本发明专利技术涉及α-羟丁酸脱氢酶检测技术领域，其中试剂R1中包含缓冲液、BSA、2-酮丁酸、EMULGEN-707、羟基乙叉二膦酸、十八烷基三甲基溴化铵、烷基酚聚氧乙烯醚(APEO）、抗坏血酸氧化酶、胆红素氧化酶、蔗糖、海藻糖、黄原胶、防腐剂；试剂R2的组分为：缓冲液、NADH黄原胶、防腐剂。在试剂R1中加入了羟基乙叉二膦酸，能够有效的螯合重金属离子，并且能够较好的提高试剂的准确性；本发明专利技术还在试剂中加入了抗化学酸氧化酶和胆红素氧化酶，能去除抗坏血酸和胆红素的干扰。添加稳定剂BSA、蔗糖、海藻糖、黄原胶，增强试剂稳定性；采用新型表面活性剂烷基酚聚氧乙烯醚(APEO)，显著改善测定的性能，且增强试剂的稳定性。
【技术实现步骤摘要】
本专利技术涉及α-羟丁酸脱氢酶检测
，特别涉及一种稳定性好、准确度高的α-羟丁酸脱氢酶检测试剂。
技术介绍
α-羟丁酸脱氢酶(α-HBDH)是心肌酶谱中的一种酶，在哺乳动物体内普遍存在，主要分布在心肌红细胞、白细胞及肾脏等组织或器官中。α-羟丁酸脱氢酶不是一个独立的特异酶，而是含有H亚基的LD-1和LD-2的总称。α-羟丁酸脱氢酶（α-HBDH）与LDH关系十分密切，实际上它是LDH同工酶Ⅰ型，因其对α-酮丁酸亲合力高，所以当以α-酮丁酸作为底物时，所测LDH的活性特称为α-羟丁酸脱氢酶活性。α-HBDH活性升高常见于急性心肌梗塞、骨骼肌损伤、急性肝炎、白血病及恶性肿瘤等。目前用于α-羟基丁酸脱氢酶的检测方法主要有酶学比色法、连续检测法等。比色法的原理是α-HBDH作用于过量的α-酮丁酸生成α-羟丁酸，则剩余的α-酮丁酸与2，4-二硝基苯肼作用形成α-酮丁酸苯腙，在碱性溶液中呈棕色，其颜色深浅或吸光度与酶活性成反比。连续监测法的原理是α-HBDH催化α-酮丁酸还原成α-羟丁酸，同时使NADH氧化成NAD，在340nm监测NADH的氧化速率与该酶活性成正比。该方法简单灵敏，是目前临床检测α-羟丁酸脱氢酶的主要方法。但市面上的试剂一般存在稳定性不好，准确度不高的问题。因此，本发明根据这一问题，在连续监测法的基础上建立一种稳定性好、准确度高的检测试剂。
技术实现思路
本专利技术的目的是提供一种用于α-羟丁酸脱氢酶（α-HBDH）的试剂。基本原理：α-HBDH催化α-酮丁酸还原成α-羟丁酸，同时使NADH氧化成NAD，在340nm监测NADH的氧化速率与该酶活性成正比:α-HBDH2-酮丁酸+NADH+H+2-羟丁酸+NAD+本专利技术是通过以下步骤得到的：一种稳定性好、准确度高的α-羟丁酸脱氢酶检测试剂，包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：试剂R1中含有缓冲液·················································100mmol/L，BSA····················································1g/L2-酮丁酸···············································4.4mmol/L，EMULGEN-707············································0.7ml/L羟基乙叉二膦酸·········································1ml/L-5ml/L十八烷基三甲基溴化铵···································0.1g/L-1g/L烷基酚聚氧乙烯醚(APEO)································2g/L抗坏血酸氧化酶·········································1KU/L-5KU/L，胆红素氧化酶···········································1KU/L-3KU/L，蔗糖···················································5g/L海藻糖·················································5g/L黄原胶·················································3.8g/L，防腐剂·················································0.5ml/L-1ml/L；2）试剂R2的组分为：缓冲液·················································100mmol/L，NADH···················································4.4mmol/L，黄原胶·················································3.8g/L，防腐剂·················································0.5ml/L-1ml/L；所述的α-羟丁酸脱氢酶检测试剂，试剂R1中缓冲液为25℃，pH=7.5的磷酸盐缓冲液；所述的α-羟丁酸脱氢酶检测试剂，试剂R2中缓冲液为25℃，pH为3.5的酒石酸缓冲液；所述的α-羟丁酸脱氢酶检测试剂，所述防腐剂为PC-300；所述的α-羟丁酸脱氢酶检测试剂来检测α-羟丁酸脱氢酶的检测方法，使用全自动生化分析仪利用速率法进行测定，检测主波长为340nm；所述的检测方法，R1试剂和R2试剂的比例为4:1。本专利技术的创新处：1）在试剂的R1中采用新型表面活性剂烷基酚聚氧乙烯醚(APEO)，不仅显著改善测定的性能，而且增强了试剂的稳定性和抗干扰能力；2）除此之外，加入的十八烷基三甲基溴化铵和EMULGEN-707两种表面活性剂，能较好的提高试剂的乳化作用，从而提高试剂的准确度和特异性；3）在试剂R1中加入了羟基乙叉二膦酸，能够有效的螯合重金属离子，并且能够较好的提高试剂的准确性；4）在R1和R2中添加黄原胶作为稳定剂，能有效的改善试剂的稳定性；5）本专利技术还在试剂中加入了抗化学酸氧化酶和胆红素氧化酶，能够有效的去除抗坏血酸和胆红素的干扰。附图说明图1为两种试剂的相关性曲线图；图2为两种试剂效期稳定性曲线图；图3实施例1试剂检测方法；图4实施例1试剂与对照试剂检测α-羟丁酸脱氢酶对比检测结果；图5实施例1与对照试剂的准确度实验结果。具体实施方式下面结合具体实施例对本专利技术进行进一步说明：1）一种准确度高的α-羟丁酸脱氢酶检测试剂，包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：试剂R1中含有缓冲液···············································100mmol/L，BSA··················································1g/L，2-酮丁酸·············································4.4mmol/L，EMULGEN-707········本文档来自技高网...
【技术保护点】
一种稳定性更好、准确度高的α‑羟丁酸脱氢酶检测试剂，其特征在于包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：1）试剂R1中含有缓冲液····················································100mmol/L，BSA·······················································1g/L2‑酮丁酸··················································4.4mmol/L，EMULGEN‑707···············································0.7ml/L羟基乙叉二膦酸············································1ml/L‑5 ml/L十八烷基三甲基溴化铵······································0.1g/L‑1g/L烷基酚聚氧乙烯醚( APEO)···································2g/L抗坏血酸氧化酶············································1KU/L‑5 KU/L，胆红素氧化酶··············································1KU/L‑3 KU/L，蔗糖······················································5g/L海藻糖····················································5g/L黄原胶····················································3.8g/L，防腐剂····················································0.5ml/L‑1 ml/L；2）试剂R2的组分为：缓冲液····················································100mmol/L，NADH······················································4.4mmol/L，黄原胶····················································3.8g/L，防腐剂····················································0.5ml/L‑1 ml/L。...
【技术特征摘要】
1.一种稳定性更好、准确度高的α-羟丁酸脱氢酶检测试剂，其特征在于包括试剂R1和
试剂R2，所述试剂R1和试剂R2的组成如下：
1）试剂R1中含有
缓冲液····················································100mmol/L，
BSA·······················································1g/L
2-酮丁酸··················································4.4mmol/L，
EMULGEN-707···············································0.7ml/L
羟基乙叉二膦酸············································1ml/L-5ml/L
十八烷基三甲基溴化铵······································0.1g/L-1g/L
烷基酚聚氧乙烯醚(APEO)···································2g/L
抗坏血酸氧化酶············································1KU/L-5KU/L，
胆红素氧化酶··············································1KU/L-3KU/L，
蔗糖······················································5g/L
海藻糖····················································5g/L
黄原胶·····················...
【专利技术属性】
技术研发人员：甘宜梧，赵新，王绮，
申请(专利权)人：山东博科生物产业有限公司，
类型：发明
国别省市：山东;37
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