CN104502554A - Immunoassay method for nano-colloidal gold marker of tadalafil and analogs thereof - Google Patents
Immunoassay method for nano-colloidal gold marker of tadalafil and analogs thereof Download PDFInfo
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- CN104502554A CN104502554A CN201410777149.0A CN201410777149A CN104502554A CN 104502554 A CN104502554 A CN 104502554A CN 201410777149 A CN201410777149 A CN 201410777149A CN 104502554 A CN104502554 A CN 104502554A
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Abstract
The invention discloses an immunoassay method for a nano-colloidal gold marker of tadalafil and analogs thereof. The determination method comprises the following steps: coupling oximate tadalafil with bovine serum albumin by adopting an activated ester method, synthesizing artificial immunity antigen which is used for immunizing animals, and preparing specific tadalafil and analogs thereof; in addition, coupling the oximate tadalafil with egg albumin to be used for construct an immunity detection method; separating, purifying and labeling the tadalafil and analogs thereof to the nano-colloidal gold, and then respectively curing the tadalafil and analogs detection antibodies thereof and goat-anti-rabbit IgG on a carrier, and adding a detection antigen into a detection solution. According to the method, the lowest detection limit of the tadalafil and analogs thereof legally added into health products and Chinese patent medicines is 10mu g/kg, and the fast detection can be completed within 5 minutes. The method is stable, fast and accurate, and suitable for one-step fast detection on legally added medicines.
Description
Technical field
The present invention relates to food and drug safety detection technique field, particularly relate to the nano colloid gold label immunoassay method of a kind of tadalafil and analog thereof.
Background technology
The functional food having plant material to produce and Chinese patent drug are considered to safety and have no side effect, and are extensively subject to consumers in recent years in the whole world.Wherein, natural establishing-Yang medicine (naturalaphrodisiacs) is the product type wherein enlivened the most.But illegal interpolation phosphodiesterase 5 inhibitor (PDE-5inhibitors) in natural establishing-Yang product, increases Yang-strengthening effect with promotion, seeks the behavior of unlawful interests, become global problem.
In the phase at the beginning of the nineties in last century, one of Pfizer is used for the treatment of the PDE-5 inhibitor medicaments of coronary heart disease, and in II, III phase, clinical middle discovery effectively can treat impotence, " sildenafil citrate " in Here it is the fashionable world afterwards.Afterwards, another two PDE-5 inhibitor medicaments Vardenafils and tadalafil commercially obtain huge success in succession, lawless person has synthesized PDE-5 inhibitor medicaments and analogue thereof, and illegal adding in natural drug increases powder charge curative effect, trys to gain illegitimate benefits with promotion.
At present, the PDE-5 inhibitor that US-FDA approval listing is used for the treatment of impotence has: sildenafil citrate (Viagra, Pfizer), Vardenafil hydrochloric acid (Levitra, Baeyer) and tadalafil (Cialis, gift Lay).The PDE-5 inhibitor medicaments of illegal interpolation in Chinese patent drug, health products, not only comprises above three kinds of medicines, also comprises the analogue (structural analogues) the PDE-5 medicine getting permission to go on the market having been carried out to structure derivatization.
The harmfulness of illegal interpolation PDE-5 inhibitor medicaments is embodied in: first, PDE-5 inhibitor medicaments can cause series of side effects, as headache, flush, indigestion, eye-blurred and DOMS.The adverse drug reaction information displaying that U.S. FDA official Internet page (http://www.fda.gov/medwatch/report.htm.) issues, take Viagra may cause blind (2005), take Viagra, Levitra and Cialis and hearing can be caused to decline suddenly even become deaf (2007 years).Therefore, countries in the world all in strict accordance with prescription medicine management, are not having this similar drug taking illegal interpolation under physician guidance to be dangerous to PDE-5 medicine.Secondly, the interaction of PDE-5 inhibitor and nitrate drug can cause serious low blood pressure.Nitrate drug is widely used in diabetes, hypertension, the treatment of high fat of blood and coronary heart disease, suffers from these diseases and the patient with impotence symptom, often seeks help from natural establishing-Yang medicine.If containing the PDE-5 medicine of illegal interpolation in the natural medicine that these patients take, will terrible consequences be caused.
Wherein, tadalafil and analogue thereof add in health food, are having the trend obviously risen in the recent period.The structural formula of analog being tadalafil as follows and being found, the structure of Blocked portion is tadalafil drug effect essential group.
Teach the harmfulness comprising tadalafil PDE-5 inhibitor above, for numerous tadalafil analogue, problem seems more complicated.First, because PDE-5 inhibitor medicine did not carry out toxicity and safety experiment, there is a lot of unknown hazard factor.Secondly, silaenafil analog has stronger pharmacologically active than the similar drugs getting permission to go on the market, as tadalafil analog amino tadalafil with go first tadalafil compared with tadalafil, all lower to 50% inhibition concentration of PDE-5, pharmacologically active is higher.Moreover PDE-5 inhibitor analogue is modified due to structure, cause medicine biodistribution in vivo and pharmacokinetics to change, thus add hazards.Infer that from structure the metabolic stability of tadalafil analog normal-butyl tadalafil and octyl group tadalafil and fat-soluble enhancing can maintain long high blood concentration in vivo, and be more prone to produce serious dysopia by blood-brain barrier.
The PDE-5 inhibitor analogue of illegal interpolation is larger to the health hazard degree of consumer, must strengthen the supervision of PDE-5 inhibitor analogue and examination.In recent years, the detection department of countries in the world and scientific research institution are that the illegal PDE-5 drug sieve checking method of exploitation has done a large amount of work, mainly contain following several technology: 1. thin-layer chromatography (TLC) technology.TLC technology is simple, and testing cost is cheap, is suitable for extensive Preliminary Identification.T.S.Reddy etc. use bismuth potassium iodide colour developing, establish High Performance Thin Layer Chromatography (HPTLC) technology, and under the reference of standard items, the PDE-5 medicine of illegal interpolation in examination natural medicine achieves good effect.2. high performance liquid chromatography (HPLC) and LC-MS (LC-MS) technology.High performance liquid chromatography (HPLC) technology is widely used in the examination of PDE-5 medicine, and its maximum advantage to realize quantitative detection.LC-MS (LC-MS) can carry out confirmation inspection in conjunction with the PDE-5 medicine of the Information in Mass Spectra of inspection product to illegal interpolation.3. HPLC and LC-MS can only detect known PDE-5 medicine.For molecular structure by the analog of emerging PDE-5 being deliberated to modify, because chromatographic behavior and mass spectrum behavior and known drug there are differences, liquid phase/tandem ion-trap mass spectrum (LC/ESI-MS/MS) technology is only had to detect.This technology is separated LC the inspection product electron spray ionisation (ESI) obtained and is converted to quasi-molecular ion, detects at second order ms (MS2) through collision cell (CID) the characteristic ion fragment produced that carries out dissociating.The compound structure information that comprehensive analysis inspection product liquid phase separation result and tandem mass spectrum obtain, can detect PDE-5 medicine that is known and the unknown in same process.4. except the LC/ESI-MS/MS of costliness, only have by hydrolysising product analysis, infer the molecular structure of the analog of emerging PDE-5.By carrying out acidolysis to inspection product, with LC-MS, hydrolysate is analyzed, grasp post retention time and the m/z value of each hydrolysate of these inspection product, compare with the LC-MS testing result of the hydrolysate of known PDE-5 drug standards, the precision architecture of target inspection product can be inferred.
Although above instrument detection method is sensitive, accurate, reliable, apparatus expensive is complicated, service condition is harsh, high to personnel qualifications, can not carry out Site Detection, and its testing effect of cracking down on counterfeit goods exists limitation.Quick, sensitive, reliable, inexpensive in-situ check and test method must be had to be supplemented.
Existing tadalafil and analog method for quick thereof mainly chemical detection method and thin-layered chromatography detection method, the needs that the sensitivity of detection and antijamming capability are improved.Immunological detection method is sensitive, special, quick and inexpensive, in environmental monitoring and field of food safety widespread use, in food and drug safety detects fast, is also more and more given great expectations.
Illegal businessman, in order to evade supervision, constantly releases tadalafil analogue and carries out illegal interpolation, must develop a kind of immunological method that simultaneously can detect tadalafil and analogue thereof, effectively can hit such illegal interpolation behavior.
The present invention relates to the synthesizing artificial antigen of outstanding tadalafil and analog common group thereof, the specific antibody that can identify tadalafil and analog common group thereof is produced with the induction of artificial immunity antigen-immunized animal, exploitation detects the colloidal gold immunochromatographimethod method of illegal interpolation tadalafil and analog thereof, for large-scale special screening provides efficient technical tool.
By literature search, do not find to utilize colloidal gold immunochromatographimethod method to detect the relevant report of tadalafil and analog thereof.Patent of the present invention provides new instrument for food and drug safety testing.
Summary of the invention
The object of the invention is to overcome the deficiency that existing immunoassay technology can only detect a kind of target substance, by ANTIGEN DESIGNThe targetedly, immune animal induction generation can either identify tadalafil, the universal antibody of all potential tadalafil analogues can also be identified, there is provided a kind of can the nano colloid gold label immunoassay method of tadalafil and this series material of analog thereof, be applied to food and drug safety detection field, change traditional cumbersome test problems of illegal interpolation multistep, realize the supervision detection that single stage method detects tadalafil and the illegal interpolation of analog thereof fast.
Realize the present invention, emphasis is the artificial antigen of the outstanding tadalafil of synthesis and analog common group thereof.Tadalafil covalency must be connected to protein carrier, synthesis artificial immunity antigen, the chemical group that square frame in tadalafil and the structural formula of analog that is found thereof identifies is exposed to the outside of artificial immunity antigen, induced animal immune response could produces the universal antibody identifying tadalafil drug effect essential group.
As long as tadalafil analog, just certainly containing the tadalafil drug effect essential group identified in tadalafil and the structural formula of analog that is found thereof, with regard to one surely identify by the universal antibody that obtains by above-mentioned design proposal.Thus realize the technical though simultaneously detecting this whole class material.
The present invention adopts following technical scheme:
The nano colloid gold label immunoassay method of tadalafil of the present invention and analog thereof is by oximate tadalafil and bovine serum albumin(BSA) (BSA) coupling with active ester method, synthesis artificial immunity antigen, and for immune animal, the preparation anti-tadalafil of specificity and analog antibody thereof.In addition by oximate tadalafil and oralbumin (OVA) coupling, for building immunologic detection method.Anti-tadalafil and analog antibody thereof are marked on nano colloid gold after separation and purification, then detect antibody with tadalafil and analog thereof and goat anti-rabbit igg is solidificated on a carrier respectively, in detection solution, add detectable antigens.In negative sample testing process, form " sandwich " formula antigenic compound that " antibody-detectable antigens-antibody " forms in chromatographic film detection line region, collaurum gathers colour developing at surveyed area, thus produces detection signal.In positive testing process, according to Competitive assays immunochromatography principle, insolubilized antibody and colloidal gold labeled monoclonal antibody all can tested product competition binding, " antibody-detectable antigens-antibody " immune complex in chromatographic film detection line region is produced and suppresses, collaurum can not gather colour developing at surveyed area, thus can not produce detection signal.
The signal that this method produces is clear and stable, and detection sensitivity is high, reliability is strong.Carry out the semi-quantitative analysis of tadalafil and analog thereof, the minimum detectability degree of the detection system set up to interpolation tadalafil illegal in health products, Chinese patent drug and analog thereof is 10 μ g/kg, and completes quick detection in 5 minutes.Method is stable, quick, accurate, is suitable for the quick detection carrying out single stage method illegal interpolation medicine.
Further illustrate technical scheme of the present invention below, its concrete step is:
The preparation of artificial immunity compound, haptenic derivative reaction.Make tadalafil connect reactive group by oximation reaction, and retain tadalafil and analog common structure thereof, principle is as follows:
That is non-ly dissolved in 2mL anhydrous pyridine solution to take 2mg tadalafil, and add 5mg ethyloic azanol, room temperature is rocked reaction and spent the night.Next day, pyridine Rotary Evaporators evaporate to dryness, with 5mL sodium bicarbonate solution solubilizing reaction thing, discards precipitation.Then use salt acid for adjusting pH value to 3.0, use ethyl acetate extracting.Aqueous phase discarded.By the ethyl acetate Rotary Evaporators evaporate to dryness collected, obtain oximate tadalafil.
Outstanding tadalafil and analog thereof have group artificial immunity antigen synthetic method, and principle is as follows: oximate tadalafil and NHS condensation under EDC effect, form the active ester of oximate tadalafil:
The active ester of oximate tadalafil in the basic conditions with protein amino condensation, the immunizing antigen of the outstanding tadalafil of synthesis and analog common structure thereof:
Adopt modified activity ester process synthesis immunizing antigen, 0.5mg haptens is dissolved in 0.5mL absolute methanol, add 0.05g carbodiimide (EDC) and 0.01g N-hydroxy-succinamide (NHS) again, under room temperature, reaction is spent the night, centrifuging sediment, get supernatant, dropwise add the carbonic acid buffer (pH8.5) being dissolved in 1.0mL containing 5mg Cationic bovine serum albumin (MBSA) solution in, room temperature concussion reaction 4 react hour.Normal saline dialysis removes unreacted oximate tadalafil for 72 hours, and within every 6 hours, change dislysate once, packing is frozen in-20 DEG C of refrigerators.
Outstanding tadalafil and analog thereof have group manual detection antigen synthetic method, and principle is as follows: the active ester of oximate tadalafil lower with protein amino condensation, the immunizing antigen of the outstanding tadalafil of synthesis and analog common structure thereof:
Adopt carbodlimide method synthesis detectable antigens, 0.2mg oximate tadalafil is dissolved in 0.5mL absolute methanol, join 1.0mL containing in carbonic acid buffer (pH8.5) solution of 5mg cationization oralbumin (MOVA), add 0.05g carbodiimide (EDC) and 0.01g NHS again, shake reaction under room temperature to spend the night, centrifuging sediment, gets supernatant, magnetic agitation 4 reaction hour.Normal saline dialysis removes the active ester of unreacted oximate tadalafil for 72 hours, and within every 6 hours, change dislysate once, packing freeze-drying is for subsequent use.
Possess the Absorption Characteristics of bovine serum albumin(BSA) (BSA) and tadalafil and analog thereof through scanning qualification tadalafil and analog-BSA conjugate thereof, tadalafil and analog-OVA conjugate thereof possess the Absorption Characteristics of oralbumin (OVA) and tadalafil and analog thereof.Prove that tadalafil and analog artificial immunity antigen and manual detection antigen thereof synthesize successfully.
The preparation of anti-tadalafil and analog specific antibody thereof: with tadalafil and the analog-BSA immunization experiment new zealand white rabbit thereof of synthesis, after every White Rabbit initial immunity 0.2mg/0.2mL immunizing antigen adds the Freund's complete adjuvant emulsification of equivalent, hypodermic injection is immune.Initial immunity is separated by 28 days, with 0.1mg/0.1mL immunizing antigen with hypodermic injection after freund 's incomplete adjuvant emulsification antigen, carries out booster immunization.Once every 21 days with same dosage booster immunization, carry out four booster immunizations altogether later.After last booster immunization, within 7 days, get blood with arteria carotis and collect rabbit serum ,-20 DEG C frozen for subsequent use.
The preparation of collaurum: the gold chloride adding 1.0mL 1% concentration in 100mL ultrapure water, is heated to boiling, then adds 1% citric acid three sodium solution 1mL, continue to boil 10 minutes, after cooling, 4 DEG C save backup, and sampling sweep measuring maximum absorption band also carries out transmission electron microscope observing particle size.
The anti-tadalafil of colloid gold label and analog specific antibody thereof: the pH value regulating the colloidal gold solution (100mL) as above prepared is 8.2, anti-tadalafil and analog specific antibody 400 μ g thereof is added under rapid stirring, its final concentration is made to be that 4 μ g/mL room temperature reactions are after 15 minutes, adding polyglycol (PEG) to final concentration is 1%, continues stirring 15 minutes.With 15000r/min centrifugal 30 minutes, after sucking supernatant, gained precipitation was the golden labelled antibody of purifying, and this golden labelled antibody is suspended from conserving liquid again, 4 DEG C of preservations.
The process of nitrocellulose filter and glass fibre element film: the pH value regulating the 100mL colloidal gold solution as above prepared is 8.2, purified anti-tadalafil and analog antibody 400 μ g thereof is added under rapid stirring, its final concentration is made to be 4 μ g/mL, room temperature reaction is after 15 minutes, adding polyglycol PEG to final concentration is 1%, continue stirring 15 minutes, with 15000r/min centrifugal 30 minutes, after sucking supernatant, gained precipitation is the golden labelled antibody of purifying, this golden labelled antibody is suspended from conserving liquid again, 4 DEG C of preservations.
The preparation of immuno-chromatographic test paper strip.In the one side glue PVC board of 60mm × 300mm, paste upper glass fibre element film successively, be fixed with the glass fibre element film of the gold anti-tadalafil of mark and analog antibody thereof, parallel bag is by nitrocellulose filter, the water adsorption glass cellulose membrane of tadalafil and analog antibody and anti-mouse IgG antibody.With cutting cutter, the PVC board pasted longitudinally is cut into the test strips of 5mm × 60mm.
Sample test and result determination methods: get 0.2g (or 0.2mL) sample, be dissolved in abundant extracting target inspection product in 2.0mL absolute ethyl alcohol.Extract makes impurity fully precipitate in static 10 minutes, gets 0.2mL supernatant and joins 20mL at the sample detection liquid containing 0.05mg/mL tadalafil detectable antigens, fully shake up and become sample detection liquid.In sample pad, 3-4 dropping liquid sample detection is dripped with dropper, goat anti-rabbit igg and tadalafil and analog coupling OVA bond thereof are sprayed on nature controlling line (C) and the p-wire (T) of NC film respectively, move to the other end according to chromatographic theory through S end containing tadalafil and analog testing sample thereof, and successively cross p-wire and nature controlling line, owing to there is detectable antigens in sample detection solution, the antibody that insolubilized antibody on NC film T line and collaurum mark is being combined with detectable antigens respectively, double-antibody sandwich immune conjugate is formed at NC film T line position, T line is developed the color and produces detection signal.When there are target inspection product in sample, immune response will be blocked by competition, and the existing color of T can disappear.
The principle of nature controlling line (C) is identical with list of references report method, and be that whether effective the method for inspection itself is and set, colour developing effectively, does not develop the color and shows that method itself is invalid.
Sample not containing tadalafil and analog thereof shows obvious two lines, and be negative result.Containing the sample of 2ng/mL tadalafil and analog thereof, detection line is most of suppressed, in the weak positive.Tadalafil and analog content thereof are greater than the sample of 2ng/mL, and detection line is all suppressed, in strong positive.
Accompanying drawing explanation
Fig. 1 is gold-immunochromatographyreagent reagent for assay wiring layout;
In figure: 1. base plate; 2. sample pad; 3. colloidal gold pad; 4. nitrocellulose filter; 5. adsorptive pads; 6. detection line; 7. nature controlling line.
Embodiment
The following examples describe in further detail of the present invention.
Embodiment one:
Outstanding tadalafil and analog thereof have group artificial immunity antigen synthetic method: (1) takes 2mg tadalafil that is non-ly dissolved in 2mL anhydrous pyridine solution, and add 5mg ethyloic azanol, room temperature is rocked reaction and spent the night.Next day, pyridine Rotary Evaporators evaporate to dryness, with 5mL sodium bicarbonate solution solubilizing reaction thing, discards precipitation.Then use salt acid for adjusting pH value to 3.0, use ethyl acetate extracting.Aqueous phase discarded.By the ethyl acetate Rotary Evaporators evaporate to dryness collected, obtain oximate tadalafil.(2) modified activity ester process synthesis immunizing antigen is adopted, 0.5mg haptens is dissolved in 0.5mL absolute methanol, add 0.05g carbodiimide (EDC) and 0.01gN-N-Hydroxysuccinimide (NHS) again, under room temperature, reaction is spent the night, centrifuging sediment, get supernatant, dropwise add the carbonic acid buffer (pH8.5) being dissolved in 1.0mL containing 5mg Cationic bovine serum albumin (MBSA) solution in, room temperature concussion reaction 4 react hour.Normal saline dialysis removes unreacted oximate tadalafil for 72 hours, and within every 6 hours, change dislysate once, packing is frozen in-20 DEG C of refrigerators.(3) outstanding tadalafil and analog thereof have group manual detection antigen synthetic method: adopt carbodlimide method synthesis detectable antigens, 0.2mg oximate tadalafil is dissolved in 0.5mL absolute methanol, join 1.0mL containing in carbonic acid buffer (pH8.5) solution of 5mg cationization oralbumin (MOVA), add 0.05g carbodiimide (EDC) and 0.01g NHS again, shake reaction under room temperature to spend the night, centrifuging sediment, get supernatant, magnetic agitation 4 reaction hour.Normal saline dialysis removes the active ester of unreacted oximate tadalafil for 72 hours, and within every 6 hours, change dislysate once, packing freeze-drying is for subsequent use.
Embodiment two:
Then by the 400 μ g through separation and purification, monoclonal antibody adds in previously prepared 40nm size colloidal gold solution (pH is 8.2), room temperature reaction 15 minutes, and adding polyglycol (PEG) to final concentration is 1%, continues stirring 15 minutes.With 8000r/min centrifugal 30 minutes, after sucking supernatant, gained precipitation was the golden labelled antibody of purifying.This golden labelled antibody bond is sprayed on glass fibre element film, tadalafil and analog coupling-OVA bond thereof are sprayed at NC film, as p-wire, goat anti-rabbit igg is sprayed at NC film, as nature controlling line, then by absorption of sample pad, be coated with colloidal gold antibody glass fibre, the NC film being sprayed with p-wire and nature controlling line and water adsorption glass fiber be pasted on a plastic bottom board (5mm × 6mm) successively.
During test sample, get 0.2g (or 0.2mL) sample, be dissolved in abundant extracting target inspection product in 2.0mL absolute ethyl alcohol.Extract makes impurity fully precipitate in static 10 minutes, gets 0.2mL supernatant and joins 20mL at the sample detection liquid containing 0.05mg/mL tadalafil detectable antigens, fully shake up and become sample detection liquid.In sample pad, 3-4 dropping liquid sample detection is dripped with dropper, goat anti-rabbit igg and tadalafil and analog coupling OVA bond thereof are sprayed on nature controlling line (C) and the p-wire (T) of NC film respectively, move to the other end according to chromatographic theory through S end containing tadalafil and analog testing sample thereof, and successively cross p-wire and nature controlling line, owing to there is detectable antigens in sample detection solution, the antibody that insolubilized antibody on NC film T line and collaurum mark is being combined with detectable antigens respectively, double-antibody sandwich immune conjugate is formed at NC film T line position, T line is developed the color and produces detection signal.When there are target inspection product in sample, immune response will be blocked by competition, and the existing color of T can disappear.
The principle of nature controlling line (C) is identical with list of references report method, and be that whether effective the method for inspection itself is and set, colour developing effectively, does not develop the color and shows that method itself is invalid.
Although illustrate and describe embodiments of the invention, for the ordinary skill in the art, be appreciated that and can carry out multiple change, amendment, replacement and modification to these embodiments without departing from the principles and spirit of the present invention, scope of the present invention is by claims and equivalents thereof.
Claims (9)
1. the nano colloid gold label immunoassay method of a tadalafil and analog thereof, it is characterized in that: described method is by oximate tadalafil with active ester method, artificial immunity antigen is synthesized by active ester method and Cationic bovine serum albumin coupling, tadalafil and analog common structure thereof are exposed to artificial antigen outside, and for immune animal, the antibody of the preparation anti-tadalafil of specificity and analog common structure thereof;
By oximate tadalafil carbodlimide method and oralbumin coupling, synthesis detectable antigens, guarantee that tadalafil and analog common structure thereof are exposed to artificial antigen outside, the antibody obtained is coordinated to build with detectable antigens and detects colloidal gold immunochromatographimethod detection system, for building the immunologic detection method detecting tadalafil and analogue thereof simultaneously;
Then the antibody of anti-tadalafil and analog common structure thereof is marked on nano colloid gold after separation and purification, again by the antibody labeling of anti-tadalafil and analog common structure thereof on the detection line (T line) of nitrocellulose filter (NC film) carrier, goat anti-rabbit igg is solidificated on nature controlling line, builds colloidal-gold detecting-card;
In sample detection solution, detectable antigens is added when detecting sample, the antibody that insolubilized antibody on NC film T line and collaurum mark is combined at the tadalafil respectively on detectable antigens and analog common structure thereof, double-antibody sandwich immune conjugate is formed at NC film T line position, T line is developed the color and produces detection signal, there are target inspection product in sample, immune response will be blocked by competition, the existing color of T can disappear, thus makes collaurum at solid phase and the semi-quantitative analysis carrying out tadalafil and analog thereof.
2. the nano colloid gold label immunoassay method of tadalafil as claimed in claim 1 and analog thereof, is characterized in that: the concrete steps of described method are as follows:
(1) oximate derivatization method is carried out to tadalafil, and derivatization tadalafil is connected to different protein carriers, build and tadalafil and analog common structure thereof be exposed to outside outstanding tadalafil and analog has group artificial immunity antigen and manual detection antigen;
(2) use the artificial immunity antigen immune rabbit of outstanding tadalafil and analog common structure thereof, induction produces anti-tadalafil and analog polyclonal antibody thereof, collects serum and uses immune-affinity chromatography purification specificity antibody;
(3) preparation of collaurum;
(4) anti-tadalafil and analog specific antibody mark collaurum thereof;
(5) the antibody labeling of anti-tadalafil and analog common structure thereof on the detection line (T line) of nitrocellulose filter (NC film) carrier, goat anti-rabbit igg is solidificated on nature controlling line, builds colloidal-gold detecting-card;
(6) antibody that the insolubilized antibody in testing process on NC film T line and collaurum mark is combined at the tadalafil respectively on detectable antigens and analog common structure thereof, form double-antibody sandwich immune conjugate at NC film T line position, T line is developed the color and produces detection signal.
3. the nano colloid gold label immunoassay method of tadalafil as claimed in claim 2 and analog thereof, it is characterized in that: in step (1), the concrete steps of described tadalafil oximate derivatization method are as follows: get 2mg tadalafil and be dissolved in 2mL anhydrous pyridine solution, add 5mg ethyloic azanol, room temperature is rocked reaction and is spent the night, next day is pyridine Rotary Evaporators evaporate to dryness, with 5mL sodium bicarbonate solution solubilizing reaction thing, discard precipitation, then use salt acid for adjusting pH value to 3.0, use ethyl acetate extracting, aqueous phase discarded, by the ethyl acetate Rotary Evaporators evaporate to dryness collected, obtain oximate tadalafil.
4. the nano colloid gold label immunoassay method of tadalafil as claimed in claim 2 and analog thereof, it is characterized in that: in step (1), the synthetic method that described outstanding tadalafil and analog thereof have group artificial immunity antigen is as follows: adopt modified activity ester process synthesis immunizing antigen, 0.5mg haptens is dissolved in 0.5mL absolute methanol, add 0.05g carbodiimide and 0.01g N-hydroxy-succinamide again, under room temperature, reaction is spent the night, centrifuging sediment, get supernatant, dropwise add the carbonic acid buffer being dissolved in 1.0mL containing 5mg Cationic bovine serum albumin solution in, room temperature concussion reaction 4 reaction hour, normal saline dialysis removes unreacted tadalafil and analog thereof in 72 hours, within every 6 hours, change dislysate once, packing is frozen in-20 DEG C of refrigerators.
5. the nano colloid gold label immunoassay method of tadalafil as claimed in claim 2 and analog thereof, it is characterized in that: in step (1), the synthetic method that described outstanding tadalafil and analog thereof have group manual detection antigen is as follows: adopt carbodlimide method synthesis detectable antigens, 0.2mg oximate tadalafil is dissolved in 0.5mL absolute methanol, join 1.0mL containing in the carbonic acid buffer solution of 5mg cationization oralbumin, add 0.05g carbodiimide and 0.01g NHS again, shake reaction under room temperature to spend the night, centrifuging sediment, get supernatant, magnetic agitation 4 reaction hour, normal saline dialysis removes the active ester of unreacted oximate tadalafil for 72 hours, within every 6 hours, change dislysate once, packing freeze-drying is for subsequent use.
6. the nano colloid gold label immunoassay method of the tadalafil as described in claim 4 or 5 and analog thereof, is characterized in that: the pH=8.5 of described carbonic acid buffer.
7. the nano colloid gold label immunoassay method of tadalafil as claimed in claim 2 and analog thereof, is characterized in that: in step (3), and collaurum adopts conventional citric acid reducing process preparation.
8. the nano colloid gold label immunoassay method of tadalafil as claimed in claim 2 and analog thereof, it is characterized in that: the concrete steps of step (5) are: spray two parallel lines on nitrocellulose filter with micro-quantitation nozzle, anti-tadalafil and analog antibody thereof are as p-wire, goat anti-rabbit igg is as nature controlling line, with quantitation nozzle spraying gold mark anti-tadalafil and analog antibody in glass fibre element film on, dry nitrocellulose filter and glass fibre element film after for subsequent use.
9. the nano colloid gold label immunoassay method of tadalafil as claimed in claim 2 and analog thereof, it is characterized in that: the concrete steps of step (6) are: get 0.2g or 0.2mL sample, be dissolved in abundant extracting target inspection product in 2.0mL absolute ethyl alcohol, extract makes impurity fully precipitate in static 10 minutes, get 0.2mL supernatant and join 20mL at the sample detection liquid containing 0.05mg/mL tadalafil detectable antigens, fully shake up and become sample detection liquid, in sample pad, 3-4 dropping liquid sample detection is dripped with dropper, goat anti-rabbit igg and tadalafil and analog coupling OVA bond thereof are sprayed on nature controlling line (C) and the p-wire (T) of NC film respectively, move to the other end according to chromatographic theory through S end containing tadalafil and analog testing sample thereof, and successively cross p-wire and nature controlling line, owing to there is detectable antigens in sample detection solution, the antibody that insolubilized antibody on NC film T line and collaurum mark is being combined with detectable antigens respectively, double-antibody sandwich immune conjugate is formed at NC film T line position, T line is developed the color and produces detection signal, when there are target inspection product in sample, immune response will be blocked by competition, the existing color of T can disappear.
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