CN107192832A - A kind of immunochromatography reagent bar of fluorogenic quantitative detection AMH and preparation method thereof - Google Patents
A kind of immunochromatography reagent bar of fluorogenic quantitative detection AMH and preparation method thereof Download PDFInfo
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Abstract
A kind of fluorogenic quantitative detection AMH immuno-chromatographic test paper strip and preparation method thereof, the invention belongs to immune diagnostic technique field, it is an object of the invention to for the deficiencies in the prior art, the present invention uses following technical scheme：A kind of fluorogenic quantitative detection AMH immuno-chromatographic test paper strip, is sequentially overlapped on PVC bottom plates by sample pad, label pad, coated film, blotting paper and constituted, Avidin is coated with label pad；Nature controlling line is coated with the rabbit-anti Avidin antibody of specific recognition Avidin.The present invention has the following advantages：Detection line of the present invention and nature controlling line use independent reaction system, do not interfere with each other and influence, and calibrated by the way of T/C values, it is ensured that the degree of accuracy of test result.
Description
Technical field
The invention belongs to immune diagnostic technique field, and in particular to a kind of anti-Miao Le Shi pipe hormones of fluorogenic quantitative detection
（AMH）Immuno-chromatographic test paper strip and preparation method thereof.
Background technology
Anti- Miao Le Shi pipes hormone (anti-M ü llerian hormone, AMH) is transforming growth factor-β (TGF-β)
Superfamily member, is the glycoprotein that a kind of homodimer by 2 55kDa N-terminals and 2 12.5kDa C-terminals is constituted, for knot
Close the non-covalent bond of disulfide bond.For male, AMH hormones are generated by sustentacular cell of testis, start from embry ogenesis, and pass through
Wear life all the time, during embryonic development, AMH can cause Miao Le Shi pipes to be degenerated, and form normal male genetic pipeline.For female
Property for, AMH hormones are generated by follicular cell, until Menopause, and AMH hormonal readinesses are gradually decreased down can not
The level of detection.
Research shows：AMH maintains to play a significant role in Ovary reserve in regulation and control ovarian follicular growth and development, AMH water
Flat is in obvious positive correlation with Ovary reserve.Relative to the method for traditional prediction ovarian reserve and ovary responsiveness
（Age and Antral follicles mesh（AFC）, follicular stimulating hormone（FSH）And estradiol（E2）Deng）, detect that serum AMH has following advantage：
（1）Can earlier, the change of sensitiveer low reaction Ovary reserve；（2）Do not influenceed by pituitary gonadotropic hormone and menstrual period,
Numerical value change is little in the whole menstrual cycle；（3）In monitoring ovarian reserve power, predict joint embryo transfer technology in vitro fertilization
（IVF）Ovary responsiveness, prevention ovarian hyperstimulation syndrome（OHSS）, polycystic ovary syndrome（PCOS）Diagnosis in have
The incomparable advantage of other indexs of body.AMH is current prediction ovary responsiveness, assesses the optimal life of Ovary reserve
Thing mark.
Clinically AMH detection method has ELISA at present（ELISA）, Electrochemiluminescince and chemoluminescence method,
These methods have respective advantage and deficiency.ELISA method detection step is more, time-consuming, and the influence factor of operating process is more,
Easily cause false positive and false negative result.Therefore progressively substituted at present by chemoluminescence method, but this kind of method is totally-enclosed system,
Expensive maintenance and testing cost are high, it is necessary to special training instrument user of service, and are not suitable for single part and small lot inspection
Survey and use, be unfavorable for the extensive development of AMH detections at home at present.
In view of there is no the method for fast and accurately quantitatively detecting AMH at present, it can be used for it is an object of the invention to provide one kind
Quantitative detection AMH immuno-chromatographic test paper strip, the Ovary reserve for assessing and monitoring women, the reagent has
The advantages of simple, convenient quick, economy, accurate quantitative analysis, it is more suitable for extensively carrying out in each medical institutions.
The content of the invention
It is an object of the invention to for the deficiencies in the prior art, there is provided the simple, convenient quick, warp of one kind
Help, determine accurate AMH test strips.
To achieve the above object, the present invention uses following technical scheme：
A kind of fluorogenic quantitative detection AMH immuno-chromatographic test paper strip, is sequentially overlapped by sample pad, label pad, coated film, blotting paper
Constituted on PVC bottom plates, described label pad and described sample pad connect, described coated film and described label pad phase
Connect, described blotting paper and described coated film connect；The AMH monoclonals that fluorescent microsphere mark is coated with the label pad resist
The Avidin of body and fluorescent microsphere mark；The coated film include detection line and nature controlling line, the detection line be coated with it is described
The AMH monoclonal antibodies of fluorescent microsphere mark are in another AMH monoclonal antibodies of different epitopes, and the nature controlling line is coated with
The rabbit-anti Avidin antibody of specific recognition Avidin.
Preferably, the concentration of the AMH monoclonal antibodies of the fluorescent microsphere mark is 0.1 ~ 1.0 mg/mL, dilution ratio
For 5% ~ 20%.The concentration of the Avidin of the fluorescent microsphere mark is 0.1 ~ 1.0 mg/mL, and dilution ratio is 0.5% ~ 5%.It is described
The discharge rate of the label pad treatment fluid of the Avidin of AMH monoclonal antibodies and the fluorescent microsphere mark marked containing fluorescent microsphere is 3 ~ 6
μL/cm。
Preferably, the particle diameter of the fluorescent microsphere is 100 ~ 500 nm.The excitation wavelength of the fluorescent microsphere is 310 ~ 550
Nm, launch wavelength is 340 ~ 620 nm.
Preferably, the concentration of coated AMH monoclonal antibodies is 0.5 ~ 2 mg/mL, discharge rate in the coated film detection line
0.1~0.2μL/mm.The concentration of coated rabbit-anti Avidin antibody is 0.5 ~ 2 mg/mL, the μ of discharge rate 0.1 ~ 0.2 on the nature controlling line
L/mm。
The present invention also provides a kind of method of fluorogenic quantitative detection AMH immuno-chromatographic test paper strip, comprises the following steps：
（1）The preparation of fluorescent microsphere labelled protein
A certain amount of fluorescent microsphere is taken, 10000 ~ 15000 rpm are centrifuged 5 ~ 15 minutes, are centrifugally separating to obtain for the first time for the first time
Sediment is 0.1% ~ 1% with the phosphate buffers of 10 ~ 100 mM pH 6.0 ~ 7.0 regulation concentration, and ultrasonic disperse；Add dense eventually
Spend for 0.1 ~ 5 mg/mL carbodiimide（EDC）, mix, the N- hydroxysuccinimidyls acyl for adding final concentration of 0.1 ~ 5 mg/mL is sub-
Amine（NHS）, mix；10000 ~ 15000 rpm, second of centrifugation 5 ~ 15 minutes, second of centrifugation after being incubated at room temperature 20 ~ 40 minutes
The isolated sediment phosphate buffers of 10 ~ 100 mM pH 6.0 ~ 7.0 dissolve.By the fluorescent microsphere ultrasound after redissolution
It is scattered, it is separately added into room temperature after AMH monoclonal antibodies and Avidin, mixing according to the ratio of 0.1 ~ 1.0 mg/mL fluorescent microspheres
Rotate hybrid reaction 1.5 ~ 3 hours, 10000 ~ 15000 rpm third times centrifugation 5 ~ 15 minutes is centrifugally separating to obtain for the third time
Tris-HCl of the sediment containing 10 ~ 40 mM monoethanolamines and 0.05%-1% caseins（10 ~ 40mM, pH 7.0-8.0）Redissolve, surpass
Rotation hybrid reaction 0.5 ~ 1 hour after sound is scattered.The 4th centrifugation of 10000 ~ 15000 rpm 5 ~ 15 minutes, the 4th centrifugation point
Liquid is preserved from obtained sediment with microballoon to redissolve, 2 ~ 8 DEG C of preservations.
（2）The pretreatment of sample pad
After sample pad is soaked 5 minutes with confining liquid, humidity ＜ 20% 40 ~ 50 DEG C of baking oven is placed in, 12 ~ 24 h are dried after 2
~ 30 DEG C of sealing preserves；The confining liquid contains 0.1 ~ 1% Tris, 0.1 ~ 1% Tween20,0.1 ~ 1% casein, 0.1 ~ 2%
PEG20000,0.005 ~ 0.05% mouse anti-human RBC.
（3）The preparation of label pad
The Avidin for AMH monoclonal antibodies and the fluorescent microsphere mark that fluorescent microsphere is marked is respectively by 5% ~ 20% and 0.5% ~ 5%
Dilution ratio be sprayed on label pad treatment fluid in label pad, discharge rate be 3 ~ 6 μ L/cm.Contain in the label pad treatment fluid
0.2 ~ 2% casein, 5% ~ 20% sucrose, 0.1 ~ 1% Tween-20,0.1 ~ 0.5% PVP40,0.02 ~ 0.05%
Proclin300,0.01 ~ 0.05M, pH 7.4 PBS.The label pad prepared is placed in the 40 ~ 50 of humidity ＜ 20%
DEG C baking oven, dry 12 ~ 24 h after 2 ~ 30 DEG C of sealing preserves.
（4）The preparation of coated film
It is respectively 0.5 ~ 2 mg/mL by another AMH monoclonal antibodies and rabbit-anti Avidin coating buffer solution regulation concentration, will
AMH monoclonal antibodies are sprayed onto coated film（3）On detection line, rabbit-anti Avidin antibody is sprayed onto coated film（3）On nature controlling line,
The consumption of AMH monoclonal antibodies and rabbit-anti the Avidin antibody is 0.1 ~ 0.2 μ L/mm by film coating liquid measure, detection line and
The mm of nature controlling line interval 4 ~ 8, humidity ＜ 20% 40 ~ 50 DEG C of baking oven, dry 24 ~ 72 h after 2 ~ 30 DEG C of sealing preserves, standby.
Sequentially mutually pasting sample pad, label pad, coated film and blotting paper obtains test paper plate overlap joint on bottom plate, according to
Split requirement cuts into the test strips of 3 ~ 4 mm width.
The Cleaning Principle of AMH of the present invention immuno-chromatographic test paper strip is the AMH in double antibody sandwich method, sample pad
The AMH antibody bindings that antigen is marked under chromatography effect with fluorescent microsphere in label pad form compound, and the compound is in chromatography
The detection line of coated film is moved under effect, the antibody of identification another epitope of AMH antigens, shape are coated with coated film detection line
Into double-antibody sandwich compound, compound is gathered at the detection line of coated film, and respective wavelength is discharged by light source activation
Antigen concentration is higher in transmitting light, sample, and the intensity of detection line transmitting light is higher, is converted optical signal by fluorescence detecting system
For data signal, using concentration point as abscissa, detection line signal value is than nature controlling line signal value（T/C）It is bent that standard is drawn for ordinate
Line, so as to the concentration for calculating AMH in sample of accurate quantitative analysis.
The present invention compared with prior art, has the following advantages：
Detection line of the present invention and nature controlling line use independent reaction system, do not interfere with each other and influence, and are entered by the way of T/C values
Row calibration, it is ensured that the degree of accuracy of test result.
The present invention uses fluorescence immune chromatography method, and the detection method sensitivity is high, simple to operate, cost is low.Detectable blood
Concentration as little as 0.1 ng/mL anti-Miao Le Shi pipe hormones in liquid sample, the detector used was without professional operator, 15 minutes
It can obtain testing result.
Fluorescence immune chromatography technology is introduced into AMH detection by the present invention, with reference to fluorescence detector, realizes AMH one
The quantitative detection of part, is that Clinical practice is provided a great convenience.
Brief description of the drawings
Fig. 1 is the structural representation of the fluorogenic quantitative detection AMH immuno-chromatographic test paper strips of the present invention；
Reference：1st, sample pad；2nd, label pad；3rd, coated film；4th, blotting paper；5th, detection line；6th, nature controlling line；7th, bottom plate.
Embodiment
The present invention is described in further detail with reference to specific embodiment, it should be appreciated that following examples be in order to
Facilitate understanding of the those skilled in the art to the present invention program, but it is not as a limitation of the invention.
Embodiment 1
The preparation of AMH fluorescence immune chromatography test paper bars：
（1）The preparation of fluorescent microsphere labelled protein
The fluorescent microspheres of 0.1mL 10% are taken, 15000 rpm are centrifuged 15 minutes for the first time, the sediment phosphate of 50 mM pH 6.5
Buffer solution regulation concentration is 1%, and ultrasonic disperse；Add final concentration of 2 mg/mL carbodiimide（EDC）, mix, add
Final concentration of 5 mg/mL n-hydroxysuccinimide（NHS）, mix；After incubation at room temperature 20 minutes 15000 rpm second from
The heart 15 minutes, the sediment phosphate buffers of 50 mM pH 6.5 dissolve.By the fluorescent microsphere ultrasonic disperse after redissolution, and point
Two pipes are separately added into 0.2 mg AMH monoclonal antibodies and 0.1 mg Avidins, room temperature rotation hybrid reaction 2 hours after mixing,
15000 rpm third times centrifugation 15 minutes, Tris-HCl of the sediment containing 30 mM monoethanolamines and 0.5% casein（20 mM,
pH 8.0）Redissolve, rotation hybrid reaction 1 hour after ultrasonic disperse, the 4th centrifugation of 15000 rpm 15 minutes, precipitation microballoon
Preserve liquid to redissolve, 2 ~ 8 DEG C of preservations.
（2）The pretreatment of sample pad
After sample pad is soaked 5 minutes with confining liquid, humidity ＜ 20% 50 DEG C of baking oven is placed in, 12 h are dried after 20 ~ 30 DEG C
Sealing preserve.The confining liquid contains 0.1% Tris, 1% Tween20,0.5% casein, 1% PEG20000,0.01%
Mouse anti-human RBC.
（3）The preparation of label pad
Fluorescent microsphere is marked AMH monoclonal antibodies and fluorescent microsphere mark Avidin respectively by 10% and 1% thinner ratio
Example is sprayed in label pad with label pad treatment fluid, and discharge rate is 4 μ L/cm, contains 0.5% junket egg in the label pad treatment fluid
In vain, 5% sucrose, 1% Tween-20,0.5% PVP40,0.03% Proclin300,0.05 M, pH 7.4 PBS bufferings
Liquid.The label pad prepared is placed in humidity ＜ 20% 50 DEG C of baking oven, 24 h are dried after 20 ~ 30 DEG C of sealing preserves.
（4）The preparation of coated film
It is respectively 1.0 mg/mL by another AMH monoclonal antibodies and rabbit-anti Avidin coating buffer solution regulation concentration, by AMH
Monoclonal antibody is sprayed onto coated film（3）On detection line, rabbit-anti Avidin is sprayed onto coated film（3）On nature controlling line（6）, it is described
It is 0.1 μ L/mm, detection line that the consumption of AMH monoclonal antibodies and rabbit-anti Avidin is coated with liquid measure by film（5）And nature controlling line（6）
4 mm are spaced, humidity ＜ 20% 50 DEG C of baking oven dries 72 h after 20 ~ 30 DEG C of sealing preserves, standby.
（5）Sequentially mutually sample pad is pasted on bottom plate overlap joint（1）, label pad（2）, coated film（3）And blotting paper（4）
Test paper plate is obtained, the test strips of 4 mm width are cut into according to split requirement.
Embodiment 2
Fluorescence immune chromatography quantitatively detects blood sample moderate resistance Miao's Le Shi pipe hormones（AMH）Concentration
（1）Specification Curve of Increasing
By AMH antigens be configured to negative plasma 25 ng/mL, 10 ng/mL, 5 ng/mL, 1.0 ng/mL, 0.5 ng/mL,
0.1 ng/mL, 0 ng/mL, with a batch of reagent, each concentration point is tested 6 times.With detection line（T bands）, nature controlling line（C
Band）Fluorescence intensity ratio be ordinate, AMH reference materials concentration be abscissa, set up equation and be fitted to standard curve, will mark
Bent information is write in ID chips with burn recording software.
（2）The detection of sample：
Detector bar is taken out from kit, is torn after packaging of aluminium foil bag, detector bar is kept flat, balances 5 minutes, takes 100 μ L samples to add
Enter in well, room temperature lucifuge is reacted 15 minutes.ID chips are inserted into fluorescence detector, by detection card insertion luminoscope plug-in card
Mouthful, click on " test ", instrument calculates the concentration of AMH in sample to be tested by analysis software automatically.
（3）With the anti-Miao Le Shi pipes hormone test kit of the sub- brightness dragon in Shenzhen（Chemoluminescence method）The correlation ratio of detection
Compared with.
40 human serum samples are entered using the test strips and the anti-Miao Le Shi pipe hormone test kits of sub- brightness dragon of the present invention
Row detection, measurement result is shown, in the range of this ELISA test strip（0.1~25 ng/mL）, the correlation R of two kinds of reagents2＞
0.95（y=1.042x+0.692）.
Embodiment 3
Fig. 1 is refer to, a kind of fluorogenic quantitative detection AMH immuno-chromatographic test paper strip, described immuno-chromatographic test paper strip includes
Sample pad 1, label pad 2, coated film 3, blotting paper 4, described label pad are sequentially provided with PVC bottom plates 7, described PCV bottom plates 7
2 and described sample pad 1 connect, described coated film 3 and described label pad 2 connect, described blotting paper 4 and described bag
Envelope 3 connects；Be coated with the label pad 2 fluorescent microsphere mark AMH monoclonal antibodies and fluorescent microsphere mark it is affine
Element；The coated film 3 is provided with detection line 5 and nature controlling line 6, and detection line 5 and nature controlling line 6 are spaced 4 ~ 8 mm, and the detection line 5 is wrapped
Another AMH monoclonal antibodies for being there are the AMH monoclonal antibodies marked with the fluorescent microsphere to be in different epitopes, the matter
Control line 6 is coated with the rabbit-anti Avidin antibody of specific recognition Avidin.
AMH detection is only adapted to hospital laboratory and is used for the ELISA of batch detection on the market at present（ELISA）、
Chemiluminescence（CLIA）, Electrochemiluminescince, also be adapted to single part, quick detection, immediately go out result detection reagent.This
AMH detection reagents described in patent can greatly shorten detection time again while highly sensitive detection AMH, be clinical examination and use
Bring great convenience, be more suitable for clinical department operation.
Claims (8)
1. a kind of fluorogenic quantitative detection AMH immuno-chromatographic test paper strip, described immuno-chromatographic test paper strip includes PVC bottom plates,
Characterized in that, be sequentially provided with sample pad, label pad, coated film, blotting paper on described PCV bottom plates, described label pad and
Described sample pad connects, and described coated film and described label pad connect, described blotting paper and described coated film phase
Connect；The AMH monoclonal antibodies of fluorescent microsphere mark and the Avidin of fluorescent microsphere mark are coated with the label pad；The bag
Envelope is provided with detection line and nature controlling line, detection line and the mm of nature controlling line interval 4 ~ 8, and the detection line is coated with and the fluorescence
The AMH monoclonal antibodies of microballoon mark are in another AMH monoclonal antibodies of different epitopes, and the nature controlling line is coated with specifically
Property identification Avidin rabbit-anti Avidin antibody.
2. immuno-chromatographic test paper strip as claimed in claim 1, it is characterised in that the particle diameter of the fluorescent microsphere is 100 ~ 500
nm。
3. immuno-chromatographic test paper strip as claimed in claim 1, it is characterised in that the excitation wavelength of the fluorescent microsphere is 310 ~
550 nm, launch wavelength is 340 ~ 620 nm.
4. a kind of preparation method of fluorogenic quantitative detection AMH immuno-chromatographic test paper strip, it is characterised in that described preparation method
Comprising having the following steps：
（1）The preparation of fluorescent microsphere labelled protein
Take fluorescent microsphere, 10000 ~ 15000 rpm centrifugation 5 ~ 15 minutes for the first time, centrifuge for the first time obtained sediment with 10 ~
100 mM, pH 6.0 ~ 7.0 phosphate buffer regulation concentration be 0.1% ~ 1%, then and ultrasonic disperse adds final concentration of
0.1 ~ 5 mg/mL carbodiimide, mixes, adds final concentration of 0.1 ~ 5 mg/mL n-hydroxysuccinimide, mixes；
10000 ~ 15000 rpm, second of centrifugation 5 ~ 15 minutes, the precipitation being centrifugally separating to obtain for the second time after being incubated at room temperature 20 ~ 40 minutes
Thing with 10 ~ 100 mM, pH 6.0 ~ 7.0 phosphate buffer dissolve, by the fluorescent microsphere ultrasonic disperse after redissolution, according to 0.1 ~
The ratio of 1.0 mg/mL fluorescent microspheres is separately added into room temperature after AMH monoclonal antibodies and Avidin, mixing and rotates hybrid reaction
1.5 ~ 3 hours, 10000 ~ 15000 rpm third times centrifugation 5 ~ 15 minutes, the sediment being centrifugally separating to obtain for the third time is with 10 ~ 40
MM, pH are that Tris-HCl buffer solutions of the 7.0-8.0 containing 10 ~ 40 mM monoethanolamines and 0.05%-1% caseins redissolves, ultrasonic disperse
Rotation hybrid reaction 0.5 ~ 1 hour afterwards, 10000 ~ 15000 rpm centrifuge 5 ~ 15 minutes for the 4th time, are centrifugally separating to obtain for the 4th time
Sediment with microballoon preserve liquid redissolve, 2 ~ 8 DEG C preservation；
（2）The pretreatment of sample pad
After sample pad confining liquid is soaked, the baking oven that humidity ＜ 20%, temperature are 40 ~ 50 DEG C is placed in, 12 ~ 24 h are dried after 2
~ 30 DEG C of sealing preserves, the confining liquid contains 0.1 ~ 1% Tris, 0.1 ~ 1% Tween20,0.1 ~ 1% casein, 0.1 ~ 2%
PEG20000,0.005 ~ 0.05% mouse anti-human RBC；
（3）The preparation of label pad
The Avidin for AMH monoclonal antibodies and the fluorescent microsphere mark that fluorescent microsphere is marked is sprayed with label pad treatment fluid respectively
In label pad, discharge rate is 3 ~ 6 μ L/cm；
Containing 0.2 ~ 2% casein in the label pad treatment fluid, 5% ~ 20% sucrose, 0.1 ~ 1% Tween-20,0.1 ~
M, the pH 7.4 of 0.5% PVP40,0.02 ~ 0.05% Proclin300,0.01 ~ 0.05 PBS；
The label pad prepared is placed in humidity ＜ 20% 40 ~ 50 DEG C of baking oven, 12 ~ 24 h is dried and is protected after 2 ~ 30 DEG C of sealings
Deposit；
（4）The preparation of coated film
It is respectively 0.5 ~ 2 mg/ by another AMH monoclonal antibodies and rabbit-anti Avidin coating buffer solution regulation concentration
ML, AMH monoclonal antibodies is sprayed onto the detection line on coated film, and rabbit-anti Avidin antibody is sprayed onto to the nature controlling line on coated film,
It is 0.1 ~ 0.2 μ L/mm that the consumption of the AMH monoclonal antibodies and the rabbit-anti Avidin antibody is coated with liquid measure by film, detection
40 ~ 50 DEG C of baking oven of line and the mm of nature controlling line interval 4 ~ 8, humidity ＜ 20%, dries 24 ~ 72 h after 2 ~ 30 DEG C of sealing preserves,
It is standby；
（5）Sequentially mutually pasting sample pad, label pad, coated film and blotting paper obtains test paper plate overlap joint on bottom plate, according to
Split requirement cuts into the test strips of 3 ~ 4 mm width.
5. the preparation method of immuno-chromatographic test paper strip as claimed in claim 1, it is characterised in that step（3）Described in fluorescence
The concentration of the AMH monoclonal antibodies of microballoon mark is 0.1 ~ 1.0 mg/mL, after being diluted according to dilution ratio for 5% ~ 20%, discharge rate
It is sprayed into for 3 ~ 6 μ L/cm in label pad.
6. the preparation method of immuno-chromatographic test paper strip as claimed in claim 1, it is characterised in that step（3）Described in fluorescence
The concentration of the Avidin of microballoon mark is 0.1 ~ 1.0 mg/mL, after being diluted according to dilution ratio for 0.5% ~ 5%, and discharge rate is 3 ~ 6 μ
L/cm is sprayed into label pad.
7. the preparation method of immuno-chromatographic test paper strip as claimed in claim 1, it is characterised in that in the coated film detection line
The μ L/mm of discharge rate 0.1 ~ 0.2 of coated AMH monoclonal antibodies.
8. the preparation method of immuno-chromatographic test paper strip as claimed in claim 1, it is characterised in that coated on the nature controlling line
The μ L/mm of discharge rate 0.1 ~ 0.2 of rabbit-anti Avidin antibody.
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