CN103698505B - 一种高效稳定的tmb显色液及其制备方法 - Google Patents
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Abstract
市场上TMB 显色液存在稳定性差，保存时间短，显色背景高，灵敏度低等的缺点，本发明提供了一种高效稳定的TMB显色液。其特征在于，各组分的摩尔浓度或质量分数为：TMB的摩尔浓度为1.2mmol/L，H2O2的摩尔浓度为3.1mmol/L，曲拉通X-405的质量分数为0.3%，过氧化脲的质量分数为0.5%，醋酸钠的摩尔浓度为0.2mol/L，L(+)-酒石酸的摩尔浓度为0.027 mol/L。本发明为TMB单相显色液，使用前无需混合，可以减少人为操作过程中的误差，通过引入非离子表面活性剂，使单相TMB显色液可以长期稳定保存。
Description
技术领域
本发明涉及临床体外检测技术，特别涉及一种高效稳定的TMB显色液及其制备方法。
背景技术
自从Engvall和Perlman(1971）首次报道建立酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assays,ELISA）以来，由于ELISA具有快速、敏感、简便、易于标准化等优点，使其得到迅速的发展和广泛应用。目前酶免疫分析(EIA)技术，已被广泛应用于抗原，半抗原或抗体的定量或定性检测分析。酶联免疫吸附 (ELISA) 检测方法虽然有很多种，但各种方法都离不开酶结合物和显色剂。在 ELISA 试剂中应用最广泛的酶是辣根过氧化物酶 (HRP)，而该系统的显色剂有多种，目前市场最常用的是TMB （3,3’,5,5’- 四甲基联苯胺）。辣根过氧化物酶(HRP)及其藕联物是酶联免疫分析技术中常用的一种酶，由于3,3',5,5'-四甲基联苯胺(TMB)在HRP的显色反应体系中，比其它色原具有更高的灵敏度且无至癌性而被广泛应用。
TMB（3,3’,5,5’-四甲基联苯胺）是一种新型HRP（辣根过氧化物酶）色原底物。相对其他显色底物，TMB 以显色高效、无毒、不致癌、稳定性好等特点成为 ELISA 检测的主流显色剂。在ELISA检测时，HRP催化过氧化物释放出氧使 TMB 被氧化成蓝色的产物，加入酸性的终止液终止反应后，蓝色溶液变成橙黄色，在 450nm 波长有最大吸收峰。通过测量吸光度，便可定性或定量判断出被检测物的含量。
常见的 TMB 显色试剂多由几个组分共同组成，并且常需要现配现用，使用不便，且容易导致检测结果不太稳定。一般市场常见 TMB 显色试剂通常分为 A、B 两种贮存液，使用需将两者按比例混合，操作繁琐且容易错配，常导致实验失败。此外，市场上也有少见的单相的TMB 显色液，但常常存在稳定性差，保存时间短，显色背景高，灵敏度低等的缺点。
发明内容
针对上述技术背景，本发明提供了一种高效稳定的TMB显色液及其制备方法。
本发明的一种高效稳定的TMB显色液，其特征在于，各组分的摩尔浓度或质量分数为：TMB的摩尔浓度为1.2mmol/L，H2O2的摩尔浓度为3.1mmol/L，曲拉通X-405的质量分数为0.3%，过氧化脲的质量分数为0.5%，醋酸钠的摩尔浓度为0.2mol/L，L(+)-酒石酸的摩尔浓度为0.027 mol/L。
所述的一种高效稳定的TMB显色液的制备方法，其特征在于包括以下步骤：
a.制备A液：以10mL二甲亚砜为溶剂，缓慢加入TMB，搅拌均匀使其充分溶解，TMB最终摩尔浓度为0.12mol／L；
b.制备B液：取1L蒸馏水，加入醋酸钠16.406g使之终摩尔浓度为0.2mol/L；然后加入L(+)-酒石酸4.1g使之终摩尔浓度为0.027 mol/L，溶液最终pH值约为5.0。
c.在B液中加入适量A液、曲拉通X-405（Triton X-405）、H2O2和过氧化脲，使TMB终摩尔浓度为1.2 mmol/L，H2O2终摩尔浓度为3.1mmol/L，曲拉通X-405（Triton X-405）的终质量分数为0.3%，过氧化脲的终质量分数为0.5%。
本发明的使用方法：
用适当的干净容器(用纯净水反复冲洗)，倒出适量的单组分TMB显色液，待达到室温后即可进行如下步骤：
a) 加液：加完HRP结合物并温育一定时间后，洗板3-5次，每孔加TMB显色液100uL。 根据实验需要，在室温(15-25°C)或37°C下温育10-30分钟。
b) 终止：加等体积的1mol／L盐酸或硫酸溶液可终止反应，孔中反应液由蓝色变为黄色。
c) 读数：终止反应后30分钟内在450nm处读数。
相比市场常见TMB显色液而言，本发明为TMB单相显色液，使用前无需混合，简化操作流程，方便快捷，可以减少人为操作过程中的误差。通过引入非离子表面活性剂，促进TMB的溶解和稳定，使单相TMB显色液可以长期稳定保存，在2~8℃条件下保存有效期可达3年，并且显色终止后读数稳定。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1 实施例1方法制备的TMB显色液用酸溶液终止反应后TMB黄色产物的稳定性。
图2 实施例1方法制备的TMB显色液存放时间段后的稳定性。
具体实施方式
实施例1
本发明所述的一种高效稳定的TMB 显色液的制备方法包括如下步骤：
a制备A液：以10mL二甲亚砜为溶剂，缓慢加入TMB，搅拌均匀使其充分溶解，TMB最终摩尔浓度为0.12mol／L；
b.制备B液：取1L蒸馏水，加入醋酸钠16.406g充分溶解，使之终摩尔浓度为0.2mol/L；然后加入L(+)-酒石酸（L(+)-2,3-二羟基丁二酸）4.1g充分溶解，使之终摩尔浓度为0.027 mol/L， 溶液最终pH值约为5.0。
c.在B液中加入适量A液、曲拉通X-405（Triton X-405）、H2O2和过氧化脲，使TMB终摩尔浓度为1.2 mmol/L，H2O2终摩尔浓度为3.1mmol/L，曲拉通X-405（Triton X-405）的终质量分数为0.3%，过氧化脲的终质量分数为0.5%，醋酸钠的摩尔浓度为0.2mol/L，L(+)-酒石酸的摩尔浓度为0.027 mol/L。
实施例2
将实施例1的TMB显色液与国家食品药品监督管理局认可的某公司商品化的TMB显色液（简称为：对比显色液1）进行对比。
该对比显色液配方，其中各组分的摩尔浓度或质量分数为：
TMB的摩尔浓度为2.0mmol/L，
H2O2的摩尔浓度为4.0mmol/L，
过氧化脲的质量分数为0.1%，
磷酸氢二钠的摩尔浓度为0.1mol/L，
柠檬酸的摩尔浓度为0.013 mol/L。
以丙型肝炎病毒核心抗原试剂盒（双抗体夹心法ELISA）为例，显色15min后终止反应，在终止反应后的0，15，30，45，60min时测得OD值（λ=450nm）。用酸溶液终止反应后，计算TMB黄色产物的稳定性。
对比结果见说明书附图图1。由图1可以看出，实施例1方法得到的TMB显色液在终止反应后，TMB黄色产物的稳定性明显优于对比组TMB显色液。实施例1方法得到的TMB显色液在终止反应30分钟后，TMB换色产物几乎没有衰减；在终止反应60min时，仅有轻微衰减，稳定性明显提高。
实施例3
将用实施例1的方法制备的TMB显色液与国家食品药品监督管理局认可的某公司商品化的TMB显色液进行比较（简称为：对比显色液1）。
以丙型肝炎病毒核心抗原试剂盒（双抗体夹心法ELISA）为例，分别用实施例1的方法制备的TMB显色液和国家食品药品监督管理局认可的某公司商品化的TMB显色液（简称为：对比显色液1）做显色剂，测试丙肝核心抗原（HCV-cAg）标准品（浓度：80pg/mL），测试完成后试剂盒等均放置在2-8℃保存，每隔2个月测试一次，记录OD值。 对比结果见说明书附图图2。由图2可知，实施例1的方法制备的TMB显色液可以长期稳定存放，至少可以放置3年。
由此可见，相比市场常见TMB显色液而言，本发明为TMB单相显色液，使用时无需混合，简化操作流程，方便快捷，极大的减少人为操作误差。此外，通过加入非离子表面活性剂曲拉通X-405（Triton X-405），促进TMB的溶解和稳定，使单相TMB显色液可以长期稳定保存，在2~8℃条件下有效期3年，并且显色终止后读数稳定。可证明，本发明所涉及的TMB显色液具有高效稳定的特点，使用方便，可以满足酶联免疫试剂盒对显色底物液的各项要求。
Claims (2)
1.一种高效稳定的TMB显色液，其特征在于，各组分的摩尔浓度或质量分数为：TMB的摩尔浓度为1.2mmol/L，H2O2的摩尔浓度为3.1mmol/L，曲拉通X-405的质量分数为0.3%，过氧化脲的质量分数为0.5%，醋酸钠的摩尔浓度为0.2mol/L，L(+)-酒石酸的摩尔浓度为0.027 mol/L。
2.根据权利要求1中的一种高效稳定的TMB显色液的制备方法，其特征在于包括以下步骤：
a.制备A液：以10mL二甲亚砜为溶剂，缓慢加入TMB，搅拌均匀使其充分溶解，TMB最终摩尔浓度为0.12mol／L；
b.制备B液：取1L蒸馏水，加入醋酸钠16.406g充分溶解，使之终摩尔浓度为0.2mol/L；然后加入L(+)-酒石酸4.1g充分溶解，使之终摩尔浓度为0.027 mol/L， 溶液最终pH值约为5.0；
c.在B液中加入适量A液、曲拉通X-405、H2O2和过氧化脲，使TMB终摩尔浓度为1.2 mmol/L，H2O2终摩尔浓度为3.1mmol/L，曲拉通X-405的终质量分数为0.3%，过氧化脲的终质量分数为0.5%。
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| CN106153907A (zh) * | 2015-03-25 | 2016-11-23 | 霍普金斯(北京)医学诊断科技有限公司 | 一种基于胶体金的酶联免疫显色试剂 |
| CN105116141B (zh) * | 2015-07-02 | 2016-09-14 | 深圳市卫光生物制品股份有限公司 | 一种单组份tmb显色液及其制备方法 |
| CN107190050B (zh) * | 2016-03-14 | 2020-10-27 | 华东理工大学 | 一种hrp活性测定及h2o2浓度检测的试剂盒及其应用 |
| CN105974107B (zh) * | 2016-07-13 | 2018-01-30 | 广州捷倍斯生物科技有限公司 | 单组份tmb显色液及其配制方法 |
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