CN113156134A - 用于检测人白介素23的elisa试剂盒及检测方法 - Google Patents
用于检测人白介素23的elisa试剂盒及检测方法 Download PDFInfo
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Abstract
本申请提供了一种抗人白介素23单克隆抗体,及包含所述抗体的ELISA试剂盒及其检测方法。具体来说,本申请所述的ELISA试剂盒包括一对所述抗体,分别用作改良双抗体夹心ELISA方法中的包被抗体和检测抗体。本申请提供的试剂盒可完成人白介素23的定量检测,具有较高的检测灵敏度,开发及应用前景良好。
Description
本案是申请日为2020年11月26日、申请号为202011347406.9、发明名称为“一种抗人白介素23及包含其的试剂盒及其检测方法”的发明专利申请的分案申请。
技术领域
本申请属于免疫分析技术领域,涉及一种结合人白介素23的抗体和定量检测人白介素23的ELISA试剂盒,以及利用该试剂盒进行定量检测的方法。
背景技术
白细胞介素是一组具有重要作用的细胞因子,由免疫细胞或非免疫细胞产生,随着分子生物学和细胞生物学技术的迅速发展,目前至少发现了 38个白细胞介素,分别命名为IL-1~IL-38,功能复杂,成网络,复杂重叠;在免疫细胞的成熟、活化、增殖和免疫调节等一系列过程中均发挥重要作用,此外它们还参与机体的多种生理及病理反应。白介素23(IL-23) 是2000年发现的一种白细胞介素,目前已发现其具有多种生物学功能,并在多种疾病中发挥作用。
IL-23主要由活化的树突状细胞、巨噬细胞及单核细胞等产生,是IL- 12异源二聚体细胞因子家族中新的一员,主要由IL-23p19和IL-12/IL- 23p40两个亚基组成,其中IL-12/IL-23p40是其与IL-12共同含有的亚基。 IL-23p19与IL-12/IL-23p40两个亚基单独存在时,不具由生物学功能,只有二者相互连接形成同源二聚体,才能发挥生物学功能。
IL-23主要通过与其受体相互作用,激活下游信号通路发挥生物学功能。IL-23主要作用于Th17细胞,在Th17细胞的增殖与稳定中发挥重要作用,并能促进Th17细胞产生IL-23、IL-17F及IL-22等细胞因子,这些炎症因子作用于角质形成细胞,导致角质形成细胞活化和过度增殖。活化的角质形成细胞又通过产生大量细胞因子、趋化因子和抗菌肽等,募集并激活T细胞等免疫细胞,形成免疫应答的级联效应,引起银屑病损害的发生。IL-23在关节自身免疫炎症中,也是一个促进因子,如在风湿性关节炎中,IL-23促进IL-17的分泌,从而导致了关节的破坏。因此,在临床治疗的过程中,检测患者血清中IL-23水平及其变化情况具有非常重要的意义。
目前市场上测定白介素23含量的试剂盒产品类型少,且价格昂贵。本发明中的ELISA试剂盒可定量检测细胞培养基中白介素23的含量,操作简单方便,灵敏度高,且具有良好的特异性,对白介素23相关的自身免疫性疾病的发病机制以及药物模型等的研究具有重要的意义。
发明内容
基于上述现有技术中存在的问题,本发明提供了一种针对人白介素23 的高亲和力基于改良双抗体夹心ELISA方法研制的人白介素23ELISA试剂盒,该试剂盒包含一对抗人白介素23单克隆抗体。利用该试剂盒可实现对人白介素23的定量检测。
具体来说,本发明涉及以下内容:
1.一种分离的抗人白介素23单克隆抗体,其特征在于,所述单克隆抗体包含三个重链互补决定区(CDR-H1、CDR-H2、CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2、CDR-L3),其中:
a)CDR-H1(在本说明书中CDR-H1表示重链CDR1)的氨基酸序列如SEQ ID No:1(SYWMN)或SEQ ID No:2(SYGIS)所示;
b)CDR-H2(在本说明书中CDR-H2表示重链CDR2)的氨基酸序列如SEQ ID No:3(YIIPITGYTEYNQKFKD)或SEQ ID No:4 (KISPRSVNAYYNEKFKG)所示;
c)CDR-H3(在本说明书中CDR-H3表示重链CDR3)的氨基酸序列如SEQ ID No:5(GGGNLPY)或SEQ ID No:6(DYSNLIFDY)所示;
d)CDR-L1(在本说明书中CDR-L1表示轻链CDR1)的氨基酸序列如SEQ ID No:7(SASSSVSFTYLY)或SEQ ID No:8(SVSSSISSSNLH) 所示;
e)CDR-L2(在本说明书中CDR-L2表示轻链CDR2)的氨基酸序列如 SEQ ID No:9(SISNLAS)或SEQ ID No:10(GTSSLAS)所示;
f)CDR-L3(在本说明书中CDR-L3表示轻链CDR3)的氨基酸序列如 SEQ ID No:11(QQWSSNPPIT)或SEQ ID No:12(QQWSSYPLT)所示。
2.根据权利要求1所述的单克隆抗体,其特征在于,所述CDR-H1具有如SEQ ID No:1(SYWMN)所示的氨基酸序列,所述CDR-H2具有如 SEQ ID No:3(YIIPITGYTEYNQKFKD)所示的氨基酸序列,所述CDR-H3 具有如SEQ ID No:5(GGGNLPY)所示的氨基酸序列,所述CDR-L1具有如SEQ ID No:7(SASSSVSFTYLY)所示的氨基酸序列,所述CDR-L2具有如SEQ ID No:9(SISNLAS)所示的氨基酸序列,所述CDR-L3具有如 SEQ ID No:11(QQWSSNPPIT)所示的氨基酸序列。
3.根据权利要求1所述的单克隆抗体,其特征在于,所述CDR-H1具有如SEQ ID No:2(SYGIS)所示的氨基酸序列,所述CDR-H2具有如 SEQ ID No:4(KISPRSVNAYYNEKFKG)所示的氨基酸序列,所述CDR- H3具有如SEQ ID No:6(DYSNLIFDY)所示的氨基酸序列看,所述CDR- L1具有如SEQ ID No:8(SVSSSISSSNLH)所示的氨基酸序列,所述CDR- L2具有如SEQID No:10(GTSSLAS)所示的氨基酸序列,所述CDR-L3 具有如SEQ ID No:12(QQWSSYPLT)所示的氨基酸序列。
4.根据权利要求1~3中任一项所述的单克隆抗体,其特征在于,所述单克隆抗体包含抗体重链可变区HCVR和抗体轻链可变区LCVR,其中:
HCVR的氨基酸序列如SEQ ID No:13
(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEW IGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCAR GGGNLPYWGQGTLVTVSA)或SEQ ID No:14
(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEW IGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA RDYSNLIFDYWGQGTTLTVSS)所示;
LCVR的氨基酸序列如SEQ ID No:15
(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWI YSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITF GAGTKLELK)或SEQ ID No:16
(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIF GTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGS GTKLELK)所示。
5.根据权利要求4所述的单克隆抗体,其特征在于,所述抗体重链可变区HCVR具有如序列SEQ ID No:13
(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEW IGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCAR GGGNLPYWGQGTLVTVSA)所示的氨基酸序列,所述抗体轻链可变区 LCVR具有如SEQ ID No:15
(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWI YSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITF GAGTKLELK)所示的氨基酸序列。
6.根据权利要求4所述的单克隆抗体,其特征在于,所述抗体重链可变区HCRV具有如序列SEQ ID No:14
(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEW IGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA RDYSNLIFDYWGQGTTLTVSS)所示的氨基酸序列,所述抗体轻链可变区 LCVR具有如SEQ ID No:16
(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIF GTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGS GTKLELK)所示的氨基酸序列。
7.根据权利要求1~6任一项所述的单克隆抗体,其特征在于,所述单克隆抗体包含重链和轻链,其中:
所述重链的氨基酸序列如SEQ ID No:17
(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEW IGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCAR GGGNLPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVT CNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLT PKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSEL PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFV YSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK)或SEQ ID No:18
(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEW IGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA RDYSNLIFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCL VKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSET VTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTIT LTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVS ELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGS YFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK)所示;
所述轻链的氨基酸序列如SEQ ID No:19
(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWI YSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITF GAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC)或SEQ ID No:20
(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIF GTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGS GTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC)所示;
8.根据权利要求7所述的单克隆抗体,其特征在于,所述单克隆抗体为抗人白介素23单克隆抗体1#,其重链具有如SEQ ID No:17
(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEW IGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCAR GGGNLPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVT CNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLT PKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSEL PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFV YSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK)所示的氨基酸序列,其轻链具有如SEQ ID No:19
(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWI YSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITF GAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC)所示的氨基酸序列。
9.根据权利要求7所述的单克隆抗体,其特征在于,所述单克隆抗体为抗人白介素23单克隆抗体2#,其重链具有如SEQ ID No:18
(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEW IGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA RDYSNLIFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCL VKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSET VTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTIT LTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVS ELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGS YFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK)所示的氨基酸序列,其轻链具有如SEQ ID No:20
(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIF GTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGS GTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC)所示的氨基酸序列。
10.一种检测人白介素23的ELISA试剂盒,其特征在于,所述ELISA 试剂盒包括:
已固定至固相载体或欲固定至所述固相载体的第一抗体(包被抗体);和
经生物素标记的第二抗体(检测抗体);
其中,第一抗体和第二抗体为根据权利要求1~9中任一项所述的单克隆抗体。
11.根据权利要求10所述的ELISA试剂盒,其特征在于,所述第一抗体为抗人白介素23单克隆抗体1#,所述第二抗体为抗人白介素23单克隆抗体2#。
12.根据权利要求10或11所述的ELISA试剂盒,其特征在于,所述 ELISA试剂盒还包括过氧化物酶标记的链霉亲和素、白介素23标准品、底物、包被抗体稀释液、洗液、封闭液/样品稀释液和终止液。
13.根据权利要求10~12中任一项所述的ELISA试剂盒,其特征在于,所述试剂盒是改良双抗体夹心ELISA试剂盒。
14.权利要求10~13中任一项所述的ELISA试剂盒在检测细胞培养液、人体血清中白介素23的用途。
15.一种检测样品中白介素23含量的方法,其特征在于,使用权利要求1~9中任一项所述的抗体,利用改良双抗体夹心法进行ELISA检测。
16.根据权利要求15所述的方法,其特征在于,包括以下步骤,
使用抗人白介素23单克隆抗体1#包被酶标板;
将被测样品加入已包被的酶标板,孵育;
将被生物素标记的抗人白介素23单克隆抗体2#加入到酶标板,孵育;
将过氧化物酶标记的链霉亲和素稀释,加入到酶标板,孵育;
将底物加入酶标板,避光孵育后加入终止液,测定OD值;
利用白介素23标准品的OD值拟合出标准曲线,并将被测样品的OD 值代入方程计算出被测样品中白介素23的含量。
17.根据权利要求15或16所述的方法,其特征在于,所述白介素23 为人白介素23、人鼠嵌合白介素23或人兔嵌合白介素23中的一种。
发明效果
本申请提供了一种新的抗人白介素23单克隆抗体及包含该抗体的人白介素23ELISA试剂盒。本试剂盒应用改良双抗体夹心酶标免疫分析法测定样本中白介素23的水平,并对抗人白介素23抗体进行生物素标记,从而可以与酶标二抗形成生物素-亲合素系统(Biotin-Avidin-System,BAS),其高亲合力的牢固结合可以起到生化反应多级放大的效应,使本申请的试剂盒具有更高的检测灵敏度。同时,该所述试剂盒还能够检测人鼠嵌合白介素23、人兔嵌合白介素23,开发及应用前景良好。
附图说明
图1是抗人白介素单克隆抗体1#和2#与抗原人IL-23结合位点的竞争作用示意图
图2是人IL-23浓度检测标准曲线图
图3是人鼠嵌合IL-23检测结果
图4是人兔嵌合IL-23检测结果
具体实施方式
下面结合具体实施方式对本申请做进一步的详细描述,给出的实施例是为了能够更透彻地理解本发明,并且能够将本发明的范围完整的传达给本领域的技术人员。
本说明书中提及的科技术语具有与本领域技术人员通常理解的含义相同的含义,如有冲突以本说明书中的定义为准。
一般而言,本说明书中采用的术语具有如下含义。
在本说明书中,“分离的”抗体是已经与它的天然环境的组分分离的抗体。在某些实施方案中,将抗体纯化至大于95%或99%纯度,所述纯度通过例如电泳(例如,SDS-PAGE等电聚焦(IEF)、毛细管电泳)或色谱 (例如,离子交换或反相HPLC)来确定。关于评价抗体纯度的方法,是本技术领域公知的,例如可以参见:Flatman等人,J.Chromatogr.B848: 79-87(2007)。
在本说明书中,“单克隆抗体”表示得自基本上同源的抗体的群体的抗体,即,构成所述群体的各个抗体是相同的和/或结合相同表位,除了可能的变体抗体(例如,含有天然存在的突变或在单克隆抗体制品的生产过程中产生)以外,这样的变体通常以微量存在。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制品不同,单克隆抗体制品的每种单克隆抗体针对抗原上的单个决定簇。因而,修饰语“单克隆”指示所述抗体得自基本上同源的抗体群体的特征,并且不应解释为需要通过任何特定方法生产所述抗体。例如,要根据本发明使用的单克隆抗体可以通过多种技术来制备,所述技术包括、但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法、和使用包含人免疫球蛋白基因座的全部或部分的转基因动物的方法,本文描述了这样的方法和其它示例性的制备单克隆抗体的方法。
在本说明书中,“亲和力”表示分子(例如,抗体)的单个结合位点和它的结合配偶体(例如,抗原)之间的非共价相互作用的总和的强度。除非另外指出,否则本说明书中使用的“结合亲和力”表示反映结合对 (例如,抗体和抗原)的成员之间的1∶1相互作用的固有结合亲和力。分子X对它的配偶体Y的亲和力通常可以由平衡解离常数(KD)表示。通过本领域已知的常见方法,可以测量亲和力。
在本说明书中,“人白介素23(Human interleukin 23,hIL-23)表示一种源自人的蛋白,由p19和p40两个亚基组成的异源二聚体。p19氨基酸序列如SEQ ID NO:21所示,p40氨基酸序列如SEQ ID No:22所示,其中,下划线部分表示信号肽。
SEQ ID No:21:
MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAH PLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLI FYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPS LSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP
SEQ ID No:22:
MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVL TCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLS HSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTI STDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSA CPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNS RQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATV ICRKNASISVRAQDRYYSSSWSEWASVPCS
在本说明书中,“抗人白介素23单克隆抗体”表示这样的单克隆抗体:其能够以足够的亲和力结合人白介素23,使得所述单克隆抗体可用作靶向人白介素23的诊断剂和/或治疗剂。
本申请的抗人白介素23(IL-23)单克隆抗体与靶标无关的蛋白不结合。这里,“无关的蛋白”是指除作为靶标的人白介素23以外的其他蛋白;这里,“不结合”是指:在将本发明的抗人白介素23(IL-23)单克隆抗体与作为其靶标的人白介素23的结合能力作为100%的情况下,本申请的抗人白介素23(IL-23)单克隆抗体与所述无关蛋白的结合能力小于10%,例如9%、8%、7%、6%、5%、4%、3%、2%、1%或者0。
本申请的抗人白介素23(IL-23)单克隆抗体与其他动物种属的白介素 23不结合。这里,“其他动物种属”是指除人以外的其他动物种属,例如恒河猴、食蟹猴、大鼠、小鼠等;这里,“不结合”是指:在将本发明的抗人白介素23(IL-23)单克隆抗体与作为其靶标的人白介素23的结合能力作为100%的情况下,本发明的抗人白介素23(IL-23)单克隆抗体与其他动物种属的白介素23的结合能力小于10%,例如9%、8%、7%、6%、 5%、4%、3%、2%、1%或者0。
本申请的抗人白介素23(IL-23)单克隆抗体具有≤1μM、≤100nM、≤50nM、≤40nM的平衡解离常数(KD)。
在本说明书中,ELISA(enzyme linked immune sorbent assay,简写 ELISA),即酶联免疫吸附测定,是指利用抗体分子能与抗原分子特异性结合的特点,将游离的杂蛋白和结合于固相载体的目的蛋白结合,并利用特殊的标记物对其定性或定量分析的一种检测方法。其原理是抗原或抗体能物理性地吸附于固相表面,并且保持其免疫活性;抗原或抗体能与酶通过共价键形成酶结合物,同时保持各自的免疫活性或酶活性;酶结合物与相应的抗原或抗体结合后,能通过加入底物的颜色反应来确定免疫反应的发生,颜色反应的深浅与标本中相应抗原或抗体的量成正比。根据待检测物质以及检测的具备条件,可设计出各种不同类型的检测方法,双抗体夹心法是检测抗原最常用的方法。其是将含有已知抗体的抗血清吸附在微量滴定板上的小孔里,洗涤一次;加待测抗原,如两者是特异的,则发生结合,然后把多余抗体洗除;加入与待测抗原呈特异反应的酶联抗体,使形成“夹心”;加入该酶的底物,若看到有色的酶解产物产生,说明在存在相应的抗原。
在本说明书中,“亲和素(avidin)”是一种糖蛋白,每个分子由4个亚基组成,可以和4个生物素分子亲密结合。使用更多的是从链霉菌中提取的链霉亲和素(streptavidin)。
在本说明书中,生物素是动植物体内广发分布的一种小分子生长因子,又名辅酶R或维生素H。亲和素是卵白蛋白中提取的一种碱性糖蛋白,对生物素有非常高的亲和力。结合了酶的亲和素分子与结合有特异性抗体的生物素分子产生反应,既起到了多级放大作用,又由于酶在遇到相应底物时的催化作用而显色,达到测定其所结合的抗体量的作用。因此把亲和素和生物素与ELISA结合起来,就可大大提高ELISA的灵敏度。生物素-亲合素系统在ELISA中的应用有多种形式,可用于间接包被,亦可用于终反应放大。在常规ELISA中的酶标抗体也可用生物素化的抗体替代,然后连接亲和素-酶偶联物,以放大反应信号。
具体的,本申请涉及一种分离的抗人白介素23单克隆抗体,所述单克隆抗体包含三个重链互补决定区(CDR-H1、CDR-H2、CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2、CDR-L3),其中:CDR-H1的氨基酸序列如SEQ ID No:1(SYWMN)或SEQ ID No:2(SYGIS)所示;CDR- H2的氨基酸序列如SEQ ID No:3(YIIPITGYTEYNQKFKD)或SEQ ID No:4(KISPRSVNAYYNEKFKG)所示;CDR-H3的氨基酸序列如SEQ ID No:5(GGGNLPY)或SEQ ID No:6(DYSNLIFDY)所示;CDR-L1的氨基酸序列如SEQ ID No:7(SASSSVSFTYLY)或SEQ ID No:8(SVSSSISSSNLH)所示;CDR-L2的氨基酸序列如SEQ ID No:9 (SISNLAS)或SEQ ID No:10(GTSSLAS)所示;CDR-L3的氨基酸序列如SEQ ID No:11(QQWSSNPPIT)或SEQ ID No:12(QQWSSYPLT)所示。
在一个具体的实施方式中,本申请所提供的抗体的CDR-H1具有如 SEQ ID No:1(SYWMN)所示的氨基酸序列,CDR-H2具有如SEQ ID No:3(YIIPITGYTEYNQKFKD)所示的氨基酸序列,CDR-H3具有如SEQ ID No:5(GGGNLPY)所示的氨基酸序列,CDR-L1具有如SEQ IDNo:7 (SASSSVSFTYLY)所示的氨基酸序列,CDR-L2具有如SEQ ID No:9 (SISNLAS)所示的氨基酸序列,CDR-L3具有如SEQ ID No:11 (QQWSSNPPIT)所示的氨基酸序列。
在一个具体的实施方式中,本申请所提供的单克隆抗体的CDR-H1具有如SEQ IDNo:2(SYGIS)所示的氨基酸序列,CDR-H2具有如SEQ ID No:4(KISPRSVNAYYNEKFKG)所示的氨基酸序列,CDR-H3具有如 SEQ ID No:6(DYSNLIFDY)所示的氨基酸序列看,CDR-L1具有如SEQ ID No:8(SVSSSISSSNLH)所示的氨基酸序列,CDR-L2具有如SEQ ID No:10(GTSSLAS)所示的氨基酸序列,CDR-L3具有如SEQ ID No:12 (QQWSSYPLT)所示的氨基酸序列。
在一个具体的实施方式中,上述单克隆抗体包含抗体重链可变区 HCVR和抗体轻链可变区LCVR,其中:HCVR的氨基酸序列如SEQ ID No:13
(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEW IGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCAR GGGNLPYWGQGTLVTVSA)或SEQ ID No:14
(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEW IGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA RDYSNLIFDYWGQGTTLTVSS)所示;LCVR的氨基酸序列如SEQ ID No:15
(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWI YSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITF GAGTKLELK)或SEQ ID No:16
(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIF GTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGS GTKLELK)所示。
在一个具体的实施方式中,本申请所提供的单克隆抗体的HCVR具有如序列SEQ IDNo:13
(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEW IGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCAR GGGNLPYWGQGTLVTVSA)所示的氨基酸序列,LCVR具有如SEQ ID No:15
(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWI YSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITF GAGTKLELK)所示的氨基酸序列。
在一个具体的实施方式中,本申请所提供的单克隆抗体的HCRV具有如序列SEQ IDNo:14
(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEW IGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA RDYSNLIFDYWGQGTTLTVSS)所示的氨基酸序列,LCVR具有如SEQ ID No:16
(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIF GTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGS GTKLELK)所示的氨基酸序列。
在一个具体的实施方式中,上述单克隆抗体包含重链和轻链,其中:
所述重链的氨基酸序列如SEQ ID No:17
(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEW IGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCAR GGGNLPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVT CNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLT PKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSEL PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFV YSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK)或SEQ ID No:18
(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEW IGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA RDYSNLIFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCL VKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSET VTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTIT LTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVS ELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGS YFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK)所示;所述轻链的氨基酸序列如SEQ ID No:19
(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWI YSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITF GAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC)或SEQ ID No:20
(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIF GTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGS GTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC)所示。
在一个具体的实施方式中,本申请的抗人白介素23(IL-23)单克隆抗体为抗人白介素23单克隆抗体1#,其重链具有如SEQ ID No:17
(VQLQQSGAELAKPGASVKMSCKASGYTFSSYWMNWIRQRPGQGLEW IGYIIPITGYTEYNQKFKDMATLTADKSSSTAFMQLSSLTSEDSAVYYCAR GGGNLPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVT CNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLT PKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSEL PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFV YSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK)所示的氨基酸序列,其轻链具有如SEQ ID No:19
(QIVLTQSPAIMSASPGERVTMTCSASSSVSFTYLYWYQQKSGSSPKLWI YSISNLASGVPARFSGSGSGTSYSLTINTMEAEDAATYYCQQWSSNPPITF GAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC)所示的氨基酸序列。
在一个具体的实施方式中,本申请的抗人白介素23(IL-23)单克隆抗体为抗人白介素23单克隆抗体2#,其重链具有如SEQ ID No:18
(QVHLQQSGAELARPGASMKLSCKASGYTFTSYGISWVKQRTGQGLEW IGKISPRSVNAYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA RDYSNLIFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCL VKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSET VTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTIT LTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVS ELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGS YFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK)所示的氨基酸序列,其轻链具有如SEQ ID No:20
(EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIF GTSSLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGS GTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC)所示的氨基酸序列。
实验结果显示,本申请的抗人白介素23(IL-23)单克隆抗体可以特异性结合人白介素23(IL-23)的p19亚基。
本申请的抗人白介素23(IL-23)单克隆抗体在诸多生物活性方面与上市同类单抗产品相当、或优于上市同类单抗产品。所述生物活性例如抑制 IL-23诱导的细胞中STAT3磷酸化的活性、抑制IL23诱导的小鼠脾脏细胞释放IL-17A的活性、抑制IL-23诱导的人NK细胞释放IFN-γ活性。
进一步的,本申请中的抗人白介素23单克隆抗体均为鼠单克隆抗体。
本申请还涉及一种检测人白介素23的ELISA试剂盒,其特征在于,所述ELISA试剂盒包括:已固定至固相载体或欲固定至所述固相载体的第一抗体(包被抗体);经生物素标记的第二抗体(检测抗体)。
在一个具体的实施方式中,本申请所述ELISA试剂盒中的抗人白介素单克隆抗体包括第一抗体和第二抗体,其中第一抗体为包被抗体,其已固定至固相载体或欲固定至所述固相载体上;第二抗体为检测抗体,其由生物素进行标记。优选的,所述固相载体为酶标板。进一步优选的,为96孔酶标板,具体采用美国Corning Coster的聚苯乙烯塑料板。
具体的,所述第一抗体和第二抗体为为本申请所提供的抗人白介素23 单克隆抗体。
本申请所提供的试剂盒为双抗夹心测定试剂盒,即利用双抗体夹心法来检测样本中的细胞和/或血清中白介素23物质。具体的方法为,将已知量的第一抗体,即包被抗体,与固相载体表面结合。然后将被测样品施加至所述表面,使得其中所存在的任何细胞和/或血清中的白介素23物质被已固定的第一抗体俘获。通过一个或多个洗涤步骤优选地移除未结合的物质。然后加入由生物素进行标记的第二抗体,即检测抗体,并且允许结合被所述第一抗体所俘获的任何细胞和/或血清中的白介素23物质,使每一单位的白介素23同时结合两种抗体形成“夹心”。然后在直接或间接检测方法中测定所结合的第二抗体的量。具体的,标记或酶可以直接地或经由诸如生物素-链霉亲和素或生物素-亲和素连结之类的连结间接地连接至所述第二抗体。
在一个具体的实施方式中,所述ELISA试剂盒中的第一抗体为抗人白介素23单克隆抗体1#,其重链具有如SEQ ID No:17所示的氨基酸序列,轻链具有如SEQ ID No:19所示的氨基酸序列;第二抗体为抗人白介素23 单克隆抗体2#,其重链具有如SEQ ID No:18所示的氨基酸序列,轻链具有如SEQ ID No:20所示的氨基酸序列。
进一步的,所述ELISA试剂盒中还包括对于检测白介素23所必需的装置或试剂。
具体的,所述ELISA试剂盒中还包括过氧化物酶标记的链霉亲和素、白介素23标准品、底物、包被抗体稀释液、洗液、封闭液/样品稀释液和终止液。
在一个具体的实施方式中,所述过氧化物酶优选为辣根过氧化物酶 (HRP),其标记的链霉亲和素为链霉亲和素-辣根过氧化物酶偶联物 (SA-HRP)。
在一个具体的实施方式中,所述试剂盒中的白介素23标准品为人IL- 23蛋白;底物为3,3’,5,5’-四甲基联苯胺(TMB);包被抗体稀释液为磷酸盐缓冲液(PBS)pH7.4;洗液为磷酸盐缓冲液(PBS)含0.05% Tween20;封闭液/样品稀释液为磷酸盐缓冲液(PBS)含0.5%BSA、0.05% Tween20和0.05%Proclin300;终止液为磷酸。
优选的,所述ELISA试剂盒包括以下成分:
1)固相载体:酶标板,1块;
2)包被抗体:抗人白介素23单克隆抗体1#(即第一抗体),10μg/ 管,1管;
3)检测抗体:抗人白介素23单克隆抗体2#(即第二抗体)(以生物素标记),10μg/管,1管;
4)标准品:人IL-23蛋白,10ng/管,1管;
5)链霉亲和素-辣根过氧化物酶偶联物(Streptavidin-HRP,简称SA- HRP),500ng/管,1管;
6)底物:3,3’,5,5’-四甲基联苯胺(TMB),5ml/管,A液、B液各1 管;
7)包被抗体稀释液:磷酸盐缓冲液(PBS),pH 7.4,50ml/瓶,1 瓶;
8)洗液:磷酸盐缓冲液(PBS),含0.05%Tween20,200ml/瓶,1 瓶;
9)封闭液/样品稀释液:磷酸盐缓冲液(PBS)含0.5%BSA、0.05% Tween20和0.05%Proclin300,200ml/瓶,1瓶;
10)终止液:1M磷酸,10ml/管,1管。
本申请还涉及一种检测样品中白介素23含量的方法,其特征在于,使用本申请所提供的抗人白介素23单克隆抗体抗体,利用改良双抗体夹心法进行ELISA检测。
具体的,所述方法包括以下步骤,
使用抗人白介素23单克隆抗体1#(即第一抗体)包被酶标板;
将被测样品加入已包被的酶标板,孵育;
将被生物素标记的抗人白介素23单克隆抗体2#(即第二抗体)加入到酶标板,孵育;
将过氧化物酶标记的链霉亲和素稀释,加入到酶标板,孵育;
将底物加入酶标板,避光孵育后加入终止液,测定OD值;
利用白介素23标准品的OD值拟合出标准曲线,并将被测样品的OD 值代入方程计算出被测样品中白介素23的含量。
在一个具体的实施方式中,可采用本申请所述的ELISA试剂盒定量检测样品中的人白介素23含量,包括以下步骤:
(1)包被:采用包被抗体稀释液(磷酸盐缓冲液(PBS),pH 7.4) 将包被抗体抗人白介素23单克隆抗体1#(即第一抗体)配制成浓度为1 μg/ml的包被抗体工作液,然后按照50μl/孔的用量加入到酶标板(96孔 Coster酶标板)中,于2~8℃静置过夜;
(2)封闭:弃掉酶标板中的包被抗体工作液,用洗液(磷酸盐缓冲液 (PBS)含0.05%Tween20)洗板3次,然后按照100μl/孔的用量加入封闭液(磷酸盐缓冲液(PBS)含0.5%BSA、0.05%Tween20和0.05% Proclin300),置于摇床(120rpm)室温封闭2小时;
(3)蛋白标准品配制:取8个EP管并依次编号,从第2管起每管加入200μl样品稀释液并置于EP管架上;用样品稀释液将标准品蛋白(人 IL-23蛋白)配制成浓度为25ng/ml的液体,吸取400μl加入到第1个EP 管中,然后从第1个EP管中吸取200μl液体,加入到第2个EP管中进行二倍稀释,依此类推至第10个EP管,其中25ng/ml、0.049ng/ml点作为锚定点;
(4)质控样品配制:取5个EP管,分别从25ng/ml取样,配制成 12.5ng/ml、10ng/ml、5ng/ml、0.2ng/ml、0.098ng/ml的质控样品;
(5)加样:弃掉酶标板中的封闭液,用洗液洗板3次,然后按照50 μl/孔的用量加入蛋白标准液和质控样品,置于摇床(120rpm)室温孵育2 小时;
(6)加检测抗体:弃掉酶标板中的蛋白标准液和待测样品,用洗液洗板3次,用样品稀释液将检测抗体(抗人白介素23单克隆抗体2#(即第二抗体),以生物素标记)配制成浓度为0.2μg/ml的检测抗体工作液,然后按照50μl/孔的用量加入酶标板中,置于摇床(120rpm)孵育反应2小时;
(7)加酶标二抗:弃掉酶标板中的检测抗体工作液,用洗液洗板3 次,用样品稀释液将酶标二抗(链霉亲和素-辣根过氧化物酶偶联物(SA- HRP))配制成浓度为100ng/ml的酶标二抗工作液,然后按照50μl/孔的用量加入酶标板中,置于摇床(120rpm)室温孵育1小时;
(8)显色:将底物的A液和B液(3,3’,5,5’-四甲基联苯胺 (TMB),5ml/管,A液、B液各1管)等体积混合;弃掉酶标板中的酶标二抗工作液,用洗液洗板3次,然后按照50μl/孔的用量加入底物,室温避光反应5~10分钟;
(9)终止显色:将终止液(1M磷酸)按50μl/孔的用量加入酶标板中终止反应,然后应用酶标仪在450nm波长下测定酶标板中各孔的OD 值,根据标准品的OD值拟合出标准曲线,并将质控样品的OD值代入方程,计算出质控样品的浓度。
本申请还涉及将本申请提供的抗人白介素23单克隆抗体、包含所述抗人白介素23单克隆抗体的ELISA试剂盒以及所述检测样品中白介素23含量的方法应用于检测细胞培养液、人体血清中白介素23的用途。
与现有技术相比,本发明的人IL-23ELISA试剂盒不仅具有较高的检测灵敏度,并且能够检测人鼠嵌合IL-23、人兔嵌合IL-23,具有良好的开发及应用前景。
实施例
以下将结合具体实施方式说明本申请内容,但本申请范围不限于此。
如果没有特殊说明,以下实施方式中使用的试剂和仪器都是本领域常规试剂和仪器,可以通过商购方式获得。所使用的方法均为常规实验方法,本领域技术人员根据实施例内容可以毫无疑问地实施所述方案并获得相应结果。
实施例1抗人白介素23单克隆抗体的筛选和制备
制备人鼠嵌合白介素23(IL-23),用于免疫Balb/c小鼠,运用杂交瘤技术进行细胞融合,筛选出单克隆细胞株,进而扩大培养获得单克隆抗体。首先,用Binding ELISA检测细胞上清,挑选出与IL-23结合的克隆,再用Blocking ELISA和细胞法进行检测,挑选出具有IL-23抑制活性的克隆,从而筛选获得本发明的第一抗体和第二抗体。本发明所获得的第一抗体和第二抗体为鼠单克隆抗体,未进行人源化改造。以上免疫和筛选过程委托给睿智化学完成。
通过测序获得第一抗体和第二抗体的重轻链完整序列,然后根据Kabat 规则(参考文献:Kabat E.A.,Wu T.T.,Bilofsky H.Attempts to locate residues incomplementarity-determining regions of antibody combining sites that makecontact with antigen.Proc.Natl.Acad.Sci.USA.1976;73:617–619.)即可得到其CDR序列。
实施例2抗人白介素23单克隆抗体的特性鉴定
1、抗体识别抗原位点的竞争实验:
用第一抗体和第二抗体分别包被酶标板,2-8℃过夜,次日室温封闭 2h。梯度稀释的Bio IL-23分别与10μg/ml、1μg/ml、0μg/ml的第一抗体和第二抗体等体积相加后于室温孵育1.5小时。洗板后加入孵育好的抗体与 Bio IL-23复合物,室温孵育2h。洗板后加入稀释好的酶标二抗,于室温孵育1小时。最后洗板后进行显色,并读取OD450处的吸光度。结果如图1 所示,第一抗体和第二抗体与人IL-23抗原的结合位点不竞争,在第一抗体包被酶标板的实验中,加入第二抗体未影响第一抗体与Bio IL-23的结合,同样第二抗体包被酶标板的实验中,加入第一抗体未影响第二抗体与Bio IL-23的结合。可将第二抗体作为人IL-23试剂盒的检测抗体。
2、第二抗体生物素标记:
第二抗体(检测抗体)的生物素标记使用Sulfo-NHS-LC- Biotin试剂盒,具体步骤严格按照试剂盒说明书进行。标记完成后,使用 ZtbaTM Spin DeaslingColums过滤除去游离的生物素,所得溶液即为生物素标记的第二抗体。
实施例3人IL-23ELISA定量检测试剂盒的组成
将所述第一抗体作为包被抗体、所述第二抗体(进行生物素标记)作为检测抗体制备ELISA试剂盒,该试剂盒中的试剂组成、规格、来源以及贮存条件等信息如表1所示。
表1:ELISA试剂盒成分组成
实施例4人IL-23ELISA试剂盒对人IL-23的定量检测
采用实施例3中的人IL-23ELISA试剂盒对质控样品中的人IL-23进行定量检测。
1、样品处理:
用样品稀释液稀释人IL-23蛋白配制蛋白标准品,用细胞培养液稀释人 IL-23蛋白配制质控样品作为待检样品。根据蛋白标准品的吸光值拟合出标准曲线。将待检样品的OD值代入标准曲线,得出待检样品中人IL-23的理论浓度值,然后计算出待检样品的回收率(回收率=理论浓度值/实际浓度值*100),从而分析实施例3中的ELISA试剂盒用于细胞培养液中人IL-23的定量检测的可行性。
2、定量检测操作步骤:
(1)包被:采用包被抗体稀释液将包被抗体配制成浓度为1μg/ml的包被抗体工作液,然后按照50μl/孔的用量加入酶标板中,于4℃静置过夜;
(2)封闭:弃掉酶标板中的包被抗体工作液,用洗液洗板3次,然后按照100μl/孔的用量加入封闭液,置于摇床(120rpm)室温封闭2小时;
(3)蛋白标准品配制:取8个EP管并依次编号,从第2管起每管加入 200μl样品稀释液并置于EP管架上;用样品稀释液将标准品蛋白配制成浓度为25ng/ml的液体,吸取400μl加入到第1个EP管中,然后从第1个 EP管中吸取200μl液体,加入到第2个EP管中进行二倍稀释,依此类推至第10个EP管,其中25ng/ml、0.049ng/ml点作为锚定点;
(4)质控样品配制:取5个EP管,分别从25ng/ml取样,配制成 12.5ng/ml、10ng/ml、5ng/ml、0.2ng/ml、0.098ng/ml的质控样品;
(5)加样:弃掉酶标板中的封闭液,用洗液洗板3次,然后按照50μl/孔的用量加入蛋白标准液和质控样品,置于摇床(120rpm)室温孵育2小时;
(6)加检测抗体:弃掉酶标板中的蛋白标准液和待测样品,用洗液洗板 3次,用样品稀释液将检测抗体(生物素标记)配制成浓度为0.2μg/ml的检测抗体工作液,然后按照50μl/孔的用量加入到酶标板中,置于摇床(120rpm) 孵育反应2小时;
(7)加酶标二抗:弃掉酶标板中的检测抗体工作液,用洗液洗板3次,用样品稀释液将酶标二抗配制成浓度为100ng/ml的酶标二抗工作液,然后按照50μl/孔的用量加入到酶标板中,置于摇床(120rpm)室温孵育1小时;
(8)显色:将底物的A液和B液等体积混合;弃掉酶标板中的酶标二抗工作液,用洗液洗板3次,然后按照50μl/孔的用量加入底物,室温避光反应5~10分钟;
(9)终止显色:将终止液按50μl/孔的用量加入到酶标板中终止反应,然后应用酶标仪在450nm波长下测定酶标板中各孔的OD值,根据标准品的 OD值拟合出标准曲线,并将质控样品的OD值代入方程,计算出质控样品的浓度。
3、检测结果:
(1)人IL-23标准品的检测数据如表2所示:
表2人IL-23标准品的检测数据
人IL-23的检测曲线在0.049-25ng/ml浓度范围均能够回收。
(2)曲线方程:4-P Fit:y=(0.0603-2.36)/(1+(x/4.22)^1.1+2.36,R2=1(见图2)。
(3)待测样品检测数据如表3所示:
表3待测样品检测数据
4、准确度、精密度考察:
(1)在2批不同的实验中,每个分析批包含2套待测样品,每套待测样品包含5个浓度(LLOQ=0.098,LQC=0.2,MQC=5,HQC=10和ULOQ =12.5ng/mL)。方法的精密度用变异系数CV%表示,CV%=SD/平均值×100。准确度用相对误差RE%表示,RE%=(平均观测浓度-名义浓度)/名义浓度×100。通过上述参数分析该试剂盒检测的准确性。分析结果如表4所示。
表4:对人IL-23的定量检测准确度及精准度实验数据
(2)结果与讨论:
表4结果显示,5个浓度水平的批内变异系数CV%≤13.238%。 ULOQ、HQC、MQC和LQC的批内相对误差RE%范围在- 4.949%~15.000%之间。LLOQ的批内相对误差RE%范围在0%~2.551%之间。上述结果表明,使用本申请提供的人IL-23ELISA试剂盒定量检测样品的人IL-23可以满足批内变异系数和相对误差均≤20%(LLOQ和ULOQ ≤25%),说明本申请提供的检测方法具有良好的准确性。
实施例5利用ELISA试剂盒进行人鼠嵌合IL-23、人兔嵌合IL-23的检测
1、样品说明:
本实验中所用人鼠嵌合IL-23为人p19亚基与鼠p40亚基嵌合而成、人兔嵌合IL-23为人p19亚基与兔p40亚基嵌合而成(两种嵌合蛋白均来自江苏荃信生物医药有限公司),用稀释液稀释至5μg/ml作为首孔浓度,以 1/5进行梯度稀释11孔,最后一孔为零,备用;所涉及市售试剂盒为R&D 公司Human IL-23DuoSet ELISA试剂盒(Cat:DY1290,Lot:P148670)。具体信息如表5:
表5:Human IL-23DuoSet ELISA试剂盒组成
名称 | 规格 | 工作浓度 | Lot# |
捕获 | 360μg | 6.00μg/ml | JM1417051 |
检测 | 24.0μg | 50.0ng/ml | CCEM0417051 |
标准 | 520ng | 125-8000pg/ml | 1346567 |
Streptavidin-HRP | NA | 40倍稀释 | P141286 |
2、检测操作步骤:
(1)包被:采用包被抗体稀释液将包被抗体配制成浓度为1μg/ml的包被抗体工作液,取R&D试剂盒中捕获抗体,稀释至6μg/ml,然后按照50μl/ 孔的用量加入酶标板中,于4℃静置过夜;
(2)封闭:弃掉酶标板中的包被抗体工作液,用洗液洗板3次,然后按照100μl/孔的用量加入封闭液,置于摇床(120rpm)室温封闭2小时;
(4)加样:弃掉酶标板中的封闭液,用洗液洗板3次,然后按照50μl/孔的用量加入稀释好的人鼠嵌合IL-23、人兔嵌合IL-23,置于摇床(120rpm) 室温孵育2小时;
(6)加检测抗体:弃掉酶标板中的蛋白标准液和待测样品,用洗液洗板 3次,用样品稀释液将检测抗体(生物素标记)配制成浓度为0.2μg/ml的检测抗体工作液,取R&D试剂盒中Detection antibody,稀释至50ng/ml;然后按照50μl/孔的用量加入酶标板中,置于摇床(120rpm)孵育反应2小时;
(7)加酶标二抗:弃掉酶标板中的检测抗体工作液,用洗液洗板3次,用样品稀释液将酶标二抗配制成浓度为100ng/ml的酶标二抗工作液,取 R&D试剂盒中SA-HRP,稀释40倍至工作浓度,然后按照50μl/孔的用量加入到酶标板中,置于摇床(120rpm)室温孵育1小时;
(8)显色:将底物的A液和B液等体积混合;弃掉酶标板中的酶标二抗工作液,用洗液洗板3次,然后按照50μl/孔的用量加入底物,室温避光反应5~10分钟;
(9)终止显色:将终止液按50μl/孔的用量加入酶标板中终止反应,然后应用酶标仪在450nm波长下测定酶标板中各孔的OD值,根据标准品的 OD值拟合出曲线。
检测结果如图3、图4所示。
3、检测结果:
根据图3的检测结果所示,本申请的方法能够检测人鼠嵌合IL-23, R&D试剂盒未能检测人鼠嵌合IL-23;根据图3的检测结果所示,本申请的方法能够检测人兔嵌合IL-23且检测灵敏度远高于R&D试剂盒。
人鼠嵌合IL-23是人IL-23p19-鼠IL-23p40,人兔嵌合IL-23是人IL- 23p19-兔IL-23p40,本方法中的第一抗体和第二抗体均特异性识别人IL- 23p19,故可用于检测人鼠嵌合IL-23和人兔嵌合IL-23。
以上所述,仅是本申请的较佳实施例而已,并非是对本申请作任何形式的限制。任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本申请技术方案内容,依据本申请的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本申请技术方案的保护范围。
SEQUENCE LISTING
<110> 江苏荃信生物医药有限公司
<120> 用于检测人白介素23的ELISA试剂盒及检测方法
<130> TPD01056
<141> 2020-12-24
<160> 20
<170> PatentIn version 3.5
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Tyr Ile Ile Pro Ile Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe Lys
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Gln Gln Trp Ser Ser Tyr Pro Leu Thr
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Arg Gly Gly Gly Asn Leu Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr
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Gln Val His Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
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Gly Lys Ile Ser Pro Arg Ser Val Asn Ala Tyr Tyr Asn Glu Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala Ser
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Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
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Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
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Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
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Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
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Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
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Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
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Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
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Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser
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Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
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Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
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Leu Ser His Ser Pro Gly Lys
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Gly Lys Ile Ser Pro Arg Ser Val Asn Ala Tyr Tyr Asn Glu Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
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Ala Arg Asp Tyr Ser Asn Leu Ile Phe Asp Tyr Trp Gly Gln Gly Thr
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Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
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Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
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Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
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Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
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Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Pro
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Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
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Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
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Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
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Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
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Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
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His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
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Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
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Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
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Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
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Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
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Glu Lys Ser Leu Ser His Ser Pro Gly Lys
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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
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Glu Arg Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Phe Thr
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Ile Tyr Ser Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
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Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asn Thr Met Glu
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Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
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Pro Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp
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Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr
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Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys
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Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly
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Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser
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Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn
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Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val
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Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly
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Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser
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Asn Leu His Trp Tyr Gln Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp
35 40 45
Ile Phe Gly Thr Ser Ser Leu Ala Ser Gly Val Pro Val Arg Phe Ser
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Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
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Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro
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Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala
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Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser
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Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp
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Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val
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Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met
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Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser
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Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys
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Claims (10)
1.一种检测人白介素23的ELISA试剂盒,其特征在于,所述ELISA试剂盒包括:
已固定至固相载体或欲固定至所述固相载体的第一抗体(包被抗体)和经生物素标记的第二抗体(检测抗体);
其中,第一抗体和第二抗体为分离的抗人白介素23单克隆抗体,所述单克隆抗体包含三个重链互补决定区(CDR-H1、CDR-H2、CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2、CDR-L3),其中:
a)CDR-H1的氨基酸序列如SEQ ID No:1或SEQ ID No:2所示;
b)CDR-H2的氨基酸序列如SEQ ID No:3或SEQ ID No:4所示;
c)CDR-H3的氨基酸序列如SEQ ID No:5或SEQ ID No:6所示;
d)CDR-L1的氨基酸序列如SEQ ID No:7或SEQ ID No:8所示;
e)CDR-L2的氨基酸序列如SEQ ID No:9或SEQ ID No:10所示;
f)CDR-L3的氨基酸序列如SEQ ID No:11或SEQ ID No:12所示。
2.根据权利要求1所述的检测人白介素23的ELISA试剂盒,其特征在于,所述单克隆抗体的CDR-H1具有如SEQ ID No:1所示的氨基酸序列,CDR-H2具有如SEQ ID No:3所示的氨基酸序列,CDR-H3具有如SEQ ID No:5所示的氨基酸序列,CDR-L1具有如SEQ ID No:7所示的氨基酸序列,CDR-L2具有如SEQ ID No:9所示的氨基酸序列,CDR-L3具有如SEQ ID No:11所示的氨基酸序列。
3.根据权利要求1所述的检测人白介素23的ELISA试剂盒,其特征在于,所述单克隆抗体的CDR-H1具有如SEQ ID No:2所示的氨基酸序列,CDR-H2具有如SEQ ID No:4所示的氨基酸序列,CDR-H3具有如SEQ ID No:6所示的氨基酸序列,CDR-L1具有如SEQ ID No:8所示的氨基酸序列,CDR-L2具有如SEQ ID No:10所示的氨基酸序列,CDR-L3具有如SEQ ID No:12所示的氨基酸序列。
4.根据权利要求1~3中任一项所述的检测人白介素23的ELISA试剂盒,其特征在于,所述单克隆抗体包含抗体重链可变区HCVR和抗体轻链可变区LCVR,其中:
HCVR的氨基酸序列如SEQ ID No:13或SEQ ID No:14所示;
LCVR的氨基酸序列如SEQ ID No:15或SEQ ID No:16所示。
5.根据权利要求4所述的检测人白介素23的ELISA试剂盒,其特征在于,所述单克隆抗体的抗体重链可变区HCVR具有如SEQ ID No:13所示的氨基酸序列,抗体轻链可变区LCVR具有如序列SEQ ID No:15所示的氨基酸序列。
6.根据权利要求4所述的检测人白介素23的ELISA试剂盒,其特征在于,所述单克隆抗体的抗体重链可变区HCVR具有如SEQ ID No:14所示的氨基酸序列,抗体轻链可变区LCVR具有如序列SEQ ID No:16所示的氨基酸序列。
7.根据权利要求1~6中任一项所述的检测人白介素23的ELISA试剂盒,其特征在于,所述单克隆抗体包含重链和轻链,其中:
所述重链的氨基酸序列如SEQ ID No:17或SEQ ID No:18所示;
所述轻链的氨基酸序列如SEQ ID No:19或SEQ ID No:20所示。
8.根据权利要求7所述的检测人白介素23的ELISA试剂盒,其特征在于,所述单克隆抗体为抗人白介素23单克隆抗体1#,其重链具有如SEQ ID No:17所示的氨基酸序列,轻链具有如SEQ ID No:19所示的氨基酸序列。
9.根据权利要求7所述的检测人白介素23的ELISA试剂盒,其特征在于,所述单克隆抗体为抗人白介素23单克隆抗体2#,其重链具有如SEQ ID No:18所示的氨基酸序列,轻链具有如SEQ ID No:20所示的氨基酸序列。
10.根据权利要求1~9中任一项所述的ELISA试剂盒,其特征在于,所述第一抗体为抗人白介素23单克隆抗体1#,所述第二抗体为抗人白介素23单克隆抗体2#。
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