CN108956982A - A kind of rheumatoid arthritis marker joint quantitative testing test paper and preparation method thereof - Google Patents
A kind of rheumatoid arthritis marker joint quantitative testing test paper and preparation method thereof Download PDFInfo
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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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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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
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/10—Magnetic particle immunoreagent carriers the magnetic material being used to coat a pre-existing polymer particle but not being present in the particle core
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Abstract
A kind of rheumatoid arthritis marker joint quantitative testing test paper, the rheumatoid arthritis marker is rheumatoid factor, Anti-cyclic critrullinated polypeptide antibody and C reactive protein, test strips include sample pad, rheumatoid arthritis marker antibody magnetism rare-earth fluorescent microballoon label pad, coated film and water absorption pad, coated film is equipped with three rheumatoid arthritis marker quantitative detection lines and a quality control region C line, it will be after the measured matter concentration in sample using magnetic field, utilize low background, highly sensitive fluorescence immune chromatography method, carry out detection measured matter, solve the problems, such as that POCT class product sensitivity is low.When can detect blood sample to avoid immune lateral chromatography reagent, for the interference for reducing other substances in blood.
Description
Technical field
The invention belongs to biomedicine field, be related to based on use magnetic rare-earth fluorescent microballoon as the class wind of trace labelling object
Wet arthritis marker combines quantitative testing test paper and preparation method thereof, specifically, a kind of measurement blood (serum, blood
Slurry, whole blood) in rheumatoid factor/Anti-cyclic critrullinated polypeptide antibody/C reactive protein content detection reagent card.
Background technique
Rheumatoid arthritis (rheumatoid arthritis, RA) is the polyphyly involved based on periarticular
The inflammatory autoimmunity disease of system property, the specific cause of disease is unclear, may be related with infectant and two kinds of factors of genetic predisposition,
RA is the rheumatic disease with the characteristics of the variability of arthritis, destruction of joint, progress sex dysfunction, and China's illness rate is about
It is one of the Etiological for causing population of China disability He disabling for 0.3%-0.6%.The disease early stage often because of symptom not
Typical case is by mistaken diagnosis or cannot make a definite diagnosis in time, and the treatment postponed keeps the prognosis of the disease very poor, if it is possible to this disease is made a definite diagnosis in early days, and
Rational therapy is given in time, then can be obviously improved prognosis.RA clinical diagnosis rheumatoid factor (rheumatoid factor,
RF it is) common classical serological index, but is a lack of specificity, other than patient, is also shown in connective tissue disease, liver
The others disease such as inflammation, tumour, infectious diseases and normal healthy people.Although RF is non-specific, high titre to RA
RF and RA disease it is closely related.Especially RF titre is higher, prompts the prognosis of patient poorer, is most strong in damage criterion
Prognostic factor, other autoantibodies cannot replace diagnosis and Index for diagnosis in status.So since RF is in RA disease
Have many advantages, such as early, high sensitivity occur, is still now one of the main foundation of clinical early diagnosis.
Anti-cyclic critrullinated polypeptide antibody (cyclic citrullonated peptide, CCP) is in rheumatoid arthritis
Early stage occurs, and with the joint erosion situation of RA patient and prognosis there are close correlation, antiCCP antibody is to rheumatoid joint
Though scorching sensibility is lower than rheumatoid factor, the specificity of diagnosis is close to 95%, and being significantly higher than rheumatoid factor etc., other face
Bed diagnoses common index, by the antiCCP antibody of ELISA method detection RA patient, and proves that antiCCP antibody has very RA diagnosis
High specificity and sensibility, domestic antiCCP antibody at present are just gradually being applied to precious to the clinic of RA disconnected.AntiCCP antibody be compared with
Good RA early diagnosis marker, and identify not aggressive, aggressivity RA sensitive indexes.
C reactive protein (C-reactive protein, CRP) is infected in body or when tissue damage in blood plasma
Some protein (acute protein) steeply risen, activating complement play opsonic action with the phagocytosis for reinforcing phagocyte, remove
Invade pathogenic microorganism and the damage of body, necrosis, the histocyte of apoptosis.CRP starts a few hours in inflammation and just increases, and 48 is small
When can reach to peak value, with lesion subside, tissue, structure and function recovery be down to normal level.This reaction is not by radiotherapy, change
It treats, the influence of corticosteroid therapy.Therefore, the detection of CRP is quite extensive in clinical application, examining including acute infectious diseases
Disconnected and antidiastole, the monitoring of post-operative infection；The observation of antibiotic curative effect；Course of disease detection and Index for diagnosis etc..
When tri- RF, antiCCP antibody, CRP index joints are used to diagnose RA, sensitive property is significantly improved, with it
He compares in each individual event, greatly reduces rate of missed diagnosis；Negative predictive value also improves simultaneously, if RF, antiCCP antibody, CRP index are complete
For feminine gender, the possibility with RA can be almost excluded, there is quite high clinical value.
Clinical diagnostic applications are most widely using the rheumatoid factor in import reagent detection serum and blood plasma/anti-at present
Cyclic citrulline polypeptide antibody/C reactive protein content.Kit detection now on the market is complicated for operation, time-consuming, should not be used as general
Logical screening can only be used as confirmed diagnosis test.Meanwhile at the same time sample on the market tested after being diluted and caused by
Sensitivity is relatively low.
Summary of the invention
In order to solve the above problem, facilitate tester's serum rheumatoid factor (SRF)/Anti-cyclic critrullinated polypeptide antibody/C- reaction egg
White, the present invention provides a kind of a kind of simple, reliable, low in cost, high sensitivity rheumatoid arthritis marker joints
Quantitative testing test paper；This detection method by after the measured matter concentration in sample, utilizes low background, highly sensitive using magnetic field
Fluorescence immune chromatography method carries out detection measured matter, solves the problems, such as that POCT class product sensitivity is low.It can be to avoid immune side
When detecting blood sample to chromatography reagent, for the interference for reducing other substances in blood.
In order to reach the purpose of the present invention, technical solution provided by the invention is as follows: a kind of rheumatoid arthritis mark
Internet of Things close quantitative testing test paper, and the rheumatoid arthritis marker is rheumatoid factor, Anti-cyclic critrullinated polypeptide antibody, C-
Reactive protein, rheumatoid arthritis marker joint quantitative testing test paper includes box body, the box body include bottom plate with
Box cover, the box body is interior to be equipped with Test paper, and bottom plate is equipped with below the Test paper, is connect above Test paper with box cover；
The box cover is equipped with through hole area；The through hole area is equipped with well and result detection window；Inspection is equipped with above the Test paper
Area is surveyed, detection zone is corresponding with through hole area；The detection zone includes that sample pad, rheumatoid arthritis marker antibody magnetism are dilute
Native fluorescent microsphere label pad, coated film and water absorption pad, the coated film are equipped with three rheumatoid arthritis marker joints
Quantitative detection line and a quality control region C line, the detection line and quality control region C line are arranged in parallel.
Further, the rheumatoid arthritis marker antibody magnetism rare-earth fluorescent microballoon wraps up magnetic by high molecular material
Property rare-earth fluorescent composition, the sphere diameter of the magnetism rare-earth fluorescent microballoon is 10-500nm, and the fluorescence emission wavelengths range is
400-800nm, nanosphere surface modified active group are amino, carboxyl or sulfydryl.
Further, the coated film both ends are mutual with water absorption pad and rheumatoid arthritis marker antibody label pad respectively
Overlapping connection, the coated film are nitrocellulose filter；It is overlayed above the rheumatoid arthritis marker antibody label pad
There is sample pad, the sample pad is glass fiber sample pad.
Further, the rheumatoid arthritis marker antibody magnetism rare-earth fluorescent microballoon label pad be glass fibre or
Polyester fiber.
A kind of rheumatoid arthritis marker joint quantitative testing test paper preparation method, including rheumatoid arthritis mark
Will object antibody marks and prepares Test paper, the rheumatoid arthritis marker antibody markers step:
1. with the magnetic rare-earth fluorescent microballoon of carboxylic polystyrene microsphere package Eu3+-Fe3O4 compound preparation, partial size
200nm, solid content 1%；
2. taking above-mentioned microballoon 1mL, 7.0 0.02M 3- of 5mL pH (N- Ma Lindai) propane sulfonic acid buffer (MOPS) is added, mixes
Even, 25000rpm is centrifuged 10min, abandons supernatant；5mL MOPS is added, 90W ultrasound, work 2s, and interval 5s is repeated 2 times,
25000rpm is centrifuged 10min, abandons supernatant；
3. 5mL MOPS is added again, 90W ultrasound, work 2s, and interval 5s is repeated 2 times, and completes the cleaning to microballoon；
4. 15mg EDC and 50mg sulfo-NHS is added in microballoon after cleaning, it is vortexed after mixing, 37 DEG C of reaction 15min；
5. 8.6 μ l of 2 mercapto ethanol after the reaction was completed, is added, the activation of carboxyl is terminated；
6. being separately added into rheumatoid factor, Anti-cyclic critrullinated polypeptide antibody, C reactive protein corresponds to each 1mg of antibody, it is placed in rotation
On incubator, 37 DEG C of 90~120min of reaction；
7. 100 μ L, 1% PEG20000 of 5% BSA and 100 μ L is then added, unlabelled site is closed, rotation is placed in
Turn on incubator, 37 DEG C of reaction 60min；
8. aforesaid liquid 2000rpm after reaction, is centrifuged 15min, supernatant is abandoned, is added and saves liquid (containing 1% BSA, 10% sugarcane
The pH8.0 0.2M boric acid borax buffer of sugar, 2% trehalose), 5mL, ultrasonic 2s；
9. the microballoon after label is diluted 50~100 times, every 200 μ L packing, as single part semi-finished product, with rheumatoid because
Son, Anti-cyclic critrullinated polypeptide antibody, C reactive protein measurement test paper are used cooperatively.
Rheumatoid arthritis marker combines quantitative testing test paper preparation step:
1. with BIODOT three-dimensional metal spraying point film instrument, with 1 μ L/cm parameter, rheumatoid factor being coated on nitrocellulose filter, is resisted
The corresponding antibody three detections line of cyclic citrulline polypeptide antibody, C reactive protein and a sheep anti mouse/goat anti-rabbit igg nature controlling line,
37 DEG C drying 16 hours, as reaction film.
2. impregnating glass fibre with the 0.1 M PBS solution containing 0.5% Tween-20, then 37 DEG C drying 16 hours, cut out
Cutting 17mm × 300mm is as sample pad.
3. pasting and being assembled in bottom plate sample pad, reaction film and blotting paper (22mm × 300mm) in the form laminated layer by layer
On.
Compared with prior art, the invention has the following beneficial effects: include sample pad, rheumatoid pass by detection zone
It saves scorching marker antibody magnetism rare-earth fluorescent microballoon label pad, coated film and water absorption pad, coated film and is equipped with three rheumatoids
Arthritis marker combines quantitative detection line and a quality control region C line, after the measured matter in sample is concentrated using magnetic field, benefit
With low background, highly sensitive fluorescence immune chromatography method, detection measured matter is carried out, it is low to solve POCT class product sensitivity
Problem.When can detect blood sample to avoid immune lateral chromatography reagent, for the interference for reducing other substances in blood.
Detailed description of the invention
Fig. 1 is rheumatoid factor test curve of the present invention；
Fig. 2 is Anti-cyclic critrullinated polypeptide antibody test curve of the present invention；
Fig. 3 is C reactive protein test curve of the present invention；
Fig. 4 is rheumatism factor determination method precision test result of the present invention；
Fig. 5 is Anti-cyclic critrullinated polypeptide antibody measuring method Precision test result of the present invention；
Fig. 6 is C reactive protein measuring method Precision test result of the present invention；
Fig. 7 is structural schematic diagram of the invention；
In figure: 1, bottom plate；2, sample pad；3, rheumatoid arthritis marker antibody magnetism rare-earth fluorescent microballoon label pad；4,
Coated film；5, water absorption pad；6, box body；61, box cover；71, well；72, result watch window；8, rheumatoid arthritis mark
Internet of Things close quantitative detection line；9, quality control region C line.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
The description of specific distinct unless the context otherwise, the present invention in detectable substance title and parameter, quantity both can be single
A form exists, and form that can also be multiple exists, and the present invention is defined not to this.Although step in the present invention with
Label is arranged, but is not used to limit the precedence of step, unless expressly stated the order of step or certain step
Based on rapid execution needs other steps, otherwise the relative rank of step is adjustable.It is appreciated that institute herein
The term "and/or" used one of is related to and covers associated listed item or one or more of any and all possibility
Combination.
A kind of rheumatoid arthritis marker joint quantitative testing test paper, the rheumatoid arthritis marker are class
The rheumatism factor, Anti-cyclic critrullinated polypeptide antibody, C reactive protein, the rheumatoid arthritis marker joint quantitative detection examination
Paper includes box body, and the box body includes bottom plate and box cover, and Test paper is equipped in the box body, is equipped with below the Test paper
Bottom plate, Test paper top are connect with box cover；The box cover is equipped with through hole area；The through hole area is equipped with well and result detects
Window；Detection zone is equipped with above the Test paper, detection zone is corresponding with through hole area；The detection zone includes sample pad, class
Rheumatic arthritis marker antibody magnetism rare-earth fluorescent microballoon label pad, coated film and water absorption pad, the coated film are equipped with
Three rheumatoid arthritis marker joint quantitative detection lines and a quality control region C line, the detection line and quality control region C line are flat
Row arrangement.The rheumatoid arthritis marker antibody magnetism rare-earth fluorescent microballoon is glimmering by high molecular material coated magnetic rare earth
Photoreactivation object, the sphere diameter of the magnetism rare-earth fluorescent microballoon are 10-500nm, and the fluorescence emission wavelengths range is 400-
800nm, nanosphere surface modified active group are amino, carboxyl or sulfydryl.The coated film both ends respectively with water absorption pad and
The mutually overlapping connection of rheumatoid arthritis marker antibody label pad, the coated film is nitrocellulose filter；The class wind
Sample pad is overlayed above wet arthritis marker antibody label pad, the sample pad is glass fiber sample pad.The class
Rheumatic arthritis marker antibody magnetism rare-earth fluorescent microballoon label pad is glass fibre or polyester fiber.
Embodiment 1, the foundation of detection method:
The preparation of 1.1 polystyrene microsphere seeds
1.1.1 it in the styrene and separatory funnel for measuring 100ml, is washed 3 times, is removed in styrene repeatedly with 1 M NaOH
Then impurity is placed in drying basin more than drying for 24 hours then with purifying water washing to neutrality；
1.1.2 argon gas is filled in reaction unit, the oxygen in removing device；
1.1.3 the ethyl alcohol of 100ml is added in reaction unit, 5g PVP is then added, stirs 30min；
1.1.4 40ml washed styrene and 5ml azodiisobutyronitrile (AIBN) is added, warming-in-water is to 45 DEG C, reaction
Then reaction temperature is increased to 75 DEG C by 30min, react 12h；
1.1.5 after reaction, with the above-mentioned reaction solution of screen filtration, centrifugation 3~5 times then is washed with dehydrated alcohol, then will
Polymer after preliminary removal of impurities makes its natural sedimentation, weighs Sediment weight in 40% ethyl alcohol.
1.2 the carboxylated of polystyrene surface is modified
1.2.1 using the resulting polystyrene microsphere of above-mentioned dispersion as seed 1g, dispersed with 0.25% SDS of 10ml；
1.2.2 the hexamethylene of 2 times of seed weights, 35 DEG C of 16~18h of swelling and then are in the above system added；
1.2.3 it weighs 0.02g benzoyl peroxide, measure 10 μ l ethylene glycol monoemethyl ethers and 10 μ l dimethyl benzene olefin(e) acid ethylene glycol
Ester is added separately in above-mentioned reaction solution, continues to be swollen 12h；
1.2.4 after being swollen, 2.5mg PVA is added, improves reaction temperature to 75 DEG C, the reaction was continued 10~12h；
1.2.5 it is washed repeatedly with ethyl alcohol 3 times, removes unreacted monomer and oligomer, then 20000rpm centrifugal sedimentation is micro-
Ball.
The preparation of 1.3 magnetism-fluorescent nanometer microsphere
1.3.1 by FeCl2、FeCl3、EuCl3, NaOH solution, example Fe in molar ratio2+: Fe3+: Eu3+: Na+=1:1:2:5, by it
Solution is mixed, and is stirred to react, and is precipitated, and is filtered after being aged 3~5h, with purifying water washing 2~3 times, is dried, after grinding
Obtain Eu3+-Fe3O4Compound.
1.3.2 the ultrasonic disperse into 20ml isopropanol is added in the polystyrene microsphere for taking 0.1g carboxylated to modify
10min；
1.3.3 Eu is weighed3+-Fe3O4The ultrasonic disperse 20min into 5ml chloroform is added in compound 0.5mg；
1.3.4 two kinds of liquid in 1.3.2 and 1.3.3 are mixed, 5~6h is placed in control in vacuum environment, is then placed in normal pressure again
2~3h；
1.3.5 it by the microballoon after swelling with ethanol washing 3 times, is removed with magnetic field and fails and wrap up the microballoon of magnetic particle, with suitable
Amount is resuspended containing 0.05% sodium azide aqueous solution to get magnetic rare-earth fluorescent microballoon, and 2~8 DEG C of preservations are forbidden to freeze.
The label of 1.4 antibody
1.4.1 it takes the 200nm of above-mentioned preparation, the microballoon 1ml that solid content is 1%, 7.0 0.02M 3- (N- Ma of 5ml pH is added
Beautiful jade generation) propane sulfonic acid buffer (MOPS), it mixes, 25000rpm is centrifuged 10min, abandons supernatant；
1.4.2 5ml MOPS is added, 90W ultrasound, work 2s, and interval 5s is repeated 2 times, 25000rpm centrifugation 10min, in abandoning
Clearly；
1.4.3 5ml MOPS is added again, 90W ultrasound, work 2s, and interval 5s is repeated 2 times, and completes the cleaning to microballoon；
1.4.4 15mg EDC and 50mg sulfo-NHS is added in microballoon after cleaning, is vortexed after mixing, 37 DEG C of reactions
15min；
1.4.5 after the reaction was completed, 8.6 μ l of 2 mercapto ethanol is added, terminates the activation of carboxyl；
1.4.6 be separately added into rheumatoid factor, Anti-cyclic critrullinated polypeptide antibody, C reactive protein each 1mg of corresponding antibody,
It is placed on rotating and culturing case, 37 DEG C of 90~120min of reaction；
1.4.7 100 μ l, 1% PEG20000 of 5% BSA and 100 μ l is then added, unlabelled site is closed, is set
In on rotating and culturing case, 37 DEG C of reaction 60min；
1.4.8 after reaction, by aforesaid liquid 2000rpm be centrifuged 15min, abandon supernatant, be added save liquid (containing 1% BSA,
The pH8.0 0.2M boric acid borax buffer of 10% sucrose, 2% trehalose), 5ml, ultrasonic 2s；
1.4.9 the microballoon after label is diluted 50~100 times, every 200 μ l are dispensed, as single part semi-finished product, with rheumatoid
The factor/Anti-cyclic critrullinated polypeptide antibody/C reactive protein measurement test paper is used cooperatively.
The preparation of 1.5 test papers
1.5.1 rheumatoid factor is coated on nitrocellulose filter with 1 μ l/cm parameter with BIODOT three-dimensional metal spraying point film instrument
The corresponding antibody three detections line of antibody, Anti-cyclic critrullinated polypeptide antibody antibody, C reactive protein and a sheep anti mouse/goat-anti
Rabbit igg nature controlling line, 37 DEG C drying 16 hours, as reaction film.
1.5.2 glass fibre is impregnated with the 0.1 M PBS solution containing 0.5% Tween-20, then 37 DEG C of dryings 16 are small
When, cutting 17mm × 300mm is as sample pad.
1.5.3 sample pad, reaction film and blotting paper (22mm × 300mm) are carried out pasting group in the form laminated layer by layer
On PVC bottom plate, after slitting, as rheumatoid factor/Anti-cyclic critrullinated polypeptide antibody/C reactive protein measures test paper, sets
Enter in plastics cartridge, as rheumatoid factor/Anti-cyclic critrullinated polypeptide antibody/C reactive protein measures reagent card.
Embodiment 2, rheumatoid factor, Anti-cyclic critrullinated polypeptide antibody, C reactive protein detection method
2.1 the range of linearity
2.1.1 at least five kinds of concentration will be diluted to according to a certain percentage close to the high level sample of the range of linearity upper limit respectively, wherein
The sample of low value concentration must be close to the lower limit of the range of linearity；
2.1.2 above-mentioned each concentration samples respectively take 100 μ l, in the label microballoon after being added separately to packing, are vortexed and mix；
2.1.3 the above reaction solution is placed on magnetic field, after 1min, is discarded supernatant, then every 200 μ l PBS(0.02M of addition,
PH7.4)；
2.1.4 remove magnetic field, be vortexed again for mixing, above each concentration samples are taken into 100 μ l respectively, be added to rheumatoid factor/
In Anti-cyclic critrullinated polypeptide antibody/C reactive protein measurement test paper, every concentration retest 3 times；
2.1.5 after reacting 10min, test paper is put into dry type immunofluorescence analysis instrument, T/C value is read, calculates the T/ of each concentration
C mean value, using double-log straight line fitting concentration-response curve, (X-axis is lg (concentration)；Y-axis is lg (T/C)), as shown, class
Rheumatism factor response curve is y=1.0581x -1.1134, R=0.9964；Anti-cyclic critrullinated polypeptide antibody response curve be y=
1.3833x -2.4438, R=0.9905；C reactive protein response curve is y=0.5475x-0.1391, R=0.9907.
2.2 minimum detectability
Negative serum is operated by 2.1.2~2.1.4, retest 20 times, calculates T/C mean value, it is bent to be substituted into 2.1.5 test
In line, the minimum detectability of this method is obtained, wherein rheumatoid factor minimum detectability is 3IU/mL；Anti- cyclic citrulline polypeptide is anti-
Body minimum detectability is 7U/mL；C reactive protein minimum detectability is 0.2mg/L.
2.3 precision
Calibration object is diluted to high, medium and low three concentration, is operated by 2.1.2~2.1.4, every concentration retest 10 times.As a result
For shown in Fig. 1-Fig. 3, it can thus be seen that the coefficient of variation of this method test is respectively less than 10%, therefore precision with higher
Degree.
2.4 compared with other products
This method tests 92 parts of clinical serums, with other product measured value correlations in the market, rheumatoid factor result are as follows: and y=
0.9631x+0.5134, related coefficient 0.975, Anti-cyclic critrullinated polypeptide antibody result are as follows: y=0.9426x+
0.7012, related coefficient 0.984, C reactive protein result are as follows: y=0.9755x+0.6312, related coefficient are
0.967, show that this reagent has good consistency with the detection method listed.
It is comprehensive it is found that this method can detect rheumatoid factor, Anti-cyclic critrullinated polypeptide antibody, C reactive protein, have compared with
Good precision tests the equal < 10% of the coefficient of variation.It is had good correlation through clinical trial, and with commercialized product.This hair
It is bright using magnetic field by after the measured matter concentration in sample, using low background, highly sensitive fluorescence immune chromatography method, examined
Measured matter is surveyed, solves the problems, such as that POCT class product sensitivity is low.Blood sample can be detected to avoid immune lateral chromatography reagent
This when, for reduce blood in other substances interference, tested after being usually diluted sample and caused by sensitivity it is inclined
Low problem.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (6)
1. a kind of rheumatoid arthritis marker combines quantitative testing test paper, the rheumatoid arthritis marker is class wind
The wet factor, Anti-cyclic critrullinated polypeptide antibody, C reactive protein, it is characterised in that: the rheumatoid arthritis marker joint
Quantitative testing test paper includes box body, and the box body includes bottom plate and box cover, and Test paper, the detection examination are equipped in the box body
It is equipped with bottom plate below paper, is connect above Test paper with box cover；The box cover is equipped with through hole area；It is equipped with above the Test paper
Detection zone, detection zone are corresponding with through hole area；The detection zone includes sample pad, rheumatoid arthritis marker antibody magnetism
Rare-earth fluorescent microballoon label pad, coated film and water absorption pad, it is fixed that the coated film is equipped with three rheumatoid arthritis markers
It measures detection line and a quality control region C line, the detection line and quality control region C line is arranged in parallel.
2. a kind of rheumatoid arthritis marker according to claim 1 combines quantitative testing test paper, it is characterised in that:
The rheumatoid arthritis marker antibody magnetism rare-earth fluorescent microballoon is to be answered by high molecular material coated magnetic rare-earth fluorescent
Object is closed, the sphere diameter of the magnetism rare-earth fluorescent microballoon is 10-500nm, and the fluorescence emission wavelengths range is 400-800nm, is received
Rice microsphere surface modification activities group is amino, carboxyl or sulfydryl.
3. a kind of rheumatoid arthritis marker according to claim 1 combines quantitative testing test paper, it is characterised in that:
The coated film both ends mutually overlap connection with water absorption pad and rheumatoid arthritis marker antibody label pad respectively；The packet
Envelope is nitrocellulose filter；Sample pad, the sample have been overlayed above the rheumatoid arthritis marker antibody label pad
Product pad is glass fiber sample pad.
4. a kind of rheumatoid arthritis marker according to claim 1 combines quantitative testing test paper, it is characterised in that:
The rheumatoid arthritis marker antibody magnetism rare-earth fluorescent microballoon label pad is glass fibre or polyester fiber.
5. a kind of rheumatoid arthritis marker combines quantitative testing test paper preparation method, including rheumatoid arthritis mark
Object antibody marks and prepares Test paper, the rheumatoid arthritis marker antibody markers step:
1. wrapping up Eu with carboxylic polystyrene microsphere3+-Fe3O4The magnetic rare-earth fluorescent microballoon of compound preparation, partial size
200nm, solid content 1%；
2. taking above-mentioned microballoon 1mL, 7.0 0.02M 3- of 5mL pH (N- Ma Lindai) propane sulfonic acid buffer (MOPS) is added, mixes
Even, 25000rpm is centrifuged 10min, abandons supernatant；5mL MOPS is added, 90W ultrasound, work 2s, and interval 5s is repeated 2 times,
25000rpm is centrifuged 10min, abandons supernatant；
3. 5mL MOPS is added again, 90W ultrasound, work 2s, and interval 5s is repeated 2 times, and completes the cleaning to microballoon；
4. 15mg EDC and 50mg sulfo-NHS is added in microballoon after cleaning, it is vortexed after mixing, 37 DEG C of reaction 15min；
5. 8.6 μ l of 2 mercapto ethanol after the reaction was completed, is added, the activation of carboxyl is terminated；
6. being separately added into rheumatoid factor, Anti-cyclic critrullinated polypeptide antibody, C reactive protein corresponds to each 1mg of antibody, it is placed in rotation
On incubator, 37 DEG C of 90~120min of reaction；
7. 100 μ L, 1% PEG20000 of 5% BSA and 100 μ L is then added, unlabelled site is closed, rotation is placed in
Turn on incubator, 37 DEG C of reaction 60min；
8. aforesaid liquid 2000rpm after reaction, is centrifuged 15min, supernatant is abandoned, is added and saves liquid (containing 1% BSA, 10% sugarcane
The pH8.0 0.2M boric acid borax buffer of sugar, 2% trehalose), 5mL, ultrasonic 2s；
9. the microballoon after label is diluted 50~100 times, every 200 μ L packing, as single part semi-finished product, with rheumatoid because
Son, Anti-cyclic critrullinated polypeptide antibody, C reactive protein measurement test paper are used cooperatively.
6. a kind of rheumatoid arthritis marker according to claim 5 combines quantitative testing test paper preparation method, institute
State rheumatoid arthritis marker joint quantitative testing test paper preparation step:
1. with BIODOT three-dimensional metal spraying point film instrument, with 1 μ L/cm parameter, rheumatoid factor being coated on nitrocellulose filter, is resisted
The corresponding antibody three detections line of cyclic citrulline polypeptide antibody, C reactive protein and a sheep anti mouse/goat anti-rabbit igg nature controlling line,
37 DEG C drying 16 hours, as reaction film；
2. impregnating glass fibre with the 0.1 M PBS solution containing 0.5% Tween-20, then 37 DEG C drying 16 hours, cut
17mm × 300mm is as sample pad；
3. sample pad, reaction film and blotting paper (22mm × 300mm) are pasted and are assembled on bottom plate in the form laminated layer by layer.
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| CN111868073A (en) * | 2019-05-31 | 2020-10-30 | 广州市雷德生物科技有限公司 | Specific polypeptide related to rheumatoid arthritis and application thereof |
| CN112285364A (en) * | 2020-11-05 | 2021-01-29 | 南京申基医药科技有限公司 | Kit for combined detection of three items of rheumatism and preparation method thereof |
| CN114062666A (en) * | 2021-11-24 | 2022-02-18 | 山东康华生物医疗科技股份有限公司 | Preparation method of detection plate for quantitatively detecting RF, ASO, CRP and CCP in one card mode |
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| CN111868073A (en) * | 2019-05-31 | 2020-10-30 | 广州市雷德生物科技有限公司 | Specific polypeptide related to rheumatoid arthritis and application thereof |
| CN112285364A (en) * | 2020-11-05 | 2021-01-29 | 南京申基医药科技有限公司 | Kit for combined detection of three items of rheumatism and preparation method thereof |
| CN114062666A (en) * | 2021-11-24 | 2022-02-18 | 山东康华生物医疗科技股份有限公司 | Preparation method of detection plate for quantitatively detecting RF, ASO, CRP and CCP in one card mode |
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