CN112255421B - Lipoprotein a detection kit and detection method - Google Patents
Lipoprotein a detection kit and detection method Download PDFInfo
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- CN112255421B CN112255421B CN202011087841.2A CN202011087841A CN112255421B CN 112255421 B CN112255421 B CN 112255421B CN 202011087841 A CN202011087841 A CN 202011087841A CN 112255421 B CN112255421 B CN 112255421B
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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
- G01N33/92—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
- G01N33/54346—Nanoparticles
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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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a lipoprotein a detection kit and a detection method, the kit comprises a reagent R1, a reagent R2 and a calibrator, and is characterized in that the reagent R1 comprises 50-250 mmol/L of a first buffer solution, 1-2 wt% of an anti-interference agent, 1-2.5 wt% of an accelerant and 0.01-0.1 wt% of a preservative; the reagent R2 comprises 50-250 mmol/L of second buffer solution, 0.5-5 wt% of stabilizing agent, 0.1-2 g/L of anti-human lipoprotein a monoclonal antibody coated latex particles and 0.02-0.2 wt% of preservative. The detection kit can solve the problem of nonspecific interference caused by factors such as hydrophobic effect, complement, rheumatoid factors and the like, has the advantages of strong anti-interference capability, high specificity, high accuracy, good stability and the like, and is convenient to operate and popularize and apply.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnostic reagents, in particular to a lipoprotein a detection kit and a detection method.
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. Mainly secreted into the blood after liver synthesis and playing an extremely important role in the process of activating atherosclerosis. Lipoprotein (a) is closely related to coronary heart disease, calcified aortic stenosis, familial hypercholesterolemia, peripheral vascular disease, etc., and has become a recognized independent predictor of coronary heart disease.
At present, known methods for detecting the concentration of lipoprotein (a) include enzyme-linked immunosorbent assay, radioimmunoassay, immunoturbidimetry and other detection methods, and all of the methods have certain defects. Such as: the radioimmunoassay has radioactive contamination; the enzyme-linked immunosorbent assay is complex in operation, long in time consumption and has certain skill requirements on operators; immunoturbidimetry cross-contaminates plasmin and therefore the accuracy of the assay is low and needs to be improved. The latex particle enhanced immunoturbidimetry is a relatively stable and accurate method for detecting the humoral protein homogeneous phase immunoturbidimetry, and the principle is as follows: monoclonal or polyclonal antibodies are crosslinked on the surfaces of the polymer latex microspheres, and after the microspheres crosslinked with the antibodies are combined with antigens, the microspheres can be rapidly gathered together in a short time, so that the light-scattering performance or the light-transmitting performance of reaction liquid is changed, the change of the light-transmitting performance (namely absorbance) of the reaction liquid has strong correlation with the concentration of the detected antigens, and the concentration of the detected antigens can be reflected in a certain range.
Research shows that the latex particle enhanced immunoturbidimetry is interfered by various factors to generate false positive results, such as: 1) hydrophobic substances existing in the reaction environment can be combined with the antigen/antibody or the latex through hydrophobic interaction, so that false positive is generated when the latex particles are used for enhancing the immunoturbidimetry to detect the lipoprotein (a); 2) complement molecules in serum and tissue fluid bind to the Fc fragment of the antibody and the complement binding site of C1q, resulting in false positives; 3) binding of rheumatoid factor in serum to the Fc fragment of the coated or labeled antibody produced a false positive result.
Although the prior art has developed many detection kits based on the principle of latex particle enhanced immunoturbidimetry for detecting the content of lipoprotein (a), such as chinese patent publication No. CN 103149370A, CN 106990234A, CN 107607729A, CN 111239422A, CN 109541241 a, the above patents do not solve the problem of non-specific interference caused by factors such as hydrophobic effect, complement, and rheumatoid factor in vitro diagnostic reagents.
On the other hand, the stability of the antibody coated latex particles prepared by the prior art is poor, the antibody is coupled to the carboxyl polystyrene latex particles, the instant detection effect is good, but after the latex particles are placed at 37 ℃ for 2 days, the activity of the antibody is greatly reduced, and the latex is subjected to an agglutination phenomenon, so that the repeatability of the use of the kit is poor, the variation coefficient is increased, the detection result of the reagent is inaccurate, and the use requirement cannot be met.
Disclosure of Invention
The invention aims to provide a lipoprotein a detection kit and a detection method, the detection kit can solve the problem of nonspecific interference caused by factors such as hydrophobic effect, complement, rheumatoid factors and the like, has the advantages of strong anti-interference capability, high specificity, high accuracy, good stability and the like, and is convenient to operate and popularize and apply.
The invention is realized by the following technical scheme:
a lipoprotein a detection kit comprises a reagent R1, a reagent R2 and a calibrator, and is characterized in that the reagent R1 comprises 50-250 mmol/L of a first buffer solution, 1-2 wt% of an anti-interference agent, 1-2.5 wt% of an accelerant and 0.01-0.1 wt% of a preservative; the reagent R2 comprises 50-250 mmol/L of second buffer solution, 0.5-5 wt% of stabilizing agent, 0.1-2 g/L of anti-human lipoprotein a monoclonal antibody coated latex particles and 0.02-0.2 wt% of preservative.
Preferably, the first buffer solution and the second buffer solution are respectively and independently selected from at least one of MOPS buffer solution, 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, phosphate buffer solution, glycine buffer solution, Tris buffer solution and Tris-HCl buffer solution. The pH value range of the buffer solution is as follows: 6.5 to 8.5.
Preferably, the anti-interference agent is Ergothioneine (L-Ergothionine, EGT, chemical name is 2-sulfydryl histidine trimethyl inner salt) and TCEP-HCl (tri- (2-formyl ethyl) phosphine hydrochloride) which are composed according to the mass ratio of 1 (1-2).
More preferably, the anti-interference agent is ergothioneine and TCEP-HCl in a mass ratio of 1: 1.5.
Preferably, the stabilizer is at least one selected from the group consisting of mannooligosaccharide, carboxymethyl dextran, sucrose, trehalose, glycerol, mannitol, and sorbitol.
More preferably, the stabilizer is composed of mannan-oligosaccharide and glycerol in a mass ratio of 1 (2-4).
Preferably, the accelerant is polyethylene glycol 6000, polyethylene glycol 8000, tween-20 or tween-80.
Preferably, the preservative is Proclin300 or NaN3。
Preferably, the anti-human lipoprotein a monoclonal antibody is coated on the latex particles, the anti-human lipoprotein a monoclonal antibody is covalently bonded with the latex particles, and the monoclonal antibody is coated in the latex particles by a chemical crosslinking method. Wherein, the anti-human lipoprotein a monoclonal antibody is a goat anti-human, mouse anti-human or rabbit anti-human lipoprotein a monoclonal antibody; the latex particles are polystyrene latex particles with carboxyl on the surfaces, and the particle size of the particles is 50-300 nm. Preferably, the ratio (mass ratio) of the latex particles with the particle diameters of 50nm, 100nm and 300nm in the latex particles is respectively as follows: 2:3:1.
The anti-human lipoprotein a monoclonal antibody coated latex particle is prepared by adopting a conventional chemical crosslinking method, and the specific steps are as follows:
(1) preparation of anti-human lipoprotein a antibody solution: dissolving the anti-human lipoprotein a monoclonal antibody in a Tris-HCl buffer solution, wherein the pH value is 7.2, and preparing an anti-human lipoprotein a antibody solution with the concentration of 1.0 mg/mL;
(2) preparing a latex microsphere suspension: dissolving polystyrene latex particles with carboxyl on the surface in a Tris-HCl buffer solution, wherein the concentration is 1% (w/v), adding NHS and 100mM EDAC with the final concentration of 50mg/mL, incubating at room temperature for 30min, centrifuging (15000rpm,30min), discarding the supernatant, washing latex microspheres twice with the same volume of Tris-HCl buffer solution, removing the redundant cross-linking agent, adding the Tris-HCl buffer solution, and oscillating for re-suspension to prepare a latex microsphere suspension with the concentration of 1% (w/v);
(3) preparing anti-human lipoprotein a monoclonal antibody coated latex particles: and (2) uniformly mixing the anti-human lipoprotein a antibody solution obtained in the step (1) and the latex microsphere suspension obtained in the step (2) according to the volume ratio of 1:1, incubating for 3h at 37 ℃, adding 2mL of ethanolamine into each mL of reaction liquid, uniformly mixing, sealing at 37 ℃ for 30min, centrifuging (15000rpm,30min), discarding supernatant, washing twice with the same volume of Tris-HCl buffer solution, removing unbound antibody, adding Tris-HCl buffer solution, and oscillating and resuspending to obtain the anti-human lipoprotein a monoclonal antibody coated latex particle.
The invention also provides a method for detecting the content of lipoprotein a by using the kit for non-disease diagnosis and treatment purposes, which comprises the following steps:
(1) adding the reagent R1 into a sample to be tested, reacting for 4-6 min, adding the reagent R2, uniformly mixing, reading the absorbance A1, continuing to react for 4-6 min, reading the absorbance A2, and calculating delta A (A2-A1);
(2) and drawing a standard curve by using the lipoprotein a calibrator, and obtaining the content of the lipoprotein a in the sample to be detected according to the delta A and the standard curve.
Preferably, in the detection method, the volume ratio of the sample to be detected, the reagent R1 and the reagent R2 is 2 (120-180): (30-60), the reaction temperature is 37 ℃, and the detection wavelength is 600 nm.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention creatively uses the anti-interference agent which is formed by compounding 2-sulfydryl histidine trimethyl inner salt (ergothioneine) and tris- (2-formyl ethyl) phosphine hydrochloride (TCEP-HCl) according to a certain mass ratio, can effectively eliminate the problem of nonspecific interference caused by factors such as complement, rheumatoid factors and the like, and obviously improves the sensitivity, accuracy and specificity of reagent detection.
2. The anti-interference agent used by the invention not only can obviously enhance the anti-interference capability of the detection of the kit, but also can improve the stability of the detection reagent by cooperating with the stabilizer, and the ergothioneine and TCEP-HCl have certain ion chelation and antioxidation effects, thereby effectively protecting the activity of the antibody, keeping the space structure of the antibody protein stable, avoiding the inactivation of the antibody in the preservation process and improving the accuracy of the detection result.
3. The invention uses the stabilizer composed of mannan-oligosaccharide and glycerol according to a certain mass ratio, can effectively protect the activity of the antibody and prevent the latex particles from agglutinating and sinking, prolongs the service life of the detection reagent, improves the repeatability of the detection of the kit and improves the stability of the detection.
In conclusion, the detection kit can solve the problem of nonspecific interference caused by factors such as hydrophobic effect, complement, rheumatoid factors and the like, has the advantages of strong anti-interference capability, high specificity, high accuracy, good stability and the like, and is convenient to operate and popularize and apply.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof. The reagents in the following examples are all commercially available products unless otherwise specified.
Examples below the antibody-coated latex particles in each kit were: the preparation method of the goat anti-human lipoprotein a monoclonal antibody coated latex particles comprises the following steps:
(1) preparing a goat anti-human lipoprotein a antibody solution: dissolving goat anti-human lipoprotein a monoclonal antibody (Beijing Aspius biotechnology, Inc.) in Tris-HCl buffer solution with pH of 7.2 to obtain goat anti-human lipoprotein a antibody solution with concentration of 1.0 mg/mL;
(2) preparing a latex microsphere suspension: dissolving polystyrene latex particles with carboxyl on the surface in a Tris-HCl buffer solution, wherein the concentration is 1% (w/v), adding NHS and 100mM EDAC with the final concentration of 50mg/mL, incubating at room temperature for 30min, centrifuging (15000rpm,30min), discarding the supernatant, washing latex microspheres twice with the same volume of Tris-HCl buffer solution, removing the redundant cross-linking agent, adding the Tris-HCl buffer solution, and oscillating for re-suspension to prepare a latex microsphere suspension with the concentration of 1% (w/v);
(3) preparing goat anti-human lipoprotein a monoclonal antibody coated latex particles: and (2) uniformly mixing the goat anti-human lipoprotein a antibody solution obtained in the step (1) and the latex microsphere suspension obtained in the step (2) according to the volume ratio of 1:1, incubating at 37 ℃ for 3h, adding 2mL of ethanolamine into each mL of reaction liquid, uniformly mixing, sealing at 37 ℃ for 30min, centrifuging (15000rpm,30min), discarding supernatant, washing twice with the same volume of Tris-HCl buffer solution, removing unbound antibody, adding Tris-HCl buffer solution, and oscillating and resuspending to obtain goat anti-human lipoprotein a monoclonal antibody coated latex particles.
Example 1: lipoprotein a's assay kit
A lipoprotein a detection kit comprises a reagent R1, a reagent R2 and a calibrator.
Reagent R1: MOPS buffer solution 50mmol/L, ergothioneine 0.4 wt%, TCEP-HCl 0.6 wt%, Tween-201 wt%, and Proclin 3000.01wt%;
reagent R2: 50mmol/L of glycine buffer solution, 0.17 wt% of mannan oligosaccharide, 0.33 wt% of glycerol, 0.5g/L of goat anti-human lipoprotein a monoclonal antibody coated latex particles and 3000.02wt% of Proclin.
Calibration products: lipoprotein a antigen was dissolved in standard dilutions (glycine buffer 40mmol/L, Tween-201 wt%, Proclin 3000.03wt%, mannan oligosaccharide 0.17 wt%, glycerol 0.33 wt%), assayed with commercially available control reagents and adjusted to 150mg/L, stored at-20 ℃. The samples were taken out before use and diluted with standard dilutions to different concentrations of lipoprotein a standard (lipoprotein a antigen concentrations: 0mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, 100 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
The pH values of the MOPS buffer solution and the glycine buffer solution are as follows: 7.5.
example 2: lipoprotein a's assay kit
A lipoprotein a detection kit comprises a reagent R1, a reagent R2 and a calibrator.
Reagent R1: 150mmol/L phosphate buffer solution, 0.5 wt% ergothioneine, TCEP-HCl 1 wt%, polyethylene glycol 80002 wt%, NaN3 0.1wt％；
Reagent R2: 100mmol/L phosphate buffer solution, 0.5 wt% mannan oligosaccharide, 2 wt% glycerol, 1g/L, NaN latex particle coated with goat anti-human lipoprotein a monoclonal antibody3 0.1wt％。
Calibration products: standard dilution (phosphate buffer solution 100mmol/L, polyethylene glycol 80002 wt%, NaN) for lipoprotein a antigen30.2 wt%, manno-oligosaccharide 0.5 wt%, glycerol 2 wt%) were dissolved, detected with a commercially available control reagent and adjusted to 150mg/L, dispensed and stored at-20 ℃. The samples were taken out before use and diluted with standard dilutions to different concentrations of lipoprotein a standard (lipoprotein a antigen concentrations: 0mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, 100 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
The pH value of the phosphate buffer solution is as follows: 8.0.
example 3: lipoprotein a's assay kit
A lipoprotein a detection kit comprises a reagent R1, a reagent R2 and a calibrator.
Reagent R1: 200mmol/L of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, 1 wt% of ergothioneine, 1 wt% of TCEP-HCl, 60002 wt% of polyethylene glycol and NaN3 0.1wt％；
Reagent R2: 200mmol/L phosphate buffer solution, 1 wt% mannan oligosaccharide, 3 wt% glycerol, 1.5g/L, NaN latex particle coated with anti-human lipoprotein a monoclonal antibody3 0.2wt％。
Calibration products: standard dilution (phosphate buffer solution 100mmol/L, polyethylene glycol 60002 wt%, NaN) for lipoprotein a antigen30.3 wt% mannan oligomerSugar 1 wt%, glycerol 3 wt%) was dissolved, detected with a commercially available control reagent and adjusted to 150mg/L, stored at-20 ℃. The samples were taken out before use and diluted with standard dilutions to different concentrations of lipoprotein a standard (lipoprotein a antigen concentrations: 0mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, 100 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
The pH values of the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution and the phosphate buffer solution are as follows: 8.5.
example 4: lipoprotein a's assay kit
A lipoprotein a detection kit comprises a reagent R1, a reagent R2 and a calibrator.
Reagent R1: 250mmol/L phosphate buffer solution, 0.8 wt% ergothioneine, 1.2 wt% TCEP-HCl, 60002.5 wt% polyethylene glycol, and NaN3 0.01wt％；
Reagent R2: 250mmol/L of glycine buffer solution, 1.25 wt% of mannan oligosaccharide, 3.75 wt% of glycerol and 2g/L, NaN of anti-human lipoprotein a monoclonal antibody coated latex particles3 0.02wt％。
Calibration products: lipoprotein a antigen standard dilution (glycine buffer solution 100mmol/L, polyethylene glycol 60002 wt%, NaN)30.03 wt%, manno-oligosaccharide 1.25 wt%, glycerol 3.75 wt%) was dissolved, detected with a commercially available control reagent and adjusted to 150mg/L, dispensed and stored at-20 ℃. The samples were taken out before use and diluted with standard dilutions to different concentrations of lipoprotein a standard (lipoprotein a antigen concentrations: 0mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, 100 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
The pH values of the phosphate buffer solution and the glycine buffer solution are as follows: 7.0.
comparative example 1: lipoprotein a's assay kit
The kit of comparative example 1 differs from example 4 in that: the remaining kit components were the same as in example 4, except that the anti-interference agent was not contained.
Comparative example 2: lipoprotein a's assay kit
The kit of comparative example 2 differs from example 4 in that: the anti-interference agent is ergothioneine, and the composition of the rest kit is the same as that of the example 4.
Comparative example 3: lipoprotein a's assay kit
The kit of comparative example 3 differs from example 4 in that: the anti-interference agent is TCEP-HCl, and the rest of the kit components are the same as the example 4.
Comparative example 4: lipoprotein a's assay kit
The kit of comparative example 4 differs from example 4 in that: the ergothioneine was replaced with EDTA-2Na and the rest of the kit was composed in the same manner as in example 4.
Comparative example 5: lipoprotein a's assay kit
The kit of comparative example 5 is the kit described in chinese patent publication CN106990234 a.
Example 5: detection of lipoprotein a
Detecting by using a full-automatic biochemical analyzer according to the following steps:
(1) drawing a standard curve: performing multi-point calibration by using a calibrator, and drawing a standard curve by using the blank of the absorbance change value delta A of the calibrator as A standard-A as a vertical coordinate and the corresponding concentration C standard as a horizontal coordinate;
(2) detecting a sample to be detected: adding 120 mu L of reagent R1 into 2 mu L of human serum sample, uniformly mixing, reacting for 5min at 37 ℃, adding 30 mu L of reagent R2, uniformly mixing, standing for 30s, reading absorbance A1 at the wavelength of 600nm, continuing to react for 5min at 37 ℃, reading absorbance A2 at the wavelength of 600nm, and calculating delta A-A2-A1;
(3) and calculating according to the delta A and the standard curve to obtain the content of the lipoprotein a in the sample to be detected.
(1) And (3) accuracy detection: the quality control materials having a lipoprotein a antigen concentration of 80mg/L were measured using the kits described in examples 1 to 4 and comparative examples 1 to 5 according to the above-mentioned detection procedures, and the measurement was repeated 5 times, and the average value and the relative deviation of the measured values were calculated, where the relative deviation is [ (measured average value-theoretical value)/theoretical value ] × 100, and the detection results are shown in table 1.
TABLE 1 accuracy test results
| Reagent kit | Mean value (mg/L) | Relative deviation of |
| Example 1 | 79.31 | -0.86％ |
| Example 2 | 80.59 | 0.74％ |
| Example 3 | 80.42 | 0.52％ |
| Example 4 | 80.30 | 0.38％ |
| Comparative example 1 | 84.34 | 5.42％ |
| Comparative example 2 | 82.21 | 2.76％ |
| Comparative example 3 | 81.78 | 2.22％ |
| Comparative example 4 | 81.63 | 2.04％ |
| Comparative example 5 | 83.74 | 4.68％ |
The results show that the relative deviation of detection by using the kit provided by the invention is less, which shows that the kit provided by the invention has higher accuracy and better effect than the kits of comparative examples 1-5.
(2) And (3) detecting the precision: the content was repeatedly measured a plurality of times for the same sample having a lipoprotein a concentration of 40mg/L, the measurement was performed according to the above procedure, the sample was repeatedly tested 20 times, and the average value, Standard Deviation (SD), and Coefficient of Variation (CV) of the measured values were calculated, where CV is (standard deviation/average) × 100%, and the test results are shown in table 2.
TABLE 2 results of precision measurement
The result shows that the coefficient of variation CV detected by the kit provided by the invention is less than 5 percent, which shows that the kit provided by the invention has higher precision and better effect than the kits of comparative examples 1-5.
(3) Linear detection:
the test results are shown in Table 3, in which the test results are obtained by measuring lipoprotein a standards at 6 different concentrations, 0mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L and 100mg/L, 5 times for each gradient concentration, averaging the mean values of the measured values and the theoretical concentration of the standard, and the X-axis represents the theoretical concentration and the Y-axis represents the mean value of the measured values, respectively, using the kit of example 4.
TABLE 3 Linear test results
| Serial number | Theoretical concentration of lipoprotein a (mg/L) | Mean value of measured concentration of lipoprotein a (mg/L) |
| 1 | 0 | 0 |
| 2 | 10 | 9.7 |
| 3 | 20 | 19.5 |
| 4 | 40 | 39.2 |
| 6 | 80 | 79.0 |
| 7 | 100 | 99.3 |
The linear regression equation obtained was: 0.9929x-0.2536, correlation coefficient: r21. The result shows that the kit provided by the invention has good linear correlation relationship.
(4) And (3) stability detection:
the same serum sample (lipoprotein a concentration of 40mg/L) was assayed at 2-8 ℃ for 1 month, 3 months and 12 months using the kits of examples 1-4 and comparative examples 1-5, respectively, the sample was repeatedly assayed 10 times, and the average value, Standard Deviation (SD) and Coefficient of Variation (CV) of the assay of the sample concentration were calculated, and the assay results are shown in Table 4.
TABLE 4 stability test results
The results show that the reagent kit of the embodiment 1-4 is used for measuring the same serum sample in 1 month, 3 months and 12 months, the measured coefficient of variation CV is less than 5 percent, and the reagent kit of the embodiment 1-4 has good repeatability, stable performance and accurate measurement. CV of the kit of the comparative example 1 is more than 5 percent in 3-month detection, and CV of the kits of the comparative examples 2 and 5 is more than 5 percent in 12-month detection, which shows that the kits of the comparative examples 1, 2 and 5 have poor repeatability, unstable performance and inaccurate measurement. The CV of the kits of comparative examples 3 and 4 was close to 5% at 12 months of detection, and the stability was inferior to that of the kits of examples 1 to 4.
(5) Detecting the interference of anti-rheumatoid factor:
the kit of example 4 and the kits of comparative examples 1 to 5 were used to detect the concentration of lipoprotein a in each sample, and the same sample was tested 3 times, compared with a blank control (serum without rheumatoid factor added), and the relative deviation was calculated (the absolute value of the relative deviation is 10% or less, indicating no effect on the measured value), with the results shown in Table 5.
TABLE 5 results of anti-rheumatoid factor interference assays
The result shows that the kit of the embodiment 4 has no influence on the measured value when the rheumatoid factor is less than or equal to 1200 IU/mL; the kits of comparative examples 1 and 5 had no effect on the assay value when the rheumatoid factor was not more than 800 IU/mL. The kit of the comparative example 2 has no influence on the measured value when the rheumatoid factor is less than or equal to 1000 IU/mL. The reagent kits of comparative examples 3 and 4 have no influence on the measured value when the rheumatoid factor is less than or equal to 1200IU/mL, but the anti-interference capability is obviously inferior to that of the reagent kit of example 4.
(5) Anti-complement interference assay:
0.4, 0.8, 1.2, 1.6 and 2.0mg/mL of complement C3 was added to the same human serum sample, the concentration of lipoprotein a in each sample was measured using the kits of example 4 and comparative examples 1 to 5, the same sample was tested 3 times, and compared with a blank control (serum without complement C3), the relative deviation (absolute value of the relative deviation is 10% or less indicating no effect on the measured value) was calculated, and the results are shown in Table 6.
TABLE 6 results of anticomplementary interference detection
The result shows that the kit of the example 4 has no influence on the measured value when the complement C3 is less than or equal to 2.0 mg/mL; the kit of comparative example 1 has no influence on the measured value when complement C3 is less than or equal to 1.6 mg/mL. The kit of comparative example 5 had no effect on the assay value at complement C3 ≤ 1.2 mg/mL. The kits of comparative examples 2, 3 and 4 had no influence on the measured value when complement C3 was not more than 2.0mg/mL, but the anti-interference ability was significantly inferior to that of the kit of example 4.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (9)
1. A lipoprotein a detection kit comprises a reagent R1, a reagent R2 and a calibrator, and is characterized in that the reagent R1 comprises 50-250 mmol/L of a first buffer solution, 1-2 wt% of an anti-interference agent, 1-2.5 wt% of an accelerant and 0.01-0.1 wt% of a preservative; the reagent R2 comprises 50-250 mmol/L of second buffer solution, 0.5-5 wt% of stabilizing agent, 0.1-2 g/L of anti-human lipoprotein a monoclonal antibody coated latex particles and 0.02-0.2 wt% of preservative;
the anti-interference agent is composed of ergothioneine and TCEP-HCl in a mass ratio of 1 (1-2).
2. The lipoprotein a detection kit of claim 1, wherein the first buffer and the second buffer are each independently selected from at least one of MOPS buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, phosphate buffer, glycine buffer, Tris buffer, and Tris-HCl buffer.
3. The lipoprotein a detection kit of claim 1, wherein the anti-interference agent is ergothioneine and TCEP-HCl at a mass ratio of 1: 1.5.
4. The lipoprotein a detection kit of claim 1 wherein the stabilizer is at least one selected from the group consisting of mannooligosaccharide, carboxymethylglucan, sucrose, trehalose, glycerol, mannitol, and sorbitol.
5. The lipoprotein a detection kit of claim 4, wherein the stabilizer is composed of mannan-oligosaccharide and glycerol in a mass ratio of 1 (2-4).
6. The lipoprotein a detection kit of claim 1, wherein the accelerant is polyethylene glycol 6000, polyethylene glycol 8000, tween-20 or tween-80; the preservative is Proclin300 or NaN3。
7. The lipoprotein a detection kit of claim 1, wherein the anti-human lipoprotein a monoclonal antibody is covalently bound to the anti-human lipoprotein a monoclonal antibody in the latex particles coated with the anti-human lipoprotein a monoclonal antibody, wherein the anti-human lipoprotein a monoclonal antibody is goat anti-human, mouse anti-human or rabbit anti-human lipoprotein a monoclonal antibody; the latex particles are polystyrene latex particles with carboxyl on the surfaces, and the particle size of the particles is 50-300 nm.
8. A method for detecting the content of lipoprotein a using the kit of any one of claims 1 to 7 for non-disease diagnostic and therapeutic purposes, comprising the steps of:
(1) adding the reagent R1 into a sample to be tested, reacting for 4-6 min, adding the reagent R2, uniformly mixing, reading the absorbance A1, continuing to react for 4-6 min, reading the absorbance A2, and calculating delta A (A2-A1);
(2) and drawing a standard curve by using the lipoprotein a calibrator, and obtaining the content of the lipoprotein a in the sample to be detected according to the delta A and the standard curve.
9. The method of claim 8, wherein the volume ratio of the sample to be detected, the reagent R1 and the reagent R2 is 2 (120-180): (30-60), the reaction temperature is 37 ℃, and the detection wavelength is 600 nm.
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| CN113985028A (en) * | 2021-11-25 | 2022-01-28 | 济南百博生物技术股份有限公司 | Diluent for colloidal gold method antigen detection |
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| CN103149370A (en) * | 2013-02-27 | 2013-06-12 | 宁波美康生物科技股份有限公司 | Lipoprotein (a) detection kit |
| CN109541241A (en) * | 2019-01-25 | 2019-03-29 | 浙江夸克生物科技有限公司 | A kind of assay kit of lipoprotein (a) |
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| CN105675891A (en) * | 2016-01-26 | 2016-06-15 | 宁波天康生物科技有限公司 | Kit for testing lipoprotein a(Lp(a)) |
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| CN206540923U (en) * | 2017-03-17 | 2017-10-03 | 广州市伊川生物科技有限公司 | A kind of LP(a) determines kit |
| CN106950363A (en) * | 2017-03-31 | 2017-07-14 | 四川迈克生物科技股份有限公司 | Suppress the latex enhancing immune of rheumatoid factor interference than turbid reagent |
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| CN109541241A (en) * | 2019-01-25 | 2019-03-29 | 浙江夸克生物科技有限公司 | A kind of assay kit of lipoprotein (a) |
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