CN107449902A - 单组分tmb底物浓缩液及其制备方法与应用 - Google Patents

单组分tmb底物浓缩液及其制备方法与应用 Download PDF

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CN107449902A
CN107449902A CN201710598991.1A CN201710598991A CN107449902A CN 107449902 A CN107449902 A CN 107449902A CN 201710598991 A CN201710598991 A CN 201710598991A CN 107449902 A CN107449902 A CN 107449902A
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Abstract

本发明提供了单组分TMB底物浓缩液及其制备方法与应用，每1ml所述浓缩液中：甘油0.1ml,聚乙烯甘醇2000(PEG2000)0.1g,二甲基亚砜(DMSO)0.1ml,TMB 0.013g,β‑环糊精0.02g,乙二胺四乙酸(EDTA)0.0024g,柠檬水0.02g,过氧化氢0.22％，已丙醇0.05ml,反丁烯二酸0.01g,其余为去离子水，所述浓缩液pH值为3.6±0.2，通过混合方式获得，具有极高的稳定性和TMB的溶解度。

Description

单组分TMB底物浓缩液及其制备方法与应用
技术领域
本发明涉及分子技术领域，尤其是单组分TMB底物浓缩液及其制备方法与应用。
背景技术
TMB的中文名称为3,3’,5,5’–四甲基对二氨基联苯，是一种新型安全的色原试剂；TMB已在逐步取代强致癌物联苯胺和其他致癌性的联苯胺衍生物，应用于临床化验、法医检验、刑事侦破及环境监测等领域；尤其是在临床生化检验方面，TMB作为过氧化酶的新底物，在酶免疫分析法(EIA)和酶联免疫吸附检验法(ELISA)中获得了广泛的应用。TMB作为与过氧化物酶一起进行酶联免疫测定的底物，其作用机理在几十年前就已经确立，阻止TMB被氧化以及形成沉淀的方法也早就得到了阐明。
随着在酶联抗体检测和免疫诊疗中以TMB为底物、辣根过氧化物酶(HRP)为活性物的免疫测定中TMB被得到广泛应用，TMB已成为广泛接受的良好的过氧化物底物酶，TMB的需求量也与日剧增。然而使用浓度的TMB底物浓度较小，体积庞大，为TMB的运输造成极大不便。
发明内容
本发明所要解决的技术问题在于提供一种单组分TMB底物浓缩液。
本发明所要解决的另一技术问题在于提供上述单组分TMB底物浓缩液的制备方法。
本发明所要解决的另一技术问题在于提供上述单组分TMB底物浓缩液的应用。
为解决上述技术问题，本发明的技术方案是：
一种单组分TMB底物浓缩液，每1ml浓缩液中：甘油0.1ml,聚乙烯甘醇2000(PEG2000)0.1g,二甲基亚砜(DMSO)0.1ml,TMB 0.013g,β-环糊精0.02g,乙二胺四乙酸(EDTA)0.0024g,柠檬水0.02g,过氧化氢0.22％，已丙醇0.05ml,反丁烯二酸0.01g,其余为去离子水，所述浓缩液pH值为3.6±0.2。
优选的，上述单组分TMB底物浓缩液，所述去离子水为电阻18MΩ的去离子水。
上述单组分TMB底物浓缩液为浓缩20倍的TMB溶液。
上述单组分TMB底物浓缩液的制备方法，具体步骤如下：
(1)按组方量准备如下组分：甘油、聚乙烯甘醇2000、二甲基亚砜、TMB、β-环糊精、乙二胺四乙酸、柠檬酸、过氧化氢、异丙醇、反丁烯二酸和去离子水；
(2)配置β-环糊精：将β-环糊精加入部分去离子水中，搅拌至完全溶解；
(3)继续加入除DMSO、TMB和H2O2外的其他全部剩余试剂；
(4)在不断搅拌下将TMB加入到DMSO中；
(5)将步骤(4)中溶液、H2O2与步骤(3)中溶液混合，最终混合溶液包含组方中所有的化学试剂，调节混合溶液的pH值至3.6±0.2；
(6)继续加入剩余去离子水进行补足，所得到的TMB浓缩液在40-50℃条件下搅拌直至所有化学物质全部溶解，即得。
优选的，上述单组分TMB底物浓缩液的制备方法，所述步骤(5)中用于条件pH值的溶液为盐酸溶液。
上述单组分TMB底物浓缩液在检测辣根过氧化物酶方面的应用。
本发明的有益效果是：
上述单组分TMB底物浓缩液，采用多羟基有机化合物甘油和聚乙烯甘醇2000增加TMB溶解度，采用乙二胺四乙酸在特定pH值条件下避免TMB被重金属氧化，采用β-环糊精进一步提高TMB长期储存过程中的稳定性，采用多羟基有机溶剂增加TMB在有机溶剂和水的混合物中溶解度及稳定性，各组分协同作用，使TMB溶液在浓缩20倍的情况下TMB仍然具有极高的溶解度，进而大大提高了TMB的敏感度，选择特定电阻值的去离子水进一步增强了浓缩液在在室温下运输和储存过程中的稳定性，防止溶液运输和存储过程中变色，便于长途运输，其制备方法工艺简单，可以增加20倍TMB底物的浓度，使所述浓缩液在2-8℃条件下至少具有36个月的保质期。
附图说明
图1为单组分TMB底物浓缩液稳定性测试图。
具体实施方式
除非另有定义，本文所用的全部技术和科学术语是与在本领域的普通技术人员所通常理解的术语含义是相同的。
实施例1
一种单组分TMB底物浓缩液，为用于酶联免疫吸附测定的浓缩20倍的TMB溶液，配置100ml所述浓缩液所需试剂的重量或体积如下：
所述单组分TMB底物浓缩液的制备方法如下：
(1)按组方量准备如下组分：甘油、聚乙烯甘醇2000、二甲基亚砜、TMB、β-环糊精、乙二胺四乙酸、柠檬酸、过氧化氢、异丙醇、反丁烯二酸和电阻为18MΩ的去离子水；
(2)配置β-环糊精：将β-环糊精加入10ml去离子水中，搅拌至完全溶解；
(3)继续加入除DMSO、TMB和H2O2外的其他全部剩余试剂；
(4)在不断搅拌下将TMB加入到DMSO中；
(5)将步骤(4)中溶液、H2O2与步骤(3)中溶液混合，最终混合溶液包含组方中所有的化学试剂，混合溶液用6N HCl(盐酸)将pH值调节至3.6±0.2；
(6)继续加入去离子水使总体积达100ml，所得到的TMB浓缩液在40-50℃条件下搅拌直至所有化学物质全部溶解(一般约5-10小时)，即得。
计算单组分TMB底物浓缩液中TMB的浓度：每100ml含有1.3gTMB,TMB的分子量为240g/mol，经计算浓缩液中的TMB浓度可高达54.17mM。市场上采购的TMB底物浓度在1mM-2.5mM范围内，相比之下，所述浓缩液浓度大大提高，进而证明TMB的溶解度大大提高。
实施例2
TMB测试实验
对实施例1所述单组分TMB底物浓缩液进行稀释后得到单倍TMB底物，与市场上采购的TMB底物进行比对。先加入不同浓度的辣根过氧化物酶标准品到96孔酶标板，再加入100ul TMB底物到各个微孔，孵育10分钟，之后加入50ul 1N的盐酸停止反应，静止1分钟后，在450nm波长下读取数值。结果见下表1。
表1
可见，由本发明实施例1所述TMB浓缩液稀释得来的单倍TMB底物，与市场上其它商家提供的TMB底物比较，具有更高的敏感度。
实施例3
TMB浓缩液的稳定性
将实施例1所述单组分TMB底物浓缩液放在40℃的温箱中进行高温加速测试，周期为60天。每间隔15天，测量其效力。测量方法如下：
辣根过氧化物酶标准品被加入96孔酶标板，之后加入由20倍浓缩液稀释制成的单倍TMB底物100ul，孵育10分钟，加入50ul 1N盐酸停止反应，静止1分钟后，在波长450nm下测量结果。对照为正常条件保存的新配置TMB，并设置为效力100％。结果如图1所示，TMB在高温下60天内仍可保有80％以上的效力。
上述参照具体实施方式对该单组分TMB底物浓缩液及其制备方法与应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。

Claims (5)

1.一种单组分TMB底物浓缩液，其特征在于：每1ml浓缩液中：甘油0.1ml,聚乙烯甘醇2000 0.1g,二甲基亚砜0.1ml,TMB 0.013g,β-环糊精0.02g,乙二胺四乙酸0.0024g,柠檬水0.02g,过氧化氢0.22％，已丙醇0.05ml,反丁烯二酸0.01g,其余为去离子水，所述浓缩液pH值为3.6±0.2。
2.根据权利要求1所述的单组分TMB底物浓缩液，其特征在于：所述去离子水为电阻18MΩ的去离子水。
3.权利要求1或2所述单组分TMB底物浓缩液的制备方法，其特征在于：具体步骤如下：
(1)按组方量准备如下组分：甘油、聚乙烯甘醇2000、二甲基亚砜、TMB、β-环糊精、乙二胺四乙酸、柠檬酸、过氧化氢、异丙醇、反丁烯二酸和去离子水；
(2)配置β-环糊精：将β-环糊精加入部分去离子水中，搅拌至完全溶解；
(3)继续加入除DMSO、TMB和H2O2外的其他全部剩余试剂；
(4)在不断搅拌下将TMB加入到DMSO中；
(5)将步骤(4)中溶液、H2O2与步骤(3)中溶液混合，最终混合溶液包含组方中所有的化学试剂，调节混合溶液的pH值至3.6±0.2；
(6)继续加入剩余去离子水进行补足，所得到的TMB浓缩液在40-50℃条件下搅拌直至所有化学物质全部溶解，即得。
4.根据权利要求3所述的单组分TMB底物浓缩液的制备方法，其特征在于：所述步骤(5)中用于条件调节pH值的溶液为盐酸溶液。
5.权利要求1或2所述单组分TMB底物浓缩液在检测辣根过氧化物酶方面的应用。
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