CN113774105A - 一种稳定的游离脂肪酸测定试剂盒 - Google Patents
一种稳定的游离脂肪酸测定试剂盒 Download PDFInfo
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- CN113774105A CN113774105A CN202111166752.1A CN202111166752A CN113774105A CN 113774105 A CN113774105 A CN 113774105A CN 202111166752 A CN202111166752 A CN 202111166752A CN 113774105 A CN113774105 A CN 113774105A
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- 235000021588 free fatty acids Nutrition 0.000 title claims abstract description 49
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 63
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims abstract description 38
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 38
- 102000009027 Albumins Human genes 0.000 claims abstract description 37
- 108010088751 Albumins Proteins 0.000 claims abstract description 37
- 102000004190 Enzymes Human genes 0.000 claims abstract description 37
- 108090000790 Enzymes Proteins 0.000 claims abstract description 37
- 241000283690 Bos taurus Species 0.000 claims abstract description 36
- 239000005862 Whey Substances 0.000 claims abstract description 36
- 102000007544 Whey Proteins Human genes 0.000 claims abstract description 36
- 108010046377 Whey Proteins Proteins 0.000 claims abstract description 36
- 239000004094 surface-active agent Substances 0.000 claims abstract description 29
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000005516 coenzyme A Substances 0.000 claims abstract description 28
- 229940093530 coenzyme a Drugs 0.000 claims abstract description 28
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000007853 buffer solution Substances 0.000 claims abstract description 27
- 239000003774 sulfhydryl reagent Substances 0.000 claims abstract description 27
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 claims abstract description 26
- -1 casein sodium salt Chemical class 0.000 claims abstract description 25
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- 102000004539 Acyl-CoA Oxidase Human genes 0.000 claims abstract description 19
- 108020001558 Acyl-CoA oxidase Proteins 0.000 claims abstract description 19
- 102000005870 Coenzyme A Ligases Human genes 0.000 claims abstract description 19
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims abstract description 19
- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 claims abstract description 19
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 19
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims abstract description 19
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 19
- 229920000151 polyglycol Polymers 0.000 claims abstract description 19
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- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 claims abstract description 18
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 claims abstract description 18
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- 229940093632 flavin-adenine dinucleotide Drugs 0.000 claims abstract description 18
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims abstract description 17
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229910052708 sodium Inorganic materials 0.000 claims abstract description 17
- 239000011734 sodium Substances 0.000 claims abstract description 17
- 239000003755 preservative agent Substances 0.000 claims abstract description 16
- 230000002335 preservative effect Effects 0.000 claims abstract description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 42
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 32
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- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- HDARHUHTZKLJET-UHFFFAOYSA-M sodium;3-(n-ethyl-3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC(OC)=CC(OC)=C1 HDARHUHTZKLJET-UHFFFAOYSA-M 0.000 claims description 11
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 9
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 9
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 9
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000004471 Glycine Substances 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims description 5
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 5
- 239000004302 potassium sorbate Substances 0.000 claims description 5
- 235000010241 potassium sorbate Nutrition 0.000 claims description 5
- 229940069338 potassium sorbate Drugs 0.000 claims description 5
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 4
- 235000010234 sodium benzoate Nutrition 0.000 claims description 4
- 239000004299 sodium benzoate Substances 0.000 claims description 4
- 239000005515 coenzyme Substances 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- QLBHNVFOQLIYTH-UHFFFAOYSA-L dipotassium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O QLBHNVFOQLIYTH-UHFFFAOYSA-L 0.000 claims 1
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract description 6
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 abstract 1
- 150000003839 salts Chemical class 0.000 abstract 1
- 102000011632 Caseins Human genes 0.000 description 21
- 108010076119 Caseins Proteins 0.000 description 21
- 238000000034 method Methods 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 16
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 10
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- 238000006243 chemical reaction Methods 0.000 description 8
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- MGDKBCNOUDORNI-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;potassium Chemical compound [K].[K].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O MGDKBCNOUDORNI-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- 229910021645 metal ion Inorganic materials 0.000 description 6
- 229920000136 polysorbate Polymers 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 239000003925 fat Substances 0.000 description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 4
- 150000002632 lipids Chemical group 0.000 description 4
- 229940080237 sodium caseinate Drugs 0.000 description 4
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 3
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- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 2
- IEQAICDLOKRSRL-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-dodecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO IEQAICDLOKRSRL-UHFFFAOYSA-N 0.000 description 2
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 2
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 2
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 2
- 239000005642 Oleic acid Substances 0.000 description 2
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- 235000021314 Palmitic acid Nutrition 0.000 description 2
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- 150000001413 amino acids Chemical class 0.000 description 2
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- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 2
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- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 2
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- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 1
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- 150000005830 nonesterified fatty acids Chemical class 0.000 description 1
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- 150000003904 phospholipids Chemical class 0.000 description 1
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- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
本发明公开了一种稳定的游离脂肪酸测定试剂盒,属于医疗设备领域。所述试剂盒包括试剂R1和试剂R2,试剂R1中含有以下成分:缓冲液、辅酶A、ATP、酰基辅酶A合成酶、牛乳清白蛋白、酪蛋白钠盐、正辛酸钠、Trinder底物、表面活性剂、酶保护剂;试剂R2中含有以下成分:缓冲液、黄素腺嘌呤二核苷酸、4‑氨基安替比林、过氧化物酶、酰基辅酶A氧化酶、表面活性剂、防腐剂、复合巯基试剂、色原、十二烷基聚乙二醇醚、浓盐。本发明在试剂盒中添加复合巯基试剂、牛乳清白蛋白、酪蛋白钠盐,其中复合巯基试剂可以与辅酶A的巯基反应而去除辅酶A对色原的干扰,酪蛋白钠盐能够与牛乳清白蛋白结合,提高试剂盒的稳定性。
Description
技术领域
本发明属于医疗设备领域,具体涉及一种稳定的游离脂肪酸测定试剂盒。
背景技术
游离脂肪酸(free fatty acid)FFA,也称非酯化脂肪酸,临床上一般是指C10以上的脂肪酸,是脂肪代谢的中间产物,同时也是合成细胞膜脂质结构和***素的供体以及机体能量代谢的重要能源物质之一。脂质物质是细胞膜的主要组成,具有多种重要的生物学功能。存在于人体内的脂质,大致可以分为胆固醇、中性脂肪(三酸甘油脂)、磷脂质、游离脂肪酸等4种。游离脂肪酸是中性脂肪分解成的物质。当肌肉活动所需能源——肝酪耗尽时,脂肪组织会分解中性脂肪成为游离脂肪酸来充当能源使用。所以,游离脂肪酸可说是进行持久活动所需的物质。正常血清中的游离脂肪酸主要包括油酸(oleic acid占54%)、软脂酸(palmitic acid,占34%)、硬脂酸(stearic acid,占6%)等,其与清蛋白结合在血液中运输。通过检测血浆中的脂肪酸水平,可以研究某些重要疾病与脂质及脂肪酸的关系。特别是长链不饱和脂肪酸,由于它们是合成***素的原料,而且还能促使胆固醇的转变和***,从而降低血中胆固醇的浓度,因此更具有重要的意义。
尽管已有多种FFA检测试剂盒上市,但检测原理及方法各有不同,对疾病预测的灵敏度、特异度等指标不尽相同,尚缺乏对各种检测方法和不同试剂间的***的方法学和诊断效能的评价,测定方法尚未统一,因此函需对现有检测方法的快速和准确特性进行研究和评价,这将有利于临床工作的开展。血清FFA的测定方法主要有滴定法、比色法、化学发光法、气相色谱和酶法,酶法反应原理:游离脂肪酸在ATP、辅酶A的存在下,经酰基辅酶A合成酶作用,生成酰基辅酶A,酰基辅酶A被酰基辅酶A氧化酶氧化,生成2,3-trans-烯酞辅酶A和过氧化氢,而生成的过氧化氢在过氧化物酶存在下,与Trinder色原及4-氨基安替比林生成有色物质,通过测定其吸光度即可得到样品中游离脂肪酸的浓度。
中国专利文献“一种游离脂肪酸测定试剂盒及其制备方法(专利申请号为CN201610831420.3)”公开了一种游离脂肪酸测定试剂盒,含有试剂R1和试剂R2,试剂R1中含有以下成分:pH为7.0的磷酸二氢钠50mmol/L、辅酶A 0.05mmol/l、三磷酸腺苷3mmol/L、酰基辅酶A合成酶0.4KU/L、MgCl2 2mmol/L、Trinder底物0.5g/L、吐温20<1mL/L,pH为7.2;试剂R2中含有以下成分:pH为7.0的磷酸二氢钠60mmol/L、黄素腺嘌呤二核苷酸2mmol/L、4氨基安替比林10mmol/L、过氧化物酶40KU/L、酰基辅酶A氧化酶30KU/L、吐温20<1mL/L、甘油20mL/L-300mL/L、brij35(十二烷基聚乙二醇醚)1-1.5g/L、乙二胺四乙酸二钠1g/L,pH为7.2。该发明改进试剂的粘度,使试剂的粘度降低,这样溶液更容易快速的过滤特点,但是仍然存在试剂盒稳定性有待提高的问题。
发明内容
本发明的目的是提供一种稳定的游离脂肪酸测定试剂盒,以解决在中国专利文献“一种游离脂肪酸测定试剂盒及其制备方法(专利申请号为CN201610831420.3)”公开的基础上,如何优化组分、用量、工艺等,从而提高稳定的游离脂肪酸测定试剂盒的稳定性的问题。
为了解决以上技术问题,本发明采用以下技术方案:
一种稳定的游离脂肪酸测定试剂盒,包括试剂R1和试剂R2,
试剂R1中含有以下成分:缓冲液20-80mmol/L、辅酶A 1-10mmol/l、ATP 1-4mmol/L、酰基辅酶A合成酶0.4-1KU/L、牛乳清白蛋白10-20g/L、酪蛋白钠盐5-15g/L、正辛酸钠5-15g/L、Trinder底物0.8-1.5g/L、表面活性剂5-80ml/L、酶保护剂2-10ml/L;
试剂R2中含有以下成分:缓冲液20-80mmol/L、黄素腺嘌呤二核苷酸1-4mmol/L、4-氨基安替比林2-8mmol/L、过氧化物酶20-35KU/L、酰基辅酶A氧化酶5-10KU/L、表面活性剂5-80ml/L、防腐剂1-3ml/L、复合巯基试剂100-200ml/L、色原0.1-1mmol/L、十二烷基聚乙二醇醚1-1.5g/L、浓盐酸1-5ml/L。
优选地,所述试剂R1中含有以下成分:缓冲液50mmol/L、辅酶A6mmol/l、ATP3mmol/L、酰基辅酶A合成酶0.8KU/L、牛乳清白蛋白15g/L、酪蛋白钠盐10g/L、正辛酸钠10g/L、Trinder底物1.2g/L、表面活性剂45ml/L、酶保护剂6ml/L;
试剂R2中含有以下成分:缓冲液60mmol/L、黄素腺嘌呤二核苷酸2mmol/L、4-氨基安替比林6mmol/L、过氧化物酶28KU/L、酰基辅酶A氧化酶7KU/L、表面活性剂35ml/L、防腐剂2ml/L、复合巯基试剂150ml/L、色原0.5mmol/L、十二烷基聚乙二醇醚1.3g/L、浓盐酸3ml/L。
优选地,所述缓冲液为磷酸二氢钠或甘氨酸中的一种。
优选地,所述试剂R1配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入ATP、酰基辅酶A合成酶、牛乳清白蛋白、酪蛋白钠盐、正辛酸钠、Trinder底物、表面活性剂、酶保护剂。
优选地,所述试剂R2配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入黄素腺嘌呤二核苷酸、4-氨基安替比林、过氧化物酶、酰基辅酶A氧化酶、表面活性剂、防腐剂、复合巯基试剂、色原、十二烷基聚乙二醇醚、浓盐酸。
优选地,所述表面活性剂为Tween 20、EMULGEN A-60、EMULGEN A-90中的一种。
优选地,所述色原为TOPS和DAOS的混合物。
优选地,所述TOPS和DAOS的用量比为1:1。
优选地,所述防腐剂为叠氮化钠、山梨酸钾、苯甲酸钠中的一种。
优选地,所述酶保护剂为乙二胺四乙酸二钾和硫酸镁的浓度比为1:1的混合物。
本发明具有以下有益效果:
(1)由实施例1和对比例1-4的数据可见,在游离脂肪酸测定试剂盒中添加复合巯基试剂、牛乳清白蛋白、酪蛋白钠盐,其中复合巯基试剂可以与辅酶A的巯基反应而去除辅酶A对色原的干扰,并形成在546nm有吸收的物质,而试剂盒的主反应在500~700nm范围内有最大吸收。在采用TOPS和DAOS的混合物色原的基础上,其能完全消除辅酶A带来的干扰,同时试剂盒的稳定性也大大提高,2-8℃可稳定1年。牛乳清白蛋白为酶激活剂和酶稳定剂,其结构中有34个二硫键和一个巯基,巯基的化学反应很活泼,二硫键有抗氧化还原的作用,因此,可与多种阳离子、阴离子和小分子结合,酪蛋白钠盐能够与牛乳清白蛋白结合,能够在牛乳清白蛋白螯合酶的过程中引入金属离子,从而解除金属离子对酶的抑制作用,提高试剂盒的稳定性。此外,辅酶A的活性中心含有大量的巯基的还原态氨基酸,其反应活性较高,容易被氧化,牛乳清白蛋白可以提高辅酶A的催化效率,从而提高酶的稳定性。
(2)背景技术所引用的专利文献“一种游离脂肪酸测定试剂盒及其制备方法(专利申请号为CN201610831420.3)”,虽然该发明通过添加不同组合的蛋白保护剂,brij35(十二烷基聚乙二醇醚)、甘油、吐温20和EDTA-2Na(乙二胺四乙酸二钠),延长酶活稳定性,酶的37℃稳定性延长,由7天延长到14天,但是仍然存在在低温环境下,稳定性有待提高的问题,基于为了解决以上技术问题,本发明才对该发明的配方和工艺进行进一步的优化和改良,经过多次试验研究发现,当游离脂肪酸测定试剂盒中添加复合巯基试剂、牛乳清白蛋白、酪蛋白钠盐时,其中复合巯基试剂可以与辅酶A的巯基反应而去除辅酶A对色原的干扰,酪蛋白钠盐能够与牛乳清白蛋白结合,能够在牛乳清白蛋白螯合酶的过程中引入金属离子,从而解除金属离子对酶的抑制作用,提高试剂盒的稳定性;能够解决背景技术文件中出现的技术问题,产生了意想不到的效果。
具体实施方式
为了更好地理解本发明,现采用以下实施例加以说明,以下实施例属于本发明的保护范围,但不限制本发明的保护范围。
一种稳定的游离脂肪酸测定试剂盒,包括试剂R1和试剂R2,
试剂R1中含有以下成分:缓冲液20-80mmol/L、辅酶A 1-10mmol/l、ATP 1-4mmol/L、酰基辅酶A合成酶0.4-1KU/L、牛乳清白蛋白10-20g/L、酪蛋白钠盐5-15g/L、正辛酸钠5-15g/L、Trinder底物0.8-1.5g/L、表面活性剂5-80ml/L、酶保护剂2-10ml/L;
试剂R2中含有以下成分:缓冲液20-80mmol/L、黄素腺嘌呤二核苷酸1-4mmol/L、4-氨基安替比林2-8mmol/L、过氧化物酶20-35KU/L、酰基辅酶A氧化酶5-10KU/L、表面活性剂5-80ml/L、防腐剂1-3ml/L、复合巯基试剂100-200ml/L、色原0.1-1mmol/L、十二烷基聚乙二醇醚1-1.5g/L、浓盐酸1-5ml/L。
所述试剂R1配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入ATP、酰基辅酶A合成酶、牛乳清白蛋白、酪蛋白钠盐、正辛酸钠、Trinder底物、表面活性剂、酶保护剂。
所述试剂R2配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入黄素腺嘌呤二核苷酸、4-氨基安替比林、过氧化物酶、酰基辅酶A氧化酶、表面活性剂、防腐剂、复合巯基试剂、色原、十二烷基聚乙二醇醚、浓盐酸。
所述缓冲液为磷酸二氢钠或甘氨酸中的一种。
所述表面活性剂为Tween 20、EMULGEN A-60、EMULGEN A-90中的一种。
所述色原为用量比为1:1的TOPS和DAOS的混合物。
所述防腐剂为叠氮化钠、山梨酸钾、苯甲酸钠中的一种。
所述酶保护剂为乙二胺四乙酸二钾和硫酸镁的浓度比为1:1的混合物。
下面通过更具体的实施例加以说明。
实施例1
一种稳定的游离脂肪酸测定试剂盒,包括试剂R1和试剂R2,
试剂R1中含有以下成分:磷酸二氢钠50mmol/L、辅酶A 6mmol/l、ATP3mmol/L、酰基辅酶A合成酶0.8KU/L、牛乳清白蛋白15g/L、酪蛋白钠盐10g/L、正辛酸钠10g/L、Trinder底物1.2g/L、Tween 20 45ml/L、酶保护剂6ml/L;酶保护剂为乙二胺四乙酸二钾和硫酸镁的浓度比为1:1的混合物;
试剂R2中含有以下成分:磷酸二氢钠60mmol/L、黄素腺嘌呤二核苷酸2mmol/L、4-氨基安替比林6mmol/L、过氧化物酶28KU/L、酰基辅酶A氧化酶7KU/L、Tween 20 35ml/L叠氮化钠2ml/L、复合巯基试剂150ml/L、色原0.5mmol/L、十二烷基聚乙二醇醚1.3g/L、浓盐酸3ml/L。所述色原为用量比为1:1的TOPS和DAOS的混合物。
所述试剂R1配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入ATP、酰基辅酶A合成酶、牛乳清白蛋白、酪蛋白钠盐、正辛酸钠、Trinder底物、表面活性剂、酶保护剂。
所述试剂R2配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入黄素腺嘌呤二核苷酸、4-氨基安替比林、过氧化物酶、酰基辅酶A氧化酶、表面活性剂、防腐剂、复合巯基试剂、色原、十二烷基聚乙二醇醚、浓盐酸。
实施例2
一种稳定的游离脂肪酸测定试剂盒,包括试剂R1和试剂R2,
试剂R1中含有以下成分:甘氨酸40mmol/L、辅酶A 1mmol/l、ATP 1mmol/L、酰基辅酶A合成酶1KU/L、牛乳清白蛋白15g/L、酪蛋白钠盐15g/L、正辛酸钠15g/L、Trinder底物1.1g/L、EMULGEN A-60 55ml/L、酶保护剂10ml/L;所述酶保护剂为乙二胺四乙酸二钾和硫酸镁的浓度比为1:1的混合物;
试剂R2中含有以下成分:甘氨酸40mmol/L、黄素腺嘌呤二核苷酸1mmol/L、4-氨基安替比林6mmol/L、过氧化物酶35KU/L、酰基辅酶A氧化酶9KU/L、EMULGEN A-60 5ml/L、山梨酸钾1ml/L、复合巯基试剂145ml/L、色原0.1mmol/L、十二烷基聚乙二醇醚1.5g/L、浓盐酸3ml/L。所述色原为用量比为1:1的TOPS和DAOS的混合物。
所述试剂R1配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入ATP、酰基辅酶A合成酶、牛乳清白蛋白、酪蛋白钠盐、正辛酸钠、Trinder底物、表面活性剂、酶保护剂。
所述试剂R2配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入黄素腺嘌呤二核苷酸、4-氨基安替比林、过氧化物酶、酰基辅酶A氧化酶、表面活性剂、防腐剂、复合巯基试剂、色原、十二烷基聚乙二醇醚、浓盐酸。
实施例3
一种稳定的游离脂肪酸测定试剂盒,包括试剂R1和试剂R2,
试剂R1中含有以下成分:磷酸二氢钠80mmol/L、辅酶A 7mmol/l、ATP 3mmol/L、酰基辅酶A合成酶0.4KU/L、牛乳清白蛋白20g/L、酪蛋白钠盐5g/L、正辛酸钠5g/L、Trinder底物1.5g/L、EMULGEN A-90 80ml/L、酶保护剂2ml/L;所述酶保护剂为乙二胺四乙酸二钾和硫酸镁的浓度比为1:1的混合物;
试剂R2中含有以下成分:磷酸二氢钠80mmol/L、黄素腺嘌呤二核苷酸2mmol/L、4-氨基安替比林8mmol/L、过氧化物酶20KU/L、酰基辅酶A氧化酶10KU/L、EMULGEN A-90 60ml/L、苯甲酸钠2ml/L、复合巯基试剂200ml/L、色原0.7mmol/L、十二烷基聚乙二醇醚1g/L、浓盐酸5ml/L。所述色原为用量比为1:1的TOPS和DAOS的混合物。
所述试剂R1配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入ATP、酰基辅酶A合成酶、牛乳清白蛋白、酪蛋白钠盐、正辛酸钠、Trinder底物、表面活性剂、酶保护剂。
所述试剂R2配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入黄素腺嘌呤二核苷酸、4-氨基安替比林、过氧化物酶、酰基辅酶A氧化酶、表面活性剂、防腐剂、复合巯基试剂、色原、十二烷基聚乙二醇醚、浓盐酸。
实施例4
一种稳定的游离脂肪酸测定试剂盒,包括试剂R1和试剂R2,
试剂R1中含有以下成分:甘氨酸20mmol/L、辅酶A 10mmol/l、ATP 4mmol/L、酰基辅酶A合成酶0.7KU/L、牛乳清白蛋白10g/L、酪蛋白钠盐11g/L、正辛酸钠10g/L、Trinder底物0.8g/L、Tween 20 5ml/L、酶保护剂6ml/L;所述酶保护剂为乙二胺四乙酸二钾和硫酸镁的浓度比为1:1的混合物。
试剂R2中含有以下成分:甘氨酸20mmol/L、黄素腺嘌呤二核苷酸4mmol/L、4-氨基安替比林2mmol/L、过氧化物酶28KU/L、酰基辅酶A氧化酶5KU/L、Tween 20 80ml/L、山梨酸钾3ml/L、复合巯基试剂100ml/L、色原1mmol/L、十二烷基聚乙二醇醚1.2g/L、浓盐酸1ml/L。所述色原为用量比为1:1的TOPS和DAOS的混合物。
所述试剂R1配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入ATP、酰基辅酶A合成酶、牛乳清白蛋白、酪蛋白钠盐、正辛酸钠、Trinder底物、表面活性剂、酶保护剂。
所述试剂R2配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入黄素腺嘌呤二核苷酸、4-氨基安替比林、过氧化物酶、酰基辅酶A氧化酶、表面活性剂、防腐剂、复合巯基试剂、色原、十二烷基聚乙二醇醚、浓盐酸。
对比例1
与实施例1的制备方法基本相同,不同之处在于制备稳定的游离脂肪酸测定试剂盒中不添加复合巯基试剂、牛乳清白蛋白、酪蛋白钠盐。
对比例2
与实施例1的制备方法基本相同,不同之处在于制备稳定的游离脂肪酸测定试剂盒中不添加复合巯基试剂。
对比例3
与实施例1的制备方法基本相同,不同之处在于制备稳定的游离脂肪酸测定试剂盒中不添加牛乳清白蛋白。
对比例4
与实施例1的制备方法基本相同,不同之处在于制备稳定的游离脂肪酸测定试剂盒中不添加酪蛋白钠盐。
对比例5
采用中国专利文献“一种游离脂肪酸测定试剂盒及其制备方法(专利申请号为CN201610831420.3)”中实施例1中所述方法制备游离脂肪酸测定试剂盒。
按照实施例1-4和对比例1-5的制得游离脂肪酸测定试剂盒,检测其稳定性,
将试剂置于2-8℃条件下放置12个月,每隔3个月评价试剂的性状、空白吸光度和准确度;
其中试剂的性状:主要判断试剂是否出现沉淀;
空白吸光度的评价:于546nm波长处,10mm光径下,测定R1和R2试剂的吸光度并取平均值即为空白吸光度,空白吸光度应≤1.0000;
准确度的评价:测第三方质控品作为靶值,计算本发明中试剂盒的测定平均值与靶值的偏差值记为准确度,其中准确度≤10%算满足要求。
其结果见下表。
由上表可知:(1)由实施例1-4和对比例5的数据可见,实施例1-4制得的游离脂肪酸测定试剂盒的稳定性显著高于对比例5制得的游离脂肪酸测定试剂盒的稳定性,且实施例1为最优实施例。
(2)由实施例1和对比例1-4的数据可见,在游离脂肪酸测定试剂盒中添加复合巯基试剂、牛乳清白蛋白、酪蛋白钠盐,其中复合巯基试剂可以与辅酶A的巯基反应而去除辅酶A对色原的干扰,并形成在546nm有吸收的物质,而试剂盒的主反应在500~700nm范围内有最大吸收。在采用TOPS和DAOS的混合物色原的基础上,其能完全消除辅酶A带来的干扰,同时试剂盒的稳定性也大大提高,2-8℃可稳定1年。
牛乳清白蛋白为酶激活剂和酶稳定剂,其结构中有34个二硫键和一个巯基,巯基的化学反应很活泼,二硫键有抗氧化还原的作用,因此,可与多种阳离子、阴离子和小分子结合,酪蛋白钠盐能够与牛乳清白蛋白结合,能够在牛乳清白蛋白螯合酶的过程中引入金属离子,从而解除金属离子对酶的抑制作用,提高试剂盒的稳定性。此外,辅酶A的活性中心含有大量的巯基的还原态氨基酸,其反应活性较高,容易被氧化,牛乳清白蛋白可以提高辅酶A的催化效率,从而提高酶的稳定性。
以上内容不能认定本发明具体实施只局限于这些说明,对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思前提下,还可以做出若干简单推演或替换,都应当视为属于本发明由所提交的权利要求书确定的专利保护范围。
Claims (10)
1.一种稳定的游离脂肪酸测定试剂盒,其特征在于,包括试剂R1和试剂R2,
试剂R1中含有以下成分:缓冲液20-80mmol/L、辅酶A 1-10mmol/I、ATP 1-4mmol/L、酰基辅酶A合成酶0.4-1KU/L、牛乳清白蛋白10-20g/L、酪蛋白钠盐5-15g/L、正辛酸钠5-15g/L、Trinder底物0.8-1.5g/L、表面活性剂5-80ml/L、酶保护剂2-10ml/L;
试剂R2中含有以下成分:缓冲液20-80mmol/L、黄素腺嘌呤二核苷酸1-4mmol/L、4-氨基安替比林2-8mmol/L、过氧化物酶20-35KU/L、酰基辅酶A氧化酶5-10KU/L、表面活性剂5-80ml/L、防腐剂1-3ml/L、复合巯基试剂100-200ml/L、色原0.1-1mmol/L、十二烷基聚乙二醇醚1-1.5g/L、浓盐酸1-5ml/L。
2.根据权利要求1所述的稳定的游离脂肪酸测定试剂盒,其特征在于,所述试剂R1中含有以下成分:缓冲液50mmol/L、辅酶A 6mmol/I、ATP 3mmol/L、酰基辅酶A合成酶0.8KU/L、牛乳清白蛋白15g/L、酪蛋白钠盐10g/L、正辛酸钠10g/L、Trinder底物1.2g/L、表面活性剂45ml/L、酶保护剂6ml/L;
试剂R2中含有以下成分:缓冲液60mmol/L、黄素腺嘌呤二核苷酸2mmol/L、4-氨基安替比林6mmol/L、过氧化物酶28KU/L、酰基辅酶A氧化酶7KU/L、表面活性剂35ml/L、防腐剂2ml/L、复合巯基试剂150ml/L、色原0.5mmol/L、十二烷基聚乙二醇醚1.3g/L、浓盐酸3ml/L。
3.根据权利要求2所述的稳定的游离脂肪酸测定试剂盒,其特征在于,所述缓冲液为磷酸二氢钠或甘氨酸中的一种。
4.根据权利要求1所述的稳定的游离脂肪酸测定试剂盒,其特征在于,所述试剂R1配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入ATP、酰基辅酶A合成酶、牛乳清白蛋白、酪蛋白钠盐、正辛酸钠、Trinder底物、表面活性剂、酶保护剂。
5.根据权利要求4所述的稳定的游离脂肪酸测定试剂盒,其特征在于,所述试剂R2配制方法为:取900mL的水,加入pH为7.0的缓冲液,充分溶解,用NaOH调节pH到7.2,依次加入黄素腺嘌呤二核苷酸、4-氨基安替比林、过氧化物酶、酰基辅酶A氧化酶、表面活性剂、防腐剂、复合巯基试剂、色原、十二烷基聚乙二醇醚、浓盐酸。
6.根据权利要求1所述的稳定的游离脂肪酸测定试剂盒,其特征在于,所述表面活性剂为Tween 20、EMULGEN A-60、EMULGEN A-90中的一种。
7.根据权利要求1所述的稳定的游离脂肪酸测定试剂盒,其特征在于,所述色原为TOPS和DAOS的混合物。
8.根据权利要求7所述的稳定的游离脂肪酸测定试剂盒,其特征在于,所述TOPS和DAOS的用量比为1∶1。
9.根据权利要求1所述的稳定的游离脂肪酸测定试剂盒,其特征在于,所述防腐剂为叠氮化钠、山梨酸钾、苯甲酸钠中的一种。
10.根据权利要求1所述的稳定的游离脂肪酸测定试剂盒,其特征在于,所述酶保护剂为乙二胺四乙酸二钾和硫酸镁的浓度比为1∶1的混合物。
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