CN109813881A - A kind of hormone test reagent and kit - Google Patents
A kind of hormone test reagent and kit Download PDFInfo
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- CN109813881A CN109813881A CN201910117379.7A CN201910117379A CN109813881A CN 109813881 A CN109813881 A CN 109813881A CN 201910117379 A CN201910117379 A CN 201910117379A CN 109813881 A CN109813881 A CN 109813881A
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- miao
- shi pipe
- antibody
- pipe hormone
- wheat germ
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Abstract
The present invention relates to a kind of anti-Miao Le Shi pipe hormone (AMH) detection reagents, the detection reagent includes anti-Miao Le Shi pipe hormone antibody, wheat germ agglutinin and solid phase carrier, anti- Miao Le Shi pipe hormone antibody or wheat germ agglutinin alternative one are coated on the solid phase carrier, another is then marked using marker.It is mainly characterized by the detection reagent that sandwich method is formed using anti-AMH antibody and wheat germ agglutinin (WGA), and the detection reagent detection sensitivity is high, and high specificity is suitable for large-scale promotion.
Description
Technical field
The invention patent relates to detection reagent technical fields, and in particular to detection reagent field, in particular to a kind of anti-Miao
Le Shi pipe hormone (AMH) detection reagent.
Background technique
Women's fecundity and age, basic follicle stimulating hormone, inhibin B (inhibin B, INH B), anti-Miao Le Shi are managed
The many factors such as hormone (anti m μ Llerian hormone, AMH), estradiol, Antral follicles level are related, and wherein AMH is
Mostly important index.
AMH belongs to β-transforming factor by the single-stranded glycoprotein dimer formed of two 72kD that disulphide bridges connect
A member of family；AMH is secreted by the granular cell of ovarian growth ovarian follicle after the immature Sertoli cell of testis and birth, tool
Have and adjusts cell development and differentiation, the function of making male embryo Miao's Le Shi pipe degenerate.For women, AMH is in 36 week foetal period
Follicular cell generate, until Menopause, AMH is gradually decreased down can not detection level.AMH is clinically main
For Ovary reserve, prediction ovary responsiveness, male reproductive function assessment etc..
As described above, AMH is clinically mainly for assessment of the reproductive function of women, guiding clinical treatment.
Traditional AMH detection reagent is all based on the various immunological methods that double antibody sandwich method is established, including enzyme-linked
Immunoabsorption, chemoluminescence method；But AMH is the glycoprotein with unique texture, specificity is high, and sensitivity is good to be resisted
Body is very difficult to obtain；The current most AMH antibody haveing excellent performance significantly limits by external several business monopolies
The exploitation of AMH detection reagent and the progress of technology.
In order to solve the existing above problem, it is desirable to provide a kind of technology, can it is sensitive, specifically detect AMH, to fecundity
Power is detected, easy to use.
Summary of the invention
The purpose of the invention patent be overcome it is above-mentioned in the prior art the shortcomings that, a kind of anti-Miao Le Shi pipe hormone is provided
(AMH) detection reagent, anti-Miao Le Shi pipe hormone (AMH) detection reagent have non-specificity by AMH antibody and to glycoprotein
In conjunction with wheat germ agglutinin (WGA) form sandwich method, realize detection to AMH in sample, it is at low cost using simplicity, go forward side by side one
Step improves the accuracy of reproductive function detection, and testing result provides strong foundation for accurate treatment in next step, is suitable for extensive
It promotes and applies.
To achieve the goals above, anti-Miao Le Shi pipe hormone (AMH) detection reagent of the invention patent has following constitute:
A kind of anti-Miao Le Shi pipe hormone test reagent, including anti-Miao Le Shi pipe hormone antibody, wheat germ agglutinin and solid phase carry
Miao's Le Shi pipe hormone antibody or wheat germ agglutinin alternative one are coated on the solid phase carrier by body, another is then adopted
It is marked with marker.
More preferably, anti-Miao Le Shi pipe hormone antibody is coated on solid phase carrier, and with label substance markers wheat germ agglutinin.
More preferably, wheat germ agglutinin is coated on solid phase carrier, and with label the anti-Miao Le Shi pipe hormone antibody of substance markers.
More preferably, which is the antibody of Miao Le Shi pipe hormone specificity, including polyclonal antibody and monoclonal antibody.More
Excellently, Miao's Le Shi pipe hormone antibody is that the anti-Miao Le Shi pipe hormone polyclonal antibody of mouse, rabbit antibody Miao's Le Shi pipe hormone are more
Clonal antibody, goat-anti body Miao's Le Shi pipe hormone polyclonal antibody, chicken antibody Miao's Le Shi pipe hormone polyclonal antibody, the anti-Miao Le of mouse
Family name's pipe hormone monoclonal antibody, rabbit antibody Miao's Le Shi pipe hormone monoclonal antibody.
More preferably, the concentration of anti-Miao Le Shi pipe hormone antibody is 0.005ng/mL~1.5ng/mL.
More preferably, the wheat germ agglutinin is the natural extract of cereal, extracts from naked barley wheat germ, barley wheat germ, rice
Paddy or wheat.More preferably, the wheat germ agglutinin concentration is 0.01ng/mL~5ng/mL.
More preferably, the solid phase carrier is latex particle, nitrocellulose filter, nylon membrane, polyvinylidene fluoride film, micropore
One or more of plate or magnetic-particle.
More preferably, the marker be latex particle, it is colloidal gold, fluorescence, digoxin, biotin, alkaline phosphatase, peppery
Root peroxidase, acridinium ester, acridine ester derivant, luminol or derivatives thereof or one or more of tris (bipyridine) ruthenium.
The present invention also provides a kind of anti-Miao Le Shi pipe hormone test reagents, detect anti-Miao Le Shi pipe Hormone agents box in preparation
In application.
Anti- Miao Le Shi pipe hormone (AMH) detection reagent, its main feature is that, including AMH antibody and wheat germ agglutinin (WGA) shape
At sandwich method.When detecting AMH, an interlayer structure of AMH antibody-AMH-WGA is formed.
More preferably, the detection marker detection reagent is immunochromatographic method, immunoturbidimetry, ELISA method, albumen are exempted from
One or more of epidemic disease blotting, microfluidic method or chemoluminescence method.
The present invention also provides a kind of anti-Miao Le Shi pipe hormone (AMH) detection reagent, which applies the above method to examine
Anti- Miao Le Shi pipe hormone (AMH) is surveyed, auxiliary diagnosis reproduction related disease is used for.
Beneficial effects of the present invention mainly have:
(1) sufficient raw: the wheat germ agglutinin (WGA) of the invention patent is obtained by can extract in native malt,
At low cost, yield is high, and extremely strong with the affinity of AMH；
(2) detection technique is had excellent performance: the affinity of wheat germ agglutinin (WGA) is strong, and the high specificity of AMH antibody；Nothing
Specificity and the high pairing antibody of affinity need to be relied on as conventional method (double antibody sandwich method)；Therefore, which detects skill
The performance of art is better than the AMH detection technique of most double antibody sandwich method.
Therefore, the invention patent can match to form interlayer structure to detect AMH, using letter by AMH antibody and WGA
Just, and the accuracy that reproductive function detects being further increased, testing result provides strong foundation for accurate treatment in next step,
Suitable for large-scale promotion application.
Specific embodiment
In order to be more clearly understood that the technology contents of the invention patent, spy is subject to specifically in conjunction with specific embodiments
It is bright.
The preparation process of the AMH detection technique of the invention patent is summarized as follows, it should be appreciated that these embodiments are merely to illustrate
The present invention rather than limit the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to
Normal condition, such as Sambrook et al., molecule clone technology Laboratory Manual (New York:Cold Spring
Harbor Laboratory Press, 2005) condition described in, or according to the normal condition proposed by manufacturer.
1 fluorescent marker WGA of embodiment
Cleaning: fluorescent microsphere is taken (purchased from Bangs Lab, article No.: 11233) into centrifuge tube, to add 0.1M MES (2-
Quinoline is for ethanesulfonic acid) (pH 5.0) buffer mixes, and with 13000rpm, 15min, 4 DEG C of pelleted by centrifugation, abandons supernatant, use 0.1M
MES (pH 5.0) buffer is resuspended stand-by.It activates and cleans: by activator EDC (1- (3- dimethylamino-propyl) -3- ethyl carbon
Diimmonium salt hydrochlorate), NHS (n-hydroxysuccinimide) and fluorescent microsphere activated according to the amount of mass ratio ratio 2:1:2,
Concrete operations are as follows:
Weigh EDC, NHS be added 0.1M MES (pH 5.0) buffer in dissolve, take rapidly in right amount to cleaning finish it is glimmering
In light microballoon, seals with sealing film and be placed on room temperature on 200rpm shaking table and shake up 30min, 13000rpm, 30min, 4 after taking-up
DEG C it is centrifuged off supernatant, ultrasound is resuspended with equivalent 0.1M MES (pH 6.5) buffer and mixes and cleans, is repeated more than once behaviour
Make i.e. cleaning twice.It is stand-by that supernatant is discarded after the completion of centrifugation.
Label: the latex after activation is resuspended into 0.1M MES (pH 6.5) buffer, is separately added into WGA (purchase rapidly
From Sigma-Aldrich, article No.: L9640,25mg), it mixes, places room temperature, shaken up 4 hours on 200rpm shaking table.
Closing: the microballoon for taking above-mentioned label good is centrifuged off supernatant in 13000rpm, 30min, 4 DEG C, immediately into microballoon
Equivalent confining liquid is added, ultrasound is placed room temperature after being resuspended again, shaken up on 200rpm shaking table 1 hour.After the reaction was completed
13000rpm, 20min, 4 DEG C of centrifugations, take supernatant to be checked.
| Detection content | Quality standard | Detection reagent |
| Package amount | Package amount >=160 μ gWGA/mg microballoons | BCA determination of protein concentration method |
It cleans & to be resuspended: re-suspension liquid is added, ultrasound piping and druming is resuspended, with high speed freezing centrifuge 13000rpm, 20min, 4 DEG C
Centrifugation, discards supernatant；Re-suspension liquid is added, ultrasound piping and druming is resuspended.
The microspheres solution being resuspended marks, spare.
2 fluorescent marker AMH antibody of embodiment
Cleaning: fluorescent microsphere is taken (purchased from Bangs Lab, article No.: 11233) into centrifuge tube, to add 0.1M MES (pH
5.0) buffer mixes, and with 13000rpm, 15min, 4 DEG C of pelleted by centrifugation, abandons supernatant, buffered with 0.1M MES (pH 5.0)
Liquid is resuspended stand-by.It activates and cleans: activator EDC, NHS and fluorescent microsphere are lived according to the amount of mass ratio ratio 2:1:2
Change, concrete operations are as follows:
Weigh EDC, NHS be added 0.1M MES (pH 5.0) buffer in dissolve, take rapidly in right amount to cleaning finish it is glimmering
In light microballoon, seals with sealing film and be placed on room temperature on 200rpm shaking table and shake up 30min, 13000rpm, 30min, 4 after taking-up
DEG C it is centrifuged off supernatant, ultrasound is resuspended with equivalent 0.1M MES (pH 6.5) buffer and mixes and cleans, is repeated more than once behaviour
Make i.e. cleaning twice.It is stand-by that supernatant is discarded after the completion of centrifugation.
Label: the latex after activation is resuspended into 0.1M MES (pH 6.5) buffer, is separately added into the anti-of AMH rapidly
Body (mouse monoclonal antibody is purchased from Hangzhou Bo Yin Biotechnology Co., Ltd, article No.: M050501,5mg), mixes, places room temperature,
It is shaken up on 200rpm shaking table 4 hours.
Closing: the microballoon for taking above-mentioned label good is centrifuged off supernatant in 10000rpm, 30min, 4 DEG C, immediately into microballoon
Equivalent confining liquid is added, ultrasound is placed room temperature after being resuspended again, shaken up on 200rpm shaking table 1 hour.After the reaction was completed
10000rpm, 20min, 4 DEG C of centrifugations, take supernatant to be checked.
| Detection content | Quality standard | Detection reagent |
| Package amount | Package amount >=110 μ g antibody/mg microballoon | BCA determination of protein concentration method |
It cleans & to be resuspended: re-suspension liquid is added, ultrasound piping and druming is resuspended, with high speed freezing centrifuge 10000rpm, 20min, 4 DEG C
Centrifugation, discards supernatant；Re-suspension liquid is added, ultrasound piping and druming is resuspended.
The microspheres solution being resuspended marks, spare.
3 fluorescent marker goat anti-rabbit igg antibody of embodiment
Cleaning: fluorescent microsphere is taken (purchased from Bangs Lab, article No.: 11233) into centrifuge tube, to add 0.1M MES (pH
5.0) buffer mixes, and with 13000rpm, 15min, 4 DEG C of pelleted by centrifugation, abandons supernatant, buffered with 0.1M MES (pH 5.0)
Liquid is resuspended stand-by.It activates and cleans: activator EDC, NHS and fluorescent microsphere are lived according to the amount of mass ratio ratio 2:1:2
Change, concrete operations are as follows:
Weigh EDC, NHS be added 0.1M MES (pH 5.0) buffer in dissolve, take rapidly in right amount to cleaning finish it is glimmering
In light microballoon, seals with sealing film and be placed on room temperature on 200rpm shaking table and shake up 30min, 13000rpm, 30min, 4 after taking-up
DEG C it is centrifuged off supernatant, ultrasound is resuspended with equivalent 0.1M MES (pH 6.5) buffer and mixes and cleans, is repeated more than once behaviour
Make i.e. cleaning twice.It is stand-by that supernatant is discarded after the completion of centrifugation.
Label: the latex after activation is resuspended into 0.1M MES (pH 6.5) buffer, is separately added into goat-anti rabbit rapidly
IgG (purchased from the double Long Shenghua in Chengdu, article No.: J0711-6,1mg), mixes, places room temperature, shake up 4 hours on 200rpm shaking table.
Closing: the microballoon for taking above-mentioned label good is centrifuged off supernatant in 10000rpm, 30min, 4 DEG C, immediately into microballoon
Equivalent confining liquid is added, ultrasound is placed room temperature after being resuspended again, shaken up on 200rpm shaking table 1 hour.After the reaction was completed
10000rpm, 20min, 4 DEG C of centrifugations, take supernatant to be checked.
| Detection content | Quality standard | Detection reagent |
| Package amount | Package amount >=200 μ g antibody/mg microballoon | BCA determination of protein concentration method |
It cleans & to be resuspended: re-suspension liquid is added, ultrasound piping and druming is resuspended, with high speed freezing centrifuge 10000rpm, 20min, 4 DEG C
Centrifugation, discards supernatant；Re-suspension liquid is added, ultrasound piping and druming is resuspended.
The microspheres solution being resuspended marks, spare.
The specking of 4 bonding pad of embodiment
The specking method of WGA bonding pad is as follows: fluorescent marker solution dilution: with fluorescent marker re-suspension liquid by above-mentioned preparation
After fluorescent marker WGA conjugate (coming from embodiment 1) and fluorescent marker goat anti-rabbit igg conjugate (coming from embodiment 3) mixing
2 times of dilution；Point film instrument is set, the power supply of point film instrument is opened, sets specking program, specking amount is 8 μ L/cm；No. 1 pipeline is spray
Point channel；Point film instrument initialization: No. 1 pipeline is placed in fluorescent marker and is resuspended in solution, initialization program is selected, initializes 6
Circulation；Specking: bonding pad is placed in point film instrument by fixed bit horizontalization, is pressed on control panel " GO " key and is started specking, after having put
Remove, check the good bonding pad of specking, the fluorescent marker WGA band of specking uniformly, continuous and the entire bonding pad of perforation straight line
For qualified specking product, occurring breakpoint in two straight lines is unqualified specking product；A piece of bonding pad is often put, by a control panel
On " GO " key be that specking is primary (a piece of)；Specking terminates, and the bonding pad of specking is placed in room temperature and is spontaneously dried 1 hour, film
On should can't see specking trace.
The specking of 5 bonding pad of embodiment
The specking method of the antibody bonding pad of anti-AMH is as follows: fluorescent marker solution dilution: will be upper with fluorescent marker re-suspension liquid
Fluorescent labeled antibody (from embodiment 2) and the fluorescent marker goat anti-rabbit igg conjugate (from embodiment 3) for stating preparation are mixed
4 times are diluted after conjunction；Point film instrument is set, the power supply of point film instrument is opened, sets specking program, specking amount is 8 μ L/cm；No. 1 pipeline
For specking channel；Point film instrument initialization: No. 1 pipeline is placed in fluorescent marker and is resuspended in solution, selects initialization program, initially
Change 6 circulations；Specking: bonding pad is placed in point film instrument by fixed bit horizontalization, is pressed on control panel " GO " key and is started specking,
Removed after having put, check the good bonding pad of specking, the fluorescent marker WGA band of specking uniformly, the continuous and entire bonding pad of perforation
Straight line be qualified specking product, occurring breakpoint in two straight lines is unqualified specking product；A piece of bonding pad is often put, by a secondary control
" GO " key on panel is that specking is primary (a piece of)；Specking terminates, and it is small that the bonding pad of specking is placed in natural drying 1 in room temperature
When, specking trace should be can't see on film.
The preparation (AMH antibody) of 6 nitrocellulose filter of embodiment
Antibody (mouse monoclonal antibody is purchased from Hangzhou Bo Yin Biotechnology Co., Ltd, article No.: M050501,5mg) nitric acid of AMH is fine
It is as follows to tie up plain membrane preparation method: taking the 500 μ g of monoclonal antibody of the anti-AMH of mouse, is added in 5mL graduated centrifuge tube, antibody diluent to 1mL,
Container Tag T flag.Take rabbit igg (purchased from Hangzhou Bo Yin Biotechnology Co., Ltd, article No.: P200201, specification: 5mg/mL)
25 μ L, are added in 5mL graduated centrifuge tube, antibody diluent to 1mL, Container Tag C mark.Point film instrument is set, point film instrument is opened
Power supply, set specking program, specking amount be 1 μ L/cm；No. 1 pipeline is detection band specking channel, and No. 2 pipelines are control band spray
Point channel；Point film instrument initialization: No. 1 pipeline is placed in detection band solution, No. 2 pipelines is placed in control band solution, selection
Initialization program initializes 6 circulations；Specking: nitrocellulose filter is placed in point film instrument by fixed bit horizontalization, by control
" GO " key starts specking on panel, removes after having put, and checks the good nitrocellulose filter of specking, detection band and control band are two
Item uniformly, continuous and the entire nitrocellulose filter of perforation straight line be qualified specking product, occurring breakpoint in two straight lines is not conform to
Lattice specking product；A piece of nitrocellulose filter is often put, pressing " GO " key on a control panel is that specking is primary (a piece of)；Specking
Terminate, the nitrocellulose filter of specking is placed in room temperature and is spontaneously dried 1 hour, specking trace should be can't see on film.
The preparation (WGA) of 7 nitrocellulose filter of embodiment
WGA nitrocellulose filter the preparation method is as follows: take 200 μ g of WGA (purchased from Sigma-Aldrich, article No.: L9640,
25mg), it is added in 5mL graduated centrifuge tube, is diluted to 1mL, Container Tag T flag.Rabbit igg is taken (to win mattress biology section purchased from Hangzhou
Skill Co., Ltd, article No.: P200201, specification: 5mg/mL) 25 μ L, it is added in 5mL graduated centrifuge tube, antibody diluent is extremely
1mL, Container Tag C mark.Point film instrument is set, the power supply of point film instrument is opened, sets specking program, specking amount is 1 μ L/cm；1
Number pipeline is detection band specking channel, and No. 2 pipelines are control band specking channel；Point film instrument initialization: No. 1 pipeline is placed in inspection
In measuring tape solution, No. 2 pipelines are placed in control band solution, initialization program is selected, initializes 6 circulations；Specking: by nitre
Acid cellulose film is placed in point film instrument by fixed bit horizontalization, is pressed on control panel " GO " key and is started specking, removes after having put, inspection
The good nitrocellulose filter of specking is looked into, band is detected and control band is uniform for two, continuous and the entire nitrocellulose filter of perforation
Straight line is qualified specking product, and occurring breakpoint in two straight lines is unqualified specking product；A piece of nitrocellulose filter is often put, by one
" GO " key on secondary control panel is that specking is primary (a piece of)；Specking terminates, and the nitrocellulose filter of specking is placed in room temperature
It spontaneously dries 1 hour, specking trace should be can't see on film.
Embodiment 8 assembles
The protection sheet of wider portion on bottom plate is removed, along the lower edge of protection sheet above, the cellulose nitrate of line will be pulled
Plain film (comes from embodiment 6), by C line above in a manner of be attached on bottom plate plate；Bonding pad (coming from embodiment 4) is attached to T line
Lower section contacts a little with NC film；Sample pad is attached to below bonding pad, is contacted a little with bonding pad；Then top protection is removed
Blotting paper is attached to the top of NC film by paper, is contacted a little with NC film；Protection sheet and instruction band paper are attached to one by one assembled
Outside test strips, it is assembled into kilocalorie.
Embodiment 9 assembles
The protection sheet of wider portion on bottom plate is removed, along the lower edge of protection sheet above, the cellulose nitrate of line will be pulled
Plain film (comes from embodiment 7), by C line above in a manner of be attached on bottom plate plate；Bonding pad (coming from embodiment 5) is attached to T line
Lower section contacts a little with NC film；Sample pad is attached to below bonding pad, is contacted a little with bonding pad；Then top protection is removed
Blotting paper is attached to the top of NC film by paper, is contacted a little with NC film；Protection sheet and instruction band paper are attached to one by one assembled
Outside test strips, it is assembled into kilocalorie.
Embodiment 10 is cut
Cutting electromechanical source is connected, setting cuts film program, sets cutting width as 4mm；By kilocalorie (from embodiment 8 or in fact
Apply example 9) it lays flat in cutting machine platform track, face-up, " GO " key is pressed on operation panel, starts to cut；It often puts a piece of big
It is primary to press on operation panel " GO " key for card qualified product, until having cut all kilocalorie qualified products；After the completion of cutting, by test strips
It sticks on bottom plate side by side, forms test strip.
The assembling of 11 kit of embodiment
Above-mentioned test strips (coming from embodiment 10) are fitted into cartridge, detection card is formed.
Take aluminium foil bag and desiccant；Open heat sealing machine, preheating；Detection card to be packed, 1 bag of desiccant are packed into aluminium foil bag
In；According to aluminium foil bag of the length cutting equipped with detection card and desiccant of regulation；Aluminium foil bag is sealed with heat sealing machine；It is labelled.
12 HRP of embodiment marks AMH antibody
5mg HRP (being purchased from Roche, article No.: 1464325, specification: 25mg/ bottles) is dissolved in 0.5mL 0.1M NaHCO3It is molten
In liquid；Add 0.5mL 10mM NaIO4Solution mixes, and covers tightly bottle stopper, and room temperature is protected from light effect 2 hours；Add 0.75mL 0.1M
Na2CO3It mixes；Addition 0.75mL antibody (mouse monoclonal antibody is purchased from Hangzhou Bo Yin Biotechnology Co., Ltd, article No.: M050501,
5mg), it mixes.
The preservation of HRP antibody: after equivalent glycerol is added, -20 DEG C of storages of a small amount of packing prevent multigelation.
13 HRP of embodiment marks WGA
5mg HRP (being purchased from Roche, article No.: 1464325, specification: 25mg/ bottles) is dissolved in 0.5mL 0.1M NaHCO3It is molten
In liquid；Add 0.5mL 10mM NaIO4Solution mixes, and covers tightly bottle stopper, and room temperature is protected from light effect 2 hours；Add 0.75mL 0.1M
Na2CO3It mixes；0.75mLWGA (being purchased from Sigma-Aldrich, article No.: L9640,25mg) is added, mixes.
The preservation of HRP label WGA conjugate: after equivalent glycerol is added, -20 DEG C of storages of a small amount of packing prevent multigelation.
14 AMH antibody of embodiment coating
Using 0.01M, by AMH antibody, (mouse monoclonal antibody wins mattress biology section purchased from Hangzhou to the CBS (carbonate buffer solution) of pH 10
Skill Co., Ltd, article No.: M050501,5mg) it is diluted to 1 μ g/mL；96 hole microwell plates are taken, the 100 above-mentioned coatings of μ L are added in every hole
Antibody；37 DEG C of incubation 1h；It gets rid of coating buffer, is added the Block buffer (2% bovine serum albumin(BSA), pH7.4) of 200 μ L, 37 DEG C
1h is incubated, confining liquid is got rid of；In natural drying at room temperature 12h, it is put into desiccant, encloses aluminium foil bag, 2~8 DEG C spare.
15 WGA of embodiment coating
Using 0.01M, the CBS (carbonate buffer solution) of pH 10 by WGA (be purchased from Sigma-Aldrich, article No.: L9640,
25mg) it is diluted to 0.2 μ g/mL；96 hole microwell plates are taken, the above-mentioned coating WGA of 100 μ L is added in every hole；37 DEG C of incubation 1h；Get rid of packet
By liquid, the Block buffer (2% bovine serum albumin(BSA), pH7.4) of 200 μ L is added, 37 DEG C of incubation 1h get rid of confining liquid；In room
Temperature spontaneously dries 12h, is put into desiccant, encloses aluminium foil bag, 2~8 DEG C spare.
The assembling of 16 ELISA kit of embodiment
HRP is marked into AMH antibody (coming from embodiment 12) and WGA coating plate (coming from embodiment 15), is assembled into AMH inspection
The ELISA kit of survey.
The assembling of 17 ELISA kit of embodiment
HRP is marked into WGA (coming from embodiment 13) and AMH antibody coating plate (coming from embodiment 14), is assembled into AMH detection
ELISA kit.
18 kit of embodiment detects AMH
By above-mentioned detection card (coming from embodiment 11), verified with clinical sample.
Sample to be tested is added drop-wise in the sample aperture of detection card, is subsequently placed in fluorescence detector and is read.
Testing result and the goldstandard (Beckman chemoluminescence method) of AMH detection are as follows:
| Beckman chemoluminescence method is positive | Beckman chemoluminescence method is negative | It is total | |
| Detection card detection is positive | 76 | 3 | 79 |
| Detection card detection is negative | 5 | 134 | 139 |
| It is total | 81 | 137 | 218 |
According to upper Biao Ke get:
The detection sensitivity of the test strips are as follows: 76/ (76+5) × 100%=93.83%；
The detection specificity of the test strips are as follows: 134/ (134+3)=97.81%.
19 kit of embodiment detects AMH
By above-mentioned coating plate (coming from embodiment 17), verified with clinical sample.
Sample to be tested is added drop-wise in the sample aperture of coating plate, is subsequently placed in enzyme micro-plate reader and is read.
Testing result and the goldstandard (Beckman chemoluminescence method) of AMH detection are as follows:
| Beckman chemoluminescence method is positive | Beckman chemoluminescence method is negative | It is total | |
| It is positive to be coated with plate detection | 80 | 2 | 82 |
| It is negative to be coated with plate detection | 1 | 135 | 136 |
| It is total | 81 | 137 | 218 |
According to upper Biao Ke get:
The detection sensitivity of the kit are as follows: 80/ (80+1) × 100%=98.77%；
The detection specificity of the kit are as follows: 135/ (135+2)=98.54%.
Above-described embodiment the result shows that, this detection kit detect AMH accuracy rate it is very high, can satisfy clinical application
Requirement.
The sandwich method AMH detection technique that reagent manufactured in the present embodiment can be established by AMH antibody and WGA, specifically
Property, sensitivity are higher；It is remarkably improved the accuracy of reproductive function detection, and can be carried out according to the testing result of AMH
The stronger therapeutic scheme of specific aim.
Therefore, AMH detection technique provided by the invention using simplicity, and further increases the accurate of reproductive function detection
Property, it is suitable for large-scale promotion application.
In this description, the invention patent is described with reference to its specific embodiment.But it is clear that still can be with
The spirit and scope that various modification can be adapted and converts without departing from the invention patent.Therefore, specification should be considered as illustrative
And not restrictive.
Claims (10)
1. a kind of anti-Miao Le Shi pipe hormone test reagent, which is characterized in that the detection reagent include anti-Miao Le Shi pipe hormone antibody,
Anti- Miao Le Shi pipe hormone antibody or wheat germ agglutinin alternative one are coated on the solid phase by wheat germ agglutinin and solid phase carrier
On carrier, another is then marked using marker.
2. anti-Miao Le Shi pipe hormone test reagent according to claim 1, which is characterized in that anti-Miao Le Shi pipe hormone antibody
Be coated on solid phase carrier, and with label substance markers wheat germ agglutinin.
3. anti-Miao Le Shi pipe hormone test reagent according to claim 1, which is characterized in that it is characterized in that, wheat germ is solidifying
Collection element be coated on solid phase carrier, and with mark the anti-Miao Le Shi pipe hormone antibody of substance markers.
4. anti-Miao Le Shi pipe hormone test reagent according to claim 1-3, which is characterized in that anti-Miao
Le Shi pipe hormone antibody is the anti-Miao Le Shi pipe hormone polyclonal antibody of mouse, rabbit antibody Miao's Le Shi pipe hormone polyclonal antibody, goat-anti
Body Miao's Le Shi pipe hormone polyclonal antibody, chicken antibody Miao's Le Shi pipe hormone polyclonal antibody, the anti-Miao Le Shi pipe hormone monoclonal of mouse
Antibody, rabbit antibody Miao's Le Shi pipe hormone monoclonal antibody.
5. anti-Miao Le Shi pipe hormone test reagent according to claim 4, which is characterized in that anti-Miao Le Shi pipe hormone antibody
Concentration be 0.005ng/mL~1.5ng/mL.
6. anti-Miao Le Shi pipe hormone test reagent according to claim 1-3, which is characterized in that the wheat germ
Agglutinin extracts from naked barley wheat germ, barley wheat germ, paddy or wheat.
7. anti-Miao Le Shi pipe hormone test reagent according to claim 6, which is characterized in that the wheat germ agglutinin is dense
Degree is 0.01ng/mL~5ng/mL.
8. anti-Miao Le Shi pipe hormone test reagent according to claim 1, which is characterized in that the solid phase carrier is cream
One or more of glue particle, nitrocellulose filter, nylon membrane, polyvinylidene fluoride film, microwell plate or magnetic-particle.
9. anti-Miao Le Shi pipe hormone test reagent according to claim 1, which is characterized in that the marker is latex
Particle, colloidal gold, fluorescence, digoxin, biotin, alkaline phosphatase, horseradish peroxidase, acridinium ester, acridine ester derivant,
Luminol or derivatives thereof or one or more of tris (bipyridine) ruthenium.
10. anti-Miao Le Shi pipe hormone test reagent according to claim 1 detects anti-Miao Le Shi pipe Hormone agents in preparation
Application in box.
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| CN201910117379.7A CN109813881A (en) | 2019-02-15 | 2019-02-15 | A kind of hormone test reagent and kit |
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| CN201910117379.7A CN109813881A (en) | 2019-02-15 | 2019-02-15 | A kind of hormone test reagent and kit |
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Application publication date: 20190528 |