CN103364562A - Test strip for testing sulfanilamide drug residues and application method - Google Patents
Test strip for testing sulfanilamide drug residues and application method Download PDFInfo
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- CN103364562A CN103364562A CN2013103293809A CN201310329380A CN103364562A CN 103364562 A CN103364562 A CN 103364562A CN 2013103293809 A CN2013103293809 A CN 2013103293809A CN 201310329380 A CN201310329380 A CN 201310329380A CN 103364562 A CN103364562 A CN 103364562A
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Abstract
The invention provides an immunochromatographic test strip for testing sulfanilamide drug residues. The test strip is characterized in that a sample applying zone, a reaction capturing zone and a remaining liquid absorbing zone are arranged on the substrate of the test strip. Specific monoclonal antibodies of sulfanilamide drugs and labeled with quantum dots are fixed in the sample applying zone. Test strips containing specific monoclonal antibodies of sulfanilamide drugs and quality control strips containing rabbit anti rat IgG antibodies are arranged in the reaction capturing zone. The sample applying zone, the reaction capturing zone and the remaining liquid absorbing zone are located on downward-recessed micro flow channels of the substrate of the test strip.
Description
Technical field
The present invention relates to a kind of sulfa drug residue test strip and using method.
Background technology
From 1932 first synthetic contain the red azo dyestuff Prontosil of sulfanilamide (SN) group since, the history that its development has been experienced more than 80 year, sulfa drugs (sulfaonamides) is wide with its antimicrobial spectrum, stable in properties, cheap, do not consume grain, the domestic advantage such as can produce in a large number, think so far one of important drugs of fowl poultry kind anti-infective therapy.As feed addictive, after aquaculture is used and indicated the animal-use drug product of withdrawal time and contain the pastille feed addictive, if do not observe the suitable off-drug period, will be polluted by residual sulfa drugs in its musculature, egg and the milk.Heavy dose of or the long-term low dose of administration of short-term is easy to cause sulfa drugs to accumulate in each tissue of animal.After feed or drinking-water are polluted by sulfa drugs, also can cause residual quantity of sulfonamide severe overweight in the animal food.The appearance of sulfa drug residue problem has nearly 30 years, and the sulfa drug residue phenomenon that exceeds standard is all more serious than other any residues of veterinary drug in nearly 15-20.The sulfa drugs phenomenon that exceeds standard is that generation is arranged in the surperficial Pork and its products of a lot of researchs, and secondly its residual mainly occurring in the pork is residual in veal and the poultry, and particularly the sulfamethazine of carcinogenic row is residual has brought threat to human health.Studies show that sulfamido abuse of antibiotics phenomenon is very serious in the present cultivation, cause sulfa drugs severe enrichment in animal food and even potable water, use these food and drink such water source, can produce various toxic actions to the mankind, even cause organic damage, also can cause allergic reaction, cause what is more the generation of cancer.
Because people are to the growing interest of sulfa drug residue in the animal derived food, existing a large amount of technology are used for the detection of sulfa drug residue level, and these technology comprise microbial method, immunoassay, thin-layered chromatography (TLC), gas chromatography (GC), GC-MS(gas chromatography-mass spectrography) (GC-MS), capillary electrophoresis (CE), high performance liquid chromatography (HPLC) and biology sensor etc.HPLC method detection sensitivity and specificity are all higher, but its operation is more loaded down with trivial details.Physics and chemistry detection method program is complicated, and analysis cost is high, is not suitable for the detection of a large amount of samples, microbial method length consuming time, and sensitivity is not high.
At present, colloidal gold immunity chromatography is a kind of method the most commonly used in the Sulfonamides retention analysis, but because the limitation of the optical characteristics of certification mark thing collaurum molecule, the method can only realize the qualitative detection to sulfa drugs, be difficult to realize its residual observational measurement, and varying owing to test sample in the prior art, pH value wherein, ion concentration is different with protein content, this must cause on the release of pad indicator substance different impacts, the chromatograph test strip such as nitrocellulose filter forms the structure inhomogeneity in addition, can cause chromatography and the immune response of solution to produce difference, larger inhomogeneity may appear in different batches in the identical chromatograph test strip, i.e. difference between batch CV.
Quantum dot (Quantum Dots, QDs) be a kind of semiconductor nanocrystal, it is very wide to have excitation wavelength range, can contain whole spectrum, from the ultraviolet to the far infrared region, the light of Same Wavelength can excite the quantum dot of different sizes, the emissioning light spectrum peak is narrow and symmetrical, red holder tail phenomenon without organic fluorescence, emission spectrum is adjustable, launching light intensity, is 1000 times of organic fluorescent dye, and good stability, can stand repeated multiple times exciting, be difficult for the good spectral signature of cancellation and optical stability, compare with the bioluminescence dyestuff and have wide exciting line scope, the narrow spectral line of emission, luminescence efficiency is high, glow color is adjustable, the series of advantages such as good light stability are suitable for as fluorescent marker.
For the problems referred to above, the present invention selects the quantum dot conduct to marker molecules and to the improving layer analysis system, the sulfa drugs chromatograph test strip detection system that acquisition can quantitatively detect and differences between batches are little.
Summary of the invention
Technical matters to be solved by this invention provides a kind of immuno-chromatographic test paper strip that detects sulfa drug residue, and it improves marker molecules, to realize the quantitative detection to medicament residue; While improving layer analysis system is to obtain the little sulfa drugs immuno-chromatographic test paper strip detection system of differences between batches.
For solving the problems of the technologies described above, the invention provides a kind of immuno-chromatographic test paper strip that detects sulfa drug residue, it is characterized in that being provided with on the substrate of described test strips sample and apply district, reaction trapping region and remaining liq uptake zone, and described sample applies and is fixed with quantum dot-labeled sulfa drugs monoclonal antibody specific in the district, and the test strip that contains the sulfa drugs polyclonal antibody and the Quality Control band that contains rabbit anti-mouse igg antibody are set in the reaction trapping region;
Wherein, described sample applies district, reaction trapping region and remaining liq uptake zone and is positioned on the fluid channel that the test strips substrate is arranged with downwards;
Wherein, described fluid channel bottom is cast with nitrocellulose solution, forms nitrocellulose layer;
Wherein, the diameter that sample applies district, reaction trapping region and remaining liq uptake zone on the described fluid channel is not more than 2mm;
Wherein, the material of described substrate is polycarbonate, and the fluid channel on it obtains by etch process;
Wherein, the thickness of nitrocellulose layer is not less than sample and applies district and remaining liq uptake zone in the described reaction trapping region;
Wherein, described quantum dot-labeled thing is CdTe;
Wherein, the quantum dot-labeled mark sulfa drugs of described CdTe monoclonal antibody, its labeling method is to get the red quantum dot of CdTe that 5 mL concentration are 3 mg/mL, be that the sulfa drugs monoclonal antibody of 1 ng/mL is fully mixed with concentration, then under the condition of magnetic agitation, dropwise add the EDC solution that 0.5 mL concentration is 1 mg/mL, this mixed liquor shading places the reaction of rotation hybrid frame after 3 hours, 4 ℃ of hold over night.After 24 hours, get this mixed liquor 15,000 and left the heart 20 minutes, remove supernatant, the phosphate buffer (Phosphate-Buffered Saline(PBS) that contains 0.5% tween-20 (v/v) with 10.5 mL: 0.2 mg/mL KCL, 1.44 mg/mL Na2HPO4,0.24mg/mL KH2PO4,8 mg/mL NaCl, pH 7.4) resuspended centrifugal residue, use again 15,000 to leave heart washing quantum dot-monoclonal anti nanocrystal composition, 3 times repeatedly.At last, disperse quantum dot-monoclonal anti nanocrystal composition with 100 mL PBST, and place 4 ℃ of preservations;
Wherein, contain 1% sucrose component in the detection line of reaction trapping region and the nature controlling line immobile liquid, " diving phenomenon " in chromatography detects, occur to prevent quantum dot-monoclonal anti nanocrystal composition.
Description of drawings
Fig. 1 represents commercialization test strips structure in the prior art.
Fig. 2 represents chromatograph test strip structure among the present invention, and wherein 1 is remaining liq uptake zone for the Quality Control band on the reaction trapping region and 4 for sample applies district, 2 for the test strip of reaction on the trapping region, 3.
Embodiment
Describe embodiments of the present invention in detail below with reference to drawings and Examples, how the application technology means solve technical matters to the present invention whereby, and the implementation procedure of reaching technique effect can fully understand and implements according to this.
The invention provides a kind of immuno-chromatographic test paper strip that detects sulfa drug residue, it is characterized in that being provided with on the substrate of described test strips sample and apply district, reaction trapping region and remaining liq uptake zone, and described sample applies and is fixed with quantum dot-labeled sulfa drugs monoclonal antibody specific in the district, and the test strip that contains the sulfa drugs polyclonal antibody and the Quality Control band that contains rabbit anti-mouse igg antibody are set in the reaction trapping region;
Wherein, described sample applies district, reaction trapping region and remaining liq uptake zone and is positioned on the fluid channel that the test strips substrate is arranged with downwards;
Wherein, described fluid channel bottom is cast with nitrocellulose solution, forms nitrocellulose layer;
Wherein, the diameter that sample applies district, reaction trapping region and remaining liq uptake zone on the described fluid channel is about and is not more than 2mm;
Wherein, the material of described substrate is polycarbonate, and the fluid channel on it obtains by etch process;
Wherein, the thickness of nitrocellulose layer is not less than sample and applies district and remaining liq uptake zone in the described reaction trapping region;
Wherein, described quantum dot-labeled thing is CdTe;
Wherein, the quantum dot-labeled mark sulfa drugs of described CdTe monoclonal antibody, its labeling method is to get the red quantum dot of CdTe that 5 mL concentration are 3 mg/mL, be that the sulfa drugs monoclonal antibody of 1 ng/mL is fully mixed with concentration, then under the condition of magnetic agitation, dropwise add the EDC solution that 0.5 mL concentration is 1 mg/mL, this mixed liquor shading places the reaction of rotation hybrid frame after 3 hours, 4 ℃ of hold over night.After 24 hours, get this mixed liquor 15,000 and left the heart 20 minutes, remove supernatant, the phosphate buffer (Phosphate-Buffered Saline(PBS) that contains 0.5% tween-20 (v/v) with 10.5 mL: 0.2 mg/mL KCL, 1.44 mg/mL Na2HPO4,0.24mg/mL KH2PO4,8 mg/mL NaCl, pH 7.4) resuspended centrifugal residue, use again 15,000 to leave heart washing quantum dot-monoclonal anti nanocrystal composition, 3 times repeatedly.At last, disperse quantum dot-monoclonal anti nanocrystal composition with 100 mL PBST, and place 4 ℃ of preservations;
Wherein, contain 1% sucrose component in the detection line of reaction trapping region and the nature controlling line immobile liquid, " diving phenomenon " in chromatography detects, occur to prevent quantum dot-monoclonal anti nanocrystal composition.
Embodiment 1: the specificity of the immuno-chromatographic test paper strip of checking the present invention preparation as an example of sulfadimidine SMZ example
1. used SMZ monoclonal antibody specific and rabbit anti-mouse igg two are anti-in this experiment is commercialization reagent, available from SIGMA company.
2. the processing of testing sample
The disposal route of pork, chicken and crucian: take by weighing in the equal quality sample of the 2g road vial, add 4ml methyl alcohol, violent mixing 2min, Filter paper filtering.
The disposal route of milk: measure 1ml milk and add the 2mlPBST mixing, then add 60 μ l 1mol/Lde solution of zinc sulfate, mixing 1min adds 60 μ l 0.36mol/L potassium ferrocyanide solutions vibration 1min again, filters and uses.
3. select 15 kinds of sulfa drugss (STZ, SIZ, SME, SMPD, SP, SMT, SCP, SD, SMR, SM, SMX, SDZ, SDMX, SIX and SQ) and other common veterinary drugs except SMZ: chloromycetin, Ractopamine, hydrochloric acid Te Lunteluo carry out specific detection.The result is as shown in table 2, and specificity reaches 100%.
Table 1: test strips specific detection result of the present invention
Embodiment 2: adopt chromatograph test strip of the present invention that the concentration limit that detects of 16 kinds of sulfa drugss is detected, the result shows that quantum dot-labeled chromatograph test strip with having the fluid channel result all has higher sensitivity to these sulfa drugss among employing the present invention, can be widely used in the residual mensuration of multiple medicines thing.
Table 2: the concentration limit that test strips of the present invention detects 16 kinds of sulfa drugss
| The sulfa drugs title | Concentration limit μ g/L |
| STZ | 1.5 |
| SIZ | 2 |
| SME | 3 |
| SMPD | 3 |
| SP | 3 |
| SMT | 5 |
| SCP | 5 |
| SD | 5.5 |
| SMR | 6 |
| SM | 8 |
| SMX | 8 |
| SDZ | 10 |
| SMZ | 20 |
| SDMX | 50 |
| SIX | 60 |
| SQ | 90 |
Embodiment 3: test strips differences between batches CV of the present invention detects
Select the colloidal gold immuno-chromatography test paper strip buied on the market in contrast, analyze the differences between batches with chromatograph test strip of fluid channel of the present invention, analysis result is as shown in table 3, analysis result shows that the chromatograph test strip differences between batches of employing fluid channel significantly reduce, the analysis result that uses it to detect acquisition is more stable, and is reliable.
Table 3: test strips differences between batches CV analysis result
This intellecture property of primary enforcement that all are above-mentioned is not set restriction this new product of other forms of enforcement and/or new method.Those skilled in the art will utilize this important information, and foregoing is revised, to realize similar implementation status.But all modifications or transformation belong to the right of reservation based on new product of the present invention.
The above only is preferred embodiment of the present invention, is not to be the restriction of the present invention being made other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not break away to any simple modification, equivalent variations and remodeling that above embodiment does, still belongs to the protection domain of technical solution of the present invention according to technical spirit of the present invention.
Claims (9)
1. immuno-chromatographic test paper strip that detects sulfa drug residue, it is characterized in that being provided with on the substrate of described test strips sample and apply district, reaction trapping region and remaining liq uptake zone, and described sample applies and is fixed with quantum dot-labeled sulfa drugs monoclonal antibody specific in the district, and the test strip that contains the sulfa drugs polyclonal antibody and the Quality Control band that contains rabbit anti-mouse igg antibody are set in the reaction trapping region.
2. immuno-chromatographic test paper strip as claimed in claim 1 is characterized in that described sample applies district, reaction trapping region and remaining liq uptake zone and is positioned on the fluid channel that the test strips substrate is arranged with downwards.
3. immuno-chromatographic test paper strip as claimed in claim 2 is characterized in that described fluid channel bottom is cast with nitrocellulose solution, forms nitrocellulose layer.
4. immuno-chromatographic test paper strip as claimed in claim 3 is characterized in that the diameter that sample on the described fluid channel applies district, reaction trapping region and remaining liq uptake zone is not more than 2mm.
5. such as the arbitrary described immuno-chromatographic test paper strip of claim 1-4, the material that it is characterized in that described substrate is polycarbonate, and described fluid channel obtains by etch process.
6. such as the arbitrary described immuno-chromatographic test paper strip of claim 1-5, the thickness that it is characterized in that nitrocellulose layer in the described reaction trapping region is not less than sample and applies district and remaining liq uptake zone.
7. such as the arbitrary described immuno-chromatographic test paper strip of claim 1-6, it is characterized in that described quantum dot-labeled thing is CdTe.
8. immuno-chromatographic test paper strip as claimed in claim 7, it is characterized in that wherein quantum dot-labeled antibody obtains as follows: getting 5 mL concentration is the red quantum dot of CdTe of 3 mg/mL, be that the sulfa drugs monoclonal antibody of 1 ng/mL is fully mixed with concentration, then under the condition of magnetic agitation, dropwise add the EDC solution that 0.5 mL concentration is 1 mg/mL, this mixed liquor shading places the reaction of rotation hybrid frame after 3 hours, 4 ℃ of hold over night, after 24 hours, get this mixed liquor 15,000 left the heart 20 minutes, remove supernatant, the phosphate buffer (Phosphate-Buffered Saline(PBS) that contains 0.5% tween-20 (v/v) with 10.5 mL: 0.2 mg/mL KCL, 1.44 mg/mL Na2HPO4,0.24mg/mL KH2PO4,8 mg/mL NaCl, pH 7.4) resuspended centrifugal residue, use again 15,000 leaves heart washing quantum dot-monoclonal anti nanocrystal composition, 3 times repeatedly, at last, disperse quantum dot-monoclonal anti nanocrystal composition with 100 mL PBST, and place 4 ℃ of preservations.
9. such as the arbitrary described immuno-chromatographic test paper strip of claim 1-8, it is characterized in that reacting in the detection line of trapping region and the nature controlling line immobile liquid and contain 1% sucrose component.
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Cited By (5)
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| CN104502584A (en) * | 2014-12-18 | 2015-04-08 | 南京基蛋生物科技有限公司 | Dry-type immunochromatographic analysis method based on metal nanoparticle enhanced fluorescence |
| CN105866442A (en) * | 2016-04-29 | 2016-08-17 | 广州天宝颂原生物科技开发有限公司 | Immunochromatography quantitative detection test paper strip for procalcitonin of terminal blood |
| CN108717054A (en) * | 2018-04-26 | 2018-10-30 | 河南省农业科学院农业质量标准与检测技术研究所 | A kind of quantum dot-labeled antibody probe test strips and its preparation method and application |
| CN108761076A (en) * | 2018-05-24 | 2018-11-06 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PEDV immune detections chromatograph test strip and its preparation method and application in milk |
| CN109061159A (en) * | 2018-05-24 | 2018-12-21 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PEDV immune detection chromatograph test strip and its preparation method and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104502584A (en) * | 2014-12-18 | 2015-04-08 | 南京基蛋生物科技有限公司 | Dry-type immunochromatographic analysis method based on metal nanoparticle enhanced fluorescence |
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Application publication date: 20131023 |