CN110850107A - Kit for detecting lipoprotein (a) molecular concentration and application thereof - Google Patents
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- 108010033266 Lipoprotein(a) Proteins 0.000 title claims abstract description 116
- 102000057248 Lipoprotein(a) Human genes 0.000 title claims abstract 16
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 133
- 239000004816 latex Substances 0.000 claims abstract description 36
- 229920000126 latex Polymers 0.000 claims abstract description 36
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 33
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 31
- 239000004005 microsphere Substances 0.000 claims abstract description 24
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 16
- 239000004471 Glycine Substances 0.000 claims abstract description 16
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims description 41
- 238000012360 testing method Methods 0.000 claims description 31
- 239000000523 sample Substances 0.000 claims description 29
- 238000002835 absorbance Methods 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 239000012488 sample solution Substances 0.000 claims description 4
- 230000031700 light absorption Effects 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 208000024172 Cardiovascular disease Diseases 0.000 abstract description 3
- 238000004879 turbidimetry Methods 0.000 abstract description 2
- 102100040214 Apolipoprotein(a) Human genes 0.000 description 106
- 210000002966 serum Anatomy 0.000 description 25
- 239000012491 analyte Substances 0.000 description 17
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 101710115418 Apolipoprotein(a) Proteins 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 5
- 102000004895 Lipoproteins Human genes 0.000 description 4
- 108090001030 Lipoproteins Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 102000001554 Hemoglobins Human genes 0.000 description 3
- 108010054147 Hemoglobins Proteins 0.000 description 3
- 102000013566 Plasminogen Human genes 0.000 description 3
- 108010051456 Plasminogen Proteins 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 150000003278 haem Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 102000007592 Apolipoproteins Human genes 0.000 description 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 208000000563 Hyperlipoproteinemia Type II Diseases 0.000 description 1
- 102100024640 Low-density lipoprotein receptor Human genes 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 206010045261 Type IIa hyperlipidaemia Diseases 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
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- 201000001386 familial hypercholesterolemia Diseases 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 208000010125 myocardial infarction Diseases 0.000 description 1
- 238000004848 nephelometry Methods 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
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- 230000002285 radioactive effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- 150000003626 triacylglycerols Chemical class 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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- 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
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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Abstract
The invention discloses a kit for detecting the concentration of lipoprotein (a) molecules and application thereof, aiming at solving the problem that the concentration of the lipoprotein (a) molecules can not be accurately detected in the prior art. The invention provides a kit for detecting lipoprotein (a), which comprises a reagent A and a reagent B; the reagent A comprises the following components: 50-200nmol/L of glycine, 2-6g/L of bovine serum albumin, 0.5-1g/L of sodium azide, 201-3 mL/L of tween-201, and the balance of water; the reagent B comprises the following components: 50-200nmol/L of glycine, 2-6g/L of bovine serum albumin, 0.5-1g/L of sodium azide, 3-6g/L of latex microspheres coated with lipoprotein (a) antibody and the balance of water. The invention fills the blank of the existing home-made reagent for detecting lipoprotein (a) molecular concentration latex turbidimetry, provides an effective method for detecting lipoprotein (a) molecular concentration for clinicians and researchers, and provides a more reliable means for correctly judging and predicting cardiovascular disease risks for doctors.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to a kit for detecting lipoprotein (a) molecular concentration and application thereof.
Background
Lipoprotein (a), a specific macromolecular cholesterol-rich lipoprotein synthesized by the liver, is composed of a lipid core, apoB-100 molecules, and hydrophilic apo (a) at its periphery. The surface of the lipoprotein (a) is coated by cholesterol and phospholipid, and is embedded with hydrophilic apolipoprotein, so that the lipoprotein (a) can enter and deposit on the blood vessel wall, and has the function of promoting atherosclerosis. Lipoprotein (a) is structurally homologous to Plasminogen (PLG) and competes with plasminogen for binding to fibrin sites, thereby inhibiting fibrinolysis and promoting thrombosis. Therefore, the lipoprotein (a) level in blood has close correlation with atherosclerosis, myocardial infarction and familial hypercholesterolemia, and is an important factor for predicting the risk of cardiovascular diseases.
Due to the high variability of the apo (a) portion of lipoprotein (a), the number of repeated copies of the apo (a) portion K-IV-type2 region varies between individuals (between 3-40), resulting in a large difference in the molecular weight of the lipoprotein (a) protein between individuals and even within the same individual (187-. At present, monoclonal antibodies or polyclonal antibodies used by an immunoturbidimetry method commonly used in clinical laboratories mainly act on a K-IV-type2 region, so that the molecular concentration of lipoprotein (a) is difficult to accurately measure, the detection results of lipoproteins (a) with different sizes generate larger deviation, and the judgment of a doctor on the patient's condition by using the index is influenced.
Currently, the detection methods for lipoprotein (a) are: radioimmunoassay, ELISA method, immunoturbidimetry method, lipoprotein (a) -cholesterol method, and the like. Radioimmunoassays have been phased out by the market because of their radioactive contamination. Although the ELISA method can accurately measure the number of lipoprotein (a) molecules, it is not highly automated and is greatly affected by human factors. The lipoprotein (a) -cholesterol method has been recently developed, and has advantages in that it can avoid the problems of lipoprotein (a) determination due to apo (a) polymorphism in immunoassay, and disadvantages in that the method is complicated to operate, and it is necessary to separate lipoprotein (a) from plasma by ultracentrifugation or other methods and then detect lipoprotein (a) by the cholesterol enzymatic method, and thus it is difficult to widely apply the method. Compared with other methods, the immunoturbidimetry has the advantages of simplicity, rapidness, sensitivity and reliability, does not need to separate the lipoprotein (a) from the blood plasma, can be directly used for an automatic or semi-automatic biochemical analyzer, has a wider application range and good market prospect, and has the defect of larger detection deviation for apo (a) molecules with different sizes. Therefore, it is an urgent technical problem in the art to find a method for detecting the concentration of lipoprotein (a) molecules that can avoid detection deviation and is easy to operate.
Disclosure of Invention
The invention aims to provide a kit for detecting the concentration of lipoprotein (a) molecules and application thereof, so as to solve the problem that the concentration of the lipoprotein (a) molecules cannot be accurately detected in the prior art.
The invention provides a kit for detecting lipoprotein (a), which comprises a reagent A and a reagent B;
the reagent A comprises the following components: 50-200nmol/L of glycine, 2-6g/L of bovine serum albumin, 0.5-1g/L of sodium azide, 201-3 mL/L of tween-201, and the balance of water;
the reagent B comprises the following components: 50-200nmol/L of glycine, 2-6g/L of bovine serum albumin, 0.5-1g/L of sodium azide, 3-6g/L of latex microspheres coated with lipoprotein (a) antibody and the balance of water.
The reagent A comprises the following components: 170nmol/L of glycine, 5g/L of bovine serum albumin, 0.9g/L of sodium azide, 202mL/L of tween-and the balance of water.
The reagent B comprises the following components: 170nmol/L of glycine, 2g/L of bovine serum albumin, 0.9g/L of sodium azide, 5g/L of latex microspheres coated with lipoprotein (a) antibody and the balance of water.
The pH value of the reagent A is 6.8-7.2.
The pH of reagent A is 7.0.
The pH value of the reagent B is 7.2-7.6.
The pH of reagent B is specifically 7.3.
The preparation method of the latex microsphere coated with the lipoprotein (a) antibody can comprise the following steps: the latex microspheres are activated and then linked to lipoprotein (a) antibodies.
In the preparation method, the mass ratio of the lipoprotein (a) antibody to the latex microsphere can be 10: (1-2).
In the preparation method, the mass ratio of the lipoprotein (a) antibody to the latex microsphere is specifically 10: 1.2.
the latex microspheres can be latex microspheres with the particle size of 80-120 nm.
The latex microspheres can be latex microspheres with the particle size of 105 nm.
The latex microspheres may specifically be carboxyl latex microspheres. The activation may specifically be a carboxyl group activation.
The latex microspheres can be specifically the following: shanghai Huizhi Biotech, Inc., Cat number DR 105C.
The lipoprotein (a) antibody may be: lipoprotein (a) rabbit polyclonal antibody.
The lipoprotein (a) antibody can be specifically the following products: rb a Hu Lipoprotein (a), ForTurbididimetry/Nephelometry. The manufacturer is Agilent technologies (Shanghai) Inc. The seller is Shanghai echographic medical products, Inc., having a product number of O9118.
The invention also protects the application of any one of the reagent A and the reagent B in preparing a kit for detecting the lipoprotein (a).
The invention also protects the application of any one of the kits in detecting the lipoprotein (a).
Any one of the above test lipoproteins (a) is a test lipoprotein (a) in serum.
Any one of the above-mentioned test lipoproteins (a) is a test lipoprotein (a) concentration.
The invention also provides a method for detecting the concentration of the lipoprotein (a) by using the kit, which comprises the following steps:
mixing 2 μ l of sample solution with 133 μ l of reagent A, and incubating at 37 deg.C for 5 min;
then adding 33 mul reagent B and incubating for 5min at 37 ℃, and detecting and recording the light absorption value A1;
then incubating for 10min at 37 ℃, and detecting and recording the light absorption value A2;
△ A-A2-A1, △ A value was calculated, and the concentration of lipoprotein (a) in the test sample was calculated from △ A value.
The main wavelength/sub-wavelength of absorbance was 660nm/800 nm.
The kit provided by the invention can detect the molecular concentration of the lipoprotein (a), has accurate result, good sensitivity and precision, wide linear range and strong stability, is suitable for detection of various full-automatic biochemical detectors, and has the advantages of simple and convenient operation and low cost. The invention solves the problem that the concentration of lipoprotein (a) molecules can not be accurately detected in the prior art. The invention fills the blank of the existing home-made reagent for detecting lipoprotein (a) molecular concentration latex turbidimetry, provides an effective method for detecting lipoprotein (a) molecular concentration for clinicians and researchers, and provides a more reliable means for correctly judging and predicting cardiovascular disease risks for doctors.
Drawings
FIG. 1 is a graph showing the correlation between the results of detection by the reagent A and the reagent B and the results of detection by the kit in example 2.
FIG. 2 is a correlation between the results of detection by the reagent A and the reagent D1 in example 2 and the results of detection by the kit.
FIG. 3 is a correlation between the results of detection by the reagent A and the reagent D2 in example 2 and the results of detection by the kit.
FIG. 4 is a correlation between the results of detection by the reagent A and the reagent B in example 3 and the results of detection by the kit.
FIG. 5 shows the results of the concentration of lipoprotein (a) in the test sample in example 4.
FIG. 6 is a graph showing the correlation between the measured value and the theoretical value in example 6.
FIG. 7 is a graph showing the results of analysis of the prozone effect in example 7.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Lipoprotein (a): apo (a) antigen from Epoxicam Biotech, Inc., Youk-036.
Example 1 preparation of reagents
First, preparation of the reagent of the present invention
Reagent a (ph 7.0): 170nmol/L of glycine, 5g/L of bovine serum albumin, 0.9g/L of sodium azide, 202mL/L of tween-and the balance of water.
Reagent b (ph 7.3): 170nmol/L of glycine, 2g/L of bovine serum albumin, 0.9g/L of sodium azide, 5g/L of latex microspheres coated with lipoprotein (a) antibody and the balance of water.
The preparation method of the latex microsphere coated with the lipoprotein (a) antibody comprises the following steps: carboxyl groups of the carboxyl latex microspheres are activated by carboxyl groups, and then the carboxyl latex microspheres are connected with lipoprotein (a) antibodies. The mass ratio of the lipoprotein (a) antibody to the carboxyl latex microspheres is 10: 1.2. lipoprotein (a) antibody: lipoprotein (a) Rabbit polyclonal antibody, "Rb a Hu Lipoprotein (a), for Turbimidey/nephelometer" is Agilent technologies, Inc., Shanghai echocardiogram products, Inc., having a product number of O9118. Carboxyl latex microspheres: 105nm, Shanghai Huizhizi Biotech, Inc., under the product number DR 105C.
Secondly, preparation of reagent D1
Reagent D1: instead of the lipoprotein (a) antibody, the reagent B was the same.
Lipoprotein (a) antibody: dukang, an Aifukang Biotechnology Co., Ltd., Van. RTM., GK-037.
Preparation of reagent D2
Reagent D2: instead of the lipoprotein (a) antibody, the reagent B was the same.
Lipoprotein (a) antibody: polyclonal antibody, Holmes corporation, cat # HP063 BG.
Fourth, method for using reagent
The detection is carried out on a HITACHI 3500 full-automatic biochemical analyzer.
1. Preparation of a Standard Curve
Taking the lipoprotein (a) standard substance, diluting with human-like serum matrix solution to make the lipoprotein (a) have different concentrations, thus obtaining each standard substance solution. Human-like serum matrix solution: containing 0.1g/100ml BSA, 0.9g/100ml NaCl, 0.2g/100ml NaN3The balance was PBS buffer (pH 7.5, 15 mM).
Mu.l of the standard solution was mixed with 133. mu.l of reagent A, incubated for 5min at 37 ℃, then 33. mu.l of reagent B was added and incubated for 5min at 37 ℃ (absorbance A1 detected and recorded at this time), and then incubated for 10min at 37 ℃ (absorbance A2 detected and recorded at this time), △ A ═ A2-A1. the dominant/accessory wavelength of absorbance was measured at 660nm/800 nm.
A standard curve is prepared by a spline function fitting method, wherein the X axis is the content of the lipoprotein (a) and the unit is nmol/L, and the Y axis is the △ A value.
2. Test sample supply book
Mu.l of the sample solution was mixed with 133. mu.l of the reagent A, followed by incubation at 37 ℃ for 5min, then 33. mu.l of the reagent B was added and incubated at 37 ℃ for 5min (at which the absorbance A1 was detected and recorded), followed by incubation at 37 ℃ for 10min (at which the absorbance A2 was detected and recorded). △ A ═ A2-A1. the lipoprotein (a) concentration in the sample solution was then obtained according to a standard curve.
Example 2 correlation comparison
The test sample was 20 portions of human serum.
The lipoprotein (a) concentration in the test sample is detected by using a lipoprotein (a) detection kit (latex enhanced immunoturbidimetry) and operating according to the instruction. Lipoprotein (a) detection kit (latex enhanced immunoturbidimetry): roche, cat # 05852633-.
The concentration of lipoprotein (a) in a sample was measured using reagent A and reagent B prepared in example 1 and by the procedure of step four in example 1.
The concentration of lipoprotein (a) in the test sample was measured using reagent A and reagent D1 prepared in example 1, and by referring to the procedure four of example 1 (the difference is only that reagent D1 is used instead of reagent B).
The concentration of lipoprotein (a) in the test sample was measured using reagent A and reagent D2 prepared in example 1, and by referring to the procedure four of example 1 (the difference is only that reagent D2 is used instead of reagent B).
The results of the lipoprotein (a) concentration in the test sample are shown in Table 1, and the unit of the lipoprotein (a) concentration is nmol/L. The correlation between the detection results of the reagent A and the reagent B and the detection result of the kit is shown in FIG. 1. The correlation between the detection results of the reagent A and the reagent D1 and the detection result of the kit is shown in FIG. 2. The correlation between the detection results of the reagent A and the reagent D2 and the detection result of the kit is shown in FIG. 3. The result shows that the combined detection result of the reagent A and the reagent B has the best correlation with the detection result of the kit.
TABLE 1
| Test serum sample | Reagent kit | Reagent A and reagent B | Reagent A and reagent D1 | Reagent A and reagent D2 |
| 1 | 5.7 | 6.7 | 11.0 | 13.2 |
| 2 | 3.2 | 3.3 | 6.7 | 8.0 |
| 3 | 26.5 | 28.4 | 39.9 | 42.2 |
| 4 | 4.2 | 3.6 | 2.0 | 4.2 |
| 5 | 6.2 | 5.7 | 12.9 | 15.9 |
| 6 | 4.2 | 3.0 | 1.7 | 2.0 |
| 7 | 17.1 | 15.1 | 22.2 | 28.2 |
| 8 | 101.2 | 97.5 | 117 | 130.1 |
| 9 | 85.6 | 85.5 | 60.1 | 68.1 |
| 10 | 11.1 | 10.7 | 16.9 | 17.2 |
| 11 | 4.0 | 4.7 | 6.1 | 7.0 |
| 12 | 4.8 | 4.8 | 3.2 | 4.8 |
| 13 | 16.2 | 18.2 | 25.5 | 30.1 |
| 14 | 75.0 | 77.0 | 102.6 | 130.1 |
| 15 | 12.2 | 14.9 | 20.8 | 27.2 |
| 16 | 17.5 | 15.9 | 24.4 | 30.2 |
| 17 | 5.0 | 3.2 | 3.3 | 4.7 |
| 18 | 151.7 | 140.7 | 221.1 | 256.1 |
| 19 | 42.2 | 44.2 | 23.1 | 30.1 |
| 20 | 191.1 | 186.5 | 265.7 | 311.1 |
Example 3 correlation analysis of the method and imported reagents
The test sample was 90 portions of human serum.
The Lipoprotein (a) concentration in the test sample was determined using the Lipoprotein (a) detection kit (Latex enhanced immunoturbidimetry) Tina-quant Lipoprotein (a) gen.2(Latex) (LPA2) and following the protocol.
The concentration of lipoprotein (a) in a sample was measured using reagent A and reagent B prepared in example 1 and by the procedure of step four in example 1.
The results of the lipoprotein (a) concentration in the test sample are shown in Table 2, and the unit of the lipoprotein (a) concentration is nmol/L.
TABLE 2
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| Reagent kit | 18.9 | 7.3 | 6.4 | 6.6 | 6.6 | 5.9 | 12.3 | 1.5 | 49.7 | 9.6 |
| Reagent A and reagent B | 20.6 | 8.6 | 7.9 | 5.2 | 10.9 | 12.4 | 15.7 | 3.6 | 53 | 15.5 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | |
| Reagent kit | 11.1 | 4.8 | 6.5 | 48.5 | 67.6 | 45.1 | 86.9 | 41.5 | 150.4 | 12.8 |
| Reagent A and reagent B | 10.5 | 9.1 | 6.1 | 41.3 | 64.3 | 39 | 82.4 | 39.3 | 140.5 | 13.9 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | |
| Reagent kit | 10.1 | 190.3 | 40.2 | 207 | 204 | 23.8 | 10.7 | 4.1 | 6.5 | 3.4 |
| Reagent A and reagent B | 13.9 | 184.2 | 51.8 | 194 | 167.1 | 24.1 | 12.4 | 3.1 | 13.1 | 3.7 |
| 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 | |
| Reagent kit | 7.9 | 50.2 | 7.4 | 1.9 | 5.8 | 4.2 | 12.2 | 1.5 | 195.7 | 5.1 |
| Reagent A and reagent B | 9.3 | 53.1 | 3.7 | 4.7 | 4.4 | 5.6 | 14.6 | 0.3 | 175.7 | 4.3 |
| 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 | |
| Reagent kit | 4.1 | 79.8 | 24.9 | 10.4 | 6.5 | 62.3 | 160 | 56.1 | 5.6 | 83.1 |
| Reagent A and reagent B | 5.2 | 74.0 | 24.5 | 11.0 | 7.4 | 58.3 | 156.2 | 52.75 | 4.7 | 74.2 |
| 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 | |
| Reagent kit | 45.9 | 2.6 | 15.4 | 4.3 | 13.8 | 90.1 | 42.9 | 4.6 | 7.5 | 37.7 |
| Reagent A and reagent B | 46.2 | 6.4 | 20.1 | 6.9 | 16.0 | 83.7 | 46.9 | 2.9 | 7.1 | 34.8 |
| 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 | |
| Reagent kit | 4.6 | 107.9 | 84.7 | 16.5 | 6.6 | 12.9 | 7.7 | 5.5 | 30.6 | 5.9 |
| Reagent A and reagent B | 6.0 | 99.5 | 78.3 | 14.4 | 7.6 | 15.9 | 9.9 | 6.7 | 28.4 | 5.5 |
| 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 | |
| Reagent kit | 83.7 | 25.8 | 3.4 | 12.2 | 164.9 | 59.9 | 6.1 | 3.3 | 4.5 | 11.6 |
| Reagent A and reagent B | 86.2 | 26.8 | 6.5 | 13.9 | 152.1 | 64.5 | 5.3 | 1.8 | 5.5 | 11.0 |
| 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 | |
| Reagent kit | 5.7 | 53.1 | 8.3 | 10.0 | 5.3 | 1.1 | 7.5 | 30.6 | 22.1 | 10.8 |
| Reagent A and reagent B | 10.1 | 55.6 | 8.3 | 9.8 | 6.7 | 1.0 | 8.1 | 30.2 | 28.9 | 13.1 |
And after removing the samples with the concentration less than 6nmol/L or more than 220nmol/L which do not meet the requirement, carrying out linear regression analysis on the remaining samples which meet the requirement. The results are shown in FIG. 4. The result shows that the correlation between the detection result of the combination of the reagent A and the reagent B and the detection result of the kit is good, namely the reagent A and the reagent B have higher detection accuracy.
Example 4 precision analysis of the method of the invention
The sample is 2 parts of human serum. The test specimens were stored at 4 ℃.
The Lipoprotein (a) concentration in the test sample was determined using the Lipoprotein (a) detection kit (Latex enhanced immunoturbidimetry) Tina-quant Lipoprotein (a) gen.2(Latex) (LPA2) and following the protocol. The concentration of lipoprotein (a) in 1 serum was in the interval of 20-40nmol/L as a low concentration analyte. The concentration of lipoprotein (a) in 1 part of human serum was in the interval of 100-150nmol/L as a high concentration analyte.
The concentration of lipoprotein (a) in the test sample was measured every day for 20 consecutive days using reagent A and reagent B prepared in example 1 and by the procedure of step four in example 1. And calculating the standard deviation of repeatability among days, the coefficient of variation of repeatability and the percentage of the coefficient of variation of intermediate precision.
The results of the lipoprotein (a) concentration in the test sample are shown in Table 3 and FIG. 5, and the unit of the lipoprotein (a) concentration is nmol/L.
TABLE 3
The precision of reproducibility of each batch was calculated according to the following formula:
total days I
Intraday analyte batch j
Working day i
Measurement of x in ith day, jth batch of analyte, kth replicateijk
Mean of analyte results for batch j:
batch j standard deviation of reproducibility (within-batch precision):
coefficient of variation of repeatability of batch j:
the results are shown in Table 4. The result shows that the reagent has good stability of bottle opening, and good repeatability and precision.
TABLE 4
| Performance index | Calculated value |
| CVSR-1 | 1.85％ |
| CVSR-2 | 0.83％ |
Example 5 lower limit of detection analysis of the method of the invention
5 parts of physiological saline were used as blank analytes.
5 parts of human serum were subjected to the protocol of the lipoprotein (a) test kit (Latex-enhanced immunoturbidimetry) Tina-quant lipoprotein (a) Gen.2(Latex) (LPA2) to determine the concentration of lipoprotein (a) in the test sample. The concentration in serum was in the interval 10-40nmol/L as low concentration analyte.
The sample is essentially a blank analyte or a low concentration analyte. The test specimens were stored at 4 ℃.
The concentration of lipoprotein (a) in the test sample was measured every half day for 6 consecutive days using reagent A and reagent B prepared in example 1 and by the procedure of step four in example 1. The blank limit (LoB) and the detection limit (LoD) are calculated.
The results of the lipoprotein (a) concentration in the test sample are shown in Table 5, and the unit of the lipoprotein (a) concentration is nmol/L.
The results are shown in Table 5.
TABLE 5
Blank limit (LoB) calculation method:
sequencing the detection results of all blank samples from low to high (the reading value of the concentration detection result from the low to the high K position is recorded as V)rank-K) And calculating the blank limit according to the following formula:
LoB＝Vrank-57
detection limit (LoD) calculation method:
LoD＝LoB+Cp×SDtotal
the blank and detection limit results are shown in Table 6. The result shows that the reagent has better lower detection limit.
TABLE 6
| Performance index | Calculated value |
| LoB | 1.3 |
| LoD | 2.629 |
Example 6 Linear Range analysis of the method of the invention
The Lipoprotein (a) concentration in the serum of 2 healthy persons was measured using a Lipoprotein (a) detection kit (Latex enhanced immunoturbidimetry) Tina-quant Lipoprotein (a) gen.2(Latex) (LPA2) according to the protocol. The detection is carried out for 5 times continuously, and the detection results are shown in Table 7. The concentration of lipoprotein (a) in 1 serum was in the interval of 2-6nmol/L as a low concentration analyte. The concentration of lipoprotein (a) in 1 human serum was in the interval 200-240nmol/L as a high concentration analyte. And mixing the low-concentration analyte and the high-concentration analyte according to different volume ratios to obtain a sample book. The theoretical concentration of lipoprotein (a) in the test sample was calculated from the ratio, and is shown in Table 8.
The concentration of lipoprotein (a) in a sample was measured using reagent A and reagent B prepared in example 1 and following the procedure of step four of example 1, and an actual measurement value of the concentration of lipoprotein (a) was obtained. The test was repeated three times. The results are shown in Table 9, where the concentration of lipoprotein (a) is in nmol/L. The results were statistically analyzed, and the correlation coefficient (r) between the measured and theoretical values, and the average and relative deviation values for each concentration were calculated.
The correlation between measured and theoretical values is shown in FIG. 6. The result shows that the reagent has better linearity in the range of 6-200 nmol/L.
TABLE 7
| Concentration of analyte | First measurement | Second measurement | Third measurement | Fourth measurement | Fifth measurement | Mean value of |
| Low concentration of analyte | 6.4 | 5.7 | 6.0 | 5.3 | 6.4 | 5.9 |
| High concentration of analyte | 209.0 | 208.6 | 209.1 | 209.8 | 212.0 | 210.0 |
TABLE 8
| Sample supply book | Low concentration analyte ratio | High concentration analyte ratio | Theoretical value |
| Concentration-1 | 100％ | 0％ | 5.9 |
| Concentration-2 | 95％ | 5％ | 16.2 |
| Concentration-3 | 90％ | 10％ | 26.4 |
| Concentration-4 | 85％ | 15％ | 36.5 |
| Concentration-5 | 80％ | 20％ | 46.8 |
| Concentration-6 | 70％ | 30％ | 67.1 |
| Concentration-7 | 60％ | 40％ | 87.5 |
| Concentration-8 | 50％ | 50％ | 108.0 |
| Concentration-9 | 40％ | 60％ | 128.4 |
| Concentration-10 | 30％ | 70％ | 148.7 |
| Concentration-11 | 20％ | 80％ | 169.1 |
| Concentration-12 | 10％ | 90％ | 189.5 |
| Concentration-13 | 0％ | 100％ | 210.0 |
TABLE 9
Example 7 prozone Effect analysis of the method of the invention
A series of concentrations of lipoprotein (a) solutions were prepared using a degreased serum (human serum, purchased from SeraCare, cat # 1800-. The concentration gradient for the series of concentrations was 50 nmol/L. The lipoprotein (a) solution was used as a test sample.
The reagent A and reagent B prepared in example 1 were used, and absorbance was measured in accordance with 2 of step four in example 1. The theoretical concentration value of the highest absorbance point appearing at the earliest is taken as the concentration of the prozone effect.
The results are shown in Table 10 and FIG. 7. In FIG. 7, the abscissa represents the theoretical concentration, and the ordinate represents the absorbance.
The results show that the reagent of the invention does not have prozone effect in the interval of less than 450 nmol/L.
Example 8 analysis of interfering substances according to the method of the invention
The serum of healthy human was divided into three portions of 190. mu.l each. The first portion was supplemented with 10. mu.l of sterile physiological saline as normal serum. Mu.l of hemoglobin (Sigma-Aldrich, cat # H9379) solution was added in the second portion, which was serum containing hemoglobin (hemoglobin concentration 1000 mg/dL). Mu.l of a triglyceride (Sigma-Aldrich, cat # I141-100mL) solution, i.e., triglyceride-containing serum (triglyceride concentration 2000mg/dL), was added to the third aliquot.
The test samples were: normal serum, serum containing heme, or serum containing triglycerides.
The concentration of lipoprotein (a) in a sample was measured using reagent A and reagent B prepared in example 1 and by the procedure of step four in example 1.
The results are shown in Table 11, where the concentration of lipoprotein (a) is expressed in nmol/L. The results show that the reagent of the invention has good anti-interference performance on heme and triglyceride.
TABLE 11
| Numbering | Normal serum | Heme-containing serum | Serum containing triglyceride |
| Repetition of 1 | 23.3 | 22.9 | 25.1 |
| Repetition 2 | 23.5 | 22.8 | 24.6 |
| Repetition of 3 | 23.5 | 22.6 | 24.0 |
| Mean value of | 23.5 | 22.8 | 24.6 |
| Deviation CV | NA | 3％ | 4.7％ |
Claims (8)
1. A kit for detecting lipoprotein (a) comprising a reagent a and a reagent b;
the reagent A comprises the following components: 50-200nmol/L of glycine, 2-6g/L of bovine serum albumin, 0.5-1g/L of sodium azide, 201-3 mL/L of tween-201, and the balance of water;
the reagent B comprises the following components: 50-200nmol/L of glycine, 2-6g/L of bovine serum albumin, 0.5-1g/L of sodium azide, 3-6g/L of latex microspheres coated with lipoprotein (a) antibody and the balance of water.
2. The kit of claim 1, wherein:
the reagent A comprises the following components: 170nmol/L of glycine, 5g/L of bovine serum albumin, 0.9g/L of sodium azide, 202mL/L of tween-and the balance of water.
3. The kit of claim 1 or 2, wherein:
the reagent B comprises the following components: 170nmol/L of glycine, 2g/L of bovine serum albumin, 0.9g/L of sodium azide, 5g/L of latex microspheres coated with lipoprotein (a) antibody and the balance of water.
4. The use of reagent A and reagent B in the preparation of a kit for the detection of lipoprotein (a);
the reagent A comprises the following components: 50-200nmol/L of glycine, 2-6g/L of bovine serum albumin, 0.5-1g/L of sodium azide, 201-3 mL/L of tween-201, and the balance of water;
the reagent B comprises the following components: 50-200nmol/L of glycine, 2-6g/L of bovine serum albumin, 0.5-1g/L of sodium azide, 3-6g/L of latex microspheres coated with lipoprotein (a) antibody and the balance of water.
5. The use of claim 4, wherein:
the reagent A comprises the following components: 170nmol/L of glycine, 5g/L of bovine serum albumin, 0.9g/L of sodium azide, 202mL/L of tween-and the balance of water.
6. Use according to claim 4 or 5, characterized in that:
the reagent B comprises the following components: 170nmol/L of glycine, 2g/L of bovine serum albumin, 0.9g/L of sodium azide, 5g/L of latex microspheres coated with lipoprotein (a) antibody and the balance of water.
7. Use of a kit according to any one of claims 1 to 3 for the detection of lipoprotein (a).
8. A method for detecting lipoprotein (a) using the kit of any one of claims 1 to 3, comprising the steps of:
mixing 2 μ l of sample solution with 133 μ l of reagent A, and incubating at 37 deg.C for 5 min;
then 33. mu.l of reagent B was added and incubated at 37 ℃ for 5min, at which time the absorbance A1 was recorded;
then incubating for 10min at 37 ℃, and recording the light absorption value A2;
△ A-A2-A1, △ A value was calculated, and the concentration of lipoprotein (a) in the test sample was calculated from △ A value.
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