CN105606812A - Colloidal gold immunochromatographic test strip for quinolone drugs - Google Patents
Colloidal gold immunochromatographic test strip for quinolone drugs Download PDFInfo
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- CN105606812A CN105606812A CN201510883769.7A CN201510883769A CN105606812A CN 105606812 A CN105606812 A CN 105606812A CN 201510883769 A CN201510883769 A CN 201510883769A CN 105606812 A CN105606812 A CN 105606812A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention relates to a colloidal gold immunochromatographic test strip for ciprofloxacin prepared by using the technology of labeling an antibody with nanometer colloidal gold particles and the mode of detecting an antigen with competitive immunochromatography. The invention provides a colloidal gold immunochromatographic test strip for detecting 11 quinolone drugs, and a preparation method and application thereof. The test strip comprises a sample pad, a colloidal gold-labeled pad, a cellulose nitrate membrane with detection line and quality control line antibodies, an absorption pad and a base plate and is prepared by superposing and connecting the sample pad, the colloidal gold-labeled pad, the cellulose nitrate membrane and the absorption pad, pasting the above-mentioned elements on the base plate and carrying out assembling. The test strip provided by the invention has the advantages of simple operation, good specificity, high sensitivity, convenience, rapidness, no need for special equipment and professional training, intelligible and easily distinguishable results and easiness in promotion, and is applicable to detection of 11 quinolone drug residues in aquatic products, etc.
Description
Technical field
The invention belongs to field of immunodetection, the colloidal gold immunochromatographimethod that is specifically related to a kind of QNS detects examinationPaper slip.
Background technology
Quinolones (Quinolones, QNs) medicine, claims again pyridonecarboxylic acids or pyridone acids, is newer wide of a classSpectrum antimicrobial. In chemical constitution, QNs belongs to pyridonecarboxylic acid derivative (Pyridonecarboxylicacids, PCAs). QNsAs the antibacterials of the full chemical synthesis of a class, have that antibacterial activity is strong, has a broad antifungal spectrum, absorption are fast, widely distributed etc. excellent in bodyPoint, comparatively cheap compared with the antibiotic of price and therapeutic equivalence, be widely used in the multi-infection disease of humans and animalsIn treatment. Domestic animal industry is very big to the demand of QNs class medicine, and annual consumption is more than 500 tons, and the market demandAmount is still in Fast growth phase. Because environment in recent years worsens, aquiculture animal disease takes place frequently, and causes QNs medicine excessiveUse phenomenon is serious, and in aquatic products, QNs medicament residue exceeds standard and culture fishery caused to great negative effect.
At present, to the residual detection method of QNs in aquatic products comprise microbial method, high performance liquid chromatography, liquid chromatogram-Tandem mass spectrometry, capillary electrophoresis analysis method, immunoassay etc. HPLC and LC-MS detect the routine side of QNs as laboratoryMethod has high accuracy, high precision and high sensitivity, but needs instrument and the specialty of loaded down with trivial details sample treatment step, costlinessOperative skill, be therefore not suitable for for detecting gross sample and promoting the use of.
Colloidal gold immunochromatographimethod technology, because it is quick, convenient, does not need special installation, and result judges the advantages such as directly perceived, moreMore be subject to people's attention. The present invention utilizes monoclonal antibody and colloidal gold-labeled method to set up quinolones colloid gold immuneChromatography method for quick.
Summary of the invention
The object of the invention is to openly a kind of colloidal gold immunochromatographydetection detection test paper bar of QNS, be specially11 kinds of QNSs of a kind of fast detecting (Ciprofloxacin, Enrofloxacin, the husky star of single promise, Ofloxacin, methanesulfonic acid training fluorine sandStar, Danofloxacin mesylate, Abbott 56619, hydrochloric acid sarafloxacin, enoxacin and Sparfloxacin etc.) colloid gold immune layerAnalyse test strips, and the preparation method of this test strips is provided, there is excellent that special, accurate, quick, simple to operate, high flux detectsGesture. Apply this ELISA test strip QNS overall process and be no more than 15min.
The colloidal gold immunochromatographydetection detection test paper bar of a kind of QNS of the present invention, this test strips comprise sample pad,Gold mark pad, nitrocellulose filter, absorption pad, base plate; The material of sample pad is selected from glass fibre, filter paper fibre or polyester film, goldThe golden labeling antibody supporting film of mark pad is selected from glass fibre, filter paper fibre or polyester film, and absorption pad is selected absorbent filter, and base plate is selectedWith the PVC plate of adhesive sticker. On base plate taking sample pad as initiating terminal, successively overlapping stickup be fixed with sample pad, gold mark pad, withThe nitrocellulose filter of detection line and nature controlling line antibody, absorption pad. The antibody of anti-Ciprofloxacin is monoclonal antibody; Nature controlling line is anti-Body can be selected IgG or the staphylococcal protein A of sheep anti mouse, the anti-mouse of rabbit according to the source of golden labeling antibody.
Specifically by be coated with anti-monoclonal antibody against ciprofloxacin colloid gold label thing gold mark pad, be coated with CiprofloxacinBovine serum albumin(BSA) artificial complete antigen (CPFX-BSA) and rabbit anti-mouse igg or staphylococcal protein A or staphylococcal protein GThe compositions such as nitrocellulose filter, sample pad, blotting paper, base plate, on base plate, taking sample pad as initiating terminal, overlapping stickup is fixed successivelyThere are sample pad, gold mark pad, nitrocellulose filter, blotting paper with detection line and nature controlling line antibody.
To achieve these goals, the technical solution used in the present invention is as follows:
The preparation process of this test strips comprises: (1) prepares anti-monoclonal antibody against ciprofloxacin; (2) prepare nano-colloid gold grain;(3) prepare the nano colloid gold label of anti-monoclonal antibody against ciprofloxacin, form golden labeling antibody; (4) by even golden labeling antibodySpray on golden labeling antibody supporting film drying preparation gold mark pad; (5) will resist monoclonal antibody against ciprofloxacin to spray to nitric acid fibreTie up detection line position on plain film as detection line capture antibody, two anti-or staphylococcal protein As of anti-golden labeling antibody spray toNature controlling line position on nitrocellulose filter is as nature controlling line capture antibody, and seals the blank site on nitrocellulose filter;(6) absorption pad, the nitrocellulose filter with detection line and nature controlling line antibody, gold mark pad, sample pad, base plate are assembled into colloidGold immunochromatographydetecting detecting test strip.
Specifically comprise as follows:
(1) preparation of anti-monoclonal antibody against ciprofloxacin: by CPFX-BSA antigen immune Balb/c mouse, prepare splenocyte outstandingLiquid, myeloma cell's suspension; Splenocyte and myeloma cell are merged to screening positive monoclonal cell hole through cell hydridization;Through further positive colony and screening thereof, set up the anti-Ciprofloxacin bacterium positive, cell line 1 strain that specificity is good, called after5G4. Get hybridoma cell strain 5G4 and prepare ascites cultivation pneumoretroperitoneum injection Balb/c mouse, and obtain anti-ring third through purifyingHusky star monoclonal antibody 5G4;
(2) preparation of nano colloid gold: 100mL0.01% gold chloride is prepared to nano-colloid with 1mL1% trisodium citrateGold grain solution;
(3) the colloid gold label thing of anti-monoclonal antibody against ciprofloxacin 5G4 preparation: use 0.1molLK2CO3By above-mentioned colloidThe pH value of gold solution is adjusted to 8.5, and nano colloid gold particle solution and anti-monoclonal antibody against ciprofloxacin 5G4 are pressed to 100mL colloidGold solution adds the ratio of 0.2mg monoclonal antibody to mix, and it is compound to make collaurum and antibody form stable collaurumThing, is passing through purifying, the concentrated rear colloid gold label thing that forms;
(4) will resist monoclonal antibody against ciprofloxacin 5G4 colloid gold label thing to be sprayed on gold mark pad upper, by CPFX-BSA antigen and rabbitAnti-mouse IgG is sprayed on the detection line and nature controlling line position of nitrocellulose filter, dries at 37 DEG C of drying boxes;
(5) nitrocellulose filter, the gold that are coated with detection line and nature controlling line antibody are marked to pad, sample pad, blotting paper etc. sticky successivelyOn base plate;
(6) baseboard material gluing is successively cut into and cuts into wide × long rectangular Du-6859a that is for 0.35cm × 6cmThing colloidal gold immunochromatographydetection detection test paper bar, packages spare.
Described test strips is applied to and detects Ciprofloxacin, Enrofloxacin, the husky star of single promise, Ofloxacin, methanesulfonic acid training fluorine sand11 kinds of quinolones such as star, Danofloxacin mesylate, Abbott 56619, hydrochloric acid sarafloxacin, enoxacin and SparfloxacinMedicine. This detecting step is as follows: (1) gets 80-150 μ L sample to be checked and be added in the sample application zone of test strip; (2) at 20minInterior observation testing result.
Good effect of the present invention is:
(1) low price: production flow process is simple, and production cost is low;
(2) detection speed is fast: in overall process 15min, complete;
(3) can production scene detect, high flux detects;
(4) high-quality: the test strips specificity that this method is prepared from is good, highly sensitive, reproducible;
(5) simple to operate: the test strips that this method is prepared from is using collaurum as cue mark, fast qualitative, result accurately,Fast, easy and simple to handle, only need application of sample to detect and outcome record step;
(6) be easy to promote the use of: operating personnel are without professional training, and by specification gets final product complete operation.
Brief description of the drawings
Fig. 1 is the assembling assumption diagram of a kind of fast detecting QNS of the present invention colloidal gold immuno-chromatography test paper strip,Wherein: the 1st, sample pad; The 2nd, gold mark pad; The 3rd, detection line colour developing position; The 4th, nature controlling line colour developing position; The 5th, nitrocellulose filter;The 6th, blotting paper; The 7th, base plate.
Fig. 2 is the appearance assumption diagram of a kind of fast detecting QNS of the present invention colloidal gold immuno-chromatography test paper strip,Wherein: the 8th, plastic casing; The 9th, result reads window; The 10th, detect sample pipetting volume district.
Detailed description of the invention
The specific embodiment of the present invention is elaborated with reference to the following examples, but is not limited to this.
The preparation of embodiment 1, anti-monoclonal antibody against ciprofloxacin
With CPFX-BSA comlete antigen immunity Balb/c mouse, prepare splenocyte suspension and myeloma cell's suspension; By splenocyteMerge screening positive monoclonal cell hole through cell hydridization with myeloma cell; Set up anti-monoclonal antibody against ciprofloxacin thinBorn of the same parents' strain 5G4, mouse peritoneal injection preparation ascites, and the anti-monoclonal antibody against ciprofloxacin of purifying.
The preparation of embodiment 2, colloid gold particle
Get one of 250mL triangular flask, preparation 1wt% chlorauride 100mL, ebuillition of heated; The 1wt% natrium citricum of getting 1mL addsIn above-mentioned solution, mix rapidly, then keep boiling 15min, naturally cooling rear distilled water returns to 100mL volume; Glue4 DEG C of preservations of reagent bottle packing of silication for body gold solution, for subsequent use.
The preparation of embodiment 3, gold mark pad
Get the test tube of two 50mL, add respectively 10mL25nm collaurum; Add appropriate 0.05moLL-1K2CO3Adjusting pH is 8.5; Under 4 DEG C of conditions, add the anti-monoclonal antibody against ciprofloxacin 5G4 reaction 20min of optimum mark concentration, willColloidal gold solution and anti-monoclonal antibody against ciprofloxacin 5G4 add the ratio of 0.2mg monoclonal antibody in 100mL colloidal gold solutionMix 4 DEG C of 12000rmin-1Centrifugal 30min, supernatant discarded; In every centrifuge tube, add the resuspended liquid of l0mLThe same centrifugal after the precipitation that suspends, abandon supernatant, the pipe end, obtains bolarious loose shape precipitation, and resuspended liquid is 0.2moLL-1PBBuffer solution is (containing lwt%BSA, 0.02wt%PEG, 0.3wt%Tween-20,0.1moLL-1NaCl); Repeated centrifugation l time,Supernatant discarded; 0.05moLL-1PB(containing lwt%BSA, 0.02wt%PEG, 0.3wt%Tween-20,12wt% sucrose,1wt%NaN3、0.1moL·L-1NaCl) resuspended precipitation, 4 DEG C of refrigerator storage are for subsequent use.
Good immune colloid gold solution is evenly sprayed on glass fibre element film to get above-mentioned mark, spends the night dry in 37 DEG C of incubatorsDry, pack that to be placed in 4 DEG C of refrigerators for subsequent use.
Detection line on embodiment 5, nitrocellulose filter, spraying and the sealing of nature controlling line antibody
After nitrocellulose filter is soaked and wafts and wash with 1% methanol solution, softly wash rapidly dry, dry; By CPFX-BSA antigen（2mg·mL-1), rabbit anti-mouse igg antibody (lmgmL-1) add respectively the detection of bio-dot collaurum spot injection systemIn the storage bottle of line, nature controlling line, adjust the position of drawing film, spray film line; The cellulose nitrate of detection line, nature controlling line will be pulledElement film is placed in 37 DEG C of incubators dries 2h; Taking-up nitrocellulose filter is placed in after neutral protein immersion soaks and gets immediatelyGo out, be placed in 37 DEG C of incubators and seal after 4h, it is for subsequent use that sealing is kept at 4 DEG C of refrigerators.
Result of the test has been prepared and has been had immunocompetent detection line and nature controlling line, and blank site has been sealed with confining liquidImmune nitrocellulose filter.
The assembling of embodiment 6, test strips
Blotting paper, gold mark pad, sample pad are cut into respectively to strip 2.5cm, 0.5cm, 1.5cm; By immune celluloidFilm is along the long axis direction of base plate, apart from adhesive sticker base plate upper edge 2.2cm, 1.8cm position stickup below; By blotting paper and immune nitreThe overlapping 0.3cm of acid cellulose film place sticks on the top of nitrocellulose filter; By overlapping to gold mark pad and immune nitrocellulose filter0.1cm place sticks on the below of immune nitrocellulose filter; Sample pad and the overlapping 0.1cm of gold mark pad stick on gold mark pad underSide; The base plate being fixed with successively from top to bottom blotting paper, immune nitrocellulose filter, gold mark pad, sample pad is cut into wide× long rectangular for 0.35cm × 6cm, test-strips, pack in aluminium foil bag sealed type storage into together with drier.
Through assemble, cut out, be dried, the step such as sealing, can be assembled into QNS collaurum fast detecting and tryPaper slip.
Embodiment 7, detection method
The processing of sample to be checked: detected sample (animal tissue) is added to the carrene of equivalent, vortex vibration 5min, 4The centrifugal 10min of 000r/min, discards upper strata liquid and grease, extracts lower floor's liquid, adds equivalent dichloro in lower floor's liquid againMethane repeats degreasing once. Finally carefully suck upper strata liquid and grease, subnatant is final sample solution, blows to nitrogen instrumentAfter dry, redissolve; QNS colloidal gold fast detecting test paper strip is recovered to room temperature; Extract 50 μ L and detect sample, slowly addIn sample pad in test strip; The colour developing situation of observing test strip after 15min, records result.
Testing result: the negative result if nature controlling line colour developing and detection line all develop the color, detection sample do not contain Ciprofloxacin,Enrofloxacin, the husky star of single promise, Ofloxacin, pefloxacin mesilate, Danofloxacin mesylate, Abbott 56619, hydrochloric acid sandDraw 11 kinds of QNSs such as Sha Xing, enoxacin and Sparfloxacin. If nature controlling line colour developing, the detection line positive knot that do not develop the colorReally, detect sample and contain QNS.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change withModify, all should belong to covering scope of the present invention.
Claims (7)
1. a QNS colloidal gold immunochromatographydetection detection test paper bar, is characterized in that: this test strips comprise sample pad,Gold mark pad, nitrocellulose filter, absorption pad, base plate; The material of sample pad is selected from glass fibre, filter paper fibre or polyester film, goldThe golden labeling antibody supporting film of mark pad is selected from glass fibre, filter paper fibre or polyester film, and absorption pad is selected absorbent filter, and base plate is selectedWith the PVC plate of adhesive sticker; On base plate taking sample pad as initiating terminal, successively overlapping stickup be fixed with sample pad, gold mark pad, withThe nitrocellulose filter of detection line and nature controlling line antibody, absorption pad.
2. QNS colloidal gold immunochromatographydetection detection test paper bar according to claim 1, is characterized in that: anti-The antibody of Ciprofloxacin is monoclonal antibody; Nature controlling line antibody can be selected goat-anti rabbit, mouse-anti rabbit, rabbit according to the source of golden labeling antibodyIgG or the staphylococcal protein A of anti-sheep, sheep anti mouse, the anti-mouse of rabbit.
3. QNS colloidal gold immunochromatographydetection detection test paper bar according to claim 1, is characterized in that: instituteThe test strips of stating is by the gold mark pad that has been coated with anti-monoclonal antibody against ciprofloxacin colloid gold label thing, has been coated with Ciprofloxacin oxThe nitrocellulose filter of haemocyanin artificial antigen and rabbit anti-mouse igg or staphylococcal protein A or staphylococcal protein G, samplePad, blotting paper, base plate composition.
4. a preparation method for QNS colloidal gold immunochromatographydetection detection test paper bar, is characterized in that: this test stripsPreparation process comprise: (1) prepares anti-monoclonal antibody against ciprofloxacin or polyclonal antibody; (2) prepare nano-colloid gold grain;(3) prepare the nano colloid gold label of anti-monoclonal antibody against ciprofloxacin, form golden labeling antibody; (4) by even golden labeling antibodySpray on golden labeling antibody supporting film drying preparation gold mark pad; (5) will resist monoclonal antibody against ciprofloxacin to spray to nitric acid fibreTie up detection line position on plain film as detection line capture antibody, two anti-or staphylococcal protein As of anti-golden labeling antibody spray toNature controlling line position on nitrocellulose filter is as nature controlling line capture antibody, and seals the blank site on nitrocellulose filter;(6) absorption pad, the nitrocellulose filter with detection line and nature controlling line antibody, gold mark pad, sample pad, base plate are assembled into colloidGold immunochromatographydetecting detecting test strip.
5. the preparation method of a kind of QNS colloidal gold immunochromatographydetection detection test paper bar according to claim 4,It is characterized in that: concrete grammar comprises as follows:
(1) preparation of anti-monoclonal antibody against ciprofloxacin: by CPFX-BSA antigen immune Balb/c mouse, prepare splenocyte outstandingLiquid, myeloma cell's suspension; Splenocyte and myeloma cell are merged to screening positive monoclonal cell hole through cell hydridization;Through further positive colony and screening thereof, set up the anti-Ciprofloxacin bacterium positive, cell line 1 strain that specificity is good, called after5G4, gets hybridoma cell strain 5G4 and prepares ascites cultivation pneumoretroperitoneum injection Balb/c mouse, and obtain anti-ring third through purifyingHusky star monoclonal antibody 5G4;
(2) preparation of nano-colloid gold grain: by 1mL1wt% trisodium citrate preparation for 100mL0.01wt% gold chlorideNano colloid gold particle solution;
(3) the colloid gold label thing of anti-monoclonal antibody against ciprofloxacin 5G4 preparation: use 0.1molLK2CO3By above-mentioned collaurumThe pH value of particle solution is adjusted to 8.5, and nano colloid gold particle solution and anti-monoclonal antibody against ciprofloxacin 5G4 are pressed to 100mL glueBody gold grain solution adds the ratio of 0.2mg monoclonal antibody to mix, and makes collaurum and antibody form stable colloidAu composite, is passing through purifying, the concentrated rear colloid gold label thing that forms;
(4) the anti-monoclonal antibody against ciprofloxacin 5G4 of step (3) colloid gold label thing is sprayed on to gold mark pad above, by CPFX-BSAAntigen and rabbit anti-mouse igg are sprayed on respectively on the detection line and nature controlling line position of nitrocellulose filter, dry at 37 DEG C of drying boxes;
(5) nitrocellulose filter, gold mark pad, sample pad, the blotting paper that are coated with detection line and nature controlling line antibody are bonded at successivelyOn base plate;
(6) baseboard material gluing is successively cut into and cuts into wide × long rectangular Du-6859a that is for 0.35cm × 6cmThing colloidal gold immunochromatographydetection detection test paper bar, packages spare.
6. an application for QNS colloidal gold immunochromatographydetection detection test paper bar as claimed in claim 1, its featureBe: this test strips is applied to and detects Ciprofloxacin, Enrofloxacin, the husky star of single promise, Ofloxacin, pefloxacin mesilate, firstSulfonic acid Danofloxacin, Abbott 56619, hydrochloric acid sarafloxacin, enoxacin and 11 kinds of QNSs of Sparfloxacin.
7. application according to claim 6, is characterized in that: this detecting step is as follows:
(1) get 80-150 μ L sample to be checked and be added in the sample application zone of test strip;
(2) in 20min, observe testing result.
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| CN201510883769.7A CN105606812A (en) | 2015-12-07 | 2015-12-07 | Colloidal gold immunochromatographic test strip for quinolone drugs |
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| CN201510883769.7A CN105606812A (en) | 2015-12-07 | 2015-12-07 | Colloidal gold immunochromatographic test strip for quinolone drugs |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108931638A (en) * | 2017-05-23 | 2018-12-04 | 北京健平金星生物科技有限公司 | A kind of colloid gold chromatographic test paper strip and preparation method thereof |
| CN109142736A (en) * | 2018-11-15 | 2019-01-04 | 广州智汇生物科技有限公司 | A kind of test strips, preparation method and applications detecting quinolone drugs |
| CN110632296A (en) * | 2019-09-29 | 2019-12-31 | 江西省农业科学院农产品质量安全与标准研究所 | Quick detection method for ofloxacin |
| CN111308067A (en) * | 2020-02-03 | 2020-06-19 | 南京农业大学 | Quantum dot microsphere immunochromatography test strip for quantitatively detecting fluoroquinolone drugs |
| CN115420893A (en) * | 2022-07-11 | 2022-12-02 | 昂科生物医学技术(苏州)有限公司 | PSEP colloidal gold test paper, kit, preparation method and application thereof |
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| CN110632296B (en) * | 2019-09-29 | 2023-06-02 | 江西省农业科学院农产品质量安全与标准研究所 | Rapid ofloxacin detection method |
| CN111308067A (en) * | 2020-02-03 | 2020-06-19 | 南京农业大学 | Quantum dot microsphere immunochromatography test strip for quantitatively detecting fluoroquinolone drugs |
| CN115420893A (en) * | 2022-07-11 | 2022-12-02 | 昂科生物医学技术(苏州)有限公司 | PSEP colloidal gold test paper, kit, preparation method and application thereof |
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Application publication date: 20160525 |