CN107561281A - A kind of centrichromatography colloid gold immune detection technique and application thereof - Google Patents
A kind of centrichromatography colloid gold immune detection technique and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of centrichromatography colloid gold immune detection combination suit, it is characterised by, detection combination suit contain thing specificity junction mixture to be checked, colloidal gold particles, immobilon-p, centrifugal device and collaurum detector, wherein described colloidal gold particles are that directly can be formed non-specific binding using prepared by gold solution with protein and maintained the particle of stabilization；The immobilon-p is with the film quality material with protein non-specific binding characteristic；The centrifugal device is the structure that can centrifuge driving liquid phase chromatographic flow on the immobilon-p；The thing specificity junction mixture to be checked is at least one of antigen with specific binding capacity, antibody, Avidin, biotin and the like；The collaurum detector is the detector quantitatively or semi-quantitatively detected by the colorimetric analysis to collaurum color.The present invention has the characteristics of high sensitivity, stability is good, detection time is short, has a good application prospect.
Description
Technical field
The present invention relates to a kind of centrichromatography colloid gold immune detection technique and application thereof, belongs to immunoassay technology neck
Domain.
Background technology
Immunology detection technology is measure antigen, antibody, immunocyte and the chemical composition of applied immunology principle design
Deng laboratory facilities, the sample of medical diagnosis on disease and health detection can be carried out from human body and animal body and be used for by being widely used in
Environment, Pharmaceutical Analysis, food and the sample of Industrial Analysis.Conventional has immune turbidity technology, solid-phase enzyme immunoassay technology, changes
Learn luminescent detection techniques, immunofluorescence label technology, flow cytometry, colloidal gold technique etc..Immune turbidity technology, it is also referred to as immune
Nephelometry is soluble antigen, antibody specific bond in the liquid phase, produces a certain size compound, forms the refraction or suction of light
Receive, determine the transmitted light after this refraction or absorption or scattering light as unit of account, for quantitatively detecting, but detection sensitivity
It is low, it is not suitable for trace detection.The enzyme of immobilization and antigen or antibody of the solid-phase enzyme immunoassay technology based on antigen or antibody
Mark, is incorporated in the antigen of surface of solid phase carriers or antibody keeps its immunologic competence, and the enzyme conjugates of antigen or antibody was both protected
Its immunologic competence is stayed, retains the activity of enzyme again, in measure, by inspection sample（Determine antibody or antigen therein）With enzyme mark
Antigen or antibody react by the antigen or antibody of different step and surface of solid phase carriers, have high sensitivity, linear response
The remarkable advantages such as scope is wide and easy to automate, but detect reaction time length and limit its use.Chemiluminescence detection skill
Art is a kind of highly sensitive micro and Analytical Methods of Trace, has easy to operate, high sensitivity, linear response range is wide and is easy to
The remarkable advantages such as automation are realized, is widely used in environment, clinic, Pharmaceutical Analysis, food and Industrial Analysis, is also based on
The luminescence reagent labelling technique of the solid phase separation means and antigen or antibody of antigen or antibody, but it is long and to inspection to detect the reaction time
The requirement height of measurement equipment also influences its use.Immunofluorescence label technology, flow cytometry, colloidal gold technique are also conventional inspection
Survey technology is widely used, but has its corresponding deficiency, and the detection reaction time is long or sensitivity, accuracy shortcoming are generally to deposit
Deficiency.High sensitivity, quick, miniaturization, quantitative, automation is that the development of current clinical immunization detection technique product becomes entirely
Gesture, but existing above-mentioned function can not be all realized simultaneously.Colloid gold immune paper chromatography quantitative measurement technology is due to its reaction solution
The unstability that intensity changes over time, hence it is evident that affect the clinical application effect of the technology.
The content of the invention
It is an object of the invention to provide a kind of centrichromatography colloid gold immune detection technique and application thereof.The present invention has inspection
Survey the characteristics of time is short, detection stability is good, easy to use.
A kind of centrichromatography colloid gold immune detection combination suit provided by the invention, is characterised by, the detection combination
Suit contains thing specificity junction mixture to be checked, colloidal gold particles, immobilon-p, centrifugal device and collaurum detector, wherein institute
The colloidal gold particles stated are non-specific binding can directly to be formed with protein using prepared by gold solution and remained stable
Particle；The immobilon-p is with the film quality material with protein non-specific binding characteristic；The centrifugal device is can be from
The heart drives the structure of liquid phase chromatographic flow on the immobilon-p；The thing specificity junction mixture to be checked is with specific binding
At least one of the antigen of ability, antibody, Avidin, biotin and the like；The collaurum detector be by pair
The detector that the colorimetric analysis of collaurum color is quantitatively or semi-quantitatively detected.
The detection method that detection combination described in a kind of claim 1 is set with, it is characterised in that：The detection method is as follows
（A）-（B）In it is any：
（A）Comprise the following steps：
1）Colloidal gold particles solution is prepared with gold solution；
2）The colloidal gold particles described in the thing specific binding substance markers to be checked；
3）Reacted with the colloidal gold particles after the sample containing thing to be checked and the mark, the thing to be checked by with positioned at described
The thing specificity junction mixture to be checked on colloidal gold particles after mark combines, and then forms " colloidal gold particles-thing to be checked spy
Specific binding agent-thing to be checked " compound, i.e. compound 1；
4）Coating can be by forming the of specific binding with the thing to be checked on compound 1 on the immobilon-p
Two thing specificity junction mixtures to be checked；
5）With the liquid phase containing the compound 1, by chromatography, to flow through coated second thing to be checked on the immobilon-p special
Property conjugate, it is multiple to form " colloidal gold particles-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked "
Compound, i.e. compound 2, and be captured and be fixed on the immobilon-p；
6）The compound 1 being not associated with the immobilon-p and the glue of residual are cleaned with cleaning fluid using centrifugal chromatography
Body gold material；
7）The institute that the immobilon-p after cleaning is indirectly fixed on the immobilon-p with collaurum detector detection
The chromatic value of colloidal gold particles is stated, to detect the content of the thing to be checked；
（B）Comprise the following steps：
1）Colloidal gold particles solution is prepared with gold solution；
2）The colloidal gold particles described in the thing specific binding substance markers to be checked；
3）Reacted with the colloidal gold particles after the sample containing thing to be checked and the mark, the thing to be checked by with positioned at described
The thing specificity junction mixture to be checked on colloidal gold particles after mark combines, and then forms " colloidal gold particles-thing to be checked spy
Specific binding agent-thing to be checked " compound, i.e., described compound 1；
4）The second thing specificity junction mixture to be checked is marked with intermediate material A；
5）Coating can form the intermediate material B of specific binding with the intermediate material A on the immobilon-p；
6）The the second thing specificity junction mixture to be checked marked with the liquid phase containing the compound 1 with the intermediate material A is carried out
Reaction, form " colloidal gold particles-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture-intermediate to be checked
Matter A " compounds, i.e. compound 3；
7）The coated intermediate material B on the immobilon-p is flowed through by chromatography with the liquid phase containing the compound 3, formed
" colloidal gold particles-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked-intermediate material A- intermediates
Matter B " compounds, i.e. compound 4, and be captured and be fixed on the immobilon-p；
8）The compound 3 being not associated with the immobilon-p and the glue of residual are cleaned with cleaning fluid using centrifugal chromatography
Body gold material；
9）The institute that the immobilon-p after cleaning is indirectly fixed on the immobilon-p with collaurum detector detection
The chromatic value of collaurum material is stated, to detect the content of the thing to be checked.
In above-mentioned centrichromatography colloid gold immune detection method, the method for the cleaning immobilon-p is clear using being not added with
The dry method eccentric cleaning of the centrichromatography mode of washing lotion, i.e., using not tied on immobilon-p described in the direct eccentric cleaning of centrifugal chromatography
The compound 3 and the collaurum material of residual closed.
In above-mentioned centrichromatography colloid gold immune detection method, the intermediate material is with specific binding capacity
At least one of antigen, antibody, Avidin, biotin and the like.
In above-mentioned centrichromatography colloid gold immune detection method, it is described chromatograph flow through the reaction of the immobilon-p from
Completed on center device and using centrifugal chromatography driving liquid chromatographic flow on immobilon-p, i.e., centrifuged and driven using centrifugal chromatography
Move part or all of liquid phase chromatographic flow on the immobilon-p.
In above-mentioned centrichromatography colloid gold immune detection combination suit or detection method, the particle diameter of the colloidal gold particles
10nm-200nm is selected, concretely 20 ~ 150 nm, 30 ~ 100 nm or 30 ~ 80 nm.
In above-mentioned centrichromatography colloid gold immune detection combination suit, the rotating speed of the centrifugal device uses programme-control
Mode；The rotating speed of the centrifugal device is 200 ~ 10000 revs/min, concretely 500 ~ 5000 revs/min, 800 ~ 3000
Rev/min or 800 ~ 2000 revs/min.
In above-mentioned centrichromatography colloid gold immune detection method, the rotating speed of the centrifugal chromatography uses programme-control side
Formula；The rotating speed of the centrichromatography is 200 ~ 10000 revs/min, concretely 500 ~ 5000 revs/min, 800 ~ 3000
Rev/min or 800 ~ 2000 revs/min.
In above-mentioned centrichromatography colloid gold immune detection combination suit or detection method, it is fine that the immobilon-p includes nitric acid
Tie up plain film, polyvinylidene fluoride film, nylon membrane, DEAE cellulose membranes.The one or both sides of the immobilon-p carry backing.The present invention
In, the polyvinylidene fluoride film abbreviation pvdf membrane；The DEAE cellulose membranes refer to diethyl amino ethyl group (DEAE) introducing fibre
Manufactured paper-like film after plain molecule is tieed up, is a kind of weak base type anion-exchange material.
In above-mentioned centrichromatography colloid gold immune detection combination suit, the detection combination suit also includes described clear
Washing lotion, the cleaning fluid are the normal experiment cushioning liquid containing surfactant.Currently used surfactant has
TWEEN20, TritonX-100, TritonX-405 etc..
In above-mentioned centrichromatography colloid gold immune detection method, the cleaning fluid is conventional real containing surfactant
Test and use cushioning liquid.Currently used surfactant has TWEEN20, TritonX-100, TritonX-405 etc..
In above-mentioned centrichromatography colloid gold immune detection combination suit, the detection combination suit, which also includes second, to be treated
Examine at least one of thing specificity junction mixture, intermediate material A, intermediate material B.
Application of the described combined complete or detection method in immune detection product development.
For the present invention due to taking above technical scheme, it has advantages below：
1st, the present invention uses carrying carrier of the colloidal gold particles as thing to be checked and its intermediate reaction material, is chromatographed on immobilon-p
Flow and complete to react, improve capture binding ability of the immobilon-p to thing to be checked and its intermediate reaction material.
2nd, the liquid phase that the present invention is driven using centrifugal device chromatographic flow on immobilon-p, it is micro- can be effectively reduced collaurum
The non-specificity of grain and immobilon-p directly or indirectly combines, and reduces the ambient noise interference of immobilon-p, improves detection sensitivity.
3rd, existing paper chromatography colloid gold immune detection technique, using lateral flow control, detection line colloidal gold particles
Color density is not only directly proportional to thing concentration to be checked in the range of certain time, is also proportionate with the reaction time, to actual inspection
Observing and controlling system brings very big difficulty, and detection error increases.The present invention carries out colloidal gold particles face using paper chromatography centrifugation technique
Color density detects, and can significantly reduce influence of the detection time to testing result, improve the accuracy of detection.
4th, operating procedure of the present invention is simple, it is easy to accomplish automation mechanized operation.The inventive method has high sensitivity, Quan Ding
The characteristics of amount, automation, while have that detection is quick, uses the simple detection technique of equipment again；Not only easy to use, reduction original
The waste of material, while operating efficiency is also significantly improved, applied to detection and analysis, the numerous areas of separation.
Brief description of the drawings
Fig. 1 is the schematic diagram of the centrichromatography colloid gold immune detection combination of the embodiment of the present invention 1 suit.
Marked in figure as follows：
1 thing specificity junction mixture to be checked；2 colloidal gold particles；3 immobilon-ps；4 centrifugal devices；5 colloid detectors；6 second treat
Examine thing specificity junction mixture or intermediate material B；7 mark adsorbed film pads；8 absorbing membrane pads；9 support egative films.
Embodiment
The present invention is further described below in conjunction with the accompanying drawings, but the invention is not limited in following examples.
The preparation of embodiment 1, centrichromatography colloid gold immune detection combination of the present invention suit：
As shown in figure 1, centrichromatography colloid gold immune detection combination of the present invention suit includes, thing specificity junction mixture 1 to be checked, glue
Body gold particulate 2, immobilon-p 3, centrifugal device 4, collaurum detector 5, at the same also include the second thing specificity junction mixture to be checked or
Intermediate material B 6, mark adsorbed film pad 7, absorbing membrane pad 8, support egative film 9.Thing specificity junction mixture 1 and colloidal gold particles to be checked
2 combine, and are printed on mark adsorbed film pad 7.The second thing specificity junction mixture or intermediate material to be checked is printed with immobilon-p 3
B Acquisition Detections line 6.Binding mark adsorbed film pad 7, immobilon-p 3 and absorbing membrane pad 8 successively on support egative film 9, detection examination is made
Paper slip.During detection, test strip is placed on centrifugal device 4, loads on mark adsorbed film pad 7 and is treated containing thing to be checked
Sample sheet, the colloidal gold particles 2 for being now marked with thing specificity junction mixture 1 to be checked are combined with thing to be checked, and it is compound to form thing to be checked
Thing, chromatography flow through immobilon-p 3, and the thing compound to be checked formed is coated to be captured in the Acquisition Detection line 6 on immobilon-p 3,
Centrichromatography cleaning is carried out, is then caught indirectly using Acquisition Detection line 6 of the detection of collaurum detector 5 on immobilon-p 3
The chromatic value of the colloidal gold particles 2 obtained, and then the content of thing to be checked is obtained, wherein sample introduction, capture and the process of cleaning is centrifuging
Completed on device 4 by centrichromatography.
The detection method and its effect of following description of test present invention, but be not limitation of the invention.In following experiments
Used experimental method is conventional method unless otherwise specified.Material used, reagent etc. in following experiments, such as without spy
Different explanation, is commercially obtained.
Experiment one, centrichromatography colloid gold immune test experience of the present invention
Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described A.
First, experiment material
Anti-human myoglobins polyclonal antibody（Genagates companies of the U.S.）, anti-human myoglobins monoclonal antibody（The U.S.
Genagates companies）, spectrophotometer（Shanghai mountain valley with clumps of trees and bamboo China Tech skill Instrument Ltd., 752 ultraviolet-uisible spectrophotometers）, people
Myoglobins（Sigma-Aldrich products）, BioFlow die instrument（IMAGENE companies of the U.S.）, Index cutting machines（It is beautiful
A-point companies of state）, DBF-900 sealing machines（Wenzhou south of the River packing factory）, ACBO dehumidifiers（Jiangsu wuxi Austria ripple dehumidifier is public
Department）, desk centrifuge（Eppendoff companies of the U.S.）, bovine serum albumin(BSA)（Abbreviation BSA, SIGMA product）, nitrocellulose
Diaphragm（MILLIPORE companies）, multi-polyester cellulose membrane (Alstrom companies of the U.S.), absorb water paper membrane pad（S＆S companies of the U.S.）,
Gold chloride（SIGMA products）, the quantitative chromatographic analysis instrument of collaurum（Norway's Skannex products）.
It is as follows that above-mentioned material corresponds to Patent Application Request content, and the anti-human myoglobins polyclonal antibody of this experiment is second to treat
Thing specificity junction mixture is examined, anti-human myoglobins monoclonal antibody is thing specificity junction mixture to be checked, and human muscle hemoglobin is to be checked
Thing, desk centrifuge are centrifugal device, and nitrocellulose diaphragm is immobilon-p, and gold chloride is the material for preparing colloidal gold particles,
The quantitative chromatographic analysis instrument of collaurum is collaurum detector.
2nd, experimental method
The preparation of human muscle hemoglobin solution：The human muscle hemoglobin solution of concentration known is taken, uses sample dilution buffer（1%BSA,
100mM glycine, 50mM PBS, 150mM NaCl, pH7.4）Dilution configuration 3,10,30,70,100,150,200ng/ml
Serial human muscle hemoglobin solution.
Colloidal gold particles mark：10ml pure water is taken, heating stirring, it is molten that the gold chlorides of 500 μ l 10% are added when boiling water
Liquid, heating are boiled 5 minutes, add the citric acid three sodium solutions of 500 μ l 12%, are kept this solution stirring boiling 10 minutes, are dropped naturally
Warm to room temperature, produce particle diameter 50nm colloidal gold particles solution.Colloidal gold solution volume 10ml is taken, pH is adjusted extremely with 10% potassium carbonate
8.3, the anti-human μ g of myoglobins monoclonal antibody 100 are rapidly added, to 10 μ g/ml final concentrations, rock beaker mixing, room temperature is placed
30 minutes, 10% bovine serum albumin solution 100ul is rapidly added, makes final concentration of 1%, while shake beaker, room temperature places 30
Minute, 12000rpm is centrifuged 20 minutes, carefully suctions out supernatant；Add 5ml 50mM phosphate (PBS) buffer solution, pH7.4,
Precipitation is suspended, 12000rpm is centrifuged 20 minutes, suctions out supernatant, is dissolved in the bovine serum albumin(BSA) that 1.0ml contains 1% by precipitating
In the phosphate buffer of 3% sucrose, 4 DEG C are kept in dark place.
Colloidal gold particles mark absorption film preparation：Preparation contains 0.5%PVA（That is polyvinyl alcohol）, 50mM PBS liquid, 0.5%
BSA, 0.88% NaCl, pH 7.4 multi-polyester cellulose membrane pretreatment fluid, pending multi-polyester cellulose membrane is put in pre- place
Manage in liquid, soaking at room temperature 1 hour, take the film out, put and seal after 37 DEG C of dryings standby, can also be used directly as dispersion membrane.Take
Colloidal gold particles labelled antibody solution, with colloid buffer solution（1%BSA, 3% sucrose, 50mM PBS, pH7.4）It is diluted to OD530
For 30, start die instrument, load antibody, open pressurized nitrogen, take multi-polyester cellulose membrane, start die, set die condition
For：The airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 5.0, the film after printing is put into drying box, 37 DEG C dry
Dry 6 hours, it is subsequently placed in the hermetic bag containing drier and preserves use.
It is prepared by polyclonal antibody die：Anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffers
(pH 7.4) is diluted to 1mg/ml concentration.Start die instrument, load antibody, take the PVC pieces for posting nitrocellulose filter（That is polychlorostyrene
Piece of vinyl）, start die, set die condition as：Airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 1.5.Will print
The film made is put into 37 DEG C of drying boxes, dries 6 hours, then film is placed in the drying receptacle containing drier and preserves use.
Semi-finished product assemble method：Starting dehumidifier makes the humidity in operating room be reduced to less than 25%, is printed in polyclonal antibody
Water suction paper membrane pad and colloidal gold particles mark adsorbed film are pasted respectively in film both ends.The detection lug pasted is put on cutting machine, is cut
Into 3.5mm test strips.Test strips are put into the aluminium amber hermetic bag of drier, sealed on sealing machine, labelling.
Experimental group：The test strips of above-mentioned preparation are taken, with the side of colloidal gold particles mark adsorbed film upward, are placed in centrifuge
In rotor, the human muscle hemoglobin solution 80ul for the various concentrations that preparation is added dropwise on adsorbed film is marked to colloidal gold particles, stands 2 points
Clock, with 1000 revs/min centrifuge 1 minute, then to colloidal gold particles mark adsorbed film on be added dropwise pH7.4 0.05% Tween-20,
50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, take out test strips, are positioned over the quantitative chromatographic analysis of collaurum
Chromatic value is read on instrument, experiment in triplicate, results averaged, then draws concentration-chromatic value curve, and calculate phase relation
Number.
Control group：The test strips of above-mentioned preparation are taken, are placed in desktop, is marked to colloidal gold particles and is added dropwise what is prepared on adsorbed film
The human muscle hemoglobin solution 80ul of various concentrations, stand, treat the red colloid gold particulate mark on colloidal gold particles mark adsorbed film
Remember thing all chromatography flow into nitrocellulose filter, then to colloidal gold particles mark adsorbed film on be added dropwise pH7.4 0.05% tween-
20th, 50mM PBSs 80ul, stand, liquid all flows into nitrocellulose filter, removes test strips, is positioned over collaurum and determines
Chromatic value is read on amount chromatographic analysis instrument, experiment in triplicate, results averaged, then draws concentration-chromatic value curve, and
Calculate coefficient correlation.
3rd, experimental result
The present invention is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described A, as a result, the present invention is real
Test group test strips single detection process time average out to 3.6 minutes, control test group（Do not do centrifugal treating）Test strips single is examined
Survey processing time average out to 37 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-chromatic value, work as inspection
When survey higher limit is set to 150 ng/ml, correlation coefficient r 0.989, linear detection range is 3-150 ng/ml；Control group detects
Correlation r when higher limit is set to 100 ng/ml is 0.992, has reached the testing requirements more than 0.98, and linear detection range is
3-100 ng/ml.The ratio of experimental group 10ng/ml of the present invention and 3ng/ml detection data is 2.26, and control group is only 1.09.
Non-specific binding of the above-mentioned difference mainly due to colloidal gold particles on immobilon-p can not be effectively cleaned, and cause background mistake
It is high relevant.Experimental result illustrates that the technology of the present invention not only shortens detection time, while improves the linear of prior art detection
And accuracy.Experimental result is as shown in table 1.
The centrichromatography colloid gold immune detection method A experimental results of the present invention of table 1（Unit：Chromatic value）
Experiment two, dry method centrichromatography of the present invention cleaning test experience
Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described A.
First, experiment material
With experiment one.
Above-mentioned material corresponds to Patent Application Request content with experiment one.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one.
Colloidal gold particles mark：With experiment one.
Colloidal gold particles mark absorption film preparation：With experiment one.
It is prepared by polyclonal antibody die：With experiment one.
Semi-finished product assemble method：With experiment one.
Experimental group：Delete " again to 0.05% Tween-20, the 50mM of dropwise addition pH7.4 on colloidal gold particles mark adsorbed film
The step of PBS 80ul ", direct 2000 revs/min of centrifugations are cleaned for 30 seconds, other with experiment one.
Control group：Delete " again to 0.05% Tween-20, the 50mM of dropwise addition pH7.4 on colloidal gold particles mark adsorbed film
PBS 80ul, stand, liquid all flows into nitrocellulose filter ", it is other with experiment one.
3rd, experimental result
The present invention is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described A, as a result, the present invention is real
Test group test strips single detection process time average out to 3.6 minutes, control test group（Do not do centrifugal treating）Test strips single is examined
Survey processing time average out to 39 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-chromatic value, work as inspection
When survey higher limit is set to 150 ng/ml, correlation coefficient r 0.990, linear detection range is 3-150 ng/ml；Control group detects
Correlation r when higher limit is set to 100 ng/ml is 0.992, has reached the testing requirements more than 0.98, and linear detection range is
3-100 ng/ml.The ratio of experimental group 10ng/ml of the present invention and 3ng/ml detection data is 1.26, and control group is only 1.21.
Non-specific binding of the above-mentioned difference mainly due to colloidal gold particles on immobilon-p can not be effectively cleaned, and cause background mistake
It is high relevant.Experimental result illustrates that the technology of the present invention not only shortens detection time, while improves the linear of prior art detection
And accuracy.Experimental result is as shown in table 2.
The centrichromatography colloid gold immune detection dry method cleaning experiment result of the present invention of table 2（Unit：Chromatic value）
Experiment three, centrichromatography colloid gold immune detection method B of the present invention experiments
Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described B.
First, experiment material
NHS activated biotins（SIGMA）, Avidin（SIGMA）, it is other with experiment one.
Above-mentioned material corresponds to that Patent Application Request content is as follows, and this bioorganism element is intermediate material A, Avidin is middle
Substance B, it is other with experiment one.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one.
Anti-human myoglobins polyclonal antibody biotin labeling：Anti-human myoglobins polyclonal antibody is taken in 0.1M pH9.5
Dialysed overnight in sodium carbonate buffer, adjust final concentration of 2mg/ml.NHS activated biotins 20mg is taken to be dissolved in 1ml dimethyl methyls
Acid amides, 50ul is taken, be added in above-mentioned solution, reacted at room temperature 4 hours.Reaction solution is dialyzed overnight in PBS, -20 DEG C
Preserve.
Colloidal gold particles mark：With experiment one.
Colloidal gold particles mark absorption film preparation：Colloidal gold particles labelled antibody solution is taken, with colloid buffer solution（1%BSA,
3% sucrose, 50mM PBS, pH7.4）It is 30 to be diluted to OD530, adds the anti-human myoglobins polyclonal antibody 10 of biotin labeling
μ g/ml, mix.Other same experiments one.
It is prepared by Avidin die：Avidin solution is taken, it is dense to be diluted to 1mg/ml with 50mM phosphate buffers (pH 7.4)
Degree.Start die instrument, load avidin solution, it is other with experiment one.
Semi-finished product assemble method：Starting dehumidifier makes the humidity in operating room be reduced to less than 25%, in Avidin die two
Water suction paper membrane pad and colloidal gold particles mark adsorbed film are pasted in end respectively, other with experiment one.
Experimental group：With experiment one.
Control group：With experiment one.
3rd, experimental result
The present invention is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described A, as a result, the present invention is real
Test group test strips single detection process time average out to 3.6 minutes, control test group（Do not do centrifugal treating）Test strips single is examined
Survey processing time average out to 37 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-chromatic value, work as inspection
When survey higher limit is set to 150 ng/ml, correlation coefficient r 0.989, linear detection range is 3-150 ng/ml；Control group detects
Correlation r when higher limit is set to 100 ng/ml is 0.991, has reached the testing requirements more than 0.98, and linear detection range is
3-100 ng/ml.The ratio of experimental group 10ng/ml of the present invention and 3ng/ml detection data is 2.23, and control group is only 1.52.
Non-specific binding of the above-mentioned difference mainly due to colloidal gold particles on immobilon-p can not be effectively cleaned, and cause background mistake
It is high relevant.Experimental result illustrates that the technology of the present invention not only shortens detection time, while improves the linear of prior art detection
And accuracy.Experimental result is as shown in table 3.
The centrichromatography colloid gold immune detection method B experimental results of the present invention of table 3（Unit：Chromatic value）
Experiment four, influence of the centrifugal speed of the present invention to testing result
Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described A.
First, experiment material
With experiment one.
2nd, experimental method
Control group is not set, experimental group human muscle hemoglobin solution sample introduction uses 500,1000,2000,3000,4000,5000 revs/min
Centrifugation, 1 minute is stood after point sample and is centrifuged, 2000 revs/min of centrifugations of cleaning are other same to test one.
3rd, experimental result
This experiment is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described A, observes different centrifugation speed
Spend the influence to testing result.Experiment have detected the human muscle hemoglobin sample of various concentrations, and experimental result is as shown in table 4.By table 4
Understand, detection accuracy is relevant with centrifugal speed, and 500,1000,2000 revs/min of centrifugal speed obtains satisfactory detection
As a result.3000th, the testing result that 4000,5000 revs/min of centrifugal speed obtains, chromatic value significantly decrease, influence to detect
Sensitivity and accuracy.Illustrate that the optimal centrifugal speed of present invention progress myoglobins detection should be below 2000 revs/min.
Influence of the centrifugal speed of table 4 to testing result（Unit：Chromatic value）
Experiment five, influence of the colloidal gold particles particle diameter of the present invention to testing result
Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described A.
First, experiment material
Colloidal gold particles solution（Particle diameter 20,30,40,80nm, Shenzhen Xin Bosheng biologies）, it is other with experiment one.
Above-mentioned material corresponds to that Patent Application Request content is as follows, and this experiment colloidal gold particles solution is colloidal gold particles, its
It is the same as experiment one.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one.
Colloidal gold particles mark：It is other with experiment one using the colloidal gold particles solution of outsourcing.
Colloidal gold particles mark absorption film preparation：With experiment one.
It is prepared by polyclonal antibody die：With experiment one.
Semi-finished product assemble method：With experiment one.
Experimental group：With experiment one, influence of the colloidal gold particles to testing result of different-grain diameter is tested.
3rd, experimental result
The present invention is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described A, observes different-grain diameter pair
The influence of experimental result.As a result, when particle diameter of the present invention is 20nm, detection sensitivity is less than 10nm/ml；Particle diameter be 30,40,80nm
When, 3nm/ml can be reached by detecting linear lower limit, but chromatic value reduces with the reduction of particle diameter, but particle diameter is in more than 30nm
Testing requirements can be met.Experimental result is as shown in table 5.
Influence of 5 diameter of particle of the present invention of table to testing result（Unit：Chromatic value）
Experiment six, the comparative experiments of time correlation of the present invention detection stability and existing detection technique
Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described A.
First, experiment material
With experiment one.
2nd, experimental method
The preparation of human muscle hemoglobin solution：The human muscle hemoglobin solution of concentration known is taken, uses sample dilution buffer（1%BSA,
100mM glycine, 50mM PBS, 150mM NaCl, pH7.4）Dilution configuration 50ng/ml human muscle hemoglobin solution.
Colloidal gold particles mark：With experiment one.
Colloidal gold particles mark absorption film preparation：With experiment one.
It is prepared by polyclonal antibody die：With experiment one.
Semi-finished product assemble method：With experiment one.
Experimental group one：With experiment one, cleaning fluid eccentric cleaning is completed, test strips is taken out, is positioned over collaurum and quantitatively chromatographs
Chromatic value is read on analyzer, was read once every two minutes, it is continuous to read five times, test in triplicate, results averaged.
Experimental group two：With experiment one, dry-type centrifugal cleaning is completed（Without cleaning fluid）, test strips are taken out, are positioned over colloid
Gold is quantified and reads chromatic value on chromatographic analysis instrument, is read once every two minutes, continuous to read five times, and experiment in triplicate, is tied
Fruit is averaged.
Control group：With experiment one, 20 minutes are stood after point sample, takes out test strips, is positioned over the quantitative chromatographic analysis of collaurum
Chromatic value is read on instrument, was read once every two minutes, it is continuous to read five times, test in triplicate, results averaged.
3rd, experimental result
This experiment is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described A, after cleaning is completed in observation
The stability that chromatic value extends with standing time, experimental result are as shown in table 6.Experimental group cleaning fluid eccentric cleaning of the present invention and dry
Formula cleaning detection data are within 8 minutes Continuous Observation phases afterwards, and its result is basicly stable, and current art control group testing number
According within 8 minutes Continuous Observation phases afterwards, its result increases as time went on.Illustrate that the technology of the present invention can improve experiment
The stability of detection.
6 time correlation of the present invention of table detects the comparative experiments of stability and existing detection technique（Unit：Chromatic value）
Experiment seven, the of the invention and comparison of existing dot immune gold filtration assay testing result
Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described A.
First, experiment material
Sheep anti-mouse igg polyclonal antibody（Genagates companies of the U.S.）, it is other with experiment one.
2nd, experimental method
1st, standard curve is made：Take concentration known human muscle hemoglobin solution 3,10,30,70,100,150,200ng/ml people's flesh
Hemoglobin solution, of the invention and existing dot immune gold filtration assay is respectively adopted and detects and draws standard curve.With concentration known
Human muscle hemoglobin 50ng/ml is as sample to be checked.
2nd, existing dot immune gold filtration assay group：
Colloidal gold particles mark：With experiment one.
Colloidal gold particles mark absorption film preparation：With experiment one.
It is prepared by polyclonal antibody die：According to the antibody die method of experiment one sheep anti mouse is printed in the distal end of immobilon-p
IgG polyclonal antibodies, as nature controlling line（C lines）, anti-human myoglobins polyclonal antibody is printed in the proximal part of immobilon-p, as
Detection line（T lines）, it is other with experiment one.
Semi-finished product assemble method：Colloidal gold labeled monoclonal antibody adsorbed film is pasted on tip side, and absorbing membrane pad is pasted on C line sides, its
It is the same as experiment one.
The test strips of above-mentioned preparation are taken, are placed in desktop, marks to be added dropwise on adsorbed film to colloidal gold particles and prepares sample to be checked
Solution 80ul, 20 minutes are stood, be placed on the quantitative chromatographic analysis instrument of collaurum and read detection line T on polyclonal antibody printed film
With nature controlling line C chromatic value, and T/C ratios are calculated, reference standard curve calculates sample myoglobin concentration to be checked, and experiment repeats
Three times, results averaged.
3rd, detection technique group of the present invention：Material and method are the same as above-mentioned experiment two.The test strips of above-mentioned preparation are taken, with colloid
The side of golden particulate labelled antibody adsorbed film upward, is placed in centrifuge rotor, on colloidal gold particles labelled antibody adsorbed film
Sample solution 80ul to be checked is added dropwise, stands 2 minutes, 1000 revs/min centrifuge 1 minute, then are adsorbed to colloidal gold particles labelled antibody
PH7.4 0.05% Tween-20,50mM PBSs 80ul are added dropwise on film, 2000 revs/min of centrifugations are cleaned for 30 seconds, take out test paper
Bar, it is positioned on the quantitative chromatographic analysis instrument of collaurum and reads detection line T and nature controlling line C colourity on polyclonal antibody printed film
Value, and T/C ratios are calculated, reference standard curve calculates sample myoglobin concentration to be checked, and experiment in triplicate, is as a result averaged
Value.
3rd, experimental result
For the technology of the present invention through operating as above, standard curve is linearly good, and the correlation coefficient r value in 3-150ng/ml sections is 0.989,
Sample measure has been carried out immediately, three times the ng/ml of empirical average 51, detection error is within 10%.Determined using prior art, point
20 minutes of existing Product checking are stood after sample, standard curve is linearly good, and the correlation coefficient r value in 3-100ng/ml sections is
0.991, carried out sample measure according to the condition of 20 minutes immediately, average value is 52ng/ml three times, detection error 10% with
It is interior.The concrete outcome for repeating experiment three times is as shown in table 7.Compared with the prior art, the present invention significantly shorten detection time.
The present invention of table 7 and the Analysis of test results of dot immune gold filtration assay（Unit：ng/ml）
| Repeat 1 | Repeat 2 | Repeat 3 | Average value | |
| The present invention | 52 | 48 | 53 | 51 |
| Prior art | 47 | 58 | 55 | 53 |
Experiment eight, influence of the cleaning step of the present invention to testing result
Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described A.
First, experiment material
With experiment one.
2nd, experimental method
Using the human muscle hemoglobin 50ng/ml of concentration known as sample to be checked, control is not cleaned, and stands chromatography；Experiment using 0,
40th, 80,120,160ul cleanings, it is other with experiment one.
3rd, experimental result
Experimental result is as shown in table 8.Two test results compare, and it is non-specific on immobilon-p that cleaning can reduce colloidal gold particles
Property combine, and centrichromatography cleaning effect it is especially pronounced.Be not added with cleaning fluid do not do centrifugal treating background chromatic value be 69.2,
The background chromatic value of centrifugal treating is 60.1.80ul eccentric cleanings make immobilon-p background fall below 52.6 from 60.1, obtain good
Good background experimental result.Illustrate test sample introduction after with cleaning the step of it is meaningful.
Influence of 8 cleaning step of the present invention of table to testing result（Unit：Chromatic value）
Claims (13)
1. a kind of centrichromatography colloid gold immune detection combination suit, is characterised by, the detection combination suit contains thing to be checked
Specificity junction mixture, colloidal gold particles, immobilon-p, centrifugal device and collaurum detector, wherein described colloidal gold particles
Non-specific binding can be directly formed with protein for what is prepared using gold solution and maintain stable particle；The immobilon-p
For with the film quality material with protein non-specific binding characteristic；The centrifugal device drives liquid phase described for that can centrifuge
The structure of chromatographic flow on immobilon-p；The thing specificity junction mixture to be checked be the antigen with specific binding capacity, antibody,
At least one of Avidin, biotin and the like；The collaurum detector is to pass through the colourity to collaurum color
Analyze the detector quantitatively or semi-quantitatively detected.
2. the detection method that detection combination described in a kind of claim 1 is set with, it is characterised in that：The detection method is as follows
（A）-（B）In it is any：
（A）Comprise the following steps：
1）Colloidal gold particles solution is prepared with gold solution；
2）The colloidal gold particles described in the thing specific binding substance markers to be checked；
3）Reacted with the colloidal gold particles after the sample containing thing to be checked and the mark, the thing to be checked by with positioned at described
The thing specificity junction mixture to be checked on colloidal gold particles after mark combines, and then forms " colloidal gold particles-thing to be checked spy
Specific binding agent-thing to be checked " compound, i.e. compound 1；
4）Coating can be by forming the of specific binding with the thing to be checked on compound 1 on the immobilon-p
Two thing specificity junction mixtures to be checked；
5）With the liquid phase containing the compound 1, by chromatography, to flow through coated second thing to be checked on the immobilon-p special
Property conjugate, it is multiple to form " colloidal gold particles-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked "
Compound, i.e. compound 2, and be captured and be fixed on the immobilon-p；
6）The compound 1 being not associated with the immobilon-p and the glue of residual are cleaned with cleaning fluid using centrifugal chromatography
Body gold material；
7）The institute that the immobilon-p after cleaning is indirectly fixed on the immobilon-p with collaurum detector detection
The chromatic value of colloidal gold particles is stated, to detect the content of the thing to be checked；
（B）Comprise the following steps：
1）Colloidal gold particles solution is prepared with gold solution；
2）The colloidal gold particles described in the thing specific binding substance markers to be checked；
3）Reacted with the colloidal gold particles after the sample containing thing to be checked and the mark, the thing to be checked by with positioned at described
The thing specificity junction mixture to be checked on colloidal gold particles after mark combines, and then forms " colloidal gold particles-thing to be checked spy
Specific binding agent-thing to be checked " compound, i.e., described compound 1；
4）The second thing specificity junction mixture to be checked is marked with intermediate material A；
5）Coating can form the intermediate material B of specific binding with the intermediate material A on the immobilon-p；
6）The the second thing specificity junction mixture to be checked marked with the liquid phase containing the compound 1 with the intermediate material A is carried out
Reaction, form " colloidal gold particles-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture-intermediate to be checked
Matter A " compounds, i.e. compound 3；
7）The coated intermediate material B on the immobilon-p is flowed through by chromatography with the liquid phase containing the compound 3, formed
" colloidal gold particles-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked-intermediate material A- intermediates
Matter B " compounds, i.e. compound 4, and be captured and be fixed on the immobilon-p；
8）The compound 3 being not associated with the immobilon-p and the glue of residual are cleaned with cleaning fluid using centrifugal chromatography
Body gold material；
9）The institute that the immobilon-p after cleaning is indirectly fixed on the immobilon-p with collaurum detector detection
The chromatic value of collaurum material is stated, to detect the content of the thing to be checked.
3. detection method according to claim 2, it is characterised in that：The method of the cleaning immobilon-p, which uses, to be not added with
The dry method eccentric cleaning of the centrichromatography mode of cleaning fluid.
4. detection method according to claim 2, it is characterised in that：The intermediate material is with specific binding capacity
Antigen, antibody, Avidin, at least one of biotin and the like.
5. detection method according to claim 2, it is characterised in that：The reaction that the chromatography flows through the immobilon-p exists
Completed on centrifugal device and using centrifugal chromatography driving liquid chromatographic flow on immobilon-p.
6. the detection method described in combined complete according to claim 1 or claim 2, it is characterised in that：The glue
The particle diameter selection 10nm-200nm of body gold particulate.
7. detection combination suit according to claim 1, it is characterised in that：The rotating speed of the centrifugal device uses program control
Mode processed；The rotating speed of the centrifugal device is 200 ~ 10000 revs/min.
8. detection method according to claim 2, it is characterised in that：The rotating speed of the centrifugal chromatography uses programme-control
Mode；The rotating speed of the centrichromatography is 200 ~ 10000 revs/min.
9. the detection method described in combined complete according to claim 1 or claim 2 or 5, it is characterised in that：It is described
Immobilon-p includes nitrocellulose filter, polyvinylidene fluoride film, nylon membrane, DEAE cellulose membranes.
10. detection combination suit according to claim 1, it is characterised in that：The detection combination suit is also comprising
Cleaning fluid is stated, the cleaning fluid is the normal experiment cushioning liquid containing surfactant.
11. detection method according to claim 2, it is characterised in that：The cleaning fluid is to contain the normal of surfactant
Advise experiment cushioning liquid.
12. detection combination suit according to claim 1, it is characterised in that：Detection combination suit also includes the
At least one of two thing specificity junction mixtures to be checked, intermediate material A, intermediate material B.
13. the answering in immune detection product development of the combined complete or detection method any one of claim 1-12
With.
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| CN201610561226.8A CN107561281A (en) | 2016-07-16 | 2016-07-16 | A kind of centrichromatography colloid gold immune detection technique and application thereof |
| PCT/CN2017/086044 WO2017206800A1 (en) | 2016-05-31 | 2017-05-26 | Centrifugal chromatography immunoassay method |
| US16/206,726 US20190170735A1 (en) | 2016-05-31 | 2018-11-30 | Immuno chromatography method with centrifuge isolation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107525923A (en) * | 2017-04-01 | 2017-12-29 | 北京康华源科技发展有限公司 | One kind centrifuges immunochromatography detection method and device |
| CN111337689A (en) * | 2020-04-03 | 2020-06-26 | 山西医科大学 | Novel coronavirus detection kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009039239A2 (en) * | 2007-09-18 | 2009-03-26 | Idexx Laboratories, Inc. | Lateral flow assay using centrifugal force |
| CN101520454A (en) * | 2008-02-04 | 2009-09-02 | 深圳微点生物技术有限公司 | Centrifugal fluid analyzer rotor |
| US20130302830A1 (en) * | 2010-06-17 | 2013-11-14 | Rajesh K. Mehra | Rotors for immunoassays |
| CN104020286A (en) * | 2014-06-10 | 2014-09-03 | 武汉璟泓万方堂医药科技股份有限公司 | Quantitative detection instrument and detection method of immunochromatography test strip |
| CN204422537U (en) * | 2015-02-13 | 2015-06-24 | 南京天纵易康生物科技有限公司 | With the immunochromatographiassays assays instrument of centrifugal device |
| CN105353159A (en) * | 2014-08-20 | 2016-02-24 | 余波 | Microfluidic test device and method for operating the same |
| CN107192819A (en) * | 2016-03-14 | 2017-09-22 | 北京康华源科技发展有限公司 | One kind centrifuges detection method |
-
2016
- 2016-07-16 CN CN201610561226.8A patent/CN107561281A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009039239A2 (en) * | 2007-09-18 | 2009-03-26 | Idexx Laboratories, Inc. | Lateral flow assay using centrifugal force |
| CN101520454A (en) * | 2008-02-04 | 2009-09-02 | 深圳微点生物技术有限公司 | Centrifugal fluid analyzer rotor |
| US20130302830A1 (en) * | 2010-06-17 | 2013-11-14 | Rajesh K. Mehra | Rotors for immunoassays |
| CN104020286A (en) * | 2014-06-10 | 2014-09-03 | 武汉璟泓万方堂医药科技股份有限公司 | Quantitative detection instrument and detection method of immunochromatography test strip |
| CN105353159A (en) * | 2014-08-20 | 2016-02-24 | 余波 | Microfluidic test device and method for operating the same |
| CN204422537U (en) * | 2015-02-13 | 2015-06-24 | 南京天纵易康生物科技有限公司 | With the immunochromatographiassays assays instrument of centrifugal device |
| CN107192819A (en) * | 2016-03-14 | 2017-09-22 | 北京康华源科技发展有限公司 | One kind centrifuges detection method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107525923A (en) * | 2017-04-01 | 2017-12-29 | 北京康华源科技发展有限公司 | One kind centrifuges immunochromatography detection method and device |
| CN111337689A (en) * | 2020-04-03 | 2020-06-26 | 山西医科大学 | Novel coronavirus detection kit |
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