CN106706926A - Serum amyloid A testing kit and manufacturing method - Google Patents
Serum amyloid A testing kit and manufacturing method Download PDFInfo
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- CN106706926A CN106706926A CN201611165653.0A CN201611165653A CN106706926A CN 106706926 A CN106706926 A CN 106706926A CN 201611165653 A CN201611165653 A CN 201611165653A CN 106706926 A CN106706926 A CN 106706926A
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- 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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- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- 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
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Abstract
The invention relates to the technical field of fluorescence immunoassay in medical immunology, and in particular relates to a serumamyloid A (SAA) testing kit (fluorescence immunochromatographic method) and a manufacturing method. The kit is provided with a test paper card. The kit is characterized in that a PVC (Polyvinyl Chloride) plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorbing pad are sequentially arranged on the test paper card from bottom to top; a rare-earth Eu<3+> fluorescent microsphere marked serum amyloid A monoclonal antibody is adsorbed on the conjugate pad; the diameter of a rare-earth fluorescent microsphere is 200nm; the rare-earth fluorescent microsphere contains a rare-earth lanthanide element Eu<3+>; the rare-earth fluorescent microsphere is stable in a ground state and emits fluorescence of which the wavelength is 615nm under the stimulation action of laser of 337nm; and the monoclonal antibody is a monoclonal antibody which is purified and mixed, and comes from a monoclonal antibody cell strain for 2-6 different serum amyloid A epitopes. The kit has the advantages of being simple and convenient to operate, rapid in reaction, high in sensitivity, good in specificity and the like.
Description
Technical field：
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, including protein-crosslinking technology, film layer analysis skill
Art, labelling immunoassay technology etc..Specifically one kind can fast and accurately to blood in the samples such as serum, blood plasma and whole blood
The serum amyloid A protein that clear amyloid A carries out quantitative analysis determines kit and preparation method.
Background technology：
Serum amyloid A protein (SAA) is the serum precursor of main constituents amyloid A protein in amyloidosis, is
A kind of acute phase protein in serum is secreted into after being produced by liver cell, although be seen everywhere, but under normal circumstances,
Express still very low in human body, but body is once being inflamed, infect or during tissue damage, SAA levels can be in 5~6h
It is rapid to raise about 1 000 times, research display, in bacterium, fungi, viral infection, atherosclerosis, angiocardiopathy, acute
SAA is raised in can detect blood in the diseases such as graft-rejection, is declined rapidly in the convalescence of disease.Serum amyloid sample
Albumin A (SAA) is a height heterogeneity albumen, represents the family that a relative molecular mass is as 12000.In some aspects
SAA has more sensitivity than other common acute albumen, and such as in virus and bacterium infection, the serum-concentration of SAA is raised, and
In virus infection reaction, the serum-concentration of C reactive protein (CRP) is hardly raised or raised unobvious.SAA can be by activation
The release of the inflammatory mediator such as neutrophil leucocyte and the induction of other inflammatory cells and metalloproteinases, so as to strengthen the inflammatory of body
Reaction, there is immunoregulation effect.Therefore, while SAA is considered as a kind of Inflammatory Mediators, a kind of inflammatory signals are also served as
Triggering agent is present.The content concn of SAA is the sensitive indicator for reflecting infectious diseases Earlier period of inflammation, contributes to the auxiliary of inflammation to examine
Break, assess its activity, monitor its activity and treatment.
Immuno analytical method is detected such as using the immune response between trace antigen and corresponding antibody with high specificity
It is living in the organisms such as hormone, medicine, protein, polypeptide, enzyme, tumor associated antigen, micro-element, virus, bacterium and metallic element
Property material.Immuno analytical method includes labelling immunoassay, non-marked immunoassay and instrument immunoassay.This kit is utilized
The carboxyl latex microballoon labelling immunoassay technology containing rare earth element be belonging to one kind of labelling immunoassay.
Fluoroimmunoassay (FIA) and radiommunoassay (RIA) since the advent of the world, experienced the development of decades, but
It is that people increasingly feel FIA because of naturally local too high, interference detection results；RIA uses isotope marks, has pole to human body
Big harm is simultaneously made troubles to experiment.EIA enzyme immunoassay (EIA) is larger by other influences factor also because enzyme is unstable in itself, pushes away
Wide application is restricted.The beginning of the eighties, people begin one's study and replace fluorescent material and isotope-labelled protein with rare earth element
Or antibody, TIME RESOLVED TECHNIQUE is incorporated into field of biological detection, establish new ultramicron time-resolved fluoroimmunoassay point
Analysis technology (Time resolved Fluoroimmunoassay, abbreviation TrFIA).The technology uses multidisciplinary advanced technology, collection
The characteristics of having tied other immunoassays, in fields such as immunology, molecular biology, cytology and medical science, obtains significant progress
And extensive use.
TrFIA make use of trivalent rare earth ion and chelate with unique fluorescent characteristic be tracer replace fluorescent material,
Enzyme, isotope, chemiluminescent substance, labelled antibody, antigen, hormone, polypeptide, protein, nucleic acid probe and biological cell treat anti-
Answer system (such as antigen-antibody reaction, nucleic acid probe hybridization, biotin-labeled pentylamine reaction and the killing of target cell pairing effect cell
Effect etc.) occur after, with TrFIA detectors determine product in fluorescence intensity.According to product fluorescence intensity and relatively glimmering
The ratio of luminous intensity, judges the concentration of analyte in reaction system, so as to reach quantitative analysis.In common fluoremetry,
Due to containing various fluorescent components in test sample, background fluorescence (causes from the colloidal solid and solvent molecule in sample
The non-specific fluorescence that scattering light and Proteins in Serum and other compounds send) intensity is big, disturb strong, as fluorescence point
The bottleneck that analysis method is promoted on a large scale.Why TrFIA can turn into after the new sensitive detection method of the latter of EIA, RIA,
Depend primarily on the wavelength resolution and time-delay technique that are used in the unique fluorescence feature of lanthanide series, detection and dissociation-
Enhancing technology.
Lanthanide series (lanthanide, Ln) belongs to rare earth element, has in 17, be usually used in TrFIA mainly have europium (Eu),
Samarium (Sm), terbium (Tb), dysprosium (Dy).Lanthanide series has unique fluorescence radiation feature, compared with common fluorescent, lanthanide ion chela
Compound fluorescence decay time is long, is the 10 of conventional fluorescent3-106Times.If the fluorescence decay time of lanthanide ion chelate is in 60-
900 μ s, conventional Eu3+Fluorescence decay time is 714 μ s, and the fluorescence decay time of fluorogen only has in common fluorescent immunoassay
1-100 μ s, the fluorescence decay time of some protein is only 1-10 μ s in sample, therefore utilizes TIME RESOLVED TECHNIQUE, postpones one
Measured after fixing time, just can obtain Eu3+Specific fluorescence signal.Simultaneously because decay time is long, Eu3+Label is in measurement
Between in can be excited repeatedly, ground state is transitted to by excitation state quickly after exciting every time, just there is fluorescence to send, then again can be weighed
Newly excite, so it is per second have 1000 times excite so that the relative specific activity of TrFIA fluorescent markers is very high.Lanthanide series is glimmering
The maximum of light spectrum is characterized in that the Stokes displacements between exciting light and launching light are larger, Eu3+Excitation wavelength is 337nm, transmitting
Wavelength is 615nm, and Stokes displacements are up to 278nm；While Eu3+The fluorescence light belt being excited is extremely narrow, and the emission peak of fluorescence is very
Sharply, can adjust instrument to be determined in extremely narrow wave-length coverage, thus almost completely eliminate the interference of background fluorescence, after
And pass through time delay and wavelength resolution, and strong specificity fluorescent and background fluorescence are distinguished into open (therefore referred to as time resolution), make interference
Reach almost nil.
In view of the application of above labeling method and detection technique, this kit has good detection specific, higher
The fluorescent marker of sensitivity, the simplicity of operation and stabilization ensure that the accuracy of detection.
The content of the invention：
The present invention is for shortcoming and defect present in prior art, it is proposed that a kind of utilization fluorescence immune chromatography it is sensitive
Property, combined with fluorescent immunochromatographiassays assays instrument realize sensitivity it is high, fast and simple, can be with the serum amyloid protein of accurate quantitative analysis
A determines kit and preparation method.
The present invention can be reached by following measures：
A kind of serum amyloid A protein determines kit, is provided with test card, it is characterised in that the test card is from the bottom to top
It is sequentially provided with：Rare-earth fluorescent is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad
Microballoon mark antiserum amyloid A monoclonal antibody-microballoon coupled complex, the rare-earth fluorescent microballoon it is a diameter of
100-250nm, rare-earth fluorescent microballoon is stable under ground state containing one or more in rare earth lanthanide, 300-400nm's
Launch the fluorescence that wave-length coverage is 550-650nm under excitation source effect；The monoclonal antibody is the list for mixing after purification
Clonal antibody, from for the 2-6 cell strain of monoclonal antibody of different serum amyloid A protein epitopes.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is preferably 120-150nm；The rare-earth fluorescent microballoon
Preferably comprise one or more rare earth lanthanides；The antibody of rare-earth fluorescent microballoon mark is preferably derived from for 2 on pad
The monoclonal cell cell line of individual different epitopes.
Pad of the present invention is obtained using following steps：Glass fibre membrane is soaked in 150mM Tris-HCL treatment
In liquid (X-100 containing 1.0%Triton, 2.5%BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 small
When, it is standby, glass fibre membrane is placed on Bio-DotXYZ3050 three-dimensional specking platforms, connect with Bio-Jet Quanti300 are non-
The antiserum amyloid A monoclonal antibody coupled complex that the micro- quantitation nozzle of touch marks rare-earth fluorescent microballoon is sprayed onto glass
Glass tunica fibrosa, 37 DEG C drying 1 hour after be obtained.
The antiserum amyloid A monoclonal antibody of the rare-earth fluorescent microballoon mark in the present invention on pad is adopted
It is obtained with following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：With serum amyloid A protein sterling immune mouse, using standard
Method for preparing monoclonal antibody prepares the cell strain of monoclonal antibody of specific high-affinity, and the monoclonal antibody cell line to being obtained enters
Row pairing screening, the monoclonal antibody cell line for kit is preferably gone out according to pairing result and affinity data；
Step 2：The preparation of monoclonal antibody：Antiserum amyloid egg is prepared and purified using the ascites production technology of standard
White A monoclonal antibodies, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：5mg rare-earth fluorescent microballoons are taken, with 20mM, the carbonate of pH 9.5 delays
Fliud flushing, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in the above-mentioned carbon of 100 μ l
In phthalate buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In washing and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Step 4：Rare earth Eu3+The preparation of the antiserum amyloid A monoclonal antibody of fluorescent microsphere mark：Selection comes from
2 monoclonal antibodies of the monoclonal cell cell line of different epitopes, according to mass ratio 1:1 by 2mg serum amyloid sample eggs
, in 4 DEG C of dialysed overnights, then the rare-earth fluorescent microballoon with above-mentioned aldehyde radical is mixed for the above-mentioned carbonate buffer solution of white A monoclonal antibodies
Close, 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM is added, 4 DEG C are reacted 4 hours；Add isometric confining liquid
(50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 50mM Tris-HCL, pH7.4 are used
Buffer solution washed 3 times using centrifugal process, in being resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l (containing 1.2%NaCL,
0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.
The nitrocellulose filter for being coated with detection line and nature controlling line of the present invention is obtained by following steps：
Step 1：Using the cell different from antiserum amyloid A cell strain of monoclonal antibody used on pad
Strain, antiserum amyloid A monoclonal antibody is prepared and purified using the ascites production technology of standard, be stored in -20 DEG C it is standby
With；
Step 2：Coating dilution is used respectively by above-mentioned mouse source antiserum amyloid A monoclonal antibody and sheep anti-Mouse
To 1-3mg/ml, film liquid amount is 1-3 μ l/cm to IgG antibody adjustment concentration, using them as detection line spray parallel with nature controlling line
Spill in being coated with nitrocellulose filter, detection line and nature controlling line are subsequently placed in baking oven at intervals of 3-7mm, 37 DEG C of drying 2
Hour.
Sample pad of the present invention is obtained by following steps：Glass fibre membrane is soaked in and contains 1.0%Triton X-
100,2.5%BSA, 0.15M Tris buffer solutions, in the treatment fluid of pH7.5,4 hours are soaked in 4 DEG C, are subsequently placed in baking oven
In, 37 DEG C dry 2 hours.
Present invention also offers the serum amyloid A protein preparation method that a kind of kit as described above is realized, its feature
It is to comprise the following steps：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, kept flat；
Step 2：Card Reader：IC-card is placed on dry type fluorescence immunity analyzer labeling position, relevant information is read, it is described glimmering
Light immunochromatographiassays assays instrument is a kind of Systems for optical inspection, and the measurement range to serum amyloid A protein is 5.00-200mg/L；
Step 3：Sample-adding：Serum/plasma：Take 100 μ L serum/plasmas samples vertically to drop at test card sample-adding, whole blood：
150 μ L whole blood samples are taken vertically to drop at test card sample-adding；It is careful not to suck bubble during sampling；
Step 4：Detection, can be detected, automatic test using automatic test or immediately test both of which：By test card
Insert on the carrier of dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned analysis detection to test card automatically；
Immediately test：After test card room temperature places 15min, insert on the carrier of dry type fluorescence immunity analyzer, click on test immediately.
The present invention provides a kind of using rare earth Eu3+Serum prepared by the fluorescence immune chromatography technology of carboxyl latex microballoon mark
Amyloid A (SAA) determines kit (fluorescence immune chromatography method), while being adapted to serum, blood plasma and whole blood sample, and is adapted to
Clinically single part detection, relative to the qualitative colloid gold reagent of serum amyloid A protein, the serum in energy quantitative determination sample forms sediment
Powder sample Protein A content, with more specific Clinical significance of MG, with easy to operate, reaction it is quick, sensitivity is high, specificity
By force, be adapted to Site Detection and it is economical and practical the advantages of.
Brief description of the drawings：
Accompanying drawing 1 is the structural representation of test card in the present invention.
Accompanying drawing 2 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 3 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 4 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Reference：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4, adsorptive pads 5.
Specific embodiment：
The present invention is further illustrated with reference to the accompanying drawings and examples：
As shown in Figure 1, kit is determined present invention firstly provides a kind of serum amyloid A protein, examination is provided with box
Paper card, the test card is sequentially provided with from the bottom to top：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4 and adsorptive pads
5, rare earth Eu is wherein adsorbed with pad3+The antiserum amyloid A monoclonal antibody of fluorescent microsphere mark-microballoon coupling
Compound, a diameter of 150nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+, it is steady under ground state
It is fixed, the fluorescence of wavelength 615nm is launched under the excitation source effect of 337nm；The monoclonal antibody mixes after purification
Monoclonal antibody, from for the 2-6 cell strain of monoclonal antibody of different serum amyloid A protein epitopes.
The diameter of the rare-earth fluorescent microballoon of the pad 3 is preferably 150nm；The rare-earth fluorescent microballoon preferably comprises dilute
Native lanthanide series europium (Eu3+)；The antibody of rare-earth fluorescent microballoon mark is preferably derived from for 2 different epitopes on pad
Monoclonal cell cell line.
Embodiment 1：
Each part that serum amyloid A protein determines test card in kit can be obtained by following measures：
1st, the preparation of sample pad 2：
Glass fibre membrane is soaked in containing 1.0%Triton X-100,2.5%BSA, 0.15M Tris buffer solutions,
In the treatment fluid of pH7.5,4 hours are soaked in 4 DEG C, be subsequently placed in baking oven, 37 DEG C dry 2 hours.
2nd, the preparation of the pad 3 of absorption fluorescent microsphere labelled antibody：
Glass fibre membrane is soaked in (X-100 containing 1.0%Triton, 2.5% in 150mM Tris-HCL treatment fluids
BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 hours, standby, and glass fibre membrane is placed on into Bio-
On DotXYZ3050 three-dimensional specking platforms, quantitation nozzle is declined by rare earth Eu with Bio-Jet Quanti300 noncontacts3+Fluorescence is micro-
The antiserum amyloid A monoclonal antibody coupled complex of ball mark is sprayed onto glass fibre membrane, and 37 DEG C of drying are made after 1 hour
.
The aldehyde radical of rare-earth fluorescent Nano microsphere：5mg rare-earth fluorescent Nano microspheres are taken, with 20mM, the carbonate of pH9.5 delays
Fliud flushing, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in the above-mentioned carbon of 100 μ l
In phthalate buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In washing and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Rare earth Eu3+The preparation of the antiserum amyloid A monoclonal antibody of fluorescent microsphere mark：Choose from 2 not
The monoclonal antibody of the monoclonal cell cell line of synantigen epitope, according to mass ratio 1:1 by 2mg antiserum amyloid As
Then the above-mentioned carbonate buffer solution of monoclonal antibody mixes in 4 DEG C of dialysed overnights with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical,
4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM is added, 4 DEG C are reacted 4 hours；Add isometric confining liquid
(50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 50mM Tris-HCL, pH7.4 are used
Buffer solution washed 3 times using centrifugal process, in being resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l (containing 1.2%NaCL,
0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.
3rd, it is coated with the preparation of the nitrocellulose filter 4 of detection line and nature controlling line：
Using the cell line different from antiserum amyloid A cell strain of monoclonal antibody used on pad, use
The ascites production technology of standard is prepared and purifies antiserum amyloid A monoclonal antibody, be stored in -20 DEG C it is standby；
Above-mentioned mouse source antiserum amyloid A monoclonal antibody and goat anti-mouse igg are resisted with coating dilution respectively
Body adjusts concentration to 1.5mg/ml, and film liquid amount is 1.5 μ l/cm, and they are sprayed on as detection line is parallel with nature controlling line
It is coated with nitrocellulose filter, detection line and nature controlling line are subsequently placed in baking oven at intervals of 4mm, 37 DEG C dry 2 hours.
The assembling of test card：Paste treated sample pad 2 successively in PVC board 1, be adsorbed with rare-earth fluorescence labeling
The pad 3 of antibody, the nitrocellulose filter 4 and adsorptive pads 5 that are coated with detection line and nature controlling line, obtain test paper big after assembling
Plate, it is wide to cut into 4mm as requested, loads in plastic clip and form test card test paper.
The equipment and raw material selected in above steps preferably following raw material：
Serum amyloid A protein specific pairs antibody；Serum amyloid A protein quality-control product：Britain's Landau laboratory diagnosis
Co., Ltd；Rare-earth fluorescent microballoon：Shanghai Zhen Zhun bio tech ltd；Nitrocellulose (NC) film：Millipore companies
Product；Bovine serum albumin(BSA) (BSA), polyethylene glycol PEG20000, caseinhydrolysate：Sigma products, other common agents are
AR.
Embodiment 2：Accuracy test
From above-mentioned test card and fluorescence immune chromatography analyzer (model：NEO-007),
The setting of fluorescence immunity analyzer parameter：After test card technological parameter is set on fluorescence immunity analyzer, take
The above-mentioned test card for assembling, respectively with 5,20,40,60,80,100, the serum amyloid A protein calibration object of 200mg/L, use
Test card is measured, and obtains the fluorescence intensity level of each calibration object, and result is input in the parameter of analyzer, completes analyzer
Parameter setting.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 300 parts of latex enhancing immune turbidimetry definite value samples
Sheet, wherein 100 parts of serum sample, 100 parts of plasma sample, 100 parts of whole blood sample, serum amyloid A protein content distribution are interval
For between 5.00-200mg/L.
Detection method：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, kept flat；
Step 2：Card Reader：IC-card is placed on dry type fluorescence immunity analyzer labeling position, relevant information is read；
Step 3：Sample-adding：Serum/plasma：100 μ L serum/plasmas samples are taken vertically to drop at test card sample-adding；Whole blood：
Take 150 μ L whole blood samples vertically to drop at test card sample-adding, be careful not to suck bubble during sampling；
Step 4：Detection, can be detected, automatic test using automatic test or immediately test both of which：By test card
Insert on the carrier of dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned analysis detection to test card automatically,
Immediately test：After test card room temperature places 15min, insert on the carrier of dry type fluorescence immunity analyzer, click on test immediately.
Test result analysis：
After the completion of prepared by clinical sample detection reagent, all clinical samples are detected by detection method, and analyze inspection
Survey result.
Result of the test：
As shown in Figure 2, as Y-axis, the test value with contradistinction system draws scatterplot to the detected value with experimental system as X-axis
Figure, and carry out correlation analysis.Clinical sample detection is less than to 300 parts of clinical definite value pattern detections, sample mean deviation
10%, maximum deviation is less than 20%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection kit for preparing
Can be good, it is suitable for clinical detection, meet the differentiation needs of the different detection occasions of different clients.
The present invention provides a kind of serum amyloid A protein fast quantification of utilization rare-earth fluorescent immunochromatography technique preparation and exempts from
Epidemic disease chromatographs detection kit, while being adapted to serum/plasma and whole blood sample, and is adapted to clinically single part detection, relative to blood
The qualitative colloid gold reagent of clear amyloid A, the serum amyloid A protein content in energy quantitative determination sample, with clearer and more definite
Clinical significance of MG, with easy to operate, reaction quick, sensitivity high, high specificity, be adapted to Site Detection and economical and practical
The advantages of.
Claims (7)
1. a kind of serum amyloid A protein determines kit, is provided with test card, it is characterised in that the test card from the bottom to top according to
It is secondary to be provided with：Rare earth Eu is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad3+Fluorescence
Microballoon mark antiserum amyloid A monoclonal antibody-microballoon coupled complex, the rare-earth fluorescent microballoon it is a diameter of
100-200nm, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+, the stabilization under ground state, under the excitation source effect of 337nm
Launch the fluorescence of wavelength 615nm；The monoclonal antibody is the monoclonal antibody for mixing after purification, from for 2-6
The cell strain of monoclonal antibody of different serum amyloid A protein epitopes.
2. a kind of serum amyloid A protein according to claim 1 determines kit, it is characterised in that the pad
The diameter of rare-earth fluorescent microballoon is 120-150nm；The rare-earth fluorescent microballoon is doped with rare earth lanthanide Eu3+。
3. a kind of serum amyloid A protein according to claim 2 determines kit, it is characterised in that the rare-earth fluorescent
Microballoon Eu containing rare earth lanthanide3+；The antibody sources of rare-earth fluorescent microballoon mark are in for 2 different epitopes on pad
Monoclonal cell cell line.
4. a kind of serum amyloid A protein according to claim 1 determines kit, it is characterised in that the pad is adopted
It is obtained with following steps：During glass fibre membrane is soaked in into 150mM Tris-HCL treatment fluids (X-100 containing 1.0%Triton,
2.5%BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 hours, standby, and glass fibre membrane is placed on
On Bio-DotXYZ3050 three-dimensional specking platforms, quantitation nozzle is declined by rare earth Eu with Bio-Jet Quanti300 noncontacts3+It is glimmering
The antiserum amyloid A monoclonal antibody coupled complex of light microballoon mark is sprayed onto glass fibre membrane, and 37 DEG C dry 1 hour
After be obtained.
5. a kind of serum amyloid A protein according to claim 4 determines kit, it is characterised in that the institute on pad
The serum amyloid A protein monoclonal antibody for stating rare-earth fluorescent microballoon mark is obtained using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：With serum amyloid A protein sterling immune mouse, using the Dan Ke of standard
Grand preparation method for antibody prepares the cell strain of monoclonal antibody of specific high-affinity, and the monoclonal antibody cell line to being obtained is matched somebody with somebody
To screening, the monoclonal antibody cell line for kit is preferably gone out according to pairing result and affinity data；
Step 2：The preparation of monoclonal antibody：Prepared and purified blood serum amyloid A list using the ascites production technology of standard
Clonal antibody, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：5mg rare-earth fluorescent microballoons are taken, with 20mM, the carbonate buffer solution of pH9.5,
Washed 3 times using centrifugal process, centrifugal speed is 12000rpm, the time is 5 minutes, is finally resuspended in the above-mentioned carbonate of 100 μ l
In buffer solution, the glucan of 500 μ l aldehyde radicals is added, mixed, at room temperature dark reaction 4 hours, washed using same centrifugal process
Be resuspended in the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Step 4：The preparation of the serum amyloid A protein monoclonal antibody of rare-earth fluorescent microballoon mark：Choose and resist from 2 differences
The monoclonal antibody of the monoclonal cell cell line of former epitope, according to mass ratio 1:1 by 2mg serum amyloid A protein monoclonals
Then the above-mentioned carbonate buffer solution of antibody mixes in 4 DEG C of dialysed overnights with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical, and 4 DEG C anti-
Should overnight；Then, sodium borohydride to final concentration 5mM is added, 4 DEG C are reacted 4 hours；Add isometric confining liquid (50mM
Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then the buffering of 50mM Tris-HCL, pH7.4 is used
Liquid is washed 3 times using centrifugal process, in being resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l (containing 1.2%NaCL, 0.5%BSA,
0.1%Tween 20), 4 DEG C keep in dark place it is standby.
6. a kind of serum amyloid A protein according to claim 1 determines kit, it is characterised in that described to be coated with inspection
The nitrocellulose filter of survey line and nature controlling line is obtained by following steps：
Step 1：Using the cell line different from serum amyloid A protein cell strain of monoclonal antibody used on pad, use
The ascites production technology of standard is prepared and purified blood serum amyloid A monoclonal antibody, be stored in -20 DEG C it is standby；
Step 2：Coating dilution is used respectively by above-mentioned mouse source antiserum amyloid A monoclonal antibody and goat anti-mouse igg
To 1.5mg/ml, film liquid amount is 1.5 μ l/cm to antibody adjustment concentration, using them as detection line sprinkling parallel with nature controlling line
In being coated with nitrocellulose filter, detection line and nature controlling line are subsequently placed in baking oven at intervals of 4mm, and 37 DEG C of drying 2 are small
When.
7. a kind of serum amyloid A protein according to claim 1 determines kit, it is characterised in that the sample pad is led to
Following steps are crossed to be obtained：Glass fibre membrane is soaked in containing 1.0%Triton X-100,2.5%BSA, 0.15M Tris delay
Fliud flushing, in the treatment fluid of pH7.5,4 hours is soaked in 4 DEG C, is subsequently placed in baking oven, and 37 DEG C dry 2 hours.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6136549A (en) * | 1999-10-15 | 2000-10-24 | Feistel; Christopher C. | systems and methods for performing magnetic chromatography assays |
| JP2009115822A (en) * | 2009-02-23 | 2009-05-28 | Furukawa Electric Co Ltd:The | Label silica nano-particle for immuno-chromatography reagent, immuno-chromatography reagent, test strip for immuno-chromatography using it, and fluorescence detection system for immuno-chromatography |
| CN102890155A (en) * | 2012-09-12 | 2013-01-23 | 暨南大学 | Fluorescent test strip based on resonance energy transfer, and preparation method and application for fluorescent test strip |
| CN104714033A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | Procalcitonin detection kit and detection method |
| CN104730245A (en) * | 2014-11-28 | 2015-06-24 | 威海纽普生物技术有限公司 | D-dimer detection kit and D-dimer detection method |
| CN105628932A (en) * | 2016-02-01 | 2016-06-01 | 苏州东尼生物技术有限公司 | SAA (serum amyloid A) immunochromatographic test strip and preparation method and test method thereof |
| CN105675879A (en) * | 2015-12-31 | 2016-06-15 | 苏州市博纳泰科生物技术有限公司 | Fluorescence immunochromatographic assay method of serum amyloid protein A and kit |
| CN105717303A (en) * | 2016-01-29 | 2016-06-29 | 山东康力医疗器械科技有限公司 | Method and reagent kit for detecting phosphatidylinositol proteoglycan 3 with fluorescence immunochromatographic method |
-
2016
- 2016-12-16 CN CN201611165653.0A patent/CN106706926A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6136549A (en) * | 1999-10-15 | 2000-10-24 | Feistel; Christopher C. | systems and methods for performing magnetic chromatography assays |
| JP2009115822A (en) * | 2009-02-23 | 2009-05-28 | Furukawa Electric Co Ltd:The | Label silica nano-particle for immuno-chromatography reagent, immuno-chromatography reagent, test strip for immuno-chromatography using it, and fluorescence detection system for immuno-chromatography |
| CN102890155A (en) * | 2012-09-12 | 2013-01-23 | 暨南大学 | Fluorescent test strip based on resonance energy transfer, and preparation method and application for fluorescent test strip |
| CN104714033A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | Procalcitonin detection kit and detection method |
| CN104730245A (en) * | 2014-11-28 | 2015-06-24 | 威海纽普生物技术有限公司 | D-dimer detection kit and D-dimer detection method |
| CN105675879A (en) * | 2015-12-31 | 2016-06-15 | 苏州市博纳泰科生物技术有限公司 | Fluorescence immunochromatographic assay method of serum amyloid protein A and kit |
| CN105717303A (en) * | 2016-01-29 | 2016-06-29 | 山东康力医疗器械科技有限公司 | Method and reagent kit for detecting phosphatidylinositol proteoglycan 3 with fluorescence immunochromatographic method |
| CN105628932A (en) * | 2016-02-01 | 2016-06-01 | 苏州东尼生物技术有限公司 | SAA (serum amyloid A) immunochromatographic test strip and preparation method and test method thereof |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107907676A (en) * | 2017-10-30 | 2018-04-13 | 威海纽普生物技术有限公司 | Alzheimer related neural silk-fibroin detection kit and preparation method thereof |
| CN111344571A (en) * | 2017-11-13 | 2020-06-26 | 斯泰伦博什大学 | Method for detecting inflammation |
| CN110275015A (en) * | 2018-03-16 | 2019-09-24 | 优志旺电机株式会社 | Immune chromatograph measurement method, immune chromatograph dilution and immunologic test box |
| CN108680751A (en) * | 2018-04-04 | 2018-10-19 | 苏州遵道生物科技有限公司 | Quantitatively detect the time-resolved fluoroimmunoassay chromatograph test strip and preparation method thereof of serum amyloid A protein |
| CN108387748A (en) * | 2018-05-04 | 2018-08-10 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting cat serum amyloid A protein chromatographs detection card and preparation method |
| CN109085358A (en) * | 2018-07-02 | 2018-12-25 | 威海纽普生物技术有限公司 | Serum amyloid A protein assay kit and production method |
| CN109633162A (en) * | 2018-11-28 | 2019-04-16 | 浙江聚康生物工程有限公司 | S-100 β protein detection kit |
| CN109991409A (en) * | 2018-12-03 | 2019-07-09 | 浙江聚康生物工程有限公司 | Pepsinogen I/pepsinogen I I detection kit |
| CN109991409B (en) * | 2018-12-03 | 2022-03-04 | 浙江聚康生物工程有限公司 | Pepsinogen I/pepsinogen II detection kit |
| CN110702900A (en) * | 2019-10-10 | 2020-01-17 | 南京欧凯生物科技有限公司 | Immunofluorescence chromatography detection card for serum amyloid A |
| CN110907639A (en) * | 2019-12-05 | 2020-03-24 | 四川新健康成生物股份有限公司 | Serum amyloid protein A detection kit and preparation method thereof |
| CN112924686A (en) * | 2019-12-05 | 2021-06-08 | 复旦大学 | Immunochromatography test strip for detecting serum amyloid A and preparation and detection methods thereof |
| CN112924686B (en) * | 2019-12-05 | 2024-08-23 | 复旦大学 | Immunochromatography test strip for detecting serum amyloid A and preparation and detection methods thereof |
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