CN102023210A - Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof - Google Patents
Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof Download PDFInfo
- Publication number
- CN102023210A CN102023210A CN2009100920652A CN200910092065A CN102023210A CN 102023210 A CN102023210 A CN 102023210A CN 2009100920652 A CN2009100920652 A CN 2009100920652A CN 200910092065 A CN200910092065 A CN 200910092065A CN 102023210 A CN102023210 A CN 102023210A
- Authority
- CN
- China
- Prior art keywords
- enrofloxacin
- fluorescent
- detection
- card
- quality control
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and a preparation method thereof. The detection card sequentially comprises filter paper, a sample pad, a glass fiber film, a nitrocellulose film and absorbent paper, wherein a fluorescent microsphere-labeled antibody is sprayed on the glass fiber film; a detection area and a quality control area are fixed on the nitrocellulose film; a conjugate of enrofloxacin and carrier protein is sprayed in the detection area; and an anti-mouse antibody is sprayed in the quality control area. By taking core-shell bistructure luminous nano particles complexed by silica and fluorescent substances as labels and adopting immunochromatography in a competition blocking mode, the invention realizes rapid immunoassay of the enrofloxacin. In the detection process, an optimized excitation light source of the fluorescent microsphere is adopted for excitation, the emitted fluorescence passes through a light filter device, and that whether a detection line is provided with fluorescent substances is observed by naked eyes. The invention has the characteristics that: the detection card has high sensitivity and is rapid in detection, convenient to operate, and economic and practical.
Description
Technical field
The invention belongs to the food safety detection field, specifically relate to a kind of test card of utilizing fluorescent micro-ball immune chromatography technology qualitative detection Enrofloxacin and preparation method thereof.
Background technology
(Enrofloxacin ENR) has another name called the ethyl Ciprofloxacin to Enrofloxacin, is the fluoroquinolones of first livestock and poultry special use, and nineteen eighty-three is is at first researched and developed success by Bayer A.G, and China put on market in 1994.Because of it can suppress the DNA of bacteria helicase, has a broad antifungal spectrum, efficient, low toxicity, tissue penetration are strong, become the animal doctor face examine with aquaculture in one of most important anti-infectives, be used for the treatment of in a large number, prevent disease and promotion growth.This medicine is for oral administration and the intramuscular injection absorption is rapid, and metabolism is for having the Ciprofloxacin of antibacterial activity equally in vivo, and metabolin generates and eliminates all slow, widely distributed.But human edible by behind the animal foodstuff of this drug contamination, can cause normal flora generation drug resistance in the body, cause human body poisoning or allergic reaction, influence the synthetic of nucleic acid in vivo, have potential carcinogenicity.The U.S., European Union and China have all carried out strict control to the use of Enrofloxacin.The U.S. bans use of Enrofloxacin in livestock and poultry cultivation; Stipulate clearly in European Union's rules that the Enrofloxacin examination criteria is: the flesh of fish and fats portion are 0.1mg/kg, and liver, kidney partly are 0.2mg/kg.
At present, detection and the method for supervising at Enrofloxacin comprises conclusive evidence method and examination method two big classes.The conclusive evidence method comprises that mainly high performance liquid chromatography, gas chromatography, adverse current chromatogram, chromatography of ions, ion trap mass spectrometry, plasma mass spectrum, liquid matter or gas chromatography mass spectrometry etc. utilize the method for high-end devices for supplementary means.Though above method is responsive, special, stable, exist detection time long, it is not high that general condition detects flux down, need expensive device support simultaneously, so the conclusive evidence method is used to the arbitration of the incident of having a question mostly; Screening method generally is meant method for quick.ELISA (enzyme linked immunosorbent assay (ELISA)) and colloidal gold immunity chromatography are present internationally recognized mainstream technologys, and it is fast that these two kinds of methods have detection speed, low price, and simple operation and other advantages is the main method for supervising that detects both at home and abroad at present.Still need the professional but ELISA detects, and need long time showing result; Colloidal gold method can carry out qualitative analysis to Enrofloxacin generally speaking, relatively lacks detection time (10-15min).The quick reagent of present domestic use no matter adopt which kind of principle, because its sensitivity restriction can not be carried out many times of dilutions with sample, to such an extent as to the interference component in the sample is many, causes can producing false negative or false positive phenomenon in the actual sample testing process.
Summary of the invention
An object of the present invention is at above-mentioned the deficiencies in the prior art part, a kind of highly sensitive, easy and simple to handle, fluorescent micro-ball immune chromatography test card of detecting quick, cheap detection Enrofloxacin is provided; Another object of the present invention provides the preparation method of above-mentioned test card.
A technical scheme taking of the present invention is to achieve these goals:
A kind of fluorescent micro-ball immune chromatography test card that detects Enrofloxacin is provided, and this test card is the single deck tape-recorder type, is made of an immuno-chromatographic test paper strip, end card and face card.Immuno-chromatographic test paper strip is pasted filter paper, sample pad, glass fibre membrane, NC film (nitrocellulose filter) and thieving paper successively overlap joint and is made on base plate, be coated with fluorescent microsphere on the described glass fibre membrane, be fixed with detection zone and Quality Control district on the described NC film; The fluorescent microsphere that sprays on the described glass fibre membrane is the anti-Enrofloxacin antibody of fluorescent microsphere mark, and detection zone is coated with the coupled antigen of carrier protein and Enrofloxacin on the described nitrocellulose filter, and described Quality Control district all is fixed with anti-mouse antibody.
Described fluorescent microsphere is that diameter is the microballoon with silicon dioxide parcel fluorescent material of 0.01～1 μ m, and its surface is connected with reactive group, or with the Streptavidin coupling; Described fluorescent material comprises the fluorescent material of organic or inorganic or the admixture and the quantum dot of multiple fluorescent material.
Described reactive group is-CHO ,-COOH ,-OH ,-NH
2Or-SH; Described fluorescent material is a phenanthroline connection ruthenium organic dyestuff, different thiocyanate fluorescein, different thiocyanate rhodamine, 6-Fluoresceincarboxylic acid carboxylic acid amide esters, 3,6-diethoxy fluorane, 3-diethylamino-7-(2 '-chloroanilino) fluorane, Luo Danming B, Luo Danming 110, Luo Danming 123 and Luo Dan bright 10,1, the fluorescent material that 8-benzene-naphthalene diimide 4-position alkoxy or aryloxy group replace, 1, introduce sulfonic group in the 8-benzene-naphthalene diimide, the fluorescent dye of carboxyl and quaternary ammonium salt water soluble group, 1,8-naphthalene acid anhydride and the resulting Pi piceneketone of o-phenylenediamine condensation class fluorescent material, trifluoro formaldehyde cumarin, two fluorophore cumarins, 1,2,3, the admixture and the quantum dot of 3-tetramethyl indolyl radical cumarin organic fluorescent dye or multiple fluorescent material.
Another technical scheme that the present invention takes provides a kind of preparation method of fluorescent micro-ball immune chromatography test card of above-mentioned detection Enrofloxacin, specifically may further comprise the steps:
1. the preparation of immuno-chromatographic test paper strip
(1) preparation of fluorescent microsphere pad
Use the fluorescent microsphere labelled antibody, and it is sprayed on the glass fibre membrane.
Get commercially available fluorescent microsphere at the centrifugal 10～15min of 1000 * g, collecting precipitation, regulating microballoon concentration with the borate buffer solution of 0.01M pH 4.8 is OD
450=0.2, add 20～100mg/ml then respectively to ethyl-N, N-dimethyl propyl carbodiimide (EDC) 100 μ l, 2～20mg/ml nitrogen N-Hydroxysuccinimide (NHS), 100 μ l add the borate buffer solution of 0.01M pH4.8 again, the vibration mixing, behind incubated at room 10～30min, centrifugal 5～the 15min of 1000 * g, precipitation is resuspended with the borate buffer solution of 0.01M pH 7～8, and adjusting microballoon concentration is OD
450Be 0.2～1.0, in this fluorescent microsphere of 0.1ml, add the anti-Enrofloxacin antibody of 1～10 μ g, fully behind the mixing, stirring at room reaction 1～4h, ultrapure water centrifuge washing 2～5 times, precipitation 0.01M pH 7.2PBS (phosphate buffer, wherein comprise 5% sucrose and 0.05%Tween-20) redissolve be precipitated to initial volume after, be sprayed on the glass fibre membrane with the BIODOT operating platform, 25 ℃ of vacuum drying 1～2h, it is standby to be put in dry environment.
(2) preparation in detection zone and Quality Control district
With the coupled antigen of carrier protein and Enrofloxacin, anti-mouse antibody is sprayed onto on the nitrocellulose filter respectively, makes detection zone and Quality Control district; Described carrier protein is bovine serum albumin(BSA) or oralbumin.
With 0.01M pH 7.4PBS (phosphate buffer, wherein comprising 5% sucrose and 0.05% Tween-20) concentration of regulating coupled antigen and anti-mouse antibody respectively is 0.5～8.0mg/ml, spray film amount is 0.7 μ L/cm, detection zone spraying coupled antigen, Quality Control district spraying anti-mouse antibody, the two districts 5mm of being separated by, the Quality Control offset is from NC film one end 2mm, after 37 ℃ of oven dry are spent the night, under the drying at room temperature environment, preserve standby.
(3) assembling and shearing
Overlap joint ground is pasted successively on adhesive base: filter paper, sample pad, glass fibre membrane, nitrocellulose filter and thieving paper, and cut into proper width and promptly become immuno-chromatographic test paper strip.
2. the assembling of immuno-chromatographic test paper strip
To be fixed on the end card according to the Enrofloxacin test strips of method for preparing, compress with the face card on the test strips surface then, promptly become immunochromatographydetection detection card.End card and face card generally all are plastic clip, and end card can make sample pad, glass fibre membrane, nitrocellulose filter and the thieving paper on the test strips combine closely, and the face card can be protected test strips, and it is not damaged.Reserve each two of well and view windows on the face card, the position of well is corresponding with the sample pad of test strips, and the position of view window is corresponding with the nitrocellulose filter of test strips.
Fluorescent micro-ball immune chromatography test card qualitative detection Enrofloxacin with above-mentioned may further comprise the steps:
(1) adds sample to be checked in the well of immunochromatographydetection detection card, behind reaction 3～20min, test card is put into detection window;
(2) fluorescent microsphere that is trapped in detection zone and Quality Control district sends strong fluorescence band under best exciter lamp source;
(3) emitted fluorescence detects by an unaided eye whether fluorescence signal is arranged behind the filtering veiling glare; When not containing detected material in the sample, a fluorescence band all appears in detection zone and Quality Control district, and it is negative promptly to detect sample; When containing excessive detected material in the sample, detection zone does not have the fluorescence band, and there is a fluorescence band in the Quality Control district, and it is promptly positive to detect sample.
When containing inexcessive detected material matter in the sample, detection zone has a hypofluorescence band, and there is a fluorescence band in the Quality Control district, detects sample and is the weak positive.
Advantage of the present invention is as follows:
1. after fluorescent material is excited by incident light, launch fluorescence when returning ground state, can detect in the direction vertical with excitation source, therefore fluorescence is not subjected to the interference from the exciting light background, sensitivity improves greatly, and its sensitivity is 10～1000 times with conventional dyes and coloured mark substance detection method.Advantages such as in addition, the fluorescence labeling detection method also has easy and simple to handle, detects fast, and is cheap;
2. fluorescent microsphere is the nucleocapsid double structure, and the dyestuff that has overcome conventional fluorescent microsphere is revealed, and shortcomings such as anti-solution interference performance difference have increased the stability and the fluorescence lifetime of fluorescent microsphere;
3. fluorescent microsphere surface modified active group adopts the chemical coupling method to come labelled antibody or antigen, forms the stable bond of antibody or antigen and microballoon;
4. can realize qualitative detection by easy optical filter device to Enrofloxacin.
Test the chicken sample of 100 routine known injection Enrofloxacins with the fluorescent micro-ball immune chromatography test card of method preparation of the present invention, accuracy rate all reaches 100%, shows that fluorescent micro-ball immune chromatography method result provided by the invention is accurate, detects fast, easy and simple to handle, highly sensitive.The sample that the present invention detects comprises meat, liver and the kidney etc. of aquatic products and fowl poultry kind.
Description of drawings
Fig. 1 is the structural representation of immuno-chromatographic test paper strip;
Fig. 2 is the structural representation of immunochromatographydetection detection card;
Fig. 3 is that the fluorescent micro-ball immune chromatography test card detects schematic diagram.
As shown in Figure 1, constituting of this immuno-chromatographic test paper strip: on adhesive base 18, successively overlap joint ground paste filter paper 11, sample pad 12, be coated with the anti-Enrofloxacin antibody of fluorescent microsphere mark glass fibre membrane 13, be coated with the detection zone 15 of Enrofloxacin coupled antigen and be coated with nitrocellulose filter 14 and the blotting paper 17 in the Quality Control district 16 of anti-mouse antibody, detection zone 15 and Quality Control district 16 5mm of being separated by, the Quality Control offset is from NC film one end 2mm.
As shown in Figure 2, this immunochromatographydetection detection card is the single deck tape-recorder type, be fixed on the end card 21 and form by detecting the Enrofloxacin immuno-chromatographic test paper strip, concrete structure comprises end card 21, face card 22, well 23, observation window 24, NC film 25, Quality Control district 26, detection zone 27.
Shown in Fig. 1,2 and 3, the detection principle is as follows: add sample to be checked in the well 23 of test card, the anti-Enrofloxacin antibody of the fluorescent microsphere mark of spraying on the sample dissolving glass fibre membrane 13, by capillarity forward swimming on glass fibre membrane 13, the Enrofloxacin in the sample reacts with corresponding fluorescent microsphere label simultaneously; During reactant liquor process detection zone 15, with the spray reaction of detection zone 15, and richness is amassed at detection zone 15; Test card is put into detection window 31, sample 33 to be checked excites lower at light source 32, the fluorescence 34 of emission is observed the fluorescence band that whether has by watch window 36 at detection zone 15 after filtering by monochromatic filter 35, and the Quality Control district is no matter sample is that feminine gender or the positive all the fluorescence band can occur. If detection zone 15 does not have the fluorescence band, sample is positive, if detection zone 15 has the fluorescence band, sample is negative.
As shown in Figure 3, the detecting step of fluorescent micro-ball immune chromatography qualitative detection Enrofloxacin is as follows:
(1) in the well of test card, drips sample to be checked, behind reaction 3～20min, put it into detection window 31;
(2) fluorescent microsphere 33 that is trapped of detection zone and Quality Control district excites down at best excitation source 32, sends fluorescence 33 by monochromatic filter 34, at the yin and yang attribute of watch window 35 judgement samples. Described best excitation source refers to that under the 400-460nm excitation light source excites, fluorescent microsphere can send the strongest fluorescence.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but not as a limitation of the invention.
Embodiment 1
1, the preparation of fluorescent microsphere label (EDC method):
The preparation of the anti-Enrofloxacin antibody of fluorescent microsphere mark: get fluorescent microsphere that 1mg wraps up different thiocyanate rhodamine organic dyestuff at the centrifugal 10min of 1000 * g, collecting precipitation, regulating microballoon concentration with the borate buffer solution of 0.01M pH4.8 is OD
450=0.2, add 90 μ l 50mg/ml EDC then, 150 μ l 5mg/ml NHS, the vibration mixing, behind the incubated at room 20min, the centrifugal 5min of 1000 * g, precipitation is dissolved with the borate buffer solution of 0.01M pH4.8, and adjusting microballoon concentration is OD
450Be 0.5.At 0.1ml OD
450Be to add the anti-Enrofloxacin antibody of 1 μ g in this fluorescent microsphere of 0.5, fully behind the mixing, stirring at room reaction 3h, after washing centrifugal 3 times with ultrapure water, after being precipitated to initial volume with the PBS (wherein comprising 5% sucrose and 0.05%Tween-20) of 0.01M pH 7.2 is resuspended, be the anti-Enrofloxacin antibody of the fluorescent microsphere mark for preparing.
2, the preparation of fluorescent microsphere pad:
With BIODOT specking instrument, the anti-Enrofloxacin antibody of fluorescent microsphere mark is sprayed on the glass fibre membrane, spray film amount is 4 μ l/cm, and the glass fibre membrane size is 30 * 0.8cm, and 25 ℃ of vacuum drying 1～2h place dry environment standby.
3, the preparation of NC film:
The coupled antigen and the anti-mouse antibody of bovine serum albumin(BSA) and Enrofloxacin are sprayed on the nitrocellulose filter: with 0.01M pH7.4PBS (phosphate buffer, wherein comprise 5% sucrose and 0.05% Tween-20) be 1mg/ml with the concentration adjustment of the coupled antigen of bovine serum albumin(BSA) and Enrofloxacin, it is sprayed on forms detection zone on the nitrocellulose filter; The concentration of regulating anti-mouse antibody with 0.01MpH 7.2PBS (phosphate buffer wherein comprises 5% sucrose and 0.05% Tween-20) is 1mg/ml, it is sprayed on forms the Quality Control district on the nitrocellulose filter.The spray film amount in two districts is 0.7 μ L/cm, and the two districts 5mm of being separated by, Quality Control offset after 37 ℃ away from NC film one end 2mm oven dry are spent the night, down preserve standby in the drying at room temperature environment be;
4, the preparation of fluorescent micro-ball immune chromatography test card:
Assembling test strips A: overlap joint ground is pasted successively on plastic bottom board: (1) filter paper and sample pad, sample pad are a kind of glass fibre membrane through 1-5% Tween-20 immersion treatment; (2) be coated with the glass fibre membrane of the anti-Enrofloxacin antibody of fluorescent microsphere mark; (3) coupled antigen that is coated with bovine serum albumin(BSA) and Enrofloxacin is as detection zone and the anti-mouse antibody nitrocellulose filter as the Quality Control district; (4) thieving paper is assembled the width that cuts into 4mm after finishing, and promptly becomes immuno-chromatographic test paper strip.
Immuno-chromatographic test paper strip is fixed on the plastic bottom card, the test strips surface compresses with the face card, face is stuck in the sample pad of corresponding every test strips and the position of nitrocellulose filter is reserved well and view window respectively, and the immunochromatographydetection detection card of making can be used to detect Enrofloxacin.Pack into after this test card assembles in the aluminium foil bag, seal preservation behind the adding drying agent, under the drying at room temperature environment, can preserve at least 1 year.
5, the detection step of fluorescent micro-ball immune chromatography qualitative detection Enrofloxacin is as follows:
(1) keep flat test card, chicken extract sample balance to be measured drips sample 80 μ l in the well of test card to room temperature, behind reaction 10min under the room temperature, test card is put into detection window;
(2) fluorescent microsphere that is trapped of detection zone and Quality Control district sends fluorescence by monochromatic filter under best excitation light source excites, observes at watch window, if the test strips detection zone does not have the fluorescence band, there is a fluorescence band in the Quality Control district, and judgement sample is positive.If the test strips detection zone has the fluorescence band, there is a fluorescence band in the Quality Control district, and judgement sample is negative.
Embodiment 2
The preparation method of present embodiment is substantially the same manner as Example 1, and difference is:
Carrier protein is selected oralbumin for use, and the labeled vector fluorescent microsphere is quantum dot-silica core-shell double structure microballoon.
May further comprise the steps with above-mentioned fluorescent micro-ball immune chromatography test card qualitative detection Enrofloxacin:
(1) keep flat test card, chicken liver extract sample balance to be measured adds sample 90 μ l in the well of test card to room temperature, react 15min under room temperature, and test card is put into detection window;
(2) fluorescent microsphere that is trapped in detection zone and Quality Control district sends strong fluorescence under the exciting of best excitation source; Observe at watch window, if the test strips detection zone has the fluorescence band, there is a fluorescence band in the Quality Control district, and judgement sample is negative; If the test strips detection zone does not have the fluorescence band, there is a fluorescence band in the Quality Control district, and judgement sample is positive.
Embodiment 3
The preparation method of present embodiment is substantially the same manner as Example 1, and difference is: 1, the labeled vector fluorescent microsphere is different thiocyanate fluorescein-silica core double structure microballoon, and its finishing is Streptavidin.2, anti-Enrofloxacin antibody can adopt EDC method and biotin coupling, and the EDC method is substantially the same manner as Example 1.
Above-described embodiment, the present invention embodiment a kind of more preferably just, the common variation that those skilled in the art carries out in the technical solution of the present invention scope and replace and all should be included in protection scope of the present invention.
Claims (8)
1. a fluorescent micro-ball immune chromatography test card that detects Enrofloxacin is characterized in that: the test strips that is used to detect Enrofloxacin.Described test strips comprises: filter paper, sample pad, glass fibre membrane, nitrocellulose filter and the thieving paper pasted successively on base plate overlap joint; Be coated with anti-Enrofloxacin antibody and fluorescent microsphere conjugate on the described glass fibre membrane; Be fixed with detection zone and Quality Control district on the described nitrocellulose filter, the detection zone on its nitrocellulose filter is coated with the coupled antigen of carrier protein and Enrofloxacin; The Quality Control district of described test strips is fixed with anti-mouse antibody.
2. the fluorescent micro-ball immune chromatography test card of detection Enrofloxacin according to claim 1, it is characterized in that: described fluorescent microsphere is that diameter is the microballoon with silicon dioxide parcel fluorescent material of 0.01～1 μ m, its surface is connected with reactive group, or with the Streptavidin coupling; Described fluorescent material comprises the fluorescent material of organic or inorganic or the admixture and the quantum dot of multiple fluorescent material.
3. the Enrofloxacin fluorescent micro-ball immune chromatography test card of detection according to claim 2 is characterized in that: described reactive group for-COOH ,-NH
2,-CHO ,-OH or-SH; Described fluorescent material is a phenanthroline connection ruthenium organic dyestuff, different thiocyanate fluorescein, different thiocyanate rhodamine, 6-Fluoresceincarboxylic acid carboxylic acid amide esters, 3,6-diethoxy fluorane, 3-diethylamino-7-(2 '-chloroanilino) fluorane, Luo Danming B, Luo Danming 110, Luo Danming 123 and Luo Dan bright 10,1, the fluorescent material that 8-benzene-naphthalene diimide 4-position alkoxy or aryloxy group replace, 1, introduce sulfonic group in the 8-benzene-naphthalene diimide, the fluorescent dye of carboxyl and quaternary ammonium salt water soluble group, 1,8-naphthalene acid anhydride and the resulting Pi piceneketone of o-phenylenediamine condensation class fluorescent material, trifluoro formaldehyde cumarin, two fluorophore cumarins, 1,2,3, the admixture and the quantum dot of 3-tetramethyl indolyl radical cumarin organic fluorescent dye or multiple fluorescent material.
4. the preparation method of detection Enrofloxacin fluorescent micro-ball immune chromatography test card according to claim 1 is characterized in that the immuno-chromatographic test paper strip preparation process comprises:
(1) preparation of fluorescent microsphere pad:, and it is sprayed on obtains the fluorescent microsphere pad on the glass fibre membrane with the anti-Enrofloxacin antibody of commercially available fluorescent microsphere mark;
(2) preparation in detection zone and Quality Control district: the coupled antigen of carrier protein and Enrofloxacin is sprayed to detection zone scope on the nitrocellulose filter, make detection zone; Anti-mouse antibody is sprayed to Quality Control district scope on the nitrocellulose filter, make the Quality Control district;
(3) assembling and shear: on adhesive base successively overlap joint ground paste filter paper, sample pad, be coated with the fluorescent microsphere labelled antibody glass fibre membrane, be fixed with the nitrocellulose filter and the thieving paper in detection zone and Quality Control district, and cut into required width and promptly become immuno-chromatographic test paper strip;
Make detection Enrofloxacin test strips according to above-mentioned steps.
5. the preparation method of the fluorescent micro-ball immune chromatography test card of detection Enrofloxacin according to claim 4 is characterized in that, the preparation method of described fluorescent microsphere labelled antibody is as follows:
Get fluorescent microsphere at the centrifugal 10～15min of 1000 * g, centrifugal back collecting precipitation, regulating microballoon concentration with the borate buffer solution of 0.01M pH 4.8 is OD
450=0.2, add 20～100mg/ml then respectively to ethyl-N, N-dimethyl propyl carbodiimide 100 μ l, 2～20mg/ml nitrogen N-Hydroxysuccinimide, 100 μ l add the borate buffer solution of 0.01M pH 4.8 again, the vibration mixing, behind incubated at room 10～30min, centrifugal 5～the 15min of 1000 * g, precipitation is resuspended with the borate buffer solution of 0.01M pH 7～8, and adjusting microballoon concentration is OD
4500.2～1.0, in this fluorescent microsphere of 0.1ml, add the anti-Enrofloxacin antibody of 1～10 μ g, fully behind the mixing, stirring at room reaction 1～4h, ultrapure water centrifuge washing 2～5 times, with 0.01M pH 7.2 phosphate buffers redissolve be precipitated to initial volume after, be sprayed on the glass fibre membrane with the BIODOT operating platform, 25 ℃ of vacuum drying 1～2h place dry environment standby.
6. the preparation method of the fluorescent micro-ball immune chromatography test card of detection Enrofloxacin according to claim 4 is characterized in that, the preparation method in detection zone and Quality Control district is as follows on the described nitrocellulose filter:
Regulate the coupled antigen of carrier protein and Enrofloxacin respectively and the concentration of anti-mouse antibody is 0.5～8.0mg/ml with 0.01M pH 7.4 phosphate buffers, spray film amount is 0.7 μ l/cm, the coupled antigen of detection zone spraying carrier protein and Enrofloxacin, Quality Control district spraying anti-mouse antibody, the two districts 5mm of being separated by, the Quality Control offset after 37 ℃ away from nitrocellulose filter one end 2mm oven dry are spent the night, is preserved standby be under the drying at room temperature environment.
7. the preparation method of the fluorescent micro-ball immune chromatography test card of detection Enrofloxacin according to claim 4 is characterized in that, the preparation method of immunochromatographydetection detection card is as follows:
Test strips is fixed on the end card, the test strips surface compresses with the face card, reserves well and view window on described the card, and the position of well is corresponding with the sample pad of test strips, the position of view window is corresponding with the nitrocellulose filter of test strips, and card of the described end and face card are plastic clip.
8. with the method for claim 1,2 or 3 described test card qualitative detection Enrofloxacins, it is characterized in that, may further comprise the steps:
(1) in the well of immunochromatographydetection detection card, adds sample to be checked, behind reaction 3～20min, test card is put into simple and easy fluorescence analyser windows detecting;
(2) fluorescent microsphere sends strong fluorescence band under the exciter lamp source;
(3) emitted fluorescence detects by an unaided eye whether fluorescence signal is arranged behind the filtering veiling glare; When not containing detected material in the sample, a fluorescence band all appears in detection zone and Quality Control district, and it is negative promptly to detect sample; When containing excessive detected material in the sample, detection zone does not have the fluorescence band, and there is a fluorescence band in the Quality Control district, and it is promptly positive to detect sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100920652A CN102023210A (en) | 2009-09-21 | 2009-09-21 | Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100920652A CN102023210A (en) | 2009-09-21 | 2009-09-21 | Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102023210A true CN102023210A (en) | 2011-04-20 |
Family
ID=43864761
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100920652A Pending CN102023210A (en) | 2009-09-21 | 2009-09-21 | Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102023210A (en) |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102393463A (en) * | 2011-10-21 | 2012-03-28 | 广州万孚生物技术有限公司 | Kit for fluorescence quantitative detection of salbutamol and preparation method of fluorescence labeling liquid |
| CN102830230A (en) * | 2012-08-28 | 2012-12-19 | 暨南大学 | Ractopamine-clenbuterol fluorescent microsphere detection test strip and preparation method and application |
| CN103207270A (en) * | 2013-04-09 | 2013-07-17 | 江西中德生物工程有限公司 | Cimaterol colloidal gold test strip as well as preparation method and application thereof |
| CN103293312A (en) * | 2013-06-14 | 2013-09-11 | 张峰 | Ultrasensitive lateral chromatography rapid detection method based on luminescent quantum dots, and preparation method of test strip |
| CN103525695A (en) * | 2012-07-04 | 2014-01-22 | 长沙中生众捷生物技术有限公司 | Portable kidney function detecting system |
| CN103901193A (en) * | 2012-12-26 | 2014-07-02 | 深圳先进技术研究院 | Tony red immunodetection test paper and preparation method thereof |
| CN103901194A (en) * | 2012-12-26 | 2014-07-02 | 深圳先进技术研究院 | Goat toxoplasmosis immunodetection test paper and preparation method thereof |
| CN104101713A (en) * | 2013-04-11 | 2014-10-15 | 南昌大学 | Apparatus for rapidly detecting soybean proteins in food, and making method thereof |
| CN104101703A (en) * | 2013-04-11 | 2014-10-15 | 南昌大学 | Apparatus for rapidly detecting cow's milk allergens in food, and making method thereof |
| CN104101581A (en) * | 2013-04-11 | 2014-10-15 | 南昌大学 | Apparatus for rapidly detecting allergic peanut proteins in food, and making method thereof |
| CN105044365A (en) * | 2015-07-10 | 2015-11-11 | 天水师范学院 | Preparation method of time-resolved fluoroimmunoassay test paper for detection of Enrofloxacin residue |
| CN106526192A (en) * | 2016-09-13 | 2017-03-22 | 江苏量点科技有限公司 | Quantum dot-antibody fluorescent probe, preparation method, probe and test paper strip |
| CN107991487A (en) * | 2017-11-29 | 2018-05-04 | 洛阳现代生物技术研究院有限公司 | A kind of fluorescent microsphere immune test paper card, preparation and detection method for detecting carbostyril antibiotic |
| CN108957009A (en) * | 2017-02-21 | 2018-12-07 | 南昌大学 | Detect the Nano silver grain delustring immuno-chromatographic test paper strip of Enrofloxacin in chicken |
| CN110133260A (en) * | 2019-06-11 | 2019-08-16 | 上海雄图生物科技有限公司 | A kind of time-resolved fluoroimmunoassay chromatography kit detecting meat, eggs and milk veterinary drug residue |
| CN113238037A (en) * | 2021-04-29 | 2021-08-10 | 浙江工业大学 | Detection system and detection method for detecting pick-level enrofloxacin |
| CN113406048A (en) * | 2021-06-11 | 2021-09-17 | 北京吉森生物科技有限公司 | Fluorescence excitation type detection pen |
| CN113533279A (en) * | 2021-07-15 | 2021-10-22 | 河北农业大学 | Method for detecting enrofloxacin by using fluorescent dipeptide nano microspheres/nucleic acid aptamer |
-
2009
- 2009-09-21 CN CN2009100920652A patent/CN102023210A/en active Pending
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102393463A (en) * | 2011-10-21 | 2012-03-28 | 广州万孚生物技术有限公司 | Kit for fluorescence quantitative detection of salbutamol and preparation method of fluorescence labeling liquid |
| CN103525695A (en) * | 2012-07-04 | 2014-01-22 | 长沙中生众捷生物技术有限公司 | Portable kidney function detecting system |
| CN103525695B (en) * | 2012-07-04 | 2015-05-20 | 长沙中生众捷生物技术有限公司 | Portable kidney function detecting system |
| CN102830230A (en) * | 2012-08-28 | 2012-12-19 | 暨南大学 | Ractopamine-clenbuterol fluorescent microsphere detection test strip and preparation method and application |
| CN103901193A (en) * | 2012-12-26 | 2014-07-02 | 深圳先进技术研究院 | Tony red immunodetection test paper and preparation method thereof |
| CN103901194A (en) * | 2012-12-26 | 2014-07-02 | 深圳先进技术研究院 | Goat toxoplasmosis immunodetection test paper and preparation method thereof |
| CN103207270A (en) * | 2013-04-09 | 2013-07-17 | 江西中德生物工程有限公司 | Cimaterol colloidal gold test strip as well as preparation method and application thereof |
| CN104101713A (en) * | 2013-04-11 | 2014-10-15 | 南昌大学 | Apparatus for rapidly detecting soybean proteins in food, and making method thereof |
| CN104101703A (en) * | 2013-04-11 | 2014-10-15 | 南昌大学 | Apparatus for rapidly detecting cow's milk allergens in food, and making method thereof |
| CN104101581A (en) * | 2013-04-11 | 2014-10-15 | 南昌大学 | Apparatus for rapidly detecting allergic peanut proteins in food, and making method thereof |
| CN103293312A (en) * | 2013-06-14 | 2013-09-11 | 张峰 | Ultrasensitive lateral chromatography rapid detection method based on luminescent quantum dots, and preparation method of test strip |
| CN105044365A (en) * | 2015-07-10 | 2015-11-11 | 天水师范学院 | Preparation method of time-resolved fluoroimmunoassay test paper for detection of Enrofloxacin residue |
| CN106526192A (en) * | 2016-09-13 | 2017-03-22 | 江苏量点科技有限公司 | Quantum dot-antibody fluorescent probe, preparation method, probe and test paper strip |
| CN108957009A (en) * | 2017-02-21 | 2018-12-07 | 南昌大学 | Detect the Nano silver grain delustring immuno-chromatographic test paper strip of Enrofloxacin in chicken |
| CN107991487A (en) * | 2017-11-29 | 2018-05-04 | 洛阳现代生物技术研究院有限公司 | A kind of fluorescent microsphere immune test paper card, preparation and detection method for detecting carbostyril antibiotic |
| CN110133260A (en) * | 2019-06-11 | 2019-08-16 | 上海雄图生物科技有限公司 | A kind of time-resolved fluoroimmunoassay chromatography kit detecting meat, eggs and milk veterinary drug residue |
| CN113238037A (en) * | 2021-04-29 | 2021-08-10 | 浙江工业大学 | Detection system and detection method for detecting pick-level enrofloxacin |
| CN113238037B (en) * | 2021-04-29 | 2023-09-26 | 浙江工业大学 | Detection system and detection method for detecting pick-level enrofloxacin |
| CN113406048A (en) * | 2021-06-11 | 2021-09-17 | 北京吉森生物科技有限公司 | Fluorescence excitation type detection pen |
| CN113533279A (en) * | 2021-07-15 | 2021-10-22 | 河北农业大学 | Method for detecting enrofloxacin by using fluorescent dipeptide nano microspheres/nucleic acid aptamer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102023210A (en) | Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof | |
| CN101556280B (en) | Immunochromatography detection card for detecting fluorescent microspheres of ketamine and methyl amphetamine and preparation method thereof | |
| CN105765388B (en) | Improved pregnancy tests apparatus and method | |
| US4645646A (en) | Luminescence immunoassay for haptens and chemiluminescence labelled hapten conjugates | |
| CN111537748B (en) | Test strip and kit for detecting novel human coronavirus IgM antibody and preparation method thereof | |
| CN101900728A (en) | Multi-test-line immunochromatographic test strip for semi-quantitatively detecting aflatoxin B1 and preparation method thereof | |
| CN101435823A (en) | Fluorescent micro-ball immune chromatography test paper strip for detecting residual animal medicine and preparing method thereof | |
| KR101894753B1 (en) | Chromatograph kit and chromatograph method | |
| CN102253222B (en) | Method and special test paper for estrogen detection | |
| CN106680488A (en) | Preparation and application of immunochromatography method and test paper for quantitative detection of prealbumin | |
| CN101726596A (en) | Fluorescent microsphere immunochromatographic testing card for testing five indexes of hepatitis b and method for preparing same | |
| CN109765384A (en) | A kind of canine coronavirus antibody fluorescence test strip and its preparation method and application | |
| WO2017206800A1 (en) | Centrifugal chromatography immunoassay method | |
| KR20160124905A (en) | Immunochromatographic assay method | |
| CN108845128A (en) | Immunofluorescence for detecting canine distemper virus antigen chromatographs detection card and preparation method | |
| JPWO2019138898A1 (en) | Immunochromatographic test piece and measurement kit and measurement method | |
| CN102565382B (en) | Immunochromatography method for detecting allergen-specific IgE antibodies in blood samples | |
| CN102435731A (en) | Viewable gold nanorod test strip for rapid detection of ochratoxin A and preparation method thereof | |
| CN113567666A (en) | Fluorescent microsphere labeled immunochromatography novel coronavirus detection test strip and preparation method and application thereof | |
| CN113156105A (en) | A type botulinum toxin rapid quantitative detection card | |
| CN102680697A (en) | Reagent kit for detecting troponin I and preparation and use method thereof | |
| CN108535486A (en) | A kind of chloramphenicol immunofluorescence assay method based on europium label | |
| CN114544564A (en) | Morphine and methamphetamine combined determination reagent based on AIE nano-microspheres and preparation method and application thereof | |
| CN110372789A (en) | CDs/SiO2- SFTSV monoclonal antibody conjugate and its preparation method and application | |
| CN109856406A (en) | A kind of canine parvovirus antibody fluorescence test strip and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: ZHONGDE BIO-ENGRG CO., LTD., JIANGXI Free format text: FORMER OWNER: ZHONGDE-DADI FOOD SAFETY TECHNOLOGY DEVELOPMENT CO., LTD., BEIJING Effective date: 20121226 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 100081 HAIDIAN, BEIJING TO: NANCHANG, JIANGXI PROVINCE |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20121226 Address after: High tech Development Zone, Jiangxi, Nanchang high and new way (Chang Jian Industrial Park) Applicant after: Zhongde Bio-Engrg Co., Ltd., Jiangxi Address before: 100081, room 107, Granville Technology Building, 8 Zhongguancun South Street, Beijing, Haidian District Applicant before: Zhongde-Dadi Food Safety Technology Development Co., Ltd., Beijing |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110420 |