CN108398563A - A kind of double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits - Google Patents
A kind of double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits Download PDFInfo
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Abstract
The invention discloses a kind of double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively to detect cTnI and H FABP kits, includes H FABP monoclonal antibody solutions, cleaning solution and the enhancement solution of the immunomagnetic beads of coating cTnI monoclonal antibodies, the coating immunomagnetic beads of H FABP monoclonal antibodies, the calibration object solution of cTnI and H FABP, the cTnI monoclonal antibody solutions of europium label and samarium label.The described coating cTnI and H FABP immunomagnetic beads are the super-paramagnetic bead antibody covalent coupling object with cTnI, H FABP respectively of 1 ~ 3 μm of the diameter with modified with functional group.The sensitivity of the high sensitivity of the present invention, cTnI is 0.05ng/mL, and the sensitivity of H FABP is 1ng/mL, and serum (slurry) sample need not dilute；Detection time is short, and 30min goes out report；Sample requirements are few, and a loading only needs 100 μ L；Mating full-automatic Timed-resolved fluoroimmunoassay instrument, it is easy to operate, save manpower.
Description
Technical field
The present invention relates to external reagent detection technique fields, specifically, the present invention relates to a kind of double labelling time resolutions
Fluorescence immunoassay magnetic particle quantitatively detects cTnI and H-FABP kits.
Background technology
It falls ill within the scope of acute myocardial infarction (acute myocardial infarction, AMI) world in recent years and persistently rises
Height, and to the treatment of AMI, the effect of reperfusion as treatment and time in significantly negatively correlated, early detection AMI to it is timely, correctly control
It treats very crucial.The biochemical indicator of common diagnosis AMI has myoglobins (myoglobin, MYO), troponin at present
(troponin I, cTnI or troponin T, cTnT) and creatine kinase isozyme (creatine kinasemass, CK-
MB).All there is certain defect, MYO half-life shorts in these indexs, peak value is arrived to the sensitivity or specificity that early diagnose AMI
Up to morning, but poor specificity is easy to be interfered, and obtains the inspection result of false positive；CK-MB specificity is relatively good, but partly declines
Phase with respect to MYO long, needs the longer time that could feed back the state of an illness, postpones the diagnosing and treating of doctor.CTnI is the current diagnosis heart
The goldstandard of flesh infarct, but cTnI is released into evening time of blood in myocardial damage, just has diagnosis after the 6h that generally also to fall ill
Value.For AMI patient, time is life, in order to realize the EARLY RECOGNITION to chest pain patients and carry out risk stratification,
We need faster to provide the detecting instrument of accurate result and faster myocardial damage early diagnosis marker.
H-FABP (heart fatty acid-binding protein, H-FABP) is a kind of new
The Applications of Cardiac Markers of type, and study the more sensitive biochemical indicators high, specificity is high of early diagnosis AMI both at home and abroad at present.
After myocardial cell injury, H-FABP can be released into blood circulation rapidly prior to cardiac troponin.Most sent out early in AMI
After 30 minutes after being ill, H-FABP can be detected in blood plasma.Therefore, joint-detection H-FABP, cTnI makes H-FABP, cTnI
The time window phase of diagnosis is complementary, makes up the deficiency of individual event detection, will greatly improve AMI diagnosis, in AMI early diagnosis, treats
It plays an increasingly important role in effect observation and Index for diagnosis.
For this purpose, double labelling Time-resolved fluoroimmunoassay is combined by the present invention with magnetic particle, one kind is provided
Double labelling time resolved fluoro-immunoassay kit based on magnetic particle and preparation method thereof and detection method, can not only be same
When detection sample in H-FABP and cTnI contents, but also ensure that testing result is more accurate and reliable, there is higher detection
Sensitivity and specificity, and reached preferable performance parameter.
Invention content
The object of the present invention is to provide a kind of double labelling magnetic bead time resolutions of low cost, easy to operate, accurate, sensitive
Fluorescence immunoassay quantitatively detects cTnI and H-FABP kits.
To achieve the above object, present invention employs following technical solutions：
A kind of double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits, including：Coating
The calibration object of the immunomagnetic beads of cTnI monoclonal antibodies, the immunomagnetic beads for being coated with H-FABP monoclonal antibodies, cTnI and H-FABP
H-FABP monoclonal antibody solutions, cleaning solution and the enhancement solution of solution, the cTnI monoclonal antibody solutions of europium label and samarium label.
In above-mentioned double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits, the packet
By cTnI and H-FABP immunomagnetic beads be 1~3 μm of the diameter with modified with functional group super-paramagnetic bead respectively with cTnI, H-FABP
Antibody covalent coupling object, wherein used in magnetic bead be carboxyl magnetic bead, amino magnetic bead, hydroxyl magnetic bead, tosyl magnetic bead,
In NHS magnetic beads, streptavidin magnetic bead, albumin A magnetic bead, Protein G magnetic bead, anti-mouse IgG magnetic beads, hydrophilic magnetic bead, hydrophobic magnetic bead
One or more.
It is described to exempt from above-mentioned double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits
The preparation method of epidemic disease magnetic bead is that the magnetic particle of the activation of 1~3 μm of diameter is washed with the corresponding monoclonal antibody, activates, is even
Connection, closing are prepared.
The preparation method of the cTnI immunomagnetic beads is：It draws in carboxyl magnetic bead to centrifuge tube, centrifuge tube is placed on magnetic
In power frame, the separation of magnetic bead and buffer solution is carried out using magnetic frame, is washed with 2- (N- morpholines) ethanesulfonic acid of 0.1M pH6.0
Magnetic bead 3~5 times；2- (N- morpholines) ethanesulfonic acid of 0.1M pH6.0 is added in the magnetic bead good to above-mentioned washing, 20mg/mL is added
1- (3- dimethylamino-propyls) -3- ethyl carbodiimide hydrochlorides solution and 20mg/ml n-hydroxysuccinimides, room temperature shake
Swing reaction；Activated magnetic bead is placed on magnetic frame, supernatant is abandoned, collects activated magnetic bead；Monoclonal antibody, room temperature is added
Lasting rotation is incubated 3~5 hours, and the reaction time depends on the dentate and concentration of magnetic bead；Above-mentioned magnetic bead is placed on magnetic frame, is abandoned
Supernatant collects immunomagnetic beads；The Tris-HCl buffer solutions containing 5%BSA 0.1M pH 8.0 are added, magnetic bead are resuspended, closing is not
The activated carboxyl site of conjugated monoclonal antibodies is reacted 30~60 minutes；Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, is added
Immunomagnetic beads preserve liquid.The preservation liquid is containing 5% (w/v) BSA, 10% (w/v) trehalose, 0.1% (v/v) Tween-20
0.05M pH 8.0 Tris-HCl buffer solutions.
The preparation method of the H-FABP immunomagnetic beads is：It draws in carboxyl magnetic bead to centrifuge tube, centrifuge tube is placed on
In magnetic frame, the separation of magnetic bead and buffer solution is carried out using magnetic frame, with 2- (N- morpholines) second sulphur of 1ml 0.1M pH5.0
Acid elution magnetic bead 3~5 times；2- (N- morpholines) ethanesulfonic acid of 1ml 0.1M pH5.0 is added in the magnetic bead good to above-mentioned washing,
1- (3- the dimethylamino-propyls) -3- ethyl carbodiimide hydrochlorides solution and 30mg/ml N- hydroxysuccinimidyl acyls of 10mg/mL is added
Imines, room temperature concussion reaction；Activated magnetic bead is placed on magnetic frame, supernatant is abandoned, collects activated magnetic bead；Dan Ke is added
Grand antibody, room temperature persistently rotate incubation 5~8 hours, and the reaction time depends on the dentate and concentration of magnetic bead；Above-mentioned magnetic bead is placed in
On magnetic frame, supernatant is abandoned, collects immunomagnetic beads；The Tris-HCl buffer solutions containing 5%BSA 0.05M pH 8.0 are added, are resuspended
Magnetic bead closes the activated carboxyl site of non-conjugated monoclonal antibodies, reacts 30~60min；Above-mentioned magnetic bead is placed on magnetic frame, is abandoned
Supernatant is added immunomagnetic beads and preserves liquid.The preservation liquid is containing 8% (w/v) BSA, 5% (w/v) sucrose, 0.5% (v/v)
The Tris-HCl buffer solutions of the 0.05M pH 8.0 of Tween-20.
In the preparation method that the europium marks cTnI antibody：CTnI antibody and Eu3+Mass ratio be 5:1.
In the preparation method that the samarium marks H-FABP antibody：H-FABP antibody and Sm3+Mass ratio be 2:1.
The formula of the cleaning solution is：0.8~1.5%Tween-20,0.02%Proclin300, pH 7.2~
7.50.05M Tris-Hcl buffer solutions.
The formula of the enhancement solution is：0.03% sodium acetate, 0.0002~0.0009% β-NTA, 0.0024%TOPO,
0.08% acetic acid, 0.1% absolute ethyl alcohol, 0.05% Triton X-100 aqueous solution.
The preparation method of the cTnI calibration objects is：With containing 1.5%Tween-20,0.02%Proclin300, pH7.5
0.02M Tris-Hcl buffer solutions are by cTnI antigen diluents to 0,0.05,0.1,0.5,1,5,25,50ng/ml.
The preparation method of the H-FABP calibration objects is：Delayed with containing 0.02%Proclin300,7.5 0.02M PBS of pH
Fliud flushing is by H-FABP antigen diluents 0,1,5,10,50,100,180ng/ml.
The excitation wavelength of the fluorescent material is 300~350nm, and launch wavelength is 500~650nm.
Double labelling magnetic bead time-resolved fluoroimmunoassay of the present invention quantitatively detects the inspection of cTnI and H-FABP kits
Survey principle is sandwich method, is preinstalled with cTnI and H-FABP immunomagnetic beads in the reacting hole of reagent strip, when test, first by sample to be tested
(serum or blood plasma) or calibration object are added in the reacting hole of reagent, add europium mark cTnI antibody and samarium mark H-FABP antibody,
Oscillation incubation 20min forms immunomagnetic beads-antigen-europium mark/samarium labeling antibody compound.After washing, enhancement solution is added, is being swashed
Under the action of shining, fluorescent material emits the optical signal of certain wavelength, identifies that measured object is got in sample by Immunofluorescence test instrument
More, the fluorescence signal intensity of generation is stronger.By cTnI and H-FABP concentration and fluorescence signal value fitted dose-response curve, i.e.,
The concentration of cTnI and H-FABP in unknown sample can be obtained by this measured value.
Compared with prior art, the present invention has the advantages that：
(1) magnetic separation technique is used, the immune complex of the formation Direct precipitation in externally-applied magnetic field, being not required to centrifugation can
Immune complex is detached with unbonded material.Due to magnetic bead has the bonded area of bigger and can disperse in the liquid phase fully instead
It answers, greatly improves detection range, shorten the reaction time, improve sensitivity.Since magnetic bead and antigen or antibody are covalent coupling, gram
The unstability of physical absorption is taken, therefore the immunomagnetic beads holding time is long and more stable.
(2) Time-resolved fluoroimmunoassay is used, using lanthanide chelate europium, terbium, samarium, dysprosium as marker,
It has wider excitation spectrum, relatively narrow emission spectrum, advantageously reduces background, improves sensitivity；Ultraviolet excitation have compared with
High quantum production rate, larger Stokes displacements, 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.
(3) mating full-automatic detecting instrument realizes automation mechanized operation, can detect simultaneously in one or more parts sample cTnI and
H-FABP contents, sample dosage is few, it is easy to operate quickly (result can be gone out within 30 minutes), for early diagnose AMI provide it is important according to
According to having saved valuable time for patient.
Description of the drawings
Fig. 1 be double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detect cTnI and H-FABP kits using reagent strip into
Row detection, the structure schematic top plan view of reagent strip.
Fig. 2 be double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detect cTnI and H-FABP kits using reagent strip into
Row detection, the structural schematic diagram of reagent strip.
1, instrument connection 1,2, instrument connection 2,3, fluorescent marker Isosorbide-5-Nitrae, fluorescent marker 2,6, washing lotion, 7, washing lotion, 8, sample
Dilution 1,9, Sample dilution 2,12, enhancement solution, 5,10,11,13 be preparation hole.
Specific implementation mode
CTnI and H-FABP coating magnetic bead of the present invention is the superparamagnetic of 1~3 μm of the diameter with modified with functional group
The covalent coupling object of pearl and albumen, wherein used magnetic bead has carboxyl magnetic bead, amino magnetic bead, hydroxyl magnetic bead, tosyl
Magnetic bead, NHS magnetic beads, streptavidin magnetic bead, albumin A magnetic bead, Protein G magnetic bead, anti-mouse IgG magnetic beads, hydrophilic magnetic bead, hydrophobic magnetic
Pearl etc. is one such or several.
Below in conjunction with the drawings and specific embodiments, the present invention will be described in detail.
Embodiment 1
In this embodiment, double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits by trying
Agent item and cTnI, H-FABP calibration object composition.
Wherein, by instrument connection 1, (cTnI monoclonal antibodies are coated with carboxyl magnetic bead to reagent strip, H-FABP monoclonal antibodies are coated with
Carboxyl magnetic bead), the 3rd hole (europium mark cTnI monoclonal antibodies, samarium mark H-FABP monoclonal antibodies),ClearlyWashing lotion hole 6 and 7, enhancement solution
Hole 12 forms, as depicted in figs. 1 and 2.
The preparation process that the double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits is as follows,
Wherein
(1) preparation method of cTnI immunomagnetic beads：
1. washing magnetic bead
It draws in 1mg 1 μm of carboxyl magnetic bead to 1.5ml centrifuge tubes of diameter, centrifuge tube is placed in magnetic frame, magnetic force is utilized
Frame carry out magnetic bead and buffer solution separation, with 2- (N- morpholines) ethanesulfonic acids (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 are added
Methylaminopropyl) -3- ethyl carbodiimide hydrochlorides 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, abandons supernatant, collects activated magnetic bead.0.05~1mg is added
CTnI monoclonal antibodies, room temperature persistently rotate incubation 3~5 hours, 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 0.1M pH8.0's
Magnetic bead is resuspended in Tris-HCl buffer solutions, and closing is not coupled the activated carboxyl site of cTnI monoclonal antibodies, 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 preserve liquid.
The preservation liquid is containing 5% (w/v) BSA, 10% (w/v) trehalose, the 0.05M of 0.1% (v/v) Tween-20
The Tris-HCl buffer solutions of pH 8.0.
By the above-mentioned immunomagnetic beads dilution prepared, make final concentration of 0.0025~0.0100g/ of cTnI monoclonal antibodies
L, in packing to the 1st hole (instrument connection 1) of reagent strip.
(2) preparation method of H-FABP immunomagnetic beads：
1. washing magnetic bead
It draws in 1mg 3 μm of carboxyl magnetic beads to 1.5ml centrifuge tubes of diameter, centrifuge tube is placed in magnetic frame, magnetic force is utilized
Frame carry out magnetic bead and buffer solution separation, with 2- (N- morpholines) ethanesulfonic acids (MES) of 1ml 0.1M pH5.0 wash magnetic bead 3~
5 times.
2. magnetic bead activates
The MES of 1ml 0.1M pH5.0 is added in the magnetic bead good to above-mentioned washing, the 1- (3- bis- of 20 μ L 10mg/mL are added
Methylaminopropyl) -3- ethyl carbodiimide hydrochlorides solution (EDC) and 20 μ L 30mg/ml n-hydroxysuccinimides (NHS),
Room temperature concussion reaction 0.5~1 hour.
3. the coupling of H-FABP monoclonal antibodies and magnetic bead
Above-mentioned activated magnetic bead is placed on magnetic frame, abandons supernatant, collects activated magnetic bead.0.05~1mg H- are added
FABP monoclonal antibodies, room temperature persistently rotate incubation 5~8 hours, 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 solutions, are resuspended magnetic bead, and closing is not coupled the activated carboxyl sites of H-FABP monoclonal antibodies, and reaction 30~
60min。
5. storing
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, and 200 μ L immunomagnetic beads are added and preserve liquid.
The preservation liquid is containing 8% (w/v) BSA, 5% (w/v) sucrose, the 0.05M pH of 0.5% (v/v) Tween-20
8.0 Tris-HCl buffer solutions.
The immunomagnetic beads dilution prepared above-mentioned, make H-FABP monoclonal antibodies final concentration of 0.0080~
0.0200g/L, in packing to the 1st hole (instrument connection 1) of reagent strip.
(3) europium marks cTnI preparation method for antibody：
1. the preparation of europium mark cTnI monoclonal antibodies
(1) 1mg cTnI monoclonal antibodies are taken out and are placed in 30KD ultra-filtration centrifuge tubes, 10000rpm centrifuges 10min, discards filter
Liquid.
(2) 200 μ L, 10000rpm centrifugation 10min of label buffer solution (0.05M pH9.0 carbonate buffer solutions) are added, abandon
Go filtrate.Repeat this operation 4~5 times.
(3) centrifuge tube filter membrane is inverted, 3000rpm centrifuges 6min, collects the antibody of concentration.200 μ L label bufferings are added
Liquid stands 3~5min, then centrifuge tube filter membrane is inverted, and 3000rpm centrifuges 6min, collects the SA of concentration.
(4) according to mass ratio cTnI monoclonal antibodies：Europium chelate DTTA-Eu=5:1 ratio mixes well, and is put into rotation
Turn incubator, reacts at room temperature 16~24 hours.
2. the purifying of europium mark cTnI monoclonal antibodies
With SepHadex TM G-75 gel column purification europium mark cTnI monoclonal antibodies, europium mark cTnI monoclonal antibody preservative agents are 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.
By the above-mentioned europium mark cTnI monoclonal antibodies dilution prepared, make final concentration of 0.005~0.010g/L of cTnI monoclonal antibodies, point
It is filled in the 3rd hole of reagent strip.
The excitation wavelength of the fluorescent material europium is 340nm, launch wavelength 615nm.
(4) samarium marks H-FABP preparation method for antibody：
1. the preparation of samarium mark H-FABP monoclonal antibodies
(1) 1mg H-FABP monoclonal antibodies are taken out and are placed in 30KD ultra-filtration centrifuge tubes, 10000rpm centrifuges 10min, discards filtrate.
(2) 200 μ L, 10000rpm centrifugation 10min of label buffer solution (0.05M pH9.0 carbonate buffer solutions) are added, abandon
Go filtrate.Repeat this operation 4~5 times.
(3) centrifuge tube filter membrane is inverted, 3000rpm centrifuges 6min, collects the antibody of concentration.200 μ L label bufferings are added
Liquid stands 3~5min, then centrifuge tube filter membrane is inverted, and 3000rpm centrifuges 6min, collects the SA of concentration.
(4) according to mass ratio H-FABP monoclonal antibodies：Samarium chelate DTTA-Sm=2:1 ratio mixes well, and is put into rotation training
Device is supported, is reacted at room temperature 20~24 hours.
2. the purifying of samarium mark H-FABP monoclonal antibodies
With SepHadex TM G-75 gel column purification samarium mark H-FABP monoclonal antibodies, samarium mark H-FABP monoclonal antibodies are then added and preserve
Agent (10% (w/v) BSA+3% (w/v) Proclin300), makes BSA and Proclin300 final concentration of 0.5%, is filtered through 0.22 μm
Membrane filtration, 4 DEG C store for future use.
By the above-mentioned samarium mark H-FABP monoclonal antibodies dilution prepared, make final concentration of 0.010~0.030g/ of H-FABP monoclonal antibodies
L, in packing to the 4th hole of reagent strip.
The excitation wavelength of the fluorescent material europium is 340nm, launch wavelength 642nm.
(5) preparation method of washing lotion：
It prepares and contains 0.8~1.5%Tween-20,7.2~7.5 0.05M Tris- of 0.02%Proclin300, pH
Hcl buffer solutions dispense prepared solution into washing lotion hole 6 and 7 with 2000 μ L of every hole.
(6) preparation method of enhancement solution：
Prepare containing 0.03% sodium acetate, 0.0002~0.0009% β-NTA, 0.0024%TOPO, 0.08% acetic acid,
0.1% absolute ethyl alcohol, 0.05% Triton X-100 aqueous solution.Prepared solution is dispensed with 400 μ L of every hole to examination
The 12nd hole of agent article (enhancing fluid apertures).
(7) calibration object preparation method：
The preparation method of the cTnI calibration objects is：With containing 1.5%Tween-20,0.02%Proclin300, pH7.5
0.02M Tris-Hcl buffer solutions are by cTnI antigen diluents to 0,0.05,0.1,0.5,1,5,25,50ng/ml.
The preparation method of the H-FABP calibration objects is：Delayed with containing 0.02%Proclin300,7.5 0.02M PBS of pH
Fliud flushing is by H-FABP antigen diluents 0,1,5,10,50,100,180ng/ml.
The making and detection of 2 reagent strip of embodiment
Reagent strip in the present embodiment, semi-finished product are assembled by following process：It is dispensed respectively the 1st, 3,6,7,12
50 μ LcTnI immunomagnetic beads and 50 μ LH-FABP immunomagnetic beads, 100 μ L europiums mark cTnI antibody and 100 μ L samarium mark H-FABP antibody,
2000 μ L cleaning solutions, 2000 μ L cleaning solutions, 400 μ L enhancement solutions, are then sealed with film sealing machine, be coated on the sealing plate film for
The product information mark of full-automatic fluorimetric analysis instrument scanning recognition includes company standard curve, batch, the date of manufacture, effective
Phase.Finished product reagent strip, the cTnI calibration objects dispensed and H-FABP calibration objects and other assembling fittings are at kit.
Pattern detection：
1. being loaded：
Sample to be tested or calibration object are put into the Load System of full-automatic instrument, reagent strip is inserted into reagent clamp bar slot,
The product information of instrument automatic identification sealing plate film.100 μ L samples to be tested are added in reagent strip instrument connection 1,50 μ L are then added
Europium mark cTnI antibody and 50 μ L samarium mark H-FABP antibody.
2. being incubated：
37 DEG C of concussions are incubated 20min after the completion of sample-adding.
3. washing：
After the completion of incubation, the automatic hole flushing of instrument 5 times, 200 holes μ L/ of each washing lotion.
4. enhancement solution is added：
After the completion of washing, 100 holes μ L/ of enhancement solution, 37 DEG C of incubation 3min are added.
5. detecting：
Reagent strip is pushed into darkroom, and instrument detects and goes out result automatically.
Full-automatic magnetic beads time-resolved fluorescence immunoassay instrument for test agent item includes sample loading system, bar code
Reading system, sample adding system, incubation system, fluorescent light source detecting system and automatic software analysis and Control system.
Double labelling magnetic bead time-resolved fluoroimmunoassay of the present invention quantitatively detects cTnI and H-FABP and kit, inspection
Reagent strip need to be only inserted into full-automatic instrument by this when of test sample, and instrument is automatically performed sample-adding, incubation, Magneto separate, detection process, entirely
Journey only needs 30min to read examining report.
The comparison of embodiment 3 and commercial reagent box
The quick troponin-i assay kit of commercially available height (chemiluminescence particulate immunodetection) (Abbott Laboratories are respectively adopted
Trade Co., Ltd) and H-FABP immue quantitative detection reagent box (time-resolved fluoroimmunoassay) (Soviet Union
Zhou Xin waves Bioisystech Co., Ltd) (cTnI concentration ranges are 0.05 in the sample with the method detection sample in embodiment 1
~50ng/mL, H-FABP concentration range are 1~180ng/mL) in triplicate, verifying the testing result of the kit of the present invention just
True property, as a result see the table below 1.
1 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.Illustrate this
The invention double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits, has following advantage：
1) sensitivity of high sensitivity, cTnI is 0.05ng/mL, and the sensitivity of H-FABP is 1ng/mL, and serum (slurry) sample does not need
Dilution；2) detection time is short, and 30min goes out report；3) sample requirements are few, and a loading only needs 100 μ L；4) when mating full-automatic
Between resolved immuno analyzer, it is easy to operate, save manpower.
Claims (9)
1. a kind of double labelling magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI and H-FABP kits, it is characterised in that packet
It includes：It is coated with the immunomagnetic beads of cTnI monoclonal antibodies, the immunomagnetic beads for being coated with H-FABP monoclonal antibodies, cTnI and H-FABP
Calibration object solution, europium label cTnI monoclonal antibody solutions and samarium label H-FABP monoclonal antibody solutions, cleaning solution and
Enhancement solution.
2. double labelling magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects cTnI and H-FABP kits,
It is characterized in that, the coating cTnI and H-FABP immunomagnetic beads are the superparamagnetic of 1 ~ 3 μm of the diameter with modified with functional group
The pearl antibody covalent coupling object with cTnI, H-FABP respectively, wherein used in magnetic bead be carboxyl magnetic bead, amino magnetic bead, hydroxyl
Magnetic bead, tosyl magnetic bead, NHS magnetic beads, streptavidin magnetic bead, albumin A magnetic bead, Protein G magnetic bead, anti-mouse IgG magnetic beads,
One or more of hydrophilic magnetic bead, hydrophobic magnetic bead.
3. double labelling magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects cTnI and H-FABP kits,
It is characterized in that, the preparation method of the immunomagnetic beads of the coating cTnI monoclonal antibodies is：Carboxyl magnetic bead is drawn to centrifuge tube
In, centrifuge tube is placed in magnetic frame, the separation of magnetic bead and buffer solution is carried out using magnetic frame, with the 2- of 0.1M pH6.0
（N- morpholines）Ethanesulfonic acid washs magnetic bead 3 ~ 5 times；The 2- of 0.1M pH6.0 is added in the magnetic bead good to above-mentioned washing（N- morphines
Quinoline）1- (3- the dimethylamino-propyls) -3- ethyl carbodiimide hydrochlorides solution and 20mg/ml N- of 20mg/mL is added in ethanesulfonic acid
HOSu NHS, room temperature concussion reaction；Activated magnetic bead is placed on magnetic frame, supernatant is abandoned, collects activated magnetic
Pearl；Monoclonal antibody is added, room temperature persistently rotates incubation 3 ~ 5 hours；Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, and collection is exempted from
Epidemic disease magnetic bead；The Tris-HCl buffer solutions containing 5%BSA 0.1M pH 8.0 are added, magnetic bead is resuspended, it is anti-to close non-conjugated monoclonal
The activated carboxyl site of body is reacted 30 ~ 60 minutes；Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, and immunomagnetic beads are added and preserve
Liquid.
4. double labelling magnetic bead time-resolved fluoroimmunoassay as claimed in claim 3 quantitatively detects cTnI and H-FABP kits,
It is characterized in that, the preservation liquid is containing 5%（w/v）BSA, 10%（w/v）Trehalose, 0.1%（v/v）Tween-20's
The Tris-HCl buffer solutions of 0.05M pH 8.0.
5. double labelling magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects cTnI and H-FABP kits,
It is characterized in that, the preparation method of the immunomagnetic beads of the coating H-FABP monoclonal antibodies is：Carboxyl magnetic bead is drawn to centrifugation
Centrifuge tube is placed in magnetic frame by Guan Zhong, the separation of magnetic bead and buffer solution is carried out using magnetic frame, with 1ml 0.1M pH5.0
2-（N- morpholines）Ethanesulfonic acid washs magnetic bead 3 ~ 5 times；The 2- of 1ml 0.1M pH5.0 is added in the magnetic bead good to above-mentioned washing
（N- morpholines）Ethanesulfonic acid, be added 10mg/mL 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide hydrochlorides solution and
30mg/ml n-hydroxysuccinimides, room temperature concussion reaction；Activated magnetic bead is placed on magnetic frame, supernatant is abandoned, is collected
Activated magnetic bead；Monoclonal antibody is added, room temperature persistently rotates incubation 5 ~ 8 hours；Above-mentioned magnetic bead is placed on magnetic frame, is abandoned
Clearly, immunomagnetic beads are collected；The Tris-HCl buffer solutions containing 5%BSA 0.05M pH 8.0 are added, magnetic bead is resuspended, closing is not even
30 ~ 60min is reacted in the activated carboxyl site of receipts or other documents in duplicate clonal antibody；Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, is added immune
Magnetic bead preserves liquid.
6. double labelling magnetic bead time-resolved fluoroimmunoassay as claimed in claim 5 quantitatively detects cTnI and H-FABP kits,
It is characterized in that, the preservation liquid is containing 8%（w/v）BSA, 5%（w/v）Sucrose, 0.5%（v/v）The 0.05M of Tween-20
The Tris-HCl buffer solutions of pH 8.0.
7. double labelling magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects cTnI and H-FABP kits,
It is characterized in that, in the cTnI monoclonal antibody solutions of the europium label, cTnI antibody and Eu3+Mass ratio be 5:1；Institute
In the H-FABP monoclonal antibody solutions for the samarium label stated, H-FABP antibody and Sm3+Mass ratio be 2:1.
8. double labelling magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects cTnI and H-FABP kits,
It is characterized in that, the formula of the cleaning solution is：The .5% of 0 .8 ~ 1 Tween-20,0.02%Proclin300, pH 7.2 ~ 7.5
0.05M Tris-Hcl buffer solutions；
The formula of the enhancement solution is：0.03% sodium acetate, 0.0002 ~ 0.0009% β-NTA, 0.0024% TOPO, 0.08% vinegar
Acid, 0.1% absolute ethyl alcohol, 0.05% Triton X-100 aqueous solution.
9. double labelling magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects cTnI and H-FABP kits,
It is characterized in that, the preparation method of the cTnI calibration objects is：With containing 1.5% Tween-20,0.02%Proclin300, pH
7.5 0.02M Tris-Hcl buffer solutions are by cTnI antigen diluents to 0,0.05,0.1,0.5,1,5,25,50ng/ml；
The preparation method of the H-FABP calibration objects is：With containing 0.02%Proclin300,7.5 0.02M PBS buffer solution of pH will
H-FABP antigen diluents 0,1,5,10,50,100,180ng/ml.
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| CN111122859A (en) * | 2019-11-14 | 2020-05-08 | 南方医科大学 | Dual-labeled anti-HIV and HCV time-resolved fluorescence immunoassay 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 |
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| JPWO2023276298A1 (en) * | 2021-07-01 | 2023-01-05 | ||
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