CN113248609B - 针对再生胰岛衍生蛋白1α的抗体组合以及包含其的检测试剂盒 - Google Patents
针对再生胰岛衍生蛋白1α的抗体组合以及包含其的检测试剂盒 Download PDFInfo
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Abstract
本发明提供一种可用于基于ELISA检测再生胰岛衍生蛋白1α(REG1A)的抗体组合。本发明提供的抗体组合中的每个抗体均能够以高亲和力特异性结合REG1A,且可形成双抗体夹心模式,从而能够对人REG1A进行定性和定量检测。相应地,本发明还提供一种包含所述抗体组合的ELISA检测试剂盒。
Description
技术领域
本发明涉及生物检测领域,具体而言,本发明涉及一种血清学标志物的检测技术,特别涉及一种可用于定量检测血清中再生胰岛衍生蛋白1α的抗体组合以及包含其的试剂盒。
背景技术
再生胰岛衍生蛋白1α(regenerating family member 1alpha,REG1A)属于再生基因(regenerating gene,REG)家族成员,该家族成员的蛋白质拥有部分共同结构,主要在消化***内起作用,与细胞的生长功能密切相关,可影响胰岛细胞、神经细胞、上皮细胞的生长。正常情况下,REG1A主要表达于胰腺,在胃粘膜和肾脏里也有少量表达;在健康的肠道中没有表达,但发生病变时,REG1A表达明显升高。
目前针对REG1A与不同疾病类型之间的关联性开展了大量研究。例如已有研究表明,人血清REG1A可以作为阿尔茨海默病的临床早期诊断标志物;此外,发现该基因在鼻咽癌、膀胱癌、原发性肝癌、乳腺癌、皮肤黑色素瘤等恶性肿瘤的发生发展中具有临床检测意义。而更多研究表明REG1A与胃肠***炎性疾病及肿瘤形成相关。
双抗体夹心ELISA具有操作简单、高效、可靠、适用于大批样本快速筛选的优势,尤其基于单克隆抗体的双夹心ELISA较基于多克隆抗体更稳定,特异性更强。因此,可以尝试针对血清中REG1A,开发能够特异性结合REG1A且能得到有效检测的抗体或抗体组合,从而用于制备可定性或定量检测血清中再生胰岛衍生蛋白1α浓度的检测或诊断试剂。
发明内容
为了解决上述技术问题,本发明的目的是提供一种能够以高灵敏和特异性检测血清中REG1A存在或其浓度的新试剂与方法。
具体地,本发明的目的是提供一种靶向再生胰岛衍生蛋白1α(REG1A)不同表位的抗体组合,该抗体组合中的每个抗体需以高亲和力和特异性结合REG1A,且可形成双抗体夹心模式,从而能够对人REG1A进行定性和定量检测。
基于上述抗体组合,本发明的另一个目的是提供包含所述抗体组合的ELISA试剂盒,例如双抗体夹心ELISA试剂盒,用于待测样本中REG1A的定性和定量检测。
本发明的技术方案如下。
一方面,本发明提供一种抗体组合,所述抗体组合包括:
(i)抗体1,所述抗体1包含重链(HC)和轻链(LC),其中所述重链包含SEQ ID NO. 1所示的重链CDR1(H-CDR1)、SEQ ID NO. 2所示的重链CDR2(H-CDR2)和SEQ ID NO. 3所示的重链CDR3(H-CDR3),所述轻链包含SEQ ID NO. 6所示的轻链CDR1(L-CDR1)、SEQ ID NO. 7所示的轻链CDR2(L-CDR2)和SEQ ID NO. 8所示的轻链CDR3(L-CDR3);和
(ii)抗体2,所述抗体2包含重链(HC)和轻链(LC),其中所述重链包含SEQ ID NO.11所示的重链CDR1(H-CDR1)、SEQ ID NO. 12所示的重链CDR2(H-CDR2)和SEQ ID NO. 13所示的重链CDR3(H-CDR3),所述轻链包含SEQ ID NO. 16所示的轻链CDR1(L-CDR1)、SEQ IDNO. 17所示的轻链CDR2(L-CDR2)和SEQ ID NO. 18所示的轻链CDR3(L-CDR3)。
在本发明的上下文中,所述抗体1和抗体2是能够特异性结合再生胰岛衍生蛋白1α(REG1A)的结合剂。
优选地,所述抗体1的重链包含重链可变区(VH),轻链包含轻链可变区(VL),并且所述抗体1的重链可变区包含SEQ ID NO. 4所示的氨基酸序列,轻链可变区包含SEQ IDNO. 9所示的氨基酸序列;和/或,所述抗体2的重链包含重链可变区(VH),轻链包含轻链可变区(VL),并且所述抗体2的重链可变区包含SEQ ID NO. 14所示的氨基酸序列,轻链可变区包含SEQ ID NO. 19所示的氨基酸序列。
优选地,所述抗体1和抗体2选自鼠抗体、嵌合抗体和人源化抗体。优选地,所述抗体1和抗体2选自单克隆抗体、Fab、Fv和ScFv等抗体形式。
优选地,在本发明提供的抗体组合中,所述抗体1和/或抗体2为单克隆抗体,其由两条重链和两条轻链组成,优选为鼠IgG1/Kappa同种型。在这一方面,抗体的结构示意见图1。
根据本发明的具体实施方式,所述抗体1的重链包含SEQ ID NO. 5所示的氨基酸序列,轻链包含SEQ ID NO. 10所示的氨基酸序列;和/或,所述抗体2的重链包含SEQ IDNO. 15所示的氨基酸序列,轻链包含SEQ ID NO. 20所示的氨基酸序列。
根据本发明的具体实施方式,所述抗体1和/或抗体2带有可检测标记。优选地,抗体2带有可检测标记。所述可检测标记例如酶标记、放射性标记、发光标记、显色标记、半抗原(例如地高辛、生物素)、金属络合物和金属(例如胶体金)。
另一方面,本发明提供所述抗体组合在制备用于检测再生胰岛衍生蛋白1α(REG1A)的试剂中的用途。或者,本发明提供所述抗体组合在制备用于辅助诊断再生胰岛衍生蛋白1α(REG1A)相关疾病的试剂或用于预测再生胰岛衍生蛋白1α(REG1A)相关疾病患病风险的试剂中的用途。优选地,在本发明的上下文中,所述再生胰岛衍生蛋白1α为人再生胰岛衍生蛋白1α(NCBI参考序列:NP_002900.2)。
在本发明的上下文中,所述REG1A相关疾病中REG1A表达升高。优选地,所述REG1A相关疾病为消化***疾病。优选地,所述疾病为消化道疾病,例如炎症性肠病;消化性溃疡;腺瘤性息肉;1期、2期、3期或4期肠癌;胃肠炎;或胃癌。或者,所述疾病为胰腺炎,例如急性胰腺炎。
在本发明的上下文中,所述试剂可以是能够检测来自受试者的生物学样本中REG1A存在或浓度的试剂。在本发明的上下文中,“浓度”是指所述蛋白在生物学样本中的可检测到的量,并且在本文中,“浓度”可与“水平”或“量”互换使用。根据本发明的具体实施方式,REG1A的存在或浓度在全血、血清或血浆中检测得到。
上述检测结果可以用于辅助诊断所述受试者是否患有再生胰岛衍生蛋白1α(REG1A)相关疾病,或者用于预测所述受试者是否处于罹患所述相关疾病的风险。在本发明的上下文中,所述受试者是哺乳动物,优选为灵长类动物,更优选人;所述生物学样本为选自来自受试者的全血、血浆、血清、血细胞、腹水、淋巴液、唾液、痰液、汗液、尿液、粘液、间质液、组织活检和细胞中的一种或多种,优选全血、血清或血浆。
优选地,所述试剂可用于选自以下的检测方法:化学发光免疫分析法、免疫比浊、酶联免疫吸附测定(ELISA)、蛋白质印迹(Western blotting)、抗体微阵列、免疫沉淀、放射免疫测定(RIA)等。优选地,所述试剂为用于ELISA的检测试剂,例如用于双抗体夹心ELISA的检测试剂,其中所述ELISA用于检测全血、血清或血浆中的再生胰岛衍生蛋白1α。优选地,在所述ELISA中,所述抗体1作为捕获抗体(包被抗体),所述抗体2作为检测抗体。
另一方面,本发明还提供一种试剂盒,所述试剂盒包含本发明所述的抗体组合。
所述试剂盒用于检测来自受试者的生物学样本中REG1A的存在或浓度,因此能够用于辅助诊断所述受试者是否患有再生胰岛衍生蛋白1α(REG1A)相关疾病,或者预测所述受试者是否处于罹患所述相关疾病的风险。优选地,所述再生胰岛衍生蛋白1α为人再生胰岛衍生蛋白1α。所述受试者是哺乳动物,优选为灵长类动物,更优选人;所述生物学样本为选自来自受试者的全血、血浆、血清、血细胞、腹水、淋巴液、唾液、痰液、汗液、尿液、粘液、间质液、组织活检和细胞中的一种或多种,优选全血、血清或血浆。所述REG1A相关疾病中REG1A表达升高。优选地,所述REG1A相关疾病为消化***疾病。优选地,所述疾病为消化道疾病,例如炎症性肠病;消化性溃疡;腺瘤性息肉;1期、2期、3期或4期肠癌;胃肠炎;或胃癌。或者,所述疾病为胰腺炎,例如急性胰腺炎。
所述试剂盒可用于选自以下的检测方法:化学发光免疫分析法、免疫比浊、酶联免疫吸附测定(ELISA)、蛋白质印迹(Western blotting)、抗体微阵列、免疫沉淀、放射免疫测定(RIA)等。优选地,所述试剂盒为ELISA检测试剂盒,例如双抗体夹心ELISA检测试剂盒,所述检测试剂盒包含本发明所述的抗体组合。优选地,所述ELISA检测试剂盒包括所述抗体1作为捕获抗体,包括所述抗体2作为检测抗体。优选地,抗体2带有可检测标记。所述可检测标记例如酶标记、放射性标记、发光标记、显色标记、半抗原(例如地高辛、生物素)、金属络合物和金属(例如胶体金)。
更优选地,所述检测试剂盒还包括采用ELISA检测生物学样本中REG1A的存在或浓度所需的其他试剂。例如,所述检测试剂盒还包括以下中的一种或多种,甚至全部:再生胰岛衍生蛋白1α的校准品和/或质控品;抗体稀释液;洗涤液;封闭液;待测样本稀释液;酶标板;显色液;终止液。
还一方面,本发明提供一种检测来自受试者的生物学样本中再生胰岛衍生蛋白1α的方法,所述方法包括:采用本发明所述的抗体组合或试剂盒,检测来自受试者的生物学样本中再生胰岛衍生蛋白1α的存在或浓度。
或者,本发明提供一种用于辅助诊断再生胰岛衍生蛋白1α(REG1A)相关疾病的方法或用于预测再生胰岛衍生蛋白1α(REG1A)相关疾病患病风险的方法,所述方法包括:采用本发明所述的抗体组合或试剂盒,检测来自受试者的生物学样本中再生胰岛衍生蛋白1α的浓度。并且,所述方法还包括,将检测到的再生胰岛衍生蛋白1α的浓度与参考水平相比较,在所述生物学样本中REG1A的更高浓度指示着所述受试者患有再生胰岛衍生蛋白1α(REG1A)相关疾病或处于罹患再生胰岛衍生蛋白1α(REG1A)相关疾病的风险。
在这一方面,再生胰岛衍生蛋白1α(REG1A)、REG1A相关疾病、受试者、生物学样本、检测方法等等,如上文所定义。
在本发明提供的上述方法中,所述参考水平是指就再生胰岛衍生蛋白1α在生物学样本中的浓度而言,通过对健康人群和疾病人群的ROC分析和临床需求的综合考虑而确定的值。根据本发明的具体实施方式,所述生物学样本为血清,参考水平为60ng/ml。
根据本发明的具体实施方式,所述方法采用本发明提供的ELISA检测试剂盒,例如双抗体夹心ELISA检测试剂盒进行。根据本发明的具体实施方式,可包括以下步骤:
(1)在酶标板上包被捕获抗体(抗体1),并封闭;
(2)稀释待测样本;
(3)分别向酶标板的不同板孔中加入待测样本,可选地加入校准品和/或质控品,再加入经标记的检测抗体(抗体2),孵育;
(4)洗板;
(5)针对检测抗体上的标记进行检测,例如显色;
(6)终止反应;
(7)读取结果,例如OD值;
(8)以校准品浓度为横坐标,以校准品的检测结果例如OD值为纵坐标,制作标准曲线,然后基于待测样本的检测结果计算样本中再生胰岛衍生蛋白1α的浓度。
具体而言,本发明提供一种针对人再生胰岛衍生蛋白1α(REG1A)的抗体组合,该抗体组合中的每个抗体能够以高亲和力和特异性结合REG1A,且结合该蛋白的不同表位,因此可形成双抗体夹心模式,从而可组合用于REG1A的定性和定量检测。相应地,本发明还提供一种采用该抗体组合的ELISA检测方法以及相应的检测试剂盒。
目前临床上消化***疾病的诊断仍以病理学诊断为主。例如,本领域已知癌胚抗原(CEA)和糖类抗原19-9(CA19-9)可作为肠癌的生物标志物,糖类抗原72-4(CA72-4)可作为胃癌的生物标志物,但是肠癌、胃癌的最终诊断仍需要依靠肠镜、胃镜、超声、成像等再加上病理学检查。
本发明的发明人经过大量前期研究,发现REG1A这种蛋白与肠癌、腺瘤性息肉、胃癌、炎症性肠病、消化性溃疡等多种消化***疾病存在相关性,因此可以通过对生物学样本中存在的REG1A水平的准确检测,实现对这类疾病的辅助诊断或患病风险预测。在这一方面,实验证明,本发明提供的抗体1(捕获抗体)和抗体2(检测抗体)配对能够在血清中准确、特异地定性和定量检测人REG1A。特别是相对于其他抗体组合,本发明的抗体1和抗体2组合时,检测的人REG1A的最佳线性范围超过1000pg/mL,对人血清进行稀释后,可有效测定其中的REG1A浓度,对于浓度极高或极低的样本,可调整样本稀释度,以保证测量结果的准确性,因此完全满足准确检测样本中REG1A水平的要求。
本发明的发明人利用包括炎症性肠病、消化性溃疡、腺瘤性息肉、1期至4期肠癌、胃肠炎、胃癌和急性胰腺炎患者在内的大量血清样本进行了验证。结果表明,采用本发明提供的抗体组合,进行双抗体夹心ELISA,对患者血清样本的检测总灵敏度可达到75%,最高可达到100%,检测结果非常准确,证明本发明的抗体组合以及基于该抗体组合的ELISA(例如双抗体夹心ELISA)检测方法与试剂盒具有良好的临床应用价值。
附图说明
以下,结合附图来详细说明本发明的实施方案,其中:
图1为本发明提供的抗体组合中抗体为单克隆抗体时的结构示意图。
图2显示了抗体配对筛选结果。
图3显示了抗体配对筛选结果。
图4为根据不同血清样本的REG1A检测结果绘制的ROC曲线,其中:
4-1:1期肠癌患者;4-2:2期肠癌患者;4-3:3期肠癌患者;4-4:4期肠癌患者;4-5:全部肠癌患者;4-6:胃癌患者;4-7:胃肠炎患者;4-8:腺瘤性息肉患者;4-9:炎症性肠病患者;4-10:消化性溃疡患者;4-11:急性胰腺炎患者。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解,这些实施例仅用于说明本发明,其不以任何方式限制本发明的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的原料、试剂材料等,如无特殊说明,均为市售购买产品。
实施例中采用的人REG1A重组蛋白为根据NCBI参考序列NP_002900.2重组制备得到。
实施例1 针对人再生胰岛衍生蛋白1α(REG1A)的单克隆抗体的获得
免疫:用人REG1A重组蛋白,采用皮下多点注射的方式对小鼠进行3~4次免疫。免疫后取少量小鼠血液样本进行效价测定,选择效价高的小鼠脾脏进行下一步融合。
细胞融合:取小鼠脾细胞,研磨后过细胞筛,离心并用培养液清洗,然后制成脾淋巴细胞悬液;将骨髓瘤细胞与脾细胞按一定比例混合,加入融合剂使二者进行融合。
筛选:用HAT选择培养液培养,筛选出杂交瘤细胞,同时用ELISA测定培养上清液,筛选出能产生特异性识别人REG1A的抗体的杂交瘤细胞。将筛选得到的阳性杂交瘤细胞用有限稀释法进行克隆,得到稳定的单克隆杂交瘤细胞。
抗体生产:取选定的杂交瘤细胞进行扩大培养,取培养上清液,采用亲和层析的方式进行纯化,得到单克隆抗体。
获得5株单克隆抗体,经检验为鼠IgG1/Kappa同种型,本发明中分别命名为:抗体4H8F1;抗体12F12A10;抗体11D11B11;抗体21B11D2;抗体25F3C4。抗体序列如下:
(i) 4H8F1:
重链(SEQ ID NO. 5(去掉了信号肽);其中,重链可变区为SEQ ID NO. 4;CDR依次为SEQ ID NO. 1/SEQ ID NO. 2/SEQ ID NO. 3):
MGRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSLS TSGMSVG WIRQPSGKGLEWL A HIWWNDDKFYNPALKS RLTISKDTSKNQIFLKIASVVTADSATYYCAR IEEGWFAY WGQGTLVTVST AKTTPPSVY PLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAH PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKV SLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK
轻链(SEQ ID NO. 10(去掉了信号肽);其中,轻链可变区为SEQ ID NO. 9;CDR依次为SEQ ID NO. 6/SEQ ID NO. 7/SEQ ID NO. 8):
MKSQTQVFVFLLLCVSAAHGSIVMTQTPKFLLVSVGDRVTITC KASQTMSNDVA WYQQKPGQSPKLLIY YASNRYT GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC QQDYSSPLT FGAGTKLELK RADAAPTVSIFPPSSEQLT SGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTS PIVKSFNRNEC
(ii) 12F12A10:
重链(SEQ ID NO. 15(去掉了信号肽);其中,重链可变区为SEQ ID NO. 14;CDR依次为SEQ ID NO. 11/SEQ ID NO. 12/SEQ ID NO. 13):
MERHWIFLFLLSITAGVHSQVQLQQSATELARPGASVKMSCKASGYTFT SYMMH WVKQRPGQGLEWIG Y INPSSGYTDYNQKFKD KTTLTADKSSSTAYMQLSSLTSEDSAVYYCAR YRYPHYFDY WGQGTTLTVSS AKTTPPSVY PLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAH PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKV SLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK
轻链(SEQ ID NO. 20(去掉了信号肽);其中,重链可变区为SEQ ID NO. 19;CDR依次为SEQ ID NO. 16/SEQ ID NO. 17/SEQ ID NO. 18):
MDFQVQIFSFLLISASVIISRGQIVLTQSPAIMSASPGEKVTMTC SASSSVSYIH WYQQKSGTSPKRWI Y DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAAIYYC QQWSSNPPT FGAGTKLELK RADAAPTVSIFPPSSEQL TSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTST SPIVKSFNRNEC
(iii) 抗体11D11B11:
重链(SEQ ID NO. 21(去掉了信号肽)):
MSSEHRPLTMNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGSLKLSCAAS GFTFSSFS MSWFRQTP DKRLEWVAT ISSGGSST YYPDSVKGRFTISRDNAKNTLYLQMTSLKSEDTAIFYC RGGYYGNYDPLDY WGQGTSVTV SA AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTW PSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSED DPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP QVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSY SCSVVHEGLHNHHTTKSFSRTPGK
轻链(SEQ ID NO. 22(去掉了信号肽)):
MGIKMESQTQVFVYMLLWLSGVDGDIVMIQSQKFMSTSVGDRVSITCTAS HNVDSN VAWYQQKPGQSPQ ALIY SAS YRYSGVPDRFTGSGSGTDFTLTINNVQSEDLAEYFC QQYNSYPLT FGGGTRLEIK RADAAPTVSIFPPSS EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHK TSTSPIVKSFNRNEC
(iv) 抗体21B11D2:
重链(SEQ ID NO. 23(去掉了信号肽)):
MEWSWVFLFLLSVIAGVQSQAQLQQSGAELVRPGASVTLSCKAS GYIFTDYE MHWVKQTPVHGLEWIGA IDPETGGT AYNQKFKGKARLTADKSSSTAYMELRSLTSEDSAVYYC TIYYTNHFDY WGQGTTLTVSS AKTTPPSVYP LAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHP ASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRI SWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILP PPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRH EGLKNYYLKKTISRSPGK
轻链(SEQ ID NO. 24(去掉了信号肽)):
MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSS QSLVYSNGDTY LYWYLQKPGQSPK LLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPLT FGAGTKLELK RADAAPTVSIFPPSS EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHK TSTSPIVKSFNRNEC
(v) 抗体25F3C4
重链(SEQ ID NO. 25(去掉了信号肽)):
MEWSWVFLFLLAVIAGVQSQVQLQQSGAELVRPGASVTLSCKAS GYTFTDYE VHWVKQTPVHGLEWIGA IDPETGGT AYNLKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYF TIYYTNNFDY WGQGTALTVSS AKTTPPSVYP LAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHP ASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRI SWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILP PPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRH EGLKNYYLKKTISRSPGK
轻链(SEQ ID NO. 26(去掉了信号肽)):
MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSS QSLVYSDGNTY LHWYLQKPGQSPK LLIY KVS YRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPLT FGAGTKLELK RADAAPTVSIFPPSS EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHK TSTSPIVKSFNRNEC
上述序列中,粗体部分为信号肽;斜体部分为可变区,其中带有下划线的为CDR;可变区之后带下划线的为恒定区。
实施例2 捕获抗体与检测抗体的筛选与验证
(一)第一次配对筛选
将实施例1中获得的5个单克隆抗体两两配对,分别作为捕获抗体和检测抗体,测定对抗原人REG1A重组蛋白的检测效果。过程如下:
包被:用稀释液(10mM pH 7.4 PBS)将捕获抗体稀释至2.5μg/mL,作为包被液,向酶标板中加入包被液,100μL/孔,4℃孵育过夜,然后弃掉孔中液体,用洗涤液(10mM pH7.4PBS+0.5%吐温20,PBST)洗板2次;
封闭:向酶标板中加入封闭液(10mM pH7.4 PBS+1% BSA+0.5%吐温20),250μL/孔,37℃孵育2h,然后弃掉孔中液体,用PBST洗板2次;
抗原孵育:用基础稀释液(10mM pH7.4 PBS+0.5% BSA+0.5%吐温20)将REG1A重组蛋白制备不同浓度的溶液,加入酶标板中,100μL/孔,37℃孵育1h,然后弃掉孔中液体,用PBST洗板3次;
检测抗体孵育:用基础稀释液将生物素标记的检测抗体稀释至1μg/mL,加入酶标板中,100μL/孔,37℃孵育1h,然后弃掉孔中液体,用PBST洗板3次;
二抗孵育:用基础稀释液将HRP标记的链霉亲和素(SIGMA)稀释20000倍,加入酶标板中,100μL/孔,37℃孵育45min,然后弃掉孔中液体,用PBST洗板3次;
显色:向酶标板中加入TMB底物液(湖州英创生物科技有限公司),100μL/孔,37℃避光显色15min,然后加入50μL终止液(0.2M硫酸)终止反应;
读数:使用酶标仪在450nm主波长、620/630nm次波长下测定各孔的OD值(450nm主波长下的OD值减去620/630nm次波长下的OD值)。
结果见表1。
根据表1,相比其他组配对抗体,如下三组配对抗体可以得到更好的检测效果:分别作为捕获抗体的4H8F1、21B11D2和25F3C4,作为检测抗体的12F12A10。
(二)第二次配对筛选
对第一次配对筛选得到的3组配对抗体做进一步筛选。过程如下:
包被:用PBS将捕获抗体稀释至1μg/mL,作为包被液,向酶标板中加入包被液,100μL/孔,4℃孵育过夜,然后弃掉孔中液体,用PBST洗板2次;
封闭:向酶标板中加入封闭液,250μL/孔,37℃孵育2h,然后弃掉孔中液体,用PBST洗板2次;
抗原孵育:用基础稀释液将REG1A重组蛋白制备成不同浓度的溶液,加入酶标板中,100μL/孔,37℃孵育1h,然后弃掉孔中液体,用PBST洗板3次;
检测抗体孵育:用基础稀释液将HRP标记的检测抗体稀释30000倍,加入酶标板中,100μL/孔,37℃孵育1h,然后弃掉孔中液体,用PBST洗板3次;
显色:向酶标板中加入TMB底物液,100μL/孔,37℃避光显色15min,然后加入50μL终止液(0.2M硫酸)终止反应;
读数:使用酶标仪在450nm主波长、620/630nm次波长下测定各孔的OD值(450nm主波长下的OD值减去620/630nm次波长下的OD值)。
结果见表2和图2。
从结果可以看出,抗体对21B11D2-12F12A10和25F3C4-12F12A10在REG1A浓度小于500pg/mL的范围内线性较好,抗体对4H8F1-12F12A10最佳线性范围超过1000pg/mL,考虑到人血清样本中的REG1A浓度为ng级,最终选择抗体对4H8F1-12F12A10作为捕获抗体和检测抗体。
(三)捕获抗体和检测抗体的验证
分别用4H8F1、21B11D2和25F3C4作为包被抗体,12F12A10作为检测抗体,对血清样本进行测试。按照上文“(二)第二次配对筛选”所述进行,在检测血清样本时将抗原换成血清样本,以100μL/孔直接加入。相对于标准曲线计算血清样本中的蛋白浓度。
结果见表3和图3。
从标准曲线可以看到,21B11D2-12F12A10、25F3C4-12F12A10这两对抗体在抗原浓度≥10ng/mL时,OD值变化很小,推测此时抗原抗体的反应已达到饱和。这一点也可以由血清样本的检测结果得到证明,这两对抗体测得的血清样本中蛋白浓度均在10ng/mL以内;并且以样本232和242为例,4H8F1-12F12A10检测到这两个样本的蛋白浓度相差较大,但是21B11D2-12F12A10、25F3C4-12F12A10这两对抗体检测到的浓度则无明显差异。
实施例3 采用4H8F1-12F12A10配对抗体对不同样本中REG1A的测定
获取805例正常人血清样本,1924例肠癌(CRC)患者血清样本(1期344例,2期669例,3期567例,4期344例),89例胃癌患者血清样本,198例腺瘤性息肉,34例炎症性肠病,80例消化性溃疡,158例胃肠炎和7例急性胰腺炎。血清样本来自中山大学附属肿瘤医院和南方医科大学珠江医院。
根据上文所述,采用本发明提供的4H8F1-12F12A10抗体组合检测血清样本,4H8F1作为包被抗体(捕获抗体),12F12A10作为检测抗体,制成ELISA试剂盒。以正常人作为对照组,分别与疾病组数据对比,做ROC分析,综合ROC分析结果和临床应用的需求,确定人血清中REG1A浓度的cutoff值为60ng/ml,在此基础上计算得到相应的灵敏度和特异性。结果见表4。
根据以上测得数据,使用GraphPad Prism进行受试者工作曲线分析(ROC),结果见图4。
以上对本发明具体实施方式的描述并不限制本发明,本领域技术人员可以根据本发明作出各种改变或变形,只要不脱离本发明的精神,均应属于本发明所附权利要求的范围。
序列表
<110> 深圳市盛波尔生命科学技术有限责任公司
<120> 针对再生胰岛衍生蛋白1α的抗体组合以及包含其的检测试剂盒
<130> LC20110098
<160> 26
<170> PatentIn version 3.3
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Claims (37)
1.一种抗体组合,所述抗体组合包括:
(i)抗体1,所述抗体1包含重链(HC)和轻链(LC),其中所述重链包含SEQ ID NO. 1所示的重链CDR1(H-CDR1)、SEQ ID NO. 2所示的重链CDR2(H-CDR2)和SEQ ID NO. 3所示的重链CDR3(H-CDR3),所述轻链包含SEQ ID NO. 6所示的轻链CDR1(L-CDR1)、SEQ ID NO. 7所示的轻链CDR2(L-CDR2)和SEQ ID NO. 8所示的轻链CDR3(L-CDR3);和
(ii)抗体2,所述抗体2包含重链(HC)和轻链(LC),其中所述重链包含SEQ ID NO. 11所示的重链CDR1(H-CDR1)、SEQ ID NO. 12所示的重链CDR2(H-CDR2)和SEQ ID NO. 13所示的重链CDR3(H-CDR3),所述轻链包含SEQ ID NO. 16所示的轻链CDR1(L-CDR1)、SEQ ID NO.17所示的轻链CDR2(L-CDR2)和SEQ ID NO. 18所示的轻链CDR3(L-CDR3)。
2.根据权利要求1所述的抗体组合,其特征在于,所述抗体1和抗体2是能够特异性结合再生胰岛衍生蛋白1α(REG1A)的结合剂。
3.根据权利要求1所述的抗体组合,其特征在于,所述抗体1的重链包含重链可变区(VH),轻链包含轻链可变区(VL),并且所述抗体1的重链可变区包含SEQ ID NO. 4所示的氨基酸序列,轻链可变区包含SEQ ID NO. 9所示的氨基酸序列;和,
所述抗体2的重链包含重链可变区(VH),轻链包含轻链可变区(VL),并且所述抗体2的重链可变区包含SEQ ID NO. 14所示的氨基酸序列,轻链可变区包含SEQ ID NO. 19所示的氨基酸序列。
4.根据权利要求3所述的抗体组合,其特征在于,所述抗体1和抗体2选自鼠抗体、嵌合抗体和人源化抗体。
5.根据权利要求3所述的抗体组合,其特征在于,所述抗体1和抗体2选自单克隆抗体、Fab、Fv和ScFv的抗体形式。
6.根据权利要求5所述的抗体组合,其特征在于,所述抗体1和抗体2均为单克隆抗体,其由两条重链和两条轻链组成。
7.根据权利要求6所述的抗体组合,其特征在于,所述抗体1和抗体2均为鼠IgG1/Kappa同种型。
8.根据权利要求6所述的抗体组合,其特征在于,所述抗体1的重链包含SEQ ID NO. 5所示的氨基酸序列,轻链包含SEQ ID NO. 10所示的氨基酸序列;和,所述抗体2的重链包含SEQ ID NO. 15所示的氨基酸序列,轻链包含SEQ ID NO. 20所示的氨基酸序列。
9.根据权利要求1至8中任一项所述的抗体组合,其特征在于,所述抗体1或抗体2带有可检测标记。
10.根据权利要求9所述的抗体组合,其特征在于,所述抗体2带有可检测标记。
11.根据权利要求10所述的抗体组合,其特征在于,所述可检测标记为酶标记、放射性标记、发光标记、显色标记、半抗原、金属络合物或金属。
12.根据权利要求11所述的抗体组合,其特征在于,所述半抗原为地高辛或生物素。
13.根据权利要求11所述的抗体组合,其特征在于,所述金属为胶体金。
14.权利要求1至13中任一项所述的抗体组合在制备用于检测再生胰岛衍生蛋白1α(REG1A)的试剂中的用途。
15.权利要求1至13中任一项所述的抗体组合在制备用于辅助诊断再生胰岛衍生蛋白1α(REG1A)相关疾病的试剂中的用途或用于预测再生胰岛衍生蛋白1α(REG1A)相关疾病患病风险的试剂中的用途。
16.根据权利要求14或15所述的用途,其特征在于,所述再生胰岛衍生蛋白1α为人再生胰岛衍生蛋白1α。
17.根据权利要求15所述的用途,其特征在于,所述REG1A相关疾病中REG1A表达升高。
18.根据权利要求17所述的用途,其特征在于,所述REG1A相关疾病为消化***疾病。
19.根据权利要求18所述的用途,其特征在于,所述消化***疾病为消化道疾病。
20.根据权利要求19所述的用途,其特征在于,所述消化道疾病为炎症性肠病;消化性溃疡;腺瘤性息肉;1期、2期、3期或4期肠癌;胃肠炎;或胃癌。
21.根据权利要求18所述的用途,其特征在于,所述消化***疾病为胰腺炎。
22.根据权利要求21所述的用途,其特征在于,所述胰腺炎为急性胰腺炎。
23.根据权利要求14或15所述的用途,其特征在于,所述试剂用于选自以下的检测方法:化学发光免疫分析法、免疫比浊、酶联免疫吸附测定(ELISA)、蛋白质印迹(Westernblotting)、抗体微阵列、免疫沉淀和放射免疫测定(RIA)。
24.根据权利要求23所述的用途,其特征在于,所述试剂为用于ELISA的检测试剂。
25.根据权利要求24所述的用途,其特征在于,所述检测试剂为用于双抗体夹心ELISA的检测试剂;其中,所述ELISA用于检测全血、血清或血浆中的再生胰岛衍生蛋白1α。
26.根据权利要求25所述的用途,其特征在于,在所述ELISA中,所述抗体1作为捕获抗体,所述抗体2作为检测抗体。
27.一种试剂盒,所述试剂盒包含权利要求1至13中任一项所述的抗体组合。
28.根据权利要求27所述的试剂盒,其特征在于,所述试剂盒为用于选自以下的检测方法的试剂盒:化学发光免疫分析法、免疫比浊、酶联免疫吸附测定(ELISA)、蛋白质印迹(Western blotting)、抗体微阵列、免疫沉淀和放射免疫测定(RIA)。
29.根据权利要求28所述的试剂盒,其特征在于,所述试剂盒为ELISA检测试剂盒。
30.根据权利要求29所述的试剂盒,其特征在于,所述检测试剂盒为双抗体夹心ELISA检测试剂盒。
31.根据权利要求30所述的试剂盒,其特征在于,所述试剂盒包括所述抗体1作为捕获抗体,包括所述抗体2作为检测抗体。
32.根据权利要求31所述的试剂盒,其特征在于,所述抗体2带有可检测标记。
33.根据权利要求32所述的试剂盒,其特征在于,所述可检测标记为酶标记、放射性标记、发光标记、显色标记、半抗原、金属络合物或金属。
34.根据权利要求33所述的试剂盒,其特征在于,所述半抗原为地高辛或生物素。
35.根据权利要求33所述的试剂盒,其特征在于,所述金属为胶体金。
36.根据权利要求27至35中任一项所述的试剂盒,其特征在于,所述试剂盒还包括采用ELISA检测生物学样本中REG1A的存在或浓度所需的其他试剂。
37.根据权利要求36所述的试剂盒,其特征在于,所述其他试剂为以下中的一种或多种:
再生胰岛衍生蛋白1α校准品和/或质控品;抗体稀释液;洗涤液;封闭液;待测样本稀释液;酶标板;显色液;终止液。
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