CN105974143A - Enzyme-linked immunosorbent assay kit for detection of human oxidized low-density lipoprotein and preparation method thereof - Google Patents
Enzyme-linked immunosorbent assay kit for detection of human oxidized low-density lipoprotein and preparation method thereof Download PDFInfo
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- 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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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/545—Synthetic resin
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/104—Lupus erythematosus [SLE]
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/321—Arterial hypertension
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
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- G—PHYSICS
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- 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
Abstract
An enzyme-linked immunosorbent assay (ELISA) kit for detection of human oxidized low-density lipoprotein and a preparation method thereof are disclosed. The invention belongs to the field of in vitro test diagnostic reagents and specifically relates to a kit for content measurement of Ox-LDL in human body and preparation and application of the kit. The kit comprises an ELISA plate for immobilization of a recombinant protein P.rbeta2-GPI-DV or E.coli recombinant protein rbeta2-GPI-DV with a novel ligand structure which can specifically bind oxidized low-density lipoprotein, a standard control sample of different concentrations of oxidized low-density lipoprotein, a quality control, an enzyme-labeled antibody, a diluent, a wash buffer, a developing solution A, a developing solution B and a stop buffer. Ox-LDL detected by combining yeast recombinant protein P.rbeta2-GPI-DV or E.coli recombinant protein rbeta2-GPI-DV used in the invnetion and oxLDL novel ligand recognition sites is pathogenic Ox-LDL which causes atherosclerosis, cardiovascular disease and nonalcoholic fatty liver disease's inflammatory response. The kit has application value for accurate assistant diagnoses of diseases accompanied by atherosclerosis, abnormal lipids metabolism and inflammatory response. The detecting instrument used in the invention is simple; detection cost is low; and non-professionals can realize detection operation.
Description
Technical field
The invention belongs to vitro detection diagnostic reagent field, the OxLDL ELISA Ox-LDL being specifically related in a kind of human body
The test kit of content detection and preparation and application.
Background technology
Low density lipoprotein, LDL LDL is a kind of apolipoprotein being present in blood plasma, and core component is triglyceride and cholesterol ester,
Surface distributed Genetyping PO-B, free cholesterol and phospholipid.Active oxygen, cell, transition metal, covellite egg in vivo
In vain, peroxidase, haemachrome, chlorophyll and more extraneous sensible factors such as, smoking, medicine, diabetes and hypertension
Deng oxidative modification effect under formed Ox-LDL.In ox-LDL oxidized derivatives, oxidized phospholipids, oxidation sterin and modification change
ApoB antibody be that it plays pathogenic key factor.
It is to cause atherosclerosis, cardiovascular disease, autoimmunity cardiovascular disease and non-wine that oxLDL is pointed in numerous researchs
The biomarker of essence fatty liver hepatitis.Ox-LDL as the independent risk factor of atherosclerotic agent cardiovascular disease,
It is widely present in disease focus and blood samples of patients, by causing endothelial injury, vessel wall thickening, lipidosis, hematic acid to be formed
The course of disease through whole disease is changed Deng case series；Research in recent years finds, the anti-phospholipid syndrome in autoimmune disease
(APS) containing substantial amounts of Ox-LDL and in systemic lupus erythematosus (sle) (SLE) blood samples of patients, can be with the glycoprotein in blood
β 2-GPI, anti-β2-GPI antibody form immunity ternary complex, play the pathomechanism promoting foam wanshing, participate in tremulous pulse medicated porridge
The formation [1,2] of sample hardening stove, research in recent years more shows that oxLDL is probably in fatty liver especially non-alcoholic fatty liver disease hepatopathy
Therefore initiated oxidation, stress cause the lipid metabolism risk factor of hepatitis after inflammation, the Ox-LDL such as human blood, serum and body fluid
The accurately detection of content is great to cardiovascular disease and the prevention of lipoidosis disease, monitoring and early stage diagnostic significance.
At present, in prior art, have been disclosed for some relevant detection methods, by sxemiquantitative or quantitative by the way of realize
Ox-LDL detects, such as: number of patent application is: 200710202084.7, and 201210160797.2,201510263037.8,
In invention disclosed above technology, the critical packet that detection is used is the Dan Ke of anti-oxidation low-density lipoprotein by solid phase main body
Grand antibody.But OxLDL ELISA oxLDL mobility between blood and focus and oxidative modification degree, oxidative modification
The feature of derivant complicated, oxLDL exists with the inhomogenous composite form of composition in vivo, its oxidative modification derivant
It is that it plays new key factor of causing a disease, the most accurately reaches to identify the oxidized derivatives part of pathogenic oxLDL, reach precisely to examine
Survey oxLDL and to atherosclerosis, cardiovascular disease, autoimmunity APS and SLE and non-alcoholic fatty liver disease etc.
Assistance application in disease development process is the purpose of the present invention.
Summary of the invention
Technical scheme provide a kind of can specific binding cause macrophage foam cell formation and produce inflammatory factor cause
The new technique of characteristic of disease Ox-LDL detection, the core of this technology is a kind of yeast expression P.r β 2-GPI DV recombiant protein specific recognition
Pathogenic oxLDL surface ligand, this surface ligand has the pathogenic oxLDL of mediation and excites the surface such as macrophage and hepatocyte
Receptor and intracellular receptor, the ANOMALOUS VARIATIONS of trigger cell lipid metabolism causes the diseases such as atherosclerosis, cardiovascular and lipid metabolism
Sick generation.This inventive technique Cleaning Principle is clear, and experimental apparatus simple to operate, that relate to is single, cheap and has essence
The speciality of quasi-detection oxLDL recognition site is clinical cardiovascular disease, the atherosclerosis autoimmune that occurs together disease and fat
Dysbolismus medical diagnosis on disease valuable auxiliary diagnostic test kits.
The enzyme-linked immunologic detecting kit of a kind of human body OxLDL ELISA oxLDL, has specific binding including immobilization
The ELISA Plate of the recombiant protein of oxLDL, the oxLDL standard control sample of variable concentrations, quality-control product, the antibody of enzyme labelling, dilute
Release liquid, cleanout fluid, nitrite ion A, nitrite ion B and stop buffer.
Further, described ELISA Plate is 96 microwell plates of polystyrene；Described immobilised specific recombinant proteins is ferment
Female recombinant expressed P.r β 2-GPI-DV albumen；The antibody of described enzyme labelling is the anti-of the goat-anti people of horseradish peroxidase-labeled
ApoB antibody；Described diluent is phosphate buffer；Described cleanout fluid is the phosphate buffer containing polysorbas20；
The described citrate buffer that nitrite ion A is hydrogen peroxide；Described nitrite ion B is o-phenylenediamine (OPD) solution；
Described stop buffer is sulfuric acid solution.
Further, on described test kit microwell plate, the sample size of immobilised recombiant protein is 50ug/ hole；Described oxidation is low close
Degree lipoprotein oxLDL standard reference material is 6, each 2ml；Described quality-control product 5ml, 2；Described Radix Cochleariae officinalis peroxidating
The goat-anti people anti-apoB antibody 1 of thing enzyme labelling, 10ul；Described diluent 10ml, 1；Described cleanout fluid 50ml, 1；
Described nitrite ion A5ml, 1, nitrite ion B 10ml, 1；Described stop buffer 10ml, 1.
Further, described OxLDL ELISA oxLDL standard reference material be the sterling of OxLDL ELISA with
OxLDL ELISA standard dilutions proportionally dilutes and forms.The difference of described quality-control product and standard reference material is the denseest
Degree difference, described quality-control product set up purpose is by whether quality-control product detected value comes standard substance matched curve in the range of Quality Control
Carrying out checking and approving check and correction, if quality-control product is qualified, then detection sample is fitted calculating oxidized low density in sample by available standards curve
The concentration of lipoprotein, if quality-control product is defective, then test kit cannot accurately carry out detecting matching.
Further, described OxLDL ELISA standard dilutions is in the phosphate buffer of PH 7.4, respectively
Add sucrose and the OxLDL ELISA of 1U, 2U, 4U, 8U, 16U of 5%；Described OxLDL ELISA Quality Control
Product are the OxLDL ELISA sterlings of 0.5U and 20U；Described 1U is defined as 25 μ g.
Further, described diluent is the phosphate buffer of PH7.4；Described cleanout fluid is every liter of tween containing 5ml
20；Described nitrite ion A is that addition mass fraction is the hydrogen peroxide of the 30% of 1% in the citrate solution of 1M；Nitrite ion
B contains the OPD aqueous solution of 10ug in being every liter；Described stop buffer is the concentrated sulfuric acid solution of 2N.
The kit method that preparation is as above the most described, including following preparation method, regardless of tandem:
The preparation of the P.r β 2-GPI-DV recombiant protein of specific binding OxLDL ELISA oxLDL: genes of interest is recombinated
In the yeast recombinant strains of X-33, under methanol induction, produce recombinant expression protein r β2-GPI-DV, nickel post is affine layer
Analysis obtains purifying protein and is lyophilized into after powder in-80 DEG C of preservations；
The preparation of OxLDL ELISA sterling: take low density lipoprotein, LDL, is dissolved in the phosphate buffer of 1M, low close
The concentration of degree lipoprotein is 1mg/ml；Add copper sulfate in this solution so that it is whole content is 1%, 37 DEG C；8h.
Immobilization has a preparation of recombiant protein microwell plate: by the weight of specific binding OxLDL ELISA that concentration is 1mg/ml
It is 50ug/ml that histone P.r β 2-GPI-DV PH9.6 concentration 1M carbonate buffer solution is diluted to concentration, joins in microwell plate
50ul/ hole, 4 DEG C of immobilization 12h；Then use cleanout fluid to clean microwell plate 4 times, shake 2min every time；Incline and be coated liquid, get rid of
Dry plate, then in microwell plate, add 1%gelatin solution, 200ul/ hole carries out 37 DEG C, and 1h closes；Incline deblocking liquid, waits micro-
Orifice plate is dried, adds the vacuum bag encapsulation of desiccant, and both having obtained immobilization has the ELISA Plate of recombiant protein, is placed in 4 DEG C of preservations.
OxLDL ELISA reference standards and the preparation of quality-control product:
(1) preparation of OxLDL ELISA sterling:
1.. take low density lipoprotein, LDL certain volume quantity (filtration sterilization) of actual concentrations (100ug/ml), join sterilizing culture bottle
In；
2.. add 500uMCuSO4·5H2O so that it is final concentration reaches 5uM (filtration sterilization)；
3.. adding the phosphate buffer (PBS, filtration sterilization) of 1M, the volume fraction of addition is 89%；Cultivate bottle cap half pine situation
Under, 37 DEG C, hatch 8h.After hatching 4h, take out culture bottle rock and continue the most afterwards to hatch to 8h；
4.. the EDTA adding copper sulfate volume half amount after hatching 8h terminates oxidation；
5.. the oxidation solution terminating oxidation is loaded in ready bag filter (100 DEG C boiling water boiling 3 times, 3min/ time)；
6.. put into (the 1M phosphate buffer containing 0.5%200mM EDTA) in dialysis solution；
7.. bag filter and dialysis solution being put under 4 DEG C of environment, every 4h changes a dialysis solution, changes 5 times；
8.. by the filtration sterilization 4 DEG C preservation after super filter tube concentration, TBARS, BCA measure of the liquid in bag filter after completing dialysis；
(2) preparation of OxLDL ELISA standard substance:
Standard substance 1: containing the sucrose of 5%；
Standard substance 2: containing the sucrose of 5%, the OxLDL ELISA sterling of 1U/ml；
Standard substance 3: containing the sucrose of 5%, the OxLDL ELISA sterling of 2U/ml；
Standard substance 4: containing the sucrose of 5%, the OxLDL ELISA sterling of 4U/ml；
Standard substance 5: containing the sucrose of 5%, the OxLDL ELISA sterling of 8U/ml；
Standard substance 6: containing the sucrose of 5%, the OxLDL ELISA sterling of 16U/ml；
(3) OxLDL ELISA quality-control product:
Quality-control product 1: containing the sucrose of 5%, the OxLDL ELISA sterling of 2.5U/ml；
Quality-control product 2: containing the sucrose of 5%, the OxLDL ELISA sterling of 10U/ml；
The phosphate buffer of the preparation of Sample dilution: 1M；
The preparation of cleanout fluid: containing the phosphate buffer of the 1M of 0.05%；
Nitrite ion A: the concentration containing 1% is the 1M citrate buffer of the hydrogen peroxide of 30%；
Nitrite ion B: every liter contains the aqueous solution of 10ugOPD；
Stop buffer: 2N sulfuric acid solution.
The invention provides the using method of test kit described in early stage, it comprises the steps of
(1) diluted sample: use diluent as requested by testing sample with the dilution proportion of 1:10-1:100；
(2) solution preparation:
Standard substance and quality-control product, redissolve；
The anti-apoB antibody working solution of enzyme labelling: enzyme labelled antibody is diluted 1000～5000 times with diluent；
Cleaning liquid: with ultra-pure water by the cleaning liquid of the cleaning solution dilution of 10X to 1X；
(3) take immobilization and have the ELISA Plate of specific recombiant protein, after equilibrium at room temperature 30min, wash 4 times with cleaning liquid,
Standby；
(4) sample-adding: take standard substance, quality-control product, sample that dilution is good are separately added into microwell plate, every hole 100ul, hatch 1h at 37 DEG C,
Wash 4 times with cleanout fluid；
(5) antibody of enzyme labelling is added: after being diluted by the anti-apoB antibody 1:1000 of enzyme labelling bottom vertical addition microwell plate, 50ul/
Hole, hatches 1h at 37 DEG C, washs 4 times with cleanout fluid；
(6) developer is added: nitrite ion A, nitrite ion B are separately added into microwell plate with the ratio of 1:9,100ul/ hole altogether, room temperature
Lucifuge hatches 15min；
(7) stop buffer is added: 100ul/ hole；
(8) measure absorbance: under microplate reader, measure the absorbance under 492nm, measure and require after terminating reaction in 30min
Inside complete.
Beneficial effects of the present invention: test kit of the present invention provides the recombiant protein specific binding with Ox-LDL, this albumen is
A kind of recombination human source P.r β 2-GPI-DV albumen, this albumen can recognition site oxLig-1 novel with Ox-LDL specific binding.
Ox-LDL aoxidizes novel recognition site oxLig-1 as macrophage and the affinity ligand of surface of hepatocytes scavenger donee CD 36
And the part of intracellular nuclear factor PPARs, mediation oxLDL activates the activation of downstream inflammatory factor NF-κ B, oxidation should
Swash and the outflow regulation of cholesterol, and then promote disease to occur and the generation of inflammation foci.Therefore, restructuring used in the present invention
Albumen P.r β 2-GPI-DV is to have cause arteriosclerosis and the cause of inflammatory reaction by combining the Ox-LDL of novel recognition site detection
Characteristic of disease Ox-LDL；There is the characteristic of precisely detection, for anti-with atherosclerosis hardening, abnormalities of sugar/lipid metabolism and inflammation
The diagnosis of the diseases such as cardiovascular disease and the non-alcoholic fatty liver disease of answering speciality has using value and good market application foreground,
Meanwhile, due to the fact that detecting instrument used is relatively simple, be conventionally used instrument, therefore testing cost is relatively low, be beneficial to push away
Extensively；And this test kit easily operates, it is not necessary to the personnel of specialty can realize；Planning standard product diluent in test kit simultaneously
With Sample Buffer formula of liquid, it is favorably improved standard reliability and the accuracy of testing result.
Accompanying drawing explanation
Fig. 1 is the molecular docking figure of embodiment 1 recombiant protein and oxLDL ligands specific recognition site oxLig-1
Fig. 2 embodiment 2, the cold wind of embodiment 4 dry up immobilization figure
Fig. 3 is the microwell plate immobilization figure of embodiment 3
Fig. 4 is embodiment 6 OxLDL ELISA standard reference material Specification Curve of Increasing figure
Fig. 5 is the optimum enzyme labelled antibody Cigarette dilution detection sample graph of embodiment 7
Fig. 6 is the clinical practice result figure of embodiment 8 invention test kit.
Detailed description of the invention
Embodiment 1: recombiant protein and the molecular docking of specific recognition site oxLig-1:
(1) from Protein Data Bank (Protein Data Bank), the β containing part is obtained2The crystal knot of-GPI-DV complex
Structure (PDB numbering is respectively 3OP8).Part is peeled off from complex extraction, receptor is imported to AUTODOCK 4.2.6
Software kit carries out the structural modification of necessity, including removing hydrone, adding hydrogen atom and add Gasteiger-Marsili electricity
Lotus etc.；
(2) utilize AutoDock 4.2.6 software kit to receptor β2-GPI-DV and oxLig-1 molecule carry out semi-flexible docking；
In molecular docking, β 2-GPI-DV and oxLig-1 molecule is combined in 1:1 ratio；Before carrying out semi-flexible docking simulation, make
Calculate grid with AUTOGRID, dock grid length and width used each time and height is 100 mesh points, grid bag
The all reactive amino acid residues that may act on containing oxLig-1 molecule；
(3) β in simulation docking operation2-GPI-DV structure is considered as rigidity, and oxLig-1 molecule is flexible, oxLig-1 intramolecular
Rotation key by AUTODOCK procedure identification and arbitrarily can rotate in search procedure, molecular docking is at AutoDock
Carrying out in software kit, docking uses Lamarchian genetic algorithm to combine (GALS) to β with local energy search
2-GPI-DV-oxLig-1 complex conformation scans for.
Embodiment 2: immobilization has recombinant beta2The preparation of the microwell plate of-GPI-DV albumen
(1) preparation of P.r β 2-GPI-DV recombiant protein
P.r β 2-GPI-DV recombiant protein is the recombiant protein that Pichia sp. system is expressed, and recombinant expressed process is as follows: will be stored in-80
X-33 bacterial strain take out, be inoculated in growth medium (BMGY) with the ratio of 1:10, through 29 DEG C, 240 to shake bacterium raw
Long；Again with the ratio amplification culture of 1:50, after the same terms cultivates 24h；It is concentrated in inducing culture (BMMY) induction
Express, add every day concentrated solution 1% methanol, after continuous induction 72h, collect bacterium solution supernatant；Obtain through affinity chromatography
Highly purified β2-GPI-DV albumen, is prepared as lyophilized powder ,-80 DEG C of preservations by P.r β 2-GPI-DV.
(2) immobilization of P.r β 2-GPI-DV microwell plate
By the P.r β 2-GPI-DV recombiant protein PH9.6 concentration of specific binding OxLDL ELISA that concentration is 1mg/ml
It is 50ug/ml that 1M carbonate buffer solution is diluted to concentration, joins 50ul/ hole in microwell plate, 4 DEG C of immobilization 12h；Then make
Clean microwell plate 4 times with cleanout fluid, shake 2min every time；Incline and be coated liquid, dry plate, then in microwell plate, add 1%gelatin
Solution, 200ul/ hole carries out 37 DEG C, and 1h closes；Incline deblocking liquid, waits that microwell plate is dried, adds the vacuum bag of desiccant
Encapsulation, both having obtained immobilization has the ELISA Plate of recombiant protein, is placed in 4 DEG C of preservations.
Embodiment 3: immobilization has the preparation of the microwell plate of E.coli restructuring r β 2-GPI-DV albumen
Preparation process inside this embodiment is substantially the same manner as Example 2, except for the difference that:
Step (1) β2The preparation of-GPI-DV recombiant protein, β2The recombiant protein that-GPI-DV recombiant protein protokaryon system is expressed,
Recombinant expressed process is as follows: will be stored in the protokaryon β of-802-GPI-DV protein expression strain takes out, and inoculates with the ratio of 1:10
In growth medium LB, through 37 DEG C, 4h shakes bacteria growing；Add the IPTG induction of final concentration 5mg/ml, continuously
After induction 5h, abandon supernatant and collect thalline；After ultrasonication, obtain highly purified r β 2-GPI-DV albumen through affinity chromatography.
Embodiment 4: immobilization has the preparation of the microwell plate of E.coli restructuring r β 2-GPI-DV albumen
This embodiment is substantially the same manner as Example 2, except for the difference that:
In step (2) microwell plate immobilization, after r β 2-GPI-DV recombiant protein is joined microwell plate, dry up with cold wind, freeze
Dry mode carries out the immobilization of albumen.
Embodiment 5: be prepared by the following method this test kit
Immobilization recombiant protein β2The preparation of-GPI-DV: same as in Example 2；
Recombinant beta2The preparation of the immobilised microwell plate of-GPI-DV albumen: same as in Example 2；
The preparation of Sample dilution: 1M phosphate buffer, PH7.4；By sodium chloride 8g, potassium chloride 0.2g, hydrogen sulfate disodium
1.42g, potassium dihydrogen phosphate 0.27g are dissolved in 1L ultra-pure water, are configured to the phosphate buffer of concentration 1M；
The preparation of cleanout fluid: containing 0.05% polysorbas20, concentration is the phosphate buffer of 1M；
Nitrite ion A:1M citrate buffer solution: contain the citric acid of 86.1g, the sodium citrate aqueous solution of 173.46g in every liter；
The hydrogen peroxide of volume fraction is 1% 30% is added in 1M Fructus Citri Limoniae buffer；
Nitrite ion B: the OPD of 10ug is dissolved in the ultra-pure water of 1L；
The concentrated sulphuric acid of stop buffer: 54ml is poured slowly in the ultra-pure water of 946ml and obtains 2N sulfuric acid solution；
Embodiment 6: the drafting of the OxLDL ELISA standard control sample standard curve of variable concentrations:
(1) according to the concentration requirement of OxLDL ELISA standard control sample 0U, 1U, 2U, 4U, 8U, 16U,
Use the phosphate buffer of 1mol/L, PH7.4 by stand-by for the dilution of OxLDL ELISA sterling；
(2) immobilization has the microwell plate of specific binding OxLDL ELISA recombiant protein to prepare: concentration is the spy of 1mg/ml
The recombiant protein PH9.6 concentration 1M carbonate buffer solution of anisogamy OxLDL ELISA is diluted to concentration and is
50ug/ml, joins 50ul/ hole in microwell plate, 4 DEG C of immobilization 12h；Then cleanout fluid is used to clean microwell plate 4 times, every time
Shaking 2min；Inclining and be coated liquid, dry plate, then add 1%gelatin solution in microwell plate, 200ul/ hole carries out 37 DEG C,
1h closes；Incline deblocking liquid；Using cleanout fluid to clean microwell plate 4 times, shake 2min every time, incline cleanout fluid, dries plate；
(3) sample is added: OxLDL ELISA standard reference material ready in (1) is added separately in (2) standard
In the microwell plate got ready, three holes of each concentration, hatch 1h for 37 DEG C；
(4) use cleanout fluid to clean microwell plate 4 times, shake 2min every time；Incline cleanout fluid, dries plate；
(5) add the anti-apoB antibody of enzyme labelling, hatch 1h for 37 DEG C；
(6) use cleanout fluid to clean microwell plate 4 times, shake 2min every time；Incline cleanout fluid, dries plate；
(7) by nitrite ion A, B with the proportions of 1:9, fully mix, join microwell plate 100ul, color development at room temperature 15mim altogether；
(8) the 2N sulphuric acid stop buffer of 100ul is added；
(9) absorbance is measured at microplate reader 492nm；
The absorbance of the OxLDL ELISA standard reference material of (10) 6 concentration draws standard curve, its standard curve
Correlation coefficient be 0.987
Embodiment 7: carry out the detection of quality-control product and sample by the following method:
(1) prepare before detection:
(1) before measuring premise, 30min takes out test kit from 4 DEG C, under room temperature after balance, washs 4 times with cleaning liquid, standby
With；
(2) by cleaning liquid: with ultra-pure water by the cleaning liquid of the cleaning solution dilution of 10X to 1X；
(3) standard substance and quality-control product, redissolves；
(4) the anti-apoB antibody working solution of enzyme labelling: enzyme labelled antibody is diluted 1000 times with diluent；
(5) nitrite ion preparation: by nitrite ion A, B with the proportions of 1:9, fully mix, as 1ml nitrite ion A adds
9ml nitrite ion B, prepared before use.
(2) measure
(6) diluted sample: with diluent by diluted sample 10-100 times；
(7) sample-adding: take standard substance, quality-control product, sample that dilution is good are separately added into microwell plate, every hole 100ul, incubate at 37 DEG C
Educate 1h；
Plate washing method:
Automatically plate is washed: require that cleaning mixture 300ul is injected in the most every hole, inject and sucking-off time interval 1min；
Manual plate washing: liquid in hole of inclining, every hole adds 300ul cleaning mixture, after shaking 1min, gets rid of liquid in hole, inhaling
Pat dry on water paper；
(8) plate is washed 4 times with the cleanout fluid diluted；
(9) antibody of enzyme labelling is added: vertically add after the dilution proportion of anti-apoB antibody 1:1000～1:5000 of enzyme labelling
Enter bottom microwell plate, 50ul/ hole, hatch 1h at 37 DEG C；
(10) cleanout fluid wash plate is used 4 times；
(11) developer is added: the nitrite ion mixture configured is added microwell plate, altogether 100ul/ hole, and room temperature lucifuge is incubated
Educate 15min；
(12) stop buffer is added: 100ul/ hole；
(13) absorbance is measured: under microplate reader, measure the absorbance under 492nm, measure and require 30min after terminating reaction
Complete in.
(3) measurement result calculates
(14) standard substance, quality-control product and the mean absorbance values of detection sample are calculated；
(15) with the standard substance of 0U/ml as blank, the absorbance value of standard substance, Quality Control thing and sample should deduct blank right
According to absorbance value；
(16) using log-linear drawing, with the logarithm of standard concentration as transverse axis, the logarithm of absorbance value is the longitudinal axis, draws
Ox-LDL concentration-absorbance value double-log dose-response curve；
(17) from dose-response curve, the OxLDL concentration of dilute sample is found according to the testing sample absorbance value of dilution；
(18) in sample, the ultimate density of Ox-LDL is multiplied by extension rate equal to the concentration of dilute sample；
(19) if the absorbance value of sample is higher than 16U/ml or the absorbance value less than 1U/ml standard substance, can proper extension song
Line calculates, and extrapolation factor (Extrapolation Factor) is less than 2；
Table 1 embodiment 5 sample detection result
| 0.842 | 0.641 | 0.846 | 0.655 | 0.759 | 0.793 | 0.989 | 0.841 |
| 0.722 | 0.732 | 0.755 | 0.812 | 0.858 | 0.88 | 0.876 | 0.893 |
As it can be seen from table 1 the detected value of quality-control product 1,2 is all in the range of target value, it is possible to determine credible result；, sample
The detected value standard deviation of product compares, and CV% is not higher than 15%, illustrates that this technological invention has good repeatability.
Embodiment 8: the clinical practice of test kit of the present invention
Use the test kit of this inventive technique, have detected 100 example normal subjectses and 50 example heart infarction patients serum Ox-LDL, knot
Fruit display, 100 example experimenter's serum Ox-LDL content are 25.79 ± 3.89U/ml, and heart infarction patients serum's Ox-LDL content is
46.58 ± 6.03U/ml, the two has significant difference (0.003), can fast and accurate diagnosis of coronary heart disease, heart infarction patients serum
OxLDL level, experiment display simultaneously is proportional with the diagnosis of the sign of heart infarction patient.
Above embodiment is preferred embodiment, not in order to limit the present invention, it is understood that all based on foregoing of the present invention
Any modification, equivalent substitution and improvement made etc. belong to the scope of the present invention.
Claims (4)
1. the enzyme-linked immunologic detecting kit of a human body OxLDL ELISA, it is characterised in that it includes that immobilization has spy
The ELISA Plate of the recombiant protein of anisogamy OxLDL ELISA, the OxLDL ELISA standard control sample of variable concentrations
Product, quality-control product, the antibody of enzyme labelling, diluent, cleanout fluid, nitrite ion A, nitrite ion B, stop buffer；
Wherein, immobilization have specific binding OxLDL ELISA recombiant protein ELISA Plate in ELISA Plate be polystyrene
96 microwell plates, immobilised specific recombinant proteins are recombiant protein P.r β 2-GPI-DV or the E.coli restructuring that yeast is recombinant expressed
Albumen r β 2-GPI-DV, on test kit microwell plate, the quantity of immobilised recombiant protein is 50ug/ hole；
Quality-control product is each 2 of the OxLDL ELISA sterling of 0.5U and 20U containing 5% sucrose, every 5ml, wherein 1U
It is defined as 2.5ug；
The antibody of enzyme labelling is the goat-anti people anti-apoB antibody of horseradish peroxidase-labeled, the anti-apoB of horseradish peroxidase-labeled
Antibody is 1,10ul；
The OxLDL ELISA standard control sample of variable concentrations is 6, each 2ml, OxLDL ELISA standard reference material
Be the sterling of OxLDL ELISA and OxLDL ELISA standard dilutions according to 0U, 1U, 2U, 4U, 8U,
16U dilution forms, OxLDL ELISA standard dilutions is in the phosphate buffer of PH 7.4, is separately added into
The sucrose of 5% and the OxLDL ELISA of 1U, 2U, 4U, 8U, 16U, 1U is defined as 25 μ g；
Diluent is to be the phosphate buffer of PH7.4,10ml, 1；
Cleanout fluid is every liter of phosphate buffer containing 5ml polysorbas20,50ml, 1；
Nitrite ion A be add in the citrate solution of 1M mass fraction is 1% 30% hydrogen peroxide, 5ml, 1；
Nitrite ion B be every liter in containing the OPD aqueous solution of 10ug, 10ml, 1；
Stop buffer is the concentrated sulfuric acid solution of 2N, 10ml, 1.
The enzyme-linked immunologic detecting kit of a kind of human body OxLDL ELISA the most according to claim 1, its feature
Being, its preparation method is:
(1) preparation of OxLDL ELISA sterling:
1.. take the low density lipoprotein, LDL certain volume quantity of actual concentrations, join in sterilizing culture bottle；
2.. add 500uMCuSO4·5H2O so that it is final concentration reaches 5uM；
3.. adding the phosphate buffer (PBS) of 1M, the volume fraction of addition is 89%；In the case of cultivating bottle cap half pine,
37 DEG C, hatch 8h, after cultivating 4h, take out culture bottle rock；
4.. the EDTA adding copper sulfate volume half amount after hatching 8h terminates oxidation；
5.. the oxidation solution terminating oxidation is loaded in ready bag filter, 100 DEG C of boiling water boilings 3 times, 3min/ time；
6.. putting in dialysis solution, dialysis solution is the 1M phosphate buffer containing 0.5%200mMEDTA；
7.. bag filter and dialysis solution being put under 4 DEG C of environment, every 4h changes a dialysis solution, changes 5 times；
8.. by the 4 DEG C of preservations after super filter tube concentration, TBARS, BCA measure of the liquid in bag filter after completing dialysis；
(2) preparation of OxLDL ELISA standard substance:
Standard substance 1: containing the sucrose of 5%；
Standard substance 2: containing the sucrose of 5%, the OxLDL ELISA sterling of 1U/ml；
Standard substance 3: containing the sucrose of 5%, the OxLDL ELISA sterling of 2U/ml；
Standard substance 4: containing the sucrose of 5%, the OxLDL ELISA sterling of 4U/ml；
Standard substance 5: containing the sucrose of 5%, the OxLDL ELISA sterling of 8U/ml；
Standard substance 6: containing the sucrose of 5%, the OxLDL ELISA sterling of 16U/ml；
(3) preparation of OxLDL ELISA quality-control product:
Quality-control product 1: containing the sucrose of 5%, the OxLDL ELISA sterling of 2.5U/ml；
Quality-control product 2: containing the sucrose of 5%, the OxLDL ELISA sterling of 10U/ml；
(4) immobilization has recombiant protein P.r β 2-GPI-DV or the E.coli recombiant protein of specific binding OxLDL ELISA
The preparation of r β 2-GPI-DV microwell plate: the recombiant protein of the specific binding OxLDL ELISA that concentration is 1mg/ml is used
It is 50ug/ml that PH9.6 concentration 1M carbonate buffer solution is diluted to concentration, joins 50ul/ hole in microwell plate, changes 12h mutually for 4 DEG C；
Then use cleanout fluid to clean microwell plate 4 times, shake 2min every time；Incline and be coated liquid, dry plate, then add in microwell plate
1%gelatin solution, 200ul/ hole carries out 37 DEG C, and 1h closes；Incline deblocking liquid, waits that microwell plate is dried, adds desiccant
Vacuum bag encapsulation, both obtained immobilization have specific binding OxLDL ELISA recombiant protein P.r β 2-GPI-DV or
The microwell plate of E.coli recombiant protein r β 2-GPI DV, is placed in 4 DEG C of preservations.
The enzyme-linked immunologic detecting kit of a kind of human body OxLDL ELISA the most according to claim 1 and 2, it is special
Levying and be, the using method of test kit comprises the steps of
(1) diluted sample: use diluent as requested by testing sample with the dilution proportion of 1:10-1:100；
(2) solution preparation:
Standard substance and quality-control product, redissolve；
The anti-apoB antibody working solution of enzyme labelling: enzyme target antibody is diluted 1000～5000 times with diluent；
Cleaning liquid: with ultra-pure water by the cleaning liquid of the cleaning solution dilution of 10X to 1X；
(3) take immobilization and have recombiant protein P.r β 2-GPI-DV or the E.coli recombiant protein of specific binding OxLDL ELISA
The microwell plate of r β 2-GPI DV, after equilibrium at room temperature 30min, washs 4 times with cleaning liquid, standby；
(4) sample-adding: take standard substance, quality-control product, sample that dilution is good are separately added into microwell plate, every hole 100ul, hatch 1h at 37 DEG C,
Wash 4 times with cleanout fluid, shake 1min every time；
(5) antibody of enzyme labelling is added: after being diluted by the anti-apoB antibody 1:1000 of enzyme labelling bottom vertical addition microwell plate, 50ul/
Hole, hatches 1h at 37 DEG C, washs 4 times with cleanout fluid, shakes 1min every time；
(6) developer is added: nitrite ion A, nitrite ion B are separately added into microwell plate with the ratio of 1:9,100ul/ hole altogether, room temperature
Lucifuge hatches 15min；
(7) stop buffer is added: 100ul/ hole；
(8) measure absorbance: under microplate reader, measure the absorbance under 492nm, measure and require after terminating reaction in 30min
Inside complete.
The enzyme-linked immunologic detecting kit of a kind of human body OxLDL ELISA the most according to claim 3, its feature exists
In, test kit is preparing antiphospholipid syndrome, systemic lupus erythematosus (sle), atherosclerosis, acute myocardial infarction, non-ethanol
The application of the reagent for disease diagnosis such as property fatty liver disease, hyperlipemia, diabetes and hypertension.
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