CN109387628A - It is centrifugated detection method - Google Patents
It is centrifugated detection method Download PDFInfo
- Publication number
- CN109387628A CN109387628A CN201811176124.XA CN201811176124A CN109387628A CN 109387628 A CN109387628 A CN 109387628A CN 201811176124 A CN201811176124 A CN 201811176124A CN 109387628 A CN109387628 A CN 109387628A
- Authority
- CN
- China
- Prior art keywords
- detection
- checked
- immobilon
- film
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of centrifuge separation detection methods.It includes the following steps: to flow through immobilon-p using centrifugal device driving liquid phase, the liquid phase includes the measuring samples containing object to be checked and detection phase, and the detection is mutually the substance containing the detection indicator that can directly or indirectly form specific binding with the object to be checked；When the liquid phase flows through immobilon-p, the compound of detection indicator-object-to be checked object specificity junction mixture to be checked is formed in conjunction with object specificity junction mixture to be checked coated on the immobilon-p, the compound is captured to be fixed on the immobilon-p, the amount for the detection indicator that detector is indirectly fixed on the immobilon-p by detection, to detect the content of the object to be checked.The present invention has the characteristics that high sensitivity, detection time are short.
Description
It is on 03 14th, 2016 that the application, which is application No. is the 201610143694.3, applying date, invention and created name is
The divisional application of " a kind of centrifuge separation detection method "
Technical field
The present invention relates to a kind of centrifuge separation detection methods, belong to technical field of immunoassay.
Background technique
Immunology detection technology is measurement antigen, antibody, immunocyte and the chemical component 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.There are commonly immune turbidity technology, solid-phase enzyme immunoassay technology, change
Learn luminescent detection techniques, immunofluorescence label technology, flow cytometry, colloidal gold technique etc..
Immune turbidity technology, also referred to as immune turbidimetry is soluble antigen, antibody specific bond in the liquid phase, is generated certain
The compound of size forms the refraction or absorption of light, and the transmitted light or scattering light after measuring this refraction or absorption are used as and calculate
Unit is used for quantitative detection, but detection sensitivity is low, is not suitable for trace detection.Solid-phase enzyme immunoassay technology is based on antigen
Or the immobilization and antigen of antibody or the enzyme of antibody mark, the antigen or antibody for being incorporated in surface of solid phase carriers keep its immunology
The enzyme conjugates of activity, antigen or antibody had not only retained its immunologic competence, but also retained the activity of enzyme, in measurement, being marked by inspection
This (measuring antibody or antigen therein) and enzyme-labelled antigen or antibody by different step and surface of solid phase carriers antigen or resist
Body reacts, and has the remarkable advantages such as high sensitivity, linear response range be wide and easy to automate, but detect the reaction time
Length limits its use.Immunochemiluminescence detection technique is a kind of highly sensitive micro and Analytical Methods of Trace, has operation
The remarkable advantages such as convenience, high sensitivity, linear response range be wide and easy to automate, be widely used in environment, clinic,
In Pharmaceutical Analysis, food and Industrial Analysis, it is also based on the solid phase separation means and antigen of antigen or antibody or shining for antibody
Reagent labelling technique, but the detection reaction time is long and the requirement height on detection device also influences its use.Immunofluorescence label skill
Art, flow cytometry, colloidal gold technique are also that common detection technique is widely used, but has it corresponding insufficient, are detected
Reaction time is long or sensitivity, accuracy shortcoming are generally existing deficiencies.
It is highly sensitive, quickly, miniaturization, Quan Dingliang, automation be that the development of current clinical immunization detection technique product becomes
Gesture, but existing all not can be implemented simultaneously above-mentioned function.
Summary of the invention
The object of the present invention is to provide a kind of centrifuge separation detection methods.The present invention has high sensitivity, detection time short
The characteristics of.
Centrifuge separation detection method provided by the invention includes the following steps: to flow through using centrifugal device driving liquid phase solid
Phase film, the liquid phase include the measuring samples containing object to be checked and detection phase, it is described detection mutually for containing can with it is described to be checked
Object directly or indirectly forms the substance of the detection indicator of specific binding；When the liquid phase flows through immobilon-p, with the solid phase
Coated object specificity junction mixture combination to be checked forms detection indicator-object-to be checked object specificity junction mixture to be checked and answers on film
Object is closed, the compound is captured to be fixed on the immobilon-p, and detector is indirectly fixed to the immobilon-p by detection
On detection indicator amount, that is, detect the content of the object to be checked.
In above-mentioned method, in the device that the method uses, the rotary part of the centrifugal device using plane or
Type is inclined outwardly by center.
In above-mentioned method, in the device that the method uses, set in the middle part of the rotary part plane of the centrifugal device
Have: the measuring samples detect mutually respective storage device, the sampling device of the measuring samples and the detection phase with described
And driving device；
The outer of the rotary part plane of the centrifugal device is equipped with the apparatus for placing of the immobilon-p.
In the present invention, the sample introduction proximal end of the immobilon-p is connect with liquid adsorption dispersal device, and sample introduction distal end is inhaled with liquid
Adsorption device connection；；
The liquid adsorption dispersal device uses the solid phase material with quick liquid dispersing characteristic, the solid phase material
For multi-polyester cellulose membrane and/or glass fibre element film, concretely colloid gold label adsorbed film, fluorescent labeled antibody are adsorbed
Film, chemiluminescent labeling adsorbed film and/or dispersion membrane；The liquid adsorption device uses water-absorbing material, the water imbibition material
Material is blotting paper and/or water absorbent gel.
In above-mentioned method, in the device that the method uses, the detector is set to the side or two of the immobilon-p
Side.
In above-mentioned method, the sample introduction speed of the revolving speed of the centrifugal device and the sampling device is all made of process control
Mode；
The revolving speed of the centrifugal device be 200~10000r/min, concretely 500~5000r/min, 800~
3000r/min or 800~2000r/min.
In above-mentioned method, the immobilon-p is nitrocellulose filter, polyvinylidene fluoride film, nylon membrane, DEAE cellulose
Film, and the one or both sides of the immobilon-p have backing；
The detector includes any in absorbance, fluorescence, chemiluminescence and the detector of color of image digital processing
Kind.
In the present invention, the polyvinylidene fluoride film abbreviation pvdf membrane；
The DEAE cellulose membrane refers to that manufactured paper-like is thin after diethyl amino ethyl group (DEAE) is introduced cellulosic molecule
Film is a kind of weak base type anion-exchange material.
In above-mentioned method, the object to be checked is to generate immune live with immunocompetent protein or with protein coupling
The substance of property；
The object specificity junction mixture to be checked is the antigen or antibody with the object specific binding to be checked；
The detection indicator is at least one of colloidal metal, dyestuff, fluorescein and chemiluminescent substance.
In above-mentioned method, the colloidal metal is at least one of colloidal gold, electroselenium and colloid gold-magnetic particles；
The fluorescein is fluorescein isothiocynate (abbreviation FITC), RB 200, tetramethyl isothiocyanate Luo Dan
Bright, phycoerythrin (PE), perdinin phyllochlorin (PerCP), propidium iodide (PI), other phycocyanin (APC) and europium
At least one of compound, wherein europium compound concretely europium oxide；
The chemiluminescent substance is luminol and different luminol and its derivative species, acridinium ester and a word used for translation shallow lake amides, (gold
Steel alkane) -1,2- dichloroethane and its at least one of derivative and tris (bipyridine) ruthenium.
In above-mentioned method, the compound is formed with following any:
1) the object specificity junction mixture to be checked includes while mutually forming specificity with the object to be checked and the detection
In conjunction with level-one interphase；
2) the object specificity junction mixture to be checked includes while mutually being formed with the level-one interphase and the detection special
The second level interphase that the opposite sex combines；
The level-one interphase and the second level interphase be antigen with specific binding capacity, antibody, affine
At least one of element, biotin and the like；
Further include the steps that mutually cleaning the immobilon-p with cleaning after the association reaction in the method, it is described clear
It washes mutually as at least one of phosphate buffer, carbonate buffer solution and Tris buffer.
The present invention is centrifugated detection method and is applied in the content detection of immunoassay product.
The invention adopts the above technical scheme, which has the following advantages:
1, the present invention is flowed and is cleaned on immobilon-p using the liquid phase of centrifugal device driving detection, improves object to be checked
Capture binding ability, reduce the ambient noise interference of immobilon-p, improvement method detection sensitivity realizes with existing detection
The highly sensitive detection of reagent.
2, the present invention is flowed on immobilon-p using the liquid phase of centrifugal device driving detection, changes existing film detection skill
Art by nature flow and liquid with the extension of process on film reduced status, be able to maintain liquid and at the uniform velocity flowed on film
It is dynamic, it ensure that the homogeneity that object to be checked combines on film, detection accuracy can be improved.
3, existing film detection technique is flowed by nature, and flowing velocity of the liquid on film slows down with the time, is completed
One detection generally requires 15 minutes or more time.And the present invention is flowed on immobilon-p using centrifugal device driving detection liquid phase
It is dynamic, it maintains liquid and is at the uniform velocity flowed on film, it is quick to shorten detection time.
4, existing highly sensitive detection technique, is all made of multi-step, too many levels drive control, is related to detecting sample, inspection
Survey the transposition and movement of phase and reaction carriers.And the present invention is using centrifugal device driving liquid phase flowing and sampling pump sample introduction, behaviour
It is simple to make step.
5, operation of the present invention step is simple, it is easy to accomplish automatic operation.The method of the present invention has highly sensitive, Quan Ding
Amount, automation the characteristics of, while again have detection quickly, use the simple detection technique of equipment；Not only easy to use, reduction original
The waste of material, while working efficiency is also significantly improved, applied to detection and analysis, the numerous areas of separation.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
Material, reagent etc., are commercially available unless otherwise specified.
Embodiment 1, the present invention are compared with the detection accuracy of existing detection technique
One, experimental material
Anti-human myoglobins polyclonal antibody (Genagates company, the U.S., catalog number GP301042) is anti-human
Myoglobins monoclonal antibody (Genagates company, the U.S., article No. GP300616), spectrophotometer (Shanghai mountain valley with clumps of trees and bamboo China Tech skill instrument
Device Co., Ltd, 752 ultraviolet-uisible spectrophotometers), (Sigma-Aldrich product, catalog number are human muscle hemoglobin
F3879-1G), BioFlow die instrument (IMAGENE company, the U.S.), Index cutting machine (A-point company, the U.S.), DBF-900
Sealing machine (Wenzhou Jiangnan packing factory), ACBO dehumidifier (jiangsu wuxi Ao Bo dehumidifier company), the desk centrifuge (U.S.
Eppendoff company), bovine serum albumin(BSA) (abbreviation BSA, SIGMA product, article No.: B8894), nitrocellulose diaphragm (AE
99, provided by Genagates company, the U.S.), multi-polyester cellulose membrane (Reemay 2033, U.S.'s Alstrom Products),
It absorbs water paper membrane pad (Grade 470, U.S.'s S&S Products), gold chloride (SIGMA product, article No.: B8894), colloidal gold is quantitative
Chromatographic analysis instrument (Norway's Skannex product).
Two, experimental method
The preparation of human muscle hemoglobin solution: taking the human muscle hemoglobin solution of known concentration, with sample dilution buffer (1%
BSA, 100mM glycine, 50mM PBS, 150mM NaCl, pH7.4) dilution configuration 3.125,6.25,12.5,25,50,
The serial human muscle hemoglobin solution of 100ng/ml.
The preparation of the anti-human myoglobins monoclonal antibody of colloid gold label: 10ml pure water, heating stirring, to water boiling are taken
When be added 500 μ l, 10% chlorauric acid solution, heating is boiled 5 minutes, and 500 μ l, 12% citric acid three sodium solution is added, keeps this
Solution stirring boiling 10 minutes, it is naturally cooling to room temperature, i.e. colloidal gold solution.Colloidal gold solution volume 10ml is taken, with 10% carbon
Sour potassium tune pH to 8.3 is rapidly added anti-human 100 μ g of myoglobins monoclonal antibody, until 10 μ g/ml final concentrations, it is mixed to shake beaker
It is even, 30 minutes are placed at room temperature for, 10% bovine serum albumin solution 100ul is rapidly added, makes final concentration of 1%, while shaking burning
Cup, is placed at room temperature for 30 minutes, and 12000rpm is centrifuged 20 minutes, and supernatant is carefully sucked out；Add 5ml 50mM phosphate (PBS)
Buffer, pH7.4 suspend precipitating, and 12000rpm is centrifuged 20 minutes, and supernatant is sucked out, precipitating is dissolved in 1.0ml and contains 1%
Bovine serum albumin(BSA) and 3% sucrose phosphate buffer in, 4 DEG C are kept in dark place.
Colloid gold label adsorbs film preparation: preparing and contains 0.5%PVA (i.e. polyvinyl alcohol), 50mM PBS liquid, 0.5%
BSA, 0.88%NaCl, the multi-polyester cellulose membrane pretreatment fluid of pH 7.4 set multi-polyester cellulose membrane to be processed in pre- place
It manages in liquid, soaking at room temperature 1 hour, takes the film out, set and seal after 37 DEG C of dryings spare, can also be used directly as dispersion membrane.It takes
Colloidal gold labeled monoclonal antibody solution, being diluted to OD530 with colloid buffer (1%BSA, 3% sucrose, 50mM PBS, pH7.4) is
30, start die instrument, load antibody, open pressurized nitrogen, take multi-polyester cellulose membrane, start die, sets die condition are as follows:
Airbrush movement speed 30mm/ seconds, the film after printing was put into drying box by 3.0 μ l/cm of liquid fltting speed, and 37 DEG C of dryings 6 are small
When, it is subsequently placed in the hermetic bag containing desiccant and saves use.Colloid gold label adsorbed film and dispersion membrane are also the present invention simultaneously
The liquid adsorption dispersal device.
The preparation of polyclonal antibody die: anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffer (pH
7.4) it is diluted to 1mg/ml concentration.Start die instrument, loads antibody, take PVC piece (the i.e. polyvinyl chloride for posting nitrocellulose filter
Piece), start die, sets die condition are as follows: airbrush movement speed 30mm/ seconds, 0.5 μ l/cm of liquid fltting speed.It will produce
Film be put into 37 DEG C of drying boxes, dry 6 hours, then film is placed in save in the drying receptacle containing desiccant and used.
Semi-finished product assemble method: starting dehumidifier makes to operate indoor humidity and is reduced to 25% hereinafter, in polyclonal antibody
Water suction paper membrane pad and colloid gold label adsorbed film are pasted in die both ends respectively, then use Pressure sensitive adhesive tape sealing label surface.It sets and pastes
Detection lug on cutting machine, being cut into 3.5mm test strips.Paper slip is put into the aluminium amber hermetic bag of desiccant, in sealing machine
Upper sealing, labelling.
The test strips of above-mentioned preparation are taken, upward with the side of colloid gold label adsorbed film, being placed in centrifuge rotor, (extroversion is inclined
Oblique type) in, the human muscle hemoglobin solution 80ul of the various concentration of preparation is added dropwise on colloid gold label adsorbed film, stands 1-15 points
Clock, 2000 revs/min are centrifuged 1 minute, then the 50mM PBS buffer solution 80ul of pH7.4 is added dropwise on colloid gold label adsorbed film,
2000 revs/min are cleaned for centrifugation 1 minute, are taken out test strips, are placed in colloidal gold and quantify and read on chromatographic analysis instrument (i.e. detector)
The digital picture of polyclonal antibody trace band carries out image procossing and obtains corresponding chroma value.Control stripes item do not do from
Heart processing, after the above-mentioned identical time of repose of setting, then stands 2.5 minutes, then reads corresponding chroma value.
In triplicate, results are averaged for experiment.The detected value of statistical calculations difference pre-dwell time test strips.
Three, experimental result
The present invention shows the correlation coefficient r of the technology of the present invention detection using colloidal gold as the detection technique measurement result of indicator
Average value is 0.9884, and the correlation coefficient r that the prior art detects (not doing centrifugal treating) is 0.957, P < 0.05, is significantly better than
The testing result of the prior art illustrates that the technology of the present invention improves the accuracy of prior art detection.Experimental result such as 1 institute of table
Show.
1 present invention of table is using colloidal gold as the testing result analysis of the accuracy (unit: pattern colour angle value) of indicator
Embodiment 2, the present invention are compared with the limit of identification of existing detection technique
One, experimental material
With embodiment 1
Two, experimental method
With embodiment 1
Three, experimental result
For the present invention using colloidal gold as the measurement result of indicator, analysis develops common correlation coefficient r using Related product
Requirement of the value greater than 0.98 has carried out statistical disposition to data, and minimum value when being greater than 0.98 with r value is set to limit of identification.Knot
Under the same experiment condition of fruit, the pre-dwell time 1-15 minutes, use the limit of identification of the prior art for 25 or > 25ng/
Ml is 3.125ng/ml using limit of identification of the invention, and detection sensitivity is significantly higher than the prior art, illustrates the present invention
Technology improves the detection sensitivity of prior art detection.Experimental result is as shown in table 2.
2 present invention of table analyzes (unit: pattern colour angle value) by the detection sensitivity of indicator of colloidal gold
The influence of embodiment 3, the present invention to detection specificity
One, experimental material
With embodiment 1
Two, experimental method
With embodiment 1
Specific detection sample used is A:50ng/ml myoglobins, B:10ng/ml Troponin I, C:30ng/ml flesh
Acid kinase isodynamic enzyme, D:80mg/ml human serum albumins, E:20mg/ml cholesterol.
Three, experimental result
For the present invention using colloidal gold as the above-mentioned specific detection sample of the detection of indicator, experimental result is as shown in table 3, existing
It is respectively 50.3 and 51.0ng/ml that technology and the technology of the present invention, which repeat detection average value to myoglobins sample, other not contain
The detected value of the sample of myoglobins detection method detection sensitivity lower limit hereinafter, be feminine gender, and without apparent colour developing
Reaction.
3 present invention of table is compared with the result of current art detection myoglobins specificity (unit: ng/ml)
Embodiment 4, the present invention are compared with the detection performance of existing chemiluminescence detection technology
One, experimental material
Anti-human myoglobins polyclonal antibody (Genagates company, catalog number GP301042), horseradish peroxide
Compound enzyme marks anti-human myoglobins monoclonal antibody (Genagates company, the U.S., article No. GP300616), magnetic particle (MP-
COOH-20020, Zhengzhou Ying Nuo Biotechnology Co., Ltd), pico luminescence reagent (Thermo scientific), chemiluminescence
Detector (Promega, Glomax Multi JRDetectionSystem), human muscle hemoglobin (Sigma-Aldrich product,
Catalog number is F3879-1G), BioFlow die instrument (IMAGENE company, the U.S.), Index cutting machine (U.S. A-point
Company), DBF-900 sealing machine (Wenzhou Jiangnan packing factory), ACBO dehumidifier (jiangsu wuxi Ao Bo dehumidifier company) is desk-top
Centrifuge (Eppendoff company, the U.S.), bovine serum albumin(BSA) (SIGMA product, article No.: B8894), nitrocellulose diaphragm
(AE 99 is provided by Genagates company, the U.S.), (Reemay 2033, Alstrom company, the U.S. produce multi-polyester cellulose membrane
Product), it absorbs water paper membrane pad (Grade 470, U.S.'s S&S Products).
Two, experimental method
The preparation of human muscle hemoglobin solution: taking the human muscle hemoglobin solution of known concentration, with sample dilution buffer (1%
BSA, 100mM glycine, 50mM PBS, 150mM NaCl, pH7.4) dilution configuration 3.125,6.25,12.5,25,50,
100ng/ml human muscle hemoglobin solution.
1, existing chemiluminescence detection technology group
The label of magnetic particle, using conventional labels method, with the anti-human myoglobins polyclonal antibody of 1mg/ml to magnetic particle
It is marked, the amount ratio (w/w) of anti-human myoglobins polyclonal antibody and magnetic particle is 3:1.Each Concentration Testing takes three to put down
The 100 μ l of magnetic particle marked with anti-human myoglobins polyclonal antibody is added in row pipe, every pipe, then it is corresponding dense for being separately added into concentration
Each 100 μ l of the human muscle hemoglobin solution of degree, association reaction shake incubation at 37 DEG C 60 minutes, micro- with magnetic separator adsorbing separation magnetic
Grain abandons supernatant, and 200 μ l of PBS cleaning is added three times, with magnetic separator adsorbing separation magnetic particle, abandons supernatant, horseradish peroxide is added
Compound enzyme marks anti-human 200 μ l of myoglobins monoclonal antibody, and association reaction shakes incubation at 37 DEG C 60 minutes, uses magnetic separator
Adsorbing separation magnetic particle abandons supernatant, and 200 μ l of PBS cleaning is added three times, with magnetic separator adsorbing separation magnetic particle, abandons supernatant,
Magnetic particle is shifted to glow cup, sets chemiluminescence detector, 100 μ l luminous substrate working solutions are added, when reaction carries out 2 minutes,
Record luminous quantity 6 seconds.
2, of the present invention group
The pretreatment of multi-polyester cellulose membrane with embodiment 1, set seal after 37 DEG C of dryings it is spare.
Polyclonal antibody die is placed in the drying receptacle containing desiccant with embodiment 1 and saves use.
The preparation of chemiluminescent labeling adsorbed film: taking pretreated multi-polyester cellulose membrane, with 50mM PBS buffer solution (pH
7.4) the anti-human myoglobins monoclonal antibody 0.15mg/ml of horseradish peroxidase-labeled is diluted, die instrument is started, is loaded anti-
Body opens pressurized nitrogen, takes multi-polyester cellulose membrane, starts die, sets die condition are as follows: and airbrush movement speed 30mm/ seconds,
3.0 μ l/cm of liquid fltting speed, by after printing film be freeze-dried, set 4 DEG C be sealed it is spare.
Semi-finished product assemble method: starting dehumidifier makes to operate indoor humidity and is reduced to 25% hereinafter, in polyclonal antibody
Water suction paper membrane pad and chemiluminescent labeling adsorbed film are pasted in die both ends respectively, then use Pressure sensitive adhesive tape sealing label surface.Set stickup
Good detection lug is on cutting machine, being cut into 3.5mm test strips.Paper slip is put into the aluminium amber hermetic bag of desiccant, is being sealed
It is sealed on machine, labelling.
The test strips of above-mentioned preparation are taken, upward with the side of chemiluminescent labeling adsorbed film, are placed in centrifuge rotor, to
The human muscle hemoglobin solution 80ul of the various concentration of preparation is added dropwise on chemiluminescent labeling adsorbed film, stands 2 minutes, 2000 turns/
It separates the heart 1 minute, then the PBS buffer solution 80ul of pH7.4 is added dropwise on chemiluminescent labeling adsorbed film, 2000 revs/min are centrifuged 1 point
100 μ l luminous substrate working solutions are added dropwise to chemiluminescent labeling adsorbed film in clock cleaning, and 800 revs/min are centrifuged 30 seconds, from PVC egative film
On strip nitrocellulose filter, set chemiluminescence detector, record luminous quantity 6 seconds.
Three, experimental result
Use the present invention with chemiluminescence detection technology and existing chemiluminescence detection technology to human muscle hemoglobin solution into
Row detection, experimental result is as shown in table 4, as shown in Table 4, good concentration linear relationship, correlation coefficient r value point is both presented
It Wei 0.993 and 0.992.Illustrate that the present invention has detection effect similar with existing chemiluminescence, but significantly shortens
Detection time.
4 present invention of table is compared with the detection performance of existing chemiluminescence detection technology (unit: mV)
Embodiment 5, the present invention are compared with existing chemiluminescence detection technology testing result
One, experimental material
With embodiment 4.
Two, experimental method
Production standard curve: human muscle hemoglobin solution 3.125,6.25,12.5,25,50,100ng/ml of known concentration are taken
Human muscle hemoglobin solution, is respectively adopted the present invention and existing chemiluminescence detection technology detects and draws standard curve.With known
The human muscle hemoglobin 10ng/ml of concentration is as sample to be examined.Other methods are the same as embodiment 4.
Three, experimental result
The concrete outcome of three repeated experiments is as shown in table 5.Existing chemiluminescence detection technology measurement result shows to be checked
The content of sample human muscle hemoglobin is 9.52ng/ml, and measurement result of the present invention shows that the content of sample to be examined human muscle hemoglobin is
9.82ng/ml, two kinds of experimental method acquired results are almost the same, no difference of science of statistics (P > 0.05), but completion of the invention is real
Testing the time is significantly shorter than current art.
5 present invention of table is compared with existing chemiluminescence detection technology testing result (unit: ng/ml)
Embodiment 6, the present invention are compared with existing fluorescence immunoassay detection technique detection performance
One, experimental material
Anti-human myoglobins polyclonal antibody (Genagates company, catalog number GP301042), anti-human flesh are red
Protein monoclonal antibody (Genagates company, the U.S., article No. GP300616), fluorescent microsphere (fluorescein used be europium compound,
Article No. JY-SJ126, Shanghai outstanding person one biotech firm), EDC (Pierce product, article No. 22980), NHS (Pierce product, article No.
24500), human muscle hemoglobin (Sigma-Aldrich product, catalog number F3879-1G), quantitative fluorescence analysis instrument (Shanghai
Woman biotech firm, HG-98), BioFlow die instrument (IMAGENE company, the U.S.), (U.S. A-point is public for Index cutting machine
Department), DBF-900 sealing machine (Wenzhou Jiangnan packing factory), ACBO dehumidifier (jiangsu wuxi Ao Bo dehumidifier company), it is desk-top from
Scheming (Eppendoff company, the U.S.), bovine serum albumin(BSA) (SIGMA product, article No.: B8894), nitrocellulose diaphragm (AE
99, provided by Genagates company, the U.S.), multi-polyester cellulose membrane (Reemay 2033, U.S.'s Alstrom Products),
It absorbs water paper membrane pad (Grade 470, U.S.'s S&S Products).
Two, experimental method
The preparation of human muscle hemoglobin solution: taking the human muscle hemoglobin solution of known concentration, with sample dilution buffer (1%
BSA, 100mM glycine, 50mM PBS, 150mM NaCl, pH7.4) dilution configuration 3.125,6.25,12.5,25,50,
100ng/ml human muscle hemoglobin solution.
Fluorescent microsphere label: taking 0.5ml fluorescent microsphere, using 0.1M PB eccentric cleaning 4 times of PH7.2,13000rpm from
The heart is redissolved to 1ml with the 0.1M PB of pH7.2, the anti-human myoglobins monoclonal antibody of 150ug is added, mixed, be added pH7.2's
The EDC aqueous solution of the 40mg/ml of 250ul is added in 0.1M PB to 1.5ml, and the NHS aqueous solution of 250ul 40mg/ml is added, and mixes
It is even, it reacts at room temperature 60 minutes.The BSA of 20mg is added, mixes, reacts at room temperature 60 minutes.Centrifugation, which is inhaled, abandons supernatant, uses 0.05M
After Tris pH7.6 eccentric cleaning 4 times, 4 DEG C preservations stand-by to 10ml are redissolved with 1%BSA, 0.05M Tris pH7.6.
The preparation of fluorescent labeled antibody adsorbed film: it prepares and contains 0.5%PVA, 50mM PBS liquid, 0.5%BSA, 0.88%
The multi-polyester cellulose membrane pretreatment fluid of NaCl, pH 7.4, sets multi-polyester cellulose membrane to be processed in pretreatment fluid, room temperature
Impregnate 1 hour, take the film out, set seal after 37 DEG C of dryings it is spare.Fluorescent microsphere labelled antibody solution is taken, with 1%BSA, 0.05M
Tris pH7.6 buffer dilutes 3 times, starts die instrument, loads antibody, opens pressurized nitrogen, takes multi-polyester cellulose membrane, open
Beginning die sets die condition are as follows: and airbrush movement speed 30mm/ seconds, 5.0 μ l/cm of liquid fltting speed, by the film after printing,
Be put into drying box, 37 DEG C drying 6 hours, be subsequently placed in the hermetic bag containing desiccant save use.
The preparation of polyclonal antibody die: anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffer (pH
7.4) it is diluted to 1mg/ml concentration.Start die instrument, load antibody, take the PVC piece for posting nitrocellulose filter, starts die,
Set die condition are as follows: airbrush movement speed 30mm/ seconds, the film produced was put into 37 DEG C by 0.5 μ l/cm of liquid fltting speed
It is 6 hours dry in drying box, then film is placed in the drying receptacle containing desiccant and saves use.
Semi-finished product assemble method: starting dehumidifier makes to operate indoor humidity and is reduced to 25% hereinafter, in polyclonal antibody
Water suction paper membrane pad and fluorescent labeled antibody adsorbed film are pasted in die both ends respectively, then use Pressure sensitive adhesive tape sealing label surface.Set stickup
Good detection lug is on cutting machine, being cut into 3.5mm test strips.Paper slip is put into the aluminium amber hermetic bag of desiccant, is being sealed
It is sealed on machine, labelling.
The test strips of above-mentioned preparation are taken, upward with the side of fluorescent labeled antibody adsorbed film, are placed in centrifuge rotor, to
The human muscle hemoglobin solution 80ul of the various concentration of preparation is added dropwise on fluorescent labeled antibody adsorbed film, stands 2 minutes, 2000 turns/
It separates the heart 1 minute, then the PBS buffer solution 80ul of pH7.4 is added dropwise on fluorescent labeled antibody adsorbed film, 2000 revs/min are centrifuged 1 point
Clock cleaning, takes out test strips, and the fluorescent value of polyclonal antibody trace band is read on quantitative fluorescence analysis instrument.
Current art control stripes item does not do centrifugal treating, after 2 minutes time of repose of setting, then stands 2.5 minutes,
Then fluorescent value is read.
In triplicate, results are averaged for experiment.
Three, experimental result
Concrete outcome is as shown in table 6.The technology of the present invention is as above operated, and linear response is good, and correlation coefficient r value is
0.995.It is measured using the prior art, 2 points is stood after point sample, then stand 2.5 points, linear bad, 12.5ng/ml sample below
Product, for luminous quantity close to background level, correlation coefficient r value is 0.937.The existing detection reaction time of this test strips should be 15
Minute, 4.5 minutes are stood after this experiment point sample, detection reaction is not yet completed, therefore, linear bad.Compared with the prior art, originally
Invention significantly shortens detection time.
6 present invention of table is compared with the detection performance of existing Immunofluorescence test technology (unit: mV)
Embodiment 7, the present invention are compared with existing Immunofluorescence test technology testing result
One, experimental material
Sheep anti-mouse igg polyclonal antibody (Genagates company, the U.S. provides, article No. GP301231), other same embodiments
6。
Two, experimental method
The preparation of human muscle hemoglobin solution: taking the human muscle hemoglobin solution of known concentration, with sample dilution buffer (1%
BSA, 100mM glycine, 50mM PBS, 150mM NaCl, pH7.4) dilution configuration 3.13,6.25,12.5,25,50,100ng/
Ml human muscle hemoglobin solution.Prepare the human muscle hemoglobin measuring samples of 10ng/ml known concentration.
Fluorescent microsphere label: with embodiment 6.
The printing of fluorescent microsphere film: with embodiment 6.
The preparation of fluorescent labeled antibody adsorbed film: with embodiment 6.
The preparation of polyclonal antibody die: anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffer (pH
7.4) it is diluted to 1mg/ml concentration.Sheep anti-mouse igg Anti-TNF-α liquid solution is taken, is diluted to 50mM phosphate buffer (pH 7.4)
1mg/ml concentration.Start die instrument, load antibody, take the PVC piece for posting nitrocellulose filter, start die, same nitric acid is fine
It ties up and prints anti-human myoglobins polyclonal antibody on plain film as detection line T, sheep anti-mouse igg polyclonal antibody as nature controlling line C,
Set die condition are as follows: airbrush movement speed 30mm/ seconds, the film produced was put into 37 DEG C by 0.5 μ l/cm of liquid fltting speed
It is 6 hours dry in drying box, then film is placed in the drying receptacle containing desiccant and saves use.
Semi-finished product assemble method: starting dehumidifier makes to operate indoor humidity and is reduced to 25% hereinafter, in polyclonal antibody
Die detects line end and pastes fluorescent labeled antibody adsorbed film, and Quality Control line end pastes water suction paper membrane pad, then uses Pressure sensitive adhesive tape sealing label
Surface.The detection lug that pastes is set on cutting machine, being cut into 3.5mm test strips.Paper slip is put into the aluminium amber sealing of desiccant
In bag, sealed on sealing machine, labelling.
The test strips of above-mentioned preparation are taken, upward with the side of fluorescent labeled antibody adsorbed film, are placed in centrifuge rotor, to
The human muscle hemoglobin solution and each 80ul of sample to be tested of the various concentration of preparation are added dropwise on fluorescent labeled antibody adsorbed film, stands 2
Minute, 2000 revs/min are centrifuged 1 minute, then the PBS buffer solution 80ul of pH7.4 is added dropwise on fluorescent labeled antibody adsorbed film, and 2000
Rev/min centrifugation 1 minute clean, take out test strips, on quantitative fluorescence analysis instrument read polyclonal antibody printed film on detection line T
With the fluorescent value of nature controlling line C, and T/C ratio is calculated, draw standard curve, calculates measuring samples myoglobin concentration.
Current art control stripes item does not do centrifugal treating, after the time of repose of setting, then stands 2.5 minutes, then
Fluorescent value is read, and calculates T/C ratio, draws standard curve, calculates measuring samples myoglobin concentration.
In triplicate, results are averaged for experiment.
Three, experimental result
The technology of the present invention is as above operated, and standard curve is linearly good, and correlation coefficient r value is 0.995, has carried out sample immediately
Product measurement, empirical average 10.84ng/ml, detection error meet the requirements within 10% three times.It is measured using the prior art, point
2 points are stood after sample, then stands 2.5 points, and examination criteria curve linear is bad, and correlation coefficient r value is 0.937.Then by total point
Time of repose extends to 15 minutes of existing product testing after sample, and standard curve is linearly good, and correlation coefficient r value is 0.989, with
Sample measurement is carried out according to 15 minutes conditions, average value is 9.49ng/ml three times, and detection error is within 10%, symbol
It closes and requires.The concrete outcome of three repeated experiments is as shown in table 7.Compared with the prior art, when the present invention significantly shortens detection
Between.
7 present invention of table is with the Analysis of test results (unit: ng/ml) of immune detection technique of fluorescence
Embodiment 8, fluorescence detection of the present invention are compared with existing enzyme linked immunosorbent detection technology testing result
One, experimental material
Anti-human myoglobins polyclonal antibody (Genagates company, catalog number GP301042), anti-human flesh are red
Protein monoclonal antibody (Genagates company, the U.S., article No. GP300616), fluorescent microsphere (all fluoresceins be europium compound,
Article No. JY-SJ126, Shanghai outstanding person one biotech firm), EDC (Pierce product, article No. 22980), NHS (Pierce product, article No.
24500), human muscle hemoglobin (Sigma-Aldrich product, catalog number F3879-1G), quantitative fluorescence analysis instrument (Shanghai
Woman biotech firm, HG-98), BioFlow die instrument (IMAGENE company, the U.S.), (U.S. A-point is public for Index cutting machine
Department), DBF-900 sealing machine (Wenzhou Jiangnan packing factory), ACBO dehumidifier (jiangsu wuxi Ao Bo dehumidifier company), it is desk-top from
Scheming (Eppendoff company, the U.S.), bovine serum albumin(BSA) (SIGMA product, article No.: B8894), nitrocellulose diaphragm (AE
99, provided by Genagates company, the U.S.), multi-polyester cellulose membrane (Reemay 2033, U.S.'s Alstrom Products),
It absorbs water paper membrane pad (Grade 470, U.S.'s S&S Products), the anti-human myoglobins monoclonal of horseradish peroxidase-labeled resists
Body (Genagates company, the U.S., article No. GP300616), o-phenylenediamine, enzyme-linked immunosorbent assay instrument (Bio-Rad, Model
550), (Genagates company, the U.S. provides, goods for Healthy Human Serum (healthy premenopausal volunteers donations), sheep anti-mouse igg polyclonal antibody
Number GP301231).
Two, experimental method
The preparation of human muscle hemoglobin solution: taking the human muscle hemoglobin solution of known concentration, with PBS solution configuration 3.125,
6.25, the serial human muscle hemoglobin solution of 12.5,25,50,100ng/ml, for making standard curve.With known concentration
8.2ng/ml human muscle hemoglobin Healthy Human Serum is as measuring samples.
Experiment detects human muscle hemoglobin solution with existing enzyme linked immunosorbent detection technology using fluorescence detection of the present invention and draws
Then standard curve takes measuring samples to measure, the concentration of myoglobins in measuring samples is calculated with standard curve.Each sample is done
3 parallel pipes.
1, existing enzyme linked immunosorbent detection technology groups
Using 96 hole elisa plates, anti-human 100 μ l of myoglobins polyclonal antibody is added in every pipe, and 4 DEG C are coated with overnight, cleaning three
It is secondary, then it is separately added into 100 μ l of human muscle hemoglobin solution or measuring samples, association reaction incubates 120 minutes at 37 DEG C, cleaning three
It is secondary, anti-human 100 μ l of myoglobins monoclonal antibody is added, association reaction incubates 60 minutes at 37 DEG C, and cleaning three times, abandons supernatant,
100 μ l of horseradish peroxidase-labeled sheep anti-mouse igg polyclonal antibody is added, association reaction incubates 60 minutes at 37 DEG C, cleaning
Three times, supernatant is abandoned, 100 μ l developing solutions (formula: 0.1M citric acid 2.43ml, 0.2M disodium hydrogen phosphate 2.57ml, adjacent benzene two is added
Amine 5mg, 5 μ l of hydrogen peroxide), it is protected from light 5 minutes, 2M sulfuric acid is added and terminates reaction.It sets and reads OD490 suction on enzyme-linked immunosorbent assay instrument
Light value, 3.125,6.25,12.5,25,50,100ng/ml concentration correspond to OD value be respectively 0.182,0.215,0.256,0.398,
0.791,1.212, standard curve is drawn, r=0.993 calculates human muscle hemoglobin content in measuring samples.
2, of the present invention group
Fluorescent microsphere label: with embodiment 6.
The printing of fluorescent microsphere film: with embodiment 6.
The preparation of fluorescent labeled antibody adsorbed film: with embodiment 6.
The preparation of polyclonal antibody die: anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffer (pH
7.4) it is diluted to 1mg/ml concentration.Sheep anti-mouse igg Anti-TNF-α liquid solution is taken, is diluted to 50mM phosphate buffer (pH 7.4)
1mg/ml concentration.Start die instrument, load antibody, take the PVC piece for posting nitrocellulose filter, start die, same nitric acid is fine
It ties up and prints anti-human myoglobins polyclonal antibody on plain film as detection line T, sheep anti-mouse igg polyclonal antibody as nature controlling line C,
Set die condition are as follows: airbrush movement speed 30mm/ seconds, the film produced was put into 37 DEG C by 0.5 μ l/cm of liquid fltting speed
It is 6 hours dry in drying box, then film is placed in the drying receptacle containing desiccant and saves use.
Semi-finished product assemble method: starting dehumidifier makes to operate indoor humidity and is reduced to 25% hereinafter, in polyclonal antibody
Die detects line end and pastes fluorescent labeled antibody adsorbed film, and Quality Control line end pastes water suction paper membrane pad, then uses Pressure sensitive adhesive tape sealing label
Surface.The detection lug that pastes is set on cutting machine, being cut into 3.5mm test strips.Paper slip is put into the aluminium amber sealing of desiccant
In bag, sealed on sealing machine, labelling.
The test strips of above-mentioned preparation are taken, upward with the side of fluorescent labeled antibody adsorbed film, are placed in centrifuge rotor, to
The human muscle hemoglobin solution and each 80ul of sample to be tested of the various concentration of preparation are added dropwise on fluorescent labeled antibody adsorbed film, stands 2
Minute, 2000 revs/min are centrifuged 1 minute, then the PBS buffer solution 80ul of pH7.4 is added dropwise on fluorescent labeled antibody adsorbed film, and 2000
Rev/min centrifugation 1 minute clean, take out test strips, on quantitative fluorescence analysis instrument read polyclonal antibody print printed film on detects
The fluorescent value of line T and nature controlling line C, and T/C ratio is calculated, 0.001,0.005,0.024,0.138,0.373,0.683, r=
0.991, standard curve is drawn, measuring samples myoglobin concentration is calculated.
Three, experimental result
Concrete outcome is as shown in table 8.Existing enzyme linked immunosorbent detection technology measurement result shows sample to be examined human muscle hemoglobin
Content be 7.89ng/ml, measurement result of the present invention shows that the content of sample to be examined human muscle hemoglobin is 8.19ng/ml, two kinds
Experimental method acquired results are almost the same, no difference of science of statistics (P > 0.05), but completion experimental period of the invention is significantly shorter than
Current art.
The fluorescence detection of the present invention of table 8 (unit: ng/ml) compared with existing enzyme linked immunosorbent detection technology testing result
The influence of embodiment 9, centrifugal speed of the present invention to testing result
One, experimental material
With embodiment 1
Two, experimental method
With embodiment 1
Three, experimental result
The present invention, using different centrifugal speeds, has detected the human muscle hemoglobin sample of various concentration using colloidal gold as indicator
Product, experimental result are as shown in table 9.As shown in Table 9, detection accuracy is related with centrifugal speed, 500,1000,2000 revs/min
Centrifugal speed obtains satisfactory testing result, and correlation coefficient r value is all larger than 0.98.3000,4000,5000 revs/min from
The testing result that heart speed obtains, correlation coefficient r value are below 0.98, do not reach coherent detection requirement.Illustrate the present invention
The best centrifugal speed for carrying out myoglobins detection should be at 2000 revs/min or less.
Influence of 9 centrifugal speed of table to testing result
The influence of embodiment 10, input mode of the present invention to testing result
One, experimental material
Small ep decelerating motor (1000 revs/min of revolving speed of output, power 100w), stainless steel plate, (Baoding wound is sharp for peristaltic pump
Pump industry, model BT100LC), the other the same as in Example 1.
Two, experimental method
Small ep decelerating motor is uprightly installed, shaft is upward.Diameter 30mm circular slab, center are made with stainless steel plate
Punching.Stainless steel plate level is fixed on small DC arbor, peristaltic pump is installed to the center of stainless steel plate.It will
Direct current generator and peristaltic pump are connect with battery.Sample to be tested and soda liquor container are fixed on to the top of peristaltic pump.By preparation
Test strips are outside with absorbing membrane pad, and colloid gold label adsorbed film inward direction affixes in stainless steel plate.Peristaltic pump imbibition
Pipe one end is placed in sample to be tested and soda liquor container, and the other end is fixed on colloid gold label adsorbed film.Peristaltic pump imbibition
Pipe liquid feeding side, which is equipped with, can change the three-way cock for inhaling liquid flow path direction.Three-way cock is first set when experiment in sample to be tested side,
Sample to be tested 40ul is added dropwise to colloid gold label adsorbed film, opens centrifuge, opens peristaltic pump, with 20ul/min speed to colloid
Gold label adsorbed film sample-adding, after 2 minutes, swivel tee is switched to cleaning solution side, and wriggling pump speed is adjusted to 160ul/min, and 30
Peristaltic pump is closed when the second, continues centrifugation 30 seconds, closes centrifuge, takes test strips colloidal gold to quantify chromatographic analysis instrument measurement flesh red
The pattern colour angle value at protein band position.The other the same as in Example 1.
The input mode that control stripes item of the present invention is added dropwise using sample loading gun and is centrifuged on centrifuge is loaded for the first time
1 minute is stood after 80ul, is then centrifuged 1 minute for 1000 revs/min, cleaning solution 80ul is added, 1000 revs/min are centrifuged 1 minute, then read
Take pattern colour angle value.
Three, experimental result
Experimental result is as shown in table 10.Two test results compare, and the testing result of automatic sampling mode is better than manual point
Sample, correlation coefficient r value are respectively 0.996 containing 0.983.Illustrate automatic sampling better than hand sampling.
Influence (unit: pattern colour angle value) of the input mode of the present invention of table 10 to testing result
The influence of embodiment 11, detection mode of the present invention to testing result
One, experimental material
With embodiment 6
Two, experimental method
The preparation of human muscle hemoglobin solution: taking the human muscle hemoglobin solution of known concentration, with sample dilution buffer (1%
BSA, 100mM glycine, 50mM PBS, 150mM NaCl, pH7.4) dilution configuration 3.125,6.25,12.5,25,50,
100ng/ml human muscle hemoglobin solution.
Fluorescent microsphere label: with embodiment 6.
The printing of fluorescent microsphere film: with embodiment 6.
The preparation of fluorescent labeled antibody adsorbed film: with embodiment 6.
The preparation of polyclonal antibody die: anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffer (pH
7.4) it is diluted to 1mg/ml concentration.Sheep anti-mouse igg Anti-TNF-α liquid solution is taken, is diluted to 50mM phosphate buffer (pH 7.4)
1mg/ml concentration.Start die instrument, load antibody, take the PVC piece for posting nitrocellulose filter, start die, same nitric acid is fine
It ties up and prints anti-human myoglobins polyclonal antibody on plain film as detection line T, sheep anti-mouse igg polyclonal antibody as nature controlling line C,
Set die condition are as follows: airbrush movement speed 30mm/ seconds, the film produced was put into 37 DEG C by 0.5 μ l/cm of liquid fltting speed
It is 6 hours dry in drying box, then film is placed in the drying receptacle containing desiccant and saves use.
Semi-finished product assemble method: starting dehumidifier makes to operate indoor humidity and is reduced to 25% hereinafter, in polyclonal antibody
Die detects line end and pastes fluorescent labeled antibody adsorbed film, and Quality Control line end pastes water suction paper membrane pad, then uses Pressure sensitive adhesive tape sealing label
Surface.The detection lug that pastes is set on cutting machine, being cut into 3.5mm test strips.Paper slip is put into the aluminium amber sealing of desiccant
In bag, sealed on sealing machine, labelling.
The test strips of above-mentioned preparation are taken, upward with the side of fluorescent labeled antibody adsorbed film, are placed in centrifuge rotor, to
The human muscle hemoglobin solution and each 80ul of sample to be tested of the various concentration of preparation are added dropwise on fluorescent labeled antibody adsorbed film, stands 2
Minute, 2000 revs/min are centrifuged 1 minute, then the PBS buffer solution 80ul of pH7.4 is added dropwise on fluorescent labeled antibody adsorbed film, and 2000
Rev/min centrifugation 1 minute clean, take out test strips, on quantitative fluorescence analysis instrument read polyclonal antibody printed film on detection line
Fluorescent value.
Bilateral detection is to install another fluorescence in existing fluorescence detection probe opposite side in quantitative fluorescence analysis
Detection probe, when detection, tear PVC egative film, and other conditions are constant, read the fluorescent value of detection line on polyclonal antibody printed film.
Three, experimental result
Concrete outcome is as shown in table 11.Compared with conventional unilateral detection, the present invention can be significantly improved using bilateral detection
The fluorescence volume of reading, can be improved detection sensitivity.
Influence (unit: mV) of the detection mode of the present invention of table 11 to testing result
The influence of embodiment 12, cleaning step of the present invention to testing result
One, experimental material
With embodiment 1
Two, experimental method
Control experiment cleaning step is stood, and is not cleaned.The other the same as in Example 1.
Three, experimental result
Experimental result is as shown in table 12.Two test results compare, and the testing result of cleaning is better than not cleaning, related coefficient
R value is respectively 0.996 containing 0.979.Illustrate to be necessary after testing sample introduction with the step of cleaning.
Influence (unit: pattern colour angle value) of the cleaning step of the present invention of table 12 to testing result
Claims (3)
1. a kind of centrifuge separation detection method includes the following steps: to flow through immobilon-p, the liquid using centrifugal device driving liquid phase
It mutually include the measuring samples containing object to be checked and detection phase, the detection is mutually for containing can be direct or indirect with the object to be checked
Form the substance of the detection indicator of specific binding；When the liquid phase flows through immobilon-p, on the immobilon-p it is coated to
Inspection object specificity junction mixture combines the compound for forming detection indicator-object-to be checked object specificity junction mixture to be checked, described compound
Object is captured to be fixed on the immobilon-p, the detection indicator that detector is indirectly fixed on the immobilon-p by detection
Amount, to detect the content of the object to be checked；
The object to be checked is to generate immunocompetent substance with immunocompetent protein or with protein coupling；It is described to be checked
Object specificity junction mixture is the antigen or antibody with the object specific binding to be checked；The detection indicator be colloidal metal,
At least one of dyestuff, fluorescein and chemiluminescent substance；
The immobilon-p is at least one of nitrocellulose filter, polyvinylidene fluoride film, nylon membrane and DEAE cellulose membrane；
The detector includes any one of absorbance, fluorescence, chemiluminescence and detector of color of image digital processing；Institute
State the one or both sides that detector is set to the immobilon-p；
The colloidal metal is at least one of colloidal gold, electroselenium and colloid gold-magnetic particles；The fluorescein is different sulphur cyanogen
Sour fluorescein, RB 200, tetramethylisothiocyanate rhodamine, phycoerythrin, perdinin phyllochlorin, iodate
Third pyridine, other at least one of phycocyanin and europium compound；The chemiluminescent substance be luminol and different luminol and its
In derivative species, acridinium ester and a word used for translation shallow lake amides, (golden steel alkane) -1,2- dichloroethane and its derivative and tris (bipyridine) ruthenium extremely
Few one kind.
2. according to the method described in claim 1, it is characterized by: the rotary part of the centrifugal device using plane or by
Center is inclined outwardly type；The revolving speed of the centrifugal device and the sample introduction speed of sampling device are all made of program controlled mode；It is described
The revolving speed of centrifugal device is 200~10000r/min；The time being centrifuged through the centrifugal device is 1~5min；
The one or both sides of the immobilon-p have backing.
3. application of the method for any of claims 1 or 2 in the content detection of immune detection product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811176124.XA CN109387628A (en) | 2016-03-14 | 2016-03-14 | It is centrifugated detection method |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811176124.XA CN109387628A (en) | 2016-03-14 | 2016-03-14 | It is centrifugated detection method |
| CN201610143694.3A CN107192819A (en) | 2016-03-14 | 2016-03-14 | One kind centrifuges detection method |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610143694.3A Division CN107192819A (en) | 2016-03-14 | 2016-03-14 | One kind centrifuges detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109387628A true CN109387628A (en) | 2019-02-26 |
Family
ID=59871262
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610143694.3A Pending CN107192819A (en) | 2016-03-14 | 2016-03-14 | One kind centrifuges detection method |
| CN201811176124.XA Pending CN109387628A (en) | 2016-03-14 | 2016-03-14 | It is centrifugated detection method |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610143694.3A Pending CN107192819A (en) | 2016-03-14 | 2016-03-14 | One kind centrifuges detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN107192819A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107543922A (en) * | 2016-06-27 | 2018-01-05 | 北京康华源科技发展有限公司 | A kind of centrichromatography fluorescence immunoassay detection technique and application thereof |
| CN107561281A (en) * | 2016-07-16 | 2018-01-09 | 北京康华源科技发展有限公司 | A kind of centrichromatography colloid gold immune detection technique and application thereof |
| 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 |
| CN107727850B (en) * | 2017-10-10 | 2021-08-27 | 常州博闻迪医药股份有限公司 | Lateral flow chromatography detection reaction start control method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86108549A (en) * | 1985-12-20 | 1987-06-17 | 古甘·简 | Extract the method and apparatus of the blood plasma of scheduled volume from the blood sample that is used for analyzing |
| CN1638871A (en) * | 2002-02-26 | 2005-07-13 | 拜尔健康护理有限责任公司 | Method and apparatus for precise transfer and manipulation of fluids by centrifugal, and/or capillary forces |
| WO2009093838A2 (en) * | 2008-01-21 | 2009-07-30 | Thin-film layered centrifuge device and analysis method using the same | |
| CN102135535A (en) * | 2010-01-25 | 2011-07-27 | 刘凤鸣 | Immune colloidal metal detection technology capable of directly performing semi-quantitative analysis, preparation method and application |
| CN102472739A (en) * | 2010-01-29 | 2012-05-23 | 三星电子株式会社 | Centrifugal micro-fluidic device and method for detecting analytes from liquid specimen |
| CN104862216A (en) * | 2014-02-21 | 2015-08-26 | 宁波大学 | High-throughput visual totally-enclosed split-type LAMP-LFD detection chip device |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1916524A1 (en) * | 2006-09-27 | 2008-04-30 | Roche Diagnostics GmbH | Rotatable test element |
| CN102115712B (en) * | 2010-12-02 | 2015-05-13 | 广州安必平医药科技股份有限公司 | Liquid handling method for nucleic acid molecular hybridization experiment and reactor thereof |
| KR101656037B1 (en) * | 2014-04-30 | 2016-09-08 | 울산과학기술원 | Rare cell isolation device, method of rare cell isolation and method of rare cell detection using thereof |
-
2016
- 2016-03-14 CN CN201610143694.3A patent/CN107192819A/en active Pending
- 2016-03-14 CN CN201811176124.XA patent/CN109387628A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86108549A (en) * | 1985-12-20 | 1987-06-17 | 古甘·简 | Extract the method and apparatus of the blood plasma of scheduled volume from the blood sample that is used for analyzing |
| CN1638871A (en) * | 2002-02-26 | 2005-07-13 | 拜尔健康护理有限责任公司 | Method and apparatus for precise transfer and manipulation of fluids by centrifugal, and/or capillary forces |
| WO2009093838A2 (en) * | 2008-01-21 | 2009-07-30 | Thin-film layered centrifuge device and analysis method using the same | |
| CN102135535A (en) * | 2010-01-25 | 2011-07-27 | 刘凤鸣 | Immune colloidal metal detection technology capable of directly performing semi-quantitative analysis, preparation method and application |
| CN102472739A (en) * | 2010-01-29 | 2012-05-23 | 三星电子株式会社 | Centrifugal micro-fluidic device and method for detecting analytes from liquid specimen |
| CN104862216A (en) * | 2014-02-21 | 2015-08-26 | 宁波大学 | High-throughput visual totally-enclosed split-type LAMP-LFD detection chip device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107192819A (en) | 2017-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102759631B (en) | The latex enhancing immune of a kind of quantitative detection Procalcitonin PCT is than turbid kit | |
| US20180364227A1 (en) | Detection method with centrifuge isolation and detection device thereof | |
| CN109387628A (en) | It is centrifugated detection method | |
| CN101324579A (en) | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method | |
| WO2017206800A1 (en) | Centrifugal chromatography immunoassay method | |
| CN105195243B (en) | The magnetic microparticle chemiluminescence micro-fluidic chip that a kind of myoglobins is quantitatively detected | |
| CN101813699A (en) | Photo-induced chemiluminescence immunoassay quantitative kit and method for detecting content of milk protein | |
| CN102901812A (en) | Magnetic particle chemiluminescence immunoassay kit and assay method for human thyroglobulin antibodies (TGAb) | |
| CN107449906A (en) | A kind of paper chromatography chemical luminescence detection method | |
| CN203337668U (en) | Listeria monocytogene immunochromatography kit employing fluorescent quantitative detection | |
| CN107525923A (en) | One kind centrifuges immunochromatography detection method and device | |
| CN108761075A (en) | A kind of deoxynivalenol quantifies rapid detection card and its detection method | |
| CN106248894A (en) | A kind of paper chip detecting Residue of Antibiotics in Milk | |
| CN1885038A (en) | ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection | |
| CN104374916A (en) | Listeria monocytogenes fluorescence quantitative determination immunochromatography kit | |
| CN112415193A (en) | Novel method for quickly detecting magnetic bimetallic nanoenzyme based on polydopamine mediation | |
| CN115639366A (en) | Beta 2-microglobulin fluorescence immunochromatography assay kit and detection method thereof | |
| JP2003215126A (en) | Method for extracting microbe antigen | |
| CN109459570A (en) | The colloid gold immune test paper preparation method of lead ion in a kind of quick detection blood of human body | |
| CN205538994U (en) | Highly sensitive time -resolved fluorescence immunity chromatography detect reagent device | |
| CN110873801A (en) | Thyroid hormone immunochromatography test strip and preparation method and kit thereof | |
| CN107561281A (en) | A kind of centrichromatography colloid gold immune detection technique and application thereof | |
| CN203337664U (en) | Escherichia coli immunochromatography kit employing fluorescent quantitative detection | |
| CN102507940B (en) | Method for quickly detecting chloramphenicol in cosmetics | |
| WO2018177445A1 (en) | Centrifugation immunochromatography detection method and apparatus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190226 |
|
| RJ01 | Rejection of invention patent application after publication |