CN110133265B - 血清中肌酸激酶试剂盒及测定方法 - Google Patents
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Abstract
本发明公开了一种肌酸激酶试剂盒及测定方法，属于利用可见光，通过测试反应结果颜色的变化来测试材料的方法。本发明的技术方案是：肌酸激酶法咪唑缓冲液中溶解有N‑乙酰半胱氨酸，NADP，葡萄糖‑6‑磷酸脱氢酶，己糖激酶，葡萄糖，磷酸肌酸，5‑磷酸腺苷，ADP等，同时含有镁离子和钙离子，其中镁离子和钙离子的离子浓度更接近人体血液中离子浓度，本发明方法试剂盒不同于现有方法试剂盒，测定的肌酸激酶的活性更接近人体血液内肌酸激酶活性，其使用方法与现有方法相同，是一种快捷、真实反映体内肌酸激酶活性的检测方法。
Description
技术领域
本发明属于一种包含酶的测定方法；或是利用可见光，通过测试反应的结果产生颜色变化来测试材料的方法，特别是涉及一种用生化分析仪快捷、准确检测血清中肌酸激酶的试剂盒及测定方法。
背景技术
目前临床实验室测定肌酸激酶为推荐的IFCC法，肌酸激酶（CK）能使磷酸肌酸和ADP中的磷酸键转移生成肌酸和ATP，ATP与葡萄糖在己糖激酶（HK）己糖激酶与镁离子的作用下生成6-磷酸-葡萄糖，6-磷酸-葡萄糖与氧化型辅酶Ⅱ(NADP+)在6-磷酸-葡萄糖脱氢酶作用下生成6-磷酸-葡萄糖酸和NADPH。在340/660nm下测定NADPH形成的速率来测定CK的活性。
反应方程式为：
临床实验室常用酶法测定血清中肌酸激酶活性，肌酸激酶法试剂盒中咪唑缓冲液(pH6.5 37℃)溶解HK、镁离子、葡萄糖、EDTA、AMP、ADP、NADP、己糖激酶、葡萄糖-6-磷酸脱氢酶，磷酸腺苷、磷酸肌酸、N-乙酰半胱氨酸、催化剂、稳定剂、防腐剂，其中二磷酸腺苷、一磷酸腺苷、五磷酸腺苷为抑制腺苷酸激酶活性，N-乙酰半胱氨酸保护CK上的游离巯基，镁离子为肌酸激酶和己糖激酶的激活离子。在临床实验室常用试剂盒中，镁离子浓度大于等于10mmol/L，而且使用EDTA去除血清中钙离子，这种方法导致体外测定的肌酸激酶的活性远高于体内血液中肌酸激酶的实际活性。
发明内容
为了能准确测定血清中肌酸激酶活性，本发明提供一种经济方便易行，能够真实反映血液中肌酸激酶活性的测定方法。
本发明采用的技术方案是：咪唑缓冲液中溶解有N-乙酰半胱氨酸、氧化型辅酶Ⅱ、葡萄糖-6-磷酸脱氢酶、己糖激酶、葡萄糖、磷酸肌酸、5-磷酸腺苷、ADP等，同时溶解有镁离子和钙离子，镁离子和钙离子浓度更接近人体血液中浓度，本发明技术特性为测定的肌酸激酶的活性更接近人体血液内肌酸激酶活性，真实反映体内肌酸激酶活性的检测方法。
具体实施方式：
下面通过实施例对本发明做进一步详细说明。
实施例1
试剂的组成：
a. 试剂Ⅰ：
试剂Ⅰ咪唑缓冲液100mmol/L溶解D-葡萄糖20.0mmol/L、醋酸镁1.08mmol/L、醋酸钙2.88mmol/L、己糖激酶20KU/L、二磷酸腺苷5.0mmol/L、一磷酸腺苷7.0mmol/L、五磷酸腺苷15.0mmol/L、N-乙酰半胱氨酸22.0mmol/L、氧化型辅酶Ⅱ6.0mmol/L、Proclin-300 200µl/L。
b.试剂Ⅱ：
试剂Ⅱ咪唑缓冲液100mmol/L溶解葡萄糖-6-磷酸脱氢酶12000U/L、磷酸肌酸60.0mmol/L、Proclin-300 200µl/L。
其中，镁离子为肌酸激酶激活剂，钙离子为肌酸激酶抑制剂，Proclin-300为液体高效防腐剂。
本发明肌酸激酶法测定工作液最终浓度为：咪唑缓冲液100mmol/L溶解D-葡萄糖16.6mmol/L、醋酸镁0.90mmol/L、醋酸钙2.40mmol/L、己糖激酶16.6KU/L、二磷酸腺苷4.16mmol/L、一磷酸腺苷5.63mmol/L、五磷酸腺苷12.5mmol/L、N-乙酰半胱氨酸18.3mmol/L、氧化型辅酶Ⅱ5.0mmol/L、葡萄糖-6-磷酸脱氢酶2000U/L、磷酸肌酸10.0mmol/L、Proclin-300 200µl/L。
实施例2
测定程序
双试剂法：在贝克曼AU5800全自动化生化分析仪上，仪器自动将10µl样品加入到250µl试剂Ⅰ中混匀，37℃孵育3分钟，加入50µl试剂Ⅱ混匀，37℃孵育5.1分钟，全自动分析仪在340/660nm波长处检测，仪器自动计算出肌酸激酶活性结果，具体见表1。
表1. 本发明自动化生化分析仪测试条件
计算公式为：
CK=ΔA/minΧTVΧ1000/6.33ΧSVΧL
其中，ΔA/min为每分钟吸光度变化，TV为反应总体积，6.33为NADPH在340nm处的毫摩尔消光系数，SV为样品体积，L为比色杯光径（cm）
本发明的技术参数为：批内精密度<5.0%，批间精密度<6.0%，参考区间为2～24U/L，线性范围为1.2～946.8U/L，可报告范围为1.2～9788.4U/L，空白吸光度<0.30(0.5cm,37℃，340/660nm)，灵敏度为80U/L的样本吸光度变化为≥0.012。
肌酸激酶参与机体的能量代谢，肌酸激酶（CK）能使磷酸肌酸和ADP中的磷酸键转移生成肌酸和ATP，因此，准确测定人体中肌酸激酶活性对于判断机体能量代谢具有重要意义，目前国内实验室使用的肌酸激酶试剂盒中镁离子浓度大于等于10mmol/L，而且使用EDTA去除血清中钙离子，这种方法使体外测定的肌酸激酶的活性远高于人体内血液中肌酸激酶的实际活性，在本发明中，镁离子和钙离子浓度更接近人体血液中浓度，测定的肌酸激酶的活性更接近人体血液内肌酸激酶活性，真实反映体内肌酸激酶活性的检测方法。
Claims (1)
1. 一种检测血清中肌酸激酶的试剂在制备检测血清中肌酸激酶试剂盒中的应用，其特征在于试剂Ⅰ咪唑缓冲液80～120mmol/L溶解D-葡萄糖18.0～22.0mmol/L、醋酸镁0.98～1.18 mmol/L、醋酸钙2.68～3.08 mmol/L、己糖激酶18～22KU/L、二磷酸腺苷4.0～6.0mmol/L、一磷酸腺苷6.0～8.0 mmol/L、五磷酸腺苷14.0～16.0 mmol/L、N-乙酰半胱氨酸18.0～24.0mmol/L、氧化型辅酶Ⅱ5.0～7.0 mmol/L、Proclin-300 100～300µl/L；试剂Ⅱ咪唑缓冲液80～120mmol/L溶解葡萄糖-6-磷酸脱氢酶8000～16000U/L、磷酸肌酸40.0～80.0 mmol/L、Proclin-300 100～300µl/L ，试剂Ⅰ和试剂Ⅱ中咪唑缓冲液的pH值为6.5±0.2，测定所用各物品的体积比为：样品∶试剂I∶试剂II=10∶200～300∶40～60。
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