CN110441517A - A kind of test strips and method detecting 6-benzyladenine - Google Patents
A kind of test strips and method detecting 6-benzyladenine Download PDFInfo
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- CN110441517A CN110441517A CN201910643893.4A CN201910643893A CN110441517A CN 110441517 A CN110441517 A CN 110441517A CN 201910643893 A CN201910643893 A CN 201910643893A CN 110441517 A CN110441517 A CN 110441517A
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- G—PHYSICS
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- 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
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a kind of test strips and method for detecting 6-benzyladenine.The test strips include test paper and micropore reagent, the test paper includes reaction film, sample absorption pad, water absorption pad and bottom plate, have on the reaction film and be coated with the detection line of 6-benzyladenine hapten-carrier protein conjugate and be coated with the nature controlling line of sheep anti mouse antiantibody, freeze-drying has 6-benzyladenine monoclonal antibody-colloid gold label object on the micropore reagent.With easy, quick, intuitive, accurate, easy to carry, applied widely, at low cost, the easy popularization and use of method of test strips of the present invention detection 6-benzyladenine.
Description
Technical field
The present invention relates to a kind of test strips and method for detecting 6-benzyladenine, and in particular to one kind is for detecting 6- benzyl
The colloidal gold strip of base adenine, it is especially suitable for the remaining detections of 6-benzyladenine in bean sprouts.
Background technique
6-benzyladenine (6-benzylarninopuring, 6-BA) is a kind of artificial synthesized basic element of cell division, is
White or off-white color crystal, are insoluble in water, are slightly soluble in ethyl alcohol, stablize in acid, alkali.With inhibition leaves of plants inner chlorophyll, core
The effect of acid and breaks down proteins, is mainly used for grain, cultivation of fruit tree and gardening, each growth phase of crop can all be applied.6-
BA is widely used today as the growth regulator of rootless bean sprouts.Human body, which takes in excessive 6-BA, can stimulate skin and mucosa, cause esophagus,
There is Nausea and vomiting etc. in mucosal lesion.Original state food pharmaceuticals administration general bureau, China, the Ministry of Agriculture, national health and meter
The fertility committee is drawn about the bulletin (o.11 in 2015) for being forbidden to use the substances such as 6-benzyladenine in the production process of bean sprouts
Middle regulation: the producer must not use the objects such as 6-benzyladenine, 4-chlorophenoxyacetic acid sodium, gibberellin in the production process of bean sprouts
Matter, bean sprouts operator must not manage the bean sprouts containing substances such as 6-benzyladenine, 4-chlorophenoxyacetic acid sodium, gibberellin.
The method of detection 6-benzyladenine reported at present is mainly liquid chromatography, liquid chromatography tandem mass spectrometry
Equal instrumental methods.These methods must operate in laboratory conditions, and sample pre-treatments are cumbersome time-consuming, also need to be equipped with expensive instrument
Device equipment, testing cost is higher, time-consuming, complicated for operation, has in actual application very big restricted, it is difficult to meet big
The needs that amount sample and field sample quickly detect.Therefore, it develops a kind of simple and quick, residual suitable for 6-benzyladenine in bean sprouts
The colloidal gold strip stayed can meet a large amount of sample site screenings and monitoring, can preferably meet foods supervision department, China
Equal developments detect work.
Summary of the invention
The purpose of the present invention is to provide one kind to be able to detect the remaining colloidal gold strip of 6-benzyladenine in bean sprouts,
And provide efficient one kind, accurate, simplicity, suitable for the detection method of on-site supervision and great amount of samples screening.
The test strips of detection 6-benzyladenine provided by the present invention, including test paper and micropore reagent；The test paper packet
Bottom plate, sample absorption pad, reaction film, water absorption pad are included, is sequentially connected, has on the reaction film and is coated with 6-benzyladenine
The detection line of hapten-carrier protein conjugate and the nature controlling line for being coated with sheep anti mouse antiantibody；It is lyophilized on the micropore reagent
There is 6-benzyladenine monoclonal antibody-colloid gold label object, with micropore plug.
The 6-benzyladenine monoclonal antibody is using 6-benzyladenine hapten-carrier protein conjugate as exempting from
Epidemic focus prepares.
The 6-benzyladenine hapten-carrier protein conjugate is even by 6-benzyladenine haptens and carrier protein
Connection obtains, the carrier protein be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein or human serum albumins,
The 6-benzyladenine haptens is obtained by amino -6-benzyladenine and 4- (3- oxopropyl)-benzoic acid,
Molecular structural formula are as follows:
The bottom plate can be the material that PVC bottom plate or other hard do not absorb water；The sample absorption pad can for suction strainer paper or
Filter paper for oil；The water absorption pad is blotting paper；The reaction film can be nitrocellulose filter or cellulose acetate film.
It is a further object to provide a kind of methods for preparing above-mentioned test strips comprising step:
1) preparation freeze-drying has 6-benzyladenine monoclonal antibody-colloid gold label object micropore reagent；
2) preparation has the detection line for being coated with 6-benzyladenine hapten-carrier protein conjugate and is coated with goat-anti
The reaction film of the nature controlling line of mouse antiantibody；
3) 2) reaction film prepared and sample absorption pad, water absorption pad, bottom plate are assembled into test paper；
4) 1) there will be 6-benzyladenine monoclonal antibody-colloid gold label object micropore reagent with the freeze-drying 3) prepared
Test strips are assembled into test paper.
Specifically, step includes:
1) by amino -6-benzyladenine and 4- (3- oxopropyl)-benzoic acid, it is anti-to prepare 6-benzyladenine half
It is former；
2) by 6-benzyladenine haptens and carrier protein couplet, 6-benzyladenine hapten-carrier albumen is prepared
Conjugate；
3) mouse is immunized with 6-benzyladenine hapten-carrier protein conjugate, by mouse boosting cell and mouse bone marrow cells
Oncocyte obtains the hybridoma cell strain of secretion 6-benzyladenine monoclonal antibody by fusion, screening；
4) mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5) 6-benzyladenine hapten-carrier protein conjugate and sheep anti mouse antiantibody are coated in reaction film respectively
In detection line (T) and nature controlling line (C)；
6) it is reacted with trisodium citrate with gold chloride and prepares colloidal gold；
7) the 6-benzyladenine monoclonal antibody of preparation is added in the colloidal gold of preparation, obtains 6-benzyladenine
Monoclonal antibody-colloid gold label object；
8) after by 6-benzyladenine monoclonal antibody-colloid gold label object freeze-drying in micropore reagent, by micropore reagent
In addition micropore plug；
9) phosphate buffer containing 0.5% bovine serum albumin(BSA), pH 7.2,0.1mol/L is used to soak sample absorption pad
2h is steeped, dries 2h at 37 DEG C；
10) it pastes upper sample absorption pad, reaction film, water absorption pad in order on bottom plate, is finally cut into the small item of 3mm wide,
Add plastic casing, is vacuum-packed；
11) the micropore reagent and test paper that prepare are assembled into test strips, are saved 12 months under the conditions of 2~8 DEG C.
It is a further object to provide 6-benzyladenine residuals in a kind of application above-mentioned test strips detection bean sprouts
Method, it comprising steps of
(1) sample pre-treatments；
(2) it is detected with test strips；
(3) analysis detection result.
6-benzyladenine Rapid detection test strip of the invention is reacted and is immunized using the antibody antigen of high degree of specificity
The 6- in micropore reagent, in sample is lyophilized in 6-benzyladenine monoclonal antibody-colloid gold label object by Chromatographic techniques
Benzyladenine is first sufficiently reacted with it, forms drug-antibody-colloid gold label object；It is instilled in test strips card hole again, in sample
Drug in flow process with the 6-benzyladenine hapten-carrier protein conjugate competitive binding in reaction film detection line
6-benzyladenine monoclonal antibody-colloid gold label object, judges in analyte sample fluid according to the detection line red stripes depth
Whether remained containing 6-benzyladenine.
When detection, sample is instilled after processing in micropore reagent, is sufficiently added in test strips card hole after reaction, when 6- benzyl
The concentration of base adenine in the sample is lower than detection limit or when being zero, and monoclonal antibody-colloid gold label object is in chromatography process
It can be in conjunction with the 6-benzyladenine hapten-carrier protein conjugate being fixed on reaction film, in detection line (T) and nature controlling line
(C) one red stripes of each appearance on, and the colour developing of T line develops the color deep or develops the color with C line consistent than C line；If 6-benzyladenine
Concentration in the sample is equal to or higher than detection limit, and monoclonal antibody-colloid gold label object can all be tied with 6-benzyladenine
It closes, thus because competitive reaction will not be in conjunction with 6-benzyladenine hapten-carrier protein conjugate without occurring at T line
Red stripes are more shallow than the colour developing of C line.As shown in Figure 4.
It is negative: when nature controlling line (C) shows that red stripes, detection line (T) also show that red stripes, and (T) line simultaneously
When color is close or is deeper than (C) line, it is judged to feminine gender.
Positive: when nature controlling line (C) shows red stripes, and detection line (T) does not develop the color or (T) line color is shallower than (C) line
When, it is judged to the positive.
It is invalid:, should whether nature controlling line (C) does not show red stripes, then no matter detection line (T) shows red stripes
Test strips are judged in vain.
Test strips of the invention have that high sensitivity, high specificity, at low cost, easy to operate, detection time is short, it is each to be suitble to
The advantages of kind unit uses, storage is simple, long shelf-life.Wherein, using the 6-benzyladenine monoclonal antibody of high specific,
It ensure that the reliability of testing result；By gold labeling antibody freeze-drying in micropore reagent, in the detection process, gold labeling antibody can be made
It is come into full contact with measuring samples liquid, sufficiently reacts, to reduce error, increase the reaction sensitivity of whole system.With the present invention
Easy, quick, intuitive, accurate, applied widely, at low cost, the easy popularization and use of method of test strips detection 6-benzyladenine.
Detailed description of the invention
Fig. 1 is 6-benzyladenine hapten synthesis figure.
Fig. 2 is test paper the schematic diagram of the section structure.
Fig. 3 is micropore reagent figure.
Fig. 4 is test strips testing result process decision chart.
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not intended to limit the scope of the invention.
Embodiment 1 detects the preparation of the test strips of 6-benzyladenine
The preparation method of the test strips mainly comprises the steps that
1) preparation freeze-drying has 6-benzyladenine monoclonal antibody-colloid gold label object micropore reagent；
2) preparation has the detection line for being coated with 6-benzyladenine hapten-carrier protein conjugate and is coated with goat-anti
The reaction film of the nature controlling line of mouse antiantibody；
3) 2) reaction film prepared and sample absorption pad, water absorption pad, bottom plate are assembled into test paper；
4) 1) there will be 6-benzyladenine monoclonal antibody-colloid gold label object micropore reagent with the freeze-drying 3) prepared
Test strips are assembled into test paper.
Substep narration in detail below:
1, the synthesis of 6-benzyladenine haptens (synthetic route is shown in attached drawing 1)
Amino -6-benzyladenine 2.4g is taken, adds ethyl alcohol 70mL to dissolve, adds NaOH 0.45g, after being sufficiently stirred, add 4-
(3- oxopropyl)-benzoic acid 1.85g, 60 DEG C of heating react 4h, are cooled to room temperature, add sodium borohydride 0.38g, continue to stir
2h stops reaction, adds water 120mL, add 6mol/L salt acid for adjusting pH value to 6, add ethyl acetate 80mL, extracts, and divides and goes water phase, has
Machine is mutually rotated and is evaporated, and obtains red oil, and acetonitrile/n-hexane (1:1, V/V) 165mL recrystallization obtains 6-benzyladenine
Haptens product 1.9g, yield 47.26%.
2, the preparation of immunogene
6-benzyladenine haptens product 15mg is taken, n,N-Dimethylformamide (DMF) 1mL is added, dissolution clarification adds carbon
Change diimine (EDC) 13mg, n-hydroxysuccinimide (NHS) 9.4mg, after mixing well, react 4h, obtains haptens activation
Liquid A liquid；Bovine serum albumin(BSA) (BSA) 60mg is taken, adds 0.05mol/L PB to dissolve, obtains B liquid；A drop is added in B liquid, 4 DEG C
For 24 hours, 0.02mol/L PBS dialysis purification three days is changed liquid 3 times daily for reaction, obtains 6-benzyladenine haptens-BSA coupling
Object, as immunogene, packing, -20 DEG C of preservations.
3, the preparation of coating antigen
6-benzyladenine haptens product 9.6mg is taken, DMF 1mL is added, dissolution clarification adds EDC 7.7mg, NHS
6.4mg after mixing well, reacts 4h, obtains haptens activating solution A liquid；Ovalbumin (OVA) 50mg is taken, 0.05mol/L is added
PB dissolution, obtains B liquid；A drop is added in B liquid, for 24 hours, 0.02mol/L PBS dialysis purification three days changes liquid daily for 4 DEG C of reactions
3 times, 6-benzyladenine haptens-OVA conjugate, as coating antigen are obtained, is dispensed, -20 DEG C of preservations.
4, the preparation of 6-benzyladenine monoclonal antibody
(1) animal immune
In the immunogene injection Balb/c Mice Body that step 2 is obtained, immunizing dose is 150 μ g/, its is made to generate anti-blood
Clearly.
(2) cell fusion and cloning
Immune Balb/c mouse boosting cell is taken, is merged in 8:1 (quantitative proportion) ratio with SP2/0 myeloma cell, is used
Indirect competitive ELISA measures cell supernatant, screens positive hole.Cloning is carried out to positive hole using limiting dilution assay, until
Obtain the hybridoma cell strain of stably excreting monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma is made 1 × 10 with frozen stock solution6The cell suspension of a/mL, saves for a long time in liquid nitrogen.When recovery
Cryopreservation tube is taken out, 37 DEG C of water-bath middling speeds is immediately placed in and melts, after centrifugation removal frozen stock solution, moves into culture culture in glassware.
(4) preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, is cultivated under the conditions of 37 DEG C, with octanoic acid-
Saturated ammonium sulfate method purifies obtained culture solution, obtains monoclonal antibody, -20 DEG C of preservations.
The cell culture medium is that calf serum and sodium bicarbonate are added into RPMI1640 culture medium, and calf serum is made to exist
Final concentration of 20% (mass fraction) in cell culture medium, make sodium bicarbonate in cell culture medium final concentration of 0.2%
(mass fraction)；The pH of the cell culture medium is 7.4.
5, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized using source of mouse antibody as immunogene, it is anti-to obtain sheep anti mouse
Antibody.
6,6-benzyladenine monoclonal antibody-colloid gold label object preparation
(1) preparation of colloidal gold
1% gold chloride is diluted to 0.01% (mass fraction) with double distilled deionized water, 100mL is taken to be placed in conical flask,
It is heated to boiling with thermostatic electromagnetic blender, in continuous high temperature, is persistently added with stirring 1% trisodium citrate of 2.5mL, continue
Solution is at the uniform velocity heated with stirring in stopping when bright red, is restored to original volume, 4 DEG C of guarantors with deionized water after being cooled to room temperature
It deposits.The colloidal gold appearance prepared is pure, it is bright, without precipitating and floating material, observing color in the sunlight is claret.
(2) 6-benzyladenine monoclonal antibody-colloid gold label object preparation
Under magnetic stirring, molten by every milliliter of colloidal gold with the pH value of 0.2mol/L solution of potassium carbonate tune colloidal gold to 7.2
Above-mentioned 6-benzyladenine monoclonal antibody is added into colloidal gold solution for the standard that 20~50 μ g antibody are added in liquid, continues to stir
It mixes and mixes 30min；10%BSA is added after standing 10min, makes its final concentration of 1% in colloidal gold solution, stands 10min.
12000r/min, 4 DEG C of centrifugation 40min abandon supernatant, and precipitating is washed twice with redissolution buffer, are initial colloid gold with volume
The redissolution buffer of volume 1/10 will precipitating be resuspended, set 4 DEG C it is spare.
Redissolve buffer: the mass fraction containing BSA be 0.1%~0.3%, mass fraction 0.05% of Tween-80~
0.2%, the 0.02mol/L phosphate buffer of pH7.2.
7, the preparation of micropore reagent
100 μ L 6-benzyladenine monoclonal antibodies-colloid gold label object is added into micropore reagent microwell plate, is put into
In freeze drier, under the conditions of condenser temperature is -50 DEG C, after pre-freeze 3h, then it is dried in vacuo 15h, that is, can be taken off, be lyophilized
There is 6-benzyladenine monoclonal antibody-colloid gold label object micropore reagent, is sealed.
8, the preparation of sample absorption pad
By sample absorption pad be placed in containing 0.5% bovine serum albumin(BSA), pH 7.2,0.1mol/L phosphate buffer in
2h is impregnated, drying 2h is spare at 37 DEG C.
9, the preparation of reaction film
Detection line will be constituted in 6-benzyladenine haptens-ovalbumin conjugate coating to reaction film, by sheep anti mouse
Antiantibody, which is coated on reaction film, constitutes nature controlling line.
Coating process: being diluted to 1mg/mL for 6-benzyladenine haptens-ovalbumin conjugate with phosphate buffer,
It is coated in the detection line (T line) on nitrocellulose filter with Isoflow point film instrument, package amount is 1.0 μ L/cm；With
Sheep anti mouse antiantibody is diluted to 200 μ g/mL by the phosphate buffer of 0.01mol/L, pH7.4, with Isoflow point film instrument by its
The nature controlling line (C line) being coated on nitrocellulose filter, package amount are 1.0 μ L/cm.The reaction film being coated with is placed in 37 DEG C of items
Dry 2h, spare under part.
10, the assembling of test strips
Test paper cross-section structure shown in 2 with reference to the accompanying drawings, by sample absorption pad (1), reaction film (2), water absorption pad (3) successively by suitable
Sequence is pasted on PVC bottom plate (6)；The end of sample absorption pad is connected with the beginning of reaction film, the end of reaction film and water absorption pad
Beginning be connected, the beginning of sample absorption pad is aligned with the beginning of PVC bottom plate, the end pair of the end of water absorption pad and PVC bottom plate
Together；There are detection line (4) and nature controlling line (5) on the reaction film, detection line (T line) and nature controlling line (C line) are and the test paper
The perpendicular strip tape of length；Detection line is located at the side close to the end of sample absorption pad；Nature controlling line is located remotely from sample suction
Receive the side of the end of pad；Test paper is cut into the small item of 3mm wide with machine, in special plastics fabrication.
The figure of micropore reagent shown in 3 with reference to the accompanying drawings has micropore plug 8 on micropore reagent 7.
Above-mentioned test paper and micropore reagent are assembled into test strips, stored in 2~8 DEG C of environment, validity period 12 months.
The detection of 6-benzyladenine in 2 bean sprouts of embodiment
1, the pre-treatment of sample
With homogenizer homogeneous bean sprouts sample (being sure not to be ground into powder)；2.0g ± 0.05g sample is weighed to 10mL polyphenyl
In ethylene centrifuge tube, 3mL phosphate buffer is added, is mixed with vortex mixed instrument whirling motion 1min；Room temperature (20~25 DEG C)
4000r/min is centrifuged 5min, takes supernatant liquor as sample to be tested liquid.
2, it is detected with test strips
100 μ L sample to be tested liquid are drawn in micropore with micropipettor, are slowly aspirated and sufficiently mixed with reagent in micropore
It is even；After (20-25 DEG C) incubation 3min of room temperature, draws 90 μ L of mixing liquid and vertically drip in well；Liquid flowing starts timing, instead
8min is answered, determines result.
3, analysis detection result
Negative (-): the colour developing of T line is deeper than the colour developing of C line or consistent with the colour developing of C line, indicates 6-benzyladenine concentration in sample
It is limited lower than detection, such as Fig. 4 a, 4b.
Positive (+): the colour developing of T line is more shallow than the colour developing of C line or T line does not develop the color, and indicates that 6-benzyladenine concentration is equal in sample
Or it is higher than detection limit, such as Fig. 4 c, 4d.
It is invalid: not occur C line, show the deterioration failure of incorrect operating process or test strips, such as Fig. 4 e, 4f.
3 sample detection example of embodiment
1, detection limit test
Take blank bean sprouts sample, wherein respectively addition 6-benzyladenine to final concentration of 25 μ g/kg, 50 μ g/kg,
100 μ g/kg, take test strips to be detected, and each sample is repeated three times.
With test strips detect bean sprouts sample when, when wherein without 6-benzyladenine and its add concentration be 25 μ g/kg when, examination
It shows that the colour developing of T line develops the color deep or develops the color unanimously with C line than C line on paper slip, is negative；When wherein 6-benzyladenine addition is dense
When degree is 50 μ g/kg, 100 μ g/kg, shows that the colour developing of T line is more shallow than the colour developing of C line or T line does not develop the color in test strips, be positive, table
Bright test strips are limited to 50 μ g/kg to the detection of 6-benzyladenine in bean sprouts.
2, false positive rate, false negative rate test
It takes blank bean sprouts sample and adds the positive bean sprouts sample each 20 of 6-benzyladenine to final concentration of 50 μ g/kg
Part, it is detected respectively with the test strips that 3 batches produce, calculates its yin and yang attribute rate.It the results are shown in Table 1.
Table 1 detects bean sprouts sample results
The result shows that: when detecting positive bean sprouts sample with the test strips of 3 batch productions, as a result it is all positive, it is known that sun
Property coincidence rate be 100%, false negative rate 0；When detecting 20 parts of negative bean sprouts samples, as a result it is all negative, it is known that feminine gender meets
Rate is 100%, false positive rate 0.Illustrate that the test strips of detection 6-benzyladenine of the invention can be to 6- in the sample of bean sprouts
Benzyladenine residual is used for quickly detecting.
3, specific test
1000 μ g/kg 4-chlorophenoxyacetic acids, 2,4,5 trichlorophenoxyacetic acid, gibberellin, naphthalene second are detected with this test strips
When the other plants growth regulator such as acid, heteroauxin, indolebutyric acid, paclobutrazol, Thidiazuron, show that T line is aobvious in test strips
The colour developing of color ratio C line is deep or consistent with the colour developing of C line, is negative, illustrates this test strips to these drug no cross reactions.
Claims (4)
1. a kind of test strips for detecting 6-benzyladenine, including test paper and micropore reagent, the test paper include reaction film, sample
Absorption pad, water absorption pad and bottom plate have on the reaction film and are coated with 6-benzyladenine hapten-carrier protein conjugate
Detection line and the nature controlling line for being coated with sheep anti mouse antiantibody, freeze-drying has 6-benzyladenine monoclonal anti-on the micropore reagent
Body-colloid gold label object；The 6-benzyladenine monoclonal antibody is with 6-benzyladenine hapten-carrier albumen coupling
Object is prepared as immunogene；The 6-benzyladenine hapten-carrier protein conjugate is anti-by 6-benzyladenine half
Original is obtained with carrier protein couplet, and the carrier protein is bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein
Or human serum albumins, it is characterised in that the 6-benzyladenine haptens is by amino -6-benzyladenine and 4- (3- oxygen
For propyl)-benzoic acid obtains, molecular structural formula are as follows:
2. test strips as described in claim 1, it is characterised in that the test paper by sample absorption pad, reaction film, water absorption pad according to
Secondary paste forms on bottom plate, has micropore plug on the micropore reagent.
3. a kind of method for preparing any one of claim 1-2 test strips comprising step:
1) preparation freeze-drying has 6-benzyladenine monoclonal antibody-colloid gold label object micropore reagent；
2) preparation has the detection line for being coated with 6-benzyladenine hapten-carrier protein conjugate and is coated with sheep anti mouse and resists
The reaction film of the nature controlling line of antibody；
3) 2) reaction film prepared and sample absorption pad, water absorption pad, bottom plate are assembled into test paper；
4) 1) will there are 6-benzyladenine monoclonal antibody-colloid gold label object micropore reagent and examination with the freeze-drying 3) prepared
Paper is assembled into test strips.
4. the remaining method of 6-benzyladenine in a kind of sample of detection bean sprouts comprising step:
1) sample pre-treatments；
2) it is detected with the described in any item test strips of claim 1-2；
3) analysis detection result.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114031528A (en) * | 2021-08-24 | 2022-02-11 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Florfenicol hapten, artificial antigen, antibody and synthesis method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1657235A2 (en) * | 2004-11-10 | 2006-05-17 | Randox Laboratories Ltd. | Phenethanolamine-derived haptens, immunogens and conjugates comprising them and antibodies recognising said immunogenes and conjugates |
| US20120315625A1 (en) * | 2011-06-07 | 2012-12-13 | Dussault Patrick H | Self-assembled monolayers and methods for using the same in biosensing applications |
| CN103777015A (en) * | 2012-10-22 | 2014-05-07 | 北京勤邦生物技术有限公司 | Colloidal gold test strip and method for detecting erythromycin |
| CN105004844A (en) * | 2014-05-30 | 2015-10-28 | 北京勤邦生物技术有限公司 | Gentamicin residue diagnosis strip and application thereof |
| CN105116148A (en) * | 2014-09-17 | 2015-12-02 | 北京勤邦生物技术有限公司 | Test strip and method for detecting sodium pentachlorophenate |
| CN107022023A (en) * | 2017-04-25 | 2017-08-08 | 江南大学 | A kind of synthetic method of 6 benzyl aminoadenine artificial antigen |
| CN108398552A (en) * | 2017-12-29 | 2018-08-14 | 广东产品质量监督检验研究院 | A kind of 6-benzyladenine colloid gold immune test strip |
-
2019
- 2019-07-17 CN CN201910643893.4A patent/CN110441517B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1657235A2 (en) * | 2004-11-10 | 2006-05-17 | Randox Laboratories Ltd. | Phenethanolamine-derived haptens, immunogens and conjugates comprising them and antibodies recognising said immunogenes and conjugates |
| US20120315625A1 (en) * | 2011-06-07 | 2012-12-13 | Dussault Patrick H | Self-assembled monolayers and methods for using the same in biosensing applications |
| CN103777015A (en) * | 2012-10-22 | 2014-05-07 | 北京勤邦生物技术有限公司 | Colloidal gold test strip and method for detecting erythromycin |
| CN105004844A (en) * | 2014-05-30 | 2015-10-28 | 北京勤邦生物技术有限公司 | Gentamicin residue diagnosis strip and application thereof |
| CN105116148A (en) * | 2014-09-17 | 2015-12-02 | 北京勤邦生物技术有限公司 | Test strip and method for detecting sodium pentachlorophenate |
| CN107022023A (en) * | 2017-04-25 | 2017-08-08 | 江南大学 | A kind of synthetic method of 6 benzyl aminoadenine artificial antigen |
| CN108398552A (en) * | 2017-12-29 | 2018-08-14 | 广东产品质量监督检验研究院 | A kind of 6-benzyladenine colloid gold immune test strip |
Non-Patent Citations (1)
| Title |
|---|
| 徐霞玲 等: "猪肉中1-氨基乙内酰脲的胶体金免疫层析法快速检测", 《分析测试学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114031528A (en) * | 2021-08-24 | 2022-02-11 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Florfenicol hapten, artificial antigen, antibody and synthesis method and application thereof |
| CN114031528B (en) * | 2021-08-24 | 2024-05-07 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Florfenicol hapten, artificial antigen, antibody and synthetic method and application thereof |
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|---|---|
| CN110441517B (en) | 2023-01-20 |
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