CN112014338A - Paraoxonase-1 activity detection kit and application thereof - Google Patents
Paraoxonase-1 activity detection kit and application thereof Download PDFInfo
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- 108010008184 Aryldialkylphosphatase Proteins 0.000 title claims abstract description 101
- 230000000694 effects Effects 0.000 title claims abstract description 82
- 238000001514 detection method Methods 0.000 title claims abstract description 80
- 102000006996 Aryldialkylphosphatase Human genes 0.000 title claims abstract description 22
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 75
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 45
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 43
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 29
- 229960003237 betaine Drugs 0.000 claims abstract description 24
- 239000002738 chelating agent Substances 0.000 claims abstract description 23
- 239000004094 surface-active agent Substances 0.000 claims abstract description 23
- WYMSBXTXOHUIGT-UHFFFAOYSA-N paraoxon Chemical compound CCOP(=O)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 WYMSBXTXOHUIGT-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229960004623 paraoxon Drugs 0.000 claims abstract description 19
- 239000000276 potassium ferrocyanide Substances 0.000 claims abstract description 18
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims abstract description 18
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 17
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 15
- 235000010323 ascorbic acid Nutrition 0.000 claims abstract description 14
- 239000011668 ascorbic acid Substances 0.000 claims abstract description 14
- QLBHNVFOQLIYTH-UHFFFAOYSA-L dipotassium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical group [K+].[K+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O QLBHNVFOQLIYTH-UHFFFAOYSA-L 0.000 claims abstract description 14
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 13
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 13
- 239000007853 buffer solution Substances 0.000 claims abstract description 11
- 239000011734 sodium Substances 0.000 claims abstract description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims abstract 5
- 238000002835 absorbance Methods 0.000 claims description 63
- 210000002966 serum Anatomy 0.000 claims description 54
- 238000003149 assay kit Methods 0.000 claims description 25
- 239000000203 mixture Substances 0.000 claims description 25
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical group [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 24
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 24
- 239000006173 Good's buffer Substances 0.000 claims description 17
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 12
- 239000001103 potassium chloride Substances 0.000 claims description 12
- 235000011164 potassium chloride Nutrition 0.000 claims description 12
- 239000007983 Tris buffer Substances 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 abstract description 17
- 210000004369 blood Anatomy 0.000 abstract description 16
- 239000008280 blood Substances 0.000 abstract description 16
- 206010018910 Haemolysis Diseases 0.000 abstract description 13
- 230000008588 hemolysis Effects 0.000 abstract description 13
- 102100035476 Serum paraoxonase/arylesterase 1 Human genes 0.000 description 78
- 238000012360 testing method Methods 0.000 description 31
- 239000000243 solution Substances 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 10
- 238000011156 evaluation Methods 0.000 description 10
- 239000000758 substrate Substances 0.000 description 10
- 239000000126 substance Substances 0.000 description 7
- 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 6
- 230000008859 change Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 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 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 208000019423 liver disease Diseases 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 108010010234 HDL Lipoproteins Proteins 0.000 description 3
- 102000015779 HDL Lipoproteins Human genes 0.000 description 3
- 102000001554 Hemoglobins Human genes 0.000 description 3
- 108010054147 Hemoglobins Proteins 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- -1 lipid peroxide Chemical class 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 3
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 2
- 108010024957 Ascorbate Oxidase Proteins 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 208000000563 Hyperlipoproteinemia Type II Diseases 0.000 description 2
- 108010007622 LDL Lipoproteins Proteins 0.000 description 2
- 102000007330 LDL Lipoproteins Human genes 0.000 description 2
- 102100024640 Low-density lipoprotein receptor Human genes 0.000 description 2
- 206010045261 Type IIa hyperlipidaemia Diseases 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 208000029078 coronary artery disease Diseases 0.000 description 2
- 201000001386 familial hypercholesterolemia Diseases 0.000 description 2
- 230000037356 lipid metabolism Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- YQYGPGKTNQNXMH-UHFFFAOYSA-N 4-nitroacetophenone Chemical compound CC(=O)C1=CC=C([N+]([O-])=O)C=C1 YQYGPGKTNQNXMH-UHFFFAOYSA-N 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 208000007964 Organophosphate Poisoning Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102100022824 Serum paraoxonase/arylesterase 2 Human genes 0.000 description 1
- 101710180998 Serum paraoxonase/arylesterase 2 Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 150000007824 aliphatic compounds Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical class OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 150000004657 carbamic acid derivatives Chemical class 0.000 description 1
- 206010008118 cerebral infarction Diseases 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- DWDRNKYLWMKWTH-UHFFFAOYSA-N ethyl 2-(4-nitrophenyl)acetate Chemical compound CCOC(=O)CC1=CC=C([N+]([O-])=O)C=C1 DWDRNKYLWMKWTH-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a paraoxonase-1 activity detection kit and application thereof. The kit comprises a reagent R1 and a reagent R2; the reagent R1 comprises: buffer solution, accelerator, Proclin300, ascorbic acid oxidase, chelating agent and surfactant; the chelating agent is EDTA-2K and/or EDTA-2 Na; the surfactant is selected from one or more of Emulgen A90, Emulgen A60 or Emulgen 430; the reagent R2 comprises: buffer solution, glycerol, paraoxon, sodium azide, potassium ferrocyanide and betaine; the betaine is selected from BS-12, BS-14, BS-18 or
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a paraoxonase-1 activity detection kit and application thereof.
Background
Paraoxonase (PON) is a hydrolase found in serum and liver in 1953. The paraoxonase can catalyze and esterify organic phosphates, aliphatic compounds, aromatic carboxylic acid esters and carbamates in vitro. The PON gene family includes at least PON-1, PON-2 and PON-3. Of these, PON-1 is the only alloenzyme found in serum to date, which binds tightly to HDL in serum and is therefore thought to play an important role in HDL anti-LDL oxidation. Paraoxonase-1 (PON-1) is a hydrolases synthesized by the liver and present in the blood and liver. The research proves that the paraoxonase-1 can hydrolyze lipid peroxide, prevent the low-density lipoprotein from being modified by oxidation and can inhibit the occurrence of Atherosclerosis (AS).
Paraoxonase-1 in serum plays an important role in monitoring lipid metabolism, liver diseases, organophosphorus poisoning, and the like. In lipid metabolism, many studies have demonstrated that paraoxonase-1 in serum binds tightly to high density lipoproteins, and is able to combat the oxidative properties of low density lipoproteins. It has been reported abroad that paraoxonase-1 activity is decreased in some liver diseases, and it can be used as an index for monitoring liver function.
Related studies have demonstrated that altered paraoxonase-1 activity is associated with the development of a number of diseases, such as a significant decrease in paraoxonase-1 activity in patients with coronary heart disease, liver disease and familial hypercholesterolemia and type 2 diabetes. In addition, acute cerebral infarction is a common cerebrovascular disease, and the increase of lipid peroxide generated during hyperlipidemia and the reduction of the oxidation resistance of the organism can cause the failure of timely removal of lipid peroxide in blood, which may become one of the main causes of the disease.
Many diseases will cause the decrease of the content of paraoxonase-1, so the detection of the activity of paraoxonase-1 in serum can be used as an important detection index for various diseases such as coronary heart disease, liver disease, familial hypercholesterolemia, type 2 diabetes and the like.
At present, paranitroacetophenone ethyl acetate is mainly used as a substrate in the detection of paraoxonase activity at home and abroad, but the substrate is easily influenced by an interferent in the determination process and has low sensitivity. Meanwhile, with the improvement of living standard, clinical blood lipid samples are more and more, especially clinical hemolytic samples are common, and the samples can interfere with the detection of the activity of the phosphoesterase-1 to different degrees.
Therefore, a kit for detecting the activity of paraoxonase-1 with strong anti-interference capability is needed.
Disclosure of Invention
In view of the above problems, the present invention provides a paraoxonase-1 activity assay kit and use thereof. The basic detection principle of the paraoxonase-1 activity detection kit provided by the invention is that paraoxonase-1 is utilized to hydrolyze a specific substrate (paraoxonase) to generate paranitrophenol, the absorbance of the generated paranitrophenol at 405nm is increased, and the increasing rate is in direct proportion to the concentration of paraoxonase-1 in serum (paraoxonase-1 activity in serum) in a sample to be detected. The surfactant and the chelating agent are added into the reagent R1 of the kit, so that the anti-interference capability for blood fat and hemolysis in the detection process is improved under the condition of not influencing the detection performance (such as precision, stability and the like), and the betaine is added into the reagent R2, so that the solubility of a substrate (paraoxon) is increased, and the linear detection range of the paraoxonase-1 activity detection kit provided by the invention is widened.
The technical scheme of the invention for realizing the purpose is as follows:
in one aspect of the present invention, there is provided a paraoxonase-1 activity assay kit comprising a reagent R1 and a reagent R2 (the reagent R1 and the reagent R2 are independent of each other);
the reagent R1 comprises: buffer solution, accelerator, Proclin300, ascorbic acid oxidase, chelating agent and surfactant; the reagent R2 comprises: buffer solution, glycerol, paraoxon, sodium azide, potassium ferrocyanide and betaine;
wherein the chelating agent is EDTA-2K and/or EDTA-2 Na;
the surfactant is selected from one or more of Emulgen A90, Emulgen A60 or Emulgen 430;
the betaine is selected from BS-12, BS-14, BS-18 or
One or more kinds of BB.
In some embodiments of the invention, in the paraoxonase-1 activity assay kit according to the invention, the mass concentration of the chelating agent is 0.1-0.5 g/L.
In some embodiments of the invention, in the paraoxonase-1 activity assay kit of the invention, the mass concentration of the surfactant is 5-10 g/L.
In some embodiments of the invention, the concentration of betaine is 5-20 g/L.
In some embodiments of the invention, in the paraoxonase-1 activity assay kit of the invention, the pH value of the reagent R1 is 6-8; the pH value of the reagent R2 is 6-8.
In some embodiments of the present invention, in the paraoxonase-1 activity detection kit of the present invention, the buffer is selected from one or more of Good's buffer, Tris buffer or phosphate buffer;
the molar concentration of the buffer solution is 50-100 mmol/L.
In some embodiments of the present invention, in the paraoxonase-1 activity assay kit of the present invention, the accelerator is magnesium chloride and/or potassium chloride;
the mass concentration of the accelerator is 5-10 g/L.
In some embodiments of the invention, the concentration of glycerol in the paraoxonase-1 activity assay kit is 5-20 g/L.
In another aspect of the present invention, there is provided a method for detecting paraoxonase-1 activity in serum using the paraoxonase-1 activity detection kit of the present invention, comprising:
uniformly mixing the serum to be detected with the reagent R1, and keeping the mixture at the temperature of 37 ℃ for 5 minutes to obtain a serum mixture to be detected;
adding the reagent R2 into the serum mixture to be detected, and uniformly mixing to obtain a sample to be detected;
incubating the sample to be detected at 37 ℃ for 1min, and determining the absorbance A of the sample to be detected1(ii) a Then continuously incubating for 3min, and determining the absorbance A of the sample to be detected2；
According to the absorbance A1And the absorbance A2Calculating to obtain the concentration of paraoxonase-1 in serum (namely obtaining the detection result of paraoxonase-1 activity in serum);
wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1.
In some embodiments of the present invention, the method for detecting paraoxonase-1 activity in serum according to the absorbance A of the kit for detecting paraoxonase-1 activity of the present invention1And the absorbance A2Calculating the concentration of paraoxonase-1 in serum comprising:
according to the absorbance A1And the absorbance A2The concentration of paraoxonase-1 in serum was calculated according to the following formula:
wherein Δ a is the rate of change of absorbance per minute;
the absorbance delta A of the sample to be detected is equal to the absorbance A2-said absorbance A1；
Calibrator (commercially available) Δ a ═ calibrator absorbance a2Absorption by calibratorsDegree A1。
In still another aspect of the present invention, there are provided a kit for detecting the activity of paraoxonase-1 according to the present invention and the use of the detection method according to the present invention for detecting the activity of paraoxonase-1 in serum.
In some embodiments of the invention, in the kit for detecting paraoxonase-1 activity according to the invention, the mass concentration of Proclin300 is 0.5-1.0 g/L.
In some embodiments of the invention, the concentration of the ascorbate oxidase in the kit for detecting the activity of the paraoxonase-1 according to the invention is 2 to 5 KU/L.
In some embodiments of the invention, the paraoxonase-1 activity assay kit according to the invention comprises paraoxonase at a mass concentration of 0.5-1 g/L.
In some embodiments of the invention, in the paraoxonase-1 activity assay kit according to the invention, the mass concentration of potassium ferrocyanide is 0.02-0.05 g/L.
In some embodiments of the invention, in the paraoxonase-1 activity detection kit of the invention, the mass concentration of the sodium azide is 0.5-1.0 g/L.
In some embodiments of the present invention, in the paraoxonase-1 activity detection kit according to the present invention, the reagent R1 comprises: good's buffer solution with the molar concentration of 50mmol/L, magnesium chloride with the mass concentration of 10g/L, potassium chloride with the mass concentration of 10g/L, Proclin300 with the mass concentration of 1.0g/L, ascorbic acid oxidase with the concentration of 2KU/L, EDTA-2K with the mass concentration of 0.5g/L and Emulgen A90 with the mass concentration of 10g/L, and the pH value is 7;
the reagent R2 comprises: good's buffer solution with a molar concentration of 50mmol/L, glycerol with a mass concentration of 5g/L, paraoxon with a mass concentration of 1g/L, sodium azide with a mass concentration of 1.0g/L, potassium ferrocyanide with a mass concentration of 0.05g/L and potassium ferrocyanide with a mass concentration of 10g/L
BB, pH 7.
One or more technical embodiments of the present invention have at least the following technical effects or advantages:
(1) according to the invention, the surfactant (one or more of Emulgen A90, Emulgen A60 or Emulgen 430) and the chelating agent (EDTA-2K and/or EDTA-2Na) are added into the reagent R1 of the kit, so that the anti-interference capability for blood fat and hemolysis in the detection process is improved under the condition of not influencing the detection performance (such as accuracy, precision, stability and the like); and by adding betaine (BS-12, BS-14, BS-18 or
BB) to increase the solubility of the substrate (paraoxon) and broaden the linear detection range of the kit provided by the present invention.
(2) According to the invention, the mass concentration of the chelating agent is optimized, and the mass concentration of the chelating agent is limited to 0.1-0.5 g/L, so that the anti-interference capability for blood fat and hemolysis in the detection process is further improved under the condition of not influencing the detection performance.
(3) According to the invention, the selection of the mass concentration of the surfactant is optimized, and the mass concentration of the surfactant is limited to 5-10 g/L, so that the anti-interference capability to blood fat and hemolysis in the detection process is improved more remarkably, and the sensitivity is greatly improved.
(4) The invention optimizes the selection of the betaine mass concentration, and limits the betaine mass concentration to be 5-20g/L, thereby further widening the linear detection range of the kit.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
the invention provides a paraoxonase-1 activity detection kit, which comprises a reagent R1 and a reagent R2 (the reagent R1 and the reagent R2 are independent from each other);
the reagent R1 comprises: buffer solution, accelerator, Proclin300, ascorbic acid oxidase, chelating agent and surfactant; the reagent R2 comprises: buffer solution, glycerol, paraoxon, sodium azide, potassium ferrocyanide and betaine;
wherein the chelating agent is EDTA-2K and/or EDTA-2 Na;
the surfactant is selected from one or more of Emulgen A90, Emulgen A60 or Emulgen 430;
the betaine is selected from BS-12, BS-14, BS-18 or
One or more kinds of BB.
The kit provided by the invention improves the anti-interference capability aiming at blood fat and hemolysis in the detection process under the condition of not influencing the detection performance (such as accuracy, precision, stability and the like) by adding one or more of surfactant (Emulgen A90, Emulgen A60 or Emulgen 430), chelating agent (EDTA-2K and/or EDTA-2Na) and accelerator (magnesium chloride and potassium chloride) into R1, and betaine (BS-12, BS-14, BS-18 or betaine (BS-12, BS-18 or potassium chloride) into R2
One or two or more kinds of BB),thereby not only increasing the solubility of the substrate (paraoxon), but also widening the linear detection range of the kit.
In some embodiments, in the paraoxonase-1 activity assay kit according to the present invention, the chelating agent is present at a mass concentration of 0.1-0.5 g/L.
The inventor realizes that the selection of the mass concentration of the chelating agent has certain influence on the detection effect of the activity of the phospholipase-1; according to the invention, the mass concentration of the chelating agent is optimized, and the mass concentration of the chelating agent is limited to 0.1-0.5 g/L, so that the anti-interference capability for blood fat and hemolysis in the detection process is further improved under the condition of not influencing the detection performance.
In some embodiments, in the paraoxonase-1 activity assay kit according to the present invention, the mass concentration of the surfactant is 5 to 10 g/L.
The inventor has proved through a large number of experiments that the selection of the mass concentration of the surfactant can bring certain influence on the solubility of the substrate (paraoxon); according to the invention, the selection of the mass concentration of the surfactant is optimized, and the mass concentration of the surfactant is limited to 5-10 g/L, so that the anti-interference capability to blood fat and hemolysis in the detection process is improved more remarkably, and the sensitivity is greatly improved.
In some embodiments, the kit for detecting paraoxonase-1 activity according to the present invention, wherein the concentration of betaine is 5 to 20g/L by mass.
The inventor researches and discovers that the selection of the quality concentration of the betaine brings certain influence on the solubility of a substrate (paraoxon); the invention optimizes the selection of the betaine mass concentration, and limits the betaine mass concentration to be 5-20g/L, thereby further widening the linear detection range of the kit.
In some embodiments, in the paraoxonase-1 activity assay kit of the present invention, the buffer is selected from one or more of Good's buffer, Tris buffer or phosphate buffer;
the molar concentration of the buffer solution is 50-100 mmol/L.
In some embodiments, in the paraoxonase-1 activity assay kit of the present invention, the accelerator is magnesium chloride and/or potassium chloride;
the mass concentration of the accelerator is 5-10 g/L.
In some embodiments, the kit for detecting paraoxonase-1 activity according to the present invention comprises glycerol at a concentration of 5-20 g/L.
In some embodiments, in the kit for detecting paraoxonase-1 activity according to the present invention, the mass concentration of Proclin300 is 0.5-1.0 g/L.
In some embodiments, the concentration of the ascorbate oxidase in the kit for detecting the activity of paraoxonase-1 according to the present invention is 2 to 5 KU/L.
In some embodiments, the paraoxonase-1 activity assay kit according to the present invention comprises paraoxonase at a mass concentration of 0.5 to 1 g/L.
In some embodiments, in the paraoxonase-1 activity assay kit according to the present invention, the mass concentration of potassium ferrocyanide is 0.02-0.05 g/L.
In some embodiments, in the paraoxonase-1 activity assay kit of the present invention, the mass concentration of the sodium azide is 0.5-1.0 g/L.
In some embodiments, in the paraoxonase-1 activity assay kit according to the present invention, the reagent R1 comprises: good's buffer solution with the molar concentration of 50mmol/L, magnesium chloride with the mass concentration of 10g/L, potassium chloride with the mass concentration of 10g/L, Proclin300 with the mass concentration of 1.0g/L, ascorbic acid oxidase with the concentration of 2KU/L, EDTA-2K with the mass concentration of 0.5g/L and Emulgen A90 with the mass concentration of 10g/L, and the pH value is 7.0;
the reagent R2 comprises: good's buffer solution with a molar concentration of 50mmol/L, glycerol with a mass concentration of 5g/L, paraoxon with a mass concentration of 1g/L, sodium azide with a mass concentration of 1.0g/L, potassium ferrocyanide with a mass concentration of 0.05g/L and potassium ferrocyanide with a mass concentration of 10g/L
BB, pH 7.0.
The paraoxonase-1 activity assay kit according to the present application will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1:
in this example, 4 test groups are used to illustrate the paraoxonase-1 activity assay kit and the method for detecting paraoxonase-1 activity in serum provided by the present invention, as follows:
test group 1
1. Kit composition
The reagent R1 comprises:
good's buffer solution with the molar concentration of 50mmol/L, magnesium chloride with the mass concentration of 5g/L, Proclin300 with the mass concentration of 0.5g/L, ascorbic acid oxidase with the concentration of 5KU/L, EDTA-2K with the mass concentration of 0.1g/L and Emulgen A90 with the mass concentration of 5g/L, and the pH value is 6;
the reagent R2 comprises:
good's buffer solution with the molar concentration of 100mmol/L, glycerol with the mass concentration of 20g/L, paraoxon with the mass concentration of 0.5g/L, sodium azide with the mass concentration of 0.5g/L, potassium ferrocyanide with the mass concentration of 0.02g/L and BS-12 with the mass concentration of 5 g/L.
2. The detection method using the kit comprises the following steps:
(1) an experimental instrument: HITACHI 7180;
(2) experimental parameters:
(3) method of operation
The main detection method comprises the following steps:
uniformly mixing the serum to be detected with the reagent R1, and keeping the mixture at the temperature of 37 ℃ for 5 minutes to obtain a serum mixture to be detected;
adding the reagent R2 into the serum mixture to be detected, and uniformly mixing to obtain a sample to be detected;
incubating the sample to be detected at 37 ℃ for 1min, and determining the absorbance A of the sample to be detected1(ii) a Then continuously incubating for 3min, and determining the absorbance A of the sample to be detected2；
According to the absorbance A1And the absorbance A2The concentration of paraoxonase-1 in serum is calculated according to the following formula, i.e. obtaining the paraoxonase-1 activity in serum:
wherein Δ a is the rate of change of absorbance per minute;
the absorbance delta A of the sample to be detected is equal to the absorbance A2-said absorbance A1；
Calibrator (commercially available) Δ a ═ calibrator absorbance a2Calibrator absorbance A1。
Test group 2
1. Kit composition
The reagent R1 comprises:
phosphate buffer solution with the molar concentration of 100mmol/L, potassium chloride with the mass concentration of 10g/L, Proclin300 with the mass concentration of 1.0g/L, ascorbic acid oxidase with the concentration of 2KU/L, EDTA-2Na with the mass concentration of 0.5g/L and Emulgen430 with the mass concentration of 10g/L, and the pH value is 8;
the reagent R2 comprises:
phosphate buffer solution with the molar concentration of 50mmol/L, glycerol with the mass concentration of 5g/L, paraoxon with the mass concentration of 1g/L, sodium azide with the mass concentration of 1g/L, potassium ferrocyanide with the mass concentration of 0.05g/L and BS-18 with the mass concentration of 20 g/L.
2. The detection method using the kit comprises the following steps:
(1) an experimental instrument: HITACHI 7180;
(2) experimental parameters:
(3) method of operation
The main detection method comprises the following steps:
uniformly mixing the serum to be detected with the reagent R1, and keeping the mixture at the temperature of 37 ℃ for 5 minutes to obtain a serum mixture to be detected;
adding the reagent R2 into the serum mixture to be detected, and uniformly mixing to obtain a sample to be detected;
incubating the sample to be detected at 37 ℃ for 1min, and determining the absorbance A of the sample to be detected1(ii) a Then continuously incubating for 3min, and determining the absorbance A of the sample to be detected2；
According to the absorbance A1And the absorbance A2The concentration of paraoxonase-1 in serum is calculated according to the following formula, i.e. obtaining the paraoxonase-1 activity in serum:
wherein Δ a is the rate of change of absorbance per minute;
the absorbance delta A of the sample to be detected is equal to the absorbance A2-said absorbance A1；
Calibrator (commercially available) Δ a ═ calibrationAbsorbance of product A2Calibrator absorbance A1。
Test group 3
1. Kit composition
The reagent R1 comprises:
tris buffer solution with the molar concentration of 100mmol/L, potassium chloride with the mass concentration of 10g/L, Proclin300 with the mass concentration of 1.0g/L, ascorbic acid oxidase with the concentration of 2KU/L, EDTA-2Na with the mass concentration of 0.5g/L and Emulgen A60 with the mass concentration of 10g/L, and the pH value is 8;
the reagent R2 comprises:
tris buffer solution with the molar concentration of 50mmol/L, glycerol with the mass concentration of 5g/L, paraoxon with the mass concentration of 1g/L, sodium azide with the mass concentration of 1g/L, potassium ferrocyanide with the mass concentration of 0.05g/L and BS-14 with the mass concentration of 20 g/L.
2. The detection method using the kit comprises the following steps:
(1) an experimental instrument: HITACHI 7180;
(2) experimental parameters:
(3) method of operation
The main detection method comprises the following steps:
uniformly mixing the serum to be detected with the reagent R1, and keeping the mixture at the temperature of 37 ℃ for 5 minutes to obtain a serum mixture to be detected;
adding the reagent R2 into the serum mixture to be detected, and uniformly mixing to obtain a sample to be detected;
incubating the sample to be detected at 37 ℃ for 1min, and determining the absorbance A of the sample to be detected1(ii) a Then continuously incubating for 3min, and determining the absorbance A of the sample to be detected2；
According to the absorbance A1And the absorbance A2The concentration of paraoxonase-1 in serum is calculated according to the following formula, i.e. obtaining the paraoxonase-1 activity in serum:
wherein Δ a is the rate of change of absorbance per minute;
the absorbance delta A of the sample to be detected is equal to the absorbance A2-said absorbance A1；
Calibrator (commercially available) Δ a ═ calibrator absorbance a2Calibrator absorbance A1。
Test group 4
1. Kit composition
The reagent R1 comprises:
good's buffer solution with the molar concentration of 50mmol/L, magnesium chloride with the mass concentration of 10g/L, potassium chloride with the mass concentration of 10g/L, Proclin300 with the mass concentration of 1.0g/L, ascorbic acid oxidase with the concentration of 2KU/L, EDTA-2K with the mass concentration of 0.5g/L and Emulgen A90 with the mass concentration of 10g/L, and the pH value is 7;
the reagent R2 comprises:
good's buffer solution with a molar concentration of 50mmol/L, glycerol with a mass concentration of 5g/L, paraoxon with a mass concentration of 1g/L, sodium azide with a mass concentration of 1.0g/L, potassium ferrocyanide with a mass concentration of 0.05g/L and potassium ferrocyanide with a mass concentration of 10g/L
BB。
2. The detection method using the kit comprises the following steps:
(1) an experimental instrument: HITACHI 7180;
(2) experimental parameters:
(3) method of operation
The main detection method comprises the following steps:
uniformly mixing the serum to be detected with the reagent R1, and keeping the mixture at the temperature of 37 ℃ for 5 minutes to obtain a serum mixture to be detected;
adding the reagent R2 into the serum mixture to be detected, and uniformly mixing to obtain a sample to be detected;
incubating the sample to be detected at 37 ℃ for 1min, and determining the absorbance A of the sample to be detected1(ii) a Then continuously incubating for 3min, and determining the absorbance A of the sample to be detected2；
According to the absorbance A1And the absorbance A2The concentration of paraoxonase-1 in serum is calculated according to the following formula, i.e. obtaining the paraoxonase-1 activity in serum:
wherein Δ a is the rate of change of absorbance per minute;
the absorbance delta A of the sample to be detected is equal to the absorbance A2-said absorbance A1；
Calibrator (commercially available) Δ a ═ calibrator absorbance a2Calibrator absorbance A1。
Comparative example 1:
this comparative example employed 3 control groups, as follows:
comparative group 1
1. Kit composition
The reagent R1 comprises:
good's buffer solution with the molar concentration of 50mmol/L, magnesium chloride with the mass concentration of 10g/L, potassium chloride with the mass concentration of 10g/L, Proclin300 with the mass concentration of 1.0g/L and ascorbic acid oxidase with the concentration of 2KU/L, and the pH value is 7;
the reagent R2 comprises:
good's buffer solution with the molar concentration of 50mmol/L, ethyl p-nitrophenylacetate with the mass concentration of 1g/L, sodium azide with the mass concentration of 1.0g/L and potassium ferrocyanide with the mass concentration of 0.05 g/L.
2. The detection method using the kit was the same as in example 1 test group 1:
comparative group 2
1. Kit composition
The reagent R1 comprises:
good's buffer solution with the molar concentration of 50mmol/L, magnesium chloride with the mass concentration of 5g/L, Proclin300 with the mass concentration of 0.5g/L, ascorbic acid oxidase with the concentration of 5KU/L, EDTA-2K with the mass concentration of 0.05g/L and Emulgen A90 with the mass concentration of 4g/L, and the pH value is 7;
the reagent R2 comprises:
good's buffer solution with the molar concentration of 100mmol/L, glycerol with the mass concentration of 4g/L, paraoxon with the mass concentration of 0.5g/L, sodium azide with the mass concentration of 0.5g/L, potassium ferrocyanide with the mass concentration of 0.02g/L and BS-12 with the mass concentration of 23 g/L.
2. The detection method using the kit was the same as that of test group 1 of example 1.
Comparative group 3
1. Kit composition
The reagent R1 comprises:
good's buffer solution with the molar concentration of 50mmol/L, magnesium chloride with the mass concentration of 5g/L, Proclin300 with the mass concentration of 0.5g/L, ascorbic acid oxidase with the concentration of 5KU/L, EDTA-2K with the mass concentration of 0.6g/L and Emulgen A90 with the mass concentration of 12g/L, and the pH value is 7;
the reagent R2 comprises:
good's buffer solution with the molar concentration of 150mmol/L, glycerol with the mass concentration of 22g/L, paraoxon with the mass concentration of 0.5g/L, sodium azide with the mass concentration of 0.5g/L, potassium ferrocyanide with the mass concentration of 0.02g/L and BS-12 with the mass concentration of 4 g/L.
2. The detection method using the kit was the same as that of test group 1 of example 1.
Example 2: test for verifying detection effect of paraoxonase-1 activity detection kit
The test groups 1 to 4 in example 1 of the present invention were tested as follows:
(1) precision test: the precision of the kits obtained in test groups 1 to 4 of example 1 was measured.
Evaluation of precision: the kit obtained by the test groups 1-4 in the embodiment 1 is used for testing the same serum sample or control substance for 10 times, and CV (%) is calculated, wherein CV is less than or equal to 5% to meet the requirement.
Table 1: EXAMPLE 1 precision evaluation data of paraoxonase-1 activity assay kit obtained in test group 1
According to the determination results in Table 1, after the reagent kit groups obtained in test groups 1-4 of example 1 are stored at 2-8 ℃ for 12 months and shaken at 42 ℃ for 3 days, the same serum sample or control substance is tested for 10 times, no precipitation occurs, and CV is less than or equal to 5%, so that the result shows that the precision of the paraoxonase-1 activity detection reagent kit is excellent.
(2) Linear evaluation test:
the kit obtained from the test groups 1-4 of the example 1 is used for measuring paraoxonase-1 samples with the concentration ranging from 10U/L to 800U/L, and calculating the correlation coefficient r and the deviation of the measured mean value from the theoretical concentration, wherein r is less than or equal to 0.99, and the deviation is within +/-10 percent to meet the requirement.
Table 2: example 1 Linear evaluation data of paraoxonase-1 Activity detection kits obtained from test groups 1 to 4
According to the determination results in the table 2, the linear correlation of the paraoxonase-1 activity detection kit is excellent, the measurement is accurate, and the kit meets the clinical use requirement.
The surfactant (one or more of Emulgen A90, Emulgen A60 or Emulgen 430), the chelating agent (EDTA-2K and/or EDTA-2Na) and the accelerator (magnesium chloride and/or potassium chloride) are added into the reagent R1 of the kit, so that the anti-interference capability for blood fat and hemolysis in the detection process is improved under the condition that the detection performance (such as accuracy, precision, stability and the like) is not influenced; and by adding the betaine (BS-12, BS-14, BS-18 or BS-32) to the reagent R2 of the kit
BB) is one or more than two, the solubility of a substrate (paraoxon) is increased, and the linear detection range of the paraoxonase-1 activity detection kit provided by the invention is widened.
(3) And (3) stability test:
stability test was performed on the kits obtained in test groups 1 to 4 of example 1
Transportation stability: the kit obtained by the test groups 1-4 of the embodiment 1 is shaken on a shaking table at 42 ℃ for 3 days, and then the properties, blank absorbance and accuracy of the reagent are evaluated;
long-term stability: placing the kit obtained from the test groups 1-4 in the embodiment 1 at the temperature of 2-8 ℃ for 12 months, and evaluating the properties, blank absorbance and accuracy of the reagent every few months;
wherein:
the evaluation of the kit property is mainly used for judging whether the reagent is precipitated;
evaluation of blank absorbance: measuring the absorbance of the reagent R1 and the reagent R2 in the kit obtained from the test groups 1-4 in the embodiment 1 under the optical path of 10mm at the wavelength of 405nm, and taking the average value as blank absorbance, wherein the blank absorbance is less than or equal to 1.0000;
evaluation of accuracy: the detection result of a third-party quality control product (purchased commercially) is used as a target value, and the deviation value between the measured average value and the target value of the kit obtained by the test groups 1-4 in the embodiment 1 of the invention is calculated and recorded as the accuracy, wherein the accuracy is less than or equal to 10 percent, which meets the requirement.
Table 3: stability evaluation data of the paraoxonase-1 activity assay kit obtained in test groups 1 to 4 of example 1 of the present invention
According to the determination results in table 3, the paraoxonase-1 activity detection kits obtained in test groups 1-4 of example 1 of the invention all have no precipitate after shaking for 3 days at 42 ℃ and have significantly higher accuracy; and no precipitation is detected when the product is stored for 6 months at the temperature of 2-8 ℃, so that the product has remarkably high transportation stability, long-term stability and high accuracy.
Example 3: the anti-interference effect of the detection by adopting the paraoxonase-1 activity detection kit of the invention
Authentication
The following tests were carried out on test groups 1 and 4 in inventive example 1 and comparative groups 1 to 3 in comparative example 1:
and (3) anti-interference evaluation test: the interfering substances are diluted by purified water and are respectively added into blank control serum according to the proportion of 1:9 (the blank control serum is mixed serum of normal people, no interfering substance is added, and the value is near the medical decision level), so that the final concentration after the serum is added meets the concentration to be evaluated. The reagent kits obtained from the test groups 1 and 4 in the embodiment 1 and the comparison groups 1-3 in the comparison example 1 are respectively adopted to respectively determine blank serum and serum containing interference substances with different concentrations, each sample to be determined is determined for 3 times, the average value is calculated, and the deviation is calculated with the measured value of the blank control serum, so that the interference degree is obtained. Wherein, a plurality of interference degrees are less than 10%, the interference resistance is determined, otherwise, the interference resistance is not determined. The interfering substances and the concentrations are respectively as follows: 1.0g/L ascorbic acid, 0.5g/L bilirubin, 5.0g/L hemoglobin, and less than or equal to 10g/L triglyceride.
Table 4: anti-interference evaluation result
According to the data, the kit obtained by the test groups 1-4 in the embodiment 1 of the invention has anti-interference capability on various interference substances (such as bilirubin, ascorbic acid, hemoglobin and triglyceride) in the detection process; in contrast, the kits obtained in comparative examples 1-3 in comparative example 1 have no anti-interference ability against hemoglobin and triglyceride during the detection process.
The improved reagent has obvious anti-interference capability on hemolysis and blood fat, widens the linear detection range of the reagent, and meets the requirements of other performances.
As can be seen from the above examples, the kit of the present invention has at least the following beneficial effects:
(1) according to the invention, the surfactant (one or more of Emulgen A90, Emulgen A60 or Emulgen 430) and the chelating agent (EDTA-2K and/or EDTA-2Na) are added into the reagent R1 of the kit, so that the anti-interference capability for blood fat and hemolysis in the detection process is improved under the condition of not influencing the detection performance (such as accuracy, precision, stability and the like); and by adding betaine (BS-12, BS-14, BS-18 or
BB) to increase the solubility of the substrate (paraoxon) and broaden the linear detection range of the kit provided by the present invention.
(2) According to the invention, the mass concentration of the chelating agent is optimized, and the mass concentration of the chelating agent is limited to 0.1-0.5 g/L, so that the anti-interference capability for blood fat and hemolysis in the detection process is further improved under the condition of not influencing the detection performance.
(3) According to the invention, the selection of the mass concentration of the surfactant is optimized, and the mass concentration of the surfactant is limited to 5-10 g/L, so that the anti-interference capability to blood fat and hemolysis in the detection process is improved more remarkably, and the sensitivity is greatly improved.
(4) The invention optimizes the selection of the betaine mass concentration, and limits the betaine mass concentration to be 5-20g/L, thereby further widening the linear detection range of the kit provided by the invention.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A paraoxonase-1 activity detection kit comprises a reagent R1 and a reagent R2; characterized in that the reagent R1 comprises: buffer solution, accelerator, Proclin300, ascorbic acid oxidase, chelating agent and surfactant; the reagent R2 comprises: buffer solution, glycerol, paraoxon, sodium azide, potassium ferrocyanide and betaine;
wherein the chelating agent is EDTA-2K and/or EDTA-2 Na;
the surfactant is selected from one or more of Emulgen A90, Emulgen A60 or Emulgen 430;
the betaine is selected from BS-12, BS-14, BS-18 or
One or more kinds of BB.
2. The paraoxonase-1 activity assay kit according to claim 1, wherein the chelating agent is present at a mass concentration of 0.1 to 0.5 g/L.
3. The paraoxonase-1 activity detection kit according to claim 1 or 2, wherein the mass concentration of the surfactant is 5 to 10 g/L.
4. The paraoxonase-1 activity detection kit according to claim 1 or 2, wherein the mass concentration of betaine is 5-20 g/L.
5. The paraoxonase-1 activity assay kit according to claim 1 or 2, wherein the pH of the reagent R1 is 6-8; the pH value of the reagent R2 is 6-8.
6. The paraoxonase-1 activity detection kit according to claim 1 or 2, wherein the buffer is one or more selected from Good's buffer, Tris buffer or phosphate buffer;
the molar concentration of the buffer solution is 50-100 mmol/L.
7. The paraoxonase-1 activity detection kit according to claim 1 or 2, wherein the accelerator is magnesium chloride and/or potassium chloride;
the mass concentration of the accelerator is 5-10 g/L.
8. The paraoxonase-1 activity assay kit according to claim 1 or 2, wherein the concentration by mass of glycerol is 5 to 20 g/L.
9. A method for detecting paraoxonase-1 activity in serum using the paraoxonase-1 activity detection kit of any one of claims 1 to 8, comprising:
uniformly mixing the serum to be detected with the reagent R1, and keeping the mixture at the temperature of 37 ℃ for 5 minutes to obtain a serum mixture to be detected;
adding the reagent R2 into the serum mixture to be detected, and uniformly mixing to obtain a sample to be detected;
incubating the sample to be detected at 37 ℃ for 1min, and determining the absorbance A of the sample to be detected1(ii) a Then continuously incubating for 3min, and determining the absorbance A of the sample to be detected2；
According to the absorbance A1And the absorbance A2Calculating to obtain the concentration of paraoxonase-1 in serum;
wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1.
10. The paraoxonase-1 activity assay kit according to any one of claims 1 to 8 and the use of the method according to claim 9 for the detection of paraoxonase-1 activity in serum.
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