CN107703110A - G17 detection kit and preparation method thereof - Google Patents
G17 detection kit and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically a kind of G17 detection kit and preparation method, provided with test card, it is characterised in that the test card is provided with by lower from being above sequentially provided with：PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, the G17 monoclonal antibody of rare-earth fluorescent microballoon mark is wherein adsorbed with pad, a diameter of 60 120nm of the rare-earth fluorescent microballoon, the rare earth doped lanthanide series of rare-earth fluorescent microballoon, it is stable under ground state, launch fluorescence of the wave-length coverage in 540 600nm under 340 380nm excitation source effect；The monoclonal antibody is the monoclonal antibody mixed after purification, from the cell strain of monoclonal antibody for 26 different G17 epitopes, has the advantages that easy to operate, rapid reaction, high sensitivity, high specificity.
Description
Technical field：
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically one kind can be quick and precisely
G17 detection kit and preparation method thereof that quantitative analysis is carried out to G17.
Background technology：
Serum gastrin 17 (gastrin 17, G17) is the peptide hormone secreted by intestines and stomach G cells, itself and gall-bladder
Plain acceptor (cholecystokinin receptor, CCKR) is shunk to transduce to play biology by a series of signal with reference to after
Effect, gastric acid secretion and nutrition gastrointestinal tract mucosa are primarily involved in, some researchs in recent years show that serum G17 also has and promote to increase
Grow and suppress the effect of apoptosis.Serum G17 can prompt the functional status of patient's stomach lining, therefore in the diagnosis of gastrointestinal disease
It is significant.
In atrophic gastritis (chronic atrophic gastritis, CAG), serum G17 withers with stomach lining
Contracting position and atrophy degree have correlation.Find the serum G17 of antrum atrophy group than control in a case-control study
Group is low, and the serum G17 of corpus atrophy group is higher than control group, and the serum G17 of multifocal atrophy group is lower than control group, but is higher than antrum
Group.When to analyze its reason may be antrum atrophy, G cell quantities are less, and G17 synthesis reduces；During corpus atrophy, oxyntic cell number
Amount is reduced, and is in low acid condition in stomach, is caused gastrin to raise by gastrin hydrochloric acid in gastric juice axle negative-feedback.By stomach lining it is light, neutralize weight
Atrophy group is spent respectively compared with Normal group, and serum G17 contents significantly increase in the slight atrophy of stomach lining, during moderate atrophy
Significantly reduced without significant difference, during severe atrophy；Compared with Normal group, G cell quantities in the slight atrophy of stomach lining without
Significant difference.And then significantly reduced when in stomach lining, severe atrophy.
During patient send out currently all in research, compared with non-atrophic gastritis group, gastric ulcer group serum G17 is horizontal significantly to be raised
(P<0.05).Found by the horizontal detections of G17 to 3906 parts of blood serum samples, by normal person through superficial gastritis to gastric erosion or
Ulcer, the horizontal progressive rises of serum G17.The horizontal rises of serum G17 may have inflammatory reaction and gastric erosion with stomach lining
Ulcers are more to infect correlation with H.pylori.
Serum G17 can reflect the functional status of stomach lining.In chronic gastritis, gastric ulcer, serum G17, which has, accordingly to be changed
Become, this is that clinician provides important serology foundation in the diagnosis of gastrointestinal disease.
Immuno analytical method is using the immune response between trace antigen and corresponding antibody with high specificity, to detect such as
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 utilizes
The carboxyl latex microballoon labelling immunoassay technology containing rare earth element be to belong 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
It is big to endanger and made troubles to experiment.EIA enzyme immunoassay (EIA) is larger by other influences factor also because enzyme itself is unstable, pushes away
Wide application is restricted.At the beginning of the eighties, people begin one's study replaces fluorescent material and isotope-labelled protein with rare earth element
Or antibody, TIME RESOLVED TECHNIQUE is incorporated into field of biological detection, establishes 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, obtain significant progress
And extensive use.
TrFIA make use of trivalent rare earth ion and chelate with unique fluorescent characteristic for 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 reaction product in fluorescence intensity.According to product fluorescence intensity and relatively glimmering
The ratio of luminous intensity, the concentration of analyte in reaction system is judged, so as to reach quantitative analysis.In common fluoremetry,
Due to containing a variety of fluorescent components in test sample, background fluorescence is (caused by the colloidal solid and solvent molecule in sample
The non-specific fluorescence that scattering light and Proteins in Serum and other compounds are sent) intensity is big, it is strong to disturb, turn into 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 EIA, RIA latter,
Depend primarily in the unique fluorescence feature of lanthanide series, detection the wavelength resolution used and time-delay technique and dissociation-
Enhancing technology.
Lanthanide series (lanthanide, Ln) belongs to rare earth element, in sharing 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 grown, and 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, delay one
Measured after fixing time, Eu can be obtained3+Specific fluorescence signal.Simultaneously because decay time is grown, 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 has 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 exciting light and the Stokes displacements launched between light are larger, Eu3+Excitation wavelength is 337nm, transmitting
Wavelength is 615nm, and Stokes displacements are up to 278nm；Eu simultaneously3+The fluorescence light belt being excited is extremely narrow, and the emission peak of fluorescence is very
Sharply, instrument adjustment can be made to be determined in extremely narrow wave-length coverage, thus almost completely eliminate the interference of background fluorescence, after
And passage time delay and wavelength resolution, 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
Sensitivity, the simplicity of operation and stable fluorescent marker 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 to utilize the sensitive of fluorescence immune chromatography
Property, the high sensitivity, fast and simple realized with reference to fluorescence immune chromatography analyzer can detect examination with the G17 of accurate quantitative analysis
Agent box and preparation method.
The present invention can be reached by following measures：
A kind of G17 detection kit, provided with test card, it is characterised in that the test card is set successively from the bottom to top
Have：PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, rare-earth fluorescent microballoon mark is wherein adsorbed with pad
The G17 monoclonal antibody of note, a diameter of 60-120nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon are rare earth doped
Lanthanide series, it is stable under ground state, launch wave-length coverage 540-600nm's under 340-380nm excitation source effect
Fluorescence；The monoclonal antibody is the monoclonal antibody mixed after purification, from for 2-6 different G17 antigens
The cell strain of monoclonal antibody of epitope.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is preferably 90-110nm；The rare-earth fluorescent microballoon is excellent
Choosing is doped with rare earth lanthanide, for any one or a few of the lanthanide series such as europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd)
Mixture；The preferably rare earth doped complex compound of rare-earth fluorescent microballoon；The antibody that rare-earth fluorescent microballoon marks on pad is excellent
Choosing derives from the monoclonal cell cell line for 3 different epitopes.
Pad of the present invention is made using following steps：Glass fibre membrane is soaked in 200mM Tris-HCL processing
In liquid (X-100 containing 1.5%Triton, 1.5%BSA, pH7.5), 4 DEG C are soaked 4 hours, and it is small to then take out 37 DEG C of oven for drying 4
When, it is standby, it is non-contact with Bio-Jet Quanti300 by glass fibre membrane on Bio-DotXYZ3050 three-dimensional specking platforms
The G17 monoclonal antibody that rare-earth fluorescent microballoon marks is sprayed onto glass fibre membrane by the quantitation nozzle that declines, and 37 DEG C of drying 2 are small
When after be made.
The G17 monoclonal antibody of rare-earth fluorescent microballoon mark in the present invention on pad is using following step
It is rapid to be made：
Step 1：The acquisition of cell strain of monoclonal antibody：With reference to G17 amino acid sequence, the strong position of selection antigenicity
The peptide sequence of artificial synthesized 20 amino acid of point or so, is linked on KLH, using the method for preparing monoclonal antibody system of standard
The cell strain of monoclonal antibody of standby specific high-affinity, it is real that monoclonal antibody corresponding to the cell line obtained is subjected to pairing
Test with affinity determination experiment, according to experimental result determine capture antibody and detection antibody；
Step 2：The preparation of monoclonal antibody：Prepare and purify using the ascites production technology of standard and secreted for the stomach of detection
Plain 17 monoclonal antibodies, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：3mg rare-earth fluorescent microballoons are taken, with 50mM, pH 9.5 carbonate delays
Fliud flushing, washed 3 times, centrifugal speed 12000rpm using centrifugal process, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbon
In phthalate buffer, the glucan of 300 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In the above-mentioned carbonate buffer solution for washing and being resuspended to 100 μ l, be placed in 4 DEG C it is standby；
Step 4：The preparation of the G17 monoclonal antibody of rare-earth fluorescent microballoon mark：The 2mg stomaches for being used to detect are secreted
Plain 17 monoclonal antibodies with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnights, then with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical
Mixing, 4 DEG C of reactions are overnight；Then, sodium borohydride is added to final concentration 10mM, and 4 DEG C are reacted 4 hours；Add isometric envelope
Liquid (100mM Tris-HCL, pH7.5, containing 2%BSA, 5% sucrose) is closed, 4 DEG C of closings are overnight；Then 100mM Tris-HCL are used,
PH7.5 buffer solution is washed 3 times using centrifugal process, is resuspended in 100 μ l 100mM Tris-HCL buffer solutions and (is contained 1.2%
NaCL, 0.5%BSA, 0.2%Tween 20), 4 DEG C be kept in dark place it is standby.
It is of the present invention to be coated with detection line and the nitrocellulose filter of nature controlling line is made by following steps：
Step 1：According to foregoing pairing experiment and affinity determination experiment, select for monoclonal corresponding to the antibody of capture
Antibody cell strain, prepared according to the ascites production technology of standard and purify the G17 monoclonal antibody for capture, preserved
It is standby in -20 DEG C；
Step 2：G17 monoclonal antibody and goat anti-mouse igg antibody adjustment concentration are arrived with coating dilution respectively
1-5mg/ml, film liquid amount are 1-2 μ l/cm, and nitrocellulose filter is sprayed on using them as detection line is parallel with nature controlling line
On be coated with, detection line and nature controlling line are subsequently placed in baking oven, 37 DEG C dry 2 hours at intervals of 3-7mm.
Sample pad of the present invention is made by following steps：Glass fibre membrane is soaked in containing 2.0%Triton X-
100,2%BSA, 0.1M Tris buffer solutions, in pH7.5 treatment fluid, 4 hours are soaked in 4 DEG C, are subsequently placed in baking oven, 37
DEG C drying 2 hours.
Present invention also offers a kind of G17 preparation method that kit is realized as described above, it is characterised in that including
Following steps：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, keeps flat；
Step 2：Accurate to draw 25 μ l serum samples, sample is drawn 40 μ l samples, is added in sample aperture when being whole blood, then
100 μ l Sample dilutions are added in the buffering fluid apertures of bottom immediately, Sample dilution is using physiological saline or PBS, 15-30 points
In clock result is quantitatively judged with fluorescence immune chromatography analyzer；
Step 3：After the relevant parameter for setting fluorescence immune chromatography analyzer, test card is put into storehouse and detected,
Instrument would indicate that the quantified results of sample concentration, and the fluorescence immune chromatography analyzer is a kind of Systems for optical inspection,
Detection range to G17 is 0-20ng/ml.
The present invention provides a kind of G17 fast quantification immunochromatography prepared using rare-earth fluorescent immunochromatography technique
Detection kit, while it is adapted to serum and whole blood sample, and it is adapted to clinically single part detection, relative to the qualitative glue of G17
Body gold reagent, the G17 content in detection sample can be quantified, there is more specific Clinical significance of MG, there is operation letter
Just, rapid reaction, high sensitivity, high specificity, be adapted to Site Detection and it is economical and practical the advantages that.
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.
Subordinate list 3 is the Precision Analyze result data of embodiment 3 in the present invention.
Reference：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4, adsorptive pads 5.
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 G17 detection kit, box is interior to be provided with test card, institute
Test card is stated to be sequentially provided with from the bottom to top：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4 and adsorptive pads 5, wherein tying
Close the G17 monoclonal antibody that rare-earth fluorescent microballoon mark is adsorbed with pad 3, a diameter of 60- of the rare-earth fluorescent microballoon
120nm, the rare earth doped lanthanide series of rare-earth fluorescent microballoon is stable under ground state, is issued in 340-380nm excitation source effect
Project fluorescence of the wave-length coverage in 540-600nm；The monoclonal antibody is the monoclonal antibody mixed after purification, from pin
The cell strain of monoclonal antibody of the G17 epitope different to 2-6；
The diameter of the rare-earth fluorescent microballoon of the pad 3 is preferably 90-110nm；The rare-earth fluorescent microballoon is preferably mixed
It is miscellaneous to have rare earth lanthanide, it is any one or a few mixed of the lanthanide series such as europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd)
Compound；The preferably rare earth doped complex compound of rare-earth fluorescent microballoon；The antibody that rare-earth fluorescent microballoon marks on pad preferably comes
Come from the monoclonal cell cell line for 3 different epitopes.
Embodiment 1：
Each part of test card can be made by following measures in G17 detection kit：
1st, the preparation of sample pad 2：
Glass fibre membrane is soaked in containing 2.0%Triton X-100,2%BSA, 0.1M Tris buffer solutions, pH7.5
Treatment fluid in, in 4 DEG C soak 4 hours, be subsequently placed in baking oven, 37 DEG C dry 2 hours.
2nd, the preparation of the pad 3 of fluorescent microsphere labelled antibody is adsorbed：
Glass fibre membrane is soaked in 200mM Tris-HCL treatment fluids (X-100 containing 1.5%Triton, 1.5%
BSA, pH7.5), 4 DEG C are soaked 4 hours, then take out 37 DEG C of oven for drying 4 hours, standby.By glass fibre membrane in Bio-
On DotXYZ3050 three-dimensional specking platforms, with the non-contact quantitation nozzles that decline of Bio-Jet Quanti300 by rare-earth fluorescent microballoon
The G17 monoclonal antibody of mark is sprayed onto glass fibre membrane, and 37 DEG C dry 2 hours, standby；
The aldehyde radical of rare-earth fluorescent nanoparticle：3mg rare-earth fluorescent nanoparticles are taken, with 50mM, pH9.5 carbonate delays
Fliud flushing, washed 3 times, centrifugal speed 12000rpm using centrifugal process, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbon
In phthalate buffer, the glucan of 300 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In the above-mentioned carbonate buffer solution for washing and being resuspended to 100 μ l, be placed in 4 DEG C it is standby；
Rare-earth fluorescent nanoparticle marks the preparation of G17 monoclonal antibody：The G17 that 2mg is used to detect
Monoclonal antibody in 4 DEG C of dialysed overnights, is then mixed with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical,
4 DEG C of reactions are overnight；Then, sodium borohydride is added to final concentration 10mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid
(100mM Tris-HCL, pH7.5, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 100mM Tris-HCL are used,
PH7.5 buffer solution is washed 3 times using centrifugal process, is resuspended in 100 μ l 100mM Tris-HCL buffer solutions and (is contained 1.2%
NaCL, 0.5%BSA, 0.2%Tween 20), 4 DEG C be kept 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：
According to foregoing pairing experiment and affinity determination experiment, select thin for monoclonal antibody corresponding to the antibody of capture
Born of the same parents' strain, prepared according to the ascites production technology of standard and purify the G17 monoclonal antibody for capture, be stored in -20 DEG C
It is standby；
G17 monoclonal antibody and goat anti-mouse igg antibody are adjusted into concentration to 1-5mg/ with coating dilution respectively
Ml, film liquid amount are 1-2 μ l/cm, are carried out them as be sprayed on nitrocellulose filter on parallel with nature controlling line of detection line
Coating, detection line and nature controlling line are subsequently placed in baking oven, 37 DEG C dry 2 hours at intervals of 3-7mm；
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 for being coated with detection line and nature controlling line, it is big to obtain test paper after assembling
Plate, it is wide to cut into 4mm as requested, and test paper is loaded in plastic clip and forms test card.
The preferably following raw material of equipment and raw material selected in above steps：
G17 specific pairs antibody；G17 quality-control product：Landau laboratory diagnosis Co., Ltd of Britain；Rare earth is glimmering
Light microballoon：Shanghai Zhen Zhun bio tech ltd；Nitrocellulose (NC) film：Millipore Products；Bovine serum albumin
(BSA) in vain, 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 assembled, respectively with 0.2,0.5,1,2,5,20ng/ml G17 calibration object, surveyed with test card
It is fixed, the fluorescence intensity level of each calibration object is obtained, result is input in the parameter of analyzer, the parameter for completing analyzer is set
It is fixed.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 200 parts of Roche Electrochemiluminescence immunoassay definite values
Sample, wherein 100 parts of serum sample, 100 parts of whole blood sample, G17 content distribution section is between 0-20ng/mL.
Preparation method：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, keeps flat；
Step 2：Accurate to draw 25 μ l serum samples, sample is drawn 50 μ l samples, is added in sample aperture when being whole blood, then
100 μ L Sample dilutions (physiological saline or PBS) are added in the buffering fluid apertures of bottom immediately, 15-30 minutes, interior fluorescence was exempted from
Epidemic disease chromatographic analysis instrument quantitatively judges result；
Step 3：Set test card to be put into storehouse after instrument relevant parameter and detected, instrument would indicate that sample is dense
The quantified results of degree.
Test result analysis：
After the completion of prepared by clinical sample detection reagent, all clinical samples are detected by preparation method, and analyze inspection
Survey result.
Result of the test：
As shown in Figure 2, using the detected value of experimental system as Y-axis, using the test value of contradistinction system as X-axis, scatterplot is drawn
Figure, and carry out correlation analysis.Clinical sample detection is respectively less than to 200 parts of clinical definite value pattern detections, sample mean deviation
10%, maximum deviation is less than 25%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection kit prepared
Can be good, it is suitable for clinical detection, meets the differentiation needs of the different detection occasions of different clients.
Embodiment 3：Precision test
Using the test card and measuring system of embodiment 2, the test card and fluorescence immune chromatography analyzer of the present invention are entered
Row Precision Experiment.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 2 parts of Chemiluminescence immunoassay definite value serum samples,
Wherein low value definite value sample clinical measures are 0.24ng/ml, and high level definite value sample clinical measures are 1.78ng/ml.
Preparation method：
Using the test card and measuring system of embodiment 2, replication is respectively carried out 20 times to 2 parts of definite value samples.
Test result analysis：
After the completion of prepared by clinical sample detection reagent, clinical sample is detected by preparation method, and analyzes detection knot
Fruit.
Result of the test：
As shown in table 1, the low value definite value sample and clinical measures that are 0.24ng/ml to clinical measures are 1.78ng/
Average value, standard deviation and CV, the reality of the acquired results display present invention are calculated after ml high level definite value sample replication 20 times
Check system test low value definite value sample CV is 8.13%, and test high level definite value sample CV is 5.61%.Testing result shows to prepare
Detection kit it is functional, be suitable for clinical detection, meet the differentiation needs of the different detection occasion of different clients.
The present invention provides a kind of G17 fast quantification immunochromatography prepared using rare-earth fluorescent immunochromatography technique
Detection kit, while it is adapted to serum and whole blood sample, and it is adapted to clinically single part detection, relative to the qualitative glue of G17
Body gold reagent, the G17 content in detection sample can be quantified, there is more specific Clinical significance of MG, there is operation letter
Just, rapid reaction, high sensitivity, high specificity, be adapted to Site Detection and it is economical and practical the advantages that.
Claims (7)
- A kind of 1. G17 detection kit, provided with test card, it is characterised in that the test card be provided with by it is lower from it is upper successively It is provided with：PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, rare-earth fluorescent microballoon is wherein adsorbed with pad The G17 monoclonal antibody of mark, a diameter of 60-120nm of the rare-earth fluorescent microballoon, the doping of rare-earth fluorescent microballoon are dilute Native lanthanide series, it is stable under ground state, launch wave-length coverage in 540-600nm under 340-380nm excitation source effect Fluorescence；The monoclonal antibody is the monoclonal antibody mixed after purification, is resisted from for 2-6 different G17s The cell strain of monoclonal antibody of former epitope.
- 2. a kind of G17 detection kit according to claim 1, it is characterised in that the rare earth of the pad is glimmering The diameter of light microballoon is preferably 90-110nm；The rare-earth fluorescent microballoon is doped with rare earth lanthanide.
- 3. a kind of G17 detection kit according to claim 2, it is characterised in that the rare-earth fluorescent microballoon is mixed Miscellaneous rare-earth complex；The antibody sources that rare-earth fluorescent microballoon marks on pad are in the monoclonal for 3 different epitopes Cell strain.
- 4. a kind of G17 quality detection kit according to claim 1, it is characterised in that the pad is using such as Lower step is made：Glass fibre membrane is soaked in 200mM Tris-HCL treatment fluids, 4 DEG C are soaked 4 hours, then take out 37 DEG C Oven for drying 4 hours, it is standby, by glass fibre membrane on Bio-DotXYZ3050 three-dimensional specking platforms, use Bio-Jet The G17 monoclonal antibody that rare-earth fluorescent microballoon marks is sprayed onto glass fibre by the non-contact quantitation nozzles that decline of Quanti300 Film, 37 DEG C drying 2 hours after be made.
- 5. a kind of G17 quality detection kit according to claim 4, it is characterised in that described dilute on pad The G17 monoclonal antibody of native fluorescent microsphere mark is made using following steps：Step 1：The acquisition of cell strain of monoclonal antibody：With reference to G17 amino acid sequence, the strong site people of selection antigenicity Work synthesizes the peptide sequence of 20 amino acid or so, is linked on KLH, is prepared using the method for preparing monoclonal antibody of standard special The cell strain of monoclonal antibody of different in nature high-affinity, by monoclonal antibody corresponding to the cell line obtained carry out pairing experiment and Affinity determination experiment, capture antibody and detection antibody are determined according to experimental result；Step 2：The preparation of monoclonal antibody：Prepared using the ascites production technology of standard and purify the G17 for detection Monoclonal antibody, be stored in after packing -20 DEG C it is standby；Step 3：The aldehyde radical of rare-earth fluorescent microballoon：3mg rare-earth fluorescent microballoons are taken, with 50mM, pH9.5 carbonate buffer solution, Washed 3 times, centrifugal speed 12000rpm using centrifugal process, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbonate In buffer solution, the glucan of 300 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, is washed using same centrifugal process Be resuspended in 100 μ l above-mentioned carbonate buffer solution, be placed in 4 DEG C it is standby；Step 4：The preparation of the G17 monoclonal antibody of rare-earth fluorescent microballoon mark：The G17 that 2mg is used to detect Monoclonal antibody in 4 DEG C of dialysed overnights, is then mixed with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical, 4 DEG C of reactions are overnight；Then, sodium borohydride is added to final concentration 10mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid (100mM Tris-HCL, pH7.5, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 100mM Tris-HCL are used, PH7.5 buffer solution is washed 3 times using centrifugal process, is resuspended in 100 μ l 100mM Tris-HCL buffer solutions, and 4 DEG C of lucifuges are protected Deposit standby.
- 6. a kind of G17 quality detection kit according to claim 1, it is characterised in that described to be coated with detection line It is made with the nitrocellulose filter of nature controlling line by following steps：Step 1：According to foregoing pairing experiment and affinity determination experiment, select for monoclonal antibody corresponding to the antibody of capture Cell line, prepared according to the ascites production technology of standard and purify the G17 monoclonal antibody for capture, be stored in -20 It is DEG C standby；Step 2：G17 monoclonal antibody and goat anti-mouse igg antibody are adjusted into concentration to 1- with coating dilution respectively 5mg/ml, film liquid amount are 1-2 μ l/cm, are sprayed on using them as detection line is parallel with nature controlling line on nitrocellulose filter It is coated with, detection line and nature controlling line are subsequently placed in baking oven, 37 DEG C dry 2 hours at intervals of 3-7mm.
- A kind of 7. G17 quality detection kit according to claim 1, it is characterised in that the sample pad by with Lower step is made：Glass fibre membrane is soaked in containing 2.0%Triton X-100,2%BSA, 0.1M Tris buffer solutions, In pH7.5 treatment fluid, 4 hours are soaked in 4 DEG C, are subsequently placed in baking oven, 37 DEG C dry 2 hours.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061200A (en) * | 2018-08-23 | 2018-12-21 | 上海复星长征医学科学有限公司 | Gastrin 17 time resolution micro-ball immune chromatography detection reagent card and preparation method thereof |
| CN111366724A (en) * | 2020-03-18 | 2020-07-03 | 厦门奥德生物科技有限公司 | Atrophic gastritis rare earth detection test strip, detection card, kit and preparation method |
| CN111458523A (en) * | 2020-04-07 | 2020-07-28 | 上海萨迦生物科技有限公司 | Gastrin-17 detection kit, preparation method and detection application method thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104714015A (en) * | 2014-10-28 | 2015-06-17 | 威海纽普生物技术有限公司 | Detection kit and detection method for heart-type fatty acid binding protein |
| CN104714025A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | NT-proBNP detection kit and detection method |
| 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 |
-
2017
- 2017-09-19 CN CN201710852552.9A patent/CN107703110A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104714015A (en) * | 2014-10-28 | 2015-06-17 | 威海纽普生物技术有限公司 | Detection kit and detection method for heart-type fatty acid binding protein |
| CN104714025A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | NT-proBNP detection kit and detection method |
| 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 |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061200A (en) * | 2018-08-23 | 2018-12-21 | 上海复星长征医学科学有限公司 | Gastrin 17 time resolution micro-ball immune chromatography detection reagent card and preparation method thereof |
| CN109061200B (en) * | 2018-08-23 | 2021-08-06 | 复星诊断科技(上海)有限公司 | Gastrin 17 time-resolved microsphere immunochromatography detection reagent card and preparation method thereof |
| CN111366724A (en) * | 2020-03-18 | 2020-07-03 | 厦门奥德生物科技有限公司 | Atrophic gastritis rare earth detection test strip, detection card, kit and preparation method |
| CN111366724B (en) * | 2020-03-18 | 2022-09-30 | 厦门奥德生物科技有限公司 | Rare earth detection test strip, detection card, kit and preparation method for atrophic gastritis |
| CN111458523A (en) * | 2020-04-07 | 2020-07-28 | 上海萨迦生物科技有限公司 | Gastrin-17 detection kit, preparation method and detection application method thereof |
| CN114441779A (en) * | 2022-02-14 | 2022-05-06 | 上海交通大学 | Bimodal immunochromatography kit for gastrin 17 and detection method thereof |
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