CN114214389B - Lipoprotein cholesterol detection method and kit - Google Patents
Lipoprotein cholesterol detection method and kit Download PDFInfo
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- CN114214389B CN114214389B CN202210157166.9A CN202210157166A CN114214389B CN 114214389 B CN114214389 B CN 114214389B CN 202210157166 A CN202210157166 A CN 202210157166A CN 114214389 B CN114214389 B CN 114214389B
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/32—3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate, i.e. MBTH
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
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- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
Abstract
The invention discloses a kit and a detection method for fractional determination of lipoprotein cholesterol, which are suitable for an automatic analyzer, and a reagent for the determination.
Description
Technical Field
The present invention relates to a method and a reagent for measuring cholesterol in lipoproteins, and particularly relates to a method and a reagent for detecting cholesterol in lipoproteins involved in the diagnosis of arteriosclerosis.
Background
Cholesterol is an important component of cells, and also a clinically important component, because too high levels of cholesterol lead to the conversion of macrophages to foam cells (foam cells) after their uptake in the subendothelial space and the subsequent development of primary lesions of arteriosclerosis. Low Density Lipoprotein (LDL) plays a major role in cholesterol transport in the blood and is a risk factor for arteriosclerosis. Small, dense LDLs, which are particularly small in particle size and higher in density in LDL compared to standard LDL, are known to have atherogenic capacity at several fold higher levels than normal LDL. An increase in small, dense LDL is one of the major risk factors for arteriosclerosis. Therefore, it is clinically important to perform fractional measurement (fractional measurement) of such small and dense LDLs.
Examples of conventional methods for measuring small, dense LDLs include ultracentrifugation, electrophoresis, and a method using high performance liquid chromatography. These methods are inconvenient because they require expensive equipment and a large amount of measurement time.
An example of a method for measuring small, dense LDLs using an autoanalyzer is a method involving suspending or dissolving small-particle LDLs using differences in ionic strength and then measuring the small-particle LDLs using differences in absorbance (see Japanese patent publication No. 2003-28882A). However, the difference in absorbance was determined based on turbidity according to the above method. Therefore, cholesterol in small, dense LDLs cannot be measured, and thus specificity and accuracy are insufficient.
Further, a method is known (see international patent publication WO2004/053500) which involves measuring cholesterol or triacylglycerol in small, dense LDLs by using a separating agent comprising a polyanion and a divalent cation in combination with a reagent suitable for an autoanalyzer. This method enables more convenient measurement of the lipid component in small, dense LDLs than ultracentrifugation or electrophoresis. However, this method requires pretreatment of the sample and a step of separating LDL into small, dense LDLs and LDLs other than such LDLs.
A method involving removing cholesterol from lipoproteins other than small, dense LDLs by a specific surfactant and then determining the amount of cholesterol in the remaining lipoproteins is disclosed in the prior art (see international patent publication WO2007026829a 1). The method can be used for carrying out rapid and convenient analysis without pretreating a sample. However, experiments show that the method has large deviation of low end, poor linearity and large deviation of indoor evaluation of the measured reagent, and cannot meet the requirement of accurate detection.
Disclosure of Invention
The invention relates to a method for fractional determination of lipoprotein cholesterol suitable for an automatic analyzer and reagents for such determination, which can be carried out without pretreatment of a sample, and simultaneously ensure that the determination reagents can meet the linear requirements of the reagents without influencing the clinical detection results, and have high accuracy and good stability.
The invention is realized by the following technical scheme:
the present invention provides a composition characterized in that it comprises:
3-methylbenzothiazolidone hydrazone or a salt thereof; and
a peroxidase.
According to the invention, the composition further comprises a preservative. Preferably, the preservative is one or more of sodium azide, florfenicol or PC-300. More preferably, the preservative is PC-300 and florfenicol.
The present invention also provides a composition characterized in that it comprises:
3-methylbenzothiazolidone hydrazone or a salt thereof; and
and (4) a preservative.
According to the invention, the preservative is one or more of sodium azide, florfenicol or PC-300. More preferably, the preservative is PC-300 and florfenicol.
According to the invention, the above composition is in an acidic environment.
According to the invention, the pH is preferably between 0.5 and 4.5, more preferably between 1 and 4.05.
According to the present invention, more preferably, the above composition further comprises one or more selected from the group consisting of citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and a buffer system thereof.
According to the invention, the above composition further comprises a surfactant. Wherein the surfactant is selected from polyoxyethylene derivatives, preferably polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene alkyl ether (EMULGEN 707, EMULGEN 709), polyoxyethylene alkylphenyl ether, more preferably polyethylene glycol octylphenyl ether (Triton X-100), polyoxyethylene nonylphenyl ether (EMULGEN 909).
According to the invention, the concentration of the 3-methylbenzothiazolidone hydrazone or the salt thereof is 0.1-1.0 g/L. More preferably, the concentration of the 3-methylbenzothiazolone hydrazone or the salt thereof is 0.1 to 0.4 g/L. The concentration of the peroxidase is 2000-4000U/L. The concentration of the surfactant is 20-35 g/L.
The invention also provides a kit, characterized in that it comprises at least a reagent composition (ii) comprising the above-mentioned composition.
According to the present invention, the kit further comprises a reagent composition (i) for effectively shielding interference other than the lipoprotein to be tested. Preferably, the reagent composition (i) comprises a phospholipase, preferably at least one of phospholipase a2, phospholipase C, phospholipase D, lysophospholipase (LYPL) and sphingomyelinase; preferably, the concentration of phospholipase is in the range of 0.1U/mL to 100U/mL.
The effective shielding is defined as acting on the interfering component by physical or chemical means, so that the interfering component does not substantially affect the subsequent detection steps, and the effective shielding does not require that the interfering component be completely removed or excluded from the system.
According to the invention, the reagent composition (i) also comprises a non-ionic surfactant, preferably a non-ionic surfactant of HLB 13-15, more preferably a polyoxyethylene derivative or polyoxyethylene stilbene phenyl ether, for example EMULGEN B-66, EMULGEN a-90; preferably, the concentration of the nonionic surfactant is 1.5 to 2.0 g/L.
According to the invention, said reagent composition (i) further comprises cholesterol esterase, cholesterol oxidase and a hydrogen donor.
According to the invention, the reagent composition (i) also comprises an inorganic salt ion selected from: one or more of magnesium chloride, EDTA-2 Na, EDTA-2K, sodium glutamate, sodium chloride or potassium chloride.
According to the invention, the reagent composition (i) also comprises a stabilizer selected from: one or more of BSA, sucrose or trehalose.
According to the invention, the hydrogen donor is an aniline derivative, preferably TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline).
The invention also provides a preparation method of the composition and/or the kit.
The invention also provides application of the composition and/or the kit.
According to the invention, the composition and/or the kit are used for preparing a product for detecting lipoprotein cholesterol.
Wherein the product comprises a reagent or kit. The assay for lipoprotein cholesterol comprises assays for low density lipoprotein cholesterol, small and dense low density lipoprotein cholesterol, high density lipoprotein cholesterol, remnant-like lipoprotein cholesterol, or total lipoprotein cholesterol.
According to the invention, the composition and/or the kit are also used for preparing products for diagnosing cardiovascular related diseases.
Wherein the product comprises a reagent or a kit. The diagnosis of cardiovascular related diseases, such as dyslipidemia, especially hyperlipidemia, hypercholesterolemia, atherosclerosis and the like, comprises the detection of low density lipoprotein cholesterol, small and dense low density lipoprotein cholesterol, high density lipoprotein cholesterol, remnant-like lipoprotein cholesterol or total lipoprotein cholesterol.
The invention also provides a method for improving the linearity or accuracy of lipoprotein cholesterol detection, which is characterized by using the composition and/or the kit.
The invention also provides a method for improving the stability of the 3-methylbenzothiazolidone hydrazone or the salt thereof, which is characterized by containing a preservative. Wherein the preservative is one or more of sodium azide, florfenicol or PC-300. More preferably, the preservative is PC-300 and florfenicol.
According to the present invention, the 3-methylbenzothiazolidone hydrazone or a salt thereof is preferably in an acidic environment. Preferably, the pH is 1.00-4.05. More preferably, one or more acids or buffer systems selected from citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and buffer systems thereof are included.
The present invention also provides a method for quantitatively determining lipoprotein cholesterol in a sample, which is characterized by using the above-mentioned composition and/or reagent composition (ii) in the kit.
According to the invention, the method comprises the following steps: the reagent composition (ii) added into the composition and/or the kit is used for detecting the target lipoprotein cholesterol. Further, the method comprises the steps of:
(1) interference components except the lipoprotein to be detected are effectively shielded;
(2) the reagent composition (ii) added into the composition and/or the kit is used for detecting the target lipoprotein cholesterol.
According to the present invention, it further comprises adding cholesterol esterase, cholesterol oxidase and chromogen (hydrogen donor) in step (1) or (2).
According to the invention, the reagent composition (i) comprising the above-mentioned kit is added in step (1).
According to the present invention, the kit is used for the detection of low density lipoprotein cholesterol, small and dense low density lipoprotein cholesterol, high density lipoprotein cholesterol, remnant-like lipoprotein cholesterol or total lipoprotein cholesterol.
According to the invention, the step (1) can be further subdivided into a plurality of steps to effectively shield different interference components.
Effects of the invention
The invention provides a method for measuring lipoprotein cholesterol suitable for an automatic analyzer and a reagent for measuring the lipoprotein cholesterol, wherein the method and the reagent can be carried out without pretreating a sample, have good specificity, and are rapid and convenient to analyze, and simultaneously ensure that the measuring reagent can meet the linear requirement of the reagent under the condition of not influencing the clinical detection result, and have the characteristics of high accuracy and good stability. In addition, the invention also provides a method for improving the stability of the 3-methylbenzothiazolone hydrazone or the salt thereof, and a related composition and a related kit.
Drawings
FIG. 1 is a graph of the change in 3-methylbenzothiazolidone hydrazone reactivity after acceleration with different buffer types.
FIG. 2 is a random sample correlation analysis of the reagents of example 10 with a commercially available reagent (nine strong 20-0630).
FIG. 3 is a random sample correlation analysis of the reagent of example 11 with a commercially available reagent (nine strong 20-0630).
FIG. 4 is a random sample correlation analysis of the reagent of example 12 with a commercially available reagent (nine strength 20-0630).
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
EXAMPLE 1 preparation of the kit
Taking a small and dense low-density lipoprotein cholesterol detection kit in the prior art as an example, the kit is divided into two reagent compositions of R1 and R2, and the specific formula is as follows:
example 2 measurement method
Mixing the serum sample to be detected with the reagent R1, keeping the temperature at 37 ℃ for 5min, and measuring the absorbance A1Adding the reagent R2, mixing, keeping the temperature at 37 ℃ for 5min, and measuring the absorbance A2Calculate Δ a = a2-A1
(2) Measuring absorbance difference (main wavelength 546nm, sub-wavelength 660nm, instrument reading point 16-34) after reaction with a full-automatic biochemical analyzer (Hitachi 7180);
(3) the concentration of sdLDL-C in the sample was calculated from the absorbance change values.
Preferably, in the serum sample obtained in the step (1), the volume ratio of the reagent R1 to the reagent R2 is 3:150: 50.
(4) Linear experimental method:
mixing high concentration sample near the upper limit of linear range and low concentration sample near the lower limit of linear range to 7 dilution concentrations (dilution times are shown in the table), averaging 3 times for each concentration test, and taking the average value to determine concentration value (i.e. average value y)i) Regression with the corresponding theoretical concentration values or dilution factor (xi)Analyzing, obtaining a linear regression equation (y = Ax + B), wherein a is a slope and B is an intercept, and calculating a linear regression correlation coefficient r, wherein the result is required to be satisfied.
Substituting the independent variables (xi, except the 0 value) into the linear regression equation to calculate the dependent variable (y)i) And y' and yiAbsolute and relative deviations from y' should be such that the results are satisfactory.
(1) Linear experimental requirements:
in the interval of [40, 900] mg/L, the correlation coefficient r should be more than or equal to 0.990.
Within the range of [40, 200) mg/L, the absolute deviation of linearity should not exceed plus or minus 20 mg/L;
within the interval of [200, 900] mg/L, the relative deviation of linearity should not exceed +/-10%.
Sample source: cooperative hospital (Chengdu Chinese medicine university subsidiary hospital)
(2) Terms, definitions, acronyms:
reagent blank (R): the reagent is added into purified water or specified blank liquid to be used as a sample test, and the reagent is an effective index for judging the quality of the reagent.
Calibration reactivity (S): the absorbance change (delta A) of the calibration product identification value measured in the reaction system is an important index for establishing the conversion relation between the concentration and the absorbance.
Analysis sensitivity: the slope of the calibration curve.
Linearity-the ability of a measurement to be directly proportional to the value measured in the sample.
Linear range: the test system may receive a range of linearity over which the non-linearity error is less than the specified error.
Precision: the consistency between the results of multiple consecutive measurements of the same object under defined conditions.
Interference refers to clinically significant deviations in analyte concentration in clinical chemistry due to the influence of sample properties or other components.
Interference standard: the maximum allowable interference deviation between the concentration of the analyte and the true value may be generated, and this deviation may affect the medical decision of the doctor.
Anti-interference: the interference of interfering substances is determined.
Experiments show that the correlation coefficient r of the determination kit is in accordance with the specification, but is not in accordance with the range of 40-200mg/L, and the absolute deviation of linearity is not more than +/-20 mg/L; the linear relative deviation should not exceed + -10% in the range of 200-900 mg/L. The low-end deviation is large, the linearity is poor, the indoor evaluation deviation is large, and the requirement of accurate detection cannot be met.
Example 3 concentration screening of color Source substance
1. The experimental method comprises the following steps:
according to the components and contents of the kit in table 1, reagents containing different color source substances were prepared, wherein the color source substances used were toss and the concentrations were respectively: 0.4mM, 0.8mM, 1.6mM, 2.0mM, 3.2 mM. The linearity verification was performed with reagents of different TOOS concentrations, the linearity practice and requirements being as above.
2. The experimental results are as follows:
experiments show that the correlation coefficient r of the determination kit accords with the regulation, but in the table 2, the reagent low end deviation of the kit in the table 1 is large, and the linearity is poor, the concentration of the color source substance is adjusted to be 0.4mM-3.2mM, the concentration of the color source substance does not accord with the range of 40-200mg/L, and the absolute deviation of the linearity should not exceed +/-20 mg/L; the linear relative deviation should not exceed + -10% in the concentration range of 0.4mM-3.2mM, which does not conform to the range of 200-900 mg/L. It follows that the linearity of the reagent kit can not be optimized by adjusting the concentration of the color source substance.
3. And (4) conclusion:
the reagent linearity of the kit is poor and does not meet the linear standard of the reagent, and the linearity of the reagent of the kit cannot be optimized by adjusting the concentration of the color source substance.
Example 4 surfactant concentration screening
1. Purpose of the experiment: optimizing reagent linearity
2. The experimental method comprises the following steps:
according to the kit components and contents in table 1, reagents containing different surfactants were prepared, wherein the surfactant used was polyoxyethylene distyrenated phenyl ether (a 90) with the concentrations respectively: 0.35g/L, 0.7g/L, 1.4g/L, 1.75g/L, 2.1g/L, 2.8 g/L. The linear assay was performed with reagents of different concentrations of a90, and the method of linear assay was the same as above.
3. Results of the experiment
The correlation coefficient r of the assay kit is found to be in accordance with the specification through experiments, but as can be seen from table 3, the reagent linearity can not be satisfied by adjusting the surface activity concentration.
4. And (4) conclusion:
the reagent linearity of the kit is poor and does not meet the linear standard of the reagent, and the linearity of the reagent of the kit cannot be optimized by adjusting the concentration of the surfactant.
EXAMPLE 5 screening of substrate 4-aminoantipyrine concentration
Preparing reagents containing different 4-aminoantipyrine concentrations, wherein the 4-aminoantipyrine concentrations are as follows: 2mM, 4mM, 8mM, 12mM, and carrying out linear verification by using reagents with different concentrations of 4-aminoantipyrine, wherein the linear verification method is the same as the above.
The experimental results are as follows:
the reagent has no reactivity at the concentration of 2mM of 4-aminoantipyrine. The results of the validation of the reagents for other concentrations of 4-aminoantipyrine are as follows:
experiments show that the correlation coefficient r of the determination kit meets the regulation, but as can be seen from table 4, the reagent can not meet the linear requirement even after the concentration of the 4-aminoantipyrine is increased.
4. And (4) conclusion:
the reagent linearity of the kit is poor and does not meet the linear standard of the reagent, and the linearity of the reagent of the kit can not be optimized by adjusting the concentration of the 4-aminoantipyrine.
Example 6 Triton-100 concentration Screen
Preparing reagents with different concentrations of triton-100, wherein the concentrations of triton-100 are as follows: 5g/L, 15g/L, 25g/L, 35g/L and 50 g/L. The reagents with different concentrations of triton-100 were used for linear validation, the linear validation method was the same as above.
The correlation coefficient r of the assay kit was found to be satisfactory, but as can be seen from Table 5, adjusting the amount of Triton-100 did not result in a reagent that is linear
4. And (4) conclusion:
the reagent kit in the prior art has poor reagent linearity, does not meet the linear standard of the reagent, and can not optimize the reagent linearity of the reagent kit by adjusting the concentration of triton-100.
EXAMPLE 7 replacement of the 4-aminoantipyrine reagent of example 1 with 3-methylbenzothiazolidone hydrazone
The reagent containing 3-methylbenzothiazolidone hydrazone and no 4-aminoantipyrine was prepared in R2, the 3-methylbenzothiazolidone hydrazone concentration was 0.4mol/L, and the remaining components were the same as in example 1.
Description of the experiment:
the acceleration calibration deviation calculation formula is as follows: (37 ℃ calibration reactivity-4 ℃ calibration reactivity)/4 ℃ calibration reactivity.
Results of the experiment
Experiments show that the correlation coefficient r of the determination kit meets the regulations, and as can be seen from table 6, the determination effect is good immediately after preparation, linearity meets the requirements, but reagent stability does not meet the requirements.
4. Conclusion
The inventor unexpectedly finds that the reagent linearity is optimized after 4-aminoantipyrine is replaced by a 3-methylbenzothiazolidone hydrazone substrate, and the requirement of the reagent linearity standard is met.
EXAMPLE 8 Agents 2 Agents different Agents
The preparation R2 contains 3-methylbenzothiazolidone hydrazone, original buffer type (PIPES) is used, and different preservative agents, wherein the concentration of the 3-methylbenzothiazolidone hydrazone is 0.4mol/L, and the agents are preservative-free, sodium azide is 1.0g/L, and florfenicol is 1.0g/L, PC-3001.0 g/L.
The correlation coefficient r of the assay kit was found to be satisfactory experimentally, as can be seen in table 7: the addition of different preservatives had different effects on R2 stability, with the least stability without preservative agents, followed by sodium azide and florfenicol. The inventor unexpectedly finds that PC-300 performs abnormally well in the verification of the stability of the reagent, which indicates that PC-300 not only has the function of a preservative, but also has the function of obviously improving the stability of the R2 reagent.
Example 9 reagent 2 reagents of different buffer types
R2 was prepared with 3-methylbenzothiazolidone hydrazone, PC-300, but with different buffer types, where the 3-methylbenzothiazolidone hydrazone concentration was 0.4mol/L and different types of buffers were: citric acid buffer, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, sulfuric acid and acetic acid.
The correlation coefficient r of the assay reagent was found to be satisfactory experimentally, as can be seen from table 8 and fig. 1: in the range of pH value 1.00-4.05, citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and buffer systems thereof and the like generate synergistic effect with the preservative PC-300, the stability of the reagent is obviously improved, and unexpected technical effect is obtained.
To summarize:
the reagent adopting 4-aminoantipyrine in the prior art does not meet the linear requirement of the reagent, and even if the linearity can not be optimized by adjusting the concentration of a color source substance, the concentration of a surfactant, the concentration of 4-aminoantipyrine and the concentration of triton-100, the linearity can not meet the requirement of reagent accuracy. According to the invention, the linearity of the reagent is improved after 4-aminoantipyrine is replaced by 3-methylbenzothiazolidone hydrazone, and the requirement on the accuracy of the reagent is met, but the inventor finds that the stability of the system is not high. Experiments show that PC-300 is selected as the preservative, has the function of stabilizing a reagent system and meets the requirement of performance optimization. Furthermore, the pH value is adjusted to be in the range of 1.00-4.05, the pH value and the PC-300 can generate a synergistic effect, the stability of the system is greatly improved, and an unexpected technical effect is achieved.
Example 10 reagent linearity verification
Test reagents were prepared according to the components shown in Table 9, samples of gradient concentrations were prepared by the method of example 2, and the measurements were carried out in a fully automatic biochemical analyzer using the reagents shown in Table 9. The measurement results are shown in Table 10.
As can be seen from Table 10, the correlation coefficient r using the 3-methylbenzothiazolidone assay reagent of the present invention was in accordance with the specification, and the absolute deviation and the relative deviation in the low value region were very small, and the linearity requirements were completely satisfied.
Example 11 reagent linearity verification
Test reagents were prepared according to the components shown in Table 11, samples of gradient concentrations were prepared by the method of example 2, and the measurements were carried out in a fully automatic biochemical analyzer using the reagents shown in Table 11. The measurement results are shown in Table 12.
As can be seen from Table 12, the correlation coefficient r of the measurement reagent was satisfactory, and the absolute deviation and the relative deviation in the low value region were very small, and the linearity was completely satisfied.
Example 12 reagent linearity verification
Test reagents were prepared according to the set of Table 13, samples of graded concentrations were prepared by the method of example 2, and measured in a fully automated biochemical analyzer using the reagents of Table 11. The measurement results are shown in Table 14.
As can be seen from Table 14, the correlation coefficient r of the measurement reagent was satisfactory, and the absolute deviation and the relative deviation in the low value region were very small, and the linearity was completely satisfied.
Example 13 validation of correlation of reagents of the present application with commercially available products
Using the reagents of examples 10, 11 and 12 of the present invention and a commercially available reagent (Jiuqiang 20-0630), 100 samples were measured for each parameter using Hitachi 7180 full-automatic biochemical analyzer by the method of example 2, and the measured values were subjected to correlation analysis (in mg/L in abscissa and ordinate).
As shown in FIGS. 2 to 4, the reagents of the present invention showed higher degree of consistency than the commercial reagents.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (17)
1. A kit, comprising:
a reagent composition (i) comprising cholesterol esterase, cholesterol oxidase and a hydrogen donor;
a reagent composition (ii) comprising 3-methylbenzothiazolidone hydrazone or a salt thereof;
a peroxidase;
a surfactant;
and a preservative;
the reagent composition (ii) has a pH value of 1.00-4.05, and the preservative is one or more of florfenicol or PC-300.
2. The kit of claim 1, wherein the reagent composition (i) further comprises a phospholipase.
3. The kit of claim 2, wherein said phospholipase is selected from the group consisting of phospholipase A2, phospholipase C, phospholipase D, lysophospholipase (LYPL) and sphingomyelinase.
4. The kit of claim 2 or 3, wherein the concentration of the phospholipase is from 0.1U/mL to 100U/mL.
5. The kit according to claim 1 or 2, wherein the reagent composition (i) further comprises a non-ionic surfactant.
6. The kit according to claim 5, wherein the HLB of the nonionic surfactant in the reagent composition (i) is 13 to 15.
7. The kit according to claim 5 or 6, wherein the concentration of the nonionic surfactant in the reagent composition (i) is 1.5 to 2.0 g/L.
8. The kit according to claim 5 or 6, wherein the nonionic surfactant in the reagent composition (i) is selected from polyoxyethylene derivatives or polyoxyethylene diphenylethylene phenyl ethers.
9. The kit of claim 1 or 2, wherein the hydrogen donor is TOOS.
10. The kit according to claim 1 or 2, wherein the reagent composition (ii) comprises one or more selected from the group consisting of citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and buffer systems thereof.
11. The kit according to claim 1 or 2, wherein the surfactant in reagent composition (ii) is selected from polyoxyethylene derivatives.
12. The kit according to claim 11, wherein the polyoxyethylene derivative in the reagent composition (ii) is selected from one or more of polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene alkyl ether or polyoxyethylene alkylphenyl ether.
13. The kit according to claim 1 or 2, wherein the concentration of said surfactant in the reagent composition (ii) is 20-35 g/L.
14. The kit according to claim 1 or 2, wherein the concentration of the 3-methylbenzothiazolidone hydrazone or the salt thereof is 0.1 to 1.0 g/L.
15. The kit according to claim 1 or 2, wherein the concentration of the peroxidase is 2000-4000U/L.
16. Use of a kit according to any one of claims 1 to 15 in the manufacture of a product for the detection of lipoprotein cholesterol, which is small and dense low density lipoprotein cholesterol.
17. Use of a kit according to any one of claims 1 to 15 for the manufacture of a product for the diagnosis of cardiovascular-related diseases including dyslipidemia, hypercholesterolemia or atherosclerosis.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210157166.9A CN114214389B (en) | 2022-02-21 | 2022-02-21 | Lipoprotein cholesterol detection method and kit |
| PCT/CN2022/100428 WO2023155352A1 (en) | 2022-02-21 | 2022-06-22 | Test method and test kit for lipoprotein cholesterol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210157166.9A CN114214389B (en) | 2022-02-21 | 2022-02-21 | Lipoprotein cholesterol detection method and kit |
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| CN114214389A CN114214389A (en) | 2022-03-22 |
| CN114214389B true CN114214389B (en) | 2022-07-12 |
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| WO (1) | WO2023155352A1 (en) |
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| CN114214389B (en) * | 2022-02-21 | 2022-07-12 | 北京沃森赛瑟生物技术有限公司 | Lipoprotein cholesterol detection method and kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539731A (en) * | 2012-01-09 | 2012-07-04 | 宁波天康生物科技有限公司 | Reagent and kit for quantitatively determining low-density lipoprotein cholesterol (LDL-C) in serum |
| 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 |
| CN107446989A (en) * | 2017-06-23 | 2017-12-08 | 美康生物科技股份有限公司 | Determine the method and reagent of the sdLDL-C in sample |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2600526C3 (en) * | 1976-01-08 | 1981-06-19 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method for the quantitative determination of substances forming H ↓ 2 ↓ O ↓ 2 ↓ with oxygen under the action of oxidase and the reagent for its implementation |
| DE3124590A1 (en) * | 1981-06-23 | 1983-01-27 | Boehringer Mannheim Gmbh, 6800 Mannheim | STABILIZED REAGENT TO DETECT H (DOWN ARROW) 2 (DOWN ARROW) O (DOWN ARROW) 2 (DOWN ARROW) |
| US5132086A (en) * | 1990-02-06 | 1992-07-21 | Chemtrak Corporation | Non-instrumented cholesterol assay |
| AU2008220056B2 (en) * | 2007-02-28 | 2014-05-01 | Denka Company Limited | Reagent for quantitative determination of small, dense LDLs |
| JP5766426B2 (en) * | 2010-11-10 | 2015-08-19 | デンカ生研株式会社 | Method for quantifying remnant-like lipoprotein cholesterol and kit therefor |
| JP6582369B2 (en) * | 2014-08-04 | 2019-10-02 | 東洋紡株式会社 | Method and reagent for stabilizing cholesterol esterase |
| JP7134667B2 (en) * | 2018-03-28 | 2022-09-12 | デンカ株式会社 | Method for quantifying lipoprotein cholesterol, quantitative reagent, and quantitative kit |
| CN114214389B (en) * | 2022-02-21 | 2022-07-12 | 北京沃森赛瑟生物技术有限公司 | Lipoprotein cholesterol detection method and kit |
-
2022
- 2022-02-21 CN CN202210157166.9A patent/CN114214389B/en active Active
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102539731A (en) * | 2012-01-09 | 2012-07-04 | 宁波天康生物科技有限公司 | Reagent and kit for quantitatively determining low-density lipoprotein cholesterol (LDL-C) in serum |
| CN107446989A (en) * | 2017-06-23 | 2017-12-08 | 美康生物科技股份有限公司 | Determine the method and reagent of the sdLDL-C in sample |
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| Comparison of seven different reagents of peroxidase method for small and dense low density lipoprotein cholesterol (sdLDL-C) measurement;Fan Xuesong等;《J Clin Lab Anal》;20201229;第35卷(第3期);第699-702页 * |
| 低密度脂蛋白胆固醇(LDL-C)表面活性剂法检测试剂的研制;罗军等;《中国实验诊断性》;20080630;第12卷(第6期);e23660 * |
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| CN114214389A (en) | 2022-03-22 |
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