CN116735857A - 一种碱性磷酸酶酶标缓冲液和碱性磷酸酶酶标试剂 - Google Patents
一种碱性磷酸酶酶标缓冲液和碱性磷酸酶酶标试剂 Download PDFInfo
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- CN116735857A CN116735857A CN202310214109.4A CN202310214109A CN116735857A CN 116735857 A CN116735857 A CN 116735857A CN 202310214109 A CN202310214109 A CN 202310214109A CN 116735857 A CN116735857 A CN 116735857A
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Abstract
本发明属于医用材料技术领域，具体涉及一种碱性磷酸酶酶标缓冲液和碱性磷酸酶酶标试剂。本发明技术方案中的碱性磷酸酶酶标缓冲液，包括次氮基三乙酸(NTA)、二亚乙基三胺五乙酸(DTPA)与镁离子形成的第一螯合物以及蛋氨酸与苏氨酸与锌离子形成的第二螯合物，还包括以下质量百分比的组分：0.2～2.0％还原型谷胱甘肽、1.0～2.5％玉米肽、0.5～10.0％蛋白质、0.1～2.0％人参皂苷、0.1～1.0％表面活性剂、1.0～5.0％糖类化合物、1.0～10.0％多元醇、0.05～0.1％防腐剂和0.01～0.05％抗生素，其余为非离子两性缓冲液。通过次氮基三乙酸、二亚乙基三胺五乙酸与镁离子形成的第一螯合物以及蛋氨酸与苏氨酸与锌离子形成的第二螯合物复合使用，结合谷胱甘肽、蛋白质等物质，得到具有长期稳定性的碱性磷酸酶酶标缓冲液。
Description
技术领域
本发明属于医用材料技术领域，具体涉及一种碱性磷酸酶酶标缓冲液和碱性磷酸酶酶标试剂。
背景技术
碱性磷酸酶(以下也称ALP)是一种在免疫测定法中通常作为标记使用的酶，属于同源二聚体蛋白，分子量为56KDa，每个单体由449个氨基酸组成，完整的AKP分子呈现典型的α/β的拓扑结构，同时每个单体均具有一个活性中心，活性中心区域由Asp101-Ser102-Ala103三连体、Arg166、水分子、三个金属离子及其配体氨基酸组成，AKP被phoA基因编码，与很多分泌蛋白一样，在细胞质内合成氨基末端带有信号肽的单体前体，信号肽引导前体跨内膜运输后被切除，形成同源二聚体。碱性磷酸酶是一种能够将对应底物去磷酸化的酶，通过水解磷酸单酯将底物分子上的磷酸基团除去，并生成磷酸根离子和自由的羟基，底物包括核酸、蛋白、生物碱等，该脱去磷酸基团的过程被称为去磷酸化或脱磷酸化。同时碱性磷酸酶作为一种磷酸酶，作用与激酶的作用正相反(激酶是磷酸化酶，可以利用能量分子，如ATP，将磷酸基团加到对应底物分子上)，碱性磷酸酶在碱性环境具有最大活力，对来源于细菌中的ALP来说，其最适pH是8.0，而对来源于牛的ALP，最适pH则是8.5。ALP是一种含锌的糖蛋白，在碱性环境中(最适pH为10)可以水解各种天然及人工合成的磷酸单酯化合物底物。
化学发光免疫测定(CLIA)属于标记抗体技术的一种，以化学发光剂、催化发光酶或产物间接参与发光反应的物质标记抗原或抗体，当标记物与相应物质结合后，发光底物受发光剂、催化酶或参与产物作用，发生氧化还原反应，反应中释放光信号，最后使用光电倍增管进行检测，技术特点有高通量，特异性高，灵敏度高、分离简单、快速、安全稳定、自动化等优势。
目前，通常采用化学发光酶免疫分析方法检测蛋白质(比如肿瘤标志物、CA125、HE4)的浓度，即将具有高灵敏度的酶催化化学发光技术与高特异性的抗原抗体反应相结合，用于定量检测各种抗原、半抗原、抗体、激素、酶、脂肪酸、维生素、和药物等。该技术具有特异性高、灵敏度高、分离简便快速、可自动化分析等优点。其中，所使用到的酶主要是碱性磷酸酶，碱性磷酸酶的标记物溶液是体外诊断试剂盒的组分之一，碱性磷酸酶的酶活稳定性成为试剂盒质量性能的重要影响因素。同其他生物酶一样，碱性磷酸酶半衰期短、易失活。酸、碱、盐离子、温度条件的变化都会改变甚至使酶完全失去活性。碱性磷酸酶在溶液中的长期稳定保存技术是体外诊断试剂盒产品的关键技术。
发明内容
本发明的目的是针对上述技术问题，提供一种碱性磷酸酶酶标缓冲液，提高碱性磷酸酶的检测效率和长期保存稳定性。
本发明技术方案中的碱性磷酸酶酶标缓冲液，包括次氮基三乙酸(NTA)、二亚乙基三胺五乙酸(DTPA)与镁离子形成的第一螯合物以及蛋氨酸与苏氨酸与锌离子形成的第二螯合物。
镁离子是ALP位点的特异性“效应分子”，可以与碱性磷酸酶中有关基团进行配位，稳定了酶活性部位的构象，使得自发折叠过程能够向类天然态的方向进行，与次氮基三乙酸、二亚乙基三胺五乙酸螯合后，可以有效提高碱性磷酸酶的催化效率及稳定性。
进一步地，镁离子来自于镁离子化合物，镁离子化合物可以列举为氯化镁、硫酸镁、硝酸镁、乙酸镁、乳酸镁中的一种或多种。
进一步地，锌离子来自于镁离子化合物，锌离子化合物可以列举为氯化锌、硫酸锌、硝酸锌、乙酸锌、乳酸锌中的一种或多种。
进一步地，第一螯合物中次氮基三乙酸、二亚乙基三胺五乙酸与镁离子的质量比为0.5～2.5:0.5～2.5:1.0。
进一步地，第二螯合物中蛋氨酸与苏氨酸与锌离子的质量比为5～10:5～10:1。
进一步地，第一螯合物和第二螯合物的质量百分比分别为2.0～3.0％和0.1～0.5％。
进一步地，上述碱性磷酸酶酶标缓冲液的pH为6.0～7.0。
进一步地，上述碱性磷酸酶酶标缓冲液，还包括以下质量百分比的组分：0.2～2.0％还原型谷胱甘肽、1.0～2.5％玉米肽、0.5～10.0％蛋白质、0.1～2.0％人参皂苷、0.1～1.0％表面活性剂、1.0～5.0％糖类化合物、1.0～10.0％多元醇、0.05～0.1％防腐剂和0.01～0.05％抗生素，其余为非离子两性缓冲液。
谷胱甘肽是由谷氨酸、胱氨酸及甘氨酸组成的一种三肽，它是甘油醛酸脱氢酶的辅基，又是乙二醛酶及磷酸丙糖脱氢酶的辅酶，参与体内三羧酸循环及糖代谢，谷胱甘肽在体内以还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)两种形式存在，其活性成分为还原型谷胱甘肽，能参与体内氧化还原过程，在谷胱甘肽转移酶的作用下，还原型谷胱甘肽能和过氧化物及自由基相结合，以对抗氧化剂对巯基的破坏，保护含巯基的蛋白质和含巯基的酶不被破坏，进而提高酶结合物的稳定性；玉米肽是以玉米蛋白粉为原料，经过水解环节获得的分子量小但是活性高的短肽分子组合而成的混合物质。相比较于氨基酸或者蛋白质来说，在大范围PH下还可以溶于水，无浑浊的情况和沉淀物产生，玉米肽的热稳定性好，组分不会发生变化，提高酶结合物的稳定性；人参皂苷具抗菌、抗氧化性进而提高ALP偶联物的稳定性。
进一步地，蛋白质为牛血清白蛋白(BSA)、牛乳铁蛋白(LF)、鸡卵白蛋白(OVA)中的一种或多种。优选质量比为2～3:1的牛乳铁蛋白与鸡卵白蛋白混合物。
进一步地，表面活性剂为非离子表面活性剂，列举为聚氧乙烯聚氧丙烯醚、辛基酚聚氧乙烯醚、聚氧乙烯失水山梨醇酯、失水山梨醇酯中的一种或多种。
进一步地，糖类化合物为葡聚糖，多聚果糖，果糖，木糖，鼠李糖中的一种或多种。
进一步地，多元醇为甘露醇、山梨醇、木糖醇中的一种或多种。
本发明还提供一种碱性磷酸酶酶标试剂，包括上述碱性磷酸酶酶标缓冲液。
相比现有技术，本发明的技术方案具有如下有益效果：
(1)通过次氮基三乙酸、二亚乙基三胺五乙酸与镁离子形成的第一螯合物以及蛋氨酸与苏氨酸与锌离子形成的第二螯合物复合使用，结合谷胱甘肽、蛋白质等物质，得到具有长期稳定性的碱性磷酸酶酶标缓冲液；
(2)本发明所得碱性磷酸酶酶标缓冲液，可以长期保护碱性磷酸酶的活性，维持碱性磷酸酶酶标试剂良好的检测效果。
具体实施方式
下面通过具体实施例，对本发明的技术方案作进一步描述说明，应当理解的是，此处所描述的具体实施例仅用于帮助理解本发明，不用于本发明的具体限制。如果无特殊说明，本发明的实施例中所采用的原均为本领域常用的原料，实施例中所采用的方法，均为本领域的常规方法。
以下实施例及对比例的碱性磷酸酶酶标缓冲液，以HEPES非离子两性缓冲液作为基础缓冲液，表面活性剂为PluronicF-127，防腐剂为bronidox，抗生素用硫酸庆大霉素，具体配方如下表1所示。
表1实施例碱性磷酸酶酶标缓冲液配方表
| 实施例1 | 实施例2 | 实施例3 | 实施例4 | 实施例5 | 实施例6 | |
| MgCl2 | 0.1％ | 0.1％ | 0.1％ | 0.1％ | 0.1％ | 0.1％ |
| NTA | 2.0％ | 1.5％ | 2.0％ | 2.0％ | 2.0％ | 2.0％ |
| DTPA | 0.5％ | 1.0％ | 0.5％ | 0.5％ | 0.5％ | 0.5％ |
| ZnCl2 | 0.05％ | 0.05％ | 0.05％ | 0.05％ | 0.05％ | 0.05％ |
| 蛋氨酸 | 0.1％ | 0.1％ | 0.15 | 0.1％ | 0.1％ | 0.1％ |
| 苏氨酸 | 0.1％ | 0.1％ | 0.15 | 0.1％ | 0.1％ | 0.1％ |
| 还原型谷胱甘肽 | 1.0％ | 1.0％ | 1.0％ | 1.0％ | 1.0％ | 0.1％ |
| LF | 1.5％ | 1.5％ | 1.5％ | 1.5％ | 1.5％ | - |
| OVA | 1.0％ | 1.0％ | 1.0％ | 1.0％ | - | - |
| 人参皂苷 | 0.6％ | 0.6％ | 0.6％ | 0.6％ | - | - |
| PluronicF-127 | 0.5％ | 0.5％ | 0.5％ | - | - | - |
| 鼠李糖 | 2.0％ | 2.0％ | 2.0％ | - | - | - |
| 玉米肽 | 1.5％ | 1.5％ | 1.5％ | 1.5％ | 1.5％ | - |
| 甘露醇 | 3.0％ | 3.0％ | - | - | - | - |
| bronidox | 0.08％ | 0.08％ | - | - | - | - |
| 硫酸庆大霉素 | 0.02％ | - | - | - | - | - |
| pH | 6.2 | 6.5 | 6.2 | 6.2 | 6.2 | 6.2 |
表2对比例碱性磷酸酶酶标缓冲液配方表
将碱性磷酸酶标记物加入到上述实施例的碱性磷酸酶酶标缓冲液中，配制成终浓度为2ug/mL的酶溶液，置于37℃恒温恒湿箱中进行加速试验。以酶溶液为样本，利用海尔施智造的GV300化学发光仪及配套测定试剂盒，测定溶液中碱性磷酸酶标记物的活性剩余比率(加速试验后的活性与制备后活性的比率)，结果如下表3所示。
表3碱性磷酸酶标记物的活性剩余比率数据
| 加速天数 | 第1天 | 第2天 | 第5天 | 第7天 | 第9天 |
| 实施例1 | 99.7％ | 99.7％ | 99.5％ | 99.2％ | 98.9％ |
| 实施例2 | 99.0％ | 98.5％ | 97.5％ | 97.0％ | 96.9％ |
| 实施例3 | 99.0％ | 97.0％ | 97.0％ | 96.0％ | 95.2％ |
| 实施例4 | 98.0％ | 95.0％ | 92.0％ | 88.0％ | 84.7％ |
| 实施例5 | 97.2％ | 90.0％ | 85.0％ | 76.0％ | 74.0％ |
| 实施例6 | 93.5％ | 84.0％ | 75.0％ | 65.0％ | 62.3％ |
| 对比例1 | 92.5％ | 81.0％ | 74.5％ | 62.0％ | 59.3％ |
| 对比例2 | 91.5％ | 80.2％ | 73.4％ | 64.2％ | 50.1％ |
| 对比例3 | 94.5％ | 81.2％ | 72.8％ | 60.1％ | 51.4％ |
| 对比例4 | 92.5％ | 79.5％ | 71.8％ | 65.1％ | 59.5％ |
| 对比例5 | 93.5％ | 77.8％ | 69.5％ | 60.4％ | 55.4％ |
| 对比例6 | 95.5％ | 79.2％ | 71.4％ | 62.4％ | 51.8％ |
| 对比例7 | 92.5％ | 79.5％ | 70.4％ | 60.5％ | 51.4％ |
| 对比例8 | 91.5％ | 79.1％ | 68.4％ | 59.8％ | 54.3％ |
由表3的数据可知，本发明的碱性磷酸酶酶标缓冲液具有优异的稳定性，碱性磷酸酶标记物经过加速后，一周后碱性磷酸酶活性剩余98.9％，损失了近1.01％，可以长期保护碱性磷酸酶的活性。
最后应说明的是，本文中所描述的具体实施例仅仅是对本发明精神作举例说明，而并非对本发明的实施方式的限定。本发明所属技术领域的技术人员可以对所描述的具有实施例做各种各样的修改或补充或采用类似的方式替代，这里无需也无法对所有的实施方式予以全例。而这些属于本发明的实质精神所引申出的显而易见的变化或变动仍属于本发明的保护范围，把它们解释成任何一种附加的限制都是与本发明精神相违背的。
Claims (10)
1.一种碱性磷酸酶酶标缓冲液，其特征在于，包括次氮基三乙酸、二亚乙基三胺五乙酸与镁离子形成的第一螯合物以及蛋氨酸与苏氨酸与锌离子形成的第二螯合物。
2.根据权利要求1所述的碱性磷酸酶酶标缓冲液，其特征在于，第一螯合物中次氮基三乙酸、二亚乙基三胺五乙酸与镁离子的质量比为0.5～2.5:0.5～2.5:1.0。
3.根据权利要求1所述的碱性磷酸酶酶标缓冲液，其特征在于，第二螯合物中蛋氨酸与苏氨酸与锌离子的质量比为2～5:2～5:1。
4.根据权利要求1所述的碱性磷酸酶酶标缓冲液，其特征在于，第一螯合物和第二螯合物的质量百分比分别为2.0～3.0％和0.1～0.5％。
5.根据权利要求1所述的碱性磷酸酶酶标缓冲液，其特征在于，所述碱性磷酸酶酶标缓冲液的pH为6.0～7.0。
6.根据权利要求1所述的碱性磷酸酶酶标缓冲液，其特征在于，所述碱性磷酸酶酶标缓冲液，还包括以下质量百分比的组分：0.2～2.0％还原型谷胱甘肽、1.0～2.5％玉米肽、0.5～10.0％蛋白质、0.1～2.0％人参皂苷、0.1～1.0％表面活性剂、1.0～5.0％糖类化合物、1.0～10.0％多元醇、0.05～0.1％防腐剂和0.01～0.05％抗生素，其余为非离子两性缓冲液。
7.根据权利要求6所述的碱性磷酸酶酶标缓冲液，其特征在于，蛋白质为牛血清白蛋白、牛乳铁蛋白、鸡卵白蛋白中的一种或多种。
8.根据权利要求6所述的碱性磷酸酶酶标缓冲液，其特征在于，糖类化合物为葡聚糖，多聚果糖，果糖，木糖，鼠李糖中的一种或多种。
9.根据权利要求6所述的碱性磷酸酶酶标缓冲液，其特征在于，多元醇为甘露醇、山梨醇、木糖醇中的一种或多种。
10.一种碱性磷酸酶酶标试剂，其特征在于，包括权利要求1所述的碱性磷酸酶酶标缓冲液。
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