CN106771264A - Thyrotropin assay kit and preparation method - Google Patents
Thyrotropin assay kit and preparation method Download PDFInfo
- Publication number
- CN106771264A CN106771264A CN201611162058.1A CN201611162058A CN106771264A CN 106771264 A CN106771264 A CN 106771264A CN 201611162058 A CN201611162058 A CN 201611162058A CN 106771264 A CN106771264 A CN 106771264A
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- rare
- pad
- earth
- hormone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
Abstract
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically a kind of thyrotropic hormone (TSH) determines kit (fluorescence immune chromatography method) and preparation method, it is provided with test card, it is characterised in that the test card is sequentially provided with from the bottom to top：Rare earth Eu is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad3+The thyrotropic hormone monoclonal antibody of fluorescent microsphere mark, a diameter of 150nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+, it is stable under ground state, the fluorescence of wavelength 615nm is launched under the excitation source effect of 337nm；The monoclonal antibody is the monoclonal antibody for mixing after purification, from for 26 cell strain of monoclonal antibody of different thyrotropic hormone epitopes, having the advantages that easy to operate, reaction is quick, sensitivity is high, high specificity.
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 promoting in the samples such as serum, blood plasma and whole blood
Thyroid hormone carries out the thyrotropin assay kit and preparation method of quantitative analysis.
Background technology：
Thyrotropic hormone (thyrotropin, thyroid stimulating hormone, TSH) is adenohypophysis secretion
Promotion it is thyroid growth and function hormone.The TSH of the mankind is a kind of glycoprotein, and containing 211 amino acid, carbohydrate accounts for whole
The 15% of individual molecule.Whole molecule is by two peptide chains --- and α chains and β chains are constituted.Thyrotropic hormone (TSH) is regulation and control thyroid gland
Cell growth and the Main Factors of thyroid hormone synthesis and secretion, are synthesized by TSH cells of pituitary gland and are secreted, and receive
The negative-feedback regulation of thyroid hormone.Clinically, (Free Triiodothyronine, that is, dissociate triiodo first shape for TSH, FT3
Gland original ammonia acid) and FT4 (Fr ee Thyroxine, i.e. free thyroid hormone) use in conjunction, for dysthyroid
Auxiliary diagnosis：(1) FT3, FT4 are raised with TSH reductions, the exophthalmic goiter that mostly thyroid gland disease in itself causes.(2)FT3、
FT4 is raised, and TSH is also raised, the secondary hyperthyroidism that mostly hypothalamus-pituitary function disorder causes.(3) FT3, FT4 reduction companion
TSH is raised, and is mostly primary in thyroid hypofunction.(4) FT3, FT4 reduction, TSH is also reduced, mostly hypothalamus-hypophysis
Function is damaged the secondary hypothyroidism for causing.When thyroid function changes, the fluctuation of TSH is rapider and notable compared with thyroid hormone,
It is the sensitive indicator for reflecting hypothalamic-pituitary-thyroid axis function.By detecting that blood TSH levels can reflect thyroid function
State, contributes to auxiliary examination, auxiliary diagnosis, therapeutic effect judge and the Index for diagnosis of thyroid disease.
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, the sensitivity that combined with fluorescent immunochromatographiassays assays instrument is realized is high, fast and simple, can be surveyed with the thyrotropic hormone of accurate quantitative analysis
Determine kit and preparation method.
The present invention can be reached by following measures：
A kind of thyrotropin assay 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 antithyrotropic hormone 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 thyrotropic hormone epitopes.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is 120-200nm；The rare-earth fluorescent microballoon contains
One or more rare earth lanthanides；The antibody of rare-earth fluorescent microballoon mark is preferably derived from anti-for 2 differences on pad
The monoclonal cell cell line of former epitope.
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 antithyrotropic hormone monoclonal antibody coupled complex that the micro- quantitation nozzle of touch marks rare-earth fluorescent microballoon is sprayed onto glass
Tunica fibrosa, 37 DEG C drying 1 hour after be obtained.
The antithyrotropic hormone monoclonal antibody of the rare-earth fluorescent microballoon mark in the present invention on pad is used
Following steps are obtained：
Step 1：The acquisition of cell strain of monoclonal antibody：With thyrotropic hormone sterling immune mouse, using the list of standard
Clonal antibody preparation method prepares the cell strain of monoclonal antibody of specific high-affinity, and the monoclonal antibody cell line to being obtained is carried out
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：Prepared using the ascites production technology of standard and purify antithyrotropic hormone
Monoclonal 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 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：The preparation of the antithyrotropic hormone monoclonal antibody of rare-earth fluorescent microballoon 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 thyrotropic hormone 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.
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 line different from antithyrotropic hormone cell strain of monoclonal antibody used on pad, adopt
Prepared with the ascites production technology of standard and purify antithyrotropic hormone monoclonal antibody, be stored in -20 DEG C it is standby；
Step 2：Coating dilution is used respectively by above-mentioned mouse source antithyrotropic hormone monoclonal antibody and goat anti-mouse igg
To 1-3mg/ml, film liquid amount is 0.5-3 μ l/cm to 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 thyrotropic hormone preparation method that a kind of kit as described above is realized, it is characterised in that
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 thyrotropic hormone is 0.3-120mIU/mL；
Step 3：Sample-adding：Serum/plasma：100 μ L serum/plasmas samples are taken vertically to drop at test card sample-adding；For
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 20min, insert on the carrier of dry type fluorescence immunity analyzer, click on test immediately.
The present invention provides rush first shape prepared by a kind of fluorescence immune chromatography technology of utilization rare earth carboxyl latex microballoon mark
Glandular hormone (TSH) determines kit (fluorescence immune chromatography method), while being adapted to serum, blood plasma and whole blood sample, and is adapted to clinic
Upper single part detection, relative to the qualitative colloid gold reagent of thyrotropic hormone, the thyrotropic hormone in energy quantitative determination sample
Content, with more specific Clinical significance of MG, with easy to operate, reaction is quick, sensitivity high, high specificity, be adapted to it is existing
The advantages of field is detected and is economical and practical.
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, present invention firstly provides a kind of thyrotropin assay kit, test paper is provided with box
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,
Antithyrotropic hormone monoclonal antibody-microballoon the coupled complex of rare-earth fluorescent microballoon mark is wherein adsorbed with pad,
A diameter of 150nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+, the stabilization under ground state,
Launch the fluorescence of wavelength 615nm under the excitation source effect of 337nm；The monoclonal antibody is the monoclonal for mixing after purification
Antibody, from for the 2-6 cell strain of monoclonal antibody of different thyrotropic hormone 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 of test card can be obtained by following measures in thyrotropin assay kit：
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 antithyrotropic hormone monoclonal antibody coupled complex of ball mark is sprayed onto glass fibre membrane, and 37 DEG C of drying are obtained 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 antithyrotropic hormone monoclonal antibody of fluorescent microsphere 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 resists 2mg antithyrotropic hormone monoclonals
Then the above-mentioned carbonate buffer solution of body mixes in 4 DEG C of dialysed overnights with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical, 4 DEG C of reactions
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.
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 antithyrotropic hormone cell strain of monoclonal antibody used on pad, using standard
Ascites production technology prepare and purify antithyrotropic hormone monoclonal antibody, be stored in -20 DEG C it is standby；
Above-mentioned mouse source antithyrotropic hormone monoclonal antibody and goat anti-mouse igg antibody are adjusted with coating dilution respectively
To 2mg/ml, film liquid amount is 2 μ l/cm to whole concentration, and they are sprayed on into cellulose nitrate as detection line is parallel with nature controlling line
It is coated with plain film, 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：
Thyrotropic hormone specific pairs antibody；Thyrotropic hormone quality-control product：The limited public affairs of Britain's Landau laboratory diagnosis
Department；Rare-earth fluorescent microballoon：Shanghai Zhen Zhun bio tech ltd；Nitrocellulose (NC) film：Millipore Products；
Bovine serum albumin(BSA) (BSA), polyethylene glycol PEG20000, caseinhydrolysate：Sigma products, other common agents are analysis
Pure reagent.
Embodiment 2：Accuracy test
From above-mentioned test card and fluorescence immune chromatography analyzer (model：NEO-007), fluorescence immunity analyzer parameter
Setting：After test card technological parameter is set on fluorescence immunity analyzer, the above-mentioned test card for assembling is taken, used respectively
0.4th, 4,10,20,40,80, the thyrotropic hormone calibration object of 100mIU/L, is measured with test card, obtains each calibration object
Fluorescence intensity level, result is input in the parameter of analyzer, complete 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, thyrotropic hormone content distribution interval is
Between 0.3-120mIU/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, for
Whole blood sample, takes 150 μ L whole blood samples and vertically drops at test card sample-adding, is careful not to suck bubble during sampling；
Step 4：Detection：Can be detected using automatic test or immediately test both of which, wherein automatic test：To survey
On the carrier of examination card insertion dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned analysis to test card automatically
Detection；Immediately test：After test card room temperature places 20min, insert on the carrier of dry type fluorescence immunity analyzer, click on instant
Test.
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 thyrotropic hormone fast quantification of utilization rare-earth fluorescent immunochromatography technique preparation and is immunized
Chromatography detection kit, while being adapted to serum, blood plasma and whole blood sample, and is adapted to clinically single part detection, relative to rush first
The qualitative colloid gold reagent of shape glandular hormone, the thyrotropic hormone content in energy quantitative determination sample, refers to more specific clinic
Meaning is led, be there is easy to operate, reaction quick, sensitivity high, high specificity, be adapted to Site Detection and economical and practical.
Claims (8)
1. a kind of thyrotropin assay kit, is provided with test card, it is characterised in that the test card is from the bottom to top successively
It is provided with：Rare earth Eu is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad3+Fluorescence is micro-
Antithyrotropic hormone monoclonal antibody-microballoon the coupled complex of ball mark, a diameter of 100- of the rare-earth fluorescent microballoon
250nm, rare-earth fluorescent microballoon is stable under ground state containing one or more in rare earth lanthanide, in exciting for 300-400nm
Launch the fluorescence that wave-length coverage is 550-650nm under light source effect；The monoclonal antibody is the monoclonal for mixing after purification
Antibody, from for the 2-6 cell strain of monoclonal antibody of different thyrotropic hormone epitopes.
2. a kind of thyrotropin assay kit according to claim 1, it is characterised in that the pad it is dilute
The diameter of native fluorescent microsphere is 120-200nm；The antibody sources of rare-earth fluorescent microballoon mark are in anti-for 2 differences on pad
The monoclonal cell cell line of former epitope；Rare earth Eu is adsorbed with pad3+The antithyrotropic hormone list of fluorescent microsphere mark
Clonal antibody-microballoon coupled complex.
3. a kind of thyrotropin assay kit according to claim 1, it is characterised in that rare-earth fluorescent microballoon
A diameter of 150nm, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+, the stabilization under ground state, in the excitation source effect of 337nm
Under launch the fluorescence of wavelength 615nm.
4. a kind of thyrotropin assay kit according to claim 1, it is characterised in that the pad it is dilute
The diameter of native fluorescent microsphere is 200nm；The rare-earth fluorescent microballoon is doped with rare earth lanthanide Eu3+。
5. a kind of thyrotropin assay kit according to claim 1, it is characterised in that the pad is used
Following steps are obtained：Glass fibre membrane is soaked in 150mM Tris-HCL treatment fluids, Tris-HCL treatment fluids contain 1.0%
Triton X-100,2.5%BSA, pH7.4,4 DEG C is soaked 2 hours, then takes out 37 DEG C of oven for drying 4 hours, standby, by glass
Glass tunica fibrosa is placed on Bio-DotXYZ3050 three-dimensional specking platforms, is declined quantitative spray with Bio-Jet Quanti300 noncontacts
Head is by rare earth Eu3+The antithyrotropic hormone monoclonal antibody coupled complex of fluorescent microsphere mark is sprayed onto glass fibre membrane, 37
DEG C drying 1 hour after be obtained.
6. a kind of thyrotropin assay kit according to claim 1, it is characterised in that described on pad
The thyrotropic hormone monoclonal antibody of rare-earth fluorescent microballoon mark is obtained using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：With thyrotropic hormone sterling immune mouse, using the monoclonal of standard
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
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：Thyrotropic hormone monoclonal is prepared and purified using the ascites production technology of standard
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 thyrotropic hormone monoclonal antibody of rare-earth fluorescent microballoon mark：Choose and come from 2 not synantigens
The monoclonal antibody of the monoclonal cell cell line of epitope, according to mass ratio 1:1 uses 2mg thyrotropic hormones monoclonal antibody
Then above-mentioned carbonate buffer solution mixes in 4 DEG C of dialysed overnights with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical, and 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 the buffer solution of 50mM Tris-HCL, pH7.4 is used using centrifugation
Method is washed 3 times, (containing 1.2%NaCL, 0.5%BSA, 0.1%Tween in being resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l
20), 4 DEG C keep in dark place it is standby.
7. a kind of thyrotropin assay kit according to claim 1, it is characterised in that described to be coated with detection
The nitrocellulose filter of line and nature controlling line is obtained by following steps：
Step 1：Using the cell line different from thyrotropic hormone cell strain of monoclonal antibody used on pad, using standard
Ascites production technology prepare and purify thyrotropic hormone monoclonal antibody, be stored in -20 DEG C it is standby；
Step 2：Coating dilution is used respectively by above-mentioned mouse source antithyrotropic hormone monoclonal antibody and goat anti-mouse igg antibody
To 2mg/ml, film liquid amount is 2 μ l/cm to adjustment concentration, and they are sprayed on into nitric acid fibre as detection line is parallel with nature controlling line
It is coated with the plain film of dimension, detection line and nature controlling line are subsequently placed in baking oven at intervals of 4mm, 37 DEG C dry 2 hours.
8. a kind of thyrotropin assay kit according to claim 1, it is characterised in that the sample pad passes through
Following steps are obtained：Glass fibre membrane is soaked in and contains 1.0%Triton X-100,2.5%BSA, 0.15M Tris bufferings
Liquid, 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611162058.1A CN106771264A (en) | 2016-12-15 | 2016-12-15 | Thyrotropin assay kit and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611162058.1A CN106771264A (en) | 2016-12-15 | 2016-12-15 | Thyrotropin assay kit and preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106771264A true CN106771264A (en) | 2017-05-31 |
Family
ID=58892634
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611162058.1A Pending CN106771264A (en) | 2016-12-15 | 2016-12-15 | Thyrotropin assay kit and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106771264A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389930A (en) * | 2017-08-16 | 2017-11-24 | 孙圣荣 | Portable Thyroid hormone determination instrument |
| CN108061727A (en) * | 2017-12-13 | 2018-05-22 | 施康培医疗科技（武汉）有限公司 | A kind of thyroglobulin quantitative testing test paper item and preparation method thereof |
| CN110726848A (en) * | 2019-11-21 | 2020-01-24 | 海卫特(广州)医疗科技有限公司 | Thyroid stimulating hormone detection immune test paper and preparation method thereof |
Citations (9)
| 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 |
| CN202794191U (en) * | 2012-08-07 | 2013-03-13 | 天津中新科炬生物制药有限公司 | Thyroid stimulating hormone (TSH) quick quantitative immunochromatographic detection kit |
| CN103172941A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof |
| 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 |
| CN104880414A (en) * | 2015-05-27 | 2015-09-02 | 广州华弘生物科技有限公司 | TSH immunochromatography kit and manufacturing method thereof |
| 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
- 2016-12-15 CN CN201611162058.1A patent/CN106771264A/en active Pending
Patent Citations (9)
| 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 |
| CN103172941A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof |
| CN202794191U (en) * | 2012-08-07 | 2013-03-13 | 天津中新科炬生物制药有限公司 | Thyroid stimulating hormone (TSH) quick quantitative immunochromatographic detection kit |
| 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 |
| CN104880414A (en) * | 2015-05-27 | 2015-09-02 | 广州华弘生物科技有限公司 | TSH immunochromatography kit and manufacturing method thereof |
| 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 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389930A (en) * | 2017-08-16 | 2017-11-24 | 孙圣荣 | Portable Thyroid hormone determination instrument |
| CN108061727A (en) * | 2017-12-13 | 2018-05-22 | 施康培医疗科技（武汉）有限公司 | A kind of thyroglobulin quantitative testing test paper item and preparation method thereof |
| CN110726848A (en) * | 2019-11-21 | 2020-01-24 | 海卫特(广州)医疗科技有限公司 | Thyroid stimulating hormone detection immune test paper and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106706926A (en) | Serum amyloid A testing kit and manufacturing method | |
| CN104714015B (en) | Detection kit and detection method for heart-type fatty acid binding protein | |
| CN104714033B (en) | Procalcitonin. detection kit and detection method | |
| CN106841631A (en) | Cardiac muscle troponin I/N ends Natriuretic Peptide/D dimer is three-in-one to determine kit and preparation method | |
| CN106771128A (en) | Parathyroid hormone determines kit and preparation method | |
| CN106959372A (en) | Serum amyloid A protein and the two-in-one measure kit of C reactive proteins and preparation method | |
| CN107664700A (en) | Cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof | |
| CN106990254A (en) | 25 hydroxycholecalciferols determine kit and preparation method | |
| CN106153927A (en) | A kind of fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo simultaneously | |
| CN109975559B (en) | Kit and method for time-resolved fluorescence quantitative detection of 25-hydroxy vitamin D | |
| CN104714025A (en) | NT-proBNP detection kit and detection method | |
| CN105891508A (en) | TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and preparation method | |
| CN104849468A (en) | Chemiluminescent protein chip, kit and detection method for detection of fucose index of seroglycoid | |
| CN106248927A (en) | The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK MB and preparation method | |
| CN106771239A (en) | Serum amyloid A protein/Procalcitonin/C reactive proteins are three-in-one to determine kit and preparation method | |
| CN106872716A (en) | Serum amyloid A protein and two-in-one measure kit and the preparation method of Procalcitonin | |
| CN107703110A (en) | G17 detection kit and preparation method thereof | |
| CN106771264A (en) | Thyrotropin assay kit and preparation method | |
| CN105988008A (en) | Measurement device, kit and measurement method | |
| CN103823058B (en) | The chemiluminescence protein chip method of Antigens albumen and kit in serum | |
| JP4068148B2 (en) | Assay method | |
| CN106645689A (en) | Thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit and preparation method thereof | |
| CN107907694A (en) | Anti- mullerian duct hormone test kit and preparation method thereof | |
| CN113125696A (en) | Estradiol homogeneous phase chemiluminescence detection kit and application thereof | |
| CN106706931A (en) | Progesterone measurement kit and preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170531 |
|
| RJ01 | Rejection of invention patent application after publication |