Phospholipid detection reagent, detection kit and application thereof

Abstract

The invention discloses a phospholipid detection reagent, a detection kit and application thereof, and belongs to the technical field of detection and analysis. The phospholipid detection reagent comprises a first detection reagent and a second detection reagent, according to the invention, a chromogenic substrate 10-(carboxymethyl aminocarbonyl)-3, 7-bis (dimethylamino) phenothiazine sodium salt is added into the first detection reagent, and after the detection dominant wavelength is changed into 666nm, the drug interference resistance of the reagent is obviously improved; meanwhile, after the surfactant and the metal salt are added into the first detection reagent and the second detection reagent, the sensitivity, the precision and the linear range of the reagents are remarkably improved; the reagents have a synergistic effect, so that the detection performance of the reagent is remarkably improved; therefore, the detection reagent has a good application prospect in phospholipid detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis, and in particular relates to a phospholipid detection reagent, a detection kit and applications thereof. Background Art

[0002] Serum phospholipids are closely related to cholesterol and triglyceride metabolism. Increased serum phospholipids are commonly seen in cholestasis (possibly related to increased LP-x, which is rich in phospholipids), primary cholestatic cirrhosis, hyperlipidemia, fatty liver, LCAT deficiency, nephrotic syndrome, etc. Decreased serum phospholipids are commonly seen in severe hepatitis, acute infection fever, hyperthyroidism, malnutrition-induced cirrhosis, etc.

[0003] Phospholipids are hydrolyzed by phospholipase D to generate choline and phosphatidic acid. Choline is oxidized by choline oxidase to generate hydrogen peroxide, which is then converted into a colored product by the action of peroxidase (POD) through a chromogenic substrate. At a certain wavelength, the content of phospholipids in the sample is quantitatively detected by comparing it with a calibration solution treated in the same way. Currently, most phospholipid determination kits on the market have a relatively narrow linear range, which can only reach about 12.9mmol / L. Samples that exceed the upper limit of the linear range need to be diluted and retested, and the retest result is multiplied by the dilution factor to obtain the sample result. The higher the upper limit of the linear range of the kit, the fewer samples need to be diluted and retested, thereby reducing hospital testing costs and manpower requirements. In addition, after transportation or long-term storage, the phospholipid determination kits on the market are prone to precipitation, which affects the stability of the reagents; the colorimetric substrate used in most phospholipid determination reagents on the market is N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS), and the Trinder reaction involving DAOS is easily interfered by commonly used clinical drugs (calcium dobesilate, ethylphenolsulfonate, methyldopa, and levodopa).

[0004] Currently, the existing phospholipid determination kit (choline oxidase method) has problems such as narrow linear range, low sensitivity, low precision and drug interference. Summary of the invention

[0005] The purpose of the present invention is to provide a phospholipid detection reagent, a detection kit and its application, which are used to solve the problems of narrow linear range, low sensitivity, low precision and drug interference in the existing phospholipid determination kit (choline oxidase method).

[0006] In a first aspect, the present invention provides a phospholipid detection reagent, comprising a first detection reagent and a second detection reagent; wherein the first detection reagent comprises the following components: 25-100 mmol / L of a first buffer, 0.5-1 g / L of sodium azide, 0.5-5 g / L of a metal salt, 0.5-5 mL / L of Trion X-100, 0.3-1 g / L of EDTA-2K, 2-10 g / L of a surfactant, 0.5-5 KU / L of phospholipase D, 0.1-1 g / L of 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt (DA-67), and 0.5-2 KU / L of ascorbic acid oxidase; the second detection reagent comprises the following components: 25-100 mmol / L of a second buffer, 1-5 mL / L of Trion X-100, 0.5-2g / L 4-aminoantipyrine, 0.5-5g / L metal salt, 2-10g / L surfactant, 0.5-1mL / L ProClin 300, 0.1-0.2g / L potassium ferrocyanide, 2-10KU / L peroxidase (POD), 2-10KU / L choline oxidase.

[0007] In the present invention, the inventors have found that after adding the chromogenic substrate DA-67 to the first detection reagent and the main detection wavelength is changed to 666nm, the anti-drug interference ability of the reagent is significantly improved; at the same time, after adding surfactants and metal salts to the first detection reagent and the second detection reagent, the sensitivity, precision and linear range of the reagents are significantly improved; the above reagents significantly improve the detection performance of the reagents through synergistic effects.

[0008] It is understandable that the phospholipase D in the first detection reagent and the peroxidase and choline oxidase in the second detection reagent may be derived from animals or microorganisms, as long as the enzyme activity requirements are met.

[0009] In some embodiments, the first detection reagent further includes 0.5-20 g / L of a protective agent, and the protective agent is selected from at least one of β-cyclodextrin, trehalose, and ADP-K.

[0010] In the present invention, the inventors further discovered that after adding a protective agent to the first detection reagent, the stability of the reagent was significantly improved.

[0011] In some embodiments, the pH value of the first buffer in the first detection reagent is 7.4-7.6, and the first buffer is selected from at least one of piperazine-1,4-diethanesulfonic acid (PIPES), 3-tris(hydroxymethyl)methylamine-2-hydroxypropanesulfonic acid (TAPSO), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)methylamineethanesulfonic acid (TES), and 3-morpholinepropanesulfonic acid (MOPS).

[0012] In some embodiments, the metal salt in the first detection reagent is selected from at least one of KCl, NaCl, CaCl2, MgCl2, and BaCl2; the surfactant in the first detection reagent is selected from at least one of polyoxyethylene alkyl ether ADEKATOL SO-120, polyoxyethylene alkyl ether ADEKATOL SO-105, polyoxyethylene alkyl ether ADEKATOL SO-80, polyoxyethylene alkyl ether ADEKATOLSO-135, polyoxyethylene alkyl ether ADEKATOL SO-145, and polyoxyethylene alkyl ether ADEKATOL SO-160.

[0013] It is understandable that the first buffer, metal salt and surfactant in the first detection reagent can be conventionally selected according to actual use requirements.

[0014] In some embodiments, the second detection reagent further includes 0.5-20 g / L of a protective agent, and the protective agent is selected from at least one of β-cyclodextrin, trehalose, and ADP-K.

[0015] In the present invention, the inventors further discovered that after adding a protective agent to the first detection reagent, the stability of the reagent was significantly improved.

[0016] In some embodiments, the pH value of the second buffer in the second detection reagent is 7.4-7.6, and the second buffer is selected from at least one of piperazine-1,4-diethanesulfonic acid, 3-tris(hydroxymethyl)methylamine-2-hydroxypropanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, tris(hydroxymethyl)methylaminoethanesulfonic acid, and 3-morpholinepropanesulfonic acid.

[0017] In some embodiments, the metal salt in the second detection reagent is selected from at least one of KCl, NaCl, CaCl2, MgCl2, and BaCl2; the surfactant in the second detection reagent is selected from at least one of polyoxyethylene alkyl ether ADEKATOL SO-120, polyoxyethylene alkyl ether ADEKATOL SO-105, polyoxyethylene alkyl ether ADEKATOL SO-80, polyoxyethylene alkyl ether ADEKATOLSO-135, polyoxyethylene alkyl ether ADEKATOL SO-145, and polyoxyethylene alkyl ether ADEKATOL SO-160.

[0018] It is understandable that the second buffer, metal salt and surfactant in the second detection reagent can be routinely selected according to actual use requirements.

[0019] In a second aspect, the present invention discloses a phospholipid detection kit, comprising any one of the above-mentioned phospholipid detection reagents.

[0020] The detection principle of the phospholipid detection kit provided by the present invention is as follows: serum phospholipids are hydrolyzed to generate choline and phosphatidic acid under the action of phospholipase D, choline is oxidized by choline oxidase to generate hydrogen peroxide, and hydrogen peroxide reacts with 4-aminoantipyrine and DA-67 under the action of peroxidase to generate a color-developing complex. This complex has a maximum absorption peak at 666nm, and its absorption intensity is proportional to the content of phospholipids in the sample. Based on this, phospholipids can be quantitatively detected.

[0021] In a third aspect, the present invention discloses the use of any of the above-mentioned phospholipid detection reagents or the above-mentioned phospholipid detection kits in phospholipid detection.

[0022] In some embodiments, when a phospholipid detection reagent or a phospholipid detection kit is used for phospholipid detection, the volume ratio of the first detection reagent to the second detection reagent is (3-5):(0.5-1.5).

[0023] In some preferred embodiments, when a phospholipid detection reagent or a phospholipid detection kit is used for phospholipid detection, the volume ratio of the first detection reagent to the second detection reagent is 4:1.

[0024] The beneficial effects of the present invention are as follows: different from the prior art, the present invention significantly improves the drug interference resistance of the reagent by adding a chromogenic substrate DA-67 to the first detection reagent, and the detection main wavelength is changed to 666nm; at the same time, after adding a surfactant and a metal salt to the first detection reagent and the second detection reagent, the sensitivity, precision and linear range of the reagent are significantly improved; the above reagents significantly improve the detection performance of the reagents through synergistic effects; therefore, the above detection reagents have good application prospects in phospholipid detection. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0027] Example 1

[0028] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0029] The first detection reagent includes the following components: 100mmol / L TAPSO (pH 7.5), 0.5g / L sodium azide, 5g / L NaCl, 1mL / L Trion X-100, 0.5g / L EDTA-2K, 2g / L polyoxyethylene alkyl ether ADEKATOL SO-145, 1KU / L phospholipase D, 0.3g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt, 2KU / L ascorbic acid oxidase;

[0030] The second detection reagent includes the following components: 50mmol / L HEPES (pH 7.5), 2mL / L Trion X-100, 0.5g / L 4-aminoantipyrine, 0.5g / L CaCl2, 5g / L polyoxyethylene alkyl ether ADEKATOL SO-80, 1mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 5KU / L peroxidase, and 2KU / L choline oxidase.

[0031] Example 2

[0032] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0033] The first detection reagent includes the following components: 50mmol / L MOPS (pH 7.5), 0.5g / L sodium azide, 2g / L KCl, 1mL / L Trion X-100, 0.5g / L EDTA-2K, 4g / L polyoxyethylene alkyl ether ADEKATOLSO-160, 1KU / L phospholipase D, 0.5g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt, 2KU / L ascorbic acid oxidase;

[0034] The second detection reagent includes the following components: 50mmol / L PIPES (pH 7.5), 2mL / L Trion X-100, 0.8g / L 4-aminoantipyrine, 1g / L MgCl2, 10g / L polyoxyethylene alkyl ether ADEKATOL SO-105, 1mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 8KU / L peroxidase, and 10KU / L choline oxidase.

[0035] Example 3

[0036] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0037] The first detection reagent includes the following components: 100mmol / L TES (pH 7.5), 0.5g / L sodium azide, 3g / L KCl, 1mL / L Trion X-100, 0.5g / L EDTA-2K, 3g / L polyoxyethylene alkyl ether ADEKATOLSO-145, 5KU / L phospholipase D, 0.5g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt, 2KU / L ascorbic acid oxidase;

[0038] The second detection reagent includes the following components: 25mmol / L TAPSO (pH 7.5), 2mL / L Trion X-100, 0.5g / L 4-aminoantipyrine, 1g / L BaCl2, 3g / L polyoxyethylene alkyl ether ADEKATOL SO-120, 1mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 6KU / L peroxidase, and 10KU / L choline oxidase.

[0039] Example 4

[0040] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0041] The first detection reagent includes the following components: 50mmol / L PIPES (pH 7.5), 0.5g / L sodium azide, 3g / L KCl, 1mL / L Trion X-100, 0.5g / L EDTA-2K, 5g / L polyoxyethylene alkyl ether ADEKATOLSO-160, 3KU / L phospholipase D, 0.2g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt, 2KU / L ascorbic acid oxidase;

[0042] The second detection reagent includes the following components: 50mmol / L TES (pH 7.5), 2mL / L Trion X-100, 1g / L 4-aminoantipyrine, 0.5g / L CaCl2, 2g / L polyoxyethylene alkyl ether ADEKATOL SO-135, 1mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 10KU / L peroxidase, and 10KU / L choline oxidase.

[0043] Example 5

[0044] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0045] The first detection reagent includes the following components: 50mmol / L PIPES (pH 7.5), 0.5g / L sodium azide, 5g / L NaCl, 1mL / L Trion X-100, 0.5g / L EDTA-2K, 5g / L polyoxyethylene alkyl ether ADEKATOLSO-160, 2KU / L phospholipase D, 0.5g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt, 20g / L trehalose, and 2KU / L ascorbic acid oxidase;

[0046] The second detection reagent includes the following components: 50mmol / L PIPES (pH 7.5), 2mL / L Trion X-100, 1g / L 4-aminoantipyrine, 1g / L CaCl2, 10g / L polyoxyethylene alkyl ether ADEKATOL SO-135, 1mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 5KU / L peroxidase, and 5KU / L choline oxidase.

[0047] Example 6

[0048] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0049] The first detection reagent includes the following components: 25mmol / L TAPSO (pH 7.5), 1g / L sodium azide, 3g / L NaCl, 0.5mL / L Trion X-100, 0.3g / L EDTA-2K, 5g / L polyoxyethylene alkyl ether ADEKATOL SO-145, 0.5KU / L phospholipase D, 1g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt, 1KU / L ascorbic acid oxidase;

[0050] The second detection reagent includes the following components: 100mmol / L TES (pH 7.5), 1mL / L Trion X-100, 1g / L 4-aminoantipyrine, 1g / L BaCl2, 5g / L polyoxyethylene alkyl ether ADEKATOL SO-105, 0.5g / L ADP-K, 0.5mL / L ProClin 300, 0.2g / L potassium ferrocyanide, 2KU / L peroxidase, and 10KU / L choline oxidase.

[0051] Example 7

[0052] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0053] The first detection reagent includes the following components: 50mmol / L MOPS (pH 7.5), 0.5g / L sodium azide, 5g / L KCl, 2mL / L Trion X-100, 0.5g / L EDTA-2K, 10g / L polyoxyethylene alkyl ether ADEKATOLSO-160, 3KU / L phospholipase D, 0.5g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt, 10g / L β-cyclodextrin, 1KU / L ascorbic acid oxidase;

[0054] The second detection reagent includes the following components: 40mmol / L PIPES (pH 7.5), 1mL / L Trion X-100, 0.8g / L 4-aminoantipyrine, 1g / L CaCl2, 5g / L polyoxyethylene alkyl ether ADEKATOL SO-135, 0.5mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 5KU / L peroxidase, and 5KU / L choline oxidase.

[0055] Example 8

[0056] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0057] The first detection reagent includes the following components: 80mmol / L MOPS (pH 7.5), 0.5g / L sodium azide, 3g / L NaCl, 1mL / L Trion X-100, 0.5g / L EDTA-2K, 2g / L polyoxyethylene alkyl ether ADEKATOLSO-160, 2KU / L phospholipase D, 0.2g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt, 10g / L trehalose, and 2KU / L ascorbic acid oxidase;

[0058] The second detection reagent includes the following components: 25mmol / L PIPES (pH 7.5), 2mL / L Trion X-100, 0.8g / L 4-aminoantipyrine, 0.5g / L CaCl2, 5g / L polyoxyethylene alkyl ether ADEKATOL SO-135, 1g / L ADP-K, 1mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 8KU / L peroxidase, and 10KU / L choline oxidase.

[0059] Example 9

[0060] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0061] The first detection reagent includes the following components: 100mmol / L HEPES (pH 7.5), 1g / L sodium azide, 5g / L KCl, 2mL / L Trion X-100, 0.8g / L EDTA-2K, 3g / L polyoxyethylene alkyl ether ADEKATOLSO-145, 5KU / L phospholipase D, 0.1g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt, 10g / L β-cyclodextrin, 1KU / L ascorbic acid oxidase;

[0062] The second detection reagent includes the following components: 50mmol / L MOPS (pH 7.5), 5mL / L Trion X-100, 1g / L 4-aminoantipyrine, 1g / L MgCl2, 10g / L polyoxyethylene alkyl ether ADEKATOL SO-80, 0.5g / L ADP-K, 0.5mL / L ProClin 300, 0.2g / L potassium ferrocyanide, 5KU / L peroxidase, and 10KU / L choline oxidase.

[0063] Example 10

[0064] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0065] The first detection reagent includes the following components: 50mmol / L PIPES (pH 7.5), 0.5g / L sodium azide, 5g / L NaCl, 5mL / L Trion X-100, 0.3g / L EDTA-2K, 5g / L polyoxyethylene alkyl ether ADEKATOLSO-160, 5KU / L phospholipase D, 0.3g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt, 10g / L β-cyclodextrin, 10g / L trehalose, 2KU / L ascorbic acid oxidase;

[0066] The second detection reagent includes the following components: 80mmol / L HEPES (pH 7.5), 1mL / L Trion X-100, 0.6g / L 4-aminoantipyrine, 0.5g / L CaCl2, 8g / L polyoxyethylene alkyl ether ADEKATOL SO-120, 0.5mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 5KU / L peroxidase, and 10KU / L choline oxidase.

[0067] Embodiment 11

[0068] This embodiment provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0069] The first detection reagent includes the following components: 100mmol / L HEPES (pH 7.5), 0.5g / L sodium azide, 5g / L KCl, 1mL / L Trion X-100, 0.5g / L EDTA-2K, 5g / L polyoxyethylene alkyl ether ADEKATOL SO-160, 5KU / L phospholipase D, 0.5g / L 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt, 10g / L β-cyclodextrin, 10g / L trehalose, 2KU / L ascorbic acid oxidase;

[0070] The second detection reagent includes the following components: 50mmol / L PIPES (pH 7.5), 2mL / L Trion X-100, 1g / L 4-aminoantipyrine, 0.5g / L BaCl2, 10g / L polyoxyethylene alkyl ether ADEKATOL SO-135, 2g / L ADP-K, 1mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 5KU / L peroxidase, and 5KU / L choline oxidase.

[0071] Comparative Example 1

[0072] This comparative example provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0073] The first detection reagent includes the following components: 50mmol / L TAPSO (pH 7.5), 0.5g / L sodium azide, 1mL / L Trion X-100, 0.5g / L EDTA-2K, 2KU / L phospholipase D, 0.2g / L DASO, and 2KU / L ascorbate oxidase;

[0074] The second detection reagent includes the following components: 25mmol / L PIPES (pH 7.5), 2mL / L Trion X-100, 1g / L 4-aminoantipyrine, 1mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 10KU / L peroxidase, and 5KU / L choline oxidase.

[0075] Comparative Example 2

[0076] This comparative example provides a phospholipid detection kit, which includes a phospholipid detection reagent, and the phospholipid detection reagent includes a first detection reagent and a second detection reagent;

[0077] The first detection reagent includes the following components: 50mmol / L TAPSO (pH 7.5), 0.5g / L sodium azide, 1mL / L Trion X-100, 0.5g / L EDTA-2K, 2KU / L phospholipase D, 0.2g / L DA67, and 2KU / L ascorbate oxidase;

[0078] The second detection reagent includes the following components: 25mmol / L PIPES (pH 7.5), 2mL / L Trion X-100, 1g / L 4-aminoantipyrine, 1mL / L ProClin 300, 0.1g / L potassium ferrocyanide, 10KU / L peroxidase, and 5KU / L choline oxidase.

[0079] The experimental parameters for phospholipid detection using the kits in Examples 1-11 and Comparative Examples 1-2 are shown in Table 1 below.

[0080] Table 1 Experimental parameters

[0081] Method: Endpoint method Temperature: 37℃ Main wavelength: 666 or 600nm Sub-wavelength: 700nm Sample volume: 2 μL First detection reagent: 200μL Second detection reagent: 50μL Reaction direction: forward Reaction time: 10min Calibration method: two-point calibration

[0082] Furthermore, the HITACHI 7180 fully automatic biochemical analyzer was used for the determination, and the instrument operation is shown in Table 2 below.

[0083] Table 2 Instrument operating parameters

[0084]

[0085] After the test is completed, ΔA=A2-A1 is calculated to obtain the phospholipid concentration.

[0086] Application Test Example 1

[0087] This application test example tests the anti-drug interference ability of the kit in Example 1-11 and Comparative Example 1-2. Specifically, the interfering substances (calcium dobesilate, phensulfonylamine, levodopa, methyldopa) are diluted with purified water in a gradient, and added to the blank control serum at a ratio of 1:9 (the blank control serum is a normal mixed serum without any interfering substances, and its value is near the medical determination level), so that the final concentration after adding the serum meets the concentration to be evaluated, and then the blank serum and the serum with different concentrations of interfering substances are measured respectively with the kit of each group. Each sample is measured 3 times, and the mean is calculated. The deviation is calculated from the blank control serum measurement value, which is the interference degree. If the interference degree is less than 10%, it is anti-interference, otherwise it is not anti-interference; the results are shown in Tables 3-6 below.

[0088] Table 3 Anti-calcium dobesilate interference evaluation data results

[0089]

[0090]

[0091] Table 4 Anti-phenolsulfonamide interference evaluation data results

[0092]

[0093]

[0094] Table 5 Anti-levodopa interference evaluation data results

[0095]

[0096]

[0097] Table 6 Anti-methyldopa interference evaluation data results

[0098]

[0099]

[0100] As can be seen from Tables 3-6, the chromogenic substrate DAOS in Comparative Example 1 is not resistant to interference from commonly used clinical drugs (calcium dobesilate, ethylphenolsulfonamide, methyldopa, levodopa). After the chromogenic substrates in Examples 1-11 and Comparative Example 2 are replaced with DA-67 and the detection dominant wavelength is changed to 666 nm, the reagent can resist interference from commonly used clinical drugs. Therefore, in the present invention, by adding the chromogenic substrate DA-67, the ability of the reagent to resist drug interference can be significantly improved.

[0101] Application Test Example 2

[0102] This application test example tests the precision and linear range of the kits in Examples 1-11 and Comparative Example 2. The results are shown in Tables 7 and 8 below.

[0103] Table 7 Precision performance evaluation data

[0104]

[0105] Table 8 Linear range evaluation data

[0106]

[0107]

[0108] It can be seen from Tables 7 and 8 that, compared with Comparative Example 2, the addition of surfactant and metal salt to the reagents in Examples 1-11 significantly improved the sensitivity, precision and linear range of the reagents.

[0109] Application Test Example 3

[0110] This application test example tests the stability of the kits in Examples 4-11. Specifically, the reagents in the kits in each example were treated as follows: 1) 37°C heat-break for 7 days, 2) 42°C and 130 rpm shaking for 3 days; then the precision, linear range, accuracy, appearance change and anti-drug interference results of the reagents after the above different treatments were tested, and the results are shown in Tables 9-16 below.

[0111] Table 9 Stability evaluation data of the kit in Example 4

[0112]

[0113]

[0114] Table 10 Stability evaluation data of the kit in Example 5

[0115]

[0116] Table 11 Stability evaluation data of the kit in Example 6

[0117]

[0118] Table 12 Stability evaluation data of the kit in Example 7

[0119]

[0120] Table 13 Stability evaluation data of the kit in Example 8

[0121]

[0122] Table 14 Stability evaluation data of the kit in Example 9

[0123]

[0124]

[0125] Table 15 Stability evaluation data of the kit in Example 10

[0126]

[0127] Table 16 Stability evaluation data of the kit in Example 11

[0128]

[0129] As can be seen from Tables 9-16, compared with Example 4, the reagents in the kits in Examples 5-11 are added with one or more of β-cyclodextrin, trehalose, and ADP-K, which significantly improves the stability of the kit, and the reagents are not precipitated after heat breaking and oscillation treatment, and the precision, linear ratio, accuracy, and anti-drug interference performance remain basically unchanged. In addition, when the reagents in the kit contain β-cyclodextrin, trehalose, and ADP-K (Example 11) at the same time, the stability of the kit is the best. The results show that the above three protective agents have a synergistic effect and can significantly improve the stability of the kit.

[0130] In summary, the present invention significantly improves the drug interference resistance of the reagent by adding the chromogenic substrate DA-67 to the first detection reagent and changing the detection main wavelength to 666nm; at the same time, by adding surfactants and metal salts to the first detection reagent and the second detection reagent, the sensitivity, precision and linear range of the reagent are significantly improved; in addition, by adding a protective agent, the stability of the reagent is significantly improved.

[0131] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0132] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A phospholipid detection reagent, characterized in that: including a first detection reagent and a second detection reagent; Wherein, the first detection reagent comprises the following components: 25-100mmol / L of the first buffer, 0.5-1g / L of sodium azide, 0.5-5g / L of metal salt, 0.5-5mL / L of Trion X-100, 0.3-1g / L of EDTA-2K, 2-10g / L of surfactant, 0.5-5KU / L of phospholipase D, 0.1-1g / L of 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt, and 0.5-2KU / L of ascorbic acid oxidase; The second detection reagent includes the following components: 25-100mmol / L of a second buffer, 1-5mL / L of Trion X-100, 0.5-2g / L of 4-aminoantipyrine, 0.5-5g / L of a metal salt, 2-10g / L of a surfactant, 0.5-1mL / L of ProClin 300, 0.1-0.2g / L of potassium ferrocyanide, 2-10KU / L of peroxidase, and 2-10KU / L of choline oxidase.

2. The phospholipid detection reagent according to claim 1, characterized in that The first detection reagent also includes 0.5-20 g / L of a protective agent, and the protective agent is selected from at least one of β-cyclodextrin, trehalose, and ADP-K.

3. The phospholipid detection reagent according to claim 1, characterized in that The pH value of the first buffer in the first detection reagent is 7.4-7.6, and the first buffer is selected from at least one of piperazine-1,4-diethanesulfonic acid, 3-tris(hydroxymethyl)methylamine-2-hydroxypropanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, tris(hydroxymethyl)methylamineethanesulfonic acid, and 3-morpholinepropanesulfonic acid.

4. The phospholipid detection reagent according to claim 1, characterized in that The metal salt in the first detection reagent is selected from at least one of KCl, NaCl, CaCl2, MgCl2, and BaCl2; The surfactant in the first detection reagent is selected from at least one of polyoxyethylene alkyl ether ADEKATOL SO-120, polyoxyethylene alkyl ether ADEKATOL SO-105, polyoxyethylene alkyl ether ADEKATOL SO-80, polyoxyethylene alkyl ether ADEKATOL SO-135, polyoxyethylene alkyl ether ADEKATOL SO-145, and polyoxyethylene alkyl ether ADEKATOL SO-160.

5. The phospholipid detection reagent according to claim 1, characterized in that The second detection reagent also includes 0.5-20 g / L of a protective agent, and the protective agent is selected from at least one of β-cyclodextrin, trehalose, and ADP-K.

6. The phospholipid detection reagent according to claim 1, characterized in that The pH value of the second buffer in the second detection reagent is 7.4-7.6, and the second buffer is selected from at least one of piperazine-1,4-diethanesulfonic acid, 3-tris(hydroxymethyl)methylamine-2-hydroxypropanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, tris(hydroxymethyl)methylamineethanesulfonic acid, and 3-morpholinepropanesulfonic acid.

7. The phospholipid detection reagent according to claim 1, characterized in that The metal salt in the second detection reagent is selected from at least one of KCl, NaCl, CaCl2, MgCl2, and BaCl2; The surfactant in the second detection reagent is selected from at least one of polyoxyethylene alkyl ether ADEKATOL SO-120, polyoxyethylene alkyl ether ADEKATOL SO-105, polyoxyethylene alkyl ether ADEKATOL SO-80, polyoxyethylene alkyl ether ADEKATOL SO-135, polyoxyethylene alkyl ether ADEKATOL SO-145, and polyoxyethylene alkyl ether ADEKATOL SO-160.

8. A phospholipid detection kit, characterized in that: The invention comprises the phospholipid detection reagent according to any one of claims 1 to 8.

9. Use of the phospholipid detection reagent according to any one of claims 1 to 8 or the phospholipid detection kit according to claim 9 in phospholipid detection.

10. The use according to claim 9, characterized in that: When the phospholipid detection reagent or the phospholipid detection kit is used to perform phospholipid detection, the volume ratio of the first detection reagent to the second detection reagent is (3-5):(0.5-1.5).

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