CN111321199B - 一种谷氨酸浓度测定试剂 - Google Patents
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Abstract
本发明提供了一种谷氨酸浓度测定试剂,涉及医学检验技术领域,该测定试剂包括试剂1和试剂2,其中,试剂1包括:Tris缓冲液、丙三醇、曲拉通‑100和NAD+;试剂2包括:Tris缓冲液、乙二醇、谷氨酸脱氢酶、二磷酸腺苷、曲拉通‑100、L‑亮氨酸和硫酸镁。该试剂是由试剂1和试剂2组成的双试剂,通过合理的选择和配比,具有操作简便、灵敏度高、准确度高、性能稳定等优点,适用于市场中绝大多数的全自动生化分析仪。
Description
技术领域
本发明涉及医学检验技术领域,具体涉及一种谷氨酸浓度测定试剂。
背景技术
谷氨酸是一种常见的氨基酸,哺乳动物血液中的常见成分,对哺乳动物具有特殊生理作用的重要物质。谷氨酸大量存在于哺乳动物的脑组织中,作为重要的兴奋性神经递质,谷氨酸参与了中枢神经的一系列活动,是代谢过程中较为活跃的成分,人体血清参考范围为14-192μmol/L。人体中谷氨酸的浓度对癫痫、帕金森症、脑卒中、阿尔茨海默症等神经性疾病的诊断、病情进展以及治疗等具有指导性意义。目前,血清中谷氨酸浓度的测定主要有高效液相色谱法和分光光度法,其中,高效液相色谱法选择性好、灵敏度高,但设备投入大,检测成本高是其明显的缺点。而目前常规的分光光度法虽然设备投入小、检测成本低,但准确度和灵敏度较低,无法满足精准测定血清中谷氨酸浓度的要求。近年来,找寻一种成本低、投入小且准确度、灵敏度高的试剂盒或试剂成为目前谷氨酸浓度检测的新思路。
中国专利CN101082572公开了一种谷氨酸浓度的测定方法及谷氨酸诊断试剂盒,该试剂盒由缓冲液、稳定剂、辅酶和谷氨酸脱氢酶组成,稳定剂为硫酸铵、甘油、丙二醇、乙二醇中的至少一种,辅酶是NADP+、NAD+或thio-NAD+中的一种。该测定方法特异性高,不受内、外源物质的污染,测试结果精确、准确性好。中国专利CN101324571公开了一种利用酶比色法及酶联法技术的谷氨酸诊断/测定试剂盒,该试剂盒包括由缓冲液、稳定剂、辅酶、谷氨酸氧化酶、NAD(P)H氧化酶组成双剂试剂,其中,试剂1由缓冲液、稳定剂、辅酶组成;试剂2由缓冲液、稳定剂、谷氨酸氧化酶、NAD(P)H氧化酶组成。在该发明中的稳定剂为硫酸铵、甘油(Glycerol)、丙二醇、乙二醇及防腐剂中的至少一种。该试剂盒中的试剂不仅可以在紫外/可见光分析仪或半、全自动生化分析仪上进行谷氨酸浓度测定,而且测定速度快、准确度高,因而可以得到切实的推广应用。但上述两项专利中的试剂盒均仍存在线性范围相对较窄、灵敏度相对较低的问题。
针对谷氨酸浓度测定试剂存在的线性范围窄、灵敏度低的问题,亟需寻找一种试剂,使得该试剂灵敏度高、线性范围广的同时,能够保证操作简便且具备较高的准确度和稳定性,进而适用于市场中绝大多数的全自动生化分析仪,进而得到推广应用。
发明内容
本发明针对现有技术存在的问题,提供了一种谷氨酸浓度测定试剂。该试剂是由试剂1和试剂2组成的双试剂,具有操作简便、灵敏度高、准确度高、性能稳定等优点,适用于市场中绝大多数的全自动生化分析仪。
为实现上述目的,本发明采用的技术方案如下:
本发明提供了一种谷氨酸浓度测定试剂,包括试剂1和试剂2,所述试剂1包括:Tris缓冲液、丙三醇、曲拉通-100和NAD+;所述试剂2包括:Tris缓冲液、乙二醇、谷氨酸脱氢酶、二磷酸腺苷、曲拉通-100、L-亮氨酸和硫酸镁。
进一步地,所述试剂1和试剂2的体积比为2-5:1。
优选地,所述试剂1和试剂2的体积比为4:1。
进一步地,所述试剂1包括:50-300mmol/L Tris缓冲液、50-300mmol/L丙三醇、0.1%-1%(v/v)曲拉通-100和2-6mmol/L NAD+。
进一步地,所述试剂2包括:50-300mmol/L Tris缓冲液、50-300mmol/L乙二醇、5-10KU/L谷氨酸脱氢酶、0.1-1mmol/L二磷酸腺苷、0.1%-1%(v/v)曲拉通-100、1-10mmol/L的L-亮氨酸和5-50mmol/L硫酸镁。
优选地,所述试剂1包括:100mmol/L Tris缓冲液、100mmol/L丙三醇、0.5%(v/v)曲拉通-100和4mmol/L NAD+。述试剂2包括:100mmol/L Tris缓冲液、100mmol/L乙二醇、7KU/L谷氨酸脱氢酶、0.5mmol/L二磷酸腺苷、5%(v/v)曲拉通-100、5mmol/L的L-亮氨酸和20mmol/L硫酸镁。
进一步地,试剂1中所述丙三醇和NAD+的摩尔比为25-50:1。
优选地,试剂1中所述丙三醇和NAD+的摩尔比为25:1。
进一步地,试剂2中所述二磷酸腺苷和L-亮氨酸的摩尔比为1:10-50。
优选地,试剂2中所述二磷酸腺苷和L-亮氨酸的摩尔比为1:10。
进一步地,试剂2中所述谷氨酸脱氢酶和试剂1中所述NAD+的比例为5KU/L-10KU/L:2mmol/L-6mmol/L。
优选地,试剂2中所述谷氨酸脱氢酶和试剂1中所述NAD+的比例为7KU/L:4mmol/L。
进一步地,试剂1和试剂2中所述Tris缓冲液的pH均为9.0。
本发明所取得的技术效果是:
1.本发明的试剂利用酶比色法测定血清中的谷氨酸浓度,测试结果准确;
2.本发明的试剂在试剂1里面加入适量的丙三醇和曲拉通-100,在试剂2里面加入了适量的乙二醇和曲拉通-100,丙三醇、曲拉通-100和乙二醇作为试剂的稳定剂,提高了试剂的稳定性;
3.本发明的试剂在试剂2里面加入了二磷酸腺苷,而二磷酸腺苷作为谷氨酸脱氢酶的激活剂和稳定剂可恢复血清样本中葡萄糖醛酸胆红素等对谷氨酸脱氢酶的抑制作用同时试剂加入L-亮氨酸和硫酸镁作为谷氨酸脱氢酶的激活剂,增加了试剂的线性范围,即灵敏度增加;
4.本发明的试剂为试剂1和试剂2组成的双试剂,操作简便,适用于市场中绝大多数的全自动生化分析仪,是一种值得推广的检测血清谷氨酸浓度的试剂。
附图说明
图1为本发明实施例1-3配制的试剂稳定性实验结果;
图2为本发明对比例1-6配制的试剂稳定性实验结果。
具体实施方式
值得说明的是,本申请中使用的试剂均为普通市售产品,因此对其来源不做具体限定。
实施例1
一种谷氨酸浓度测定试剂,包括试剂1和试剂2;
试剂1的组分为:
试剂2的组分为:
实施例2
一种谷氨酸浓度测定试剂,包括试剂1和试剂2;
试剂1的组分为:
试剂2的组分为:
实施例3
一种谷氨酸浓度测定试剂,包括试剂1和试剂2;
试剂1的组分为:
试剂2的组分为:
对比例1
与实施例2的区别仅在于,丙三醇和NAD+的摩尔比为10:1。
对比例2
与实施例2的区别仅在于,二磷酸腺苷和L-亮氨酸的摩尔比为1:1。
对比例3
试剂1的组分为:
试剂2的组分为:
对比例4
一种谷氨酸浓度测定试剂,包括试剂1和试剂2,
试剂1的组分为:
试剂2的组分为:
对比例5
专利CN101082572中的测定试剂。
对比例6
与实施例2的区别仅在于,试剂2中不加入二磷酸腺苷和L-亮氨酸。
一、准确度和线性范围实验
配制100mmol/L pH=8.0Tris溶液50mL加22.1mg的L-谷氨酸,得到3mmol/L谷氨酸溶液母液,再用100mmol/L pH=8.0Tris溶液稀释母液,进行浓度梯度稀释,得到不同浓度的谷氨酸溶液:0μmol/L、12μmol/L、23.5μmol/L、47μmol/L、94μmol/L、188μmol/L、375μmol/L、750μmol/L、1500μmol/L、3000μmol/L。将实施例1-3以及对比例1-6配制的试剂分别对上述8个不同浓度的谷氨酸质控溶液进行检测,按照检测操作流程:(1)试剂1:试剂2:待测样品=20:5:1,即试剂1(200μL),试剂2(50μL),待测样品(10μL),37℃孵育5分钟,在主波长340nm下,读取各个检测杯吸光度A1,再孵育5分钟,读取各个检测杯吸光度A2。计算:ΔA=A2-A1;计算:谷氨酸浓度=(ΔA测定÷ΔA标准)×C标准,注:C标准为标准杯中的谷氨酸校准品:朗道MC1382,浓度为1870μmol/L,得到表1和表2。
表1实施例1-3准确度、线性范围实验检测结果
注:表中相关系数,均为与谷氨酸质控品浓度比较。
表2对比例1-6准确度、线性范围实验检测结果
注:表中相关系数,均为与谷氨酸质控品浓度比较。
如表1所示,实施例1、实施例2和实施例3所制备的试剂检测结果相关系数都大于0.999,且线性范围:12μmol/L至3000μmol/L。由实验结果可得本发明试剂测试结果准确,线性范围广,灵敏度高。如表2所示,对比例1-6制备的试剂检测结果相关系数均小于0.9,不同对比进行浓度测试时与质控品浓度偏差较大,线性范围均小于本申请保护范围内的试剂,灵敏度较低。
二、稳定性试验
配制实施例1-3的试剂,按照相同规格分成15等份,放置于2-8℃冰箱中,每月定时取出1组,同时现配谷氨酸朗道质控品(靶值1870μmol/L)。按照检测操作流程:(1)试剂1:试剂2:待测样品=20:5:1,即试剂1(200μL),试剂2(50μL),待测质控样品(10μL),37℃孵育5分钟,在主波长340nm下,读取各个检测杯吸光度A1,再孵育5分钟,读取各个检测杯吸光度A2。计算:ΔA=A2-A1;计算:血清谷氨酸浓度=(ΔA测定÷ΔA标准)×C标准,注:C标准为标准杯中的谷氨酸校准品:朗道MC1382,浓度为1870μmol/L,检测结果如图1所示。配制对比例1-6的试剂,测定方式与实施例1-3相同,检测结果如图2所示。
由图1和图2可知,实施例1-3制备的试剂2-8℃冰箱放置15个月后,所测得浓度与靶值的偏差均≤10%。由实验结果可得本发明制备得到的试剂稳定性好,而在本申请保护范围外制备出的试剂,随着时间的延长,浓度下降较快,稳定性较差。
上述实施例均为本发明较佳的实施方式,但实施方式并不受实施例的限制,其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代和简化均应为等效替换方式,都包含在本发明的保护范围之内。
Claims (4)
1.一种谷氨酸浓度测定试剂,其特征在于:包括试剂1和试剂2;所述试剂1和试剂2的原料及含量选自以下任意一种:
当所述试剂1包括:300mmol/L Tris缓冲液、300mmol/L丙三醇、1%(v/v)曲拉通-100和6mmol/L NAD+;此时,所述试剂2包括:50mmol/L Tris缓冲液、50mmol/L乙二醇、5KU/L谷氨酸脱氢酶、0.1mmol/L二磷酸腺苷、0.1%(v/v)曲拉通-100、1mmol/L的L-亮氨酸和5mmol/L硫酸镁;
当所述试剂1包括:50mmol/L Tris缓冲液、50mmol/L丙三醇、0.1%(v/v)曲拉通-100和2mmol/L NAD+;此时,所述试剂2包括:300mmol/L Tris缓冲液、300mmol/L乙二醇、10KU/L谷氨酸脱氢酶、1mmol/L二磷酸腺苷、1%(v/v)曲拉通-100、10mmol/L的L-亮氨酸和50mmol/L硫酸镁;
当所述试剂1包括:100mmol/L Tris缓冲液、100mmol/L丙三醇、0.5%(v/v)曲拉通-100和4mmol/L NAD+;此时,所述试剂2包括:100mmol/L Tris缓冲液、100mmol/L乙二醇、7KU/L谷氨酸脱氢酶、0.5mmol/L二磷酸腺苷、0.5%(v/v)曲拉通-100、5mmol/L的L-亮氨酸和20mmol/L硫酸镁。
2.根据权利要求1所述的谷氨酸浓度测定试剂,其特征在于:所述试剂1和试剂2的体积比为2-5:1。
3.根据权利要求1所述的谷氨酸浓度测定试剂,其特征在于:试剂1中所述Tris缓冲液的pH均为9.0。
4.根据权利要求1所述的谷氨酸浓度测定试剂,其特征在于:试剂2中所述Tris缓冲液的pH均为9.0。
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Citations (1)
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CN101082572A (zh) * | 2007-06-28 | 2007-12-05 | 苏州艾杰生物科技有限公司 | 谷氨酸浓度的测定方法及谷氨酸诊断试剂盒 |
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CN101082572A (zh) * | 2007-06-28 | 2007-12-05 | 苏州艾杰生物科技有限公司 | 谷氨酸浓度的测定方法及谷氨酸诊断试剂盒 |
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