CN114957473A - 一种对可溶性cd25蛋白含量检测的酶联免疫检测试剂盒 - Google Patents

一种对可溶性cd25蛋白含量检测的酶联免疫检测试剂盒 Download PDF

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CN114957473A
CN114957473A CN202210442440.7A CN202210442440A CN114957473A CN 114957473 A CN114957473 A CN 114957473A CN 202210442440 A CN202210442440 A CN 202210442440A CN 114957473 A CN114957473 A CN 114957473A
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张学光
刘翠平
薛群
古彦铮
常新
黄子逸
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Suzhou Xuguang Kexing Antibody Biotechnology Co ltd
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Abstract

本发明公开了一种用于检测分析可溶性CD25蛋白含量的酶联免疫试剂盒，试剂盒包括CD25‑5G1和CD25‑2D2两种抗人CD25单克隆抗体：所述的CD25‑5G1单克隆抗体，包括重链和轻链，其重链可变区的氨基酸序列为SEQ ID NO：1；轻链可变区的氨基酸序列为SEQ ID NO：2；所述的CD25‑2D2单克隆抗体，包括重链和轻链，其重链可变区的氨基酸序列为SEQ ID NO：3；轻链可变区的氨基酸序列为SEQ ID NO：4。本发明的酶联免疫试剂盒能够有效检测可溶性CD25含量。利用该试剂盒检测发现，健康人血清中含有一定基础水平的可溶性CD25蛋白，类风湿关节炎患者、Graves病患者、I型糖尿病患者血清可溶性CD25含量明显升高，与患者的临床分期、身抗体产生及疗效有相关性。

Description

一种对可溶性CD25蛋白含量检测的酶联免疫检测试剂盒
技术领域
本发明属于生物医学技术领域，具体涉及一种对可溶性CD25蛋白含量进行定量分析的酶联免疫检测试剂盒，该试剂盒可用于自身免疫性疾病患者体液可溶性CD25蛋白含量检测；从而用于自身免疫性疾病鉴别诊断和预后判断。
背景技术
白介素2(IL-2)是一种重要的调控免疫系统细胞增殖和分化的细胞因子，通过与细胞表面白介素-2受体(IL-2R)结合，继而激活胞质内多种非受体型蛋白质酪氨酸激酶(PTK),进一步启动Jak-STAT途径、Ras-MAPK途径以及PI3K途径，促进T细胞发生、发育、分化；在抗原抗体反应、感染性疾病和肿瘤等疾病致病中起着重要的作用。
白细胞介素2受体有三种多肽亚单位，包括CD25(IL-2Rα)、CD122(IL-2Rβ)和CD132(常见的γ链)。CD25即白介素2(interleukin 2，IL-2)异源三聚体受体复合物的α链，主要表达于CD4+T细胞膜表面，参与调节性CD4+T细胞在IL-2的生理低水平下进行分化和增殖。sCD25是膜结合形式IL-2受体α亚单位的裂解物，可与膜结合IL-2受体竞争性结合IL-2，是一种免疫抑制物。研究表明，sCD25是体现机体淋巴细胞活性的重要物质，在肾病综合征、病毒感染、肿瘤等疾病患者外周血及组织中高水平表达，且与患者的临床分期、预后等密切相关。健康人血清中含有一定基础水平的可溶性CD25蛋白(～500pg/ml)，而类风湿关节炎、Greves病患者、I型糖尿病患者血清可溶性CD25含量明显升高。且可溶性CD25蛋白含量与患者的临床分期、自身抗体产生和疗效有相关性。对自身免疫性疾病患者体液中可溶性CD25含量检测具有临床价值。
发明内容
本发明在筛选获得两种抗人CD25单克隆抗体的基础上，使用筛选获得的新的单克隆抗体来制备能够对可溶性CD25蛋白含量进行检测的酶联免疫检测试剂盒。
本发明首先提供两种抗人CD25蛋白的单克隆抗体，为CD25-5G1和CD25-2D2两种抗人CD25单克隆抗体；
所述的CD25-5G1单克隆抗体，包括重链和轻链，其重链可变区(mVH)的氨基酸序列如下：
EVHVKQSGAELAKPGASVKMSCKASGYNFTRHWIHWVKQRPGQGLEWIGYINPRTDYTEYNQKFKDKATLTADKSSSTAHMQLNSLTSEDSAVYYCARGNYFDYWGQGTTLTVSS(SEQ ID NO：1)；
CD25-5G1的轻链可变区(mVL)的氨基酸序列如下：
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNTEQEDIATYFCQQGNTLPPTFGSGTKLEIK(SEQ ID NO：2)；
所述的CD25-2D2单克隆抗体，包括重链和轻链，所述CD25-2D2的重链可变区(mVH)的氨基酸序列如下：
EVQLKQSGPGLVAPSQSLSITCTVSGFSLTSYAISWVRQPPGKGLEWLGVMWTGGGTNYNSALKSRLNISKDNSKSQVFLKMNSLQTEDTARYYCARPYYYDGSWFAYWGQGTLVTVSA(SEQ ID NO：3)；
CD25-2D2的轻链可变区(mVL)的氨基酸序列如下：
DIVLSQSPAILSASPGEKVTMTCRASSSVGYMHWYQQKPGSSPKPWIYATSNLASGVPVRFSGSESGTSYSLTISRVEAEDAATYYCQQWSSNPPTFGGGTKLEIK(SEQ ID NO：4)。
本发明所提供的两种抗人CD25蛋白的单克隆抗体可用于制备检测可溶性CD25含量的制剂；
本发明另一个方面还提供一种检测可溶性CD25含量的酶联免疫检测试剂盒，所提供的试剂盒包括CD25-5G1和CD25-2D2两种抗人CD25单克隆抗体：
所述的试剂盒中，CD25-5G1作为包被抗体；CD25-2D2作为检测抗体；
更进一步的，所述的检测抗体使用HRP进行标记。
本发明还提供所述的酶联免疫检测试剂盒的用途，是用于检测自身免疫性疾病患者可溶性CD25蛋白含量。
附图说明
图1为本发明的酶联免疫检测试剂盒标准曲线示意图。
图2为本发明的酶联免疫检测试剂盒特异性考察示意图。
图3可溶性CD25蛋白在类风湿关节炎患者外周血清中的表达水平及临床相关性分析示意图。
图4可溶性CD25蛋白在糖尿病患者外周血清中的表达水平及临床相关性分析示意图。
图5可溶性CD25蛋白在GD患者外周血清中的表达水平及临床相关性分析示意图。
具体实施方式
以下结合具体实例及附图对本发明作进一步描述。应理解，以下实施例仅适用于说明本发明而非限定本发明的范围。
实施例1：抗人CD25单克隆抗体轻重链可变区序列的测定
测定抗人CD25单克隆抗体重链和轻链可变区的方法包括以下步骤：
1.1杂交瘤细胞cDNA的获取
从目标的杂交瘤细胞中获取RNA，通过逆转录技术将获得的RNA反转录为cDNA。利用特定设计的上下游引物PCR克隆该杂交瘤细胞的重链可变区(mVH)和轻链可变区(mVL)。
1.2分别将重链可变区(mVH)和轻链可变区(mVL)与pJET cloning vector克隆载体连接。随后，将连接的产物转化至DH5a感受态细菌后，将转化后的菌液均匀涂布于LB固体培养基上。
1.3挑选LB固体培养基上边缘清晰、生长良好的菌落进行测序鉴定。
1.4根据测序结果保留候选的轻重链可变区序列，再次通过PCR克隆出可与表达载体连接的轻链和重链的可变区序列，并将可变区序列与表达载体连接，随后将连接产物转化至DH5a。将转化菌液均匀涂布LB固体培养基，培养过夜。
1.5挑选生长良好的细菌测序，并对比两次测序结果从而得到具有正确的序列的转化细菌，通过扩大培养后进行质粒抽提。
1.6将连有当克隆抗体重链和轻链可变区基因的表达载体一起共同转染真核表达细胞293。
1.7 293细胞用无血清培养基SFM4Transfx-293without L-Glutamine悬浮培养，在转染时培养基替换为无血清培养基
Figure BDA0003614524000000051
FreeStyleTM293Expression Medium。
1.8利用ELISA试剂盒检测含有目的抗体的上清液，结果显示良好。
通过上述方法来获得抗人CD25-5G1单克隆抗体重链可变区：
EVHVKQSGAELAKPGASVKMSCKASGYNFTRHWIHWVKQRPGQGLEWIGYINPRTDYTEYNQKFKDKATLTADKSSSTAHMQLNSLTSEDSAVYYCARGNYFDYWGQGTTLTVSS；
CD25-5G1单克隆抗体轻链可变区:
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNTEQEDIATYFCQQGNTLPPTFGSGTKLEIK；CD25-2D2单克隆抗体重链可变区
EVQLKQSGPGLVAPSQSLSITCTVSGFSLTSYAISWVRQPPGKGLEWLGVMWTGGGTNYNSALKSRLNISKDNSKSQVFLKMNSLQTEDTARYYCARPYYYDGSWFAYWGQGTLVTVSA；
CD25-2D2单克隆抗体轻链可变区
DIVLSQSPAILSASPGEKVTMTCRASSSVGYMHWYQQKPGSSPKPWIYATSNLASGVPVRFSGSESGTSYSLTISRVEAEDAATYYCQQWSSNPPTFGGGTKLEIK
本发明从表达抗人CD25单克隆抗体的杂交瘤细胞中提取到抗体的重链和轻链的可变区，根据测序结果保留候选的轻重链可变区序列，并通过PCR扩增出与表达载体匹配的轻重链可变区序列，连接PCR产物与双酶切预处理的表达载体。将连有目的单抗轻链和重链可变区的表达载体共同转染真核表达细胞株293，收取培养后的上清含有目的抗体，说明得到的重链和轻链可变区序列是正确的。
实施例2：一种检测可溶性CD25蛋白含量的ELISA试剂盒：
2.1试剂盒的组成
本发明的ELISA试剂盒包括包被于酶标板上的实施例1中的CD25包被抗体(CD25-5G1)、经HRP标记的CD25检测抗体(CD25-2D2-HRP)、可溶性CD25蛋白标准品(R&D Systems)、样本稀释液、洗涤液(PBST)、显色液(TMB)及终止液组成。
2.2样本的采集及处理
2.2.1收集一批医院自身免疫病患者的血清，分装后-80℃冰箱保存，并避免反复冻融。
2.2.2血清样本检测前，提前将样本常温平衡半小时，并震荡混匀，取适量病人血清用样本用稀释液稀释进行5倍稀释。
2.3可溶性CD25蛋白含量的测定方法
2.3.1使用包被液(Na2CO3和NaHCO3)稀释包被抗体CD25-5G1(1μg/ml)，随后将包被抗体加入96孔酶标板(100μl/孔)，4℃孵育过夜。
2.3.2第二天，用洗涤液(PBST)洗板3次，随后每孔加入3％牛血清封闭液100μl，37℃封闭1h。
2.3.3去除封闭液，加入100μl稀释好的待测血清样品以及梯度稀释好的可溶性CD25蛋白标准品(从1ng/ml开始倍比稀释，共设8个浓度，(1、0.5、0.25、0.125、0.0625、0.03125、0.015625、0ng/ml)，每个梯度做3个重复，37℃恒温箱孵育2h。
2.3.4 PBST洗液洗板三次，按1：8000比例加入HRP标记的检测抗体CD25-2D2(100μl/孔)，37℃恒温箱孵育1h。。
2.3.5 PBST洗液洗板六次，加入显色液TMB(100μl/孔)，室温避光孵育10min，之后加入终止液(50μl/孔)终止颜色反应，通过酶标仪测定每孔的OD450值。
2.3.6以CD25标准品浓度乘以样品稀释倍数(×5)的值为纵坐标，以相应测得的OD450值为横坐标制作标准曲线(如图1)，并得到计算公式，根据待测样品的OD450值计算出样品中可溶性CD25的含量。
本发明试剂盒按照方法学鉴定，可达到以下指标：
曲线方程：y＝0.04286+0.00528x
标准曲线线性：R2＝0.9998；最低检测限≤15.625pg/ml。
2.4准确性考察：
1)板内准确性分析：在同一次实验中，将五个已知浓度的样本(500、250、125、62.5、31.25pg/mL)分别设置10个复孔，进行sCD25检测，分析该试剂盒的准确性。
2)板间准确性分析：在不同批次实验中，将五个已知浓度的样本(500、250、125、62.5、31.25pg/mL)分别设置10个复孔，进行sCD25检测，分析该试剂盒的准确性。
3)结果与讨论：
统计学分析结果显示，sCD25蛋白5个浓度组的离散度(CV％)均<8％，且检测结果之间无显著性差异(P>0.05)(表1和表2)，进而表明，该检测人sCD25的ELISA方法在同批次和不同批次实验中均具有良好的可重复性和较高的精确性。
表1：人sCD25 ELISA检测体系的板内准确性分析数据表
Figure BDA0003614524000000091
表2：人sCD25 ELISA检测体系的板间准确性分析表
Figure BDA0003614524000000092
Figure BDA0003614524000000101
2.5特异性考察
将OX40Ig、CD28Ig、OX40Ig、LAG3Ig、B7-H4Ig、CD25Ig连续倍比稀释成不同的浓度。抗人CD25单抗(5G1)用碳酸盐缓冲液(0.01M CBS，pH9.3)调整为1μg/ml包被ELISA检测板，4℃过夜。PBS(含0.1％Tween 20)洗涤3次，3％BSA室温封闭1h。PBS洗涤3次后加入上述稀释好的商品化蛋白，室温反应2h，PBS洗涤3次。然后，加入HRP标记的单抗HRP-2D2(1:8000，100μl/孔)，室温继续反应1h，PBST洗涤6次后，再加入HRP反应底物TMB(100μl/孔)，室温反应10～15min，用2mol/L H2SO4终止反应，用酶标仪测定OD450nm，每个样品设置3个复孔。特异性分析结果如图2所示，该检测试剂盒可以特异性识别不同浓度的CD25Ig，而不识别其他无关蛋白，证明该试剂盒具有良好的特异性。
实施例3：可溶性CD25在类风湿关节炎临床诊断和预后监测中的应用
收集正常健康人及类风湿关节炎患者(RA)外周血清，其中包括健康对照组(HC)41例，RA患者49例，其中40RA患者分别测量2个时间点血清数值(初诊，生物制剂治疗6个月)。ELISA法检测外周血清中sCD25的表达，并进行相关实验室诊断指标相关性分析。比较治疗前后sCD25表达水平改变与DAS28评分、临床相关指标、病理指标的相关性，分析sCD25对于诊断和治疗RA的潜在临床意义。如图3A所示，RA患者外周血清中的sCD25表达显著高于健康对照组(P<0.0001)；生物制剂治疗6个月后，sCD25表达水平显著降低(P＝0.005)(图3B)；sCD25与DAS28评分、压痛关节数、肿胀关节数和CRP表达水平具有显著相关性(图3C)。鉴此，使用本发明的人可溶性CD25 ELISA试剂盒在RA的诊断和预后判断中具有应用价值。
实施例4：可溶性CD25在糖尿病患者外周血清中的表达及临床鉴别诊断中的应用
收集正常健康人及1型糖尿病患者(T1D)、2型糖尿病患者(T2D)外周血清，其中包括健康对照组(HC)70例，TID患者150例，T2D患者30例。ELISA法检测外周血清中sCD25的表达，并进行相关实验室诊断指标相关性分析。如图4A所示，T1D患者外周血清中的sCD25表达显著高于健康对照组(P<0.001)和T2D疾病组(P<0.05)；sCD25与GAD抗体产生、Cr、BUN和HbA1c表达水平具有显著相关性(图4B)，提示本发明的人可溶性CD25 ELISA试剂盒在糖尿病鉴别诊断中具有应用价值，同时可以用于糖尿病肾病相关疾病的检测。
实施例5：可溶性CD25在抗体阳性Graves病患者外周血清中的表达及临床鉴别诊断中的应用
收集正常健康人及Graves病(GD)患者外周血清，其中包括健康对照组(HC)30例，GD患者53例(其中TRAb抗体阳性患者43例，阴性10例)。ELISA法检测外周血清中sCD25的表达，并进行相关实验室诊断指标相关性分析。如图5A所示，抗体阳性GD患者外周血清中的sCD25表达显著高于健康对照组(P<0.0001)和抗体阴性疾病组(P<0.0001)；sCD25与TRAb抗体水平具有显著相关性(图5B)，提示本发明的人可溶性CD25 ELISA试剂盒在抗体阳性和阴性GD鉴别诊断中具有应用价值。
序列表
<110> 苏州旭光科星抗体生物科技有限公司
<120> 一种对可溶性CD25蛋白含量检测的酶联免疫检测试剂盒
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Claims (7)

1.抗人CD25蛋白的单克隆抗体，其特征在于，所述的单克隆抗体，为CD25-5G1和CD25-2D2两种抗人CD25单克隆抗体；
所述的CD25-5G1单克隆抗体，包括重链和轻链，其重链可变区的氨基酸序列为SEQ IDNO：1；轻链可变区的氨基酸序列为SEQ ID NO：2；
所述的CD25-2D2单克隆抗体，包括重链和轻链，其重链可变区的氨基酸序列为SEQ IDNO：3；轻链可变区的氨基酸序列为SEQ ID NO：4。
2.权利要求1所述的单克隆抗体在制备检测可溶性CD25含量的制品中给的应用。
3.如权利要求2所述的应用，其特征在于，所述的制品为酶联免疫检测试剂盒。
4.一种检测可溶性CD25含量的酶联免疫检测试剂盒，其特征在于，所述的试剂盒包含有权利要求1所述的单克隆抗体。
5.如权利要求4所述的酶联免疫检测试剂盒，其特征在于，所述的试剂盒中，CD25-5G1单克隆抗体作为包被抗体；CD25-2D2单克隆抗体作为检测抗体。
6.如权利要求5所述的酶联免疫检测试剂盒，其特征在于，所述的检测抗体使用HRP进行标记。
7.权利要求4-6任一项所述的酶联免疫检测试剂盒在检测自身免疫性疾病患者可溶性CD25蛋白含量中的应用。
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