CN109164257A - A kind of time-resolved fluoroimmunoassay detection method based on magnetic particle - Google Patents
A kind of time-resolved fluoroimmunoassay detection method based on magnetic particle Download PDFInfo
- Publication number
- CN109164257A CN109164257A CN201811025038.9A CN201811025038A CN109164257A CN 109164257 A CN109164257 A CN 109164257A CN 201811025038 A CN201811025038 A CN 201811025038A CN 109164257 A CN109164257 A CN 109164257A
- Authority
- CN
- China
- Prior art keywords
- antibody
- antigen
- magnetic bead
- detection method
- rare earth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
Abstract
The time-resolved fluoroimmunoassay detection method based on magnetic particle that the invention discloses a kind of forms immunomagnetic beads including the antigen of specificity or antibody to be coated on magnetic bead；The antigen or antibody for the chelate labels specificity that the more carboxyls of poly- amino and rare earth ion are formed, form fluorescent marker；Sample is added, is immunoreacted, so that immunomagnetic beads, tested sample and fluorescent marker is formed compound, fluorescence enhancement object is added after cleaning, so that fluorescent value is enhanced million times using dissociation-enhancing technology, finally detects, obtain the information such as the concentration of tested sample.The present invention can detect simultaneously one or more parts sample and once experiment can detect multiple indexs, it is easy to operate quickly.Detection sensitivity, detection repeatability and flexibility will TRFIA board-like far better than conventional microporous and chromatography methods.
Description
Technical field
The present invention relates to the detection technique fields such as biomedicine, animal epidemic, food safety, drug screening, specifically,
The time-resolved fluorescence detection method based on magnetic particle that the present invention relates to a kind of.
Background technique
Time-resolved fluoroimmunoassay (Time-resolved Fluorescence immunoassay, TRFIA) skill
Art is the new milestone that marker develops after radiommunoassay, it has also become biomedical research and clinical ultramicron
A most promising analysis means in biochemical investigation.With fluorescence lifetime, long, Stokes is displaced big rare earth ion to TRFIA
As marker, have preparation simplicity, storage time length, "dead" pollution, standard curve range wide, naturally not glimmering by sample
The advantages that light interference, multiple labeling.It overcomes radio immunoassay and easily pollutes the environment；Colloidal gold method, fluorescence immunoassay
Chromatography sensitivity is low, poor repeatability；Enzyme-linked immunization enzyme label is unstable；Chemoluminescence method can only once shine, Bu Nengshi
The disadvantages of existing multiple labeling.
Producer is all made of based on microwell plate TRFIA technology both at home and abroad at present, since the solid-liquid phase reaction area of microwell plate is small,
The immune response time needed is more long；Microwell plate coated antibody or antigen are coated with by physical absorption, coated antibody or antigen
It is difficult to standardize, so that testing result precision is poor；Microwell plate TRFIA technology is mainly semi-automatic detection, so that sample needs
Run up to it is a certain amount of after could detect, it is difficult to realize immediately detection.
Automation and manual errors are two hang-ups for restricting microwell plate TRFIA.Based on industry to automation, informationization,
Intelligent, big data detection system urgent need, realizes high precision, the high accuracy of testing result, fundamentally solves
The certainly puzzlement of reagent quality problem, while realizing the foundation of each neighborhood system big data.When " precision, automation, cloud "
Between resolved fluorometric immunoassay system will be the development trend in market future.
Patent of invention: 105911041 A of CN discloses a kind of time-resolved fluorescence detection method, it is characterized in that using two
The complex of tooth or four tooth beta-diketone compounds and europium shines, and is not using dissociation enhancing lanthanide series fluoroimmunoassay hair
Body of light system.The sensitivity of the patent is low, detects lower limit 10-10~10-11Mol/L is horizontal, it is still desirable to fluorescence synergist, fluorescence
The purity of synergist influences experimental result serious.
Summary of the invention
The purpose of the present invention is overcoming above-mentioned technical problem, a kind of time-resolved fluoroimmunoassay based on magnetic particle is provided
Detection method.
To achieve the above object, present invention employs following technical solutions:
A kind of time-resolved fluoroimmunoassay detection method based on magnetic particle, comprises the following steps:
(1) antigen of specificity or antibody are coated on magnetic bead, form immunomagnetic beads；
(2) rare earth ion formed without the more carboxyls of poly- amino of the bifunctional group of aromatic radical and rare earth ion is chelated
The antigen or antibody of substance markers specificity form fluorescent marker；
(3) tested sample is added, is immunoreacted, forms immunomagnetic beads, tested sample and fluorescent marker compound
Object；
(4) it by externally-applied magnetic field completes that free fluorescent marker is washed to immuno magnetic cell separation and fixation using cleaning solution
It goes, adds fluorescence enhancement solution to enhance fluorescent value；
(5) fluorescence is finally detected, the dose-response curve according to built-in testing concentration and fluorescence signal value fitting is logical
It crosses software and calculates tested sample concentration.
Preferably, the immunomagnetic beads are with official in above-mentioned time-resolved fluoroimmunoassay detection method
It can 0.1~3 μm of diameter of superparamagnetic particles of group's modification and the covalent coupling object of monoclonal antibody or antigen；Described in wherein
Magnetic particle is carboxyl magnetic bead, amino magnetic bead, hydroxyl magnetic bead, tosyl magnetic bead, NHS magnetic bead, albumin A magnetic bead, Protein G magnetic
One or more of pearl, anti-mouse IgG magnetic bead, hydrophilic magnetic bead, hydrophobic magnetic bead；Either immunomagnetic beads are affine by chain enzyme
Biscuit porcelain microballon reacts the immunomagnetic beads to be formed with biotinylated antibody.
Preferably, in above-mentioned time-resolved fluoroimmunoassay detection method, the preparation method of the immunomagnetic beads
For 0.1~3 μm of diameter of magnetic particle and corresponding antibody or antigen is washed, activation, coupling, closing are prepared.
Preferably, step (2) is described by the more carboxylics of poly- amino in above-mentioned time-resolved fluoroimmunoassay detection method
The antigen or antibody for the chelate labels specificity that base and rare earth ion are formed, the mode for forming fluorescent marker have: directly handle
Rare Earth Chelate is connected by chemical bond and biological raw material；Or Rare Earth Chelate is passed through chemical bond and streptavidin
It connects, antigen or antibody mark biotin, and last Rare Earth Chelate is reacted by biotin with streptavidin, with antigen
Or antibody connects.
Preferably, step (2) is described without aromatic radical in above-mentioned time-resolved fluoroimmunoassay detection method
The more carboxyls of poly- amino of bifunctional group are different sulphur nitrile acid phenyl-ethylenediamine tetra-acetic acid (EDTA), different sulphur nitrile acid-Diethylenetriamine base
Tetraacethyl (ETTA), Diethylenetriamine base pentaacetic acid (DTPA) or N- (the different sulfydryl benzyl of P-)-diethylenetriamine tetraacethyl
(DTTA)；The rare earth ion is europium (Eu3+), samarium (Sm3+), terbium (Tb3+) or dysprosium (Dy3+)。
Preferably, in above-mentioned time-resolved fluoroimmunoassay detection method, step (2) the rare earth ion chelating
Substance markers antibody or antigen preparation process are as follows: the mass ratio of antigen or antibody and rare earth ion chelate is 1:1~10:1；?
Under the carbonate buffer solution of 0.05M pH9.0, rare earth ion chelate is added and dialysed antigen or antibody, is dissolved in after mixing
The carbonate buffer solution of 0.05M pH9.0, room temperature concussion is overnight；Collect peak pipe after purification with sephadex column；By above-mentioned system
The fluorescent marker dilution got ready, it is spare.
Preferably, in above-mentioned time-resolved fluoroimmunoassay detection method, the temperature of step (2) described immune response
Degree is 25 DEG C~37 DEG C, and humidity is 30~75%RH, is shaking, is carrying out under oscillation.
Preferably, in above-mentioned time-resolved fluoroimmunoassay detection method, the formula of step (4) described cleaning solution
Are as follows: 7.2~7.5 0.025M Tris-Hcl buffer of 0.8~1.5%Tween-20,0.05%Proclin300, pH.
Preferably, in above-mentioned time-resolved fluoroimmunoassay detection method, step (4) described fluorescence enhancement solution
Formula are as follows: 0.0002~0.0009% β-NTA, 0.0024%TOPO, 0.08% acetic acid, 0.05% Triton X-100 group
At the aqueous solution for being 3.0-3.2 with appropriate Potassium Hydrogen Phthalate adjustment pH.
Present invention selection is chelating agent and rare earth ion chela without poly- aminopolycanboxylic acid's class of the bifunctional group of aromatic radical
Cooperation is marker, carries out the label of antigen or antibody on magnetic bead and rare earth ion chelate respectively, utilizes antigen-antibody
Immune response carries out analysis detection, level of the prepared reagent performance up to same chemical illuminating reagent.Meanwhile using liquid
Homogeneous reaction, each component are sufficiently mixed, so that reaction efficiency is higher and reaction is more abundant, overcome heterogeneous reaction poor repeatability
With unstable disadvantage.After immune response, completed by externally-applied magnetic field to immuno magnetic cell separation and fixation, it will be more using cleaning solution
After remaining component completely removes, adds fluorescence enhancement solution and read fluorescent value, entire reaction process whole process instrumentation realizes certainly
Dynamicization, precision, intelligence solve Conventional plate-type Time-resolved fluoroimmunoassay and are difficult to automate and manual errors
Big problem.Using the chelating agent with bifunctional group structure, make one end and rare earth ion europium (Eu3+), samarium (Sm3+), terbium
(Tb3+), dysprosium (Dy3+) in a kind connection, the other end on antibody/antigen molecule free amino or carboxyl connect, formation it is dilute
The antibody/antigen of native ion label, generates immune complex after immune response.Due to this compound in water glimmering
Luminous intensity is very weak, therefore fluorescence enhancement solution need to be added, and rare earth ion is disintegrated down from compound first, then freely dilute
Fluorescence enhancement object (chelating agent) chelating enters the hydrophobic inner core of micella in native ion and fluorescence enhancement solution, makes the fluorescence of rare earth ion
Millions of times of amplification.
Compared with prior art, the invention has the following beneficial effects:
(1) for the first time by immune magnetic particle technology and time-resolved fluorescence technological incorporation into product, it is fast to solve POCT industry
And inaccurate pain spot.Since magnetic particle and antigen or antibody are covalent coupling, the unstability of physical absorption is overcome, therefore exempt from
The epidemic disease magnetic particle holding time is long and more stable；The flexibility of the appearance and coating process of magnetic particle is bigger, can be according to different
Requirement of experiment is selected, and the quality of production is controllable；Using magnetic separation technique, the immune complex of formation is direct in externally-applied magnetic field
Precipitating, being not required to centrifugation can separate immune complex with unbonded material；Because magnetic particle have bigger bonded area with
It can disperse in the liquid phase and sufficiently react, greatly improve detection range, shorten the reaction time, improve sensitivity；Magnetic particle can be with
It is combined with different probe molecules, makes it possible to detect determinand different in same sample.
(2) reaction system of the invention realizes certainly from sample-adding, incubation, cleaning, daylighting to report testing result overall process
Dynamicization；One or more parts sample can be detected simultaneously and once tested can detect multiple indexs, quickly (20 minutes easy to operate
Result can be gone out), it overcomes the board-like time-resolved fluoroimmunoassay technology of conventional microporous and needs sample cumulative to certain amount ability
Detection, realizes the instant detection of sample.
(3) Time-resolved fluoroimmunoassay is used, using lanthanide chelate europium, terbium, samarium, dysprosium as marker,
It advantageously reduces background with wider excitation spectrum, relatively narrow emission spectrum, improves sensitivity；Ultraviolet excitation have compared with
High quantum production rate, larger Stokes displacement, the spectrum weight for avoiding excitation spectrum and fluorescence emission spectrum and bio-matrix from emitting
It is the advantages that conjunction, fluorescence decay time is long, more wider than conventional fluorescent substance detection range, specific more preferable.Due to being liquid phase reactor
System reacts more abundant, in addition the amplification system of Rare Earth Chelate fluorescent tracing object and magnetic particle is used, so that detection is sensitive
Degree and detection repeatability will TRFIA board-like far better than conventional microporous and chromatography methods.
(4) present invention also provides simultaneously for the high throughput of Timed-resolved fluoroimmunoassay and multi-channel detection certain
Basic platform, conventional sample are able to achieve the multiple project indicators of detection or single agents item when cooperating method of the invention
The effect that more parts of samples can be detected simultaneously, there is very big potential advantages.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the composite reagent item of the time-resolved fluorescence detection method based on magnetic particle.
1, instrument connection No.1,2, instrument connection two, 3, fluorescent marker No.1,4, fluorescent marker two, 6, washing lotion, 7,
Washing lotion, 8, Sample dilution No.1,9, Sample dilution two, 12, enhancement solution, 5,10,11,13 be preparation hole.
Fig. 2 is the structural schematic diagram for the reaction cup of the time-resolved fluorescence detection method based on magnetic particle individually tested.
Fig. 3 is cTnI dose-response curve figure.
Fig. 4 is cTnI kit and Abbott Laboratories' kit correlation.
Specific embodiment
Property content is described in further detail for the essence of the present invention With reference to embodiment.
Embodiment 1:
CTnI kit is prepared using the time-resolved fluoroimmunoassay detection method of the invention based on magnetic particle；
Wherein, the shape of composite reagent item is sector, is from left to right arranged successively 1~13 hole.1st, 2 holes are test
Hole, the 3rd, 4 holes be fluorescent marker hole, the 6th, 7 holes be dilution fluid apertures, the 8th, 9 be Sample Dilution fluid apertures, the 12nd for enhancing fluid apertures,
5th, 10,11,13 be preparation hole.Magnet can be stored between 1st and 2 holes carries out Magneto separate experiment；3rd, 4 holes can be from entire
It is disassembled on reagent strip and is independent component, convenient for carrying out packing storage to fluorescent marker.The structure of composite reagent item is shown in attached
Fig. 1.
The specific process is as follows:
(1) preparation method of cTnI immunomagnetic beads:
1. washing magnetic bead
1 μm of carboxyl magnetic bead of 1mg diameter is drawn into 1.5ml centrifuge tube, centrifuge tube is placed in magnetic frame, magnetic force is utilized
Frame carry out magnetic bead and buffer separation, with 2- (N- morpholine) ethanesulfonic acid (MES) of 1ml 0.1M pH6.0 wash magnetic bead 3~
5 times.
2. magnetic bead activates
The MES of 1ml 0.1M pH6.0 is added in the magnetic bead good to above-mentioned washing, the 1- (3- bis- of 20 μ L 20mg/mL is added
Methylaminopropyl) -3- ethyl carbodiimide hydrochloride solution (EDC) and 20 μ L 20mg/ml n-hydroxysuccinimides (NHS),
Room temperature concussion reaction 0.5~1 hour.
3. the coupling of cTnI antibody and magnetic bead
Above-mentioned activated magnetic bead is placed on magnetic frame, is abandoned supernatant, is collected activated magnetic bead.0.05~1mg is added
CTnI monoclonal antibody through having been dialysed with MES, room temperature persistently rotate incubation 3~5 hours, and the reaction time depends on matching for magnetic bead
Position base and concentration.
4. closing
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, collects immunomagnetic beads.Addition contains 1%BSA 0.1M pH8.0's
Magnetic bead is resuspended in Tris-HCl buffer, and closing is not coupled the activated carboxyl site of cTnI monoclonal antibody, reacts 30~60min.
5. storing
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, and 500 μ L immunomagnetic beads are added and save liquid.
The preservation liquid is containing 5% (w/v) BSA, 10% (w/v) trehalose, 2% (w/v) sucrose, 0.1% (v/v)
The Tris-HCl buffer of the 0.05M pH 8.0 of Tween-20.
By the above-mentioned immunomagnetic beads dilution prepared, make final concentration of 0.0050~0.0100g/ of cTnI monoclonal antibody
L is dispensed into the 1st hole (instrument connection 1) of reagent strip.
(2) preparation method of biotinylation cTnI monoclonal antibody:
1. sufficiently being dialysed with 8.0 sodium bicarbonate buffer liquid of 0.1M pH to 1mg cTnI monoclonal antibody.
2. dissolving 1mg biotin with 1ml DMSO.
3. 120 μ L biotin solutions are added (i.e. containing life to 1mlcTnI monoclonal antibody solution (being 1mg containing amount of antibody)
120 μ g of object element), it is continuously stirred at room temperature, keeps the temperature 2~4 hours.
4. sufficiently dialysing at 4 DEG C to PBS, to remove free biotin.
5. 5% (w/v) BSA is added in sample, 4 DEG C will be set in conjunction with product, be kept in dark place.
By the above-mentioned biotinylation cTnI monoclonal antibody dilution prepared, make the final concentration of of cTnI monoclonal antibody
0.001~0.005g/L is dispensed into the 3rd hole of reagent strip.
(3) SA marks europium preparation method:
①SA-Eu3+Preparation
(1) it takes out 1mg SA and is placed in 10KD ultra-filtration centrifuge tube, 10000rpm is centrifuged 10min, discards filtrate.
(2) label buffer (0.05M pH9.0 carbonate buffer solution) 200 μ L, 10000rpm is added and is centrifuged 10min, abandons
Go filtrate.Repeat this operation 4~5 times.
(3) centrifuge tube filter membrane is inverted, 3000rpm is centrifuged 6min, collects the antibody of concentration.200 μ L label buffering is added
Liquid stands 3~5min, then centrifuge tube filter membrane is inverted, and 3000rpm is centrifuged 6min, collects the SA of concentration.
(4) according to mass ratio SA: the ratio of Europium chelate DTTA-Eu=3:1 mixes well, and is put into rotary incubator, room
Temperature reaction 16~24 hours.
②SA-Eu3+Purifying
With Sepharose CL-6B gel column purification SA-Eu3+, SA-Eu is then added3+Preservative agent (15% (w/v) BSA+
5% (w/v) Proclin300), make BSA and Proclin300 final concentration of 0.2%, through 0.22 μm of membrane filtration, 4 DEG C of storages are standby
With.
By the above-mentioned SA-Eu prepared3+Dilution, makes final concentration of 0.005~0.010g/L of SA, packing to reagent strip the
In 4 holes.
The excitation wavelength of the fluorescent material europium is 340nm, launch wavelength 615nm.
(4) preparation method of cleaning solution:
It prepares and contains 0.8~1.5%Tween-20,7.2~7.5 0.025M Tris- of 0.05%Proclin300, pH
Hcl buffer dispenses prepared solution into washing lotion hole 6 and 7 with every 2000 μ L of hole.
(5) preparation method of enhancement solution:
Prepare containing 0.0002~0.0009% β-NTA, 0.0024%TOPO, 0.08% acetic acid, 0.05% Triton
X-100 composition, the aqueous solution for being 3.0-3.2 with appropriate Potassium Hydrogen Phthalate adjustment pH.By prepared solution with every hole 400
μ L is dispensed to the 12nd hole of reagent strip (enhancing fluid apertures).
(6) preparation method of cTnI calibration object:
The cTnI calibration object the preparation method comprises the following steps: with contain 1.5%Tween-20,0.02%Proclin300, pH
7.50.02M Tris-Hcl buffer is by cTnI antigen diluent to 0,0.03,0.1,0.5,1,5,25,50ng/ml.
Each component is dispensed into the particular bore of composite reagent item, and semi-finished product are assembled by following process: in embodiment 1
1st, 3,4 holes dispense 100 μ L immunomagnetic beads, 200 μ L biotinylated antibodies, 100 μ LSA-Eu respectively3+, cleaning solution is placed on the 6th,
7 holes, enhancement solution are placed on the 12nd hole.
Embodiment 2
In this embodiment, cTnI kit each group is dispensed into different reagent bottles, and reacting environment is reaction cup, reaction cup
Structure see attached drawing 2.
(1) preparation method of cTnI immunomagnetic beads:
It is that biotinylation cTnI monoclonal antibody is dispensed into reagent bottle by 0.0050~0.0200g/L of concentration, by SA- magnetic
Pearl dispenses into another reagent bottle.The two is linked together in reaction cup by streptavidin-biotin.
(2) europium mark cTnI preparation method for antibody:
1. the preparation of europium mark cTnI monoclonal antibody
A. it takes out 1mg cTnI monoclonal antibody and is placed in 30KD ultra-filtration centrifuge tube, 10000rpm is centrifuged 10min, discards filter
Liquid.
B. 200 μ L, 10000rpm centrifugation 10min of label buffer (0.05M pH9.0 carbonate buffer solution) is added, discards
Filtrate.Repeat this operation 4~5 times.
C. centrifuge tube filter membrane is inverted, 3000rpm is centrifuged 6min, collects the antibody of concentration.200 μ L label buffering is added
Liquid stands 3~5min, then centrifuge tube filter membrane is inverted, and 3000rpm is centrifuged 6min, collects the cTnI monoclonal antibody of concentration.
D. according to mass ratio cTnI monoclonal antibody: the ratio of Europium chelate DTTA-Eu=5:1 mixes well, and is put into rotation
Turn incubator, reacts at room temperature 16~24 hours.
2. the purifying of europium mark cTnI monoclonal antibody
With SepHadex TM G-75 gel column purification europium mark cTnI monoclonal antibody, europium mark cTnI monoclonal antibody preservative agent is then added
(15% (w/v) BSA+5% (w/v) Proclin300), makes BSA and Proclin300 final concentration of 0.3%, through 0.22 μm of filter membrane
Filtering, 4 DEG C store for future use.
Each component is dispensed into different reagent bottles.It is coated on the composite reagent item and reagent bottle for full-automatic glimmering
The product information mark of light detection analyzer scanning recognition includes company standard curve, batch, date of manufacture, validity period.
The detecting step of kit: it is mono- that immunomagnetic beads, sample, biotinylation cTnI are added in the 1st hole of composite reagent article
Anti-, SA-Eu3+Or SA- magnetic bead, biotinylation cTnI monoclonal antibody, sample, europium mark cTnI antibody in single reaction cup, 37 degree of oscillations
It is incubated for 15min, then adsorbs magnetic bead under the influence of a magnetic field, cleans the free SA-Eu of removal with cleaning solution3+, then it is added and increases
Strong liquid is incubated for 5min, is 340nm in excitation wavelength, launch wavelength acquires fluorescence signal under the conditions of being 615nm, bent according to calibration
Line obtains sample to be tested cTnI protein concentration.
Embodiment 3 prepares cTnI kit compared with the box of commercial reagent using the present invention
In embodiment 1 using the present invention preparation cTnI kit measurement calibration object cTnI, detection range be 0.03~
50ng/ml, calibration object concentration and fluorescent value are fitted double-log dose-response curve, standard curve Y=1.011X+4.9268R
=0.999, kit is functional, sees Fig. 3.
The full-automatic magnetic particle time-resolved fluoroimmunoassay system measurement cTnI standard curve of table 1
| Concentration ng/ml | Fluorescent value | Fluorescent value | Fluorescent value | Mean value | The coefficient of variation |
| 0 | 801 | 812 | 831 | 815 | 2% |
| 0.03 | 2236 | 2501 | 2304 | 2347 | 6% |
| 0.1 | 8439 | 8521 | 8301 | 8420 | 1% |
| 0.5 | 40088 | 40056 | 41023 | 40389 | 1% |
| 1 | 87569 | 86127 | 88156 | 87284 | 1% |
| 5 | 478965 | 469875 | 458791 | 469210 | 2% |
| 25 | 2256890 | 2156910 | 2069884 | 2161228 | 4% |
| 50 | 4178144 | 4152601 | 4203108 | 4177951 | 1% |
Using the quick troponin-i assay kit of commercially available height (chemiluminescence particulate immunodetection) (Abbott Laboratories' trade
Co., Ltd) testing result accuracy of the present invention is verified, is as a result seen below in triplicate with the method test sample in embodiment 1
Table 2.
2 pattern detection result of table
Compared with commercial reagent, this reagent deviation is respectively less than ± 10%, and the testing result of this reagent is accurate and reliable.
Using the quick troponin-i assay kit of commercially available height (chemiluminescence particulate immunodetection) (Abbott Laboratories' trade
Co., Ltd) with the reagent in embodiment 1 100 clinical samples are detected, testing result is shown in Fig. 4, table 3.
3 clinical sample testing result of table
This reagent is compared the testing results of 100 clinical serums with Abbott Laboratories kits, positive coincidence rate
96.05%, negative match-rate 83.33%, total coincidence rate of the two is 93.00%, and the correlation with Abbott Laboratories reagents is 0.996
(Fig. 4).
As it can be seen that the time-resolved fluorescence detection method the present invention is based on magnetic particle has the advantage that 1) high sensitivity；2)
Detection time is short, and 20min goes out to report；3) sample requirements are few, and a loading only needs 50 μ L；4) easy to operate, save manpower.It is real
Existing remote results inquiry, plant maintenance, troubleshooting, quality monitoring improve after-sales service efficiency, reduce maintenance cost.
Embodiment 4
Time-resolved fluoroimmunoassay detection method based on magnetic particle prepares antibody against swine fever virus detection kit, it includes
Main component have: reagent strip, negative control sera and positive control serum.The structural schematic diagram of the reagent strip is shown in attached drawing 1.
The reagent strip main component has the immunomagnetic beads of swine fever virus recombinant antigen E2 albumen, europium mark goat-anti pig anti-
Body, Sample dilution, cleaning solution, enhancement solution；
(1) the immunomagnetic beads preparation
1. washing magnetic bead
3 μm of carboxyl magnetic beads of 1mg diameter are drawn into 1.5ml centrifuge tube, centrifuge tube is placed in magnetic frame, magnetic force is utilized
Frame carry out magnetic bead and buffer separation, with 2- (N- morpholine) ethanesulfonic acid (MES) of 1ml 0.1M pH6.0 wash magnetic bead 3~
5 times.
2. magnetic bead activates
The MES of 1ml 0.1M pH6.0 is added in the magnetic bead good to above-mentioned washing, the 1- (3- bis- of 20 μ L 10mg/mL is added
Methylaminopropyl) -3- ethyl carbodiimide hydrochloride solution (EDC) and 20 μ L 50mg/ml n-hydroxysuccinimides (NHS),
Room temperature concussion reaction 0.5~1 hour.
3. the coupling of swine fever recombination E2 albumen and magnetic bead
Above-mentioned activated magnetic bead is placed on magnetic frame, is abandoned supernatant, is collected activated magnetic bead.0.5~1mg swine fever is added
E2 albumen, room temperature persistently rotate incubation 30~240 minutes, and the reaction time depends on the dentate and concentration of magnetic bead.
4. closing
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, collects immunomagnetic beads.Addition contains 5%BSA 0.05M pH8.0's
Tris-HCl buffer, is resuspended magnetic bead, and closing is not coupled the activated carboxyl site of swine fever recombination E2 albumen, reacts 30~60min.
5. storing
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, and 100 μ L immunomagnetic beads are added and save liquid.
The preservation liquid is containing 6% (w/v) BSA, 0.1% (v/v) Tween-20 and 0.1% (w/v) PEG20000
The Tris-HCl buffer of 0.05M pH 8.0.
The above-mentioned immunomagnetic beads prepared are diluted by a certain percentage, make final concentration of the 0.0025 of swine fever recombination E2 albumen
~0.0080g/L, 100 holes μ L/ are dispensed into the 1st hole (instrument connection 1) of reagent strip.
(2) the europium labeling antibody adds under the carbonate buffer solution of 0.05M pH9.0 using DTPA-Eu as marker
Enter DTPA-Eu and goat-anti pig secondary antibody, the carbonate buffer solution of 300 μ L 0.05M pH9.0, room are dissolved in after the mixing of 1:2 in mass ratio
Temperature concussion is overnight.Peak pipe is collected after being filtered with sephadex column.The above-mentioned europium mark secondary antibody prepared is diluted by a certain percentage,
Make final concentration of 0.0060~0.0075g/L of goat-anti pig antibody, 100 holes μ L/ are dispensed into the 3rd hole (marker 1) of reagent strip.
(3) Sample dilution described in is 0.01M pH7.4PBS, and 620 holes μ L/ dispense that (sample is dilute to the 8th hole of reagent strip
Release fluid apertures) in.
5 antibody against swine fever virus detection kit of embodiment and U.S. IDEXX hog cholera antibody detection kit testing result accord with
Conjunction rate compares
This method is compared the testing result of 100 clinical serums with IDEXX hog cholera antibody detection kit, positive
Coincidence rate 96.49%, negative match-rate 97.67%, total coincidence rate of the two are 97.00%.Testing result see the table below 4.By tying
The antibody against swine fever virus detection kit and import of time-resolved fluoroimmunoassay detection method preparation known to fruit based on magnetic particle
Kit has good correlation.
Table 4: this kit and IEDXX kit comparison result
Claims (9)
1. a kind of time-resolved fluoroimmunoassay detection method based on magnetic particle, characterized by comprising the steps of:
(1) antigen of specificity or antibody are coated on magnetic bead, form immunomagnetic beads；
(2) the rare earth ion chelate mark that will be formed without the more carboxyls of poly- amino of the bifunctional group of aromatic radical and rare earth ion
Remember the antigen or antibody of specificity, forms fluorescent marker；
(3) tested sample is added, is immunoreacted, immunomagnetic beads, tested sample and fluorescent marker is made to form compound；
(4) it by externally-applied magnetic field completes that free fluorescent marker is washed away to immuno magnetic cell separation and fixation using cleaning solution, then
Fluorescence enhancement solution is added to enhance fluorescent value；
(5) fluorescence is finally detected, is passed through according to the dose-response curve that built-in testing concentration and fluorescence signal value are fitted soft
Part calculates tested sample concentration.
2. time-resolved fluoroimmunoassay detection method according to claim 1, which is characterized in that the immunomagnetic beads are
The covalent coupling object of 0.1 ~ 3 μm of diameter of superparamagnetic particles and monoclonal antibody or antigen with modified with functional group；Wherein
The magnetic particle is carboxyl magnetic bead, amino magnetic bead, hydroxyl magnetic bead, tosyl magnetic bead, NHS magnetic bead, albumin A magnetic bead, egg
One or more of white G magnetic bead, anti-mouse IgG magnetic bead, hydrophilic magnetic bead, hydrophobic magnetic bead；Either immunomagnetic beads are to pass through chain
Enzyme is affine, and biscuit porcelain microballon reacts the immunomagnetic beads to be formed with biotinylated antibody.
3. time-resolved fluoroimmunoassay detection method according to claim 2, which is characterized in that the system of the immunomagnetic beads
Preparation Method is washed 0.1 ~ 3 μm of diameter of magnetic particle and corresponding antibody or antigen, activation, is coupled, closes and be prepared.
4. time-resolved fluoroimmunoassay detection method according to claim 1, which is characterized in that step (2) will be described to gather
The antigen or antibody for the chelate labels specificity that the more carboxyls of amino and rare earth ion are formed, form the mode of fluorescent marker
Have: directly Rare Earth Chelate being connected by chemical bond and biological raw material；Or Rare Earth Chelate by chemical bond with
Streptavidin connects, and antigen or antibody mark biotin, and last Rare Earth Chelate passes through biotin and streptavidin
Reaction, connects with antigen or antibody.
5. time-resolved fluoroimmunoassay detection method according to claim 1, which is characterized in that step (2) it is described without
The more carboxyls of poly- amino of the bifunctional group of aromatic radical are different sulphur nitrile acid phenyl-ethylenediamine tetra-acetic acid, different sulphur nitrile acid-Diethylenetriamine
Base tetraacethyl, Diethylenetriamine base pentaacetic acid or N- (the different sulfydryl benzyl of P-)-diethylenetriamine tetraacethyl；The rare earth ion is
Eu3+、Sm3+, Tb3+Or Dy3+。
6. time-resolved fluoroimmunoassay detection method according to claim 1, which is characterized in that step (2) described rare earth
Ion chelate complex labelled antibody or antigen preparation process are as follows: the mass ratio of antigen or antibody and rare earth ion chelate be 1:1 ~
10:1；Under the carbonate buffer solution of 0.05M pH9.0, rare earth ion chelate is added and dialysed antigen or antibody, mixing
It is dissolved in the carbonate buffer solution of 0.05M pH9.0 afterwards, room temperature concussion is overnight；Collect peak pipe after purification with sephadex column；It will
The above-mentioned fluorescent marker dilution prepared, it is spare.
7. time-resolved fluoroimmunoassay detection method according to claim 1, which is characterized in that step (2) is described immune
The temperature of reaction is 25 DEG C ~ 37 DEG C, and humidity is 30 ~ 75%RH, is shaking, is carrying out under oscillation.
8. time-resolved fluoroimmunoassay detection method according to claim 1, which is characterized in that step (4) described cleaning
The formula of liquid are as follows: 7.2 ~ 7.5 0.025M Tris-Hcl of 0 .8 ~ 1.5% Tween-20,0.05%Proclin300, pH buffering
Liquid.
9. time-resolved fluoroimmunoassay detection method according to claim 1, which is characterized in that step (4)
The formula of the fluorescence enhancement solution are as follows: 0.0002 ~ 0.0009% β-NTA, 0.0024% TOPO, 0.08% acetic acid, 0.05%
Triton X-100 composition, with appropriate Potassium Hydrogen Phthalate adjustment pH be 3.0-3.2 aqueous solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811025038.9A CN109164257A (en) | 2018-09-04 | 2018-09-04 | A kind of time-resolved fluoroimmunoassay detection method based on magnetic particle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811025038.9A CN109164257A (en) | 2018-09-04 | 2018-09-04 | A kind of time-resolved fluoroimmunoassay detection method based on magnetic particle |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109164257A true CN109164257A (en) | 2019-01-08 |
Family
ID=64894071
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811025038.9A Pending CN109164257A (en) | 2018-09-04 | 2018-09-04 | A kind of time-resolved fluoroimmunoassay detection method based on magnetic particle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109164257A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110836973A (en) * | 2019-09-09 | 2020-02-25 | 浙江博实生物科技有限公司 | Double-labeling kit for detecting troponin and compound, and preparation and detection methods thereof |
| CN111024940A (en) * | 2019-12-31 | 2020-04-17 | 广州源起健康科技有限公司 | Time-resolved fluorescence immunoassay method based on gold magnetic particles |
| CN111122859A (en) * | 2019-11-14 | 2020-05-08 | 南方医科大学 | Double-label anti-HIV and HCV time-resolved fluorescence immunoassay method based on immunomagnetic beads |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103575893A (en) * | 2013-10-14 | 2014-02-12 | 广州市疾病预防控制中心 | Method for rapidly detecting shellfish toxin |
| CN106771266A (en) * | 2017-01-12 | 2017-05-31 | 广州市丰华生物工程有限公司 | Inhibin A immue quantitative detection reagent boxes and preparation method thereof |
| CN207036854U (en) * | 2017-08-15 | 2018-02-23 | 广州源起健康科技有限公司 | A kind of Full-automatic chemiluminescence immunoassay analysis meter |
-
2018
- 2018-09-04 CN CN201811025038.9A patent/CN109164257A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103575893A (en) * | 2013-10-14 | 2014-02-12 | 广州市疾病预防控制中心 | Method for rapidly detecting shellfish toxin |
| CN106771266A (en) * | 2017-01-12 | 2017-05-31 | 广州市丰华生物工程有限公司 | Inhibin A immue quantitative detection reagent boxes and preparation method thereof |
| CN207036854U (en) * | 2017-08-15 | 2018-02-23 | 广州源起健康科技有限公司 | A kind of Full-automatic chemiluminescence immunoassay analysis meter |
Non-Patent Citations (2)
| Title |
|---|
| 任志奇: "基于磁珠固相载体的时间分辨荧光免疫分析技术平台的建立及临床应用", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 张洁等: "时间分辨荧光免疫分析法检测心肌肌钙蛋白Ⅰ方法学的建立及临床应用", 《中国免疫学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110836973A (en) * | 2019-09-09 | 2020-02-25 | 浙江博实生物科技有限公司 | Double-labeling kit for detecting troponin and compound, and preparation and detection methods thereof |
| CN111122859A (en) * | 2019-11-14 | 2020-05-08 | 南方医科大学 | Double-label anti-HIV and HCV time-resolved fluorescence immunoassay method based on immunomagnetic beads |
| CN111024940A (en) * | 2019-12-31 | 2020-04-17 | 广州源起健康科技有限公司 | Time-resolved fluorescence immunoassay method based on gold magnetic particles |
| CN111024940B (en) * | 2019-12-31 | 2024-01-16 | 广州源起健康科技有限公司 | Time-resolved fluorescence immunoassay method based on gold magnetic particles |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6376187B1 (en) | Electronically-indexed solid-phase assay for biomolecules | |
| CN100367034C (en) | Method for measuring immunologic colloidal gold particle fluorescence quenching | |
| US6087188A (en) | Two-site immunoassay for an antibody with chemiluminescent label and biotin bound ligand | |
| CN107884567A (en) | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit | |
| CN109541227A (en) | A kind of detection pepsinogen Cgene kit and preparation method thereof | |
| CN109164257A (en) | A kind of time-resolved fluoroimmunoassay detection method based on magnetic particle | |
| CN109541202A (en) | A kind of detection anti-HBs kit and preparation method thereof | |
| CN107831163A (en) | A kind of chemiluminescence detection kit of thyroglobulin and preparation method thereof | |
| CN108398563A (en) | A kind of double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits | |
| CN113156119A (en) | Method for detecting coronavirus by adopting angiotensin converting enzyme II (ACE2) | |
| CN105195243A (en) | Magnetic particulate chemiluminescent micro-fluidic chip for quantitatively detecting myohemoglobin | |
| CN112362867A (en) | Detection method using aggregation-induced emission combined immunomagnetic beads and kit thereof | |
| CN101551390A (en) | Immuomagnetic bead chromatographic test strip for rapidly detecting algae toxin and preparation method thereof | |
| CN205193076U (en) | Biotin - rapid detection cards of avidin system | |
| CN108181287A (en) | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects H-FABP kits | |
| CN108535486A (en) | A kind of chloramphenicol immunofluorescence assay method based on europium label | |
| CA2149340C (en) | Two-site immunoassay for an antibody with chemiluminescent label and biotin bound ligand | |
| CN109342718A (en) | A kind of magnetic microparticle chemiluminescence detection method | |
| JP3713087B2 (en) | Hapten immunoassay, hapten tracer antibody complex usable therefor, and method for producing the same | |
| CN108333370A (en) | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI kits | |
| CN109541226A (en) | A kind of pepsinogen Cgene/II bigeminy check reagent box and preparation method thereof | |
| CN105911041B (en) | A kind of time-resolved fluorescence detection method | |
| KR20010025027A (en) | Immunoassay reagents and immunoassay method | |
| CN109541228A (en) | A kind of detection Pepsinogen II kit and preparation method thereof | |
| CN109541230A (en) | A kind of detection anti-HBc kit and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190108 |