CN106324263A - Test strip for detecting estriol and applications thereof - Google Patents
Test strip for detecting estriol and applications thereof Download PDFInfo
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- CN106324263A CN106324263A CN201510373198.2A CN201510373198A CN106324263A CN 106324263 A CN106324263 A CN 106324263A CN 201510373198 A CN201510373198 A CN 201510373198A CN 106324263 A CN106324263 A CN 106324263A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/521—Single-layer analytical elements
- G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2407/00—Assays, e.g. immunoassays or enzyme assays, involving terpenes
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Abstract
The invention discloses a test strip for detecting estriol and applications thereof. The test strip comprises a sample absorbing pad (1), a combined substance releasing pad (2), a reaction membrane (3), a water absorbing pad (4), and a bottom plate (7). The reaction membrane comprises a detection line (5) embedded with an estriol semi-antigen-carrier protein conjugate and a quality control line (6) embedded with a goat-anti-mouse anti-antibody. An estriol monoclonal antibody-colloid gold marker is sprayed on the combined substance releasing pad (2). The invention also provides a method using the provided estriol test strip to detect residual estriol in fish meat, chicken, shrimp meat, and pork. The provided test strip has the characteristics of simple operation, high sensitivity, fast detection speed, and low cost, and is suitable for screening of massive samples and onsite monitoring.
Description
Technical field
The present invention relates to a kind of test strips detecting estriol and application thereof, be specifically related to a kind of gold colloidal for detecting estriol
Test strips, it is particularly well-suited to the detection of estriol residual in the animal derived foods such as the flesh of fish, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica.
Background technology
Estriol is the metabolite of internal estradiol, for a kind of natural estrogen being primarily present in urine.But, in aquaculture
It is frequently present of the phenomenon such as abuse of antibiotics and hormone, causes animal derived food remains a certain amount of estriol hormone, after eating
The mankind may be caused to be susceptible to hormone related disorders, as mammary gland, ovary, tumor of prostate, tumor that endocrine is relevant,
Birth defect and birth defects etc., also can cause have a strong impact on baby and teen-age growth promoter.
Based on above reason, we devise the one colloidal gold immunochromatographimethod technology for detection flesh of fish, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica etc.
The method of estriol in animal derived food, the method specificity is good, highly sensitive, easy and simple to handle, testing cost is low, be suitable for
In the selective mechanisms of batch sample, it is preferable rapid screening means, it is possible to preferably meet China's food enterprise, government function
Supervision departments etc. carry out detection work.
Summary of the invention
It is an object of the invention to provide a kind of highly sensitive, simple to operate, low cost, detection time short estriol residue detection
Test strips.
Detection estriol provided by the present invention residual test strips, including sample absorption pad (1), conjugate release pad (2),
Reaction film (3), adsorptive pads (4) and base plate (7)；Have on described reaction film and be coated with estriol hapten-carrier albumen occasionally
Joining the detection line (5) of thing and be coated with the nature controlling line (6) of sheep anti mouse anti antibody, described conjugate release pad (2) is coated with female
Triol monoclonal antibody-colloid gold label thing.
Described estriol hapten-carrier protein conjugate is to be obtained with carrier protein couplet by estriol hapten, described carrier egg
Bai Kewei bovine serum albumin, ovalbumin, hemocyanin, thyroprotein, human serum albumin.
Described estriol monoclonal antibody is to prepare using estriol hapten-carrier protein conjugate as immunogen, is by female
Triol monoclonal antibody hybridoma cell strain secretion obtains；Described sheep anti mouse anti antibody is to be obtained by Mus source antibody mediated immunity sheep.
Described sample absorption pad (1), conjugate release pad (2), reaction film (3), adsorptive pads (4) are pasted onto base plate (7) successively
On, described conjugate release pad 1/3～1/2 is capped under sample absorption pad.
The material that described base plate can be PVC base plate or other hard do not absorb water；Described sample absorption pad can be suction strainer paper or oil strain
Paper；Described conjugate release pad can be glass cotton or polyester material；Described adsorptive pads is absorbent paper；Described reaction film can be nitric acid
Cellulose membrane or cellulose acetate membrane.
It is a further object to provide a kind of method preparing above-mentioned test strips, it includes step:
1) preparation is coated with the conjugate release pad of estriol monoclonal antibody-colloid gold label thing；
2) preparation has the detection line being coated with estriol hapten-carrier protein conjugate and the matter being coated with sheep anti mouse anti antibody
The reaction film of control line；
3) by 1) and 2) the conjugate release pad for preparing, reaction film be assembled into examination with sample absorption pad, adsorptive pads and base plate
Paper slip.
Specifically, step includes:
1) prepared by hapten: estriol and chloroacetate reaction are obtained estriol hapten；
2) by estriol hapten and carrier protein couplet, estriol hapten-carrier protein conjugate is obtained；
3) with estriol hapten-carrier protein conjugate immune mouse, by mouse boosting cell and myeloma cell by merging,
Screening, obtains estriol monoclonal hybridoma strain；
4) extract mouse IgG immune health goat, obtain sheep anti mouse anti antibody；
5) gold colloidal is prepared with trisodium citrate and gold chloride reaction；
6) by step 3) the estriol monoclonal antibody prepared adds step 5) in the gold colloidal prepared, obtain estriol Dan Ke
Grand antibody-colloidal gold label；
7) being sprayed in conjugate release pad by estriol monoclonal antibody-colloid gold label thing, 37 DEG C are dried taking-up after 1h, are placed in
Dry environment saves backup；
8) estriol hapten-carrier protein conjugate is coated on reaction film composition detection line, sheep anti mouse anti antibody is coated
Reaction film constitutes nature controlling line；
9) by sample absorption pad be 7.2 containing 0.5% bovine serum albumin (volume fraction), pH, 0.1mol/L phosphate-buffered
Immersion bubble 2h, dries 2h at 37 DEG C；
10) on base plate, sample absorption pad, conjugate release pad, reaction film, adsorptive pads, sample absorption pad are pasted in order
Cover conjugate release pad, be finally cut into the wide little bar of 3mm, add plastic casing, vacuum packaging, can preserve under the conditions of 4～30 DEG C
12 months.
It is a further object to provide estriol in a kind of the application above-mentioned ELISA test strip flesh of fish, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica
The method of residual, it includes step:
(1) sample pre-treatments；
(2) detect by test strips；
(3) testing result is analyzed.
The estriol Rapid detection test strip of the present invention uses antibody antigen reaction and the immunochromatographiassays assays technology of high degree of specificity,
Estriol monoclonal antibody-colloid gold label thing is fixed in conjugate release pad, the estriol in sample in flow process,
Estriol monoclonal antibody-colloid gold label thing in conjugate release pad is combined, and forms drug-antibody-colloid gold label thing.
Medicine in sample and the estriol hapten-carrier protein conjugate competition binding estriol monoclonal anti on reaction film detection line
Body-colloid gold label thing, according to detection line red stripes with or without or shade judge whether analyte sample fluid contains estriol
Residual.
During detection, sample instills in test strips hole clipping after treatment, when estriol concentration in the sample less than detection limit or is zero
Time, monoclonal antibody-colloid gold label thing can be with the estriol hapten-carrier albumen being fixed on reaction film in chromatography process
Conjugate combines, and each in detection line (T) and nature controlling line (C) red stripes occur；If estriol is in the sample
Concentration equal to or higher than detection limit, monoclonal antibody-colloid gold label thing all can be combined with estriol, thus at T line because
Competitive reaction will not be combined with estriol hapten-carrier protein conjugate and occur without red stripes.As shown in Figure 2.
Negative: when nature controlling line (C) demonstrates red stripes, and detection line (T) colour developing is deep or colour developing is consistent, is judged to feminine gender.
Positive: when nature controlling line (C) demonstrates red stripes, and to detect line (T) and develop the color shallow or do not develop the color, be judged to the positive.
Invalid: when nature controlling line (C) does not demonstrate red stripes, the most no matter to detect whether line (T) demonstrates red stripes, should
It is invalid that test strips is all judged to.
The test strips of the present invention has highly sensitive, high specificity, low cost, simple to operate, the detection time is short, it is various to be suitable for
Unit uses, stores simple, the advantage of long shelf-life.Easy, quick by the method for ELISA test strip estriol of the present invention residual,
Intuitively, accurate, applied widely, low cost, easily promote the use of.
Accompanying drawing explanation
Fig. 1 is test strips cross-sectional view.
Fig. 2 is ELISA test strip result process decision chart.
Fig. 3 is estriol hapten synthesis figure.
Fig. 4 is estriol hapten hydrogen nuclear magnetic resonance spectrogram.
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment.Should be understood that these embodiments are merely to illustrate the present invention,
And it is not limited to the scope of the present invention.
The preparation of embodiment 1 estriol test strip
The preparation method of this test strips mainly comprises the steps that
1) preparation is coated with the conjugate release pad of estriol monoclonal antibody-colloid gold label thing；
2) preparation has the detection line being coated with estriol hapten-carrier protein conjugate and the matter being coated with sheep anti mouse anti antibody
The reaction film of control line；
3) by 1) and 2) the conjugate release pad for preparing, reaction film assemble with sample absorption pad, adsorptive pads and PVC base plate
Become test strips.
Substep narration in detail below:
1, the haptenic preparation of estriol
Take estriol 1.0g and add acetonitrile dissolving, add potassium carbonate 0.62g, add monoxone 0.47g, 60 DEG C of stirring 6h.Detection, raw material base
This reaction is complete, stopped reaction, and rotation is steamed, and adds water, and ethyl acetate extracts, and point goes organic facies, aqueous phase regulation pH value to 6, second
Acetoacetic ester extracts, and divides and goes aqueous phase, anhydrous sodium sulfate to be dried, is evaporated, and add methylene chloride/petroleum ether 1: 1, is recrystallized to give hapten
Product.Synthetic route such as Fig. 3.
Take above-mentioned product through nuclear magnetic resonance hydrogen spectruming determining, as shown in Figure 4,1H NMR(CDCl3, 300MHZ) δ: 11.0 (s, 1H,
COOH), 6.78 (d, 2H, ArH), 6.44 (d, 1H, ArH), 4.66 (s, 2H ,-CH2-), 3.58 (s, 2H, OH), 3.22 (m,
2H ,-CH-), 2.90 (m, 2H ,-CH2), 1.68-1.88 (m, 10H ,-CH2-), 1.04 (s, 3H ,-CH3), chemical shift δ=11 in collection of illustrative plates
For carboxyl hydrogen, 4.66 is methylene hydrogen on spacerarm, in addition to the peculiar hydrogen of raw material, the existence explanation of the resonance absorbing peak of these hydrogen
Spacerarm coupling success, the i.e. success of explanation hapten synthesis.
2, immunogenic preparation
Take 15mg hapten, be dissolved in 1mL DMF, obtain solution (1)；Take 30mg EDC and NHS 0.2ml water is abundant
In adding in (1) after dissolving, stir 24h under room temperature, i.e. can get reactant liquor A.Weigh BSA50mg, be allowed to fully dissolve
In 3.8mL PBS (PH 7.2), reactant liquor A is dropwise slowly dropped in protein solution, and stirs 24h at room temperature,
3 dialysis solution are changed every day with 0.01mol/L PBS 4 DEG C dialysis 3d.Subpackage, saves backup in-20 DEG C.
3, the preparation of coating antigen
Take 15mg hapten, be dissolved in 1mL DMF, obtain solution (1)；Take 30mg EDC and NHS 0.2ml water is abundant
In adding in (1) after dissolving, stir 24h under room temperature, i.e. can get reactant liquor A.Weigh OVA50mg, be allowed to fully dissolve
In 3.8mL PBS (PH 7.2), reactant liquor A is dropwise slowly dropped in protein solution, and stirs 24h at room temperature,
3 dialysis solution are changed every day with 0.01mol/L PBS 4 DEG C dialysis 3d.Subpackage, saves backup in-20 DEG C.
4, the preparation of estriol monoclonal antibody
(1) animal immune
Immunogen step 2 obtained is injected in Balb/c Mice Body, and immunizing dose is 150 μ g/ so that it is produce antiserum.
(2) cell merges and cloning
Take immunity Balb/c mouse boosting cell, in 8: 1 (quantitative proportion) ratio and SP2/0 myeloma cell fusion, use indirectly
Inhibition ELISA measures cell supernatant, the positive hole of screening.Utilize limiting dilution assay that positive hole is carried out cloning, until
Hybridoma cell strain to stably excreting monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma frozen stock solution is made 1 × 106The cell suspension of individual/ml, preserves in liquid nitrogen for a long time.Take out during recovery and freeze
Deposit pipe, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
(4) preparation of monoclonal antibody and purification
Increment culture method: be placed in cell culture medium by hybridoma, cultivates under the conditions of 37 DEG C, with sad-saturated
The culture fluid obtained is purified by ammonium sulfate method, obtains monoclonal antibody ,-20 DEG C of preservations.
Described cell culture medium is to add calf serum and sodium bicarbonate in RPMI1640 culture medium, makes calf serum at cell
Final concentration of 20% (mass fraction) in culture medium, sodium bicarbonate in cell culture medium final concentration of 0.2% (quality is divided
Number)；The pH of described cell culture medium is 7.4.
5, the preparation of sheep anti mouse anti antibody
Using sheep as immune animal, for immunogen, pathogen-free domestic sheep is carried out immunity with Mus source antibody, obtain sheep anti mouse anti antibody.
6, the preparation of estriol monoclonal antibody-colloid gold label thing
(1) preparation of gold colloidal
By double distilled deionized water, 1% gold chloride is diluted to 0.01% (mass fraction), takes 100ml and be placed in conical flask, by perseverance
Temperature magnetic stirrer is heated to boiling, at continuous high temperature, continuously stirred lower addition 2.5ml 1% trisodium citrate, continues at the uniform velocity to stir
Mix be heated to solution be bright red time stop, returning to original volume with deionized water after being cooled to room temperature, 4 DEG C of preservations.Preparation
Good gold colloidal outward appearance is pure, bright, without precipitation and floating thing.
(2) preparation of estriol monoclonal antibody-colloid gold label thing
Under magnetic stirring, with the pH value of 0.2mol/L solution of potassium carbonate tune gold colloidal to 7.0, by every milliliter of colloidal gold solution
The standard adding 20～50 μ g adds estriol monoclonal antibody in colloidal gold solution, continues stirring and evenly mixing 30min, adds
10%BSA so that it is final concentration of 1% (volume fraction) in colloidal gold solution, stands 10min.12000r/min、4℃
Centrifugal 40min, abandons supernatant, and precipitation uses redissolution buffer solution twice, with the redissolution that volume is initial colloid gold volume 1/10
Buffer will precipitate resuspended, put 4 DEG C standby.
Redissolution buffer: casein containing protein 0.02%～0.1% (mass fraction), tween 80 0.05%～0.2% (mass fraction), pH7.2
0.02mol/L phosphate buffer.
7, the preparation of conjugate release pad
Conjugate release pad is soaked in containing bovine serum albumin (bovine serum albumin concentration in buffer is 0.5%),
PH is 7.2, in the phosphate buffer of 0.5mol/L, uniformly soak 1h, and 37 DEG C to dry 3h standby.Will with Isoflow spray film instrument
The estriol monoclonal antibody prepared-colloid gold label thing even application in conjugate release pad, every 1cm conjugate release pad
After spraying 0.01ml estriol monoclonal antibody-colloid gold label thing, it is placed in 37 DEG C of environment and takes after (humidity ＜ 20%) 60min
Go out, be placed in dry environment (humidity ＜ 20%) and save backup.
8, the preparation of reaction film
Estriol hapten-ovalbumin conjugate is coated on reaction film composition detection line, sheep anti mouse anti antibody is coated on instead
Answer composition nature controlling line on film.
It is coated process: with phosphate buffer, estriol hapten-ovalbumin conjugate is diluted to 10mg/ml, uses Isoflow
The detection line (T line) that some film instrument is coated on nitrocellulose filter, package amount is 0.8 μ l/cm；With 0.01mol/L, pH7.4
Phosphate buffer sheep anti mouse anti antibody is diluted to 200 μ g/ml, be coated in celluloid with Isoflow point film instrument
Nature controlling line (C line) on film, package amount is 1.0 μ l/cm.2h it is dried under the conditions of the reaction film being coated is placed in 37 DEG C, standby
With.
9, the preparation of sample absorption pad
Sample absorption pad is placed in containing in 0.5% bovine serum albumin (volume fraction), pH7.2,0.1mol/L phosphate buffer
Soaking 2h, 37 DEG C of baking 2h are standby.
10, the assembling of test strips
Sample absorption pad, conjugate release pad, reaction film, adsorptive pads are pasted onto on PVC base plate the most in order；Conjugate
Release pad has 1/3 region to be absorbed by the sample pad covering from initiating terminal, and the end of conjugate release pad is connected with the top of reaction film,
The end of reaction film is connected with the top of adsorptive pads, and the top of sample absorption pad aligns with the top of PVC base plate, the end of adsorptive pads
End aligns with the end of PVC base plate；Detection line and nature controlling line, detection line (T line) and nature controlling line (C is had on described reaction film
Line) it is the strip tape perpendicular with the length of described test strips；Detection line is located close to the side of the end of conjugate release pad；
Nature controlling line is located remotely from the side of the end of conjugate release pad；Test strips machine is cut into the wide little bar of 3mm, is contained in special
Plastics fabrication in, can preserve 12 months under the conditions of 4～30 DEG C.
The detection of estriol residual in embodiment 2 sample
1, the pre-treatment of sample
Take tissue samples homogenizing 1min (10000rpm) in homogenizer removing fat, or shred with shears；Weigh 2.0 ± 0.05g
The good tissue samples of homogenate, in 10ml polystyrene centrifuge tube, adds 1ml sample extraction liquid, and concussion shakes up；Adding 1ml
Sample dilution is in polystyrene centrifuge tube, and concussion shakes up.
2, detect by test strips
With Dispette draw sample solution to be checked vertically drip 2～3 in well, liquid flowing time start timing, instead
Answering 5～10min, according to schematic diagram result of determination, it is invalid that other times judge.
3, testing result is analyzed
Negative (-): the colour developing of T line is deeper than the colour developing of C line or colour developing is consistent, represents that in sample, estriol concentration is less than detection limit, such as Fig. 2
(a)、(b)。
Positive (+): the colour developing of T line is more shallow than the colour developing of C line or T line does not develops the color, and represents that in sample, estriol concentration is equal to or higher than detection
Limit, such as Fig. 2 (c), (d).
Invalid: C line does not occurs, show the deterioration failure of incorrect operating process or test strips, such as Fig. 2 (e), (f).
In the case, should again read over description, and retest by new test strips.
Embodiment 3 sample detection example
1, detection limit test
Take the blank flesh of fish, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica sample, add estriol extremely final concentration of 5,10,20 μ g/kg the most respectively,
Taking test strips to detect, each sample is repeated three times.
During with the ELISA test strip flesh of fish, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica sample, when wherein estriol interpolation concentration is 5 μ g/kg, in
Negative；When wherein estriol interpolation concentration is 10,20 μ g/kg, it is positive, shows that this test strips is to the flesh of fish, Carnis Gallus domesticus, shrimp
In meat, Carnis Sus domestica, the detection of estriol is limited to 10 μ g/kg.
2, false positive rate, false negative rate test
Take known estriol content more than the flesh of fish of 10 μ g/kg, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica positive sample each 20 parts (numberings 1-20)
With content less than the flesh of fish of 10 μ g/kg, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica negative sample each 20 parts (numberings 21-40), with three batches of reagent paper
Bar detects, and calculates its yin and yang attribute rate.The results are shown in Table 1-4.
Table 1 detection flesh of fish sample results
Table 2 detects Carnis Gallus domesticus sample results
Table 3 detects Macrobrachium nipponensis sample results
Table 4 detects Carnis Sus domestica sample results
Result shows: during with the ELISA test strip positive flesh of fish of 3 batches productions, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica sample, result is complete
For the positive, it is known that positive sample coincidence rate is 100%, false negative rate is 0；Detect 20 portions of negative flesh of fish, Carnis Gallus domesticus, Macrobrachium nipponensis,
During Carnis Sus domestica sample, result is all negative, it is known that negative match-rate is 100%, and false positive rate is 0.Illustrate that the detection of the present invention is female
Estriol residual in the flesh of fish, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica can be used for quickly detecting by the test strips of triol.
3, specific test
With estriol ELISA test strip 1000 μ g/kg estradiol benzoate, tilmicosin, sulfamethazine, enrofloxacin,
The medicines such as tylosin.Result shows, test strips is all negative.Illustrate this test strips to 1000 μ g/kg estradiol benzoates,
The medicine no cross reactions such as tilmicosin, sulfamethazine, enrofloxacin, tylosin, specificity is good.
Claims (8)
1. detect a test strips for estriol, including sample absorption pad (1), conjugate release pad (2), reaction film (3),
Adsorptive pads (4) and base plate (7), it is characterised in that have on described reaction film and be coated with estriol hapten-carrier albumen coupling
The detection line (5) of thing and be coated with the nature controlling line (6) of sheep anti mouse anti antibody, described conjugate release pad (2) is coated with female three
Alcohol monoclonal antibody-colloid gold label thing, it is characterised in that described estriol hapten is to take estriol 1.0g to add acetonitrile dissolving, adds
Potassium carbonate 0.62g, adds monoxone 0.47g, 60 DEG C of stirring 6h.Detection, raw material fundamental reaction is complete, stopped reaction, and rotation is steamed,
Adding water, ethyl acetate extracts, and divides and goes organic facies, and aqueous phase regulation pH value is to 6, and ethyl acetate extracts, and divides and removes aqueous phase, anhydrous sulfur
Acid sodium is dried, and is evaporated, and add methylene chloride/petroleum ether 1: 1, is recrystallized to give hapten product.
2. test strips as claimed in claim 1, it is characterised in that described sample absorption pad (1), conjugate release pad (2),
Reaction film (3), adsorptive pads (4) are pasted onto on base plate (7) successively.
3. the test strips as described in any one of claim 1-2, it is characterised in that described conjugate release pad 1/3～1/2 is capped
Under sample absorption pad.
4. test strips as claimed in claim 1, it is characterised in that described estriol hapten-carrier protein conjugate is by estriol
Hapten obtains with carrier protein couplet, and described carrier protein can be bovine serum albumin, ovalbumin, hemocyanin, first shape
Gland albumen, human serum albumin.
5. the test strips as described in any one of claim 1 or 4, it is characterised in that described estriol hapten be by estriol with
Obtaining chloroacetate reaction, its molecular structural formula is:
6. test strips as claimed in claim 1, it is characterised in that described estriol monoclonal antibody be with estriol hapten-
Carrier protein couplet thing prepares as immunogen, and described sheep anti mouse anti antibody is to be obtained by Mus source antibody mediated immunity sheep.
7. preparing a method for test strips described in any one of claim 1-6, it includes step:
1) preparation is coated with the conjugate release pad of estriol monoclonal antibody-colloid gold label thing；
2) preparation has the detection line being coated with estriol hapten-carrier protein conjugate and the matter being coated with sheep anti mouse anti antibody
The reaction film of control line；
3) by 1) and 2) the conjugate release pad for preparing, reaction film be assembled into examination with sample absorption pad, adsorptive pads and base plate
Paper slip.
8. the method detecting the residual of estriol in the flesh of fish, Carnis Gallus domesticus, Macrobrachium nipponensis, Carnis Sus domestica, it includes step:
1) Sample pretreatment；
2) detect by the test strips described in any one of claim 1-6；
3) testing result is analyzed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510373198.2A CN106324263A (en) | 2015-07-01 | 2015-07-01 | Test strip for detecting estriol and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510373198.2A CN106324263A (en) | 2015-07-01 | 2015-07-01 | Test strip for detecting estriol and applications thereof |
Publications (1)
| Publication Number | Publication Date |
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| CN106324263A true CN106324263A (en) | 2017-01-11 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201510373198.2A Pending CN106324263A (en) | 2015-07-01 | 2015-07-01 | Test strip for detecting estriol and applications thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108931640A (en) * | 2018-09-27 | 2018-12-04 | 北京勤邦生物技术有限公司 | A kind of test strips and its application detecting organophosphorus pesticide |
| CN109239336A (en) * | 2018-09-19 | 2019-01-18 | 北京勤邦生物技术有限公司 | A kind of test strips and its application detecting Mobucin |
| CN111856000A (en) * | 2020-06-04 | 2020-10-30 | 北京勤邦生物技术有限公司 | Test strip and method for detecting chlordane |
| CN112444631A (en) * | 2019-09-05 | 2021-03-05 | 北京动物园 | Kit for detecting sex of crane |
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2015
- 2015-07-01 CN CN201510373198.2A patent/CN106324263A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239336A (en) * | 2018-09-19 | 2019-01-18 | 北京勤邦生物技术有限公司 | A kind of test strips and its application detecting Mobucin |
| CN109239336B (en) * | 2018-09-19 | 2023-02-24 | 北京勤邦生物技术有限公司 | Test strip for detecting isoprocarb and application thereof |
| CN108931640A (en) * | 2018-09-27 | 2018-12-04 | 北京勤邦生物技术有限公司 | A kind of test strips and its application detecting organophosphorus pesticide |
| CN108931640B (en) * | 2018-09-27 | 2022-10-18 | 北京勤邦生物技术有限公司 | Test strip for detecting organophosphorus pesticide and application thereof |
| CN112444631A (en) * | 2019-09-05 | 2021-03-05 | 北京动物园 | Kit for detecting sex of crane |
| CN111856000A (en) * | 2020-06-04 | 2020-10-30 | 北京勤邦生物技术有限公司 | Test strip and method for detecting chlordane |
| CN111856000B (en) * | 2020-06-04 | 2023-07-07 | 北京勤邦科技股份有限公司 | Test strip and method for detecting chlordane |
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