CN104215771B - A kind of High-sensitivity C reactive protein detection kit of amplifying based on liposome signal - Google Patents
A kind of High-sensitivity C reactive protein detection kit of amplifying based on liposome signal Download PDFInfo
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- CN104215771B CN104215771B CN201410401483.6A CN201410401483A CN104215771B CN 104215771 B CN104215771 B CN 104215771B CN 201410401483 A CN201410401483 A CN 201410401483A CN 104215771 B CN104215771 B CN 104215771B
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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
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
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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
Abstract
The present invention relates to a kind of High-sensitivity C reactive protein detection kit of amplifying based on liposome signal, this kit comprises R1 reagent, R2 reagent and calibration object; Described R1 reagent is glycine buffer system; Described R2 reagent is CRP antibody sensitized particle suspension liquid; Described calibration object is different containing the recombinant protein concentration of 6 CRP, solute and all identical cow's serum matrix of content thereof.Kit of the present invention is a kind of kit utilizing Immunoturbidimetry, CRP antibody by chemical bonds in surface of liposome.Compared with existing commercially available prod, High-sensitivity C reactive protein detection kit of the present invention has more high sensitivity, wider linear.
Description
Technical field
The present invention relates to a kind of High-sensitivity C reactive protein detection kit of amplifying based on liposome signal, be specifically related to a kind of CRP detection kit taking liposome as signal and amplify carrier.
Background technology
Hs-CRP (hypersensitive3C-reactiveprotein, hs-CRP) is a kind of c reactive protein in blood plasma.C reactive protein is the non-specific markers of a kind of systemic inflammatory reaction acute stage of being synthesized by liver, is one of the most strong predictor of cardiovascular event danger.Hs-CRP hs-CRP is the sensitive indexes distinguishing low-level inflammatory conditions, and the generation of level of serum hs-CRP and atherosclerotic and acute cerebral infarction (ACI), the order of severity and prognosis are closely related.Clinical labororatory, by hypersensitization detection technique, detects low concentration c reactive protein accurately, improves sensitivity and the accuracy of test.In recent years, along with the progress of detection technique, a large amount of article research displays, it plays an increasingly important role in the medicals diagnosis on disease such as coronary heart disease, apoplexy, peripheral vessels embolism and prediction.
CRP is only containing trace in the human body of health, and before new stimulation occurs, its concentration can maintain a long-term stability, long half time (about 18 ~ 20 hours), and not by sex, food or impact round the clock.When occurring infectious diseases, in serum, namely CRP starts to raise after infection occurs for 5 ~ 8 hours, within 48 hours, reach peak value, and peak value can reach hundreds of times normally.Along with infection control, it can decline rapidly at 24 ~ 48 hours, recovered normal in 1 week.The level of CRP and duration and gradient of infection are proportionate, when CRP continues to raise or when raising once again, point out the clinical change should paying attention to the state of an illness.
Have research display, in acute cerebral infarction gerontal patient, CRP rising person prognosis is not good; Hs-CRP content and infarct size, neurological functional deficit are relevant, are one of indexs of Patients with Cerebral Infarction lesion degree; And CRP also take part in thrombosis and arteriosclerotic pathologic process, be one of hazards of cerebral apoplexy.The inflammatory reaction of atherosclerotic plaque is plaque rupture and unstable major reason, in the forming process of atherosclerotic plaque, CRP complement complex and foam cells etc. are deposited in arterial wall, CRP can be combined with lipoprotein, activating complement system, produces a large amount of inflammatory mediator, release oxygen radical, cause blood vessel internal membrane damage, vasopasm and Vulnerable plaque come off, and increase the weight of the generation of luminal stenosis caused by atherosclerotic and ACI.Increasing evidence shows, other hazards of low-level CRP and angiocardiopathy are closely related, as hypertension, hyperlipidemia; CRP raises the incidence of disease that can increase hyperpietic's heart disease, cerebral apoplexy simultaneously; Therefore CRP occurs with atherosclerotic, develops and develop relevant proinflammatory factor.Epidemiology survey also shows, and the probability that acute apoplexy occurs hs-CRP level rising person is 2 times of normal healthy people, and the probability that myocardial infarction occurs is 3 times of normal person.European hypertension prevention and control guide (ESH/ESC) formal recommendation in 2003, hyperpietic need detect hs-CRP level.
The clinical guidance effect of hs-CRP is mainly manifested in angiocardiopathy, neonates with bacterial infections, the aspects such as kidney transplant.
At present, the CRP detection kit on market, there is sensitivity low, the range of linearity is narrow, the shortcoming that background is high.This is mainly because CRP detects reagent need to possess higher sensitivity and linear measurement range simultaneously, conventional Immunoturbidimetry adopts polystyrene microsphere bag quilt, because polystyrene microsphere is the opaque bead of milky, photopermeability is poor, therefore has and is difficult to ensure to possess high sensitivity with linear simultaneously simultaneously.And liposome is as the bead of homogeneous phase, there is transparency high, strong with antigen binding capacity, prepare the advantages such as simple and easy.But domestic at present also do not have about adopting liposome as the report of immunoturbidimetry signal amplifying system.The present invention is domestic adopts transparent liposome to have as antibody bag loaded body the advantage that background is low, antibody coating rate is high first, adopts liposome to have high sensitivity and High Linear feature as bag loaded body.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of High-sensitivity C reactive protein (hs-CRP) detection kit of amplifying based on liposome signal, set up a kind of latex-enhanced turbidimetry to measure the method for the content of CRP in human serum, use the reagent of the method should have simple to operate, accuracy is high, reproducible, highly sensitive, and can use on automatic clinical chemistry analyzer or special proteins instrument and spectrophotometer.
The present invention is achieved by the following technical solutions:
The invention provides a kind of High-sensitivity C reactive protein (hs-CRP) detection kit of amplifying based on liposome signal, comprise R1 reagent, R2 reagent and calibration object;
Described R1 reagent is glycine buffer system;
Described R2 reagent is the CRP antibody sensitized particle suspension liquid comprised based on liposome; Described liposome is 1:(1 ~ 20 of phosphatid ylcholine and synthetic phospholipid) potpourri; Described synthetic phospholipid is by one or more synthesis in dipalmitoyl phosphatidylcholine, DPPE, distearoyl phosphatidylcholine;
Described calibration object is different containing the recombinant protein concentration of 6 CRP, solute and all identical cow's serum matrix of content thereof.
Preferably, described glycine buffer system, solvent is 10 ~ 50mM, pH6.0 ~ 6.5 glycine buffer, solute and the content in described glycine buffer system thereof are NaCl200 ~ 400mM, Tween200.5 ~ 1.5ml/L, EDTA100 ~ 200mM, accelerator 200 ~ 500mM, antiseptic 0.1 ~ 2 ‰.
Preferably, described accelerator comprise glucosan series, PEG series, PVP serial in one; More preferably, described accelerator is glucosan series.
Preferably, described antiseptic comprises one or more in sodium azide, Sodium Benzoate, potassium sorbate, thimerosal, Proclin-300, phenol.
Preferably, described CRP antibody sensitized particle suspension liquid, solvent is the glycine buffer of 10 ~ 50mM, pH6.0 ~ 6.5, solute and the content in suspending liquid thereof are: the CRP antibody sensitized particle 10 ~ 50mg/L comprised based on liposome, disodium ethylene diamine tetraacetate 0.5 ~ 5mmol/L, bovine serum albumin(BSA) (BSA) 0.1 ~ 4mg/L, NaN
30.1 ~ 2 ‰; In the described CRP antibody sensitized particle comprised based on liposome CRP antibody by chemical bonds in surface of liposome.
Preferably, the preparation method of the described CRP antibody sensitized particle suspension liquid comprised based on liposome is: by surface with 150 ~ 300nm of chemical based liposome and anti-human CRP antibody with 10: 1.5 mass ratio mix, be dissolved in the phosphate buffer of 0.02M, pH5.0 ~ 6.0, the percent by volume of liposome is 0.1 ~ 1.5%, and the concentration of antibody is 10mg/L; Incubated at room half an hour, then add activator solution activation, make the concentration of activator in reaction system reach 50 ~ 100 μ g/ml; Mix, 37 DEG C in conjunction with 8 hours; Dialysis removes the antibody that do not connect, adds confining liquid and closes 2 hours, centrifugally removes supernatant, with pH6.0 ~ 6.5 containing 0.5 ~ 5mmol/L disodium ethylene diamine tetraacetate, 0.1 ~ 4% bovine serum albumin(BSA) (BSA), 0.1 ~ 2%NaN
3glycine buffer dilution; Described confining liquid is the MOPSO damping fluid of the pH7.4 containing 0.1% bovine serum albumin(BSA).
Preferably, described chemical based comprises one or more in carboxyl, hydroxyl, amino, sulfonic group.
Preferably, described anti-human CRP antibody is one or more in goat anti-human crp's antibody, the anti-human CRP antibody of rabbit, mouse-anti Human C-reactiveprotein antibody.
Preferably, the process of described activation is specially: in 100ml1% concentration liposome, adds 0.15 ~ 1ml glutaraldehyde and 30mg activator respectively, shaken at room temperature 1 hour, the dialysis unnecessary glutaraldehyde of removing and activator.
Preferably, described activator comprises carbodiimides (EDC), EDC/ (sulfo) NHS, N, one or more in N'-carbonyl dimidazoles (CDI), paratoluensulfonyl chloride, cyanogen bromide, N, N'-bis-succinimidyl carbonate (DSC), bis-epoxy based compound, hydroboration cyanogen.
Preferably, in described calibration object, the recombinant protein concentration difference of CRP specifically refers to that the concentration of the recombinant protein of CRP in calibration object is respectively 0mg/ml, 20mg/ml, 40mg/ml, 80mg/ml, 160mg/ml, 320mg/ml.
Preferably, the solute in described calibration object and the volume content in described calibration object thereof are Sodium azide 0.2 ~ 2.2 ‰, Tween-20 1 ~ 10%, bovine serum albumin(BSA) (BSA) 1 ~ 3%.
Liposome (liposome) is a kind of artificial rust.In water, phospholipid molecule hydrophilic head inserts in water, and liposome hydrophobic tail stretches to air, forms the spherical liposomes of double-deck fat molecule after stirring, and diameter 25 ~ 1000nm is not etc.Under the effect of carbodiimide, the activated carboxylic of outer surface of liposome, the carboxyl after activation and albumen are by forming acid amides and coupling.Internal reagent adopts the Nano-grade latex microballoon coated antibody of White-opalescent usually, and the sensitivity of the larger reagent of Nano microsphere will be higher, but simultaneously, reagent light transmission will be poorer, causes reagent background higher, receives the restriction of detecting instrument greatly.And adopt liposome to have the high advantage of light transmission, the liposome that diameter is larger thus can be used to carry out bag quilt.Reach and improve sensitivity and do not improve the beneficial effect of reagent background.
Compared with prior art, the present invention has following beneficial effect:
(1) the present invention is a kind of detection kit of amplifying based on liposome signal, on the highly sensitive basis of guarantee, obviously expand the detection range of linearity, achieve the unification of c reactive protein detection kit in high sensitivity and the wide range of linearity, meet the requirement of hs-CRP clinical measurement;
(2) microballoon coupling CRP polyclonal antibody preparation latex beads strengthens immunoturbidimetry reagent, there is background higher, the shortcoming of detection sensitivity difference, cannot CRP content under Simultaneously test high concentration and low concentration level, and the liposome of 150 ~ 300nm of transparent surface carboxyl groups and anti-human CRP antibody are obtained R2 reagent aaerosol solution by chemical crosslinking by the present invention, capture reagent background too high, the difficult problem that detection sensitivity is inadequate;
(4) the present invention adopts liposome bag by CRP monoclonal antibody, and by optimizing solvent, solute species and content, coating rate can be made to reach more than 95%, ensure that Antibody stability and kit sensitivity, this is that prior art is incomparable;
(5) the present invention adopts natural grease plastid and synthetic fat plastid to be mixed with to wrap by CRP monoclonal antibody, strengthen the affine performance of antibody and carrier, increase mobile equilibrium between liposome, increase embedding rate, Contrast agent box reagent natural attribute simultaneously, make it in detection sample, not produce repulsion with sample, make testing result more accurate.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the typical curve of the hs-CRP normative reference of display 6 kinds of different contents;
Wherein, each point represents the normative reference of different content hs-CRP, and X-axis represents the content of hs-CRP, and Y-axis represents absorbance;
Fig. 2 verifies the range of linearity of this kit;
Wherein, what X-axis represented is the actual test result of reagent of the present invention, and what Y-axis represented is dilution theoretical concentration, and related coefficient is R
2=0.9996, regression equation is y=1.0218x+0.6204.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
embodiment 1
The present embodiment relates to a kind of High-sensitivity C reactive protein (hs-CRP) detection kit, and it is prepared as follows:
Reagent R1:
| Glycine buffer | pH6.0～6.5 25mM |
| NaCl | 200mM |
| Tween20 | 0.9ml/L |
| EDTA | 100mM |
| Dextran 60 00 | 300mM |
| NaN 3 | 1‰ |
Various composition can add under room temperature successively, and sealing is preserved, for subsequent use.
Reagent R2: be dissolved in by EDC in purified water, is configured to 5mg/ml solution, and before configuration, half an hour is placed in equilibrium at room temperature.The amount of to be liposome (phosphatid ylcholine: DPPC=1:1) and the mouse-anti Human C-reactiveprotein monoclonal antibody of 180 ~ 235nm by the diameter of surface carboxyl groups with mass ratio be 10:1.5 mixes, add the phosphate buffer of 0.02M, pH5.0 ~ 6.0, DPPC concentration of liposomes is 0.1 ~ 1.5% (volume ratio), mixing, incubated at room half an hour, add the EDC solution (containing glutaraldehyde) of 5mg/ml again, make the concentration of EDC in reaction system reach 80 μ g/ml; Mix rear 37 DEG C of absorption 8 hours, dialysis afterwards removes the antibody do not connected, and adds confining liquid 0.1% bovine serum albumin(BSA), closes 2 hours, centrifugally remove supernatant, with 10mMpH6.0 ~ 6.5 containing 1.2mmol/L disodium ethylene diamine tetraacetate, 0.3%BSA, 0.9%NaN
3glycine buffer dilution.
The preparation of hs-CRP calibration object:
Calibration object dilution solvent is cow's serum matrix, and comprise Sodium azide 1 ‰, Tween-20 5%, bovine serum albumin(BSA) (BSA) 2%, above-mentioned number percent is the number percent of cow's serum matrix volumetric usage.
The recombinant protein of CRP is dissolved in above-mentioned cow's serum matrix, prepares the calibration object (0mg/ml, 25mg/ml, 50mg/ml, 100mg/ml, 200mg/ml, 400mg/ml) of variable concentrations.
embodiment 2
The hs-CRP detection kit that the present embodiment describes, be applicable to various types of full automatic biochemical apparatus, this sentences Hitachi 7170 full automatic biochemical apparatus is example.
Analytical approach: Two point end assay, namely the consumption of reagent R1, reagent R2 is respectively 140ul and 140ul, sample size 2ul.Adopt this reagent and said determination method, the curve (as shown in Figure 1) of the hs-CRP calibration object (self-control) of the 6 kinds of different contents adopting Hitachi 7170 Biochemical Analyzer to record, each point represents the reference calibrations product of a content, and wherein X-axis represents hs-CRP content (mg/ml); Y-axis represents absorbance.
embodiment 3: the range of linearity
By the high concentration sample (320.62mg/ml) of the hs-CRP close to the range of linearity upper limit, 1/2 is pressed, 1/10,1/15 with hs-CRP dilution, 1/30,1/60 dilution and low value sample (0.11mg/ml), the solution of totally 7 variable concentrations, detects each concentration by method described in embodiment 2, every concentration replication 3 times, the mean value and theoretical concentration that measure concentration are carried out linear regression analysis, and calculating regression equation is y=1.0218x+0.6204, and related coefficient is R
2=0.9996, show kit of the present invention good relationship in 0.1mg/ml ~ 400mg/ml range of linearity, see Fig. 2.
embodiment 4: the impact of interfering material
Be 320.62mg/mlhs-CRP calibration object solution by concentration, add each interfering material solution of same volume respectively, add rear concentration and be respectively cholerythrin (50mg/L), haemoglobin (400mg/L), chyle (formal hydrazine) turbidity is 4000 and rheumatoid factor (RF) (400IU/ml), each sample replication 3 times, average, compare with the distilled water adding same volume, observe the relative error of RBP measured value after adding interfering material and adding distilled water.Result shows: add above concentration interfering material with add same volume distilled water after the relative error of measured value be no more than 2%, can think that, when mild or moderate haemolysis, jaundice or chyle, the testing result of this assay method is substantially interference-free.
The interfering material impact experiment of table 1 kit of the present invention
| The anti-interference material added | Measure average (mg/L) | Relative error |
| Distilled water | 123.56 | |
| Cholerythrin (50mg/L) | 121.35 | -1.79％ |
| Haemoglobin (400mg/L) | 125.24 | 1.36％ |
| Chyle (formal hydrazine) turbidity is 4000 | 123.26 | -0.24％ |
| Rheumatoid factor (RF) (500IU/ml) | 122.31 | -1.01％ |
embodiment 5
The overall technological scheme of the present embodiment is identical with embodiment 1, and involved parameter has on summary of the invention basis, place and changes to some extent, described in specific as follows:
(1) reagent R1:
| Glycine buffer | pH6.0～6.5 10mM |
| NaCl | 300mM |
| Tween20 | 0.5ml/L |
| EDTA | 150mM |
| Dextran 60 00 | 200mM |
| Sodium Benzoate | 0.1‰ |
(2) reagent R2:
Solvent is the glycine buffer of 25mM, pH6.0 ~ 6.5, and solute and the content in suspending liquid thereof are: CRP antibody sensitized particle 30mg/L, disodium ethylene diamine tetraacetate 0.5mmol/L, BSA0.1mg/L, NaN
30.1 ‰;
In the process of activation: in 100ml1% concentration liposome, add 0.5ml glutaraldehyde and 30mg activator respectively;
Activator is EDC/ (sulfo) NHS, and the concentration in preparation system is 50 μ g/ml;
Surface of liposome with hydroxyl, phosphatid ylcholine: DPPE (DPPE) is 1:10;
Antibody type is goat anti-human crp's antibody.
(3) calibration object:
Solute in calibration object and the volume content in described calibration object thereof are Sodium azide 1.5 ‰, Tween-20 1%, bovine serum albumin(BSA) (BSA) 1%.
embodiment 6
The overall technological scheme of the present embodiment is identical with embodiment 1, and involved parameter has on summary of the invention basis, place and changes to some extent, described in specific as follows:
(1) reagent R1:
| Glycine buffer | pH6.0～6.5 50mM |
| NaCl | 400mM |
| Tween20 | 1.5ml/L |
| EDTA | 200mM |
| Dextran 60 00 | 500mM |
| Thimerosal | 2‰ |
(2) reagent R2:
Solvent is the glycine buffer of 50mM, pH6.0 ~ 6.5, and solute and the content in suspending liquid thereof are: CRP antibody sensitized particle 50mg/L, disodium ethylene diamine tetraacetate 5mmol/L, BSA4mg/L, NaN
32 ‰;
In the process of activation: in 100ml1% concentration liposome, add 1ml glutaraldehyde and 30mg activator respectively;
Activator is paratoluensulfonyl chloride; Concentration in preparation system is 100 μ g/ml;
Surface of liposome with sulfonic group, phosphatid ylcholine: distearoyl phosphatidylcholine (DSPC) is 1:20;
Antibody type is the anti-human CRP antibody of rabbit.
(3) calibration object:
Solute in calibration object and the volume content in described calibration object thereof are Sodium azide 2.2 ‰, Tween-20 10%, bovine serum albumin(BSA) (BSA) 3%.
embodiment 7
The present embodiment detects reagent for contrast with Japanese Sheng Yan Co., Ltd. C RP, and detect kit of the present invention and the performance of contrast agents box in the range of linearity, sensitivity and background three, detection method adopts common detection methods, and testing result is as shown in the table.Result shows, and the range of linearity of the present invention is wide, highly sensitive, background level is low.
| The present invention | Contrast agent box | |
| The range of linearity | 0.01～400mg/L | 0.05～320mg/L |
| Sensitivity | 0.01mg/L | 0.05 |
| Background | 0.5A | 1.5A |
embodiment 8
The present embodiment is identical with embodiment 2 technical scheme, not adds glutaraldehyde unlike the present embodiment in reactivation process.Result shows, the add range of linearity to kit, sensitivity and the embedding rate of glutaraldehyde create beyond thought effect, and glutaraldehyde, by the interaction with each component, obviously expands the range of linearity of kit, sensitivity and embedding rate.
| Embodiment 2 reagent (adding glutaraldehyde) | Embodiment 8 reagent (not adding glutaraldehyde) | |
| The range of linearity | 0.01～400mg/L | 0.1～250mg/L |
| Sensitivity | 0.01mg/L | 0.1mg/L |
| Coating rate | 97％ | 60％ |
embodiment 9
The present embodiment is identical with embodiment 2 technical scheme, only carrier is done with phosphatid ylcholine or synthetic phospholipid unlike the present embodiment, detect the range of linearity, sensitivity and embedding rate as in the table below, result shows the better effects if that the mixing ratio of natural grease plastid and synthetic fat plastid is used alone.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (6)
1., based on the High-sensitivity C reactive protein detection kit that liposome signal amplifies, it is characterized in that, comprise R1 reagent, R2 reagent and calibration object;
Described R1 reagent is glycine buffer system;
Described R2 reagent is the CRP antibody sensitized particle suspension liquid based on liposome bag quilt; Described liposome is 1:(1 ~ 20 of phosphatid ylcholine and synthetic phospholipid) potpourri; Described synthetic phospholipid is by one or more synthesis in dipalmitoyl phosphatidylcholine, DPPE, distearoyl phosphatidylcholine;
Described calibration object is different containing the recombinant protein concentration of 6 CRP, solute and all identical cow's serum matrix of content thereof;
Described glycine buffer system, solvent is 10 ~ 50mM, pH6.0 ~ 6.5 glycine buffer, and solute and the content in described glycine buffer system thereof are NaCl200 ~ 400mM, Tween200.5 ~ 1.5ml/L, EDTA100 ~ 200mM, accelerator 200 ~ 500mM, antiseptic 0.1 ~ 2 ‰;
The described CRP antibody sensitized particle suspension liquid based on liposome bag quilt, solvent is the glycine buffer of 10 ~ 50mM, pH6.0 ~ 6.5, solute and the content in suspending liquid thereof are: based on the CRP antibody sensitized particle 10 ~ 50mg/L of liposome bag quilt, disodium ethylene diamine tetraacetate 0.5 ~ 5mmol/L, bovine serum albumin(BSA) 0.1 ~ 4mg/L, NaN
30.1 ~ 2 ‰; Described based on CRP antibody in the CRP antibody sensitized particle of liposome bag quilt by chemical bonds in surface of liposome;
In described calibration object, the recombinant protein concentration difference of CRP specifically refers to that the concentration of the recombinant protein of CRP in calibration object is respectively 0mg/ml, 20mg/ml, 40mg/ml, 80mg/ml, 160mg/ml, 320mg/ml; Solute in described calibration object and the volume content in described calibration object thereof are Sodium azide 0.2 ~ 2.2 ‰, polysorbas20 1 ~ 10%, bovine serum albumin(BSA) 1 ~ 3%;
The preparation method of the described CRP antibody sensitized particle suspension liquid based on liposome bag quilt is: by surface with 150 ~ 300nm of chemical based liposome and CRP antibody with 10: 1.5 mass ratio mix, be dissolved in the phosphate buffer of 0.02M, pH5.0 ~ 6.0, the percent by volume of liposome is 0.1 ~ 1.5%, and the concentration of antibody is 10mg/L; Incubated at room half an hour, then add activator solution activation, make the concentration of activator in reaction system reach 50 ~ 100 μ g/mL; Mix, 37 DEG C in conjunction with 8 hours; Dialysis removes the antibody that do not connect, adds confining liquid and closes 2 hours, centrifugally removes supernatant, with pH6.0 ~ 6.5 containing 0.5 ~ 5mmol/L disodium ethylene diamine tetraacetate, 0.1 ~ 4% bovine serum albumin(BSA), 0.1 ~ 2%NaN
3glycine buffer dilution; Described confining liquid is the MOPSO damping fluid of the pH7.4 containing 0.1% bovine serum albumin(BSA).
2. High-sensitivity C reactive protein detection kit of amplifying based on liposome signal according to claim 1, it is characterized in that, described accelerator comprises the one in glucosan series, PVP series; Described antiseptic comprise in sodium azide, Sodium Benzoate, potassium sorbate, thimerosal, Proclin-300, phenol one or more.
3. High-sensitivity C reactive protein detection kit of amplifying based on liposome signal according to claim 1, it is characterized in that, in the preparation method of the described CRP antibody sensitized particle suspension liquid based on liposome bag quilt, chemical based comprises one or more in carboxyl, hydroxyl, amino, sulfonic group.
4. High-sensitivity C reactive protein detection kit of amplifying based on liposome signal according to claim 1, it is characterized in that, in the preparation method of the described CRP antibody sensitized particle suspension liquid based on liposome bag quilt, reactivation process is specially: in 100ml1% concentration liposome, add 0.15 ~ 1ml glutaraldehyde and 30mg activator respectively, shaken at room temperature 1 hour, the dialysis unnecessary glutaraldehyde of removing and activator.
5. the High-sensitivity C reactive protein detection kit of amplifying based on liposome signal according to claim 1 or 4, it is characterized in that, described activator is carbodiimides, EDC/ (sulfo) NHS, N, one or more in N'-carbonyl dimidazoles, paratoluensulfonyl chloride, cyanogen bromide, N, N'-bis-succinimidyl carbonate, bis-epoxy based compound, hydroboration cyanogen.
6. High-sensitivity C reactive protein detection kit of amplifying based on liposome signal according to claim 1, is characterized in that, described CRP antibody comprise in goat anti-human crp's antibody, the anti-human CRP antibody of rabbit, mouse-anti Human C-reactiveprotein antibody one or more.
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| CN109975555B (en) * | 2019-03-11 | 2022-03-08 | 杭州利安生物科技有限公司 | Reagent and method for improving thermal stability of C-reactive protein kit and high-thermal stability C-reactive protein kit |
| CN112014574A (en) * | 2020-08-25 | 2020-12-01 | 上海睿康生物科技有限公司 | Serum amyloid protein A detection kit coated by double-liposome nanoparticles |
| CN112180079B (en) * | 2020-09-25 | 2024-04-19 | 上海睿康生物科技有限公司 | Stable liposome particle and application thereof in immune turbidimetry detection |
| CN113406335A (en) * | 2021-06-15 | 2021-09-17 | 安徽大千生物工程有限公司 | Complement valence liposome immunoassay kit and preparation and use method thereof |
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| US5756363A (en) * | 1993-02-03 | 1998-05-26 | Nissui Pharmaceutical Co., Ltd. | Liposome reagent for immunoagglutination and immunoanalytical method |
| CN101881777A (en) * | 2010-06-30 | 2010-11-10 | 深圳市国赛生物技术有限公司 | Assay method of high sensitivity C-reactive protein (HS-CRP) and HS-CRP assay kit |
| CN102749454A (en) * | 2012-06-11 | 2012-10-24 | 宁波鼎鑫生物科技有限公司 | Full-scale C-reactive protein (CRP) colloidal gold immunoturbidimetric assay kit |
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2014
- 2014-08-14 CN CN201410401483.6A patent/CN104215771B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0303980A2 (en) * | 1987-08-20 | 1989-02-22 | Hoechst Japan Limited | Carrier coated with antigen or antibody |
| US5756363A (en) * | 1993-02-03 | 1998-05-26 | Nissui Pharmaceutical Co., Ltd. | Liposome reagent for immunoagglutination and immunoanalytical method |
| CN101881777A (en) * | 2010-06-30 | 2010-11-10 | 深圳市国赛生物技术有限公司 | Assay method of high sensitivity C-reactive protein (HS-CRP) and HS-CRP assay kit |
| CN102749454A (en) * | 2012-06-11 | 2012-10-24 | 宁波鼎鑫生物科技有限公司 | Full-scale C-reactive protein (CRP) colloidal gold immunoturbidimetric assay kit |
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