CN111321199A - 一种谷氨酸浓度测定试剂 - Google Patents
一种谷氨酸浓度测定试剂 Download PDFInfo
- Publication number
- CN111321199A CN111321199A CN202010157063.3A CN202010157063A CN111321199A CN 111321199 A CN111321199 A CN 111321199A CN 202010157063 A CN202010157063 A CN 202010157063A CN 111321199 A CN111321199 A CN 111321199A
- Authority
- CN
- China
- Prior art keywords
- reagent
- glutamic acid
- acid concentration
- tris buffer
- triton
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 131
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 239000004220 glutamic acid Substances 0.000 title claims abstract description 37
- 235000013922 glutamic acid Nutrition 0.000 title claims abstract description 37
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 45
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 41
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 28
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims abstract description 24
- 239000007983 Tris buffer Substances 0.000 claims abstract description 17
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 14
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 claims abstract description 13
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 claims abstract description 13
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims abstract description 13
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims abstract description 13
- 239000004395 L-leucine Substances 0.000 claims abstract description 12
- 235000019454 L-leucine Nutrition 0.000 claims abstract description 12
- 229960003136 leucine Drugs 0.000 claims abstract description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 7
- -1 triton-100 Chemical compound 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 8
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 238000002558 medical inspection Methods 0.000 abstract description 2
- 229960002989 glutamic acid Drugs 0.000 description 28
- 230000000052 comparative effect Effects 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 239000003381 stabilizer Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- 238000003908 quality control method Methods 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 239000005515 coenzyme Substances 0.000 description 4
- 229940049906 glutamate Drugs 0.000 description 4
- 229930195712 glutamate Natural products 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 102000004722 NADPH Oxidases Human genes 0.000 description 2
- 108010002998 NADPH Oxidases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- CXONXVMMINSQBV-NNYOXOHSSA-N (2r,3r,4s,5r)-5-[[[[(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxymethyl]-2-(3-carbamothioylpyridin-1-ium-1-yl)-4-hydroxyoxolan-3-olate Chemical compound NC(=S)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)[O-])=C1 CXONXVMMINSQBV-NNYOXOHSSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 230000002964 excitative effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
- G01N2333/90605—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4)
- G01N2333/90611—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1) in general
- G01N2333/90616—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1) in general with a definite EC number (1.4.1.-)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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+的摩尔质量比为25-50:1。
优选地,试剂1中所述丙三醇和NAD+的摩尔质量比为25:1。
进一步地,试剂2中所述二磷酸腺苷和L-亮氨酸的摩尔质量比为1:10-50。
优选地,试剂2中所述二磷酸腺苷和L-亮氨酸的摩尔质量比为1:10。
进一步地,试剂1中所述谷氨酸脱氢酶和试剂2中所述NAD+的比例为5KU/L-10KU/L:2mmol/L-6mmol/L。
优选地,试剂1中所述谷氨酸脱氢酶和试剂2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+的摩尔质量比为10:1。
对比例2
与实施例2的区别仅在于,二磷酸腺苷和L-亮氨酸的摩尔质量比为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 (9)
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。
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:所述试剂1包括:50-300mmol/LTris缓冲液、50-300mmol/L丙三醇、0.1%-1%(v/v)曲拉通-100和2-6mmol/LNAD+。
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: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。
5.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中所述丙三醇和NAD+的摩尔质量比为25-50:1。
6.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中所述二磷酸腺苷和L-亮氨酸的摩尔质量比为1:10-50。
7.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中所谷氨酸脱氢酶和试剂2中所述NAD+的比例为5KU/L-10KU/L:2mmol/L-6mmol/L。
8.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。
9.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。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010157063.3A CN111321199B (zh) | 2020-03-09 | 2020-03-09 | 一种谷氨酸浓度测定试剂 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010157063.3A CN111321199B (zh) | 2020-03-09 | 2020-03-09 | 一种谷氨酸浓度测定试剂 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111321199A true CN111321199A (zh) | 2020-06-23 |
CN111321199B CN111321199B (zh) | 2021-03-26 |
Family
ID=71169090
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010157063.3A Active CN111321199B (zh) | 2020-03-09 | 2020-03-09 | 一种谷氨酸浓度测定试剂 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111321199B (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
-
2020
- 2020-03-09 CN CN202010157063.3A patent/CN111321199B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
Non-Patent Citations (6)
Title |
---|
EDWIN PAHLICH ET AL.: "The effect of neutral salt anions on the oxidative deamination activity of plant glutamate dehydrogenase", 《PLANTA》 * |
GRAHAM ELLIS, DAVID M GOLDBERG: "Optimal Conditions for the Kinetic Assay of Serum Glutamate Dehydrogenase Activity at 37°C", 《CLINICAL CHEMISTRY》 * |
LA FAHIEN ET AL.: "Regulation of glutamate dehydrogenase by Mg2+ and magnification of leucine activation by Mg2+", 《MOLECULAR PHARMACOLOGY》 * |
MI-KYUNG LEE ET AL.: "Extremely thermostable glutamate dehydrogenase (GDH) from the freshwater archaeon Thermococcus waiotapuensis: cloning and comparison with two marine hyperthermophilic GDHs", 《EXTREMOPHILES》 * |
徐德林: "《表面活性剂合成及应用》", 31 August 1995 * |
王燕等: "谷氨酸生产菌S9114中依赖于NADPH 谷氨酸脱氢酶的纯化及性质", 《食品与发酵工业》 * |
Also Published As
Publication number | Publication date |
---|---|
CN111321199B (zh) | 2021-03-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Moss et al. | Kinetic enzymatic method for determining serum creatinine | |
Sampson et al. | A coupled-enzyme equilibrium method for measuring urea in serum: optimization and evaluation of the AACC study group on urea candidate reference method. | |
Masaru et al. | A direct method for the estimation of ornithine carbamoyltransferase activity in serum | |
CN110308282B (zh) | 一种稳定的同型半胱氨酸循环酶法检测试剂盒 | |
CN111808921A (zh) | 一种基于Trinder反应的检测试剂盒及其应用 | |
Sampson et al. | Chemical inhibition used in a kinetic urease/glutamate dehydrogenase method for urea in serum. | |
CN109517813B (zh) | 一种肌酸激酶及其同工酶激活剂、测定试剂及试剂盒 | |
CN112029817B (zh) | 一种肌酐检测试剂盒及其使用方法 | |
Wendel et al. | Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase | |
Ishihara et al. | Enzymatic determination of ammonia in blood plasma | |
EP0575610B1 (en) | Highly sensitive determination of ammonia, alpha-amino acid or alpha-keto acid and composition therefor | |
CN112710853B (zh) | 一种抗干扰、稳定的血清直接胆红素(酶法)测定试剂盒及其制备方法和应用 | |
CN116497084B (zh) | 一种抗干扰、稳定的血清单胺氧化酶测定试剂盒及其制备方法和应用 | |
CN111321199B (zh) | 一种谷氨酸浓度测定试剂 | |
CN111662955A (zh) | 一种腺苷脱氨酶测定试剂盒 | |
CN109517879B (zh) | 一种肌酸激酶及其同工酶测定试剂及其试剂盒 | |
US5618684A (en) | Method of determination of calcium | |
CN113984689A (zh) | 一种测定谷胱甘肽还原酶的试剂盒 | |
CN105861631A (zh) | 血清中肌酐的紫外分光测定方法 | |
CN111593091A (zh) | 一种用于检测同型半胱氨酸的试剂盒 | |
US5888828A (en) | Kit for measuring urea nitrogen | |
JPH05111396A (ja) | クレアチンキナ−ゼの活性測定方法 およびそれに用いる試薬 | |
Sherwin et al. | Serum and erythrocyte argininosuccinate lyase assay by NADH fluorescence generated from formed fumarate | |
EP0266905B1 (en) | Reagents for assay of gamma-glutamyl-transpeptidase | |
CN115420694A (zh) | 谷氨酸脱氢酶测定试剂盒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A reagent for measuring glutamate concentration Effective date of registration: 20231013 Granted publication date: 20210326 Pledgee: Bank of Ningbo Co.,Ltd. Shaoxing Branch Pledgor: ZHEJIANG DELTA BIOLOGICAL TECHNOLOGY CO.,LTD. Registration number: Y2023980060928 |