CN106771128A - Parathyroid hormone determines kit and preparation method - Google Patents
Parathyroid hormone determines kit and preparation method Download PDFInfo
- Publication number
- CN106771128A CN106771128A CN201611161992.1A CN201611161992A CN106771128A CN 106771128 A CN106771128 A CN 106771128A CN 201611161992 A CN201611161992 A CN 201611161992A CN 106771128 A CN106771128 A CN 106771128A
- Authority
- CN
- China
- Prior art keywords
- parathyroid hormone
- monoclonal antibody
- rare
- pad
- earth
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically a kind of parathyroid hormone determines kit and preparation method, 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 parathyroid hormone monoclonal antibody of fluorescent microsphere mark, a diameter of 200nm 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 parathyroid 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 first in the samples such as serum, blood plasma and whole blood
The parathyroid hormone that shape side glandular hormone carries out quantitative analysis determines kit and preparation method.
Background technology：
Parathyroid hormone (PTH) is synthesized in parathyroid gland, and is secreted into blood, maintains the weight of body calcium balance
One of hormone is wanted, it directly acts on bone and kidney, promote bone calcium mobilization and kidney to the reabsorption of calcium, made by promoting 1 α hydroxylases
25-OH-D3 is converted into activity 1,25- (OH) 2D3, and the function of strengthening intestines calcium uptake is played indirectly.PTH not only plays decomposition to bone
Effect, also has synthesis to bone, is to improve bone amount, improve bone mass, the new way for the treatment of osteoporosis.
Complete PTH is constituted containing 84 single polypeptide chains of amino acid, and molecular weight is about 9500 dalton, complete first
The selective determination of shape side glandular hormone can be directly used for judging the secretion capacity of parathyroid gland.PTH combines vitamin D and calcitonin
The calcium and phosphorus of skeletal system are mobilized, is promoted small intestine to the absorption of calcium and is increased excretion of the kidney to phosphorus.PTH and calcitonin it is mutual
Effect ensures the stabilization of blood calcium concentration.Hypercalcemia concentration suppresses PTH secretions, and low blood calcium concentration then promotes PTH to secrete.Parathyroid gland
Dysfunction influence PTH secretions, can cause blood calcium concentration to be raised and lowered (hypercalcinemia and hypocalcemia).
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 parathyroid hormone of accurate quantitative analysis
Determine kit and preparation method.
The present invention can be reached by following measures：
A kind of parathyroid hormone 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 anti-parathyroid 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 parathyroid hormone epitopes.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is preferably 120-200nm；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 anti-parathyroid hormone monoclonal antibody coupled complex that rare-earth fluorescent microballoon is marked is sprayed onto glass by the micro- quantitation nozzle of touch
Tunica fibrosa, 37 DEG C drying 1 hour after be obtained.
The anti-parathyroid 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 parathyroid 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：Anti- parathyroid hormone is prepared and purified using the ascites production technology of standard
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 anti-parathyroid 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 parathyroid 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 different cell line of anti-parathyroid hormone cell strain of monoclonal antibody used from pad, adopt
Anti- parathyroid hormone monoclonal antibody is prepared and purified with the ascites production technology of standard, be stored in -20 DEG C it is standby；
Step 2：Coating dilution is used respectively by the anti-parathyroid hormone monoclonal antibody in above-mentioned mouse source and goat anti-mouse igg
Antibody adjusts concentration to 0.5-3mg/ml, and film liquid amount is 0.5-3 μ l/cm, and they are parallel as detection line with nature controlling line
It is sprayed on nitrocellulose filter and is coated with, detection line and nature controlling line is subsequently placed in baking oven at intervals of 3-7mm, 37 DEG C of bakings
It is dry 2 hours.
Sample pad of the present invention is obtained by following steps：Glass fibre membrane is soaked in and contains 1.0%TritonX-
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 parathyroid 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 that a kind of Systems for optical inspection is 8.0-1200pg/mL to the measurement range of parathyroid hormone；
Step 3：Sample-adding：Serum/plasma：100 μ L serum/plasmas samples are taken 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 is provided by first shape prepared by a kind of fluorescence immune chromatography technology of utilization rare earth carboxyl latex microballoon mark
Glandular hormone determines kit, while being adapted to serum, blood plasma and whole blood sample, and is adapted to clinically single part detection, relative to first
The shape side qualitative colloid gold reagent of glandular hormone, the parathyroid hormone cellulose content in energy quantitative determination sample, with more specific clinic
Directive significance, with easy to operate, reaction quick, sensitivity high, high specificity, is adapted to Site Detection and economical and practical etc. excellent
Point.
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 parathyroid hormone, 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,
The anti-parathyroid hormone monoclonal antibody-microballoon 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 parathyroid 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 that parathyroid hormone (PTH) determines test card in kit (fluorescence immune chromatography method) can lead to
Following measures are crossed to be obtained：
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 anti-parathyroid 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 anti-parathyroid 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 the anti-parathyroid hormone monoclonals of 2mg
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 different cell line of anti-parathyroid hormone cell strain of monoclonal antibody used from pad, using standard
Ascites production technology prepare and purify anti-parathyroid hormone monoclonal antibody, be stored in -20 DEG C it is standby；
The anti-parathyroid hormone monoclonal antibody in above-mentioned mouse source and goat anti-mouse igg antibody are adjusted with coating dilution respectively
To 1.5mg/ml, film liquid amount is 1.5 μ l/cm to whole concentration, and they are sprayed on into nitric acid as detection line is parallel with nature controlling line
It is coated with cellulose membrane, 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：
Parathyroid hormone specific pairs antibody；Parathyroid 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),
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 10,100,200,500,600,800, the parathyroid hormone of 1000pg/mL calibrates
Product, are measured with test card, obtain the fluorescence intensity level of each calibration object, and result is input in the parameter of analyzer, are completed
The setting of the parameter of analyzer.
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, parathyroid hormone content distribution interval is
Between 10.0-1000pg/mL.
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：
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 using automatic test or test both of which is detected immediately；Automatic test：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 2-4, the detected value with experimental system as Y-axis, as X-axis draw and dissipate by the test value with contradistinction system
Point diagram, 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 parathyroid hormone fast quantification of utilization rare-earth fluorescent immunochromatography technique preparation and is immunized
Chromatography detection kit, while being adapted to serum/plasma and whole blood sample, and is adapted to clinically single part detection, relative to first shape
The other qualitative colloid gold reagent of glandular hormone, the parathyroid hormone cellulose 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 (7)
1. a kind of parathyroid hormone determines 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-
The anti-parathyroid hormone monoclonal antibody-microballoon 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 parathyroid hormone epitopes.
2. a kind of parathyroid hormone according to claim 1 determines kit, 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, the rare-earth fluorescent microballoon is doped with rare earth lanthanide Eu3+。
3. a kind of parathyroid hormone according to claim 1 determines kit, it is characterised in that the pad it is dilute
The diameter of native fluorescent microsphere is 200nm.
4. a kind of parathyroid hormone according to claim 1 determines kit, it is characterised in that the pad is used
Following steps are obtained：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 anti-parathyroid hormone monoclonal antibody coupled complex of light microballoon mark is sprayed onto glass fibre membrane, after 37 DEG C dry 1 hour
It is obtained.
5. a kind of parathyroid hormone according to claim 1 determines kit, it is characterised in that described on pad
The parathyroid 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 parathyroid 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：Parathyroid 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 parathyroid 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 parathyroid 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.
6. a kind of parathyroid hormone according to claim 1 determines kit, 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 parathyroid hormone cell strain of monoclonal antibody used on pad, using standard
Ascites production technology prepare and purify parathyroid hormone monoclonal antibody, be stored in -20 DEG C it is standby；
Step 2：Coating dilution is used respectively by the anti-parathyroid hormone monoclonal antibody in above-mentioned mouse source and goat anti-mouse igg antibody
To 0.5-3mg/ml, film liquid amount is 0.5-3 μ l/cm to 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 3-7mm, and 37 DEG C of drying 2 are small
When.
7. a kind of parathyroid hormone according to claim 1 determines kit, 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 |
|---|---|---|---|
| CN201611161992.1A CN106771128A (en) | 2016-12-15 | 2016-12-15 | Parathyroid hormone determines kit and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611161992.1A CN106771128A (en) | 2016-12-15 | 2016-12-15 | Parathyroid hormone determines kit and preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106771128A true CN106771128A (en) | 2017-05-31 |
Family
ID=58892537
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611161992.1A Pending CN106771128A (en) | 2016-12-15 | 2016-12-15 | Parathyroid hormone determines kit and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106771128A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107255726A (en) * | 2017-08-02 | 2017-10-17 | 江苏省原子医学研究所 | Quantitatively detect fluorescence immune chromatography test paper of human parathyroid hormone and preparation method thereof |
| CN107402300A (en) * | 2017-08-02 | 2017-11-28 | 江苏省原子医学研究所 | A kind of method of quick discriminating human parathyroid |
| CN108445237A (en) * | 2018-06-20 | 2018-08-24 | 广州市康润生物科技有限公司 | A kind of new application of PTH tachysynthesises detecting system |
| CN109307663A (en) * | 2018-09-13 | 2019-02-05 | 广州俊通生物科技有限公司 | A kind of micro reaction plate preparation method, the detection method of kit and kit |
| CN111175521A (en) * | 2020-01-07 | 2020-05-19 | 上海市第十人民医院 | Preparation method of fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside |
| CN114184796A (en) * | 2021-12-03 | 2022-03-15 | 广州达泰生物工程技术有限公司 | Kit and method for quantitatively detecting full-segment parathyroid hormone |
Citations (7)
| 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 |
| CN1838968A (en) * | 2003-08-08 | 2006-09-27 | 艾伯吉尼斯公司 | Antibodies aimed to parathyroid hormone (PTH) and uses thereof |
| 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 |
| 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 CN201611161992.1A patent/CN106771128A/en active Pending
Patent Citations (7)
| 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 |
| CN1838968A (en) * | 2003-08-08 | 2006-09-27 | 艾伯吉尼斯公司 | Antibodies aimed to parathyroid hormone (PTH) and uses thereof |
| 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 |
| 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 (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107255726A (en) * | 2017-08-02 | 2017-10-17 | 江苏省原子医学研究所 | Quantitatively detect fluorescence immune chromatography test paper of human parathyroid hormone and preparation method thereof |
| CN107402300A (en) * | 2017-08-02 | 2017-11-28 | 江苏省原子医学研究所 | A kind of method of quick discriminating human parathyroid |
| CN107255726B (en) * | 2017-08-02 | 2018-09-14 | 江苏省原子医学研究所 | Quantitatively detect the fluorescence immune chromatography test paper and preparation method thereof of human parathyroid hormone |
| CN108445237A (en) * | 2018-06-20 | 2018-08-24 | 广州市康润生物科技有限公司 | A kind of new application of PTH tachysynthesises detecting system |
| CN109307663A (en) * | 2018-09-13 | 2019-02-05 | 广州俊通生物科技有限公司 | A kind of micro reaction plate preparation method, the detection method of kit and kit |
| CN111175521A (en) * | 2020-01-07 | 2020-05-19 | 上海市第十人民医院 | Preparation method of fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside |
| CN111175521B (en) * | 2020-01-07 | 2024-01-30 | 上海市第十人民医院 | Preparation method of fluorescent immunochromatography test paper for rapid identification of thyroid medullary carcinoma and lymph node metastasis bedside |
| CN114184796A (en) * | 2021-12-03 | 2022-03-15 | 广州达泰生物工程技术有限公司 | Kit and method for quantitatively detecting full-segment parathyroid hormone |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106771128A (en) | Parathyroid hormone determines kit and preparation method | |
| 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 | |
| CN104714025B (en) | NT-proBNP 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 | |
| CN104730245B (en) | D-dimer detection reagent kit and detection method | |
| CN106990254A (en) | 25 hydroxycholecalciferols determine kit 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 | |
| CN106959372A (en) | Serum amyloid A protein and the two-in-one measure kit of C reactive proteins and preparation method | |
| CN104730251A (en) | Troponin I detection kit and troponin I detection method | |
| CN106771239A (en) | Serum amyloid A protein/Procalcitonin/C reactive proteins are three-in-one to determine kit and preparation method | |
| CN109975559B (en) | Kit and method for time-resolved fluorescence quantitative detection of 25-hydroxy vitamin D | |
| CN106153927A (en) | A kind of fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo simultaneously | |
| CN105891508A (en) | TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and preparation method | |
| CN106248927A (en) | The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK MB and preparation method | |
| CN104849468A (en) | Chemiluminescent protein chip, kit and detection method for detection of fucose index of seroglycoid | |
| CN107703110A (en) | G17 detection kit and preparation method thereof | |
| CN106872716A (en) | Serum amyloid A protein and two-in-one measure kit and the preparation method of Procalcitonin | |
| CN106855572A (en) | A kind of gastrin-releasing peptide precursor chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN107328938A (en) | Propepsin and helicobacter pylori antibody detection method and its kit | |
| CN103823058B (en) | The chemiluminescence protein chip method of Antigens albumen and kit in serum | |
| CN106771264A (en) | Thyrotropin assay kit and preparation method | |
| CN109239326A (en) | Based on the micro-fluidic immuno-chip analysis method of magnetic particle nano enzyme and application | |
| CN112505322A (en) | Alzheimer disease marker p-Tau217 detection kit and manufacturing method thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170531 |