CN116067949A - 一种单组份tmb显色液及制备方法 - Google Patents
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Abstract
本发明提供了一种单组份TMB显色液及制备方法。解决现有技术中涉及的单组份TMB配制使用试剂多、配制过程复杂且不易控制的缺点，还解决了现市售部分单组份TMB试剂加入终止液后容易产生沉淀的问题。本发明所提供显色液配制过程简单，为即用型产品，方便快捷，稳定性好，2‑8°C保存，有效期不低于24个月，显色终止后读数稳定，即使室温也可以20小时放置，也没有沉淀形成；TMB单组分显色液在370nm检测时OD值小于0.2。
Description
技术领域
本发明涉及生物体外诊断试剂，具体涉及一种单组份TMB显色液。
背景技术
TMB中文名是3,3',5,5'-四甲基联苯胺，为白色结晶粉末，无嗅、无味，难溶于水，易溶于丙酮、乙醚、二甲亚砜、二甲基甲酰胺等有机溶剂。属于一种新型安全的显色试剂。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；主要应用于:潜血指纹检测；唾液中酒精的快速检测；尿联试纸的配制；肝炎病毒检测；妊娠检测试验；血液和尿液中葡萄糖、血红蛋白、白蛋白的快速测定；粪便隐血试验；血液中粒细胞值的测定、类固醇、性激素的检测；酶活性的测定；抗原、抗体及遗传物质的分析、检测；生物样品的染色；水质检测(余氯、亚硝酸盐等)。
TMB显色机理：其氧化产物联苯醌在波长450nm处有最大消光系数，如果HRP量少，过氧化氢溶液和TMB过量时，则形成蓝色的阳离子根。降低pH，即可使蓝色的阳离子根转变为黄色的联苯醌。
经对现有技术比较研究分析发现，组份不同缓冲液都能支持完成酶促反应，完成显色反应，使用多种缓冲体系可以进行单组分TMB显色液的配制，TMB溶解多使用二甲基亚砜；但在实际配制过程中，因各种组份及配伍不同，导致在配制TMB使用时，可能会有沉淀析出，影响检测结果的可靠性。终止之后的溶液的稳定性也不太理想。
发明内容
本发明的目的是提供一种显色效果并且稳定性好的单组分TMB显色液体系，解决现行显色剂高成本，稳定性差，终止显色后容易有沉淀的问题。
本发明提供技术方案如下：
一种单组份TMB显色液，每1L的单组份TMB显色液包括以下组分：TMB 50-70mg，DMSO 5-10mL，甘油0.5-1.0mL，醋酸钠·3H2O 0.023-0.046g，柠檬酸·H2O 0.21-0.42g，30％H2O20.2-0.3mL；终止液为0.05-0.1mol/L H2SO4。
优选的，每1L的单组份TMB显色液包括以下组分：TMB 50mg，DMSO 5mL，甘油0.5mL，醋酸钠·3H2O 0.023g，柠檬酸·H2O 0.21g，30％H2O20.2mL；终止液为0.05mol/L H2SO4。
上述单组份TMB显色液按以下配比配制：1L蒸馏水中，加入2-4mL TMB底物缓冲液，0.2-0.3mL 30％H2O2，5-7mL TMB储存液。所述TMB储存液由以下原料按如下重量体积比配制：TMB 5g,DMSO 450mL,甘油50mL，加蒸馏水定容到1L。所述TMB底物缓冲液由以下原料按如下重量体积比配制：醋酸钠·3H2O 112.7g，柠檬酸·H2O 105.0g，蒸馏水800mL，用浓盐酸调节PH至3.6，加蒸馏水定容到1000mL。
优选的，单组份TMB显色液1L按以下配比配制：1L蒸馏水中，加入2mL TMB底物缓冲液，0.2mL30％H2O2，5mLTMB储存液；终止液为0.05mol/L H2SO4。
对上述单组分TMB显色液进行稳定性验证，并和其他市售产品进行对照实验，显示了经所述单组分TMB显色液显色并终止，避免了沉淀形成，避光保存在4℃有效使用期限至少为2年。
本发明中单组份显色液为即用型，无需混合，方便快捷，有效的减少操作误差。稳定性好：2-8℃保存，有效期不低于24个月，显色终止后读数稳定，即使室温也可以20小时放置，且没有沉淀形成。背景低：单组分显色液在370nm检测时OD值小于0.2。
附图说明：
图1、加终止液前后TMB显色溶液的光谱变化图
图2、单组分TMB显色液的4℃稳定性验证,OD(450nm)值的变化图
图3、加入单组份TMB显色液，OD(370nm)值的变化图
图4、加入终止液，OD(450nm)值的变化图
图5、TMB显色与HRP浓度之间的量效关系图
图6、本专利描述的单组分TMB显色液的显色和终止情况
图7、产品A的显色和终止情况
图8、产品B的显色和终止情况
具体实施方式
试剂及设备
TMB、DMSO、甘油、醋酸钠·3H2O、柠檬酸·H2O、蒸馏水、浓盐酸、鼠抗人IgG(Fc)-HRP抗体、96孔酶标板、酶标仪。
配制单组份TMB显色液：单组份TMB显色液按以下配比配制：1L蒸馏水中，加入2-4mL TMB底物缓冲液，0.2-0.3mL 30％H2O2，5-7mL TMB储存液；所述TMB储存液由以下原料按如下重量体积比配制：TMB 5g,DMSO 450mL,甘油50mL，加蒸馏水定容到1L；所述TMB底物缓冲液由以下原料按如下重量体积比配制：醋酸钠·3H2O 112.7g，柠檬酸·H2O 105.0g，蒸馏水800mL，用浓盐酸调节PH至3.6，加蒸馏水定容到1000mL。终止液：0.05-0.1mol/LH2SO4。
实施例一：加终止液前后TMB显色溶液的吸收光谱变化
将鼠抗人IgG(Fc)-HRP抗体(Southern Biotech，货号9040-05)稀释2000倍，取1μL加入到96孔酶标板的底部，加入上述单组份TMB显色液100μL，显色20分钟，用酶标仪进行可见光扫描。加入50μL终止液(0.1mol/L H2SO4)，再用酶标仪进行可见光扫描。记录加终止液前后TMB显色溶液的光谱变化(图1)。
加入终止液前TMB显色溶液的最大吸收在370nm，加入终止液后TMB显色溶液的最大吸收在450nm。在后续实施例中，加终止液前使用370nm进行检测，加入加终止液后使用450nm进行检测。
实施例二：单组分TMB显色液稳定性验证(37℃加速稳定性验证)
将上述单组份TMB显色液放入37℃恒温箱中，第1、5、10、15天，每个样品取100μL，利用酶标仪测定溶液的OD370nm，以考察溶液稳定性。
表1.单组份TMB显色液的37℃加速稳定性验证结果(OD370nm)
结果显示，在37℃加速稳定性验证中，被考察单组份TMB显色液15天的稳定性良好，溶液的OD370nm正常波动，最大值没有超过0.2。15天37℃加速稳定性考察结果，可以视同为24个月4℃的稳定性结果。因此，本发明中的单组分TMB显色液的避光保存在4℃，保质期为24个月。
实施例三：单组分TMB显色液稳定性验证(4℃稳定性验证)
将鼠抗人IgG(Fc)-HRP抗体(Southern Biotech，货号9040-05)稀释2K倍，4K倍，8K倍，16K倍，32K倍，64K倍，128K倍，蒸馏水做空白对照。取1μL加入到96孔酶标板的底部，加入上述单组份TMB显色液100μL，显色20分钟，加入50μL终止液(0.05mol/L H2SO4)，记录各孔450nm的OD值。每两个月进行一次验证实验，结果显示(图2)，单组分TMB显色液的有效使用期限至少为2年。
实施例四：单组分TMB显色液检测不同浓度的HRP
将鼠抗人IgG(Fc)-HRP抗体(Southern Biotech，货号9040-05)稀释2K倍，4K倍，8K倍，16K倍，32K倍，64K倍，128K倍，蒸馏水做空白对照。取1μL加入到96孔酶标板的底部，加入上述单组份TMB显色液100μL，显色20分钟，每30秒记录各孔的370nm的OD值(见图3)。加入50μL终止液(0.1mol/L H2SO4)，记录20分钟，每30秒记录各孔450nm的OD值(见图4)。TMB显色与HRP浓度之间的量效关系(见图5)。
实施例五：单组分TMB显色液与其他产品的对比
将鼠抗人IgG(Fc)-HRP抗体(Southern Biotech，货号9040-05)稀释2K倍，4K倍，8K倍，16K倍，32K倍，64K倍，128K倍，蒸馏水做空白对照，分别命名S1、S2、S3、S4、S5、S6、S7、B。取1μL加入到96孔酶标板的底部，加入上述单组份TMB显色液100μL，显色30分钟，加入50μL终止液(0.05mol/L H2SO4)，记录各孔450nm的OD值，同产品A和产品B进行比较。产品A为某公司的双组份TMB显色液产品，产品B为某公司的单组份产品。本发明的单组分TMB显色液的显色和终止情况见图6。产品A的显色和终止情况见图7。产品B的显色和终止情况见图8。本专利描述的单组分TMB显色液的显色稳定性和终止稳定性均优于对照产品。产品A为双组份TMB显色液产品，操作不方便。产品B终止后形成黑色沉淀。因此，本发明中，单组分TMB显色液的显色和终止情况均优于对照的上市产品。
Claims (4)
1.一种单组份TMB显色液，其特征在于，每1L单组份TMB显色液包括以下组分：TMB 50-70mg，DMSO5-10mL，甘油0.5-1.0mL，醋酸钠·3H2O0.023-0.046g，柠檬酸·H2O0.21-0.42g，30%H2O20.2-0.3mL；终止液为0.05-0.1mol/L H2SO4。
2.根据权利要求1所述的单组份TMB显色液，其特征在于，每1L的单组份TMB显色液包括以下组分：TMB50mg，DMSO5mL，甘油0.5mL，醋酸钠·3H2O0.023g，柠檬酸·H2O 0.21g，30%H2O20.2mL；终止液为0.05 mol/L H2SO4。
3.权利要求1所述的单组份TMB显色液的配制方法，其特征在于，单组份TMB显色液按以下配比配制：1L蒸馏水中，加入2-4mLTMB底物缓冲液，0.2-0.3mL 30%H2O2，5-7mLTMB储存液；所述TMB储存液由以下原料按如下重量体积比配制：TMB5g，DMSO450mL，甘油50mL，加蒸馏水定容到1L；所述TMB底物缓冲液由以下原料按如下重量体积比配制：醋酸钠·3H2O112.7g，柠檬酸·H2O105.0g，蒸馏水800mL，用浓盐酸调节PH至3.6，加蒸馏水定容到1000mL。
4.根据权利要求3所述的单组份TMB显色液的配制方法，其特征在于，单组份TMB显色液1L按以下配比配制：1L蒸馏水中，加入2mLTMB底物缓冲液，0.2mL30% H2O2，5mLTMB储存液；终止液为0.05 mol/L H2SO4。
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