CN109541241B - A kind of assay kit of lipoprotein (a) - Google Patents
A kind of assay kit of lipoprotein (a) Download PDFInfo
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- CN109541241B CN109541241B CN201910070613.5A CN201910070613A CN109541241B CN 109541241 B CN109541241 B CN 109541241B CN 201910070613 A CN201910070613 A CN 201910070613A CN 109541241 B CN109541241 B CN 109541241B
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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
- G01N33/531—Production of immunochemical test materials
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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
-
- 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
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
Abstract
The present invention provides a kind of assay kits of lipoprotein (a), are related to technical field of biomedical detection.The kit includes reagent R1, R2, calibration object and quality-control product.Reagent R1 includes trishydroxymethylaminomethane, sodium chloride, polyethylene glycol-6000, biological preservative and surfactant；Reagent R2 includes trishydroxymethylaminomethane, goat-anti human lipoprotein (a) antibody latex particle, dibutylmethyl toluene, glycine, dextran sulfate, biological preservative and surfactant.The kit uses latex immunoturbidimetry, use the emulsion reagent of mixing partial size, automatic clinical chemistry analyzer can be used to detect simultaneously to great amount of samples, good linearity, detection sensitivity and accuracy, precision are improved, detection time is shortened, it is easy to operate, economic cost is lower, and storage time is longer.
Description
Technical field
The present invention relates to technical field of biomedical detection, and in particular to a kind of assay kit of lipoprotein (a).
Background technique
Lipoprotein (a) (lipoprotein (a), Lp (a)) is a kind of special independent plasma lipoprotein, and core is
Neutral lipid and apoB-100 molecule, periphery surround hydrophilic apoa, and the two is covalently attached with disulfide bond；Wherein apoa
The characteristic sugar protein ingredient of lipoprotein (a), mainly by it is a kind of be known as Kringle characteristic structural constitute, Kringle by
80-114 amino acid residue composition, stablizes by three internal disulfide bonds, is that Norway geneticist Berg in 1963 is being studied
It is found when the Genetic Variations of low-density lipoprotein.It mainly is secreted into blood after liver synthesis, in atharosclerosis mistake
It plays an important role in journey.Lipoprotein (a) and coronary heart disease, calcific aortic stenosis, familial hypercholesterolemia
Disease, peripheral artery disease etc. are closely related, and have become generally acknowledged coronary heart disease independentpredictor.
Currently, the detection method of detection lipoprotein (a) has radioimmunology, enzyme linked immunosorbent assay, immunoturbidimetry, but
It is that radioimmunology has radioactive pollution, enzyme linked immunosorbent assay is complicated for operation, time-consuming, has certain technical ability to want operator
It asks, immunoturbidimetry and the original cross contamination of fibrinolysin, therefore the accuracy measured is also just relatively low, needs to improve.Glue
Newborn particle enhancing turbidimetry (particle-enhanced turbidimetric immunoassay, PETIA) is to go out in recent years
Existing relatively stable, the accurate homogeneous immunoturbidimetry detection method of body fluid albumen of one kind.It is handed on the surface of polymer latex microballoon
Receipts or other documents in duplicate clonal antibody can flock together rapidly in a short time, change after the microballoon and antigen binding for being crosslinked with antibody
The astigmatism performance or light transmission of reaction solution.Change and the concentration of tested antigen of reaction solution light transmission (i.e. absorbance) have compared with
Strong correlation can reflect the concentration of tested antigen in a certain range.The simplicity of PETIA detection method is quick, sensitivity
Height can more effectively avoid the interference of many extraneous factors, and stability and repeatability are all preferable, can more accurately reflect measured object
The content of matter.
Currently, Chinese patent, which announces CN106501536A, discloses a kind of latex orientation coupling technology detection lipoprotein (a)
Kit, with antibody orientation coupling technology handle lipoprotein (a) monoclonal antibody latex microsphere, which includes reagent
1 with reagent 2, reagent R1:PBS buffer (PH7.0) 20-100mmol/L, PEG 8000 (PEG 8000) 10-30g/L,
Sodium azide 0.05%；Reagent R2:PBS buffer (PH7.0) 20-100mmol/L, anti-human lipoprotein (a) latex microsphere 10-
15mg/mL, EDTA0.3-0.6g/L, sucrose 25-60mmol/L, Sodium azide 0.05%, the kit can be reserved for 6 months or more,
Its testing result accuracy relative deviation >=3.56%, precision CV >=2.25%, linearly dependent coefficient are less than r2≤0.9971。
Chinese patent announces CN106093407A and discloses a kind of kit and preparation method thereof for measuring lipoprotein (a), the kit
Including reagent R1 and reagent R2, reagent R1 includes buffer, stabilizer, accelerator, preservative, anti-human rheumatoid factor antibodies,
Reagent R2 includes buffer, surfactant, stabilizer, suspending agent, preservative, anti-human lipoprotein (a) antibody of latex coating.Its
Testing result accuracy is higher, relative deviation >=0.66%, precision CV >=2.63%.
The kit of known detection serum lipoprotein (a) is less on the market at present, and there are biggish defects, such as sensitive
Degree is insufficient, accuracy and precision are lower, it is linear it is poor, the holding time is shorter, cumbersome, expensive etc., be unfavorable for
Clinical promotion and application.Therefore, it is urgent to provide a kind of detection sensitivity and accuracies and precision height, good linearity, holding time to grow
Lipoprotein (a) assay kit.
Summary of the invention
The purpose of the present invention is to provide a kind of assay kit of lipoprotein (a) and its methods of inspection.It overcomes existing
The problem of technology, improves accuracy and precision of measurement result etc..
One aspect of the present invention provides a kind of assay kit of lipoprotein (a), the kit include: reagent R1,
Reagent R2, calibration object and quality-control product；
The reagent R1 includes: trishydroxymethylaminomethane 0.01-0.03mol/L, sodium chloride 8-10g/L, poly- second two
- 6000 35-45g/L of alcohol, biological preservative 0.4-0.6g/L and surfactant 0.5-2mL/L；
The reagent R2 includes: trishydroxymethylaminomethane 0.01-0.03mol/L, goat-anti human lipoprotein (a) antibody glue
Newborn particle 200-240mL/L；
The calibration object includes: lipoprotein (a), sodium chloride 9g/L, polyethylene glycol-6000 40g/L, dextran sulfate
8g/L, biological preservative 0.5g/L and surfactant 1mL/L；
The quality-control product includes: lipoprotein (a) 200mg/L, sodium chloride 9g/L, polyethylene glycol-6000 40g/L, sulfuric acid
Dextran 8 g/L, biological preservative 0.5g/L and surfactant 1mL/L；
The biological preservative is NaN3, sodium benzoate, in Proclin-200, Proclin-300 or Proclin500
One or more；Preferably, the biological preservative is NaN3；
The surfactant is one or more of triton x-100, polysorbas20 or polysorbate60；Preferably, institute
The surfactant stated is triton x-100.
Preferably, the reagent R1 includes: trishydroxymethylaminomethane 0.01mol/L, sodium chloride 9g/L, poly- second two
- 6000 40g/L of alcohol, biological preservative 0.5g/L and surfactant 1mL/L.
Preferably, the reagent R2 includes: trishydroxymethylaminomethane 0.01mol/L, goat-anti human lipoprotein (a) antibody
Latex particle 220mL/L；It is further preferred that the reagent R2 further include: dibutylmethyl toluene 1.0-1.5g/L, sweet ammonia
Sour 2-6g/L, dextran sulfate 6-10g/L, biological preservative 0.4-0.6g/L and surfactant 0.5-2mL/L；Further
Preferably, the reagent R2 further include: dibutylmethyl toluene 1.3g/L, glycine 4g/L, dextran sulfate 8g/L, biology
Preservative 0.5g/L and surfactant 1mL/L.
Preferably, goat-anti human lipoprotein (a) antibody latex particle is goat-anti human lipoprotein (a) antibody in the reagent R2
Coated polymethyl methacrylate latex particle, the body of goat-anti human lipoprotein (a) antibody and polymethyl methacrylate latex
Product is than being 1:1-3；It is further preferred that the body of goat-anti human lipoprotein (a) antibody and polymethyl methacrylate latex
Product is than being 1:2.
Preferably, the polymethyl methacrylate latex is the polymethyl methacrylate glue that partial size is 60-100nm
The polymethyl methacrylate latex that cream and partial size are 100-200nm mixes, the polymethyl methacrylate of 60-100nm
The mass ratio for the polymethyl methacrylate latex that latex and partial size are 100-200nm is 1:2.
Preferably, the calibration object include lipoprotein (a) content be 0mg/L, 10mg/L, 50mg/L, 100mg/L,
The standard items of 6 gradients of 200mg/L and 300mg/L.
Another aspect of the present invention provides a kind of preparation method of goat-anti human lipoprotein (a) antibody latex particle, described
Method the following steps are included:
(1) goat-anti human lipoprotein (a) antibody-solutions are prepared: goat-anti human lipoprotein (a) antibody being taken to be dissolved in Tris-HCl buffering
In liquid, goat-anti human lipoprotein (a) antibody-solutions that concentration is 1.0mL/mL are made in pH 7.2；
(2) it prepares latex microsphere suspension: polymethyl methacrylate latex particle being taken to be dissolved in Tris-HCl buffer
In, concentration is 1% (w/v), and poly-aspartate and N, N- methylene-bisacrylamide is added, and is incubated for, and supernatant is abandoned in centrifugation, is used
Tris-HCl buffer washs latex microsphere twice, and Tris-HCl buffer is added, and concussion is resuspended, and it is 1% (w/v) that concentration, which is made,
Latex microsphere suspension；
(3) goat-anti human lipoprotein (a) antibody latex particle: goat-anti human lipoprotein (a) antibody that step (1) is obtained is prepared
The latex microsphere suspension that solution and step (2) obtain is mixed, and is incubated for, and 2 hydroxy ethylamine is added and mixes, closes, centrifugation, in abandoning
Clearly, it is washed twice with Tris-HCl buffer, Tris-HCl buffer is added, concussion is resuspended, and the sheep that concentration is 220mL/L is made
Anti-human lipoprotein (a) antibody latex particle.
Another aspect of the present invention provides a kind of detection method of lipoprotein (a), and the detection method is using above-mentioned
Assay kit detection human serum in lipoprotein (a) content.
Specifically, the detection method comprises the following steps:
(1) it draws standard curve: multiple spot calibration is carried out using calibration object, with the absorbance change value Δ A=of calibration object
AStandard-ABlankFor ordinate, respective concentration C standard is that abscissa draws standard curve；
(2) it detects sample to be tested: human serum sample and reagent R1 being mixed, 37 DEG C of incubation 5min, reagent R2 is added and mixes,
30s is stood, detects absorbance A under the wavelength of 600nm1, 37 DEG C of isothermal reaction 5min, reaction was completed, under the wavelength of 600nm
Detect absorbance A2, according to the standard curve that step (1) is drawn, utilize absorbance change value Δ A=A2-A1Calculate human serum sample
The concentration of lipoprotein (a) in this；
(3) quality controls: using quality-control product monitoring operation program, replication calculates relative deviation, and relative deviation is no more than
± 15%；
The volume ratio of the human serum sample, reagent R1 and reagent R2 is 2:245:33-38；Preferably, the people
The volume ratio of serum sample, reagent R1 and reagent R2 is 2:245:35.
The human serum sample is the not haemolysis Diagnostic Value of Fasting Serum of people.
In addition, bilirubin≤300 μm ol/L, hemoglobin≤4.0g/L, triglycerides≤10mmol/L, to testing result
Nothing significantly interferes with.
Compared with prior art, positive and beneficial effect of the invention is:
A kind of lipoprotein (a) assay kit provided by the present invention uses latex immunoturbidimetry technology, uses mixing
The emulsion reagent of partial size, it is linear more preferable.It is added to dibutylmethyl toluene, glycine, dextran sulfate in the reaction system, instead
The more stable property of liquid is answered, the accuracy and precision of testing result are improved, kit storage time is longer.A certain amount of gallbladder is red
The chaff interferents such as element, hemoglobin, triglycerides are to testing result without significantly interfering with.With quality-control product monitoring operation program, detection knot
Fruit is more accurate reliable.In addition, automatic clinical chemistry analyzer measurement lipoprotein (a) can be used, great amount of samples can be carried out simultaneously
Detection, detection sensitivity is high, and as a result accurately, easy to operate, economic cost is lower.
Detailed description of the invention
Fig. 1 is the linearly related figure in embodiment 12；
Fig. 2 is the linearly related figure in embodiment 13；
Fig. 3 is the linearly related figure in embodiment 14.
Specific embodiment
The explanation of following embodiment is merely used to help understand method and its core concept of the invention.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.To disclosed implementation
The following the description of example, enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments
It will be readily apparent to those skilled in the art, the general principles defined herein can not depart from this
In the case where the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein
These embodiments in, but can be applied to meet broader model consistent with the principles and novel features disclosed in this article
It encloses.Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method and material
Material, preferred method and material are enumerated by place herein.
Unless otherwise defined, all technical and scientific terms used herein have and the technical field of the invention
The normally understood identical meaning of those of ordinary skill.
Embodiment 1: the preparation of goat-anti human lipoprotein (a) antibody latex particle
(1) goat-anti human lipoprotein (a) antibody-solutions are prepared: goat-anti human lipoprotein (a) antibody being taken to be dissolved in Tris-HCl buffering
In liquid, goat-anti human lipoprotein (a) antibody-solutions that concentration is 1.0mL/mL are made in pH 7.2；
(2) prepare latex microsphere suspension: according to mass ratio be 1:2 ratio, take respectively partial size be 60-100nm and
The polymethyl methacrylate latex particle of two kinds of specifications of 100-200nm is dissolved in Tris-HCl buffer, concentration 1%
(w/v), the N of poly-aspartate and 10mg/mL that concentration is 18mg/mL, N- methylene-bisacrylamide, in room are sequentially added
Temperature is incubated for 45 minutes, high speed centrifugation (12000rpm, 30min), is abandoned supernatant, is washed glue with the Tris-HCl buffer of same volume
Newborn microballoon twice, removes extra crosslinking agent, after washing, then latex microsphere is dissolved in Tris-HCl buffer, 25 DEG C of concussions
15min is resuspended, and the latex microsphere suspension that concentration is 1% (w/v) is made；
(3) goat-anti human lipoprotein (a) antibody latex particle: goat-anti human lipoprotein (a) antibody that step (1) is obtained is prepared
The latex microsphere suspension that solution and step (2) obtain is uniformly mixed according to the ratio that volume ratio is 1:1, and 37 DEG C are incubated for 3 hours,
The 2 hydroxy ethylamine for adding 4 μ l/mL mixes, and closes 30 minutes at 37 DEG C, high speed centrifugation (12000rpm, 30min), in abandoning
Clearly, it is washed twice with the Tris-HCl buffer of same volume, removes unbonded antibody, be added Tris-HCl buffer, 25
DEG C concussion 15min be resuspended, be made concentration be 220mL/L goat-anti human lipoprotein (a) antibody latex particle.
Embodiment 2: the preparation of goat-anti human lipoprotein (a) antibody latex particle
Compared with Example 1, only in step (3) goat-anti human lipoprotein (a) antibody-solutions and latex microsphere suspension ratio
Example is different, and volume ratio is 1:2 in the present embodiment.
Embodiment 3: the preparation of goat-anti human lipoprotein (a) antibody latex particle
Compared with Example 1, only in step (3) goat-anti human lipoprotein (a) antibody-solutions and latex microsphere suspension ratio
Example is different, and volume ratio is 1:3 in the present embodiment.
Embodiment 4: the assay kit of lipoprotein (a)
The assay kit of lipoprotein (a) is specifically formulated to point as follows:
Reagent R1: trishydroxymethylaminomethane 0.02mol/L, sodium chloride 8g/L, polyethylene glycol-6000 35g/L, biology
Preservative 0.4g/L and surfactant 0.5mL/L；
Reagent R2: trishydroxymethylaminomethane 0.02mol/L, goat-anti human lipoprotein (a) antibody latex particle 200mL/L；
Calibration object: lipoprotein (a) content is respectively 0mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L and 300mg/
Six concentration gradients of L, sodium chloride 9g/L, polyethylene glycol-6000 40g/L, dextran sulfate 8g/L, biological preservative 0.5g/
L and surfactant 1mL/L；
Quality-control product: lipoprotein (a) 200mg/L, sodium chloride 9g/L, polyethylene glycol-6000 40g/L, dextran sulfate 8g/
L, biological preservative 0.5g/L and surfactant 1mL/L；
Wherein, the biological preservative is NaN3, sodium benzoate, Proclin-200, Proclin-300 or
One or more of Proclin500；The surfactant is one of triton x-100, polysorbas20 or polysorbate60
Or it is several.Goat-anti human lipoprotein (a) antibody latex particle used is made by embodiment 2.
Embodiment 5: the assay kit of lipoprotein (a)
Compared with Example 4, only reagent R1 and reagent R2 is different.
Reagent R1: trishydroxymethylaminomethane 0.01mol/L, sodium chloride 9g/L, polyethylene glycol-6000 40g/L, biology
Preservative 0.5g/L and surfactant 1mL/L；
Reagent R2: trishydroxymethylaminomethane 0.01mol/L, goat-anti human lipoprotein (a) antibody latex particle 220mL/L.
Embodiment 6: the assay kit of lipoprotein (a)
Compared with Example 4, only reagent R1 and reagent R2 is different.
Reagent R1: trishydroxymethylaminomethane 0.03mol/L, sodium chloride 10g/L, polyethylene glycol-6000 45g/L, biology
Preservative 0.6g/L and surfactant 2mL/L；
Reagent R2: trishydroxymethylaminomethane 0.03mol/L, goat-anti human lipoprotein (a) antibody latex particle 240mL/L.
Embodiment 7: the assay kit of lipoprotein (a)
Compared with Example 5, only reagent R2 is different.
Reagent R2: trishydroxymethylaminomethane 0.01mol/L, goat-anti human lipoprotein (a) antibody latex particle 220mL/L,
Dibutylmethyl toluene 1.0g/L, glycine 6g/L, dextran sulfate 6g/L, biological preservative 0.4g/L and surfactant
0.5mL/L。
Embodiment 8: the assay kit of lipoprotein (a)
Compared with Example 5, only reagent R2 is different.
Reagent R2: trishydroxymethylaminomethane 0.01mol/L, goat-anti human lipoprotein (a) antibody latex particle 220mL/L,
Dibutylmethyl toluene 1.3g/L, glycine 4g/L, dextran sulfate 8g/L, biological preservative 0.5g/L and surfactant
1mL/L。
Embodiment 9: the assay kit of lipoprotein (a)
Compared with Example 5, only reagent R2 is different.
Reagent R2: trishydroxymethylaminomethane 0.01mol/L, goat-anti human lipoprotein (a) antibody latex particle 220mL/L,
Dibutylmethyl toluene 1.5g/L, glycine 2g/L, dextran sulfate 10g/L, biological preservative 0.6g/L and surfactant
2mL/L。
Embodiment 10: the assay kit of lipoprotein (a)
Compared with Example 8, difference is only that, goat-anti human lipoprotein (a) antibody latex particle used is made by embodiment 1
?.
Embodiment 11: the assay kit of lipoprotein (a)
Compared with Example 8, difference is only that, goat-anti human lipoprotein (a) antibody latex particle used is made by embodiment 3
?.
A kind of comparative example 1: kit of latex orientation coupling technology detection lipoprotein (a)
Chinese patent announces kit described in CN106501536A.
A kind of comparative example 2: kit and preparation method thereof measuring lipoprotein (a)
Chinese patent announces kit described in CN106093407A.
Embodiment 12: the detection of lipoprotein (a)
Detection is followed the steps below using automatic clinical chemistry analyzer:
(1) it draws standard curve: multiple spot calibration is carried out using calibration object, with the absorbance change value Δ A=of calibration object
AStandard-ABlankFor ordinate, respective concentration C standard is that abscissa draws standard curve；
(2) it detects sample to be tested: taking 2 μ l of human serum sample and 245 μ l reagent R1, be mixed, 37 DEG C of incubation 5min are added 33
μ l reagent R2 is mixed, and is stood 30s, is detected absorbance A under the wavelength of 600nm1, 37 DEG C of isothermal reaction 5min, reaction was completed,
Absorbance A is detected under the wavelength of 600nm2, according to the standard curve that step (1) is drawn, utilize absorbance change value Δ A=A2-A1
The concentration of the lipoprotein (a) in human serum sample is calculated, the human serum sample is the not haemolysis Diagnostic Value of Fasting Serum of people；
(3) quality controls: using quality-control product monitoring operation program, replication calculates relative deviation, and relative deviation is no more than
± 15%.
A. precision detects: continuous drawing 20 times in same sample are measured according to above-mentioned steps, calculate measured value
Average value, standard deviation (SD) and the coefficient of variation (CV), CV=(standard deviation/average value) * 100%, the results are shown in Table 1, CV and usually use
In the precision for measuring a measuring method, CV value is smaller, indicates that the result precision of the measuring method is better.For clinicization
For learning inspection project, method precision of the CV less than 5% is generally recognised as acceptable.
Table 1: precision testing result:
| Kit | Average value (mg/L) | Standard deviation (SD) | The coefficient of variation (CV) |
| Embodiment 4 | 175.61 | 5.13 | 2.92% |
| Embodiment 5 | 176.02 | 3.69 | 2.10% |
| Embodiment 6 | 176.15 | 4.16 | 2.36% |
| Embodiment 7 | 176.28 | 2.94 | 1.67% |
| Embodiment 8 | 176.42 | 2.31 | 1.31% |
| Embodiment 9 | 176.60 | 3.22 | 1.82% |
| Embodiment 10 | 176.54 | 4.67 | 2.65% |
| Embodiment 11 | 176.46 | 3.85 | 2.18% |
| Comparative example 1 | 75.38 | 1.7 | 2.25% |
| Comparative example 2 | 115.9 | 3.05 | 2.63% |
CV value≤2.92% in table 1 shows that kit precision provided by the invention is higher, wherein described in embodiment 8
Kit precision highest.
B. accuracy detects: being measured with the quality-control product that lipoprotein (a) antigenic content is 200mg/L, is repeated 5 times, takes
Mean value, testing result are shown in Table 2.For clinical chemistry test project, relative deviation (CB) is no more than ± 15%, is considered having
There is excellent accuracy.
Table 2: accuracy testing result
Relative deviation is no more than ± 1.17% in table 2, shows that kit accuracy provided by the invention is higher, wherein real
Apply kit accuracy highest described in example 8.
C. Detection of Stability: anti-to lipoprotein (a) at 0 month, 6 months and 12 months respectively under 2-8 DEG C of condition of storage
Former content is that the same standard items sample of 100mg/L is measured, and each sample measures 10 times take mean value, the results are shown in Table 3.
Table 3: Detection of Stability result
| Kit | 0 month | 6 months | 12 months |
| Embodiment 4 | 95.73 | 95.67 | 95.48 |
| Embodiment 5 | 103.87 | 103.91 | 104.25 |
| Embodiment 6 | 96.14 | 96.21 | 96.84 |
| Embodiment 7 | 102.53 | 102.71 | 103.54 |
| Embodiment 8 | 101.86 | 102.34 | 103.26 |
| Embodiment 9 | 97.34 | 97.12 | 96.65 |
| Embodiment 10 | 98.75 | 98.36 | 97.66 |
| Embodiment 11 | 103.21 | 103.74 | 104.35 |
As can be seen from Table 3, detected value difference is smaller, shows that stabilization of kit provided by the invention is preferable, at 2-8 DEG C
Lower storage can at least stablize 12 months or more, and wherein stabilization of kit described in embodiment 8 is best.
D. linearity test: detecting the standard items of six concentration gradients using kit described in embodiment 8, and each gradient is dense
Degree detection 3 times, is averaged, the theoretical concentration of average value and standard items to measured value makees regression analysis, and X-axis representation theory is dense
Degree, Y-axis indicate measured value mean value.General R2>=0.9900 is considered to have good linear, the result is shown in Figure 1.
Obtained equation of linear regression are as follows: y=1.0166x+0.8921, related coefficient: R2=0.9981.The result shows that
Kit provided by the invention has good linear relationship.
Embodiment 13: the detection of lipoprotein (a)
Compared with embodiment 12, difference is only that, step (2) detects sample to be tested: 35 μ l reagent R2 are added.
A. precision detects: it is identical as method described in embodiment 12, it the results are shown in Table 4.
Table 4: precision testing result:
| Kit | Average value (mg/L) | Standard deviation (SD) | The coefficient of variation (CV) |
| Embodiment 4 | 175.96 | 4.97 | 2.82% |
| Embodiment 5 | 176.51 | 3.16 | 1.79% |
| Embodiment 6 | 175.66 | 5.11 | 2.91% |
| Embodiment 7 | 176.30 | 2.87 | 1.63% |
| Embodiment 8 | 176.41 | 1.95 | 1.11% |
| Embodiment 9 | 176.59 | 3.84 | 2.17% |
| Embodiment 10 | 176.45 | 4.18 | 2.37% |
| Embodiment 11 | 176.63 | 3.29 | 1.86% |
| Comparative example 1 | 75.38 | 1.7 | 2.25% |
| Comparative example 2 | 115.9 | 3.05 | 2.63% |
CV value≤2.91% in table 4 shows that kit precision provided by the invention is higher, wherein described in embodiment 8
Kit precision highest.
B. accuracy detects: it is identical as method described in embodiment 12, it the results are shown in Table 5.
Table 5: accuracy testing result
| Kit | It measures mean value (mg/L) | Standard value (mg/L) | Relative deviation (CB) |
| Embodiment 4 | 198.96 | 200.0 | - 0.52% |
| Embodiment 5 | 202.03 | 200.0 | 1.02% |
| Embodiment 6 | 201.24 | 200.0 | 0.62% |
| Embodiment 7 | 201.26 | 200.0 | 0.63% |
| Embodiment 8 | 199.38 | 200.0 | - 0.31% |
| Embodiment 9 | 199.12 | 200.0 | - 0.44% |
| Embodiment 10 | 201.19 | 200.0 | 0.59% |
| Embodiment 11 | 198.07 | 200.0 | - 0.97% |
| Comparative example 1 | 259.0 | 250.0 | 3.56% |
| Comparative example 2 | 194.7 | 196.0 | 0.66% |
Relative deviation is no more than ± 1.02% in table 5, shows that kit accuracy provided by the invention is higher, wherein real
Apply kit accuracy highest described in example 8.
C. Detection of Stability: it is identical as method described in embodiment 12, it the results are shown in Table 6.
Table 6: Detection of Stability result
| Kit | 0 month | 6 months | 12 months |
| Embodiment 4 | 96.13 | 95.88 | 95.46 |
| Embodiment 5 | 103.45 | 103.72 | 104.55 |
| Embodiment 6 | 96.76 | 96.33 | 96.01 |
| Embodiment 7 | 101.98 | 102.35 | 102.97 |
| Embodiment 8 | 99.54 | 99.36 | 99.10 |
| Embodiment 9 | 98.45 | 98.12 | 97.95 |
| Embodiment 10 | 102.81 | 102.97 | 103.22 |
| Embodiment 11 | 103.44 | 103.56 | 103.87 |
As can be seen from Table 6, detected value difference is smaller, shows that stabilization of kit provided by the invention is preferable, at 2-8 DEG C
Lower storage can at least stablize 12 months or more, and wherein stabilization of kit described in embodiment 8 is best.
D. linearity test: it is identical as method described in embodiment 12, as a result see Fig. 2.
Obtained equation of linear regression are as follows: y=1.0306x-0.9979, related coefficient: R2=0.9994.The result shows that
Kit provided by the invention has good linear relationship.
Embodiment 14: the detection of lipoprotein (a)
Compared with embodiment 12, difference is only that, step (2) detects sample to be tested: 38 μ l reagent R2 are added.
A. precision detects: it is identical as method described in embodiment 12, it the results are shown in Table 7.
Table 7: precision testing result:
CV value≤2.93% in table 7 shows that kit precision provided by the invention is higher, wherein described in embodiment 8
Kit precision highest.
B. accuracy detects: it is identical as method described in embodiment 12, it the results are shown in Table 8.
Table 8: accuracy testing result
| Kit | It measures mean value (mg/L) | Standard value (mg/L) | Relative deviation (CB) |
| Embodiment 4 | 198.72 | 200.0 | - 0.64% |
| Embodiment 5 | 198.33 | 200.0 | - 0.83% |
| Embodiment 6 | 201.23 | 200.0 | 0.61% |
| Embodiment 7 | 198.75 | 200.0 | - 0.63% |
| Embodiment 8 | 200.96 | 200.0 | 0.48% |
| Embodiment 9 | 201.09 | 200.0 | 0.55% |
| Embodiment 10 | 198.86 | 200.0 | - 0.57% |
| Embodiment 11 | 198.01 | 200.0 | - 1.00% |
| Comparative example 1 | 259.0 | 250.0 | 3.56% |
| Comparative example 2 | 194.7 | 196.0 | 0.66% |
Relative deviation is no more than ± 1.00% in table 8, shows that kit accuracy provided by the invention is higher, wherein real
Apply kit accuracy highest described in example 8.
C. Detection of Stability: it is identical as method described in embodiment 12, it the results are shown in Table 9.
Table 9: Detection of Stability result
| Kit | 0 month | 6 months | 12 months |
| Embodiment 4 | 105.09 | 105.18 | 105.36 |
| Embodiment 5 | 97.63 | 97.54 | 97.31 |
| Embodiment 6 | 104.49 | 104.62 | 104.74 |
| Embodiment 7 | 98.68 | 98.58 | 98.56 |
| Embodiment 8 | 101.75 | 101.82 | 102.06 |
| Embodiment 9 | 102.56 | 102.66 | 102.79 |
| Embodiment 10 | 102.87 | 102.76 | 102.98 |
| Embodiment 11 | 103.07 | 103.22 | 103.54 |
As can be seen from Table 9, detected value difference is smaller, shows that stabilization of kit provided by the invention is preferable, at 2-8 DEG C
Lower storage can at least stablize 12 months or more, and wherein stabilization of kit described in embodiment 8 is best.
D. linearity test: it is identical as method described in embodiment 12, as a result see Fig. 3.
Obtained equation of linear regression are as follows: y=1.0053x+1.3572, related coefficient: R2=0.9978.The result shows that
Kit provided by the invention has good linear relationship.
Embodiment 15: interference test
Three kinds of potential interference objects are selected, bilirubin, hemoglobin, triglycerides are separately added into 320 μ in quality-control product
The bilirubin of mol/L, 340 μm of ol/L, 360 μm of ol/L；The hemoglobin of 3.0g/L, 5.0g/L, 7.0g/L；8mmol/L,
The triglycerides of 10mmol/L, 12mmol/L.Control group is the quality-control product that any chaff interferent is not added, using described in embodiment 8
Detection method described in kit and embodiment 13 is measured, and each sample measures 5 times take mean value, the results are shown in Table 10.
Table 10: chaff interferent test result
As can be seen from Table 10, bilirubin≤300 μm ol/L, hemoglobin≤4.0g/L, triglycerides≤10mmol/L,
To testing result without significantly interfering with.
To sum up, the detectable result precision of kit provided by the invention is high, precision is high, stability is good, good linearity.
It is wherein detected using detection method described in kit described in embodiment 8 and embodiment 13, testing result is best.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (4)
1. a kind of assay kit of lipoprotein (a), which is characterized in that the kit includes: reagent R1, reagent R2, school
Quasi- product and quality-control product；
The reagent R1 includes: trishydroxymethylaminomethane 0.01mol/L, sodium chloride 9g/L, polyethylene glycol-6000 40g/
L, biological preservative 0.5g/L and surfactant 1mL/L；
The reagent R2 includes: trishydroxymethylaminomethane 0.01mol/L, goat-anti human lipoprotein (a) antibody latex particle
220mL/L, dibutylmethyl toluene 1.3g/L, glycine 4g/L, dextran sulfate 8g/L, biological preservative 0.5g/L and surface
Activating agent 1mL/L；
The biological preservative is NaN3；
The surfactant is triton x-100；
The calibration object include: lipoprotein (a), sodium chloride 9g/L, polyethylene glycol-6000 40g/L, dextran sulfate 8g/L,
Biological preservative 0.5g/L and surfactant 1mL/L；
The quality-control product includes: that lipoprotein (a) 200mg/L, sodium chloride 9g/L, polyethylene glycol-6000 40g/L, sulfuric acid Portugal are poly-
Sugared 8g/L, biological preservative 0.5g/L and surfactant 1mL/L；
Goat-anti human lipoprotein (a) the antibody latex particle is the coated polymethylacrylic acid of goat-anti human lipoprotein (a) antibody
The volume ratio of methyl esters latex particle, goat-anti human lipoprotein (a) antibody and polymethyl methacrylate latex is 1:2；
The polymethyl methacrylate latex is the polymethyl methacrylate latex that partial size is 60-100nm and partial size is
The polymethyl methacrylate latex of 100-200nm mixes, the polymethyl methacrylate latex and partial size of 60-100nm
Mass ratio for the polymethyl methacrylate latex of 100-200nm is 1:2；
The preparation method of goat-anti human lipoprotein (a) the antibody latex particle, comprising the following steps:
(1) goat-anti human lipoprotein (a) antibody-solutions are prepared: goat-anti human lipoprotein (a) antibody being taken to be dissolved in Tris-HCl buffer,
PH is 7.2, and goat-anti human lipoprotein (a) antibody-solutions that concentration is 1.0mL/mL are made；
(2) it prepares latex microsphere suspension: polymethyl methacrylate latex particle being taken to be dissolved in Tris-HCl buffer, it is dense
Degree is 1% (w/v), and poly-aspartate and N, N- methylene-bisacrylamide is added, and is incubated for, and centrifugation abandons supernatant, uses Tris-HCl
Buffer washs latex microsphere twice, and Tris-HCl buffer is added, and concussion is resuspended, and it is micro- that the latex that concentration is 1% (w/v) is made
Ball suspension；
(3) goat-anti human lipoprotein (a) antibody latex particle: goat-anti human lipoprotein (a) antibody-solutions that step (1) is obtained is prepared
The latex microsphere suspension obtained with step (2) is mixed, and is incubated for, and 2 hydroxy ethylamine is added and mixes, closes, and supernatant is abandoned in centrifugation, is used
Tris-HCl buffer washes twice, and Tris-HCl buffer is added, and concussion is resuspended, and the goat-anti people that concentration is 220mL/L is made
Lipoprotein (a) antibody latex particle.
2. assay kit according to claim 1, which is characterized in that the calibration object includes containing for lipoprotein (a)
Amount is respectively six concentration gradient standard items of 0mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L and 300mg/L.
3. a kind of detection method of the lipoprotein (a) of nondiagnostic purpose, which is characterized in that the detection method is exploitation right
Benefit requires the content of lipoprotein (a) in the detection human serum of assay kit described in 1-2 any one.
4. detection method according to claim 3, which is characterized in that the detection method comprises the following steps:
(1) it draws standard curve: multiple spot calibration is carried out using calibration object, with the absorbance change value Δ A=A standard-A of calibration object
Blank is ordinate, and respective concentration C standard is that abscissa draws standard curve；
(2) it detects sample to be tested: human serum sample and reagent R1 being mixed, 37 DEG C of incubation 5min, reagent R2 is added and mixes, stands
30s detects 1,37 DEG C of isothermal reaction 5min of absorbance A under the wavelength of 600nm, and reaction was completed, detects under the wavelength of 600nm
Absorbance A 2 is calculated in human serum sample according to the standard curve that step (1) is drawn using absorbance change value Δ A=A2-A1
Lipoprotein (a) concentration；
(3) quality control: use quality-control product monitoring operation program, replication, calculate relative deviation, relative deviation be no more than ±
15%；
The volume ratio of the human serum sample, reagent R1 and reagent R2 is 2:245:33-38；
The human serum sample is the not haemolysis Diagnostic Value of Fasting Serum of people.
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| CN110553985B (en) * | 2019-08-14 | 2020-06-30 | 湖州欧邦生物科技有限公司 | Pepsinogen's assay kit |
| CN110850107A (en) * | 2019-11-22 | 2020-02-28 | 武汉市长立生物技术有限责任公司 | Kit for detecting lipoprotein (a) molecular concentration and application thereof |
| CN111426845B (en) * | 2020-04-07 | 2021-06-15 | 中拓生物有限公司 | Serum apolipoprotein B determination kit and preparation method and application thereof |
| CN112255421B (en) * | 2020-10-13 | 2021-07-23 | 北京永基恒声科技有限公司 | Lipoprotein a detection kit and detection method |
| CN112763724A (en) * | 2020-12-26 | 2021-05-07 | 北京安图生物工程有限公司 | Reagent combination and kit for simultaneously determining content of retinol binding protein in serum and urine |
| CN112763731B (en) * | 2020-12-29 | 2023-02-28 | 广州市伊川生物科技有限公司 | Lipoprotein (a) determination kit and detection method thereof |
| CN113030484B (en) * | 2021-03-10 | 2024-04-02 | 四川携光生物技术有限公司 | Procalcitonin sensitization latex, kit, preparation method and application |
| CN115078739B (en) * | 2022-08-22 | 2022-11-22 | 上海执诚生物科技有限公司 | Kit for detecting apolipoprotein A1 and application thereof |
| CN116298317A (en) * | 2023-03-14 | 2023-06-23 | 浙江夸克生物科技有限公司 | Alpha fetoprotein determination kit based on latex immunoturbidimetry |
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| CN101769932B (en) * | 2010-01-26 | 2013-09-18 | 宁波美康生物科技股份有限公司 | Full-range C-reactive protein detection kit |
| CN101819202B (en) * | 2010-05-06 | 2012-12-26 | 上海太阳生物技术有限公司 | D-Dimer measuring kit (latex immunonephelometry method) |
| US9952225B2 (en) * | 2014-01-24 | 2018-04-24 | National Jewish Health | Methods for detection of respiratory diseases |
| JP6651794B2 (en) * | 2015-11-05 | 2020-02-19 | 富士レビオ株式会社 | Cardiac troponin I measuring reagent and method |
| CN106526198A (en) * | 2016-10-03 | 2017-03-22 | 王贤俊 | Combined orientation agents optimized latex coupled antibody detection method of prealbumin (PA) |
| CN108614107A (en) * | 2016-12-12 | 2018-10-02 | 上海复星长征医学科学有限公司 | A kind of methotrexate (MTX) latex immunoturbidimetry assay kit and preparation method thereof |
| CN108593641B (en) * | 2018-04-09 | 2021-12-03 | 桂林优利特医疗电子有限公司 | Kit and method for quantitatively detecting substance to be detected in whole blood sample |
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Denomination of invention: A kit for the determination of lipoprotein (a) Effective date of registration: 20230924 Granted publication date: 20190920 Pledgee: Xinchang Zhejiang rural commercial bank Limited by Share Ltd. Pledgor: ZHEJIANG KUAKE BIOTECHNOLOGY Co.,Ltd. Registration number: Y2023330002129 |