CN202720229U - Kit for detecting furacilin metabolite - Google Patents
Kit for detecting furacilin metabolite Download PDFInfo
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- CN202720229U CN202720229U CN 201220144194 CN201220144194U CN202720229U CN 202720229 U CN202720229 U CN 202720229U CN 201220144194 CN201220144194 CN 201220144194 CN 201220144194 U CN201220144194 U CN 201220144194U CN 202720229 U CN202720229 U CN 202720229U
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- furacilin metabolite
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Abstract
The utility model provides a kit for detecting a furacilin metabolite. The kit comprises a kit body, a fixing appliance with 12 holes and 12 reagent barrels with plugs, wherein the reagent barrels are placed in the fixing appliance; the kit comprises a micro-porous reagent strip and a test paper strip; the test paper strip consists of a bottom plate, and a sample absorbing pad, a reaction film, a water absorbing pad and a protective film which are attached to the bottom plate to be closely connected in sequence; a furacilin metabolite monoclonal antibody-colloidal gold marker is freeze-dried in micro-porous reagent; the reaction film is provided with a detection line print, i.e., a vertical line, and a quality control line print, i.e., a vertical line; the detection line print is formed of hapten-carrier protein conjugate coated with the furacilin metabolite; and the quality control line print is formed by coating goat-anti-mouse antibody. The kit has the characteristics of high portability, high sensitivity, short detection time and the like, and can play an important role in residue detection of the furacilin metabolite.
Description
Technical field
The utility model relates to a kind of kit that detects Furacilin metabolite, particularly detects the kit of Furacilin metabolite in the animal tissue.
Background technology
The enterogastric diseases that itrofurans medicine Chang Zuowei wide spectrum class microbiotic is used for prevention and treats the pig, ox, poultry and the honeybee that are caused by salmonella and Escherichia.But finding that itrofurans medicine and metabolin all can make animal used as test generation canceration and gene mutation in the process of experimental for a long time, this type of drug withdrawal uses in treatment and feed.
Metabolism is rapid in vivo owing to itrofurans prototype medicine, can't detect, but its metabolic product because of and the protein bound quite stable, so the product often will analyze its metabolism when analyzing this type of medicine residual after, administrative authority just reaches the residual purpose of detection itrofurans to detect metabolic product as means.The most popular method that is used at present detecting the nitrofuran metabolite is high performance liquid chromatography (HPLC), Liquid Chromatography/Mass Spectrometry (LC-MS/MS) etc., these methods are highly sensitive, the result accurately, good reproducibility, false positive be few, but sample pretreatment process is complicated, instrumentation degree high price is expensive, by comparison, that immunization method has is highly sensitive, the detection time that sample pretreatment is simple, of short duration, the larger characteristics such as detection sample size, is fit to the examination of on-site supervision and a large amount of samples.
The utility model content
The purpose of this utility model provides a kind of kit of highly sensitive, cost is low, simple to operate, detection time is short detection Furacilin metabolite.
Kit of the present utility model, it comprises the kit box body, has holding appliance and placement 12 tool plug reagent barrels wherein in 12 holes, comprise micropore reagent strip and 8 test strips in the reagent barrel, have the micropore plug on the reacting hole of micropore reagent strip, wherein freeze-drying has Furacilin metabolite monoclonal antibody-colloid gold label thing.Test strips by base plate, be attached on the base plate absorption of sample pad, reaction film, adsorptive pads and the diaphragm that closely link to each other successively and form, have on the described reaction film and be coated with detection line trace " | " that Furacilin metabolite hapten-carrier protein conjugate consists of and the nature controlling line trace " | " of coated sheep anti mouse antiantibody formation.The absorption of sample pad is positioned at base plate top, and adsorptive pads is positioned at the base plate end, and detection line is parallel with nature controlling line, and nature controlling line is positioned at apart from adsorptive pads top and reaction film connecting place 5-8mm, and detection line is positioned at apart from nature controlling line 5-6mm.Test strips absorption of sample pad test port is pasted with diaphragm, and the MAX mark line is arranged on the diaphragm.
The test strips base plate can be the material that PVC base plate or other hard do not absorb water in the utility model kit; The absorption of sample pad can be suction strainer paper or filter paper for oil; Adsorptive pads is thieving paper; Reaction film can be nitrocellulose filter or cellulose acetate membrane; Diaphragm is PE material diaphragm; The kit box body is carton box; Reagent barrel is the plastics reagent barrel; Holding appliance is the rigid support material.
The utility model kit has following beneficial effect:
1. highly sensitive.The Furacilin metabolite detection kit is prepared from as the basis take the monoclonal antibody of colloid gold label high-affinity, priceless strong formation between gold grain and the antibody molecule in the gold labeling antibody, the two combines by the Van der Waals force between the charges of different polarity, collaurum is very little on monoclonal antibody specificity and affinity impact, and has higher mark rate.Furacilin metabolite monoclonal antibody wherein-colloid gold label thing freeze-drying in testing process, can make golden labeling antibody fully contact with sample liquid to be checked in the micropore reagent strip, fully reaction, thus reduce error, increase the reaction sensitivity of whole system.Therefore, the utility model kit has higher specificity and sensitivity.This kit is 1 μ g/L to the detection sensitivity of Furacilin metabolite.
2. show testing result image, accurately directly perceived.Test strips is to show that red line " | " and " || " trace are as the positive and negative marker during detection, namely show that at nitrocellulose filter a red line " | " trace is illustrated in the detected sample liquid Furacilin metabolite content and is higher than and equals kit to the lowest detectable limit of Furacilin metabolite, article two, red line " || " trace is illustrated in that Furacilin metabolite content is lower than kit to the lowest detectable limit of Furacilin metabolite in the detected sample, the result judges image, directly perceived, accurate, simple and clear, is not easy to occur result's erroneous judgement.
3. cost is low, small investment.Use the utility model kit, do not need to join in addition instrument and equipment and other reagent, Site Detection is settled at one go, with low cost, small investment, instant effect.
4. be easy to apply on a large scale.Kit is simple to operate, can better satisfy the demand that animal tissue is detected, and has wide market outlook and larger economical, societal benefits.
Description of drawings
Fig. 1 is the utility model test strips structural representation;
Fig. 2 is the utility model test strips vertical view;
Fig. 3 is the utility model micropore reagent figure;
Fig. 4 is as a result process decision chart of the utility model ELISA test strip;
Fig. 5 is the utility model reagent barrel structural representation;
Fig. 6 is the utility model holding appliance vertical view;
Fig. 7 is the utility model box body and holding appliance side view.
Embodiment
One, the assembling of Furacilin metabolite detection kit preparation
1) test strips
Absorption of sample pad 1; reaction film 2; adsorptive pads 3 and diaphragm 7 are pasted successively in order on described base plate 6; the absorption of sample pad is positioned at base plate top; adsorptive pads is positioned at the base plate end; the end of absorption of sample pad links to each other with reaction film top; the end of reaction film links to each other with adsorptive pads; the top of absorption of sample pad aligns with the top of base plate; the end of adsorptive pads aligns with the end of base plate; test paper absorption of sample pad test port is pasted with diaphragm; be printed on the MAX mark line on the diaphragm; detection line 4 is coated with Furacilin metabolite hapten-carrier protein conjugate, and nature controlling line 5 is coated with the sheep anti mouse antiantibody.
Base plate is the PVC base plate, and the absorption of sample pad is suction strainer paper, and reaction film is nitrocellulose filter, and adsorptive pads is thieving paper, and diaphragm is PE material diaphragm.
2) micropore reagent strip
Micropore reagent strip 8 has micropore plug 9, and freeze-drying has Furacilin metabolite monoclonal antibody-colloid gold label thing in the plate hole.
3) with 1) test strips and 2) the micropore reagent strip plastics reagent barrel 10 of packing into and having gland bonnet, the plastics reagent barrel is positioned in the holding appliance 11 in the kit box body 12.
Two, the use of kit
1, sample pre-treatments
Take by weighing the sample of 5.0g ± 0.05g homogeneous to 50ml polystyrene centrifuge tube, add 5ml 10% trichloroacetic acid, add 100 μ l derivatization reagents (adding 10ml methyl alcohol dissolving mixing in the reagent bottle that 151mg 2-nitrobenzaldehyde is housed), 3min fully vibrates again; In 60 ℃ of incubators, hatch 1.5h; Take out and to add successively 1ml 0.5mol/L dipotassium hydrogen phosphate solution, 1.5ml 2mol/L sodium hydroxide solution and 10ml ethyl acetate, vibration 10s, the sample that fat content is high slightly vibrate under the 8-10, in case sample emulsification; More than the 3000g, the centrifugal 5min of room temperature (20-25 ℃); Get 8ml ethyl acetate to the glass test tube of 10ml drying, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, whirling motion 30s adds 0.8ml sample redissolution liquid (0.2mol/L phosphate buffer), the abundant mixing of whirling motion 10s again; More than the 3000g, the centrifugal 5min of room temperature (20-25 ℃); Remove upper organic phase, take off layer 100 μ l and be used for analyzing.
2, detect with kit
Draw the sample liquid that 100 μ l redissolve with micropipettor, slowly suction and fully with micropore in the reagent mixing, in the test strips insertion micropore that mark is good---be printed on " MAX " line end down, make it fully to immerse in the solution; After room temperature (20 ℃-25 ℃) is hatched 5min, take out test strips, result of determination.
Three, Analysis of test results
The content of Furacilin metabolite in sample be higher than equal kit to its lowest detection in limited time, Furacilin metabolite monoclonal antibody-colloid gold label thing is combined with medicine, antigen binding site on the gold labeling antibody is closed, thereby in detection zone, because competitive reaction, can not be combined with Furacilin metabolite hapten-carrier protein conjugate and red stripes do not occur.Negative sample owing to lack the antigen-antibody competitive reaction, will red stripes occur at detection line and nature controlling line in testing process.As shown in Figure 4.
Positive: when red stripes of nature controlling line (C) demonstration, and detection line (T) does not develop the color, and test strips is judged to the positive, shown in Fig. 4 .a figure to show red line " | ".
Negative: when red stripes of nature controlling line (C) demonstration, detection line (T) also demonstrates a red stripes simultaneously, and test strips is to show that red line as " || ", is judged to feminine gender, shown in Fig. 4 .b.
Invalid: (C) do not demonstrate red stripes when nature controlling line, and then no matter whether detection line (T) demonstrates red stripes, and it is invalid that this test strips is judged to, shown in Fig. 4 .c, 4.d.
Four, the material preparation method of using in the detection kit
1, the synthetic and evaluation of Furacilin metabolite hapten-carrier protein conjugate
(1) the haptenic preparation of Furacilin metabolite
A. get the 1.0g parahydroxyben-zaldehyde and be dissolved in the acetonitrile, add 0.32g sal tartari, the 0.1g sodium iodide stirs.Add bromoacetate 0.44g, load onto reflux condensing tube, 80 ℃ of stirrings are spent the night.After reaction stopped, the evaporate to dryness acetonitrile was dissolved in water, ethyl acetate extraction, silicagel column on the evaporate to dryness, sherwood oil: ethyl acetate=cross post purifying get product 1.08g at 5: 1;
B. get the 1.08g product that step a obtains and be dissolved in the ethanol, hydro-oxidation potassium 0.5g, 70 ℃ are stirred 4h.After reaction stopped, evaporate to dryness ethanol was dissolved in water, and regulated pH value to 6, added 60ml * 3 ethyl acetate extractions three times, merged organic phase, evaporate to dryness, and upper silicagel column, sherwood oil: ethyl acetate=wash-out got product 0.73g in 3: 1;
C. get the 0.7g product that step b obtains and add DMF (DMF) dissolving, under agitation add the methanol solution 5ml of 0.41g Furacilin metabolite, 60 ℃ are stirred 4h.After reaction stopped, solvent evaporated added ethyl alcohol recrystallization and gets haptens product 0.53g.
(2) immunogenic preparation
Get 12mg Furacilin metabolite haptens with 0.8ml DMF dissolving, be cooled to 10 ℃, obtain solution I; Get isobutyl chlorocarbonate 10 μ l and add in the solution I, 10 ℃ of stirring reaction 10min obtain solution II; Get 40mg bovine serum albumin(BSA) (BSA) and use 3.2ml50mmol/LNa
2CO
310 ℃ of reactions of dissolving and solution II 4h, then 4 ℃ are spent the night; With 4 ℃ of dialysis of 0.01mol/LPBS 3d, change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing saves backup in-20 ℃.
(3) preparation of coating antigen
Get 12mg Furacilin metabolite haptens, be dissolved among the 1ml DMF, obtain solution I; Get 15mg carbodiimides (EDC) and fully dissolve in the rear adding solution I with 0.2ml water, stir 24h under the room temperature, can obtain solution II; Take by weighing oralbumin (OVA) 30mg, make it fully to be dissolved in the 3.8ml water, dropwise slowly be added drop-wise in the protein solution solution II, and under room temperature, stir 24h, with 4 ℃ of dialysis of 0.01mol/L PBS 3d, change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing saves backup in-20 ℃.
(4) evaluation of Furacilin metabolite hapten-carrier protein conjugate
Carrier protein, Furacilin metabolite haptens, Furacilin metabolite hapten-carrier protein conjugate are made into the solution of 0.5mg/mL with the PBS of pH7.4, return to zero with 0.01mol/L pH7.4PBS, in the interscan of wavelength 200-800nm scope, obtain the absorption curve of carrier protein, Furacilin metabolite haptens, Furacilin metabolite hapten-carrier protein conjugate with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of Furacilin metabolite haptens and carrier protein couplet.
2, the preparation of Furacilin metabolite monoclonal antibody
A. animal immune
The immunogene that step 1 is obtained is injected in the Balb/c Mice Body, and immunizing dose is 150 μ g/, makes it produce antiserum.
B. Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, merge in 8: 1 (quantitative proportion) ratios and SP2/0 myeloma cell, screening obtains the hybridoma cell strain of stably excreting Furacilin metabolite monoclonal antibody.
C. cell cryopreservation and recovery
Hybridoma is made 5 * 10 with cryopreserving liquid
6The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
D. the preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed cell culture medium, under 37 ℃ of conditions, cultivate, with sad-saturated ammonium sulfate method the nutrient solution that obtains is carried out purifying, obtain monoclonal antibody ,-20 ℃ of preservations.
Described cell culture medium is to add calf serum and sodium bicarbonate in the RPMI1640 nutrient culture media, making the final concentration of calf serum in cell culture medium is 20% (quality percentage composition), and making the final concentration of sodium bicarbonate in cell culture medium is 0.2% (quality percentage composition); The pH of described cell culture medium is 7.4.
3, the preparation of sheep anti mouse antiantibody
As immune animal, as immunogene the pathogen-free domestic sheep is carried out immunity take mouse source antibody with sheep, obtain the sheep anti mouse antiantibody.
4, the preparation of Furacilin metabolite monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
With two ionized waters that boil off 1% gold chloride (being purchased from sigma company) is diluted to 0.01% (quality percentage composition), get 100ml and place conical flask, be heated to boiling with the thermostatic electromagnetic stirrer, under continuous high temperature, the lasting stirring, add 2.5ml 1% trisodium citrate (being purchased from Guangzhou Chemical Reagent Factory), continuing at the uniform velocity to be heated with stirring to solution is bright and stops when red, return to original volume with deionized water after being cooled to room temperature, 4 ℃ of preservations.The collaurum outward appearance for preparing is pure, bright, nothing precipitates and floating thing.
(2) preparation of Furacilin metabolite monoclonal antibody-colloid gold label thing
Under magnetic agitation, transfer the pH value to 7.2 of collaurum with 0.2mol/L sal tartari, in colloidal gold solution, add above-mentioned Furacilin metabolite monoclonal antibody by the standard that adds 50～100 μ g antibody in every milliliter of colloidal gold solution, continue to stir and evenly mix 10min, adding 10% bovine serum albumin(BSA) (BSA), to make its final concentration in colloidal gold solution be 1% (volumn concentration), leaves standstill 10min.12000rpm, 4 ℃ of centrifugal 40min abandon supernatant, precipitation is with redissolving the damping fluid washed twice, it is resuspended to be with volume that the redissolution damping fluid of initial collaurum volume 1/10 will precipitate, put 4 ℃ for subsequent use.
Redissolution damping fluid: the 0.02mol/L phosphate buffer that contains bovine serum albumin(BSA) (BSA) 0.1%～0.3% (volumn concentration), Tween-80 0.05%～0.2% (quality percentage composition), pH7.2.
5, with Furacilin metabolite monoclonal antibody-colloid gold label thing freeze-drying to micropore reagent
In micropore reagent microwell plate, add 100 μ l Furacilin metabolite monoclonal antibody-colloid gold label things, put into freeze drier, be under-50 ℃ of conditions at condenser temperature, behind the pre-freeze 3h, vacuum drying 15h can take out again, obtains the micropore reagent that freeze-drying has Furacilin metabolite monoclonal antibody-colloid gold label thing, micropore reagent is added the micropore plug, and sealing is preserved.
6, the preparation of absorption of sample pad
The absorption of sample pad placed contain that bovine serum albumin(BSA) (bovine serum albumin(BSA) is 0.5% (volumn concentration) at the final concentration of damping fluid), pH are 7.2, the 0.1mol/L phosphate buffer soaks 2h, 37 ℃ of baking 2h are for subsequent use.
7, the preparation of reaction film
Coated process: with phosphate buffer Furacilin metabolite haptens-oralbumin conjugate is diluted to 10mg/mL, with Isoflow point film instrument it is coated in detection line (T) on the nitrocellulose filter, package amount is 1.0 μ g/cm
2With 0.01mol/L, pH 7.4PBS damping fluid sheep anti-mouse igg antibody is diluted to 200 μ g/mL, with Isoflow point film instrument it is coated in nature controlling line (C) on the nitrocellulose filter, package amount is 1.0 μ g/cm
2Coated good reaction film is placed dry 2h under 37 ℃ of conditions, for subsequent use.
Claims (8)
1. kit that detects Furacilin metabolite; it is characterized in that kit comprises the kit box body, has the holding appliance in 12 holes and places wherein 12 tool plug reagent barrels; comprise micropore reagent strip and 8 test strips in the reagent barrel, test strips by base plate, be attached on the base plate absorption of sample pad, reaction film, adsorptive pads and the diaphragm that closely link to each other successively and form.
2. kit as claimed in claim 1 is characterized in that having on the described reaction film and is coated with detection line trace " ︱ " that Furacilin metabolite hapten-carrier protein conjugate consists of and the nature controlling line trace " ︱ " of coated sheep anti mouse antiantibody formation.
3. kit as claimed in claim 1, it is characterized in that described absorption of sample pad is positioned at base plate top, described adsorptive pads is positioned at the base plate end, and described nature controlling line is positioned at apart from adsorptive pads top and reaction film connecting place 5-8mm, and described detection line is positioned at apart from nature controlling line 5-6mm.
4. kit as claimed in claim 1 is characterized in that the test side on the described test strips absorption of sample pad is pasted with diaphragm, and the MAX mark line is arranged on the diaphragm.
5. kit as claimed in claim 1 is characterized in that described base plate can be the PVC base plate, and the absorption of sample pad can be suction strainer paper or filter paper for oil, and adsorptive pads is thieving paper, and reaction film can be nitrocellulose filter or cellulose acetate membrane, and diaphragm is PE material diaphragm.
6. kit as claimed in claim 1 is characterized in that described micropore reagent strip has 8 reacting holes, has the micropore plug on the reacting hole.
7. kit as claimed in claim 6 is characterized in that freeze-drying has Furacilin metabolite monoclonal antibody-colloid gold label thing in the described micropore reagent.
8. kit as claimed in claim 1 is characterized in that described kit box body is carton box, and reagent barrel is the plastics reagent barrel, and holding appliance is the rigid support material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220144194 CN202720229U (en) | 2012-04-07 | 2012-04-07 | Kit for detecting furacilin metabolite |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220144194 CN202720229U (en) | 2012-04-07 | 2012-04-07 | Kit for detecting furacilin metabolite |
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| Publication Number | Publication Date |
|---|---|
| CN202720229U true CN202720229U (en) | 2013-02-06 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201220144194 Expired - Fee Related CN202720229U (en) | 2012-04-07 | 2012-04-07 | Kit for detecting furacilin metabolite |
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| CN (1) | CN202720229U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112578110A (en) * | 2020-12-16 | 2021-03-30 | 西北农林科技大学 | Biological probe and kit for detecting furacilin and furazolidone and application |
-
2012
- 2012-04-07 CN CN 201220144194 patent/CN202720229U/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112578110A (en) * | 2020-12-16 | 2021-03-30 | 西北农林科技大学 | Biological probe and kit for detecting furacilin and furazolidone and application |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130206 Termination date: 20210407 |
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| CF01 | Termination of patent right due to non-payment of annual fee |