CN102393463A - Kit for fluorescence quantitative detection of salbutamol and preparation method of fluorescence labeling liquid - Google Patents
Kit for fluorescence quantitative detection of salbutamol and preparation method of fluorescence labeling liquid Download PDFInfo
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- CN102393463A CN102393463A CN2011103222984A CN201110322298A CN102393463A CN 102393463 A CN102393463 A CN 102393463A CN 2011103222984 A CN2011103222984 A CN 2011103222984A CN 201110322298 A CN201110322298 A CN 201110322298A CN 102393463 A CN102393463 A CN 102393463A
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Abstract
The invention discloses an immunochromatography kit for fluorescence quantitative detection of salbutamol and a preparation method of a fluorescence labeling liquid. The kit comprises a test strip and a fluorescence labeling liquid, wherein the test strip is formed by sequentially lapping and sticking a sample pad, a nitrocellulose coating film and absorbent paper on a bottom plate; the nitrocellulose coating film comprises a detection area and a quality control area; the detection area is coated with an SAL-BSA conjugate; the quality control area is coated with anti-rabbit IgG; and the fluorescence labeling liquid contains a fluorescence labeling SAL antibody and a fluorescence labeling rabbit IgG. Compared with an immune colloidal gold labeling test strip, the kit disclosed by the invention has the advantages of higher sensitivity, accurate quantification and the like; and the operation is faster and more convenient than that of an enzyme coupling method.
Description
Technical field
The invention belongs to field of medical examination, specifically, the present invention relates to a kind of fluorescent quantitation and detect the immunochromatographytest test kit of salbutamol (SAL) and the preparation method of fluorescence labeling liquid.
Background technology
Salbutamol, chemical name are 1-(4-hydroxyl one 3 hydroxymethyl phenyls)-2 one (uncle's fourth is amino) ethanol, are a kind of medicines that can combine with adrenergic receptor, are mainly used in treatment asthma, bronchial spasm etc. clinically.SAL is used as feed addictive, can significantly promote growth of animal, increase feed conversion rate and lean meat percentage mouth, long-term excess is used, and then can in animal tissue, accumulate, and the people can poison after having eaten this animal product in initiation.Early 1990s, a lot of excitant poisonings took place in Europe, and China also had a lot of poisonings to take place so far from 1998, so EU countries and China all make respective specified and forbids that it uses as feed addictive.But ordered about by economic interests, still have a large amount of illegal phenomenons of using.Therefore supervision, detect in the animal product with feed in excitant just become the problem demanding prompt solution that concerns the consumer health.
What Chang Zuowei growth accelerator used in the beta-stimulants is Clenbuterol, but along with the increasing to the Clenbuterol inspecting force, illegal user begins to turn to other substitutes.Because the growth promoting function of SAL and Clenbuterol is more or less the same, its elimination time ratio Clenbuterol in animal body is short, toxicity a little less than, therefore, SAL just becomes Clenbuterol topmost substitute afterwards.But domestic immunologic detection method research for SAI also is in the starting stage; Detection method such as gas one matter coupling method (GC-MS), high performance liquid chromatography (HPLC) and liquid one matter coupling method (LC-MS) etc. to beta-stimulants; Have accurately and reliably, repeatability is strong etc. a bit; But the shortcoming of these methods is outstanding equally, like instrument costliness, complicated operation, portable difference etc., thereby is unfavorable for applying on a large scale and high throughput testing.Therefore, this patent is set up a kind of easy, reliable, quick, detection method that Sensitive Detection SAL is residual, so that monitor the SAI in the animal tissue is residual.
Application number is the patent of invention of CN201010235592.7; Relate to beta-stimulants Ractopamine, salbutamol binary detection test-strips and preparation method thereof; Can effectively solve the problem that can detect beta-stimulants Ractopamine, salbutamol fast, easily simultaneously; Method is: be disposed with diversion spun glass, carrier spun glass, nitrocellulose membrane and absorbent wool pulpboard above the base material PVC plate from back to front; Diversion spun glass, carrier spun glass and be coated with the overlay film of being made up of anterior coverlay and rear portion coverlay above the absorbent wool pulpboard can be used for detecting simultaneously beta-stimulants Ractopamine, salbutamol.But the method also is not easy realization for higher sensitivity, and cannot be as quantitative test.
Application number is the patent of invention of CN200810249803.5; A kind of chemical luminescence ELISA detection kit of salbutamol is disclosed; Comprise box body, being located at polyclonal antibody, the ELIAS secondary antibody that each hole in the box body is coated with the ELISA Plate of the envelope antigen of processing with Clenbuterol and ovalbumin coupling and is located at the salbutamol in the box body is antibody, salbutamol series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, the luminescent solution of the goat-anti rabbit of HRPO mark.That its kit has is highly sensitive, good reproducibility, easy characteristics fast and accurately; Compare with traditional colorimetric ELISA method; Sensitivity can improve an one magnitude, is expected in the salbutamolum residue of animal derived food (like milk appearance, animal tissue's appearance) and urine sample detects, play a significant role, still; This method also not the long period reaction, be unfavorable for on-the-spot test.
Summary of the invention
The objective of the invention is to overcome the defective of above-mentioned prior art, a kind of with low cost, easy and simple to handle, a kind of fluoroscopic examination salbutamol kit that sensitivity is high is provided.
For realizing above-mentioned purpose, the present invention has taked following technical scheme:
A kind of fluorescent quantitation detects salbutamol (SAL) immune chromatography reagent kit, and this kit comprises test strips and fluorescence labeling liquid, and said test strips is overlapped in order to stick on the base plate by sample pad, cellulose nitrate coated film, thieving paper and constitutes; Said cellulose nitrate coated film comprises detection zone (T district) and Quality Control district (C district); Said detection zone is coated with SAL-BSA conjugate (salbutamol-bovine serum albumin(BSA) conjugate), and said Quality Control district encapsulates anti-rabbit igg; Contain fluorescence labeling SAL antibody and fluorescence labeling rabbit igg in the said fluorescence labeling liquid.
Preferably, said cellulose nitrate coated film Quality Control district (C district), the coating buffer concentration of anti-rabbit igg is 0.2～2.0mg/ml, consumption is 90 μ l/27-35cm.
Preferably, said cellulose nitrate coated film detection zone (T district), the coating buffer concentration of salbutamol conjugate is 0.2～2.0mg/ml, consumption is 90 μ l/27-35cm.
More preferably, said cellulose nitrate coated film Quality Control district, the coating buffer concentration of anti-rabbit igg is 0.2～0.5mg/ml; The coating buffer concentration of SAL-BSA conjugate (being salbutamol antigen) is 0.2～1.5mg/ml, and consumption is 90 μ l/27-35cm.
Preferably, the concentration of the fluorescence labeling SAL antibody in the said fluorescence labeling liquid is 0.2～1ug/ml, and the concentration of fluorescence labeling rabbit igg is 0.2～1ug/ml.During use, adopt identical concentration to make the detection better effects if.The excitation wavelength of said fluorescence labeling liquid (Ex) is 310～550nm, and emission wavelength (Em) is 340～620nm.
Another goal of the invention of the present invention has provided a kind of preparation method of above-mentioned fluorescence labeling liquid.
Specifically taked following technical scheme:
The preparation method of said fluorescence labeling liquid, go step following:
The preparation method of A. carboxylic luciferin or fluorescent latex marking fluid
With 3-4mg luciferin or 90-100mg fluorescent latex label with after 14-20mg SAL antibody or rabbit igg mix; Slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS) while stirring; The content that makes EDC in its whole reaction system is 0.1mg; The content of N-hydroxy-succinamide is 0.1mg, and 2～8 ℃ of lucifuge reactions are spent the night at low temperatures; Remove impurity with dialysis or additive method, redissolve with the fluorescence protective agent; Or
B. contain the amino luciferin or the preparation method of fluorescent latex marking fluid
3-4mg luciferin or 90-100mg fluorescent latex label with after 14-20mg SAL antibody or rabbit igg mix, are placed 4～40 ℃ of environment, and regulation system pH6.8～9.0 slowly add 0.2～1% glutaraldehyde while stir; Reacted 2～5 hours, dialysis or additive method are removed impurity, redissolve with the fluorescence protective agent; Or
C. the preparation method of the luciferin of sulfur-bearing phosphoamide key or fluorescent latex marking fluid
Dissolve 14-20mg SAL antibody or rabbit igg respectively with pH8.0～pH9.6 carbonic acid buffer, with the pH8.0～dissolving of pH9.6 carbonic acid buffer or suspension 3-4mg luciferin or 90-100mg fluorescent latex; Luciferin or fluorescent latex with above-mentioned dissolving adds in the globulin solution gradually while stirring, after adding, continues lucifuge and stirs 10-14 hour, after finishing, in the bag filter of packing into, with above-mentioned carbonic acid BS dialysed overnight, redissolves with the fluorescence protective agent.
The detection principle of the immunity-chromatography test carton of salbutamol of the present invention is a competition law, fluorescein molecule or fluorescent latex particulate and SAL antibody covalent bond.To detect sample (urine sample or extract) and add in the fluorescent marker, its fluorescence labeling SAL antibody can combine with the SAL in the urine, forms compound; And the SAL-BSA conjugate that is coated on detection zone on the nitrocellulose filter (T) is also competed combined with fluorescent mark SAL antibody.After containing the SAL sample and fluorescent marker mixes, drop to test strips on; Mixed liquor moves forward along nitrocellulose filter under the chromatography effect; Contained SAL amount is many more in the sample; The fluorescently-labeled antibody that can combine with T district SAL-BSA conjugate is few more, reduces thereby make the T district record the fluorescence value of detecting.Through fluorescence detector scanning T district fluorescence signal intensity, can detect SAL content in the sample.
Salbutamol immuno-chromatographic test paper strip of the present invention is exempted from method detection salbutamol with GC/MS, HPLC isochromatic spectrum instrument and enzyme and is compared, and has easy (one step of simple operations accomplishes), is fit to varying number pattern detection and quick advantages such as (about 15 minutes the result can be arranged); Compare with the immuno-gold labeling test strips, the present invention has higher, the accurate advantage such as quantitative of sensitivity.
Salbutamol chromatography kit of the present invention, the method that adopts fluorescence labeling liquid and sample to be pre-mixed makes reaction and signal discharge homogeneous more, compares with other chromatography, and the precision and the accuracy of its batch process reach best effects.Only on the fluorescent quantitation detector, can reach and just can carry out sensitive quantitative measurement in 10 seconds, measure salbutamolum residue contained in the animal tissue sooner more accurately salbutamol; The salbutamol chromatograph test strip has good accuracy (recovery is 80%-110%), quantitatively in the deviation 20%) and high sensitivity (sensitivity reaches 0.3ug/kg), the sample size few (80ul) that needs is operated very easy.
Description of drawings
Fig. 1 is the structural representation that said fluorescent quantitation of the present invention detects test strips in the salbutamol chromatography kit;
Fig. 2 is the structural representation that said fluorescent quantitation of the present invention detects test strips in the salbutamol chromatography kit;
Fig. 3 is the structural representation that said fluorescent quantitation of the present invention detects the test card that is used to place test strips in the salbutamol chromatography kit.
Embodiment
The present invention adopts fluorescence labeling liquid to be the label of luciferin and albumen or to have fluorescent latex and the label of albumen, and wherein employed luciferin is wherein one or more such as fluorescein isothiocynate, RB 200, TRITC, phycoerythrin, the green fibroin of many dinoflagellates, lanthanide chelate, Fluoresceincarboxylic acid.
In embodiments of the present invention, the SAL antibody that is adopted is the monoclonal antibody of conventional monoclonal antibody technique preparation, and the SAL-BSA conjugate that is adopted (salbutamol antigen) is to utilize the conventional chemical synthetic method to obtain, and utilizes the competition ratio juris to detect sample.
Specify the present invention below in conjunction with accompanying drawing and specific embodiment.
Embodiment one
In this embodiment, fluorescent quantitation detects the salbutamol immune chromatography reagent kit, includes test strips and fluorescence labeling liquid.
Wherein, by the conventional method of test strips, test strips by sample pad 1, the cellulose nitrate coated film 2, the thieving paper 5 that comprise detection zone (T district) 3 and Quality Control district (C district) 4 on base plate 6, constitutes overlap each other successively stickup form, as depicted in figs. 1 and 2.
In this embodiment, the detection zone T line place of coated film is with 0.2mg/ml SAL-BSA conjugate (salbutamol antigen) coating buffer, and use amount is 90ul/27cm.Working concentration is that the anti-rabbit igg of 0.2mg/ml encapsulates at the Quality Control district C of coated film line place, and use amount is all 90ul/27cm.The rabbit igg that is used for the combined with fluorescent mark is used for the validity of test strip.
In this embodiment, fluorescence labeling liquid is excited by 310nm, and emission wavelength is 340nm.In this embodiment, the preparation method (A) of carboxylic fluorescent latex label (present embodiment use luciferin be umbelliferone latex) is adopted in the preparation of fluorescence labeling liquid, and step is following:
With the umbelliferone latex of 100mg respectively with after 14mg SAL antibody or 14mg rabbit igg mix; Slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS) while stirring; The content that makes EDC in its whole reaction system is 0.2mg; NHS content is 0.1mg, and 2～8 ℃ of lucifuge reactions are spent the night at low temperatures.Remove impurity with dialysis or additive method, redissolve with the fluorescence protective agent.
The SAL antibody and the fluorescent latex particulate mark rabbit igg of the above-mentioned fluorescent latex particulate mark for preparing are mixed by proper proportion, so that two kinds of ACs are 0.2ug/ml all respectively, packing is subsequent use.
Embodiment two
In this embodiment, fluorescent quantitation detects the salbutamol immune chromatography reagent kit, comprises test strips and fluorescence labeling liquid.
Wherein, test strips is sticked on the base plate by the conventional method of test strips by sample pad, the cellulose coated film that comprises detection zone (T district) and Quality Control district (C district), thieving paper successively each other overlap joint.
In this embodiment, the detection zone T line place of coated film is with 1.5mg/ml SAL-BSA conjugate (salbutamol antigen) coating buffer, and use amount is 90ul/35cm.Working concentration is that the anti-rabbit igg of 0.5mg/ml encapsulates at the Quality Control district C of coated film line place, and use amount is all 90ul/35cm, is used for the rabbit igg of combined with fluorescent mark, is used for the validity of test strip.
In this embodiment, after fluorescence labeling liquid was excited by 550nm, emission wavelength was 620nm.In this embodiment, the preparation method contain amino fluorescent latex label (present embodiment use luciferin be TRITC latex) is adopted in the preparation of fluorescence labeling liquid, and step is following:
100mg fluorescent latex label respectively with after 16mg SAL antibody or 16mg rabbit igg mix, is placed 4～40 ℃ of environment, and regulation system pH7.0～8.5 slowly add 0.4～0.6% glutaraldehyde while stir; Reacted 2～5 hours, dialysis or additive method are removed impurity, redissolve with the fluorescence protective agent.
The SAL antibody and the fluorescent latex particulate mark rabbit igg of the above-mentioned fluorescent latex particulate mark for preparing are mixed by proper proportion, and consequently two kinds of ACs are 0.5ug/ml all respectively, and packing is subsequent use.
Embodiment three
In this embodiment, fluorescent quantitation detects the salbutamol immune chromatography reagent kit, comprises test strips and fluorescence labeling liquid.
Wherein, test strips is sticked on the base plate by the conventional method of test strips by sample pad, the cellulose coated film that comprises detection zone (T district) and Quality Control district (C district), thieving paper successively each other overlap joint.
In this embodiment, the detection zone T line place of coated film is with 1.0mg/ml SAL-BSA conjugate (salbutamol antigen) coating buffer, and use amount is 90ul/35cm.Working concentration is that the anti-rabbit igg of 0.5mg/ml encapsulates at the Quality Control district C of coated film line place, and use amount is all 90ul/35cm.The rabbit igg that is used for the combined with fluorescent mark is used for the validity of test strip.
In this embodiment, fluorescence labeling liquid is excited by 490nm, and emission wavelength is 530nm.In this embodiment, the preparation method of the luciferin (present embodiment use luciferin be fluorescein isothiocynate) of sulfur-bearing carbon acylamino is adopted in the preparation of fluorescence labeling liquid, and step is following:
Dissolve 20mg SAL antibody or 20mg rabbit igg respectively with pH8.0～pH9.6 carbonic acid buffer, with pH8.0～dissolving of pH9.6 carbonic acid buffer or suspension 3.5mg fluorescein isothiocynate; While stirring above-mentioned fluorescein isothiocynate is added in the globulin solution gradually, after adding, continue lucifuge and stir about 12h, after finishing, in the bag filter of packing into,, redissolve with the fluorescence protective agent with above-mentioned carbonic acid BS dialysed overnight at low temperatures.
The SAL antibody and the fluorescent latex particulate mark rabbit igg of the above-mentioned fluorescent-substance markers for preparing are mixed by proper proportion, and consequently two kinds of ACs are 1ug/ml all respectively, and packing is subsequent use.
Salbutamol immunochromatographytest test kit of the present invention; In instantiation; Semi-manufacture assemble through following operation: overlap in order by sample pad, coated film, thieving paper and stick on the base plate, constitute test strips, and can be again with card shell 7 of the prior art (as shown in Figure 3); Be fixed into test card, said card shell scribbles the product information coding that can supply luminoscope scanning identification.ID chip (the quantitative Analysis formula that ID chip of the prior art adopts is semilog straight line equation or other calculation equations of detected signal value and calculating concentration, can carry out the result automatically and judge) with writing information.Divide the fluorescence labeling liquid that installs to be assembled into kit with other accessories.
Fluorescent quantitation according to the invention detects the immune chromatography reagent kit of salbutamol; In use; Be assembled in by plastics upper casing and plastics lower casing and fasten in the plastic clip shell (test card) that forms; The plastics upper casing is provided with two perforates, and well 9 and display window 8, well 9 detect the immuno-chromatographic test paper strip sample pad of salbutamol corresponding to described fluorescent quantitation; Display window 8 is corresponding to the detection zone and the Quality Control district of the immuno-chromatographic test paper strip of said fluorescent quantitation detection salbutamol as a result, and the immuno-chromatographic test paper strip that this fluorescent quantitation detects salbutamol can take out from this plastic casing.
Be used for testing the fluorescent quantitation spectral detection system (immunofluorescence detector) of immuno-chromatographic test paper strip, mainly comprise fluorescence light source system, detection system and automatic software analysis and Control system.
In one embodiment of the present of invention; Detecting sample need be through following operation: draw the sample (urine sample/extract) and fluorescence labeling liquid mixed in equal amounts of 50～150ul, draw 50～150ul behind the mixing, toward the adding of horizontal positioned test card well; Do not bring bubble into, the reaction of beginning chromatography.Reacted 15 minutes, by fluorescence detector read test result.
Described quantitative salbutamol immunochromatographytest test kit of embodiments of the invention 1-3 and immunofluorescence detector comparison shows that to the mensuration result of 200 routine samples (urine sample/extract): the accuracy of in 0.2ppb～10ppb scope, measuring salbutamol is high: quantitatively the curve linear coefficient is all greater than 0.99; Its accuracy of salbutamol that contains 1ppb and 2ppb concentration in the sample is 80%～120%, and kit detects and is limited to 0.3ppb.The general relatively colloidal gold method (qualitative) that adopts and the testing result of enzyme linked immunosorbent detection method detection kit have better sensitivity and specificity.Table specific as follows.
More than be to the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Claims (7)
1. the immune chromatography reagent kit of a fluorescent quantitation detection salbutamol is characterized in that this kit includes test strips and fluorescence labeling liquid, and said test strips is overlapped in order to stick on the base plate by sample pad, cellulose nitrate coated film, thieving paper and constitutes; Said cellulose nitrate coated film comprises detection zone and Quality Control district; Said detection zone is coated with the SAL-BSA conjugate, and said Quality Control district encapsulates anti-rabbit igg; Contain fluorescence labeling SAL antibody and fluorescence labeling rabbit igg in the said fluorescence labeling liquid.
2. fluorescent quantitation according to claim 1 detects the immune chromatography reagent kit of salbutamol, it is characterized in that, and said cellulose nitrate coated film Quality Control district, the coating buffer concentration of anti-rabbit igg is 0.2～2.0mg/ml, consumption is 90 μ l/27-35cm.
3. fluorescent quantitation according to claim 1 detects the immune chromatography reagent kit of salbutamol, it is characterized in that, and said cellulose nitrate coated film detection zone, the coating buffer concentration of SAL-BSA conjugate is 0.2～2mg/ml, consumption is 90 μ l/27-35cm.
4. detect the immune chromatography reagent kit of salbutamol according to each described fluorescent quantitation of claim 1-3, it is characterized in that, said cellulose nitrate coated film Quality Control district, the coating buffer concentration of anti-rabbit igg is 0.2～0.5mg/ml, consumption is 90 μ l/27-35cm; The coating buffer concentration of SAL-BSA conjugate is 0.2～1.5mg/ml, and consumption is 90 μ l/27-35cm.
5. detect the immune chromatography reagent kit of salbutamol according to each described fluorescent quantitation of claim 1-3; It is characterized in that; The concentration of the fluorescence labeling SAL antibody in the said fluorescence labeling liquid is 0.2～1.0ug/ml, and the concentration of fluorescence labeling rabbit igg is 0.2～1.0ug/ml.
6. fluorescent quantitation according to claim 5 detects the immune chromatography reagent kit of salbutamol, it is characterized in that the excitation wavelength of said fluorescence labeling liquid is 310～550nm, and emission wavelength is 340～620nm.
7. the preparation method of a fluorescence labeling liquid is characterized in that, its step is following:
The preparation method of A. carboxylic luciferin or fluorescent latex marking fluid
With the luciferin of 3-4mg or 90-100mg fluorescent latex label with after 14-20mg SAL antibody or rabbit igg mix; Slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide while stirring; Making 1-ethyl-3-in its whole reaction system (3-dimethyl aminopropyl)-carbodiimides content is 0.1mg; The content of N-hydroxy-succinamide is 0.1mg, spends the night 2～8 ℃ of lucifuge reactions; Remove impurity, redissolve with the fluorescence protective agent; Or
B. contain the amino luciferin or the preparation method of fluorescent latex marking fluid
The luciferin of 3-4mg or 90-100mg fluorescent latex label with after 14-20mg SAL antibody or rabbit igg mix, are placed 4～40 ℃, and regulation system pH6.8～9.0 slowly add 0.2～1% glutaraldehyde while stir; Reacted 2～5 hours, and removed impurity, redissolve with the fluorescence protective agent; Or
C. the preparation method of the luciferin of sulfur-bearing phosphoamide key or fluorescent latex marking fluid
Dissolve 14-20mg SAL antibody or rabbit igg respectively with pH8.0～pH9.6 carbonic acid buffer, with the pH8.0～dissolving of pH9.6 carbonic acid buffer or suspension 3-4mg luciferin or 90-100mg fluorescent latex; Luciferin or fluorescent latex with above-mentioned dissolving adds in the globulin solution gradually while stirring, after adding, continues lucifuge and stirs 10-14 hour, after finishing, in the bag filter of packing into, with above-mentioned carbonic acid BS dialysed overnight, redissolves with the fluorescence protective agent.
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