CN102066940B - 根据对抗Globo H及其片段的抗体量的癌症诊断 - Google Patents

根据对抗Globo H及其片段的抗体量的癌症诊断 Download PDF

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CN102066940B
CN102066940B CN200980123715.3A CN200980123715A CN102066940B CN 102066940 B CN102066940 B CN 102066940B CN 200980123715 A CN200980123715 A CN 200980123715A CN 102066940 B CN102066940 B CN 102066940B
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翁启惠
吴宗益
王正琪
陈铃津
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57415Specifically defined cancers of breast

Abstract

本发明提供一种癌症诊断的方法,其使用含有Gb5以及Globo H、Bb2、Bb3、及/或Bb4的聚糖阵列。

Description

根据对抗Globo H及其片段的抗体量的癌症诊断
相关申请
本案已向美国临时申请案第61/061,968号作过申请的优先权(2008年6月16日申请),其内容在此以其全文并入作为参考资料。
背景技术
含有六糖抗原表位的Globo H在各种癌症中表达,也在正常上皮细胞及腺体组织表达。参见Huang et al.,Proc.Natl.Acad.Sci.USA 103:15-20(2006),Wang et al.,Proc.Natl.Acad.Sci.USA 33:11661-11666(2008),以及Chang et al.,Proc.Natl.Acad.Sci.USA 33:11667-11672(2008)。已有报导指出来自乳癌病患的血清含有高量的抗-Globo H抗体。然而,单以该些抗体的量无法作为乳癌的可靠指标。
发明内容
本发明是根据无法预见的发现:对抗Globo H、其片段Bb2、Bb3、或Bb4的抗体量与对抗Gb5的抗体量的比例在乳癌病患中显著地比在无癌症人类中高。
因此,本发明提出一种癌症诊断的方法,其包括(i)提供一来自疑似患有癌症(如,乳癌、黑色素瘤、神经母细胞瘤、皮肤癌、肝癌、前列腺癌、卵巢癌、结肠癌、胃癌、肺癌、及胰脏癌)者的含有抗体的样本(如,血清样本),(ii)将该样本与Gb5以及Globo H、Bb2、Bb3、或Bb4其中一种或其中多种共同培养以容许该样本中的抗体与该些分子结合,(iii)测量Gb5-结合抗体以及GloboH-结合、Bb2-结合、Bb3-结合、或Bb4-结合抗体此两者的量,并且(iv)根据Globo H-结合、Bb2-结合、Bb3-结合、或Bb4-结合抗体的量与Gb5-结合抗体的量的比例决定该对象是否患有癌症。较高的比例则暗示该对象患有癌症。Globo H、Gb5、Bb2、Bb3、或Bb4可被固定在支持装置上以形成聚糖阵列。在一实例中,该阵列含有Gb5以及Globo H、Bb2、Bb3、及Bb4其中一种。在另一实例中,该阵列含有全部的该些分子。
以Gb5以及Globo H、Bb2、Bb3、及Bb4其中一种或其中多种作为癌症诊断以及生产用于癌症诊断的医疗装置的用途也在本发明范围的中。
本发明的各种具体实例于下文详述。本发明的其它特征将可由下文有关该等各种具体实例的详细叙述及附图以及权利要求范围而清楚呈现。
在不须进一步说明的情形下,相信所属技术领域的技术人员根据本文的叙述可将本发明应用至其最广范围。因此,下文的叙述应仅被视为说明而不以任何方式限制本发明的范围。
附图简要说明
图式首次被描述。
图1是描述Globo H(GH)中的六糖抗原表位及其片段的结构的图式。栏A:六糖抗原表位的结构。栏B:六糖抗原表位及七种其片段的结构。
图2显示聚糖阵列用途的示意图,其模拟癌细胞的表面,用以侦测抗-聚糖抗体。
具体实施方式
除非此处另有说明,否则与本发明有关的科学和技术名词与所属技术领域的技术人员通常所了解的意义相同。如此处所述,除非另有说明,否则下列名词具有其所属的意义。
除非内文需要,否则单数名词应包括复数的涵意以及复数名词应包括单数的涵意。此处所使用的冠词″a″和″an″指该冠词的一或多于一个(即,至少一个)的语法受词。例如,“一组件”意指一个组件或多于一个的组件。
已发现抗-Globo H/抗-Gb5、抗-Bb2/抗-Gb5、抗-Bb3/抗-Gb5、或抗-Bb4/抗-Gb5抗体量的比例在乳癌病患中显著地比在无癌症个体中高。因此,任一该些比例可作为癌症诊断中的可靠指标。
因此,本文中描述一种通过侦测在疑似患有癌症的对象(如,基因上癌症敏感的人类)中对抗Gb5以及Globo H、Bb2、Bb3、或Bb4其中一种或其中多种的抗体的量,并且根据任一上述的量的比例决定该个体是否患有癌症。上述抗体可为IgG、IgM、IgE、IgA、或IgD、或其混合。
Globo H是含有六糖抗原表位(示于图1,栏A),并视情形含有一非糖分子部分的聚糖。其片段(如,Gb5、Bb2、Bb3、及Bb4)是含有该六糖抗原表位以及,如适用,该非糖分子部分的片段的聚糖。七种六糖抗原表位的片段示于图1,栏B。该些寡糖可由常规方法制备。参见,如,Huang et al.,Proc.Natl.Acad.Sci.USA 103:15-20(2006))。较佳地,其可与非糖分子键接者诸如烷基胺(如,(CH2)5NH2),或烷基叠氮(如,(CH2)5N3)复合,其可共价键结至由各种物质(诸如,玻璃、塑胶、尼龙、金属、或硅)制成的支持装置(如,聚合物基板)。GloboH、Gb5、Bb2、Bb3、及Bb4可各别被点于支持装置上设定的地址以形成聚糖阵列。该阵列,其模拟表达含于Globo H、Gb5、Bb2、Bb3、及Bb4(参见图2)当中的寡糖抗体表位的癌细胞表面,可使用来侦测结合至该等寡糖抗体表位的抗体的量。
为实施本发明的方法,本文中描述的聚糖阵列与来自疑似患有癌症的对象的含有抗体的样本共同培养。该样本的实例包括,但不限于,血清、唾液、及淋巴结液。清洗该阵列以去除未结合抗体,然后与可特异性结合至所关注抗体的已标记的二级抗体共同培养,该所关注抗体可为人类IgG、IgA、IgD、IgE、或IgM。再次清洗该阵列以去除未结合的二级抗体分子,且结合的二级抗体所释放的信号对应于目标抗体的量。当在一对象中观察到较高的抗-Globo H/抗-Gb5、抗-Bb2/抗-Gb5、抗-Bb3/抗-Gb5、或抗-Bb4/抗-Gb5抗体量比例,该对象被诊断为患有癌症或具发展癌症的风险。
在无进一步阐述之下,相信本领域技术人员可根据以上说明将本发明运用至其最完全的程度。以下特定实例应被理解为仅是例示说明,而无论如何非以任何方式限制本说明书中的其它部分。所有本文中引用的文献并入本文作为参考资料。
实施例1结合抗体Vk9、Mbr1、及A488至聚糖阵列
根据描述于Huang et al.,Proc.Natl.Acad.Sci.USA 103:15-20(2006))的程序化一锅法(one-pot programmable protocol)制备示于图1的寡糖Globo H、Gb5、Gb4、Gb3、Gb2、Bb4、Bb3、及Bb2。该些寡糖通过描述于Hung et al.and Blixtet al.,Proc.Natl.Acad.Sci.USA 101:17033-17038(2004)的标准微阵列机械排印技术被共价固定于NHS-涂布的玻片(Nexterion H slide(SCHOTT NorthAmerica))上。更特定而言,各寡糖的来自储备溶液(80μM)的等分试样被以16-列(每一寡糖两列)的形式排置于玻片上。
在此研究中使用下列三种抗体:
·Mbr1,小鼠IgM抗-Globo H单克隆抗体,
·VK-9,小鼠IgG抗-Globo H单克隆抗体,及
·A488,抗-小鼠/人类Gb5单克隆抗体。
各抗体在加湿室中与上述玻片(上黏附有寡糖)于0.05%Tween 20/PBS缓冲液(pH 7.4)中共同摇晃培养1h。玻片接着(轮流)以0.05%Tween 20/PBS缓冲液(pH 7.4)清洗三次、以PBS缓冲液(pH 7.4)清洗三次、以及以水清洗三次。接着,该玻片在同一室中与Cy3-复合山羊抗-小鼠IgM(针对MBr1)或IgG(针对VK-9及A488)抗体共同摇晃培养1h。玻片再次以0.05%Tween 20/PBS缓冲液(pH 7.4)清洗三次、以PBS缓冲液(pH 7.4)清洗三次、以及以H2O清洗三次并干燥的。最后,使用微阵列萤光芯片视读器(ArrayWorx microarray reader)在595nm(针对Cy3-复合二级抗体)及488nm(针对A488抗SSEA-3抗原抗体)扫描玻片。
三种抗体全部与上述的聚糖阵列结合。VK9特异性地与Globo H及Bb4结合;Mbr1特异性地与Globo H及Bb4结合并且也与Bb3有低亲和力的结合;以及A488特异性地与Gb5结合。这些结果表明该聚糖阵列能够捕获与GloboH及/或其片段结合的抗体。
实施例2用以在乳癌病患中侦测对抗Globo H及其片段的抗体的聚糖阵列的用途
来自乳癌病患及健康个体的血浆样本以0.05%Tween20/3%BSA/PBS缓冲液(pH 7.4)1∶20稀释,然后在加湿室中与描述于上实施例1中的聚糖阵列玻片共同摇晃培养1h。以0.05%Tween 20/PBS缓冲液(pH 7.4)、PBS缓冲液(pH7.4)、以及水各清洗三次后,该玻片在加湿室中与Cy3-复合山羊抗-人类IgM或IgG抗体共同摇晃培养1h。该玻片接着以0.05%Tween 20/PBS缓冲液(pH7.4)清洗三次、以PBS缓冲液(pH 7.4)清洗三次、以及以H2O清洗三次。干燥后,以微阵列萤光芯片视读器(ArrayWorx microarray reader)在595nm(针对Cy3-复合二级抗体)扫描玻片。
如下表1及表2所示,Globo H-结合IgG/Gb-5-结合IgG(GH/Gb5IgG)以及Globo H-结合IgM/Gb-5-结合IgM(GH/Gb5IgM)的量的比例在乳癌病患的血浆样本中较在健康个体的血浆样本中高很多。如也示出于该两表中,癌症病患的Bb2/Gb5IgG、Bb4/Gb5IgM、Bb3/Gb5IgM、及Bb2/Gb5IgM的比例显著地比健康个体高。这些资料表明上列的比例是诊断癌症的可靠标记。
表1.在乳癌病患(n=58)及健康个体(n=47)中Globo H及其片段Bb2、Bb3、及Bb4与Gb5的IgG量的比例
***:p<0.001,极显著;**:p=0.001-0.01,非常显著
表2.在乳癌病患(n=57)及健康个体(n=47)中Globo H及其片段Bb2、Bb3、及Bb4与Gb5的IgM量的比例
***:p<0.001,极显著;**:p=0.001-0.01,非常显著;*:p=0.01-0.05,显著
实施例3以聚糖阵列追踪以Globo H疫苗诱发的免疫反应的用途
以Globo H-KLH疫苗(Optimer Pharmaceuticals,Inc.,圣地牙哥,加州)每周一次共三周对小鼠(六周龄雌BALB/c小鼠,BioLASCO,台湾)行皮下免疫。对照小鼠注射磷酸盐缓冲盐水(PBS)。最终次免疫后10天自经处理的小鼠收集血清样本。这些样本进行30、120、240、480、及1920倍的连续稀释然后使用描述于上实施例1中的聚糖阵列玻片或通过公知的ELISA(每孔以1.28x10-10mol的Globo H覆布)检验稀释血清样本中抗-Globo H抗体的效价。
Globo H-KLH疫苗诱发在免疫小鼠中抗-Globo H抗体的分泌。参见下表3。也发现上述的聚糖阵列分析比公知的ELISA灵敏许多。
表3.通过聚糖阵列分析及ELISA来测定以Globo H诱发的免疫反应
*计算如下:(免疫后信号强度-免疫前信号强度)/背景信号强度
其它实施例
可依任何的组合结合此专利说明书中所公开的全部特性。此专利说明书中所公开的每一特性可被用于相同、等效或类似目的的另类特性所取代。因此,除非另有说明,否则公开的每一种特性仅为一系列普通等效或类似特性的实施例。
从上述的描述,所属技术领域的技术人员可容易地确认本发明的必要特征,并可对本发明作出各种的改变和修饰以适应各种的用途和状况而不偏离其精神和范围。因此,其它实施例也在权利要求范围的中。

Claims (8)

1.Gb5以及Globo H、Bb2、Bb3及Bb4其中一种或其中多种在制备用于乳癌诊断的医疗装置中的应用,该Gb5以及该Globo H、Bb2、Bb3及Bb4其中一种或其中多种被固定于支持装置上。
2.根据权利要求1所述的应用,将一来自疑似患有乳癌者的含有抗体的样本与Gb5以及Globo H、Bb2、Bb3、及Bb4其中一种或其中多种共同培养。
3.根据权利要求1所述的应用,其特征在于,使用Gb5及Globo H。
4.根据权利要求1所述的应用,其特征在于,使用Gb5及Bb3。
5.根据权利要求1所述的应用,其特征在于,使用Gb5及Bb2。
6.根据权利要求1所述的应用,其特征在于,使用Gb5、Globo H、Bb2、Bb3、及Bb4。
7.根据权利要求2所述的应用,其特征在于,该样本为血清、唾液或淋巴结液。
8.根据权利要求2所述的应用,其特征在于,该样本为血清。
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