CN106370846A - Detection method for rapidly detecting illegally-added tadalafil in health product - Google Patents
Detection method for rapidly detecting illegally-added tadalafil in health product Download PDFInfo
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- CN106370846A CN106370846A CN201510430132.2A CN201510430132A CN106370846A CN 106370846 A CN106370846 A CN 106370846A CN 201510430132 A CN201510430132 A CN 201510430132A CN 106370846 A CN106370846 A CN 106370846A
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- 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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Abstract
The present invention provides a detection card for rapidly detecting the illegally-added tadalafil in a health product, wherein the method is achieved through the following parts: a sample pre-treatment process and a rapid tadalafil detection card. According to the present invention, the requirements on the sample are low, wherein the conventional pills, the conventional tablets, the conventional tea, the conventional capsules and other health products can be detected; the detection operation is simple, the professional staff and the professional equipment are not required, and the method is suitable for the on-site detection; and the detection cycle is short, wherein the result can be obtained within 20 min.
Description
Technical field
The invention belongs to health care of food product illegal substance detection field, the illegal detection method adding tadanafil in more particularly to a kind of quick detection health product.
Background of invention
With economic fast development, people's edible food has not been merely to absorb nourishment, sustaining life, also requires it to have the effect adjusting human physiological functions.The appearance of health food, proposes new challenge to food, is the requirement that people pursue high-quality life, is also the product of rapid economic development.China from 1996 so far, administrative department of public health ratify altogether health food reach more than 9600 kind, market scale be worth nearly hundred billion yuan, every year increase newly approval health food have as many as hundreds of.The fast development of health food brings huge economic benefit, also brings a series of safety problem simultaneously.Although country has promulgated that a series of laws and regulations are supervised successively, health food quality is very different, adulterate, adulteration happens occasionally.This not only compromises the interests of consumer, and makes people that health food is gradually lost confidence, and has a strong impact on the normal, fast-developing of health food industry.
Tadanafil is a kind of tonifying YANG class Western medicine prescription drugss, is now often illegally added to and refreshes oneself in class and tonifying YANG class health product.Because tadanafil is prescription drugss, there is certain side effect it is necessary to user can under the guidance of doctor.Tadanafil in the illegal health product adding, can cause serious health hazard to user.
The method that conventional sense illegally adds tadanafil is chemical method, hplc method, thin layer chromatography etc..Chemical method and the illegal tadanafil that adds of thin layer chromatography detection have sensitivity low, the shortcoming of poor specificity, and the detection of hplc method is illegal to add tadanafil complex operation, needs the personnel of specialty and special instrument, is not suitable for the detection at scene.Gold-immunochromatographyreagent reagent for assay is a kind of quick detection reagent based on immunological technique and chromatographic technique, this reagent typically provides testing result in 20min, sensitivity is high, specificity is good, without professional and specialized equipment, it is a kind of method being especially suitable for illegal interpolation tadanafil in field quick detection health product.
Content of the invention
It is an object of the invention to solving the defect of prior art, provide the illegal detection method adding tadanafil in a kind of quick detection health product.Solve the problems, such as the complex steps of traditional detection method, also detection accuracy is low for detection time length.
In view of this, the present invention provides the illegal detection method adding tadanafil in a kind of quick detection health product, and the method is passed through two parts and realized, i.e. sample pre-treatments technique and tadanafil rapid detection card.
Described sample pre-treatments technique includes methanol-water sample extracting solution and the pre-treating method based on this sample extracting solution.The invention provides a kind of methanol water mixed liquid sample extracting solution, this extracting solution can process the dosage form health product such as pill, tablet, medicinal tea and capsule, and can extract the tadanafil of illegal interpolation by simple pre-treatment step.
Described methanol water mixed liquid sample extracting solution, the volume of methanol-water is 1: (1～10).
The described pre-treating method based on this sample treatment liquid, comprises the steps: step 1, pill, tablet is pulverized, capsule shells opened by capsule.Step 2, weighs powder or content 1g, adds 5ml extracting solution, shakes 1min.Standing 5min treats that solution is clarified.Step 3, takes supernatant 45um membrane filtration to obtain supernatant liquid and as detects liquid.
The described pre-treating method based on sample treatment liquid, in described step 1, pill, medicinal tea and tablet can grind it is also possible to wrap pill with pan paper or tablet pulverizes in mortar, depending on the condition according to detection scene.
The described pre-treating method based on sample treatment liquid, step 3 can also be completed by centrifugal method, and parameter of noncentricity is 3000rpm, 5min.
The tadanafil rapid detection card of the present invention is based on colloidal gold immunochromatographimethod technology, it is achieved through the following technical solutions: a kind of stock body gold immunochromatography tadanafil detection card, including base plate and successively overlap joint paste with the sample pad on base plate, pad, chromatographic film and adsorptive pads thus forming multi-layer film structure, partly overlap between each film structure；The tadanafil specific antibody of colloid gold label is coated with described pad；One is had to be coated the detection line of tadanafil antigen (tadanafil-bsa complex), a nature controlling line being coated sheep anti mouse igg antibody in described chromatographic film.
Described tadanafil specific antibody can be monoclonal antibody or polyclonal antibody.
Described base plate is pvc plate；Described sample pad is a kind of glass fibre membrane or polyester fiber film, is measuring samples collecting region, and its front end overlap joint is pasted on base plate front end；The matrix of described pad is glass fibre membrane or polyester fiber film, and pad is located between sample pad and base plate, and its front end overlap joint is pasted on base plate, and certain length is extended from below sample pad rear end in rear end；Described chromatographic film is nitrocellulose filter, and its front end is inserted between pad and base plate and overlaps and is pasted on base plate, and the rear end of the straight base plate that tiles；Described adsorptive pads are absorbent filter, and its overlap joint is covered in certain length on chromatographic film rear end with base plate rear end.
Described tadanafil rapid detection card is based on colloidal gold immunochromatographimethod competition law, specific as follows: colloidal-gold detecting-card of the present invention all detects tadanafil using competition law principle, golden pad is mouse gold mark tadanafil antibody, on nitrocellulose filter, detection line is coated tadanafil antigen, and nature controlling line is coated sheep anti mouse igg polyclonal antibody.
When sample is added drop-wise in sample pad, under capillarity, liquid chromatographs to adsorptive pads direction.If containing tadanafil in sample, sample is reached gold and is combined with corresponding gold labeling antibody with reference to pad position, when then chromatographing detection line on nitrocellulose filter, then cannot be with coated tadanafil antigen binding, detection line region occurs without red stripes, so obtains positive findingses.
When not containing tadanafil in sample, sample can not react with gold labeling antibody, and with coated tadanafil antigen binding when on chromatography is to nitrocellulose filter, the red tested survey line of gold grain captures, thus showing red stripes in this region, so obtain negative findings.
Gold labeling antibody on the golden pad of negative or positive sample all many anti-bindings can produce red stripes with the sheep anti mouse of nature controlling line on nitrocellulose filter, if nature controlling line does not show explanation, testing result is invalid.
At least following several advantages of such scheme:
Present invention detection tadanafil requires the health product such as low, conventional pill, tablet, medicinal tea, capsule all can detect to sample；Detection simple to operate, need not specialty personnel and instrument, be suitable for Site Detection.Detection cycle is short, goes out result in 20min.
Brief description
The positive structure schematic of the detection card that Fig. 1 present invention is implemented.
The detection kit side sectional view that Fig. 2 present invention is implemented.
The detection kit positive structure schematic that Fig. 3 present invention is implemented.
The negative and positive sex determination schematic diagram of the detection kit that Fig. 4 present invention is implemented.
Specific embodiment
With reference to embodiment, the present invention is described in further details, but the present invention is not limited only to following embodiments.
Embodiment 1 detects blocking Preparation Method
Refer to Fig. 1-Fig. 3, the present invention provides the illegal detection card 10 adding tadanafil in a kind of quick detection health product, 12 be made up of test strip 11 and getting stuck.
Described reagent paper 11 include base plate 1 and successively overlap joint paste sample pad 2 thereon, pad 3, chromatographic film 4 and adsorptive pads 5 etc. and partly overlap between multi-layer film structure, and each film structure.Wherein, pad 3 is coated with colloid gold label tadanafil specific antibody.One is had to be coated with tadanafil Detection of antigen line 7 and a nature controlling line 6. being coated with sheep anti mouse igg antibody in chromatographic film 4
Base plate 1 is stained with the pvc plate of double faced adhesive tape for one side, carries abovementioned layers membrane structure, and it forms according to the corresponding cutting of shape of detection card 10, and such as the present embodiment is rectangle.
Sample pad 2 is a kind of glass fibre membrane or polyester fiber film, is measuring samples collecting region, and its front end overlap joint is pasted on base plate 1 front end, and rear end overlaps and pad 3 front end, and partly overlaps with pad 3.
The matrix of pad 3 is glass fibre membrane or polyester fiber film, and pad 3 is coated with the tadanafil specific antibody of colloid gold label.Pad 3 is located between sample pad 2 and base plate 1, and its front end overlap joint is pasted on base plate 1, and certain length is extended from below sample pad 2 rear end in rear end；
Described chromatographic film 4 is nitrocellulose filter, chromatographic film 4 has one be coated with tadanafil Detection of antigen line 7 and a nature controlling line 6 being coated with sheep anti mouse igg antibody.Chromatographic film 4 front end is inserted between pad 3 and base plate 1 and overlaps and is pasted on base plate 1, and the rear end of the straight base plate 1 that tiles；
Adsorptive pads 5 are absorbent filter, and it is overlapped in base plate rear end and is covered in certain length on chromatographic film 4 rear end.
Wherein, sample pad 2, pad 3, chromatographic film 4, adsorptive pads 5 width base plate 1 are identical, and overlap joint is pasted on base plate 1 successively, closely connects and partly overlap between each layer.The upper surface of each film layer tiles expansion certain area successively in test strips 11 upper surface.Shown in Fig. 2, the parallel connection of each film layer on the front of this reagent paper 11, align in side, from the front end of reagent paper 11, it is followed successively by sample pad 2, pad 3, chromatographic film 4, the front surface region of adsorptive pads 5 adjoin successively and are parallel to each other along the length direction of base plate 1, detection line 7 and nature controlling line 6 are set in parallel in chromatographic film 4 and are located between pad 3 and adsorptive pads 5 front surface region.Before detection line 7 is located at nature controlling line 6.
This detection card 10 is in strip, accordingly, gets stuck 12 also in strip.In a specific embodiment, described sample pad 2 length is 13mm, pad 3 length is 6mm, and width is 4mm；Chromatographic film 4 and adsorptive pads 5 length are 20mm, width 4mm；Overlapping region between sample pad 2, pad 3, chromatographic film 4 is 2mm length, and the overlapping region between chromatographic film 4 and adsorptive pads 5 is 3mm length.In chromatographic film 5, detection line 7 and nature controlling line 6 width are 1mm, are spaced apart 5mm between detection line 7 and nature controlling line 6, detection line 9.
Get stuck 12 and both reagent paper 11 mating shapes.After packaging, get stuck and the front surface region just to sample pad 2 for the well 14 is set on 12, the front surface region just to chromatographic film 4 for the observation window 13 and make detection line 7 and nature controlling line 6 partial-length be located at observation window 13 rear.
Prepared by embodiment 2 tadanafil rapid detection card
Quick detection tadanafil of the present invention detects blocking Preparation Method, and it specifically comprises the following steps that
Tadanafil monoclonal antibody used by this experiment and tadanafil antigen are bought from Denmark's dako diagnostic companies, sheep anti mouse igg multi-resistance is bought from Shanghai Ye Min biotech company, the auxiliary materials such as nitrocellulose filter, base plate, glass fibre membrane, polyester fiber film are bought from Shanghai Jie Ning biotech company, and the chemical reagent such as gold chloride, sodium citrate, potassium carbonate, bovine serum albumin is bought in Chinese medicines group Beijing chemical reagents corporation.
Step 1, prepared by gold colloidal
The chlorauric acid solution 98ml taking 0.01% is added in 250ml round-bottomed flask, magnetic agitation is heated to seething with excitement, rapidly join the sodium citrate solution of 2ml 1%, solution colour is by faint yellow gradually blackening to claret, treat that solution colour no longer changes, stop heating after continuing to keep boiling 5min, be cooled to purified water after room temperature and supplement volume to 100ml.
Step 2, colloid gold label
(1) traget antibody optimum ph determines
Take 5 1.5ml centrifuge tubes to be separately added into 1ml colloidal gold solution, sequentially add 3,6,9,12,15ul 2m k2co3, stand 5min.Take antibody 15ug respectively, be added in above-mentioned solution.Standing 30min.It is separately added into 100ul 10%nacl, stands 10min, observe solution colour change.Color becomes blue and illustrates that ph is improper, and the constant ph of color meets requirement.The minimum ph that color does not change is optimum ph.Optimum ph is 7.8
(2) minimum albumen consumption determines
Take 5 1.5ml centrifuge tubes to be separately added into 1ml colloidal gold solution, add necessarily k2co3To optimum ph, stand 5min.Take antibody 3-15ug respectively, be added in above-mentioned solution.Standing 30min.It is separately added into 100ul 10%nacl, stands 10min, observe solution colour change.Color becomes blue and illustrates that protein content is too low, and the constant then protein content of color meets requirement.The minimum protein content that color does not change is optimum protein consumption.Optimum protein consumption is 12ug/ml.
(3) labelling
Take 1 1.5ml centrifuge tube to add 1ml colloidal gold solution, add a certain amount of k2co3To optimum ph, stand 5min.Take the antibody of the most suitable albumen consumption, be added in above-mentioned solution.Standing 30min.Take 10ul 10%bsa to be added in above-mentioned solution, stand 15min.
Step 3, pad manufacturing process
The solution that closing is finished, is put in centrifuge after trim.Setting centrifuge parameters: rotating speed 13000rpm, centrifugation time 45min, 4 DEG C of centrifuging temperature.Start to be centrifuged.Centrifugation finishes, and abandons supernatant.Bottom precipitation is redissolved with redissolving liquid, shakes up to no visible particle, uniformly sprays to gold pad.Overnight dry for 37 DEG C.
Step 4, chromatographs film preparation
Take 100ml pbs, ph7.4,0.01m, weigh 5.0g trehalose, add dissolving to shake up.As it is coated liquid.Cleaned with purified water and draw film instrument 10 times.Tadanafil antigen and sheep anti mouse igg antibody are 0.3mg/ml and 1.0mg/ml be coated liquid being diluted to concentration respectively, rule on nitrocellulose filter film respectively with drawing film instrument, live width 1mm, makes antibody be coated on detection line and the nature controlling line as test strips on nitrocellulose filter.Stroked parameters 1ul/cm.Line finishes, and is placed in 37 DEG C and 12h is dried.Film instrument is drawn in cleaning.
Step 5, assembling detection card
Cutting consumables associated therewith.Take one piece of the pvc plate of well cutting, the nc film being coated with tadanafil antigen and sheep anti mouse igg is pasted one in pvc plate central authorities along pvc plate major diameter；Absorption pad is affixed near being coated with one end of sheep anti mouse igg (nature controlling line), and with nc film close contact, about overlapping 2mm；Pad is affixed on below nc film and about 2mm overlapping with nc film；Filter pad, sample are padded successively and is affixed on pvc plate, about 2mm overlapping with pad；Assembling finishes, and cuts into the colloidal gold immuno-chromatography test paper strip of 0.4x6cm.Test strips are positioned in detection card groove, clutch and get stuck up and down as tadanafil rapid detection card.
Embodiment 3 Sample pretreatment
Step 1, pill, tablet is pulverized, capsule shells opened by capsule.
Step 2, weighs powder or content 1g, adds 5ml extracting solution, shakes 1min.Standing 5min treats that solution is clarified.
Step 3, takes supernatant 45um membrane filtration to obtain supernatant liquid and as detects liquid.
Embodiment 4 tadanafil detects
Packaging opened by health product sample, and the method pre-treatment according to embodiment 3 obtains detection liquid.
(about 3) detection drops of 60ul are added in well 21, read result after chromatography reaction 3-8min, observe t line 8 and c line 7 judges testing result.
For relatively more sticky whole blood sample, after Deca 20ul (1) whole blood sample, then Deca 40ul (2) diluent (0.02mph7.4pbs), other steps are ibid.
Embodiment 5 detects card quality standard
1. physical behavior
1.1 outward appearance
Detection card should neatly complete, impulse- free robustness, no damaged, pollution-free；Material adhesion-tight；Label writing is clear, no damaged.
1.2 film bar width
The film bar width >=2.5mm of detection card.
1.3 liquid are divided a word with a hyphen at the end of a line speed
Liquid speed of dividing a word with a hyphen at the end of a line should be not less than 10mm/min.
2 minimum detectabilities
Minimum detectability should be not higher than 200ng/ml.
3 precision
3.1 withinrun precision
Withinrun precision cv (%) should be not higher than 10.0%.
3.2 betweenrun precision
Betweenrun precision cv (%) should be not higher than 15.0%.
4 stability
Detection is stuck in after placing 18 months in 4 DEG C～30 DEG C of environment, sampling detects 1.1,1.2,1.3,2,3.1,3.2 respectively, and result should meet the requirement of projects.
Above preferred embodiment is merely to illustrate present disclosure; in addition; the present invention also has other embodiment, as long as those skilled in the art enlighten because of technology involved in the present invention, and is all fallen within protection scope of the present invention using the technical scheme that equivalent or equivalent deformation mode are formed.
Claims (5)
1. illegally add the detection method of tadanafil in a kind of quick detection health product it is characterised in that the method passes through two
Part is realized, i.e. sample pre-treatments technique and tadanafil rapid detection card,
Described sample pre-treatments technique includes methanol-water sample extracting solution and the pre-treating method based on sample extracting solution, and the present invention carries
Supply a kind of methanol water mixed liquid sample extracting solution, this extracting solution can process the dosage forms such as pill, tablet, medicinal tea and capsule and protect
Strong product, and the tadanafil of illegal interpolation can be extracted by simple pre-treatment step,
The described pre-treating method based on sample treatment liquid, comprises the steps: step 1, and pill, tablet, medicinal tea are pulverized,
Capsule shells opened by capsule；Step 2, weighs powder or content 1g, adds 5ml extracting solution, shakes 1min；Standing 5min
Treat that solution is clarified；Step 3, takes supernatant 45um membrane filtration to obtain supernatant liquid and as detects liquid,
Described tadanafil rapid detection card is based on colloidal gold immunochromatographimethod technology, is achieved through the following technical solutions: one is brood lac
Body gold immunochromatography tadanafil detection card, include base plate and successively overlap paste with the sample pad on base plate, pad,
Chromatographic film and adsorptive pads, thus forming multi-layer film structure, partly overlap between each film structure；It is coated with colloid on described pad
The tadanafil specific antibody of golden labelling；(tadanafil-bsa is combined to have one to be coated tadanafil antigen in described chromatographic film
Thing) detection line, a nature controlling line being coated sheep anti mouse igg antibody.
2. methanol water mixed liquid sample extracting solution according to claim 1, the volume of methanol-water is 1: (1～10).
3. tadanafil rapid detection card according to claim 1, other reach and draw non-specific antibody can be monoclonal antibody
Or polyclonal antibody.
4. the pre-treating method based on sample treatment liquid according to claim 1, step 3 can also be completed by centrifugal method,
Parameter of noncentricity is 3000rpm, 5min.
5. the pre-treating method based on sample treatment liquid according to claim 1, pill, medicinal tea and tablet in described step 1
Can grind it is also possible to wrap pill with pan paper or tablet pulverizes in mortar, depending on the condition according to detection scene.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510430132.2A CN106370846A (en) | 2015-07-22 | 2015-07-22 | Detection method for rapidly detecting illegally-added tadalafil in health product |
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| CN201510430132.2A CN106370846A (en) | 2015-07-22 | 2015-07-22 | Detection method for rapidly detecting illegally-added tadalafil in health product |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267464A (en) * | 2017-07-13 | 2017-10-20 | 江南大学 | The hybridoma cell strain CIT1F2 of one plant of anti-Tadalafei monoclonal antibody specific of secretion and its application |
| CN107300617A (en) * | 2017-07-17 | 2017-10-27 | 广东志道医药科技有限公司 | The inhibitor colloidal gold immuno-chromatography test paper strips of PDE 5 and its preparation and application |
-
2015
- 2015-07-22 CN CN201510430132.2A patent/CN106370846A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267464A (en) * | 2017-07-13 | 2017-10-20 | 江南大学 | The hybridoma cell strain CIT1F2 of one plant of anti-Tadalafei monoclonal antibody specific of secretion and its application |
| CN107300617A (en) * | 2017-07-17 | 2017-10-27 | 广东志道医药科技有限公司 | The inhibitor colloidal gold immuno-chromatography test paper strips of PDE 5 and its preparation and application |
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