CN114891854A - Reagent and kit for measuring small and dense low-density lipoprotein cholesterol - Google Patents

Reagent and kit for measuring small and dense low-density lipoprotein cholesterol Download PDF

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CN114891854A
CN114891854A CN202210551217.6A CN202210551217A CN114891854A CN 114891854 A CN114891854 A CN 114891854A CN 202210551217 A CN202210551217 A CN 202210551217A CN 114891854 A CN114891854 A CN 114891854A
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lipoprotein
density lipoprotein
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王伊琳
王贤理
池万余
王友群
贾刚
邓慰
卢强
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Zhejiang Erkn Biological Technology Co ltd
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Abstract

The present invention relates to the field of biomedicine, and is especially one kind of reagent and its kit for measuring small dense low density lipoprotein cholesterol, i.e., the method of altering the solubility of non-small dense lipoprotein in the first reagent to make it easy to eliminate and quantifying small dense lipoprotein cholesterol (sdLDL-C) specifically in the second reagent. The reagent of the invention comprises a first reagent: 10 mmol/L-300 mmol/L chaotropic ionic compound, 0.1-1.0% polyethylene glycol, 1.0-90 mmol/L divalent metal ion, 1-10 KU/L cholesterol esterase, 1-10 KU/L cholesterol oxidase, 100-300 KU/L catalase, 0.2-10 mmol/L Trinder's chromogen compound, and 0.05-3% surfactant A; a second reagent: 0.1-10 mmol/L4-aminoantipyrine, 0.2-10 KU/L peroxidase, 0.01-0.3% sodium azide and 0.05-3% surfactant B, the reagent does not need to adopt phospholipase, the preparation is simple, and the reagent can be applied to specific detection of sdLDL. The determination method can be applied to a full-automatic biochemical analyzer, and can specifically and exclusively quantify the content of sdLDL in the sample.

Description

Reagent for measuring small and dense low-density lipoprotein cholesterol and kit thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a reagent for measuring small and dense low-density lipoprotein cholesterol and a kit thereof.
Background
Atherosclerosis is a disease caused by the combined action of various factors, and is often seen in middle-aged and elderly people. Atherosclerosis is also the basis of hypertension, coronary heart disease, acute myocardial infarction, cerebral apoplexy, etc. Hyperlipidemia is one of the important risk factors of atherosclerosis and coronary heart disease, and the compositional changes of Low Density Lipoprotein (LDL) are very important in analyzing the progression of coronary heart disease, especially a high proportion of small dense low density lipoprotein (sdLDL). sdLDL is more likely to cause atherosclerosis because it has a greater penetration into the intima of the artery, a lower affinity for LDL receptors, a longer plasma half-life and is susceptible to oxidation. Vascular endothelial cells are repeatedly damaged for a long time, so that lipid is continuously deposited and atherosclerosis is finally formed through a series of complex reactions, and sdLDL is closely related to the atherosclerosis.
Currently, methods for measuring sdLDL in clinical applications include electrophoresis, ultracentrifugation, and fractional precipitation. However, these methods have a long detection time, cannot perform large-scale automated detection, and require expensive equipment, and thus cannot be applied to general clinical medical detection. In recent years, the surfactant and the phospholipase are adopted to remove the non-small and dense lipoprotein cholesterol in the sample, but the method adopts the phospholipase, the price is high, and no enterprise for producing the enzyme exists in China.
Chinese patent application No. 201710485900.3 discloses a method and reagent for measuring small, dense and low-density lipoprotein cholesterol in a sample, which comprises eliminating cholesterol in lipoproteins other than sdLDL-C in the sample in the presence of cholesterol esterase and an ion selective agent; the sdLDL-C remaining after the treatment was quantitatively determined. The small and dense low-density lipoprotein cholesterol is detected by selectively eliminating lipoproteins other than the small and dense low-density lipoprotein cholesterol using an ion selective agent.
Disclosure of Invention
Aiming at the defects of the existing detection method for small and dense lipoproteins, the invention provides a small and dense low-density lipoprotein cholesterol determination reagent and a test method, namely, a surfactant A, a chaotropic ionic compound and polyethylene glycol (PEG) are adopted in a first reagent to remove non-small and dense lipoprotein cholesterol. The measurement method of the present invention can specifically and exclusively detect small, dense lipoproteins (sdLDL), is highly reliable, low in cost, and can specifically detect sdLDL in a sample on a large scale. The kit provided by the invention is easy to manufacture, simple and convenient to operate, suitable for a full-automatic biochemical analyzer and capable of accurately detecting sdLDL samples in batches.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a reagent for measuring cholesterol in small, dense and low-density lipoproteins, comprising adding a chaotropic ionic compound which disperses lipoproteins by charge action, a surfactant A and a cholesterol esterase to allow the surfactant A and the cholesterol esterase to selectively act on a specific lipoprotein, to a first reagent, thereby achieving measurement of the amount of cholesterol in the specific lipoprotein.
A kit for measuring cholesterol in small, dense low-density lipoproteins, comprising a first reagent and a second reagent, which kit achieves measurement of the amount of cholesterol in a specific lipoprotein by selectively acting on the specific lipoprotein; the ionic compound is made to disperse lipoprotein through charge so that the surfactant A and cholesterol esterase act selectively on specific lipoprotein.
The chaotropic ionic compound is selected from one or two of sodium bromide, sodium thiocyanate and potassium iodide, and the content of the chaotropic ionic compound is 10 mmol/L-300 mmol/L, preferably 10 mmol/L-200 mmol/L.
The content of Trinder's chromogen compounds in the first reagent is 0.2 to 10mmol/L, preferably 0.50 to 5.0mmol/L, and more preferably 2.0 to 3.0 mmol/L.
The content of polyethylene glycol in the first reagent of the present invention is 0.1 to 1.0%, preferably 0.1 to 0.6%, and more preferably 0.1 to 0.5%.
The divalent metal ion in the first reagent of the present invention is preferably magnesium ion or manganese ion, more preferably magnesium ion, and most preferably magnesium sulfate. The content of the divalent metal ion is 1 to 90mmol/L, preferably 2 to 30mmol/L, and more preferably 5 to 10 mmol/L.
In the first reagent of the present invention, the content of the surfactant a is 0.05 to 3.0%, preferably 0.05 to 2.0%, and more preferably 0.10 to 1.0%. Preferably, the surfactant A is a surfactant interacting with high density lipoprotein; preferably, the surfactant A is one or more of Sunnix FA-103, PTS, Nok, tocopherol polyethylene glycol succinate TPGS-750-M, TPGS-1000. Preferably Nok, tocopherol polyethylene glycol succinate TPGS-750-M, more preferably tocopherol polyethylene glycol succinate TPGS-750-M.
In the first reagent of the present invention, the content of the cholesterol esterase is 1 to 10KU/L, preferably 2 to 5 KU/L.
In the first reagent of the present invention, the content of the cholesterol oxidase is 1 to 10KU/L, preferably 2 to 5 KU/L.
In the first reagent of the present invention, the catalase content is 100 to 300KU/L, preferably 200 to 300 KU/L.
In the present invention, preferably, the first reagent further comprises a buffer. The buffer is preferably a MOPS buffer or a MOPSO buffer, more preferably a MOPSO buffer. The content of the buffer solution is preferably 25 to 120mmol/L, and more preferably 30 to 50 mmol/L.
In the present invention, preferably, the first reagent further comprises a stabilizer. The stabilizer is preferably selected from one or more of ascorbate oxidase, bovine serum albumin, sodium chloride or EDTA, more preferably one or more of bovine serum albumin, sodium chloride or EDTA, most preferably bovine serum albumin, sodium chloride and EDTA. The content of the stabilizer is preferably 1-10 KU/L of ascorbic acid oxidase; the content of the bovine serum albumin is preferably 0.1-1 g/L, more preferably 0.1-0.3 g/L; the content of the sodium chloride is preferably 5 to 150mmol/L, more preferably 5 to 100 mmol/L; preferably, the content of EDTA is 0.5 to 3 mmol/L.
In the present invention, preferably, the first agent further comprises a preservative. The preservative is preferably Proclin-300. The content of the preservative is preferably 0.01 to 0.5%.
The invention also provides a preparation method of the first reagent, which comprises the following steps: and sequentially adding the chaotropic ionic compound, polyethylene glycol, divalent metal ions, Trinder's chromogen compound and surfactant A into water, stirring until the chaotropic ionic compound, the polyethylene glycol, the divalent metal ions, the Trinder's chromogen compound and the surfactant A are completely dissolved, adjusting the pH value to 6.50-7.00, and then adding cholesterol esterase, cholesterol oxidase and catalase to obtain the first reagent.
In the present invention, preferably, the buffer, the stabilizer and the preservative are added before the pH is adjusted.
The first reagent of the present invention is in the form of a clear liquid.
In the second reagent of the present invention, the content of the peroxidase is 0.2 to 10KU/L, preferably 2.5 to 10KU/L, and more preferably 2.5 to 6 KU/L.
In the second reagent of the present invention, the content of the 4-aminoantipyrine is 1.0 to 10mmol/L, preferably 2 to 5mmol/L, and more preferably 2 to 3 mmol/L.
In the second reagent of the present invention, the content of the surfactant B is 0.05 to 3.0%, preferably 0.08 to 0.5%, and more preferably 0.1 to 0.2%. Preferably, the surfactant B is a surfactant acting on small, dense lipoproteins. Preferably, the surfactant B is one or two of Tergitol TMN-6 from the Dow company and Tergitol15-S-7 from the Dow company, and more preferably Tergitol TMN-6 from the Dow company.
In the present invention, preferably, the second reagent further comprises a buffer. The buffer is preferably a MOPS buffer or a MOPSO buffer, more preferably a MOPS buffer. The content of the buffer solution is preferably 30 to 75mmol/L, and more preferably 30 to 50 mmol/L.
In the second reagent of the present invention, the content of the sodium azide is 0.01 to 0.3%, preferably 0.1%.
In the present invention, preferably, the second reagent further comprises a stabilizer. The stabilizer is preferably bovine serum albumin. The content of the stabilizer is preferably 0.2 to 5 g/L.
The second reagent of the present invention is in the form of a clear liquid.
The invention also provides a preparation method of the second reagent, which comprises the following steps: adding 4-aminoantipyrine and a surfactant B into water, stirring until the mixture is completely dissolved, adjusting the pH value to 6.50-7.50, and then adding peroxidase to obtain a second reagent.
In the present invention, preferably, the buffer, the stabilizer, and the preservative are added before the pH is adjusted.
Further, a method for measuring a reagent for measuring small, dense and low-density lipoprotein cholesterol, comprising the steps of:
(1) mixing and reacting a sample with a first reagent to obtain a reaction solution 1;
(2) reading the absorbance values of the reaction liquid 1 obtained in the step (1) at the wavelengths of 546nm and 700 nm;
(3) mixing the reaction solution 1 obtained in the step (1) with a second reagent for reaction to obtain a reaction solution 2;
(4) reading the absorbance values of the reaction liquid 2 obtained in the step (3) at the wavelengths of 546nm and 700 nm;
(5) calculating the difference value of the absorbance obtained in the step (4) and the step (2);
(6) and comparing the absorbance value with that of a standard to determine the sdLDL content in the sample.
The apparatus used in the above determination method is a full-automatic biochemical analyzer, more preferably Hitachi 7180 full-automatic biochemical analyzer or Beckmann series full-automatic biochemical analyzer.
Wherein, the step (1) is as follows: mixing the sample with the first reagent for reaction to obtain a reaction solution 1. Wherein the mixing reaction time is the conventional condition in the field. The temperature of the mixing reaction is preferably 37 ℃. The volume ratio of the sample to the first reagent is preferably 1:75 to 1:120, more preferably 1: 110.
The step (3) is as follows: and (2) mixing the reaction solution 1 obtained in the step (1) with the second reagent for reaction to obtain a reaction solution 2. Wherein the mixing reaction time is the conventional condition in the field. The temperature of the mixing reaction is a temperature conventional in the art. The volume ratio of the reaction solution 1 to the second reagent is preferably 5:1 to 2:1, and more preferably 3: 1.
The step (4) is as follows: and (3) reading the absorbance values of the reaction liquid 2 obtained in the step (2) at the wavelengths of 546nm and 700nm, calculating the difference value of the absorbance obtained in the step (4) and the absorbance obtained in the step (2), and comparing and calculating the difference value with a standard liquid sample calibrated by the same parameters to obtain the content of the small and dense lipoprotein. Wherein the calculation method is a calculation method in an automatic biochemical analyzer, preferably a spline method.
Preferably, the assay is one for non-diagnostic or therapeutic purposes.
In the invention, the kit preferably further comprises a standard substance, and the standard substance of the kit can be a commercial standard substance of the kit which is conventionally used in the field, and is preferably Landau blood lipid standard solution, such as Landau Acusera blood lipid quality control substance.
The kit is simple and convenient to operate, and can efficiently and accurately detect small and dense lipoproteins specifically.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The invention has the following beneficial effects:
the chaotropic ionic compound, at a specific concentration, can alter the surface charge of lipoproteins, increase the water solubility of lipoproteins, and break down specific lipoproteins. The invention finds that non-sdLDL can be completely removed by reacting a serum or plasma sample with a first reagent which comprises a chaotropic ionic compound with a proper concentration and other components such as polyethylene glycol (PEG); and then reacts with a second reagent with the components and the contents thereof specially selected, thereby achieving the purpose of accurately measuring the content of sdLDL in the serum or plasma sample.
The measurement method of the present invention can specifically and exclusively detect small, dense lipoproteins (sdLDL), and the detection method has high reliability, low cost, high efficiency, and can automatically and specifically detect sdLDL on a large scale. The reagent for measuring small and dense lipoprotein is simple and convenient to prepare, and can be efficiently and accurately applied to the method for specifically measuring sdLDL. The kit provided by the invention is simple and convenient to operate, and can be used for efficiently and accurately measuring sdLDL specifically.
Drawings
FIG. 1 shows the results of the clinical specimen test according to example 1 of the present invention;
FIG. 2 shows the results of the clinical specimen test according to example 2 of the present invention;
FIG. 3 shows the results of the clinical specimen test according to example 3 of the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
The buffers described in the examples were purchased from baker's biotechnology limited, suzhou, Trinders chromogen from donnay chemical technology (shanghai), all enzymes from asahi chemicals, 4-aminoantipyrine from sigma aldrich china, and other reagents from shanghai chemical reagent stores.
The percentages mentioned in the present invention are mass percentages.
Example 1
A first reagent:
adding MOPSO buffer solution, magnesium sulfate, sodium bromide, polyethylene glycol, EDTA, BSA, TOOS and TPGS-750-M into water, stirring until completely dissolved, adjusting pH to 6.50-7.50, adding cholesterol esterase, cholesterol oxidase and catalase, and allowing the above substances to reach the following concentrations:
Figure BDA0003655088290000061
Figure BDA0003655088290000071
wherein, the molecular weight of the polyethylene glycol is 2000.
A second reagent:
adding MOPSO buffer solution, 4-aminoantipyrine, sodium azide and Tergitol TMN-6 into water, stirring until complete dissolution, adjusting pH to 6.50-7.50, adding peroxidase into water, mixing, and allowing the above substances to reach the following concentrations:
Figure BDA0003655088290000072
on a Hitachi 7180 full-automatic biochemical analyzer, 180 mu L of the first reagent reacts with 2.4 mu L of human serum sample for 5 minutes, then 60 mu L of the second reagent is added, and a two-point end point method is adopted at the main/auxiliary 546nm/700nm wavelength, and points are read for 16-34. The results of the measurement of 13 clinical specimens as shown in FIG. 1, compared with the commercial reagent using the phospholipase method, and the correlation coefficient measured in example 1 was 0.9782, which were calculated by comparison with the Landau Acusera lipid control calibration curve using the same measurement parameters, demonstrate that small, dense lipoproteins can be reliably detected using the first reagent and the second reagent described in example 1.
Example 2
A first reagent:
adding MOPS buffer solution, TOPS, polyethylene glycol, magnesium sulfate, sodium bromide, EDTA, Nok, TPGS-1000 and BSA into water, stirring until completely dissolved, adjusting pH to 6.50-7.50, adding cholesterol esterase, cholesterol oxidase and catalase, and allowing the above substances to reach the following concentrations:
Figure BDA0003655088290000081
a second reagent:
MOPS buffer, 4-aminoantipyrine, Tergitol15-S-7 and sodium azide are mixed in water, stirred until completely dissolved, the pH is adjusted to 6.50-7.50, and then peroxidase is added and the following concentrations of the above substances are achieved:
Figure BDA0003655088290000082
on a Hitachi 7180 full-automatic biochemical analyzer, 180 mu L of the first reagent reacts with 2.4 mu L of clinical human serum samples for 5min, then 60 mu L of the second reagent is added, and a two-point end point method is adopted at the main/auxiliary 546nm/700nm wavelength, and points are read for 16-34. The results of the 15 clinical samples measured in comparison to a standard calibration curve using the same measurement parameters are shown in FIG. 2, and compared to a commercially available phospholipase reagent, the correlation coefficient measured in example 2 is 0.9838. It was demonstrated that the first and second reagents described in example 2 can reliably detect small, dense lipoproteins
Example 3
A first reagent:
adding MOPS buffer, TOOS, polyethylene glycol, magnesium sulfate, sodium bromide, EDTA, Sunnix FA-103, TPGS-750-M and BSA into water, stirring until completely dissolved, adjusting pH to 6.50-7.50, then adding cholesterol esterase, cholesterol oxidase and catalase, and allowing the above substances to reach the following concentrations:
Figure BDA0003655088290000091
a second reagent:
MOPS buffer, 4-aminoantipyrine, Tergitol TMN-6 and sodium azide were added to water, stirred until completely dissolved, the pH was adjusted to 6.50-7.50, and then peroxidase was added and the following concentrations were achieved:
Figure BDA0003655088290000092
Figure BDA0003655088290000101
on a Hitachi 7180 full-automatic biochemical analyzer, 180 mu L of the first reagent reacts with 2.4 mu L of clinical human serum samples for 5min, then 60 mu L of the second reagent is added, and a two-point end point method is adopted at the main/auxiliary 546nm/700nm wavelength, and points are read for 16-34. The results of the measurement of 9 clinical specimens are shown in FIG. 3, compared with a commercial phospholipase reagent, and the correlation coefficient measured in example 3 is 0.9533, calculated by comparison with a standard calibration curve using the same measurement parameters. It was demonstrated that small, dense lipoproteins can be reliably detected by using the first reagent and the second reagent described in example 3.
Lipoproteins are mainly spherical particles formed by non-covalent bonding of lipids and proteins through hydrophobic interaction, van der waals force, electrostatic attraction and the like, and can roughly divide various components of lipoproteins existing in human blood into high-density lipoproteins, low-density lipoproteins, very-low-density lipoproteins and chylomicrons according to the density and size. The surface of lipoprotein is inlaid with a plurality of apolipoproteins, and different classes of lipoproteins are inlaid with different apolipoproteins, such as high density lipoprotein, wherein the apolipoproteins are mostly apolipoprotein A, and the low density lipoprotein contains apolipoprotein B. In the prior art, a method of combining a surfactant with other components is often adopted for measuring small and dense lipoproteins, so that other lipoproteins are eliminated or concealed so as not to participate in subsequent reactions, but the lipoproteins have the characteristic of agglomeration due to surface charges of the lipoproteins, and the measurement result is inaccurate. Therefore, the chaotropic ion compound is creatively introduced in the invention, the chaotropic ion has the function of strongly dispersing the lipoprotein, the chaotropic ion enables the surface of the protein to carry negative charges and generate stronger repulsive force through the function of changing the surface charge of the protein, so as to promote the protein to be dispersed, the collected lipoprotein is opened and dispersed into lipoprotein monomers, and the lipoprotein monomers are in a relatively free state in a sample, thereby providing a preorder advantage condition for the more effective function of a surfactant and tool enzyme in the subsequent process.
As a reagent for measuring small and dense lipoprotein, the purpose of quantifying the small and dense lipoprotein cholesterol is achieved by selecting the combination of specific cholesterol esterase, polyanionic compound and surfactant. The invention selects the specific surfactant as a medium, the tocopherol polyethylene glycol succinate is used as a novel surfactant, the biocompatibility is good, the environment is friendly, and the surfactant is harmless to people and livestock.
By adopting the mode of combining the surfactant with the chaotropic ions and utilizing the stronger function of dispersing the lipoproteins of the chaotropic ions, the method provides a preorder guarantee for the subsequent surfactant to further pointedly act on a specific part, so that the accurate removal of the specific lipoproteins and the accurate protection of the detected lipoproteins by the surfactant are realized in the subsequent process, and the accuracy of the test is ensured.
In addition, the polyanionic polyethylene glycol added in the test reagent has the function of assisting in dispersing and removing chylomicron, very low density lipoprotein and low density lipoprotein, so that the polyanionic polyethylene glycol does not participate in subsequent reaction.
According to the invention, the combination of the surfactant, the chaotropic ions, the polyanions and the enzyme is utilized to realize the removal of non-small and dense lipoproteins, the characteristic measurement of small and dense lipoprotein cholesterol is realized, and the chaotropic ions are utilized to disperse the lipoproteins when a sample is treated, so that the lipoproteins are in a relatively free state in blood, and the subsequent removal of the specific lipoproteins by the surfactant and the protection of the specific detected lipoproteins are facilitated; the combination of the surfactant, the chaotropic ions, the polyanions and the enzyme provided by the invention forms an effective characteristic method for removing and measuring, the unique combination of the surfactant, the chaotropic ions and the enzyme is selected to act on specific lipoprotein, thereby carrying out accurate measurement, and the addition of the polyethylene glycol further expands the action among the combination of the chaotropic ions, the lipoprotein esterase and the surfactant; the test method is simple to operate, can be applied to clinical biochemical analyzers, greatly shortens the test time, and is suitable for clinical detection of large-batch samples.
And (4) relevant testing:
firstly, anti-interference test:
hemolytic interference experiments: the hemoglobin solutions were added to the serum samples to make the hemoglobin concentrations 5g/L, 4g/L, 3g/L, 2g/L, and 1g/L, respectively. The samples of 5 different dilution ratios of each interferent were tested 3 times and the mean calculated. Taking the sample without hemoglobin as a reference value, recording as 100%, calculating the interference degree, and judging whether the detection item reagent is interfered or not according to the interference degree. The specified experimental result is a period without significant interference within a 90-110% interval, and the period beyond which the interference is significant interference.
Test 1
Figure BDA0003655088290000111
Figure BDA0003655088290000121
Test 2
Figure BDA0003655088290000122
Figure BDA0003655088290000131
Test 3
Figure BDA0003655088290000132
Second, reagent blank absorbance
The test was performed with a blank sample with the addition of reagents, and the results are as follows. The standard requires that: the reagent blank absorbance value should be no greater than 0.0500.
Figure BDA0003655088290000141
Third, analysis sensitivity
When the concentration of sdLDL-C in the sample was 0.65mmol/L, the absorbance change value was measured, and the results were as follows.
The standard requires that: the difference in absorbance values should be no less than 0.0100.
Figure BDA0003655088290000142
Four, linear range
Taking a high concentration sample close to the upper limit of the linear range and a low concentration sample close to the lower limit of the linear range, mixing the high concentration sample and the low concentration sample to obtain 5 diluted concentration samples (x) i ) Each diluted concentration sample was tested 3 times, and the results are as follows.
The standard requires that: in the linear range of [ 0.10-2.60 ] mmol/L, the linear correlation coefficient (r) should be not less than 0.990, in the range of [ 0.10-0.80 ] mmol/L, the absolute deviation of linearity should not exceed plus or minus 0.10mmol/L, and in the range of (0.80-2.60 ] mmol/L, the relative deviation of linearity should not exceed plus or minus 15.0%.
Test 1
Figure BDA0003655088290000143
Test 2
Figure BDA0003655088290000151
Test 3
Figure BDA0003655088290000152
Fifth, precision test
Under the repetitive condition, the quality control samples were repeatedly tested 10 times, the mean (x) and standard deviation (S) of the measured values were calculated, respectively, and the Coefficient of Variation (CV) was calculated In batch ). The standard requires that: coefficient of Variation (CV) In batch ) Should not be greater than 8.0%.
Test 1
Figure BDA0003655088290000153
Figure BDA0003655088290000161
Test 2
Number of times Sample 1 Sample 2
1 0.73 1.70
2 0.70 1.61
3 0.72 1.61
4 0.71 1.63
5 0.72 1.61
6 0.71 1.64
7 0.73 1.68
8 0.71 1.64
9 0.68 1.69
10 0.71 1.66
Mean value 0.71 1.65
Standard deviation of 0.015 0.034
CV In batch (％) 2.07 2.06
Test 3
Figure BDA0003655088290000162
Figure BDA0003655088290000171
Sixth, precision test
Recovery test: taking a proper amount of the high-concentration sample A, adding the high-concentration sample A into the low-concentration human source sample B, and mixing to obtain a mixture with a volume ratio of 1: 9. 1: the two samples of 19, each of which was tested in duplicate 3 times, were averaged to calculate the recovery. The standard requires that: the recovery rate should be between 85.0% and 115.0%.
Test 1
Figure BDA0003655088290000172
Test 2
Figure BDA0003655088290000173
Test 3
Figure BDA0003655088290000174
Seventh, limit of detection test
The measurement was repeated 20 times using a 5% Bovine Serum Albumin (BSA) solution as a measurement sample.
Figure BDA0003655088290000175
Figure BDA0003655088290000181
The lowest detection limit (mean +2SD) of the product was 0.001mmol/L by statistical data analysis.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the scope of the claims, and other substantially equivalent alternatives may be conceived by those skilled in the art and are within the scope of the present invention.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.

Claims (10)

1. A reagent for measuring cholesterol in small, dense and low-density lipoproteins, comprising a first reagent and a second reagent, wherein the first reagent comprises a chaotropic ionic compound which disperses lipoproteins by the action of electric charges so that the surfactant a and cholesterol esterase selectively act on a specific lipoprotein, thereby achieving measurement of the amount of cholesterol in the specific lipoprotein.
2. The reagent for measuring small and dense low density lipoprotein cholesterol of claim 1 in which the concentration of the chaotropic ionic compound is 10 to 300 mmol/L; the chaotropic ionic compound is one or two selected from sodium bromide, sodium thiocyanate and potassium iodide.
3. The reagent for measuring small, dense low-density lipoprotein cholesterol of claim 1, in which the first reagent further comprises cholesterol-measuring enzymes including cholesterol esterase, cholesterol oxidase, catalase.
4. The reagent for measuring small, dense and low-density lipoprotein cholesterol as set forth in claim 1, wherein said first reagent further comprises polyethylene glycol for dispersing chylomicron and very low-density lipoprotein, in cooperation with the binding action between the chaotropic ionic compound and lipoprotein esterase and surfactant a.
5. The reagent for measuring small, dense low-density lipoprotein cholesterol of claim 1, in which the first reagent further comprises Trinder's chromogen compound and divalent metal ion.
6. The reagent for measuring small, dense low-density lipoprotein cholesterol according to claim 1, wherein the first reagent further comprises one or more of a buffer, a preservative, and a stabilizer.
7. The reagent for measuring small, dense low-density lipoprotein cholesterol of claim 1 in which the second reagent comprises peroxidase, 4-aminoantipyrine, sodium azide and surfactant B.
8. The reagent for measuring small, dense low-density lipoprotein cholesterol of claim 7, in which the second reagent further comprises a buffer and/or a stabilizer.
9. A kit for measuring cholesterol in a small, dense low-density lipoprotein, comprising a first reagent and a second reagent, the kit being adapted to measure the amount of cholesterol in a specific lipoprotein by selectively acting on the specific lipoprotein; the ionic compound is made to disperse lipoprotein through charge so that the surfactant A and cholesterol esterase act selectively on specific lipoprotein.
10. The kit for measuring small, dense low-density lipoprotein cholesterol of claim 9, further comprising a standard; the standard is a freeze-dried product.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN105652021A (en) * 2016-03-11 2016-06-08 上海练佰生物技术中心 Reagent, method and kit for measuring small-and-dense lipoprotein
CN110564810A (en) * 2019-09-19 2019-12-13 武汉瀚海新酶生物科技有限公司 High-performance small and dense low-density lipoprotein cholesterol detection kit

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* Cited by examiner, † Cited by third party
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JPS5578255A (en) * 1978-12-08 1980-06-12 Kyorin Pharmaceut Co Ltd Simultaneous simple and quick measurement method for low and high specific gravity lipo protein cholesterol and its measuring kit
US20040161811A1 (en) * 1999-03-01 2004-08-19 Koji Kishi Reagent kit for detecting cholesterol in a high-density lipoprotein
CN105652021A (en) * 2016-03-11 2016-06-08 上海练佰生物技术中心 Reagent, method and kit for measuring small-and-dense lipoprotein
CN110564810A (en) * 2019-09-19 2019-12-13 武汉瀚海新酶生物科技有限公司 High-performance small and dense low-density lipoprotein cholesterol detection kit

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