CN116381217A - 检测st2的电化学发光试剂盒、制法 - Google Patents
检测st2的电化学发光试剂盒、制法 Download PDFInfo
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Abstract
本发明涉及一种从血液中检测可溶性生长刺激表达基因2蛋白(ST2)的电化学发光试剂盒、制法,制备的试剂盒包括:链霉亲和素偶联的磁性微粒工作液,生物素标记的ST2一抗工作液,三联吡啶钌标记的ST2二抗工作液,ST2校准品和/或质控品工作液,含三丙胺的电化学发光底物液,清洗液,试剂盒的发光体系为电化学发光,利用链霉亲和素‑生物素信号放大系统,检测灵敏度高、线性范围宽,检测结果重复性好,可以实现ST2的准确定量。
Description
技术领域
本发明涉及一种检测ST2的电化学发光试剂盒及用法,属于免疫分析技术领域。
背景技术
近代临床免疫诊断技术经历了从放射免疫到酶联免疫再到化学发光免疫诊断技术的转变。化学发光免疫诊断在体外诊断试剂中已成为主要研究和应用趋势。目前主流的化学发光免疫分析技术分为以下三种:酶促化学发光、直接化学发光和电化学发光。电化学发光以其电信号稳定,低值区背景信号低,灵敏度高,检测时间短等优势正逐渐成为各厂家青睐的技术平台。本试剂盒采用电化学发光法进行研制,具有背景信号低,灵敏度高、线性范围宽的优势。
ST2基因英文称为Suppression of Tumorigenicity 2,是白细胞介素1受体家族的成员。 ST2蛋白有两种异构体: 1.为可溶性形式的ST2或sST2;2.膜结合受体形式的ST2。称为ST2受体或ST2L。 ST2的配体是细胞因子白细胞介素33(IL-33)。ST2基因位于2号染色体的长臂上(2q12), 具有 40,536碱基长度,编码一个556个氨基酸的蛋白(分子量为63,358道尔顿)。已知ST2蛋白质可与MyD88的IRAK1,IRAK4和TRAF6等分子相互作用。ST2对于辅助性T细胞2类(Th2细胞)的正常功能可能具有至关重要的调节作用。
《中国心力衰竭诊断和治疗指南 2014》和 2017 年 ACC/AHA 指南均推荐 ST2 用于急性和慢性 HF 患者的临床评估,从而指导 HF 的治疗。 PRIDE 研究表明对于急性心衰(AHF)患者,ST2 测量可以预测 30 天的死亡率。ST2 水平高的患者生存较差,死亡风险更高;研究资料显示,监测 AHF 患者的 ST2 水平可以了解患者再住院的风险以及死亡风险;一项荟萃分析显示与常规的非 ST2 评估方法相比,采用 ST2 评估方法总体上对患者的全因死亡和 HF 再住院更有益。因此早期测定 ST2 水平可以帮助临床医生做出决策,考虑在患者出院前是否要加强抗纤维化的治疗。对于慢性心衰(CHF)患者,有研究表明 CHF 患者ST2 水平越高,总体上一年的死亡率明显升高;阜外医院对 1500 多例 HF 住院患者进行了随访研究,发现 ST2 水平与 3 个月、1 年、3 年全因死亡和心脏移植的发生率呈正相关,说明 ST2 的测定有利于评估 CHF 患者 1-3 年的不良事件风险。因此对于 ST2 水平高的 CHF 患者,应该加强抗纤维化的治疗。ST2 反映的是 HF 患者心肌纤维化情况,ST2 >35 ng/ml 即超出正常,ST2 水平越高,HF 患者心肌纤维化的程度越高。如果患者的 ST2水平持续居高不下,或由低到高发展,这时就考虑抗纤维化治疗,治疗药物包括血管紧张素转化酶抑制剂(ACEI)、血管紧张素 II 受体拮抗剂(ARB)、β受体阻滞剂,以及新药诺欣妥。
目前ST2检测方法主要是ELISA(酶联免疫法)法检测,ELISA固有的灵敏度较低,结果重复性较低,操作繁琐等缺点,难以实现准确、简便的ST2测定,电化学发光法具有明显优势。
发明内容
本发明要解决的技术问题是:为实现更快速、灵敏、准确地定量检测ST2的方法,提供一种检测ST2电化学发光试剂盒及制法。
本发明解决其技术问题所采用的技术方案是:
一种检测ST2的电化学发光试剂盒,包括:链霉亲和素偶联的磁性微粒工作液,生物素标记的ST2一抗工作液,三联吡啶钌标记的ST2二抗工作液,含三丙胺的电化学发光底物液,清洗液。
优选地,所述ST2抗体为外购的单克隆抗体、多克隆抗体、单克隆抗体Fab片段或多克隆抗体Fab片段,优选来源于鼠、兔、羊。
优选地,所述链霉亲和素偶联的磁性微粒工作液由pH为6.8~7.6 ,浓度为0.01mol/L~0.2 mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MOPSO缓冲液中的一种配制而成,所述缓冲液含有0.05~2 wt%的牛血清白蛋白和/或酪蛋白、0.02~1 wt%的十二烷基聚乙二醇醚(Brij35)和/或聚乙二醇对异辛基苯基醚(TritonX-100)和/或聚氧乙烯失水山梨醇单月桂酸酯(Tween20)和/或月桂醇聚氧乙烯醚、0.05~0.5 wt%的proclin 300和/或叠氮钠。
优选地,所述生物素标记的ST2一抗工作液,三联吡啶钌标记的ST2二抗工作液由pH为6.0~7.6,浓度为0.01 mol/L~0.2 mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MOPSO缓冲液中的一种配制而成,所述缓冲液含有0.5~5wt%的牛血清白蛋白、1mg/L~100mg/L的抗干扰剂A、0.5~5wt%的氯化钠、1~5wt%的蔗糖、0.05~2wt%的小牛血清、0.02~1wt%的酪蛋白、0 .02~1wt%的Brij35和/或TritonX-100和/或Tween 20和/或平平加O-20、0.05~0.5wt%的proclin300和/或叠氮钠。
优选地,所述检测ST2的电化学发光试剂盒还包括ST2校准品工作液和/或质控品工作液,所述ST2校准品和/或质控品工作液由pH为6.0~7.6,浓度为0.01 mol/L~0.2mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MES缓冲液中的一种配制而成,所述缓冲液中含有0.5~5 wt%的牛血清白蛋白、10~50 wt%的人血清、0.5~3 wt%的氯化钠、2~20 wt%的乙二醇、0.02~1 wt%的Brij 35和/或Triton X-100和/或Tween 20和/或平平加O-20、0.05~0.5 wt%的proclin 300和/或叠氮钠。
优选地,所述清洗液为pH为13以上,浓度为0 .1 mol/L~0 .5 mol/L的KOH液,所述清洗液含有0 .01~2 wt%的烷基聚乙二醇类表面活性剂。
优选地,所述化学发光底物液pH为6.0~7.2,浓度为0.05~0.4 mol/L的磷酸盐缓冲液或Tris缓冲液,所述缓冲液含有0.05~0.4 mol/L的三丙胺、0.01~2 wt%的烷基聚乙二醇类表面活性剂、0.05~0.5 wt%的proclin 300和/或叠氮钠。
本发明还提供一种上述检测ST2的电化学发光试剂盒的制法,包括:链霉亲和素偶联的磁性微粒工作液的制备,生物素标记的ST2一抗工作液,三联吡啶钌标记的ST2二抗工作液的制备,清洗液的配制,电化学发光底物液的配制,将上述制备的试剂进行分装及组装。
优选地,所述生物素标记的ST2一抗的制备方法为:将N-羟基琥珀酰亚胺活化的生物素与ST2抗体溶液按照1-20:1的摩尔比在2-8℃或室温条件下混匀反应0 .5-2小时,透析除去多余的生物素,得生物素标记的ST2抗体。
优选地,所述三联吡啶钌标记ST2抗体的制备方法为:将pH为6.5-8.0的ST2抗体溶液与Ru-NHS酯溶液混合,使ST2抗体与Ru-NHS酯的质量比为5-20: 1,37℃避光反应1-3小时,然后加入甘氨酸溶液终止反应,将反应液透析除去多余的Ru-NHS酯,得三联吡啶钌标记ST2抗体。
本发明的有益效果是:
本发明提供了一种检测ST2的电化学发光试剂盒、制法,制备的试剂盒的发光体系为电化学发光,利用链霉亲和素-生物素信号放大系统,检测灵敏度高、线性范围宽,检测结果重复性好,可以实现ST2的准确定量;尤其是将该试剂盒用于全自动电化学发光系统,加样、孵育、清洗和检测等步骤均可实现自动化,避免了人为操作带来的结果偏差,提高了工作效率,通过内置标准曲线到测试软件,只需测试样本即可定量检测样本中的ST2,使检测更快速、更可靠、更稳定。
具体实施方式
现在对本发明作进一步详细的说明。
实施例1 检测ST2的电化学发光试剂盒的制备方法。
(1)链霉亲和素偶联的磁性微粒工作液的制备
将链霉亲和素偶联的磁微粒悬浮液,磁分离去除上清,用pH为6.8,浓度为0.1mol/ L的PBS缓冲液重悬至浓度为0.5 mg/mL,所述缓冲液含有0.5 wt%的牛血清白蛋白、0.05 wt%的Triton X-100、0.05 wt% proclin300和0.05 wt%叠氮钠。
(2)生物素标记的ST2一抗的制备
取1 mg ST2抗体,加入适量PBS缓冲液(0.05 M,pH7.0-7.6)调整抗体总浓度至1mg/ml,并加入透析袋中进行透析,每隔3-4小时换一次透析液,更换3-4次,透析后将抗体转移至离心管或冻存管内;
准确称取1 mg N-羟基琥珀酰亚胺活化的生物素(NHS-Biotin),加入适量水调整其浓度为2 mg/ml,得NHS-Biotin水溶液;
将NHS-Biotin水溶液加入抗原溶液中,使NHS-Biotin与抗体按照5:1的摩尔比在室温混匀反应1小时,得生物素标记的ST2一抗溶液;将生物素标记的ST2溶液转移至透析袋进行透析(透析液为0.1 M PBS,pH7.4),每隔3-4小时换一次透析液,更换3-4次,透析后收集生物素标记的ST2抗体至离心管或冻存管内,≤-20℃冷冻保存备用。
(3)三联吡啶钌标记ST2抗体的制备
用DMSO配制浓度为10 mg/mL的Ru-NHS酯溶液;用0.1 M、pH7.4的磷酸盐缓冲液配制浓度为1 mg/mL的ST2抗体溶液;
于1 mL的ST2抗体溶液中加入10 µL配制好的Ru-NHS酯溶液,37℃避光反应2小时,然后加入20μL、2M的甘氨酸终止反应,将反应液于0.1M、pH7.4的PBS缓冲液透析过夜,期间换液3次,回收三联吡啶钌标记的ST2抗体溶液,≤-20℃冷冻保存备用。
(4)生物素标记的ST2一抗工作液以及三联吡啶钌标记的ST2二抗工作液的制备
配制pH为6.5,浓度为0.1 mol/L的磷酸盐缓冲液,所述缓冲液含有2wt%的牛血清白蛋白、0.1wt%的酪蛋白、1wt%的氯化钠、1wt%的蔗糖、2wt%的小牛血清、0.5wt%的Triton X-100、0.2wt%的proclin 300;然后用该缓冲液配制抗体浓度为1mg/L的生物素标记的ST2一抗工作液,以及抗体浓度为10mg/L三联吡啶钌标记的ST2二抗工作液。
(5)校准品和质控品工作液的配制
配制pH为7.4,浓度为0.1 mol/L的N-(2-羟乙基)哌嗪-N′(2-乙磺酸)(HEPES)缓冲液,所述缓冲液含有1wt%的牛血清白蛋白、20wt%的人血清、1wt%的氯化钠、5wt%的乙二醇、0.1wt%的Tween-20、0.2wt%的proclin 300;然后用该缓冲液配制ST2校准品和质控品工作液,校准品共5个点,ST2的浓度分别为3ng/mL、10ng/mL、50 ng/mL、100 ng/mL、200ng/ml。质控品分高、低两个浓度,ST2的浓度分别为5ng/mL、50ng/mL。
(6)清洗液的配制
配制176 mmol/L的KOH溶液,所配清洗液中含有0.15%的十二醇乙二醇醚。
(7)化学发光底物液的配制
配制pH为6.5,0.15 mol/L的三丙胺溶液,该溶液含有0.05 wt%的十二醇乙二醇醚、0.01 wt%的proclin 300。
(8)试剂分装及组装,将链霉亲和素偶联的磁性微粒工作液6.5 mL/瓶、生物素标记的ST2一抗工作液10 mL/瓶、三联吡啶钌标记的ST2一抗工作液10 mL/瓶、校准品/质控品工作液1.0 mL/瓶分装后,组装在一起,保存于2-8℃,将清洗液380 mL/瓶、化学发光底物液380 mL/瓶单独包装,保存于20℃-25℃。
实施例2 使用本发明的试剂盒检测ST2的方法以全自动电化学发光法免疫分析仪为检测仪器,将试剂盒装载到仪器上进行检测,步骤如下:
将样本(或校准品或质控品)、生物素标记的ST2一抗工作液和三联吡啶钌标记的ST2二抗工作液加入到反应杯中,37℃孵育10分钟,形成抗体-抗原-抗体复合物溶液;
于抗体-抗原-抗体复合物溶液中加入链霉亲和素包被的磁性微粒工作液,37℃孵育10分钟,形成磁性复合物悬浮液;
将磁性复合物悬浮液置于磁场内,清洗液流过,洗涤所述磁性复合物;
向洗涤后的磁性复合物中先后注入电化学发光底物液,检测其光子强度;由光子强度和定标曲线推算出ST2的含量。
本发明的试剂盒的性能评估。
实施例3 试剂灵敏度的研究
试剂灵敏度是根据最低检测限(LOD)来确定的,最低检测限按下述实验方法进行:检测零浓度校准品20次,得到20次测定结果的信号值(RLU),计算其平均值M和标准差SD,得出M+2SD所对应的RLU值,根据空白样本与低值样本5ng/mL之间的浓度-RLU值结果进行两点回归拟合得出一次方程,将M+2SD所对应的RLU值带入上述方程中,计算得出对应的浓度,即最低检测限(LOD)。
以下表1中的测定结果显示,按上述方法检测试剂最低检测限(LOD)为2.5ng/mL,根据此结果确定试剂灵敏度可达到3ng/mL以下;
表1本发明试剂盒的灵敏度测定结果。
实施例4 本发明试剂盒的重复性的研究
检测浓度为5ng/mL、50ng/mL两个浓度的样本,每个样本检测10次,分别计算每个样本的变异系数(CV),结果表明该试剂盒变异系数CV均小于4 %;该CV值明显小于ELISA等传统方法,主要是由电化学发光免疫分析仪的高性能带来的;
表2本发明试剂盒的重复性测定结果。
实施例5 本发明试剂盒的准确度测定结果
由于该指标目前尚未有国家或国际标准品,采用回收实验对试剂盒的准确度进行测定;将已知浓度的ST2高值样本(80ng/ml)加入到ST2低值血清样本(5ng/ml)中,所加入的高值血清与低值血清体积比为1:9,回收率应在85%-115%;
表3 本发明试剂盒的准确度测定结果。
披露的本发明不仅仅限于描述的特定的方法、方案和物质,本发明的范围仅受限于所附的权利要求。
本领域的技术人员还将认识到,或者能够确认使用不超过常规实验,在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。
参考文献:
1.可溶性ST2及其配体IL-33与疾病关系的研究进展,郑久荣 , 张建强 , 李莹莹《现代生物医学进展》 2021年
2.达格列净治疗老年心力衰竭的效果及对血清ST2,NT-proBNP水平的影响,缪雄,陆洋,颜永进《实用老年医学》2021年
3.血清sST2,NT-proBNP水平与血液透析患者发生心力衰竭的相关性分析,林胜 ,王华国《标记免疫分析与临床》2021年
4.The association between novel ST2, BNP, atrial fibrillation andheart failure.K Al-Wahaibi , I Khan , B Mcadam ,《European Heart Journal》 2021年。
Claims (10)
1.一种检测ST2的电化学发光试剂盒,其特征在于,包括:链霉亲和素偶联的磁性微粒工作液,生物素标记的ST2一抗工作液,三联吡啶钌标记的ST2 二抗工作液,含三丙胺的电化学发光底物液,清洗液。
2.根据权利要求1所述的检测ST2的电化学发光试剂盒,其特征在于,检测血液中可溶性生长刺激表达基因2蛋白(ST2)。
3.根据权利要求1或2所述的检测ST2的电化学发光试剂盒,其特征在于,所述链霉亲和素偶联的磁性微粒工作液由pH为6.8~7.6,浓度为0.01mol/L~0.2mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MOPS缓冲液中的一种配制而成,所述缓冲液含有0.05~2wt%的牛血清白蛋白和/或酪蛋白、0.02~1wt%的十二烷基聚乙二醇醚和/或聚乙二醇对异辛基苯基醚和/或聚氧乙烯失水山梨醇单月桂酸酯和/或月桂醇聚氧乙烯醚、0.05~0.5wt%的proclin300和/或叠氮钠。
4.根据权利要求1-3任一项所述的检测ST2的电化学发光试剂盒,其特征在于,所述生物素标记的ST2抗体以及三联吡啶钌标记的ST2抗体工作液由pH为6.0~7.6,浓度为0.01mol/L~0.2 mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MOPSO缓冲液中的一种配制而成,所述缓冲液含有0.5~5 wt%的牛血清白蛋白、1 mg/L~100 mg/L的抗干扰剂A、0.5~5 wt%的氯化钠、1~5 wt%的蔗糖、0.05~2 wt%的小牛血清、0.02~1 wt%的酪蛋白、0.02~1 wt%的十二烷基聚乙二醇醚和/或聚乙二醇对异辛基苯基醚和/或聚氧乙烯失水山梨醇单月桂酸酯和/或月桂醇聚氧乙烯醚、0.05~0.5 wt%的proclin 300和/或叠氮钠。
5.根据权利要求1-4任一项所述的检测ST2的电化学发光试剂盒,其特征在于,所述检测ST2的电化学发光试剂盒还包括ST2校准品工作液和/或质控品工作液,所述ST2校准品和/或质控品工作液由pH为6.0~7.6,浓度为0.01 mol/L~0.2 mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MES缓冲液中的一种配制而成,所述缓冲液中含有0.5~5 wt%的牛血清白蛋白、10~50 wt%的人血清、0.5~3 wt%的氯化钠、2~20 wt%的乙二醇、0.02~1 wt%的Brij 35和/或Triton X-100和/或Tween 20和/或月桂醇聚氧乙烯醚、0.05~0.5 wt%的proclin300和/或叠氮钠。
6.根据权利要求1-5任一项所述的检测ST2的电化学发光试剂盒,其特征在于,所述清洗液为pH为13以上,浓度为0.1 mol/L~0.5 mol/L的KOH,所述清洗液含有0.01~2 wt%的烷基聚乙二醇类表面活性剂。
7.根据权利要求1-6任一项所述的检测ST2的电化学发光试剂盒,其特征在于,所述电化学发光底物液为pH为6.0~7.2,浓度为0.05~0.4 mol/L的磷酸盐缓冲液或Tris缓冲液,所述缓冲液含有0.05~0.4 mol/L的三丙胺、0.01~2 wt%的烷基聚乙二醇类表面活性剂、0.05~0.5 wt%的proclin 300和/或叠氮钠。
8.一种检测ST2的电化学发光试剂盒的制法,其特征在于,包括:链霉亲和素偶联的磁性微粒工作液的制备,生物素标记的ST2一抗工作液的制备,三联吡啶钌标记的ST2二抗工作液的制备,清洗液的配制,电化学发光底物液的配制,将上述制备的试剂进行分装及组装。
9.根据权利要求8所述的检测ST2的电化学发光试剂盒的制法,其特征在于,所述生物素标记的ST2一抗的制备方法为:将N-羟基琥珀酰亚胺活化的生物素与ST2抗体按照1 ~20:1的摩尔比在2-8 ℃或室温条件下混匀反应0.5-2小时,透析或过柱除去多余的生物素,得生物素标记的ST2抗体。
10.根据权利要求8或9所述的检测ST2的电化学发光试剂盒的制法,其特征在于,所述三联吡啶钌标记的ST2抗体的制备方法为:将pH为6.5-8.0的ST2抗体溶液与Ru-NHS酯溶液混合,使ST2抗体与Ru-NHS酯的质量比为5-20: 1,37℃避光反应1-3小时,然后加入甘氨酸溶液终止反应,将反应液透析除去多余的Ru-NHS酯,得三联吡啶钌标记ST2抗体。
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