CN111551512B - High-sensitivity and high-specificity sdLDL-C colorimetric detection kit and preparation and use methods thereof - Google Patents
High-sensitivity and high-specificity sdLDL-C colorimetric detection kit and preparation and use methods thereof Download PDFInfo
- Publication number
- CN111551512B CN111551512B CN202010532055.2A CN202010532055A CN111551512B CN 111551512 B CN111551512 B CN 111551512B CN 202010532055 A CN202010532055 A CN 202010532055A CN 111551512 B CN111551512 B CN 111551512B
- Authority
- CN
- China
- Prior art keywords
- sdldl
- reagent
- mpeg
- mixing
- specificity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
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
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/775—Indicator and selective membrane
-
- 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
Abstract
The invention provides a high-sensitivity and high-specificity sdLDL-C colorimetric detection kit, which comprises a reagent R1 and a reagent R2; reagent R1: MOPS buffer, PEG 6000, BSA, naCl, naN 3 EDTA, catalase, cholesterol esterase, cholesterol oxidase, TOOS and surfactant I, wherein the solvent is purified water; reagent R2: MOPS buffer, PEG 6000, BSA, naCl, naN 3 EDTA, 4-AAP, peroxidase, mPEG-PlD, surfactant II, and purified water as solvent. The invention also provides a preparation and use method of the sdLDL-C colorimetric detection kit with high sensitivity and specificity. The kit can be used for analysis of a full-automatic biochemical analyzer, is convenient to operate and clinical application, and has higher specificity and sensitivity.
Description
Technical Field
The invention relates to the field of biochemical assay, in particular to a high-sensitivity and specificity sdLDL-C colorimetric detection kit and a preparation and use method thereof.
Background
Small, dense low density lipoprotein cholesterol (Small and dense LDL cholesterol, sdLDL-C) is a subtype of LDL-C with a large density and small particle diameter (22.0 to 25.5nm in diameter). Recent studies have demonstrated that sdLDL-C is closely related to the extent of atherosclerosis, carotid intima-media thickness (IMT), membrane island element resistance, and glucose tolerance. Numerous epidemiological studies have also shown that sdLDL-C is an independent risk factor for coronary heart disease, ischemic cerebral infarction, diabetes. Targeted intervention with plasma sdLDL-C levels in patients with coronary heart disease is more likely to prevent and reduce the occurrence of adverse cardiovascular events than just lowering LDL-C levels. The sdLDL-C level has good prediction and evaluation effects on the risk of occurrence of adverse cardiovascular events of coronary heart disease patients, has the potential of replacing invasive coronary angiography detection, has great clinical application prospect of evaluating cardiovascular related diseases, and is worthy of being widely popularized in clinical laboratories.
In recent years, methods for measuring the concentration of sdLDL-C have been reported, and examples of the method for measuring the concentration of sdLDL-C include gradient density ultracentrifugation, gradient gel electrophoresis, chemical precipitation, molecular sieve chromatography, capillary electrophoresis, nuclear magnetic resonance spectroscopy, index evaluation, dynamic light scattering, and homogeneous phase method. Among them, heparin-magnesium chemical precipitation centrifugation is the gold standard for measuring sdLDL-C at present, and although the method has better specificity and sensitivity, the operation is complex, and the method is inconvenient for large-scale clinical application. The current method is widely applied in clinic, but the reagent of each company has the problem of low specificity and/or sensitivity, which is caused by insufficient separation of sdLDL-C from other cholesterol-containing components. Accordingly, there is an urgent need for a new method for detecting sdLDL-C that is convenient to operate and has high specificity and sensitivity.
Disclosure of Invention
The invention aims to solve the technical problem of providing the sdLDL-C colorimetric method detection kit with high sensitivity and specificity and the preparation and use methods thereof, and the sdLDL-C concentration detection is carried out by adopting the kit, so that the kit is convenient to operate and clinical application, and has higher specificity and sensitivity.
The invention adopts the following technical scheme to solve the technical problems:
a high-sensitivity and high-specificity sdLDL-C colorimetric detection kit comprises a reagent R1 and a reagent R2 which are independent from each other and are double-liquid components;
the reagent R1 comprises the following components in percentage by weight: MOPS buffer solution 20-80 mM, PEG 6000100-200 g/L, BSA (bovine serum albumin) 20-30 g/L, naCl 1.8-18 g/L, naN 3 (sodium azide) 0.2-0.8 g/L, EDTA 0.1-1 g/L, catalase 500-900U/L, cholesterol esterase 200-800U/L, cholesterol oxidase 100-1000U/L, TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt) 10-15 mM, surfactant I0.05-0.1%, and the solvent is purified water;
the reagent R2 comprises the following components in percentage by weight: MOPS buffer solution 20-80 mM, PEG 6000100-200 g/L, BSA (bovine serum albumin) 20-30 g/L, naCl 1.8-18 g/L, naN 3 (sodium azide) 0.2-0.8 g/L, EDTA 0.1-1 g/L,4-AAP (4-aminoantipyrine) 0.5-2 mM, peroxidase 10-20U/L, mPEG-PlD 5-15 mg/L, surfactant II 0.05-0.8%, and pure solventDissolving water.
As one of the preferred modes of the present invention, there is also included an sdLDL calibrator; the sdLDL calibrator comprises the following components in percentage by weight: MOPS buffer solution 20-80 mM, BSA (bovine serum albumin) 20-30 g/L, sdLDL-C5-15 mg/L, naN 3 (sodium azide) 0.2-0.8 g/L, and the solvent is purified water.
As a preferred embodiment of the present invention, the surfactant I in the reagent R1 is composed of one or more polyoxyethylene styrenated phenyl ether derivatives having HBL values of 12.0 to 14.6.
As one of the preferred modes of the invention, the polyoxyethylene styrenated phenyl ether derivatives having HBL values of 12.0 to 14.6 are specifically designated as NIKKOL BT-9 and NIKOL BT-12, which are products of the sun company.
As a preferred embodiment of the present invention, the surfactant II in the reagent R2 is composed of one or more polyoxyethylene derivatives having HBL values of 15.6 to 16.0.
As one of the preferable modes of the present invention, the polyoxyethylene derivative having an HBL value of 15.6 to 16.0 is specifically Brij-35 and DTAC, which are products of Xibao corporation.
As one of the preferred modes of the present invention, the mPEG-PlD obtaining method is as follows:
(1) preparation of recombinant phospholipase D:
a. obtaining all CDS of phospholipase D in GENBANK, and adding BamHI and EcoRI cleavage sites at the head and tail respectively to obtain a target sequence, wherein the target sequence is shown in SEQ ID NO. 1;
b. after obtaining the target sequence, sending the target sequence to a gene company for synthesis;
c. double enzyme cutting, connecting to an expression vector pET32a, and introducing into an expression bacterium Ecoli;
d. coating the bacterial strain on an ampicillin flat plate, and picking positive bacteria for fermentation expression;
e. centrifuging to obtain supernatant, and subjecting to nickel column affinity chromatography to obtain target product;
(2) mPEG conjugation of recombinant phospholipase D:
a. cyanuric chloride 5.5g, anhydrous Na 2 CO 3 10g, 5g molecular sieve 5A, 5g and mPEG-5000 50g are dissolved in 400mL of anhydrousStirring and reacting benzene for 12h at normal temperature;
b. centrifuging to remove Na 2 CO 3 Suspended substances such as molecular sieve 5A and the like are precipitated by 600mL of diethyl ether, and then are dissolved and precipitated by 400mL of anhydrous benzene; repeating the precipitation and dissolution for 5 times, and removing unreacted cyanuric chloride until no light absorption is detected at 258nm wavelength; finally, vacuum drying is carried out to obtain activated CC-mPEG;
c. and (2) taking the CC-mPEG and the recombinant phospholipase D prepared in the step (1), dissolving the CC-mPEG and the recombinant phospholipase D in 10mL of borate buffer solution, reacting for 3-6 hours at room temperature on a shaking table, and dialyzing for overnight at 15kD to obtain a modified enzyme solution, namely mPEG-PlD.
As one of the preferable modes of the invention, the method for obtaining sdLDL-C is as follows: preparation of heparin-Mg 2+ Wherein, the heparin concentration is 150U/mL, mg 2+ Concentration 90mmol/L; the heparin-Mg is added in an amount of 10% 2+ Adding 50mL of human serum, uniformly mixing, standing at room temperature for 15min, and centrifuging at 1500g for 30min; 40mL of the supernatant was taken and 4mL of heparin-Mg was further added 2+ Mixing, standing at room temperature for 15min; finally, the mixture was centrifuged at 1500g for 30min, and the supernatant was the sdLDL-C product.
The preparation method of the high-sensitivity and specificity sdLDL-C colorimetric detection kit comprises the following steps:
(1) Preparation of human sdLDL-C
Preparation of heparin-Mg 2+ Wherein, the heparin concentration is 150U/mL, mg 2+ Concentration 90mmol/L; the heparin-Mg is added in an amount of 10% 2+ Adding 50mL of human serum, uniformly mixing, standing at room temperature for 15min, and centrifuging at 1500g for 30min; 40mL of the supernatant was taken and 4mL of heparin-Mg was further added 2+ Mixing, standing at room temperature for 15min; finally, centrifuging for 30min under 1500g, wherein the supernatant is the sdLDL-C product;
(2) Preparation of mPEG recombinant phospholipase D
(1) Preparation of recombinant phospholipase D:
a. obtaining all CDS of phospholipase D in GENBANK, and adding BamHI and EcoRI cleavage sites at the head and tail respectively to obtain a target sequence, wherein the target sequence is shown in SEQ ID NO. 1;
b. after obtaining the target sequence, sending the target sequence to a gene company for synthesis;
c. double enzyme cutting, connecting to an expression vector pET32a, and introducing into an expression bacterium Ecoli;
d. coating the bacterial strain on an ampicillin flat plate, and picking positive bacteria for fermentation expression;
e. centrifuging to obtain supernatant, and subjecting to nickel column affinity chromatography to obtain target product;
(2) mPEG conjugation of recombinant phospholipase D:
a. cyanuric chloride 5.5g, anhydrous Na 2 CO 3 10g, 5g of molecular sieve 5A and 50g of mPEG-5000 are dissolved in 400mL of anhydrous benzene, and stirred at normal temperature for reaction for 12h;
b. centrifuging to remove Na 2 CO 3 Suspended substances such as molecular sieve 5A and the like are precipitated by 600mL of diethyl ether, and then are dissolved and precipitated by 400mL of anhydrous benzene; repeating the precipitation and dissolution for 5 times, and removing unreacted cyanuric chloride until no light absorption is detected at 258nm wavelength; finally, vacuum drying is carried out to obtain activated CC-mPEG;
c. taking the CC-mPEG and the recombinant phospholipase D prepared in the step (1), dissolving the CC-mPEG and the recombinant phospholipase D in 10mL of borate buffer solution, reacting for 3-6 hours at room temperature on a shaking table, and dialyzing for overnight at 15kD to obtain a modified enzyme solution, namely mPEG-PlD;
(3) Preparation of sdLDL-C colorimetric detection kit
(1) Preparing a reagent R1:
according to the component content of the reagent R1, mixing the component substances in the same container, and uniformly mixing to obtain the reagent R1;
(2) preparing a reagent R2:
according to the component content of the reagent R2, mixing the mPEG-PlD prepared in the step (2) and the rest other component substances in the same container, and uniformly mixing to prepare the reagent R2;
(3) preparing an sdLDL calibrator:
the sdLDL calibrator comprises the following components in percentage by weight: MOPS buffer solution 20-80 mM, BSA 20-30 g/L, sdLDL-C5-15 mg/L, naN 3 0.2-0.8 g/L, wherein the solvent is purified water;
and (3) mixing the sdLDL-C product prepared in the step (1) and the rest other components in the same container according to the component content of the sdLDL calibrator, and uniformly mixing to prepare the sdLDL calibrator.
The application method of the high-sensitivity and specificity sdLDL-C colorimetric detection kit comprises the following specific steps:
(1) Sucking 2.4 mu L of sample, adding 180 mu L of reagent R1, and incubating for 5min at 37 ℃;
(2) Then 60 mu L of reagent R2 is added for mixing and fully reacting;
(3) Reading a light absorption value A after 5min;
(4) The calibration method is 2-point calibration, the full-automatic biochemical analyzer is adopted for detection, and the concentrations of the calibrator are respectively set as follows: 0. 45mg/dL; and measuring and calculating the sdLDL-C content in the sample according to the scaling value and the A.
Detection principle:
the invention combines a specific surfactant I (polyoxyethylene styrenated phenyl ether derivative with HBL value of 12.0-14.6) and sdLDL-C into a micelle structure, the micelle structure can form a protective layer of sdLDL-C, and the reagent R1 releases cholesterol ester and free cholesterol in other lipoprotein cholesterol and reacts with the cholesterol to eliminate all cholesterol except the protection of the surfactant I; after the reagent R2 is added, the surfactant II (polyoxyethylene derivative with HBL value of 15.6-16.0) contained in the reagent R2 can release micelle formed by the surfactant I and the sdLDL-C and release the sdLDL-C; because the high density region of sdLDL-C partially coincides with the low density region of HDL, modified phospholipase D contained in reagent R2 (modified phospholipase D contains two binding sites to better bind sdLDL-C) binds and precipitates with the released high density region of sdLDL-C and the low density region of HDL, and reagent R2 reacts with cholesterol in the remaining sdLDL-C to determine the remaining concentration of sdLDL-C. The concentration of all sdLDL-C in the sample to be detected can be obtained by comparing the sample with a standard substance, and the kit is suitable for various full-automatic biochemical analyzers based on a colorimetric method, and is convenient for clinical application.
Compared with the prior art, the invention has the advantages that: the sdLDL-C colorimetric assay kit consists of modified phospholipase D, a surfactant, a buffer solution, a preservative, a solubilizer, a protein protectant and the like, wherein the surfactant I can isolate a high-density region of idLDL-C, so that the sdLDL-C can be more specifically separated; the modified phospholipase D is better in a low density region of the precipitated HDL, so that the sensitivity and the specificity of the test are improved; the specific characteristics are as follows:
(1) The surfactant I (polyoxyethylene styrenated phenyl ether derivative with the HBL value of 12.0-14.6) and the surfactant II (polyoxyethylene derivative with the HBL value of 15.6-16.0) in the reagent form a pair of isolation systems of sdLDL-C, so that the sdLDL-C can be effectively and protectively isolated and released;
(2) The modified phospholipase D in the reagent is used for supplementing an isolation system of the surfactant I and the surfactant II, so that the sensitivity and the specificity of the kit are further improved;
(3) The reagent can be used for analysis of various full-automatic biochemical analyzers, is convenient to operate, low in cost and high in automation, and can greatly save detection time; and compared with similar products, the kit has better stability.
Drawings
FIG. 1 is a graph showing the linear relationship between the detection result of the reagent of the present invention and the detection result of the precipitation centrifugation method in example 6;
FIG. 2 is a graph showing the correlation between the reagent of the present invention and the reagent of a certain commercial company in example 6 and the result of the detection by centrifugation;
FIG. 3 is a graph showing the correlation between the reagent of the present invention and a reagent of a certain commercial company in example 6, and the result of the detection by centrifugation;
FIG. 4 is a linear regression plot of the linear range of the kit of the invention in example 6.
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
Example 1
The sdLDL-C colorimetric assay kit with high sensitivity and specificity comprises a reagent R1 and a reagent R2 which are independent from each other.
The reagent R1 comprises the following components in percentage by weight: MOPS buffer 20mM,PEG 6000 100g/L, BSA20 g/L, naCl 1.8g/L, naN 3 0.2g/L, EDTA 0.1g/L, catalase 500U/L, cholesterol esterase 200U/L, cholesterol oxidase 100U/L, TOOS 10mM, surfactant I0.05%, and purified water as solvent.
The reagent R2 comprises the following components in percentage by weight: MOPS buffer 20mM,PEG 6000 100g/L, BSA20 g/L, naCl 1.8g/L, naN 3 0.2g/L EDTA 0.1g/L,4-AAP 0.5mM, peroxidase 10U/L, mPEG-PlD 5mg/L, surfactant II 0.05%, the solvent was purified water.
In addition, the sdLDL-C colorimetric assay kit also comprises a sdLDL calibrator. The sdLDL calibrator comprises the following components in percentage by weight: MOPS buffer 20mM, BSA20 g/L, sdLDL-C5 mg/L, naN 3 0.2g/L, the solvent is purified water.
Further, the surfactant I is specifically NIKKOL BT-9, a product of sunlight company.
Further, the surfactant II is Brij-35, a product of Xibao corporation.
Example 2
The sdLDL-C colorimetric assay kit with high sensitivity and specificity comprises a reagent R1 and a reagent R2 which are independent from each other.
The reagent R1 comprises the following components in percentage by weight: MOPS buffer 80mM,PEG 6000 200g/L, BSA 30g/L, naCl 18g/L, naN 3 0.8g/L, EDTA 1g/L, catalase 900U/L, cholesterol esterase 800U/L, cholesterol oxidase 1000U/L, TOOS 15mM, surfactant I0.1%, and purified water as solvent.
The reagent R2 comprises the following components in percentage by weight: MOPS buffer 80mM,PEG 6000 200g/L, BSA 30g/L, naCl 18g/L, naN 3 0.8g/L EDTA 1g/L,4-AAP 2mM, peroxidase 20U/L, mPEG-PlD 15mg/L, surfactant II 0.8%, solvent thereforTo purify water.
In addition, the sdLDL-C colorimetric assay kit also comprises a sdLDL calibrator. The sdLDL calibrator comprises the following components in percentage by weight: MOPS buffer 80mM, BSA 30g/L, sdLDL-C15 mg/L, naN 3 0.8g/L, the solvent is purified water.
Further, surfactant I is specifically NIKKOL BT-12, a product of solar corporation.
Further, the surfactant II is DTAC (DTAC) which is a product of Xibao company.
Example 3
The sdLDL-C colorimetric assay kit with high sensitivity and specificity comprises a reagent R1 and a reagent R2 which are independent from each other.
The reagent R1 comprises the following components in percentage by weight: MOPS buffer 60mM,PEG 6000 180g/L, BSA 26g/L, naCl 13.5g/L, naN 3 0.5g/L, EDTA 0.35g/L, catalase 600U/L, cholesterol esterase 500U/L, cholesterol oxidase 800U/L, TOOS 12.5mM, surfactant I0.08%, and the solvent is purified water.
The reagent R2 comprises the following components in percentage by weight: MOPS buffer 60mM,PEG 6000 180g/L, BSA 26g/L, naCl 13.5g/L, naN 3 0.5g/L EDTA 0.35g/L,4-AAP 1.0mM, peroxidase 15U/L, mPEG-PlD 10mg/L, surfactant II 0.5%, the solvent was purified water.
In addition, the sdLDL-C colorimetric assay kit also comprises a sdLDL calibrator. The sdLDL calibrator comprises the following components in percentage by weight: MOPS buffer 60mM, BSA 26g/L, sdLDL-C10 mg/L, naN 3 0.5g/L, the solvent is purified water.
Further, surfactant I is specifically a mixture of the solar company products NIKKOL BT-9 and NIKOL BT-12.
Further, surfactant II is specifically a mixture of Brij-35 and DTAC, a product of West Bao corporation.
Example 4
The preparation method of the sdLDL-C colorimetric assay kit with high sensitivity and specificity in the above embodiments 1-3 comprises the following steps:
(1) Preparation of human sdLDL-C
Preparation of heparin-Mg 2+ Wherein, the heparin concentration is 150U/mL, mg 2+ Concentration 90mmol/L; the heparin-Mg is added in an amount of 10% 2+ Adding 50mL of human serum (from healthy donors), mixing, standing at room temperature for 15min, and centrifuging at 1500g for 30min; 40mL of the supernatant was taken and 4mL of heparin-Mg was further added 2+ Mixing, standing at room temperature for 15min; finally, centrifuging for 30min under 1500g, wherein the supernatant is the sdLDL-C product;
(2) Preparation of mPEG recombinant phospholipase D
(1) Preparation of recombinant phospholipase D:
a. obtaining all CDS of phospholipase D in GENBANK, and adding BamHI and EcoRI cleavage sites at the head and tail respectively to obtain a target sequence, wherein the target sequence is shown in SEQ ID NO. 1;
b. after obtaining the target sequence, sending the target sequence to a gene company for synthesis;
c. double enzyme cutting, connecting to an expression vector pET32a, and introducing into an expression bacterium Ecoli;
d. coating the bacterial strain on an ampicillin flat plate, and picking positive bacteria for fermentation expression;
e. centrifuging to obtain supernatant, and subjecting to nickel column affinity chromatography to obtain target product;
(2) mPEG conjugation of recombinant phospholipase D:
a. cyanuric chloride 5.5g, anhydrous Na 2 CO 3 10g, 5g of molecular sieve 5A and 50g of mPEG-5000 are dissolved in 400mL of anhydrous benzene, and stirred at normal temperature for reaction for 12h;
b. centrifuging to remove Na 2 CO 3 Suspended substances such as molecular sieve 5A and the like are precipitated by 600mL of diethyl ether, and then are dissolved and precipitated by 400mL of anhydrous benzene; repeating the precipitation and dissolution for 5 times, and removing unreacted cyanuric chloride until no light absorption is detected at 258nm wavelength; finally, vacuum drying is carried out to obtain activated CC-mPEG;
c. taking the CC-mPEG and the recombinant phospholipase D prepared in the step (1), dissolving the CC-mPEG and the recombinant phospholipase D in 10mL of borate buffer solution (40 mM, pH 7.4), reacting for 3-6 hours at room temperature on a shaking table, and dialyzing for overnight at 15kD to obtain a modified enzyme solution, namely mPEG-PlD;
(3) Preparation of sdLDL-C colorimetric detection kit
(1) Preparing a reagent R1:
according to the component content of the reagent R1, mixing the component substances in the same container, and uniformly mixing to obtain the reagent R1;
(2) preparing a reagent R2:
according to the component content of the reagent R2, mixing the mPEG-PlD prepared in the step (2) and the rest other component substances in the same container, and uniformly mixing to prepare the reagent R2;
(3) preparing an sdLDL calibrator:
and (3) mixing the sdLDL-C product prepared in the step (1) and the rest other components in the same container according to the component content of the sdLDL calibrator, and uniformly mixing to prepare the sdLDL calibrator.
Example 5
A method for testing the use of the sdLDL-C colorimetric assay kit of the present embodiment with high sensitivity and specificity in the above-described examples 1 to 3.
The analysis method comprises the following steps: a terminal method;
the reaction direction is as follows: lifting reaction;
the calibration mode is as follows: AB;
measurement wavelength: 600nm;
measuring temperature: 37 ℃;
sample: reagent R1: reagent r2=2.4: 180:60 (μl);
the testing steps are as follows: 2.4. Mu.L of the sample was aspirated, 180. Mu.L of reagent R1 was added, incubation was performed at 37℃for 5min, 60. Mu.L of reagent R2 was added, and absorbance A was read after 5 min.
The calibration method comprises the following steps: 2 point calibration, detection is carried out by adopting a Beckmann AU680 full-automatic biochemical analyzer (or other brand models), and the concentrations of the calibration products are respectively set as follows: 0. 45mg/dL.
And measuring and calculating the sdLDL-C content in the sample according to the scaling value and the A.
Example 6
This example was used to evaluate the sdLDL-C colorimetric assay kit in the example above:
(1) Linear correlation verification:
the reagent is prepared by using the formula of the example 3, and is subjected to control detection with a centrifugal precipitation method, 30 clinical serum samples are detected, the detection results are shown in table 1, and a correlation curve of the kit and the centrifugal precipitation method is obtained (see fig. 1; in fig. 1, the abscissa X represents the detection result value (unit: mg/dL) of the kit, and the ordinate Y represents the detection result value (unit: mg/dL) of the centrifugal precipitation method). The test result shows that the linear correlation curve of the two methods is shown as an equation of y=0.47739+0.97967x, R 2 = 0.99165, indicating that both have good correlation.
TABLE 1 correlation comparison of the detection results of the inventive reagents and the detection results of the centrifugal precipitation method
In addition, the abnormal serum test shows that the invention has better sensitivity and specificity compared with the product of a certain marketing company in the current market. Wherein, the comparison value of the abnormal serum detection result of the reagent of the invention and the reagent of a certain marketing company is shown in table 2, the correlation comparison diagram of the low-value serum sample of the reagent of the invention and the reagent of the marketing company and the detection result of the centrifugation method is shown in fig. 2, and the correlation comparison diagram of the high-value serum sample of the reagent of the invention and the reagent of the marketing company and the detection result of the centrifugation method is shown in fig. 3. As can be seen from FIGS. 2 and 3, the present invention has higher specificity and sensitivity than other company reagents.
TABLE 2 comparison of the detection of abnormal serum results with the reagents of the invention and with the reagents of certain marketing companies
(2) Linear range verification:
using recombinant sdLDL-C purified product and physiological saline to prepare test products having concentrations of 80mg/dL, 40mg/dL, 20mg/dL, 10mg/dL, 5mg/dL, 2.5mg/dL, 1.25mg/dL and 0mg/dL (physiological saline control), the concentrations of each test product were determined using the kit of the present invention, the linear regression equation was calculated using the measurement results as independent variables, and the relative deviations of the measurement results were calculated. The detection and calculation results are shown in Table 3, and the result shows that the linear regression equation between the measurement result and the dilution concentration is equation y= -0.06+1.0225X, and the correlation coefficient R 2 =0.9998, indicating a good linear relationship, and a linear range of 0 to 80mg/dL.
TABLE 3 Linear Range validation of the inventive kits
/>
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
SEQUENCE LISTING
<110> Anhui Daqian bioengineering Co., ltd
<120> a high sensitivity and specificity sdLDL-C colorimetric assay kit and preparation and use methods thereof
<130> 2020
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 3390
<212> DNA
<213> artificial sequence
<400> 1
ggatccatgt ctgctttcag gttgtggcct ggcctgctga tcatgttggg ttctctctgc 60
catagaggtt caccgtgtgg cctttcaaca cacgtagaaa taggacacag agctctggag 120
tttcttcagc ttcacaatgg gcgtgttaac tacagagagc tgttactaga acaccaggat 180
gcgtatcagg ctggaatcgt gtttcctgat tgtttttacc ctagcatctg caaaggagga 240
aaattccatg atgtgtctga gagcactcac tggactccgt ttcttaatgc aagcgttcat 300
tatatccgag agaactatcc ccttccctgg gagaaggaca cagagaaact ggtagctttc 360
ttgtttggaa ttacttctca catggcggca gatgtcagct ggcatagtct gggccttgaa 420
caaggattcc ttaggaccat gggagctatt gattttcacg gctcctattc agaggctcat 480
tcggctggtg attttggagg agatgtgttg agccagtttg aatttaattt taattacctt 540
gcacgacgct ggtatgtgcc agtcaaagat ctactgggaa tttatgagaa actgtatggt 600
cgaaaagtca tcaccgaaaa tgtaatcgtt gattgttcac atatccagtt cttagaaatg 660
tatggtgaga tgctagctgt ttccaagtta tatcccactt actctacaaa gtccccgttt 720
ttggtggaac aattccaaga gtattttctt ggaggactgg atgatatggc attttggtcc 780
actaatattt accatctaac aatcttcatg ttggagaatg ggaccagtga ctgcaacctg 840
cctgagaacc ctctgttcat tgcatgtggc ggccagcaaa accacaccca gggctcaaaa 900
atgcagaaaa atgattttca cagaaatttg actacatccc taactgaaag tgttgacagg 960
aatataaact atactgaaag aggagtgttc tttagtgtaa attcctggac cccggattcc 1020
atgtccttta tctacaaggc tttggaaagg aacataagga caatgttcat aggtggctct 1080
cagttgtcac aaaagcacgt ctccagcccc ttagcatctt acttcttgtc atttccttat 1140
gcgaggcttg gctgggcaat gacctcagct gacctcaacc aggatgggca cggtgacctc 1200
gtggtgggcg caccaggcta cagccgcccc ggccacatcc acatcgggcg cgtgtacctc 1260
atctacggca atgacctggg cctgccacct gttgacctgg acctggacaa ggaggcccac 1320
aggatccttg aaggcttcca gccctcaggt cggtttggct cggccttggc tgtgttggac 1380
tttaacgtgg acggcgtgcc tgacctggcc gtgggagctc cctcggtggg ctccgagcag 1440
ctcacctaca aaggtgccgt gtatgtctac tttggttcca aacaaggagg aatgtcttct 1500
tcccctaaca tcaccatttc ttgccaggac atctactgta acttgggctg gactctcttg 1560
gctgcagatg tgaatggaga cagtgaaccc gatctggtca tcggctcccc ttttgcacca 1620
ggtggaggga agcagaaggg aattgtggct gcgttttatt ctggccccag cctgagcgac 1680
aaagaaaaac tgaacgtgga ggcagccaac tggacggtga gaggcgagga agacttctcc 1740
tggtttggat attcccttca cggtgtcact gtggacaaca gaaccttgct gttggttggg 1800
agcccgacct ggaagaatgc cagcaggctg ggccatttgt tacacatccg agatgagaaa 1860
aagagccttg ggagggtgta tggctacttc ccaccaaacg gccaaagctg gtttaccatt 1920
tctggagaca aggcaatggg gaaactgggt acttcccttt ccagtggcca cgtactgatg 1980
aatgggactc tgaaacaagt gctgctggtt ggagccccta cgtacgatga cgtgtctaag 2040
gtggcattcc tgaccgtgac cctacaccaa ggcggagcca ctcgcatgta cgcactcata 2100
tctgacgcgc agcctctgct gctcagcacc ttcagcggag accgccgctt ctcccgattt 2160
ggtggcgttc tgcacttgag tgacctggat gatgatggct tagatgaaat catcatggca 2220
gcccccctga ggatagcaga tgtaacctct ggactgattg ggggagaaga cggccgagta 2280
tatgtatata atggcaaaga gaccaccctt ggtgacatga ctggcaaatg caaatcatgg 2340
ataactccat gtccagaaga aaaggcccaa tatgtattga tttctcctga agccagctca 2400
aggtttggga gctccctcat caccgtgagg tccaaggcaa agaaccaagt cgtcattgct 2460
gctggaagga gttctttggg agcccgactc tccggggcac ttcacgtcta tagccttggc 2520
tcagattgaa gatttcactg catttcccca ctctgcccac ctctctcatg ctgaatcaca 2580
tccatggtga gcattttgat ggacaaagtg gcacatccag tggagcggtg gtagatcctg 2640
atagacatgg ggctcctggg agtagagaga cacactaaca gccacaccct ctggaaatct 2700
gatacagtaa atatatgact gcaccagaaa tatgtgaaat agcagacatt ctgcttactc 2760
atgtctcctt ccacagttta cttcctcgct ccctttgcat ctaaaccttt cttctttccc 2820
aacttattgc ctgtagtcag acctgctgta caacctattt cctcttcctc ttgaatgtct 2880
ttccagtggc tggaaaggtc cctctgtggt tatctgttag aacagtctct gtacacaatt 2940
cctcctaaaa acatcctttt ttaaaaaaag aattgttcag ccataaagaa agaacaagat 3000
catgcccttt gcagggacat ggatggagct ggaggccatt atccttcata aactattgca 3060
ggaacagaaa accaaacact ccatattctc acttgtaagt gggagctaag tgagaacacg 3120
tggacacata gagggaaaca acacacactg gggcctatga gagggcggaa ggtgggagga 3180
gggagagatc aggaaaaata actaatggat acttagggtg atgaaataat ctgtgtaaca 3240
aacccccatg acacaccttt atgtatgtaa caaaccagca cttcctgcgc atgtacccct 3300
gaacttaaaa gttaaaaaaa agttgaactt aaaaataaca gattggccca tgccaatcaa 3360
agtataatag aaagcatagt atacgaattc 3390
Claims (5)
1. A high-sensitivity and high-specificity sdLDL-C colorimetric detection kit is characterized by comprising a reagent R1 and a reagent R2 which are independent from each other and are two liquid components;
the reagent R1 comprises the following components in percentage by weight: MOPS buffer 20~80 mM,PEG 6000 100~200 g/L, BSA 20-30 g/L, naCl 1.8-18 g/L, naN 3 0.2-0.8 g/L, 0.1-1 g/L EDTA, 500-900U/L catalase, 200-800U/L cholesterol esterase, 100-1000U/L cholesterol oxidase, 10-15 mM TOOS, 0.05-0.1% surfactant I, and purified water as solvent;
the reagent R2 comprises the following components in percentage by weight: MOPS buffer 20~80 mM,PEG 6000 100~200 g/L, BSA 20-30 g/L, naCl 1.8-18 g/L, naN 3 0.2-0.8 g/L, 0.1-1 g/L EDTA, 0.5-2 mM 4-AAP, 10-20U/L peroxidase, 5-15 mg/L mPEG-PlD, 0.05-0.8% surfactant II, and purified water as solvent;
wherein the surfactant I in the reagent R1 is one or more of NIKKOL BT-9 and NIKOL BT-12 products of sunlight company; the surfactant II in the reagent R2 is composed of one or more of Brij-35 and DTAC (digital television AC) which are products of Xibao corporation;
meanwhile, the mPEG-PlD acquisition method comprises the following steps:
(1) preparation of recombinant phospholipase D:
a. obtaining all CDS of phospholipase D in GENBANK, and adding BamHI and EcoRI cleavage sites at the head and tail respectively to obtain a target sequence, wherein the target sequence is shown in SEQ ID NO. 1;
b. after obtaining the target sequence, sending the target sequence to a gene company for synthesis;
c. double enzyme cutting, connecting to an expression vector pET32a, and introducing into an expression bacterium Ecoli;
d. coating the bacterial strain on an ampicillin flat plate, and picking positive bacteria for fermentation expression;
e. centrifuging to obtain supernatant, and subjecting to nickel column affinity chromatography to obtain target product;
(2) mPEG conjugation of recombinant phospholipase D:
a. cyanuric chloride 5.5g, anhydrous Na 2 CO 3 10g, 5g of molecular sieve 5A and 50g of mPEG-5000 are dissolved in anhydrous benzene of 400mL, and stirred at normal temperature for reaction 12h;
b. centrifuging to remove Na 2 CO 3 Molecular sieve 5A suspension, precipitated with 600mL diethyl ether, and then dissolved with 400mL anhydrous benzene; repeating the precipitation and dissolution for 5 times, and removing unreacted cyanuric chloride until no light absorption is detected at 258nm wavelength; finally, vacuum drying is carried out to obtain activated CC-mPEG;
c. and (2) taking the CC-mPEG and the recombinant phospholipase D prepared in the step (1), dissolving the CC-mPEG and the recombinant phospholipase D in a 10mL borate buffer solution, reacting for 3-6 hours at room temperature on a shaking table, and dialyzing overnight at 15kD to obtain a modified enzyme solution, namely mPEG-PlD.
2. The high sensitivity and specificity sdLDL-C colorimetric assay kit of claim 1, further comprising a sdLDL calibrator; the sdLDL calibrator comprises the following components in percentage by weight: MOPS buffer 20~80 mM,BSA 20~30 g/L, sdLDL-C5-15 mg/L, naN 3 0.2-0.8 g/L, wherein the solvent is purified water.
3. The high-sensitivity and specificity sdLDL-C colorimetric assay kit according to claim 2, wherein the sdLDL-C is obtained by the following method: preparation of heparin-Mg 2+ Wherein, the heparin concentration is 150U/mL, mg 2+ Concentration 90mmol/L; the heparin-Mg is added in an amount of 10% 2+ Adding 50mL human serum, mixing, standing at room temperature for 15min, and centrifuging at 1500g for 30min; taking the supernatant 40mL, and adding 4mL heparin-Mg 2+ Mixing, standing at room temperature for 15min; finally, the mixture was centrifuged at 1500g for 30min, and the supernatant was the sdLDL-C product.
4. A method for preparing the sdLDL-C colorimetric assay kit of high sensitivity and specificity according to any one of claims 1 to 3, comprising the steps of:
(1) Preparation of human sdLDL-C
Preparation of heparin-Mg 2+ Wherein, the heparin concentration is 150U/mL, mg 2+ Concentration 90mmol/L; the heparin-Mg is added in an amount of 10% 2+ Adding 50mL human serum, mixing, standing at room temperature for 15min,centrifuging at 1500g for 30min; taking the supernatant 40mL, and adding 4mL heparin-Mg 2+ Mixing, standing at room temperature for 15min; finally, centrifuging for 30min under 1500g, wherein the supernatant is the sdLDL-C product;
(2) Preparation of mPEG recombinant phospholipase D
(1) Preparation of recombinant phospholipase D:
a. obtaining all CDS of phospholipase D in GENBANK, and adding BamHI and EcoRI cleavage sites at the head and tail respectively to obtain a target sequence, wherein the target sequence is shown in SEQ ID NO. 1;
b. after obtaining the target sequence, sending the target sequence to a gene company for synthesis;
c. double enzyme cutting, connecting to an expression vector pET32a, and introducing into an expression bacterium Ecoli;
d. coating the bacterial strain on an ampicillin flat plate, and picking positive bacteria for fermentation expression;
e. centrifuging to obtain supernatant, and subjecting to nickel column affinity chromatography to obtain target product;
(2) mPEG conjugation of recombinant phospholipase D:
a. cyanuric chloride 5.5g, anhydrous Na 2 CO 3 10g, 5g of molecular sieve 5A and 50g of mPEG-5000 are dissolved in anhydrous benzene of 400mL, and stirred at normal temperature for reaction 12h;
b. centrifuging to remove Na 2 CO 3 Molecular sieve 5A suspension, precipitated with 600mL diethyl ether, and then dissolved with 400mL anhydrous benzene; repeating the precipitation and dissolution for 5 times, and removing unreacted cyanuric chloride until no light absorption is detected at 258nm wavelength; finally, vacuum drying is carried out to obtain activated CC-mPEG;
c. taking the CC-mPEG and the recombinant phospholipase D prepared in the step (1), dissolving the CC-mPEG and the recombinant phospholipase D in a 10mL borate buffer solution, reacting for 3-6 hours at room temperature on a shaking table, and dialyzing for overnight at 15kD to obtain a modified enzyme solution, namely mPEG-PlD;
(3) Preparation of sdLDL-C colorimetric detection kit
(1) Preparing a reagent R1:
according to the component content of the reagent R1, mixing the component substances in the same container, and uniformly mixing to obtain the reagent R1;
(2) preparing a reagent R2:
according to the component content of the reagent R2, mixing the mPEG-PlD prepared in the step (2) and the rest other component substances in the same container, and uniformly mixing to prepare the reagent R2;
(3) preparing an sdLDL calibrator:
the sdLDL calibrator comprises the following components in percentage by weight: MOPS buffer 20~80 mM,BSA 20~30 g/L, sdLDL-C5-15 mg/L, naN 3 0.2-0.8 g/L, wherein the solvent is purified water;
and (3) mixing the sdLDL-C product prepared in the step (1) and the rest other components in the same container according to the component content of the sdLDL calibrator, and uniformly mixing to prepare the sdLDL calibrator.
5. A method for using the sdLDL-C colorimetric assay kit of high sensitivity and specificity according to any one of claims 1 to 3, comprising the following specific steps:
(1) Sucking 2.4 mu L of sample, adding 180 mu L of reagent R1, and incubating for 5min at 37 ℃;
(2) Then 60 mu L of reagent R2 is added for mixing and fully reacting;
(3) Reading a light absorption value A after 5min;
(4) The calibration method is 2-point calibration, the full-automatic biochemical analyzer is adopted for detection, and the concentrations of the calibrator are respectively set as follows: 0. 45mg/dL; and measuring and calculating the sdLDL-C content in the sample according to the scaling value and the A.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010532055.2A CN111551512B (en) | 2020-06-11 | 2020-06-11 | High-sensitivity and high-specificity sdLDL-C colorimetric detection kit and preparation and use methods thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010532055.2A CN111551512B (en) | 2020-06-11 | 2020-06-11 | High-sensitivity and high-specificity sdLDL-C colorimetric detection kit and preparation and use methods thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111551512A CN111551512A (en) | 2020-08-18 |
| CN111551512B true CN111551512B (en) | 2023-07-25 |
Family
ID=72002274
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010532055.2A Active CN111551512B (en) | 2020-06-11 | 2020-06-11 | High-sensitivity and high-specificity sdLDL-C colorimetric detection kit and preparation and use methods thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111551512B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116990512B (en) * | 2023-09-25 | 2023-12-08 | 聚诚(北京)生物科技有限责任公司 | Matrix metalloproteinase-9 detection kit |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101663404A (en) * | 2007-02-28 | 2010-03-03 | 电化生研株式会社 | Reagent for determination of quantity of small dense low-density lipoprotein |
| CN101896620A (en) * | 2007-10-10 | 2010-11-24 | 电化生研株式会社 | Method and kit for quantification of small, dense LDL cholesterol |
| CN103180457A (en) * | 2010-10-29 | 2013-06-26 | 爱科来株式会社 | Method and kit for measuring cholesterol in low density lipoproteins |
| CN104673879A (en) * | 2015-03-07 | 2015-06-03 | 王贤俊 | Small and dense low-density lipoprotein cholesterin detection kit and preparation thereof |
| CN108410950A (en) * | 2018-03-21 | 2018-08-17 | 天津中成佳益生物科技有限公司 | A kind of efficient, special sdLDL-C detection kit |
| CN109609595A (en) * | 2019-01-24 | 2019-04-12 | 浙江夸克生物科技有限公司 | A kind of sdLDL-C assay kit and its application |
| CN109837325A (en) * | 2019-03-15 | 2019-06-04 | 安徽大千生物工程有限公司 | A kind of HDL3 colorimetric determination kit and its preparation application method based on modification sphingomyelinase optimization |
| CN111032880A (en) * | 2017-09-01 | 2020-04-17 | 日立化成诊断系统株式会社 | Method, reagent and kit for measuring cholesterol in low-density lipoprotein |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1930442B1 (en) * | 2005-08-31 | 2010-11-03 | DENKA SEIKEN Co., Ltd. | Method and kit for quantitative determination of small, dense ldl cholesterol |
-
2020
- 2020-06-11 CN CN202010532055.2A patent/CN111551512B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101663404A (en) * | 2007-02-28 | 2010-03-03 | 电化生研株式会社 | Reagent for determination of quantity of small dense low-density lipoprotein |
| CN101896620A (en) * | 2007-10-10 | 2010-11-24 | 电化生研株式会社 | Method and kit for quantification of small, dense LDL cholesterol |
| CN103952462A (en) * | 2007-10-10 | 2014-07-30 | 电化生研株式会社 | Method and kit for quantification of small, dense LDL cholesterol |
| CN103180457A (en) * | 2010-10-29 | 2013-06-26 | 爱科来株式会社 | Method and kit for measuring cholesterol in low density lipoproteins |
| CN104673879A (en) * | 2015-03-07 | 2015-06-03 | 王贤俊 | Small and dense low-density lipoprotein cholesterin detection kit and preparation thereof |
| CN111032880A (en) * | 2017-09-01 | 2020-04-17 | 日立化成诊断系统株式会社 | Method, reagent and kit for measuring cholesterol in low-density lipoprotein |
| CN108410950A (en) * | 2018-03-21 | 2018-08-17 | 天津中成佳益生物科技有限公司 | A kind of efficient, special sdLDL-C detection kit |
| CN109609595A (en) * | 2019-01-24 | 2019-04-12 | 浙江夸克生物科技有限公司 | A kind of sdLDL-C assay kit and its application |
| CN109837325A (en) * | 2019-03-15 | 2019-06-04 | 安徽大千生物工程有限公司 | A kind of HDL3 colorimetric determination kit and its preparation application method based on modification sphingomyelinase optimization |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111551512A (en) | 2020-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lyons et al. | Glycosylation of low density lipoprotein in patients with type I (insulin-dependent) diabetes: correlations with other parameters of glycaemic control | |
| EP2208794B1 (en) | Method and kit for quantification of small, dense ldl cholesterol | |
| EP1930442B1 (en) | Method and kit for quantitative determination of small, dense ldl cholesterol | |
| US20060030050A1 (en) | Assay | |
| CN111579791A (en) | Electrochemical luminescence detection kit for zinc transporter 8 islet autoantibodies | |
| CN1505759A (en) | Detection of candida | |
| EP0270039A2 (en) | Reagents for determining peptidoglycan and beta-1,3-glucan | |
| CN111551512B (en) | High-sensitivity and high-specificity sdLDL-C colorimetric detection kit and preparation and use methods thereof | |
| WO1997000971A1 (en) | Method for assaying cholesterol in high-density lipoprotein fraction and assay reagent kit | |
| CN110108889B (en) | Kit for diagnosing IgA nephropathy and application thereof | |
| AU2017277626A1 (en) | A chemical composition to stabilize extracellular vesicles in a blood sample and method of use thereof | |
| CN109837325B (en) | HDL3 colorimetric method detection kit based on modified sphingomyelinase optimization and preparation and use methods thereof | |
| Okada et al. | Hypersialyloligosacchariduria in mucolipidoses: a method for diagnosis | |
| WO2024051646A1 (en) | Method and kit for diagnosing midd | |
| Turnbull et al. | The prevalence of hereditary haemochromatosis in a diabetic population. | |
| JPH08116996A (en) | Measurement of hdl-cholesterol in serum or plasma | |
| Svens et al. | Immunocatalytic assay of pancreatic alpha-amylase in serum and urine with a specific monoclonal antibody. | |
| US6777197B1 (en) | Method and test kit for measuring immunoglobulins reactive with amylase as indication of crohn's disease | |
| JP4847668B2 (en) | Method for detecting glycated albumin | |
| AU2004212104B2 (en) | Preparation of red blood cells with a modified level of blood group antigen expression and their use in the quality control of blood typing reagents | |
| CN109298190A (en) | A kind of high-density lipoprotein cholesterol detection kit | |
| AU604211B2 (en) | Method of determining cystic fibrosis ciliostatic factor | |
| CN109295174B (en) | Reagent group, kit and detection method for detecting fungal infection | |
| CN110777137B (en) | Buffer composition for purifying tissue factor, purification preparation, PT detection composition and PT detection kit | |
| CN112176026A (en) | Detection kit for alpha-galactosidase activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |