CN116396374A - Il-12抗体和其抗原结合片段及包含其的检测试剂盒 - Google Patents
Il-12抗体和其抗原结合片段及包含其的检测试剂盒 Download PDFInfo
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Abstract
本发明公开了一种IL‑12抗体和其抗原结合片段及包含其的检测试剂盒,具体涉及抗体检测技术领域。所述抗原片段序列如SEQ ID NO:1所示。一种抗体mAb135‑HRP,是将上述抗原合成到pcDNA3.1载体上,后经过293T表达,纯化后免疫小鼠,筛选得到抗体mAb135‑HRP。本发明的试剂盒可以同时区分活性IL‑12、单体p40、二聚体p40和IL‑23,并区分IL‑12(异二聚体)和IL‑35;而且本发明的试剂盒检测范围更广,除了(15‑2000pg/mL)的低值检测,在(1‑200ng/mL)的高值检测结果也很好,可以对临床血清检测减少处理步骤。为后续在临床药物治疗中,针对IL‑12抗体治疗在肿瘤方面也有很大的参考意义。
Description
技术领域
本发明涉及抗体检测技术领域,具体涉及IL-12抗体和其抗原结合片段及包含其的检测试剂盒。
背景技术
IL-12是一种多活性细胞因子,其靶细胞是T细胞,NK细胞和骨髓祖细胞。IL-12亚单位的作用:(1)IL-12能促进激活的T细胞和NK细胞增殖,增强T细胞、NK细胞细胞毒活性并诱导其产生γ干扰素(interferon-gamma,IFN-γ)、β肿瘤坏死因子(tumor necrosisfactorbeta,TNF-β)等。(2)IL-12在Th1/Th2应答平衡中有重要作用。
IL-12p40亚单位可单独存在,二个p40形成二聚体(p40)2。p70代表了全部已知IL-12生物学活性;p40、(p40)2可拮抗p70的生物学活性,其中,(p40)2是p70强有力的竞争拮抗剂,作用强度为p40的25~50倍,其机制是竞争IL-12Rβ2链。p40:p70的比例是可变的,p40可在p70缺乏的情况下分泌,并调节p70的生物活性。
活性白细胞介素12(IL-12p70)是一种分泌性异二聚体细胞因子,含有二硫键结合的p35和p40亚基。p40也可以作为单体(IL-12p40)或同二聚体(IL-12p80)存在。p40还存在于IL-23中(IL-23是p40和p19的异二聚体)。p35也不仅是组成IL-12p70的成分,也是异二聚体IL-35(p35+EBI3)的成分。所以,使用针对p40亚单位的抗体进行的分析将无法区分活性IL-12、单体p40、二聚体p40和IL-23;同样,仅使用p35检测的分析将同时检测IL-12(异二聚体)和IL-35。
如何得到一种可以同时区分活性IL-12、单体p40、二聚体p40和IL-23,并区分IL-12(异二聚体)和IL-35的试剂盒尤为重要。
发明内容
为此,本发明提供一种IL-12抗体和其抗原结合片段及包含其的检测试剂盒,以解决如何得到一种可以同时区分活性IL-12、单体p40、二聚体p40和IL-23,并区分IL-12(异二聚体)和IL-35的试剂盒的问题。
为了实现上述目的,本发明提供如下技术方案:
根据本发明第一方面提供的一种IL-12抗原结合片段,所述片段序列如SEQ IDNO:1所示。
进一步的,所述片段是由P35片段与P40片段经GGGGS连接而成。
根据本发明第二方面提供的一种抗体mAb135-HRP,所述抗体是由如上述抗原表达而成。
进一步的,所述抗体是将如上述的抗原合成到pcDNA3.1载体上,后经过293T表达,纯化后免疫小鼠,筛选得到抗体mAb135-HRP。
根据本发明第三方面提供的一种检测试剂盒,包括IL-12抗体和抗体mAb135-HRP。
进一步的,所述试剂盒还包括显色液、封闭液、洗涤液和终止液。
进一步的,所述显色液包括显色液A:TMB和显色液B:H2O2。
根据本发明第四方面提供一种检测试剂盒在制备临床检测IL-12含量的产品中的应用。
本发明具有如下优点:
本发明的试剂盒可以同时区分活性IL-12、单体p40、二聚体p40和IL-23,并区分IL-12(异二聚体)和IL-35;与现在的检测试剂盒检测结果一致,而且本发明的试剂盒检测范围更广,除了(15-2000pg/mL)的低值检测,在(1-200ng/mL)的高值检测结果也很好,可以对临床血清检测减少处理步骤。
本发明自主生产的IL-12抗原稳定且表达量高,可以为PD-1联合用药提供原材料。
本发明中的抗体不仅可以应用于ELISA试剂盒,还可以应用在胶乳试剂盒和化学发光中检测IL-12。本发明中的抗体稳定,效价高,为后续在临床药物治疗中,针对IL-12抗体治疗在炎症和肿瘤方面也有很大的参考意义。
附图说明
实施方式或现有技术中的技术方案,下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图仅仅是示例性的,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图引伸获得其它的实施附图。
本说明书所绘示的结构、比例、大小等,均仅用以配合说明书所揭示的内容,以供熟悉此技术的人士了解与阅读,并非用以限定本发明可实施的限定条件,故不具技术上的实质意义,任何结构的修饰、比例关系的改变或大小的调整,在不影响本发明所能产生的功效及所能达成的目的下,均应仍落在本发明所揭示的技术内容得能涵盖的范围内。
图1为本发明实验例2提供的IL-12的标准曲线;
图2为本发明实验例3提供的一种检测肿瘤和非肿瘤患者血清中IL-12的含量临床统计结果图。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
一种抗体mAb135-HRP的筛选
IL-12抗原如下:
MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVS
NMLQKARQTLE
FYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCL
ASRKTSFMM
ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELM
QALNFNSETVPQK
SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSMHPQQLVVSWFSLVLLASPIVAIWELEKNVYIVELDWYPDAPGETVVLTCD
TPEEDGITW
TSDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEALSRSLLLLHKKE
DGIWSTDILKDQ
KEPKAKSFLKCEAKDYSGHFTCWWLTAISTDLKFSVKSSRGSSDPRGV
TCGAASLSAEKV
SVDHREYNKYTVECQEGSTCPAAEESLLIEVVVEAVHKLKYENYTSSF
FIRDIIKPDPPK
NLQLRPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKNKREKK
LFMDQTSAKVTCH
KDANVRVQARDRYYSSFWSEWASVSCS
其中,IL-12抗原是由P35片段与P40片段经GGGGS连接而成。
将IL-12抗原基因合成到pcDNA3.1载体上,后经过293T表达,使用镍柱纯化透析后免疫小鼠,筛选抗体mAb135-HRP,使用筛选抗体效价较高的配制ELISA试剂盒。
抗原免疫情况统计结果见表1。
表1
IL-12抗体筛选:
使用自制ELISA检测试剂盒,检测免疫后抗体效价,抗体效价检测方法:用包被液(50mM碳酸盐缓冲液pH9.6)稀释IL-12抗原进行96孔板包被,每孔100μL,4℃过夜;第二天洗涤液清洗3次,拍干;加入封闭液,每孔120μL,37℃,30min,拍干;加入筛选稀释后的IL-12抗体,100μL/孔,37℃,30min,洗涤液清洗3次,拍干;加入标记鼠抗人mAb135-HRP,每孔100μL,37℃,30min洗涤液清洗3次,拍干;显色液A液和B液1:1混合后,每孔加入100μL,10min;加入终止液,50μL/孔;酶标仪上450nm处读OD值。
抗体效价筛选结果见表2。
表2
抗体编号 | OD |
1 | 0.814 |
2 | 1.025 |
3 | 1.462 |
4 | 0.622 |
5 | 0.503 |
6 | 1.105 |
通过检测发现3号抗体效价高,4和5号抗体效价最低,试剂盒的抗体最终采用3号,即为IL-12抗体。
实施例2
本实施例提供一种IL-12 ELISA试剂盒
试剂盒组成原料:
抗体mAb135-HRP(筛选表达的鼠单抗标记HRP)、IL-12抗体;显色液A:TMB,显色液B:H2O2;封闭液:脱脂奶粉;洗涤液:1XPBST;终止液:2mol/L浓硫酸。
配对及抗体应用方法:用包被液(50mM碳酸盐缓冲液pH9.6)稀释IL-12抗体进行96孔板包被,每孔100μL,4℃过夜;第二天洗涤液清洗3次,拍干;加入封闭液,每孔120μL,37℃,30min,拍干;加入稀释后的IL-12抗原,100μL/孔,37℃,30min,洗涤液清洗3次,拍干;加入标记鼠抗人mAb135-HRP,每孔100μL,37℃,30min洗涤液清洗3次,拍干;显色液A液和B液1:1混合后,每孔加入100μL,10min;加入终止液,50μL/孔;酶标仪上450nm处读OD值。
实验例1
利用实施例2的试剂盒与abcam ELISA检测试剂IL-12p70(ab223592)相比,结果如表3所示,结果没有差异(P=0.9874),说明本发明的试剂盒配制没有问题。
表3
序号 | 实施例1(pg/mL) | abcam(pg/mL) |
1 | 100.35 | 102.27 |
2 | 187.77 | 189.00 |
3 | 492.51 | 487.65 |
4 | 195.50 | 190.78 |
5 | 1057.07 | 1063.12 |
6 | 1111.12 | 1119.38 |
7 | 1286.03 | 1277.82 |
8 | 1613.05 | 1621.34 |
9 | 184.65 | 180.47 |
10 | 1604.60 | 1612.91 |
11 | 700.54 | 708.25 |
12 | 757.18 | 762.82 |
13 | 648.59 | 652.97 |
14 | 959.62 | 966.24 |
15 | 1011.93 | 1018.19 |
16 | 1103.00 | 1098.36 |
17 | 1513.81 | 1517.19 |
18 | 1193.97 | 1193.22 |
19 | 1615.36 | 1621.93 |
20 | 1759.62 | 1766.15 |
实验例2
本实验例对实施例2的标准曲线:使用IL-12标准品进行梯度稀释,按照实施例2步骤进行标准曲线定标,制作标准曲线如图1所示,可以看到R2=0.9949,说明相关性很好,试剂盒满足使用要求,详细结果见表4。
表4
编号 | 实际值(pg/ml) | 检测值OD450 |
1 | 0.0 | 0.001 |
2 | 30.1 | 0.098 |
3 | 60.2 | 0.174 |
4 | 120 | 0.319 |
5 | 240 | 0.658 |
6 | 480 | 1.184 |
7 | 960 | 2.106 |
本发明的试剂盒检测范围更广,除了(15-2000pg/mL)的低值检测,在(1-200ng/mL)的高值检测结果也很好,另外,本发明的试剂盒可以对临床血清检测减少处理步骤。
实验例3
使用实施例2的试剂盒对临床血清检测肿瘤和非肿瘤患者血清中IL-12的含量。临床样本统计结果见表5所示。
表5
类别 | 数量 | 年龄(Mean±SD) | IL-12值(Mean±SD) |
肿瘤组 | 45(37名男性,8名女性) | 56.18±13.96 | 48.48±43.68 |
非肿瘤组 | 108(63名男性,45名女性) | 56.17±15.85 | 23.50±13.83 |
通过表5和图2的数据分析,发现两组之间差异性显著(P<0.001),这与IL-12在肿瘤发生过程中催化TNF、IFN-γ等抑制肿瘤生长、侵染和转移起作用一致。在临床药物治疗中,针对IL-12抗体治疗在肿瘤方面也有很大的参考意义。
虽然,上文中已经用一般性说明及具体实施例对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (8)
1.一种IL-12抗原结合片段,其特征在于,所述片段序列如SEQ ID NO:1所示。
2.根据权利要求1所述一种IL-12抗原结合片段,其特征在于,所述片段是由P35片段与P40片段经GGGGS连接而成。
3.一种抗体mAb135-HRP,其特征在于,所述抗体是由权利要求1或2任一抗原表达而成。
4.根据权利要求3所述一种抗体mAb135-HRP,其特征在于,所述抗体是将权利要求1或2任一抗原合成到pcDNA3.1载体上,后经过293T表达,纯化后免疫小鼠,筛选得到抗体mAb135-HRP。
5.一种检测试剂盒,其特征在于,包括IL-12抗体和抗体mAb135-HRP。
6.根据权利要求5所述一种检测试剂盒,其特征在于,所述试剂盒还包括显色液、封闭液、洗涤液和终止液。
7.根据权利要求6所述一种检测试剂盒,其特征在于,所述显色液包括显色液A:TMB和显色液B:H2O2。
8.一种检测试剂盒在制备临床检测IL-12含量的产品中的应用。
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"Interleukin-12 subunit alpha precursor (IL-12A) (IL-12 subunit p35) (Cytotoxic lymphocytematuration factor 35 kDa subunit) (CLMF p35) (NK cell stimulatory factor chain 1) (NKSF1)", UNIPROTKB/SWISS-PROT: 20141534, pages 1 - 2 * |
"Interleukin-12 subunit beta precursor (IL-12B) (IL-12 subunit p40) (Cytotoxic lymphocytematuration factor 40 kDa subunit) (CLMF p40)", UNIPROTKB/SWISS-PROT: 118595546, pages 1 - 2 * |
张梅: "重组人单链白细胞介素12融合基因的构建、真核表达及生物学活性测定", 中华血液学杂志, vol. 21, no. 12, pages 636 - 640 * |
郭伟剑等: "胃肠道肿瘤病人血清四种因子的检测", 肿瘤学杂志, no. 05, pages 275 - 276 * |
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