CN106248928A - A kind of hydrochlorothiazide rapid detection card and detection method and preparation method thereof thereof - Google Patents
A kind of hydrochlorothiazide rapid detection card and detection method and preparation method thereof thereof Download PDFInfo
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- CN106248928A CN106248928A CN201610702240.5A CN201610702240A CN106248928A CN 106248928 A CN106248928 A CN 106248928A CN 201610702240 A CN201610702240 A CN 201610702240A CN 106248928 A CN106248928 A CN 106248928A
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- hydrochlorothiazide
- gold
- line
- detection
- rabbit igg
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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
- 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
- G01N33/577—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
Abstract
The invention discloses a kind of hydrochlorothiazide rapid detection card and detection method and preparation method thereof thereof.This hydrochlorothiazide rapid detection card and detection method and preparation method thereof thereof, the sample pad that is provided with, gold-marking binding pad, coated film, after sample to be tested in sample pad and gold mark joint sheet are provided with hydrochlorothiazide monoclonal antibody colloidal gold composite and rabbit igg colloidal gold composite react, reactant and coated film be provided with successively with the solution of hydrochlorothiazide monoclonal antibodies print detect line and the nature controlling line with the printing of goat anti-rabbit igg solution reacts and develops the color.When nature controlling line and the detection equal displaing amaranth of line, testing sample is i.e. not added with hydrochlorothiazide chemical composition；When nature controlling line displaing amaranth, when detection line does not develops the color, i.e. containing hydrochlorothiazide chemical composition in testing sample；Simple to operate, detection speed is fast, and the present invention is applicable to technical field of biological.
Description
Technical field
The present invention relates to technical field of biological, particularly relate to a kind of hydrochlorothiazide rapid detection card and detection side thereof
Method and preparation method thereof.
Background technology
At present, illegal interpolation in the health-oriented products of blood pressure lowering class Chinese patent medicine and health food and other sign above-mentioned functions
Material is mainly the chemical compositions such as hydrochlorothiazide, reserpine, clonidine hydrochloride, nifedipine.Hydrochlorothiazide is diuretic, anti-height
Cardura.Be primarily adapted for use in cariacedema, hepatic edema and renal edema: as nephrotic syndrome, acute glomerulonephritis,
The edema that chronic renal failure and adrenocortical hormone cause with hyperestrogenism；Hypertension；Diabetes insipidus etc..Make for a long time
Can hyperamization cholesterol, triacylglycerol, low density lipoprotein, LDL and Very Low Density Lipoprotein raise with hydrochlorothiazide, high density fat
Albumen reduces, and has the atherosclerotic possibility of promotion.There is also weak, asthenia, dizziness, anorexia, Nausea and vomiting,
The symptoms such as diarrhoea and blood pressure reduction.Therefore the abuse controlling hydrochlorothiazide is a problem demanding prompt solution.
The technology such as thin layer chromatography, high performance liquid chromatography is commonly used in detection currently for hydrochlorothiazide, and these methods are time-consuming
Arduously, and cost is high, it is impossible to meet the requirement of conventional medicine high-volume rapid screening.
Summary of the invention
For solving the problems referred to above, the present invention provides a kind of hydrogen chlorine that can quickly detect and whether contain hydrochlorothiazide in sample
Thiazine rapid detection card and detection method and preparation method thereof thereof.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of hydrochlorothiazide rapid detection card, including detection card base plate, detection card base plate is sequentially provided with and be connected for putting
Putting the sample pad of sample, gold-marking binding pad, coated film, it is multiple that gold mark joint sheet is provided with hydrochlorothiazide monoclonal antibody-gold colloidal
Compound and rabbit igg-colloidal gold composite, coated film is provided with the detection printed successively with the solution of hydrochlorothiazide monoclonal antibodies
Line and the nature controlling line printed with goat anti-rabbit igg solution.
Being further used as the improvement of technical solution of the present invention, the consumption of rabbit igg-colloidal gold composite is 0.2～0.9 μ g/
cm2, the consumption of hydrochlorothiazide monoclonal antibody-colloidal gold composite is 0.4～0.8 μ g/cm2, the consumption of goat anti-rabbit igg is
0.03～0.05 μ g/mm, the concentration of goat anti-rabbit igg be 4mg/ml, the consumption of hydrochlorothiazide monoclonal antibodies is 0.03～0.1
μg/mm。
Being further used as the improvement of technical solution of the present invention, one end of coated film is also connected adsorptive pads, and coated film is nitre
Acid cellulose film or cellulose acetate membrane, gold mark joint sheet is glass fibre cotton.
A kind of method of quick detection hydrochlorothiazide, comprises the following steps:
A. band is detected sample and adds organic solvent dissolving, filter after adding slow releasing agent dilution, and collect filtrate；
B. by filtrate added drop-wise sample pad, filtrate is provided with hydrochlorothiazide monoclonal antibody-gold colloidal with gold mark joint sheet and is combined
Thing and rabbit igg-colloidal gold composite are reacted, it is thus achieved that reactant；
C. reactant successively with the solution of hydrochlorothiazide monoclonal antibodies on detection line and the goat anti-rabbit igg solution on nature controlling line
React and develop the color.
Be further used as the improvement of technical solution of the present invention, in step A, organic solvent be alcohol organic solvent or chlorine
Imitative, alcohol organic solvent be ethanol, slow releasing agent be PBS.
It is further used as the improvement of technical solution of the present invention, in step C, when nature controlling line and the detection equal displaing amaranth of line,
Testing sample is i.e. not added with hydrochlorothiazide chemical composition；When nature controlling line displaing amaranth, when detection line does not develops the color, i.e. testing sample
In containing hydrochlorothiazide chemical composition；When nature controlling line and detection line do not develop the color or detection line colour developing only occur, then use
New detection card detects again.
The preparation method of a kind of hydrochlorothiazide rapid detection card, comprises the following steps:
Prepare sample pad, gold-marking binding pad, coated film the most respectively；
B. on detection card base plate, sample pad, gold-marking binding pad, coated film it are connected successively.
It is further used as the improvement of technical solution of the present invention, in step A, on nitrocellulose filter or cellulose acetate membrane
Spray printing hydrochlorothiazide antigenic dilution prepares the detection line of coated film, and spray printing goat anti-rabbit igg diluent prepares coated film
Detection line；
Coating hydrochlorothiazide monoclonal antibody-colloidal gold composite and rabbit igg antibody-colloidal gold complex on the glass fibers
Diluent, puts oven for drying to prepare gold-marking binding pad.
Being further used as the improvement of technical solution of the present invention, the preparation method of coated film is to buffer with 0.01M pH7.4PBS
Liquid, 10%～20% sucrose by hydrochlorothiazide antigen diluent to 0.3～1.0mg/ml；It is sprayed onto nitrocellulose filter or cellulose acetate
On film, the discharge rate of every 300mm is 30ul, makes detection line；Goat anti-rabbit igg is diluted to 0.3mg/ml～0.5mg/ml, often
The discharge rate of 300mm is 30ul, makes nature controlling line, and the spacing of detection line and nature controlling line is 4.5mm, put into 45 degree of baking oven drying 12～
18 hours；
The preparation method of gold-marking binding pad is: first prepare gold colloidal, with high purity water, the gold chloride of 1% is diluted to 0.01%, puts
Entering magnetic stir bar, be placed in temperature constant magnetic stirring heating mantle and boil, acutely after boiling, every 100ml is disposably rapidly added
The trisodium citrate of 0.7ml, continues to boil, and keeps redness to stop heating, after being cooled to room temperature when no longer changing to color
Supply the water of loss, prepare colloid gold particle；Take the colloid gold particle that radius is 15nm～40nm, tune pH value to 7.0-
7.4, under agitation add hydrochlorothiazide monoclonal antibody or rabbit igg antibody by 20 μ g antibody/ml gold colloidal, every 1ml adds
The BSA of 100ul 10% stabilizes it, and is centrifuged 30min at 12000rpm/s, abandons supernatant, by precipitation with 1/10th initial glue
The Tris-HCl buffer of the 0.01M pH8.5 of body gold volume, 1 ‰～5 ‰ PVP-40,5%BSA, 0.1 ‰～0.5 ‰ NaN3
Resuspended；By hydrochlorothiazide monoclonal antibody-colloidal gold composite and rabbit igg antibody-colloidal gold complex 0.01M pH8.0
Tris-HCl buffer, 1 ‰～5 ‰ PVP-40,5%BSA, 1 ‰～5 ‰ TritonX-100,0.1 ‰-0.5 ‰ NaN3Dilution
To 3%～10%, take glass fibre, every coating 20ml～30ml, put baking oven and dry 18～24 hours with 37 DEG C.
Being further used as the improvement of technical solution of the present invention, in step A, the preparation method of sample pad is to take glass fibre
Coating solution is PBS, 1%BSA, 1%TWEEN～20,0.1 ‰～0.5 ‰ NaN of 0.01M pH7.43, every coating 20ml～
30ml, puts baking oven and dries 18～24 hours with 37 DEG C.
Beneficial effects of the present invention: this hydrochlorothiazide rapid detection card and detection method and preparation method thereof thereof, is provided with
Sample pad, gold-marking binding pad, coated film, the sample to be tested in sample pad is provided with hydrochlorothiazide monoclonal anti with gold mark joint sheet
After body-colloidal gold composite and rabbit igg-colloidal gold composite reaction, reactant and coated film are provided with uses hydrochlorothiazide successively
Detection line and the nature controlling line printed with goat anti-rabbit igg solution that the solution of monoclonal antibodies is printed react and develop the color.Work as nature controlling line
During with detection line equal displaing amaranth, testing sample is i.e. not added with hydrochlorothiazide chemical composition；When nature controlling line displaing amaranth, detection
When line does not develops the color, i.e. containing hydrochlorothiazide chemical composition in testing sample；Operating procedure of the present invention is simple, and detection speed is fast, knot
Fruit is easily observed, and can realize the rapid screening to batch samples, is substantially reduced testing cost, reduces workload, at food medicine
Product supervision has practical significance.The present invention is directed to testing sample complicated component problem, sample pretreating method be optimized,
Required sample size is few, it is only necessary to extracts, dilute two steps and can complete sample pre-treatments.
Accompanying drawing explanation
The invention will be further described below in conjunction with the accompanying drawings:
Fig. 1 is the overall structure schematic diagram of rapid detection card described in the embodiment of the present invention.
Detailed description of the invention
Below with reference to embodiment and accompanying drawing, the technique effect of design, concrete structure and the generation of the present invention is carried out clearly
Chu, it is fully described by, to be completely understood by the purpose of the present invention, feature and effect.Obviously, described embodiment is this
Bright a part of embodiment rather than all embodiment, based on embodiments of the invention, those skilled in the art is not paying
Other embodiments obtained on the premise of creative work, belong to the scope of protection of the invention.
With reference to Fig. 1, the present invention is a kind of hydrochlorothiazide rapid detection card, including detection card base plate 5, on detection card base plate 5
Be sequentially provided with and be connected for placing the sample pad 1 of sample, gold-marking binding pad 2, coated film 3, gold mark joint sheet 2 be provided with hydrogen
Chlorothiazide monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite, coated film 3 is provided with uses hydrochlorothiazide successively
What the solution of monoclonal antibodies was printed detects line 7 and the nature controlling line 6 printed with goat anti-rabbit igg solution.
This hydrochlorothiazide rapid detection card and detection method and preparation method thereof thereof, the sample pad 1 being provided with, gold-marking binding pad
2, coated film 3, the sample to be tested in sample pad 1 is provided with hydrochlorothiazide monoclonal antibody-gold colloidal with gold mark joint sheet 2 and is combined
After thing and rabbit igg-colloidal gold composite reaction, reactant and coated film 3 are provided with successively with hydrochlorothiazide monoclonal antibodies
Detection line 7 and the nature controlling line 6 printed with goat anti-rabbit igg solution that solution is printed react and develop the color.When nature controlling line 6 and detection line 7
All during displaing amaranth, testing sample is i.e. not added with hydrochlorothiazide chemical composition；When nature controlling line 6 displaing amaranth, detection line 7 does not shows
During color, i.e. containing hydrochlorothiazide chemical composition in testing sample；Operating procedure of the present invention is simple, and detection speed is fast, and result is easy
Observe, the rapid screening to batch samples can be realized, be substantially reduced testing cost, reduce workload, at Food and drug administration
In there is practical significance.The present invention is directed to testing sample complicated component problem, sample pretreating method is optimized, required sample
Product amount is few, it is only necessary to extracts, dilute two steps and can complete sample pre-treatments.
As the preferred embodiment of the present invention, the consumption of rabbit igg-colloidal gold composite is 0.2～0.9 μ g/cm2, hydrogen
The consumption of chlorothiazide monoclonal antibody-colloidal gold composite is 0.4～0.8 μ g/cm2, the consumption of goat anti-rabbit igg be 0.03～
0.05 μ g/mm, the concentration of goat anti-rabbit igg is 4mg/ml, and the consumption of hydrochlorothiazide monoclonal antibodies is 0.03～0.1 μ g/mm.
As the preferred embodiment of the present invention, one end of coated film 3 is also connected adsorptive pads 4, and coated film 3 is that nitric acid is fine
Dimension element film or cellulose acetate membrane, gold mark joint sheet 2 is glass fibre cotton.
A kind of method of quick detection hydrochlorothiazide, comprises the following steps:
A. band is detected sample and adds organic solvent dissolving, filter after adding slow releasing agent dilution, and collect filtrate；
B. by filtrate added drop-wise sample pad 1, it is multiple that filtrate and gold mark joint sheet 2 are provided with hydrochlorothiazide monoclonal antibody-gold colloidal
Compound and rabbit igg-colloidal gold composite are reacted, it is thus achieved that reactant；
C. reactant is molten with the goat anti-rabbit igg on the solution of the hydrochlorothiazide monoclonal antibodies on detection line 7 and nature controlling line 6 successively
Liquid reacts and develops the color.
As the preferred embodiment of the present invention, in step A, organic solvent be alcohol organic solvent or chloroform, alcohol
Class organic solvent is ethanol, slow releasing agent be PBS.
As the preferred embodiment of the present invention, in step C, when nature controlling line 6 and detection line 7 equal displaing amaranth, i.e. treat
Test sample product are not added with hydrochlorothiazide chemical composition；When nature controlling line 6 displaing amaranth, when detection line 7 does not develops the color, i.e. in testing sample
Containing hydrochlorothiazide chemical composition；When nature controlling line 6 and detection line 7 do not develop the color or only occur that detection line 7 develops the color, then use
New detection card detects again.
The preparation method of a kind of hydrochlorothiazide rapid detection card, comprises the following steps:
Prepare sample pad 1, gold-marking binding pad 2, coated film 3 the most respectively；
B. on detection card base plate 5, sample pad, gold-marking binding pad, coated film it are connected successively.
As the preferred embodiment of the present invention, in step A, spray printing hydrogen on nitrocellulose filter or cellulose acetate membrane
Chlorothiazide antigenic dilution prepares the detection line 7 of coated film 3, and spray printing goat anti-rabbit igg diluent prepares the detection of coated film 3
Line 7；
Coating hydrochlorothiazide monoclonal antibody-colloidal gold composite and rabbit igg antibody-colloidal gold complex on the glass fibers
Diluent, puts oven for drying to prepare gold-marking binding pad 2.
As the preferred embodiment of the present invention, the preparation method of coated film 3 be with 0.01M pH7.4PBS buffer,
10%～20% sucrose is by hydrochlorothiazide antigen diluent to 0.3～1.0mg/ml；It is sprayed onto nitrocellulose filter or cellulose acetate membrane
On, the discharge rate of every 300mm is 30ul, makes detection line 7；Goat anti-rabbit igg is diluted to 0.3mg/ml～0.5mg/ml, often
The discharge rate of 300mm is 30ul, makes nature controlling line 6, and detection line 7 is 4.5mm with the spacing of nature controlling line 6, puts into the drying of 45 degree of baking oven
12～18 hours；
The preparation method of gold-marking binding pad 2 is: first prepare gold colloidal, with high purity water, the gold chloride of 1% is diluted to 0.01%, puts
Entering magnetic stir bar, be placed in temperature constant magnetic stirring heating mantle and boil, acutely after boiling, every 100ml is disposably rapidly added
The trisodium citrate of 0.7ml, continues to boil, and keeps redness to stop heating, after being cooled to room temperature when no longer changing to color
Supply the water of loss, prepare colloid gold particle；Take the colloid gold particle that radius is 15nm～40nm, tune pH value to 7.0-
7.4, under agitation add hydrochlorothiazide monoclonal antibody or rabbit igg antibody by 20 μ g antibody/ml gold colloidal, every 1ml adds
The BSA of 100ul 10% stabilizes it, and is centrifuged 30min at 12000rpm/s, abandons supernatant, by precipitation with 1/10th initial glue
The Tris-HCl buffer of the 0.01M pH8.5 of body gold volume, 1 ‰～5 ‰ PVP-40,5%BSA, 0.1 ‰～0.5 ‰ NaN3
Resuspended；By hydrochlorothiazide monoclonal antibody-colloidal gold composite and rabbit igg antibody-colloidal gold complex 0.01M pH8.0
Tris-HCl buffer, 1 ‰～5 ‰ PVP-40,5%BSA, 1 ‰～5 ‰ TritonX-100,0.1 ‰-0.5 ‰ NaN3Dilution
To 3%～10%, take glass fibre, every coating 20ml～30ml, put baking oven and dry 18～24 hours with 37 DEG C.
As the preferred embodiment of the present invention, in step A, the preparation method of sample pad 1 is to take glass fibre to be coated with molten
Liquid is PBS, 1%BSA, 1%TWEEN～20,0.1 ‰～0.5 ‰ NaN of 0.01M pH7.43, every coating 20ml～30ml,
Put baking oven to dry 18～24 hours with 37 DEG C.
The preferred embodiment of the present invention one:
The method for quick of hydrochlorothiazide, for solid dosage forms, comprises the steps: successively
1), to the product that need to detect adding alcohol organic solvent or chloroform dissolves, preferably alcohol organic solvent is ethanol, then adds
Filter after the dilution of PBS slow releasing agent, collect filtrate；
Wherein: different dosage form sample dissolving method is as follows:
Hard capsule: capsule shells of outwarding winding, takes about 1 intragranular tolerant.
Soft capsule: cutting off capsule shells with shears, extrusion about 1 intragranular is tolerant.
Tablet: be crushed to powder, takes about 1 amount.
Pill: take about 1/2 each serving consumption, crushes or shreds.
2), adding organic solvent in any one of above-mentioned sample, the usage amount of organic solvent is with submergence testing sample
Being advisable, the present invention selects the usage amount of 1～2mL, substantially can cover the needs of different dosage form sample.
3), for ensureing to extract completely, the shaking time need to be no less than 30 seconds, to adapt to the needs of various dosage form.
It is found through experiments, drops to the filtrate on hydrochlorothiazide colloidal gold strip, if the concentration of organic solvent is more than
50%, then hydrochlorothiazide colloidal gold strip can not normally use, thus the PBS that need to add more than 1mL is diluted.
Simultaneously, it is contemplated that extracting solution needs to be lost certain solution in filter process, therefore the present invention add PBS be diluted to
Cumulative volume is 10mL, has both guaranteed the sensitivity of reagent paper, ensures again have enough extracting solution to be loaded.
For liquid dosage form: without pre-treatment, directly detect.
2) filtrate added drop-wise step 1) collected is on hydrochlorothiazide colloidal gold chromatographic detection card, beginning timing, and 3～5min
After, according to detection T line and the colour developing of control C line of colloidal gold strip, it is judged that testing sample is positive or negative；
3) step 2) described in colloidal gold test on control C line and detection the equal displaing amaranth of T line, then be judged as feminine gender,
Testing sample is i.e. not added with hydrochlorothiazide chemical composition；Control line C displaing amaranth on colloidal gold test, detects line T
Do not develop the color, be then judged as in the positive, i.e. testing sample containing hydrochlorothiazide chemical composition；Control line on colloidal gold test
C and detection line T does not develops the color, or detection line T colour developing only occurs, then explanation experiment is invalid, applies new colloidal gold test
Again detect.
The rapid detection card of detection hydrochlorothiazide, detection card base plate 5 uses PVC material, including PVC board, in PVC board successively
Sample pad 1, gold-marking binding pad 2, coated film 3 and the adsorptive pads 4 of linking, sees Fig. 1, and sample pad 1 is pressed in the 1/ of gold-marking binding pad 2
2～about 1/3, gold-marking binding pad 2 is pressed in the 0.1～0.2cm of coated film 3, detects line T linear distance coated film 3 lower end 6mm, matter
Control line C linear distance coated film 3 upper end 6mm, C, T spacing is 4.5mm, and adsorptive pads is pressed at coated film 0.1～0.2cm.
Wherein: gold mark joint sheet is combined for adsorbing hydrochlorothiazide monoclonal antibody～colloidal gold composite and rabbit～gold colloidal
The glass fibre cotton of thing, the orthoscopic recessiveness detection printed with the solution of hydrochlorothiazide coupling carrier albumen successively on coated film
Line T line, the orthoscopic recessiveness nature controlling line C line printed with goat anti-rabbit igg solution, two lines is arranged in parallel.
Coated film is nitrocellulose filter or cellulose acetate membrane, and the consumption of monoclonal-rabbit igg is 0.2～0.9 μ g/
Cm2, preferably 0.25～0.45 μ g/cm2, the consumption of hydrochlorothiazide monoclonal antibody～colloidal gold composite is 0.4～0.8 μ g/
Cm2, preferably 0.03 μ g/mm, the concentration of goat anti-rabbit igg is 4mg/ml, and the consumption of goat anti-rabbit igg is 0.03～0.05 μ g/mm.
The preparation method of detection card: sample pad, gold-marking binding pad, coated film and the adsorptive pads being connected successively in PVC board；
Wherein:
1) preparation method of coated film is:
With pH7.4,0.01M PBS, 10%-20% sucrose by hydrochlorothiazide antigen diluent to 0.3～1.0mg/ml, it is sprayed onto
On nitrocellulose membrane, the discharge rate of every 300mm is 30ul, for T line；Goat-anti rabbit is diluted to 0.3mg/ml～0.5mg/ml, every 300mm
Discharge rate be 30ul, running speed is 80 mm/second, pump pressure 100 pump pressure, and movable length 290cm is C line, C, T line
Spacing is 4.5mm, puts 45 DEG C of oven overnight 12～18 hours；
2) preparation method of gold-marking binding pad pad is:
With high purity water, the gold chloride of 1% is diluted to 0.01%, puts into magnetic stir bar, be placed in temperature constant magnetic stirring heating mantle and boil
Boiling, acutely after boiling, every 100ml is disposably rapidly added the trisodium citrate of 0.7ml, continues to boil, and keeps redness not to color
Change stopping heating again, supplies the water of loss after being cooled to room temperature, and qualified gold colloidal should be bright, free from admixture and floating
Thing, can preserve one month and not change.
Take the colloid gold particle that radius is 15nm～40nm, adjust pH value to 7.0-7.4, under agitation press 10 μ g antigens/ml
Gold colloidal adds hydrochlorothiazide monoclonal antibody, and the BSA that last every 1ml adds 100ul 10% stabilizes it, at 12000rpm/s
Centrifugal 30min, abandons supernatant, by the precipitation Tris-of the 0.01M pH8.5 of the 100ul of 1/10th initial colloid gold volumes
HCl buffer, 1 ‰～5 ‰ PVP-40,5%BSA, 0.1 ‰-0.5 ‰ NaN3Resuspended, put 4 DEG C and can put about 2 months；
Hydrochlorothiazide monoclonal antibody～colloidal gold composite and rabbit igg～colloidal gold composite with 0.01M pH8.0's
Tris-HCl buffer, 1 ‰～5 ‰ PVP-40,5%BSA, 1 ‰～5 ‰ TritonX-100,0.1 ‰-0.5 ‰ NaN3It is diluted to
3%～10%, take the glass fibre that area is 25 × 30cm, every coating 20ml～30ml, put baking oven, place 18～24 at 37 DEG C
Hour.
3) sample pad is prepared
Taking 25 × 30cm glass fibre, every coating 20ml～30ml coating solution is the PBS, 1%BSA, 1% of 0.01M pH7.4
TWEEN-20,0.1 ‰-0.5 ‰ NaN3, put 37 degree of baking oven 18～24 hours.
Operating procedure of the present invention is simple, and detection speed is fast, and result is easily observed, and can realize the quick sieve to batch samples
Look into, be substantially reduced testing cost, reduce workload, Food and drug administration has practical significance；The present invention is directed to treat test sample
Product complicated component problem, is optimized sample pretreating method, and required sample size is few, it is only necessary to extract, diluting two steps can be complete
Become sample pre-treatments.
Certainly, the invention is not limited to above-mentioned embodiment, and those of ordinary skill in the art are without prejudice to this
It may also be made that equivalent variations or replacement on the premise of spirit, deformation or the replacement of these equivalents are all contained in the application right
In requirement limited range.
Claims (10)
1. a hydrochlorothiazide rapid detection card, it is characterised in that: include detecting card base plate (5), on described detection card base plate (5)
Be sequentially provided with and be connected for placing the sample pad (1) of sample, gold-marking binding pad (2), coated film (3), described gold tag splice close
Pad (2) is provided with hydrochlorothiazide monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite, described coated film (3)
It is provided with the detection line (7) printed successively and the matter printed with goat anti-rabbit igg solution with the solution of hydrochlorothiazide monoclonal antibodies
Control line (6).
Hydrochlorothiazide rapid detection card the most according to claim 1, it is characterised in that: described rabbit igg-colloidal gold composite
Consumption be 0.2～0.9 μ g/cm2, the consumption of described hydrochlorothiazide monoclonal antibody-colloidal gold composite is 0.4～0.8 μ
g/cm2, the consumption of described goat anti-rabbit igg is 0.03～0.05 μ g/mm, and the concentration of described goat anti-rabbit igg is 4mg/ml, described hydrogen
The consumption of chlorothiazide monoclonal antibodies is 0.03～0.1 μ g/mm.
Hydrochlorothiazide rapid detection card the most according to claim 1 and 2, it is characterised in that: one end of described coated film (3)
Also linking has adsorptive pads (4), described coated film (3) is nitrocellulose filter or cellulose acetate membrane, described gold mark joint sheet (2)
For glass fibre cotton.
4. use the method that the hydrochlorothiazide rapid detection card described in claim 1 quickly detects hydrochlorothiazide, its feature
It is, comprises the following steps:
Band is detected sample and adds organic solvent dissolving, filter after adding slow releasing agent dilution, and collect filtrate；
By in filtrate added drop-wise sample pad (1), described filtrate is provided with hydrochlorothiazide monoclonal anti with described gold mark joint sheet (2)
Body-colloidal gold composite and rabbit igg-colloidal gold composite are reacted, it is thus achieved that reactant；
Reactant successively with the goat anti-rabbit igg on the solution of the hydrochlorothiazide monoclonal antibodies in detection line (7) and nature controlling line (6)
Solution reaction also develops the color.
The detection method of hydrochlorothiazide the most according to claim 4, it is characterised in that: in described step A, described is organic
Solvent is alcohol organic solvent or chloroform, and described alcohol organic solvent is ethanol, and described slow releasing agent is PBS.
6. according to the detection method of the hydrochlorothiazide described in claim 4 or 5, it is characterised in that: in described step C, work as matter
During control line (6) and detection line (7) all displaing amaranths, testing sample is i.e. not added with hydrochlorothiazide chemical composition；When nature controlling line (6)
Displaing amaranth, when detection line (7) does not develops the color, i.e. containing hydrochlorothiazide chemical composition in testing sample；When nature controlling line (6) and detection
When line (7) does not develops the color or detection line (7) colour developing only occurs, then new detection card is used again to detect.
7. the preparation method of the hydrochlorothiazide rapid detection card that a kind is prepared described in claim 1, it is characterised in that include with
Lower step:
Prepare sample pad (1), gold-marking binding pad (2), coated film (3) the most respectively；
B. in detection card base plate (5), sample pad, gold-marking binding pad, coated film it are connected successively.
The preparation method of hydrochlorothiazide rapid detection card the most according to claim 7, it is characterised in that: in described step A,
On nitrocellulose filter or cellulose acetate membrane, spray printing hydrochlorothiazide antigenic dilution prepares the detection line (7) of coated film (3),
And spray printing goat anti-rabbit igg diluent prepares the detection line (7) of coated film (3)；
Coating hydrochlorothiazide monoclonal antibody-colloidal gold composite and rabbit igg antibody-colloidal gold complex on the glass fibers
Diluent, puts oven for drying to prepare gold-marking binding pad (2).
The preparation method of hydrochlorothiazide rapid detection card the most according to claim 8, it is characterised in that: described coated film
(3) preparation method be with 0.01M pH7.4PBS buffer, 10%～20% sucrose by hydrochlorothiazide antigen diluent to 0.3～
1.0mg/ml；Being sprayed onto in nitrocellulose filter or cellulose acetate membrane, the discharge rate of every 300mm is 30ul, makes detection line (7)；
Goat anti-rabbit igg is diluted to 0.3mg/ml～0.5mg/ml, and the discharge rate of every 300mm is 30ul, makes nature controlling line (6), detects line
(7) spacing with nature controlling line (6) is 4.5mm, puts into 45 degree of baking oven and dries 12～18 hours；
The preparation method of described gold-marking binding pad (2) is: first prepares gold colloidal, is diluted to by the gold chloride of 1% with high purity water
0.01%, put into magnetic stir bar, be placed in temperature constant magnetic stirring heating mantle and boil, acutely after boiling, every 100ml is the rapidest
Add the trisodium citrate of 0.7ml, continue to boil, keep redness to stop heating when no longer changing to color, be cooled to room
Supply the water of loss after temperature, prepare colloid gold particle；Take the colloid gold particle that radius is 15nm～40nm, adjust pH value to arrive
7.0-7.4, under agitation adds hydrochlorothiazide monoclonal antibody or rabbit igg antibody by 20 μ g antibody/ml gold colloidal, and every 1ml adds
The BSA entering 100ul 10% stabilizes it, and is centrifuged 30min at 12000rpm/s, abandons supernatant, and precipitation is initial with 1/10th
The Tris-HCl buffer of the 0.01M pH8.5 of gold colloidal volume, 1 ‰～5 ‰ PVP-40,5%BSA, 0.1 ‰～0.5 ‰
NaN3Resuspended；By hydrochlorothiazide monoclonal antibody-colloidal gold composite and rabbit igg antibody-colloidal gold complex 0.01M
The Tris-HCl buffer of pH8.0,1 ‰～5 ‰ PVP-40,5%BSA, 1 ‰～5 ‰ TritonX-100,0.1 ‰-0.5 ‰
NaN3It is diluted to 3%～10%, takes glass fibre, every coating 20ml～30ml, put baking oven and dry 18～24 hours with 37 DEG C.
10., according to the preparation method of the hydrochlorothiazide rapid detection card described in any one in claim 7 to 9, its feature exists
In: in described step A, it is 0.01M pH7.4 that the preparation method of described sample pad (1) takes glass fibre coating solution
PBS, 1%BSA, 1%TWEEN～20,0.1 ‰～0.5 ‰ NaN3, every coating 20ml～30ml, puts baking oven and dries 18 with 37 DEG C
～24 hours.
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| CN109813897A (en) * | 2017-11-18 | 2019-05-28 | 镇江亿特生物科技发展有限公司 | Clonidine hydrochloride rapid time resolved fluorometric immunochromatographiassay assay quantitative detection test paper |
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| WO2005046735A1 (en) * | 2003-11-07 | 2005-05-26 | Newport Instruments | A reagent system and method for modifying the luminescence of lanthanide(iii) macrocyclic complexes |
| CN103969454A (en) * | 2013-12-18 | 2014-08-06 | 深圳市药品检验所 | Rapid melatonin detection method, melatonin detection card and preparation method of detection card |
| CN104447619A (en) * | 2014-10-24 | 2015-03-25 | 江南大学 | Hydrochlorothiazide semi-antigen and complete antigen as well as preparation method thereof |
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| WO2005046735A1 (en) * | 2003-11-07 | 2005-05-26 | Newport Instruments | A reagent system and method for modifying the luminescence of lanthanide(iii) macrocyclic complexes |
| CN103969454A (en) * | 2013-12-18 | 2014-08-06 | 深圳市药品检验所 | Rapid melatonin detection method, melatonin detection card and preparation method of detection card |
| CN104447619A (en) * | 2014-10-24 | 2015-03-25 | 江南大学 | Hydrochlorothiazide semi-antigen and complete antigen as well as preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109813897A (en) * | 2017-11-18 | 2019-05-28 | 镇江亿特生物科技发展有限公司 | Clonidine hydrochloride rapid time resolved fluorometric immunochromatographiassay assay quantitative detection test paper |
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