顶部,该凝胶由6.5厘米分离胶(8%聚丙烯酰胺,pH 8.8)和1.5厘米收集胶(4%聚丙烯酰胺,pH 6.8)构成,然后通过电泳进行分离。各蛋白质被收集在洗脱缓冲液(25mM Tris-HCl,0.1M甘氨酸,0.01%SDS,pH 8.3)中,每份洗脱组份为1.2毫升(0.8毫升/分钟)。洗脱组份在SDS-PAGE中仔细检查,含有所需蛋白的组份(约15毫升)被合并,用超滤法(Amicon Centriprep-50,在1,000×g)将其浓缩至总体积约为1毫升。
1.4.自身抗原的蛋白酶消化
在数种经测试的蛋白酶中,内切蛋白酶Asp-N(测序级,BoehringerMannheim)被确定为适用于片段化处理,因为它有重复性较高的切割模式,而且获得的片段可较好地分离。酶/底物的浓度调节至1∶100,消化是在37℃进行30分钟。
1.5.转移至PDVF膜
在纯化的自身抗原被消化之后,在制备性的10%Tricine凝胶上分离各个肽片段(Schagger,H.等人,分析生物化学(Anal.Biochem.)1987;166,368-379)(图2)。然后用含石墨的电极片(Fastblot B32/33,Biometra),按半干的快速印迹(fastblot)程序,在4℃将其转至PVDF膜(聚偏二氯乙烯,ImmobilonTM,Millipore)。为此,将下列各层置于阳极片上:(1)在阳极缓冲液1(300mM Tris-HCl,20%甲醇,pH10.4)中浸泡过的滤纸,(2)在阳极缓冲液2(30mM Tris-HCl,20%甲醇,pH 10.4)中浸泡过的滤纸,(3)在甲醇中活化并在阳极缓冲液2中预平衡过的PVDF膜,(4)Tricine凝胶,(5)在阴极缓冲液(25mM Tris-HCl,40mMε-氨基-正己酸,20%甲醇,pH 9.4)中浸泡过的2片滤纸,(6)阴极片。在180mA下转移进行35分钟以上。
之后,PVDF膜在0.1%考马斯蓝Serva R250,50%甲醇中染色5分钟,用50%甲醇和10%乙酸漂白,用蒸馏水充分洗涤,并空气干燥。小心地切取位于10kDa、14kDa,16kDa和25kDa处的消化后自身抗原的特征性条带,对N末端进行第一轮测序。
1.6.Edman降解法
(根据Edman和Henschen在Needleman,S.B.:蛋白质序列测定(ProteinSequence Determination),Springer Verlag,Berlin 1975;232-279)
用Applied Biosystems 4778型测序仪进行测序,结果得到3段氨基酸序列。将它们与Swiss Prot 31数据库(用PC/GENES,IntelliGenetics)进行比较。根据比较,在不一致性最小的情况下,可以明确地将该3个片段指定为人组织转谷氨酰胺酶(tTG,EC 2.3.2.13,蛋白质谷氨酰胺γ-谷氨酰基-转移酶)。以“单字符”形式表示;X表示不相同。t-转谷氨酰胺酶 28′REKLVVRRGQPFW10kDa片段 REKLVVRRGQPF(S)t-转谷氨酰胺酶 581′DLYLENPEIKIRILG14kDa片段 DLYLENPEIXIXILGt-转谷氨酰胺酶 438′DITHTYKYPE16kDa片段 DITLTYQYP(V)
对于25kDa片段没有明确序列,因为它是肽混合物。
实施例2
证实组织转谷氨酰胺酶(tTG)是口炎性腹泻的自身抗原
2.I.豚鼠tTG的免疫沉淀
可购得的并且与人tTG有序列同源性(>80%)的豚鼠肝脏tTG(Sigma),首先通过凝胶电泳进行分离,以检查其纯度。除了数种其他蛋白质之外,tTG(约50%)是主要条带中的一条。
尽管687个氨基酸的人tTG与690个氨基酸的豚鼠蛋白仅稍有差别,但是这两种蛋白质在SDS聚丙烯酰胺凝胶中的迁移行为很不相同。动物来源的蛋白如预期的那样表现为75-80kDa,而人的蛋白却表现出明显较慢的迁移,如文献中所述的那样(Gentile,V.等人,生物化学杂志(J.Biol.Chem.)1991;266,478-483),尽管明显缺乏N-糖基化却假装成表观分子量为85kDa。
通过免疫沉淀来测试口炎性腹泻血清的人自身抗体与豚鼠tTG的反应性。为此,将500微升裂解液中的4微克tTG(Sigma)和0.5%牛血清白蛋白与偶联于4BSepharose的口炎性腹泻IgA在4℃搅拌过夜,洗涤,在还原条件下在SDS分析缓冲液中煮沸,然后在10%聚丙烯酰胺凝胶上分离(cf.,4.1.2)。此时,产生特异性的预计条带的沉淀(分子量80kDa),但无杂质。
2.2.用Western印迹法证实tTG为自身抗原
在SDS凝胶上分离出2微克豚鼠tTG并转移至硝酸纤维素上之后,将印迹在用2%低脂的脱脂奶粉,0.3%Tween 20,pH 7.3的PBS缓冲液,于4℃封闭过夜。随后与相同缓冲液中的口炎性腹泻血清(1/200)温育1小时,洗涤3次,再与偶联有碱性磷酸酯酶的兔抗入IgA抗体(1/500)温育1小时。印迹用PBS洗涤,并用氮蓝四唑和以5-溴-4-氯-3-吲哚-磷酸为底物进行显色(Blake,M.S.,等人,分析生物化学(Anal.Biochem.)1984;136,175-179)。
75-80kDa条带给出了与口炎性腹泻血清反应的明确阳性信号,这再次证明了口炎性腹泻病人的血清中含有针对tTG的IgA类抗体。而对照血清确没有给出任何信号。
2.3.用间接免疫荧光法证实tTG是肌内膜的自身抗原
来自灵长动物的食管组织切片(Euroimmun,Germany),被用于间接检测口炎性腹泻血清中抗肌内膜的IgA抗体以及它们受tTG抑制的情况。在室温下,将10微升用PBS以1/320稀释过的病人血清,与0.5或10微克豚鼠tTG(Sigma)和10微克BSA(Sigma)一起预孵育1小时之后,在室温和潮湿气氛下将其与该食管切片温育1小时。将口炎性腹泻血清(1/320)和健康个体(1/50)血清分别用作阳性和阴性对照。在切片用PBS/0.2%BSA洗涤3次并空气干燥之后,使用以1/50用PBS稀释的、TRITC标记的兔抗入IgA抗体(Dianova),在室温下检测自身抗原1小时。过量的抗体通过依次用PBS/0.2%BSA,PBS和蒸馏水洗涤而除去。
病人血清显示出IgA类抗体明晰地对ECM进行了染色,该染色可通过加入更高浓度的tTG而被抑制,但是不能通过与BSA的预孵育而加以抑制。使用健康个体血清的对照,没有显示出对食管切片的染色。
实施例3
3.1.开发利用IgA抗体来诊断和随访口炎性腹泻的口炎性腹泻-特异性ELISA法
将100微升PBS中的1微克豚鼠转谷氨酰胺酶(Sigma T-5398)加至聚苯乙烯微孔板的各孔(Greiner Labortechnic,96孔)中,在轻度旋转运动下于37℃孵育2小时。通过用PBS(3×200微升)洗涤而除去未结合的tTG,未被占据的结合位点用含1%牛血清白蛋白的250微升PBS在4℃封闭过夜。在用PBS/0.1%Tween 20(3×200微升)洗涤之后,将各孔依次与PBS/0.1%Tween 20系列稀释的血清稀释液(100微升),在室温下于轻度旋转运动的情况下温育1小时,用PBS/0.1%Tween 20(3×200微升)洗涤,随后与偶联有过氧化物酶的兔抗入IgA抗体(Dianova)(100微升,用PBS/0.1%Tween 20以1/400稀释)于室温下温育1小时。在用PBS洗涤3次之后,在黑暗中于室温下用200微升0.1M柠檬酸缓冲液,17.6mM过氧化氢,5.5mM盐酸邻苯二胺(Sigma),pH 4.2温育30分钟,然后用ELISA读数仪(MRX,DynatechLaboratories)在450纳米处检测形成的染料。
在用无谷蛋白的饮食疗法治疗之前和之后(疾病的活动期和次活跃期),测试20位口炎性腹泻病人的血清。发现该测试系统的灵敏度高,并且数值与口炎性腹泻的活动期有良好的相关性。因遵守饮食而治疗成功就反映为抗tTG的IgA抗体的下降。在健康个体,溃疡性结肠炎、肝硬化、各种肿瘤,Sjoegren综合症等病人的对照血清的低消光对比下(背景水平),高特异性是很明显的(图3)。
3.2开发利用其他类别抗体ELISA法来诊断和随访口炎性腹泻,以IgG抗体为例
因为约2%口炎性腹泻病人的IgA不足,因此测试其血清抗tTG的IgG抗体的灵敏度和特异性。如3.1节中所述进行ELISA,只有偶联过氧化物酶的抗人IgA抗体(Dianova)被抗人IgG抗体(Dianova)所替换。在测试灵敏度方面,在无谷蛋白饮食之前和之后口炎性腹泻病人的数据与用IgA抗体获得的数据相符。
某些对照血清显示出数值稍增加,这与较早的发现相一致,即在IgG类的间接免疫荧光法中肌内膜抗体的特异性下降。
3.3.开发用于诊断和随访伴有针对tTG的免疫反应的其他疾病的ELISA法,以IgG抗体为例
如3.2.章节那样进行ELISA。
患有慢性炎性或自身免疫疾病(溃疡性结肠炎(C.U.),克罗恩氏病、急性自身免疫性肝炎)的病人血清,显示出从轻度到中度增加的数值。
因此,对于抗tTG的自身抗体通过使用IgG-特异性ELISA,可以诊断和随访患伴有针对tTG的免疫反应的其他疾病的病人。
实施例4
组织转谷氨酰胺酶(TGG)在麦醇溶蛋白交联过程中的新功能
尽管tTG所催化的反应有广泛的酰基受体可供利用,但是仅有少数分子可以作为酰基供体。在体外试验中,tTG介导了掺入作用(即将放射性标记的腐胺掺入到麦醇溶蛋白中),从而确立了麦醇溶蛋白作为tTG供体底物的功能。在160微升缓冲液(0.1M Tris-HCl,150mM氯化钠,5mM氯化钙,pH 7.5)中,将1微克底物(麦醇溶蛋白,或对照蛋白如白蛋白),2μCi[3H]-腐胺,和1微克tTG(豚鼠的,Sigma)在37℃下温育2小时。通过加入100微升50%三氯乙酸(TCA)而终止反应,然后蛋白质在4℃沉淀过夜。在离心之后,沉淀物用10%TCA洗涤,溶于SDS分析缓冲液中。然后一方面在SDS PAGE中分离,另一方面进行闪烁计数。在闪烁计数数据和SDS PAGE中,虽然对照被确定为没有掺入腐胺,但是麦醇溶蛋白却清楚地显示出掺入了[3H]-腐胺,这证明了麦醇溶蛋白是tTG的极好底物。
缩 写
Ab 抗体
APAAP 碱性磷酸酯酶抗碱性磷酸酯酶
BSA 牛血清白蛋白
cm 厘米
DMEM Dulbecco改进的Eagle培养基
EC 酶委员会分类
ELISA 酶联免疫吸附分析
ECM 胞外基质
FCS 胎牛血清
h 小时
H2O2 过氧化氢
HLA 人淋巴细胞抗原
IEL 上皮内淋巴细胞
Ig 免疫球蛋白
kDa 千道尔顿
M 摩尔
mA 毫安培
MHC 主组织相容性复合体
min 分钟
mM 毫摩尔
Mr 相对分子量
μg 微克
μl 微升
PAGE 聚丙烯酰胺凝胶电泳
PBS 磷酸盐缓冲液
PLA2 磷酸脂酶A2
PVDF 聚偏二氯乙烯
SDS 十二烷基磺酸钠
TCA 三氯乙酸
TGF 转化生长因子
Tris 三(羟甲基)氨基甲烷
tTG 组织转谷氨酰胺酶
Claims (7)
1.一种体外检测体液中是否存在抗-组织转谷氨酰胺酶抗体的方法,其特征在于,用组织转谷氨酰胺酶、含组织转谷氨酰胺酶的复合物、及其抗原结构物、免疫活性序列或类似物,通过免疫反应来检测,形成免疫复合物就表示存在抗-组织转谷氨酰胺酶抗体。
2.如权利要求1所述的方法,其特征在于,被检测的抗-组织转谷氨酰胺酶抗体是人IgA和/或IgG抗体。
3.如权利要求1或2所述的方法,其特征在于,组织转谷氨酰胺酶或含组织转谷氨酰胺酶的复合物来自于人、动物,或是人工合成或重组的。
4.如权利要求1所述的方法,其特征在于,检测通过将一种反应物直接或间接地偶联于易检测的标记物上而进行。
5.如权利要求4所述的方法,其特征在于,检测是在固相上进行。
6.如权利要求4所述的方法,其特征在于,检测是用酶联免疫吸附分析、放射免疫分析或免疫荧光分析法进行。
7.组织转谷氨酰胺酶,含组织转谷氨酰胺酶的复合物,及其抗原结构物、免疫活性序列或类似物的用途,其特征在于,它们被用于制备诊断自身免疫疾病的试剂。
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DE19630557.8 | 1996-07-18 | ||
DE19630557A DE19630557C2 (de) | 1996-07-18 | 1996-07-18 | Verfahren zum Nachweis von Antikörpern aus Körperflüssigkeiten durch eine Immunreaktion mit Gewebe-Transglutaminase (tTG) sowie die Verwendung von tTG in Diagnose und Therapie |
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CN1225723A CN1225723A (zh) | 1999-08-11 |
CN1138146C true CN1138146C (zh) | 2004-02-11 |
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US (2) | US6319726B1 (zh) |
EP (1) | EP0912898B1 (zh) |
CN (1) | CN1138146C (zh) |
AT (1) | ATE210296T1 (zh) |
AU (1) | AU718797B2 (zh) |
BR (1) | BR9710500B1 (zh) |
CA (1) | CA2260769C (zh) |
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DE (2) | DE19630557C2 (zh) |
DK (1) | DK0912898T3 (zh) |
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CN110055235A (zh) * | 2019-05-14 | 2019-07-26 | 深圳市亚辉龙生物科技股份有限公司 | 用于转谷氨酰胺酶抗体检测的抗原及其制备方法、试剂盒及检测方法 |
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ATE511651T1 (de) | 2007-04-06 | 2011-06-15 | Zedira Gmbh | Transglutaminase 6 als diagnostischer indikator für autoimmunerkrankungen |
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FR2949782B1 (fr) * | 2009-09-04 | 2015-10-16 | Isp Investments Inc | Peptide activateur de la transglutaminase et composition cosmetique ou pharmaceutique le contenant. |
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CN110055235A (zh) * | 2019-05-14 | 2019-07-26 | 深圳市亚辉龙生物科技股份有限公司 | 用于转谷氨酰胺酶抗体检测的抗原及其制备方法、试剂盒及检测方法 |
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