CN103364557A - Kit and method for detecting furaltadone metabolin - Google Patents
Kit and method for detecting furaltadone metabolin Download PDFInfo
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- CN103364557A CN103364557A CN2012101004551A CN201210100455A CN103364557A CN 103364557 A CN103364557 A CN 103364557A CN 2012101004551 A CN2012101004551 A CN 2012101004551A CN 201210100455 A CN201210100455 A CN 201210100455A CN 103364557 A CN103364557 A CN 103364557A
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Abstract
The invention discloses a kit and method for detecting a furaltadone metabolin. The kit comprises a microporous reagent and test paper, wherein a specific antibody of the furaltadone metabolin, which is marked by collaurum, is freeze-dried in the microporous reagent; the specific antibody of the furaltadone metabolin can be taken as a monoclonal antibody or a polyclonal antibody of the furaltadone metabolin; the test paper is formed by sequentially connecting a sample absorbing pad, a reaction membrane, a water absorbing pad, a protective membrane and a bottom plate, wherein a detecting area and a quality control area are formed on the reaction membrane; a hapten-carrier protein conjugate of the furaltadone metabolin is wrapped in the detecting area; and an antibody is wrapped in the quality control area. The kit for detecting the furaltadone metabolin is simple, rapid, visual and accurate in method, wide in application range and low in cost, thereby being easy to popularize.
Description
Technical field
The present invention relates to a kind of kit and method that detects AMOZ, be specifically related to a kind of colloidal gold strip for detection of AMOZ in the sample, it is particularly suitable for the residual detection of AMOZ 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, just causing Just because of this this type of drug withdrawal in treatment and feed, to use.
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.
Summary of the invention
An object of the present invention is to provide a kind of kit that detects AMOZ.
Kit provided by the present invention comprises test paper and micropore reagent, and micropore reagent has the micropore plug, and test paper comprises reaction film, absorption of sample pad, adsorptive pads, diaphragm, base plate; Freeze-drying has AMOZ specific antibody-colloid gold label thing in the described micropore reagent; The AMOZ specific antibody can be AMOZ monoclonal antibody or AMOZ polyclonal antibody; Comprise detection zone and Quality Control district on the reaction film; When the AMOZ specific antibody was the AMOZ monoclonal antibody, detection zone was coated with AMOZ hapten-carrier protein conjugate, and the Quality Control district is coated with the sheep anti mouse antiantibody; When the AMOZ specific antibody was the AMOZ polyclonal antibody, detection zone was coated with AMOZ hapten-carrier protein conjugate, and the Quality Control district is coated with goat-anti rabbit antiantibody.
Described AMOZ hapten-carrier protein conjugate is to be obtained by AMOZ haptens and carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), oralbumin, hemocyanin, thyroprotein, human serum albumins.
AMOZ specific antibody in described AMOZ specific antibody-colloid gold label thing is to prepare as immunogene with AMOZ hapten-carrier albumen.
Described diaphragm sticks on the absorption of sample pad, is the test side, and the above has the MAX mark line.
Described detection zone is positioned at an end of the diaphragm that is bordering on the MAX mark, and described Quality Control district is positioned at the end away from the diaphragm that the MAX mark is arranged.
Described base plate can be the material that PVC base plate or other hard do not absorb water; Described absorption of sample pad can be suction strainer paper or filter paper for oil; Described adsorptive pads is thieving paper; Described reaction film can be nitrocellulose filter or cellulose acetate membrane; Described diaphragm is PE material diaphragm.
Another object of the present invention provides a kind of method for preparing the mentioned reagent box, and it comprises step:
1) the preparation freeze-drying has the micropore reagent of AMOZ specific antibody-colloid gold label thing;
2) preparation has the reaction film in the Quality Control district of the detection zone of coated AMOZ hapten-carrier protein conjugate and coated antiantibody;
3) with 2) reaction film and absorption of sample pad, adsorptive pads, diaphragm, the base plate that prepare be assembled into test paper;
4) with 1) and 3) freeze-drying for preparing has micropore reagent and the test paper of AMOZ specific antibody-colloid gold label thing to be assembled into kit.
Specifically, step comprises:
1) preparation AMOZ haptens; With AMOZ haptens and carrier protein couplet, preparation AMOZ hapten-carrier protein conjugate;
2) with AMOZ hapten-carrier protein conjugate immune mouse, mouse boosting cell and murine myeloma cell are passed through to merge, screen, obtain secreting the hybridoma cell strain of AMOZ monoclonal antibody or with AMOZ hapten-carrier protein conjugate immunize rabbit, obtain the AMOZ polyclonal antibody;
3) extract mouse IgG or rabbit igg immune health goat, obtain sheep anti-mouse igg antibody or goat-anti rabbit antiantibody;
4) with trisodium citrate and gold chloride reaction preparation collaurum;
5) the AMOZ specific antibody with preparation joins in the collaurum of preparation, obtains AMOZ specific antibody-colloid gold label thing;
6) with after AMOZ specific antibody-colloid gold label thing freeze-drying is in micropore reagent, micropore reagent is added the micropore plug;
7) bovine serum albumin(BSA) (final concentration of bovine serum albumin(BSA) in damping fluid is 0.5%～1% volumn concentration), pH are 7.2 with containing, the 0.1mol/L phosphate buffer soaks 2h, 37 ℃ of lower oven dry 2h with the absorption of sample pad;
8) pasting in order absorption of sample pad, reaction film, adsorptive pads and diaphragm on the base plate;
9) micropore reagent and the test paper for preparing is assembled into kit, preserved 12 months under 2～8 ℃ of conditions.
Another object of the present invention provides a kind of mentioned reagent box of using and detects the residual method of AMOZ in the animal tissue, and it comprises step:
(1) sample-pretreating method;
(2) detect with kit;
(2) analyzing and testing result.
When using the kit test sample among the present invention, sample solution to be checked is dripped in micropore reagent, it is downward to indicate MAX mark line end behind the mixing, inserts micropore reagent, spreads to reaction film after the golden labeling antibody combination of sample liquid to be checked in micropore; If the content of AMOZ is high in the sample liquid to be checked, then the AMOZ in the sample liquid to be checked can combine with golden labeling antibody in the diffusion process, and then the antigen-combining site of AMOZ on the complete closed gold labeling antibody, stop golden labeling antibody AMOZ hapten-carrier protein conjugate on reaction film to be combined, detection zone does not develop the color, antiantibody then can be combined with golden labeling antibody, the colour developing of Quality Control district; If the low or nothing of the content of AMOZ in the sample liquid to be checked, then the antigen binding site on the golden labeling antibody can not be closed, and then golden labeling antibody can be combined by AMOZ hapten-carrier albumen coupling on reaction film, the detection zone colour developing, antiantibody also can be combined with golden labeling antibody simultaneously, the colour developing of Quality Control district.If the Quality Control district does not develop the color, then test paper lost efficacy.As shown in Figure 4.
Positive: (C) demonstrates band when the Quality Control district, and detection zone (T) does not develop the color, and is judged to the positive, with "+" expression.
Negative: as when Quality Control district (C) and detection zone (T) all demonstrate band, to be judged to feminine gender, to represent with "-".
Invalid: (C) do not show band when the Quality Control district, and test paper lost efficacy.
That kit of the present invention has is highly sensitive, high specificity, cost is low, simple to operate, detection time short, be fit to various units uses, stores advantage simple, long shelf-life.Wherein, adopt the AMOZ monoclonal antibody of high specific, guaranteed the reliability of testing result; In micropore reagent, in testing process, golden labeling antibody is fully contacted with sample liquid to be checked golden labeling antibody freeze-drying, fully reaction, thus reduce error, increase the reaction sensitivity of whole system.Detect the method for AMOZ with kit of the present invention, easy, quick, directly perceived, accurate, applied widely, cost is low, easily promote the use of.
Description of drawings
Fig. 1 is the test paper cross-sectional view.
Fig. 2 is the test paper vertical view.
Fig. 3 is micropore reagent figure.
Fig. 4 is as a result process decision chart of detection paper.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
One, test paper (being called test strips) (Fig. 1)
Described test paper is comprised of base plate, absorption of sample pad, reaction film, adsorptive pads, diaphragm;
Described absorption of sample pad 1, reaction film 2, adsorptive pads 3 and diaphragm 7 stick on the base plate 6 successively in order, the end of absorption of sample pad links to each other with reaction film, 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, and the end of adsorptive pads aligns with the end of base plate;
The absorption of sample pad end of described test paper is pasted with diaphragm, and diaphragm 7 covers the test side on the absorption of sample pad, is printed on MAX printed words (Fig. 2) at the test side diaphragm;
Detection zone 4 and Quality Control district 5 are arranged on the described reaction film, all be the ribbon vertical with the appearance of described test strips, detection zone is positioned at an end of the diaphragm that is bordering on the MAX mark, and the Quality Control district is positioned at the end away from the diaphragm that the MAX mark is arranged.Detection zone is coated with AMOZ hapten-carrier protein conjugate (conjugate of AMOZ haptens-oralbumin), and the Quality Control district is coated with the sheep anti mouse antiantibody;
Described base plate is the PVC base plate; Described absorption of sample pad is suction strainer paper; Described adsorptive pads is thieving paper; Described reaction film is nitrocellulose filter; Described diaphragm is PE material diaphragm.
Two, micropore reagent (Fig. 3)
Freeze-drying has AMOZ monoclonal antibody-colloid gold label thing on the described micropore reagent 8, freeze-drying amount 0.20～0.25 μ g/mL; Described micropore reagent has micropore plug 9.
Above-mentioned test paper, micropore reagent set are dressed up kit, in 2～8 ℃ of environment, preserve the term of validity 12 months.
The preparation method of kit described in embodiment 2 embodiment 1
One, the preparation of kit
The preparation method of kit mainly may further comprise the steps:
1) the preparation freeze-drying has the micropore reagent of AMOZ monoclonal antibody-colloid gold label thing;
2) preparation has the reaction film in the Quality Control district of the detection zone of coated AMOZ hapten-carrier protein conjugate and coated sheep anti mouse antiantibody;
3) with 2) reaction film and absorption of sample pad, adsorptive pads, diaphragm, the base plate that prepare be assembled into test paper;
4) with 1) and 3) freeze-drying for preparing has micropore reagent and the test paper of AMOZ monoclonal antibody-colloid gold label thing to be assembled into kit.
Following substep is described in detail:
(1) preparation of each parts
1. the synthetic and evaluation of AMOZ hapten-carrier protein conjugate
(1) the haptenic preparation of AMOZ
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 AMOZ, 60 ℃ are stirred 4h.After reaction stopped, solvent evaporated added ethyl alcohol recrystallization and gets haptens product 0.53g.
(2) immunogenic preparation
Get 12mg AMOZ haptens, be dissolved among the 1ml DMF, obtain solution I; Get 15mg dicyclohexylcarbodiimide (DCC) and 10mg N-hydroxy-succinamide (NHS) fully dissolves in the rear adding solution I with 0.2ml DMF, stir 24h under the room temperature, can obtain solution II; Take by weighing bovine serum albumin(BSA) (BSA) 30mg, make it fully to be dissolved in the 3ml 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 ℃.
(3) preparation of coating antigen
Get 12mg AMOZ haptens with 1.5ml 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 30min obtain solution II; Get 30mg oralbumin (OVA) 2.2ml 50mmol/L Na
2CO
310 ℃ of reactions of dissolving and solution II 4h, then 4 ℃ are spent the night; 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 AMOZ hapten-carrier protein conjugate
Carrier protein, AMOZ haptens, AMOZ 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 pH7.4 PBS, in the interscan of wavelength 200～800nm scope, obtain the absorption curve of carrier protein, AMOZ haptens, AMOZ hapten-carrier protein conjugate with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of AMOZ haptens and carrier protein couplet.
2. the preparation of AMOZ monoclonal antibody
(1) animal immune
The immunogene that step 1 obtains is injected in the Balb/c Mice Body, and immunizing dose is 150 μ g/, makes it produce antiserum.
(2) 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 AMOZ monoclonal hybridoma strain of stably excreting AMOZ monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma is made 2 * 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.
(4) 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 AMOZ 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 AMOZ 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 AMOZ 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) 0.3%～0.5% (volumn concentration), Tween-80 0.08%～0.2% (quality percentage composition), pH7.2.
With AMOZ monoclonal antibody-colloid gold label thing freeze-drying to micropore reagent
In micropore reagent microwell plate, add 100 μ l AMOZ 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 AMOZ monoclonal antibody-colloid gold label thing, sealing is preserved, AMOZ monoclonal antibody-colloid gold label thing freeze-drying amount 0.20～0.25 μ g/mL.
6. the preparation of absorption of sample pad
The absorption of sample pad placed contain bovine serum albumin(BSA) (bovine serum albumin(BSA) is 0.5% (volumn concentration) at the final concentration of damping fluid), pH7.2,0.1mol/L phosphate buffer and soak 2h, 37 ℃ of baking 2h are for subsequent use.
7. the preparation of reaction film
Coated process: with phosphate buffer AMOZ haptens-oralbumin conjugate is diluted to 10mg/mL, with Isoflow point film instrument it is coated in detection zone (T) on the nitrocellulose filter, package amount is 1.0 μ g/cm
2Phosphate buffer with 0.01mol/L, pH7.4 is diluted to 200 μ g/mL with sheep anti-mouse igg antibody, with Isoflow point film instrument it is coated in Quality Control district (C) on the nitrocellulose filter, and package amount is 1.0 μ g/cm
2Coated good reaction film is placed dry 2h under 37 ℃ of conditions, for subsequent use.
(2) assembling of each parts
1. the assembling of test paper
Stick in order successively described absorption of sample pad, reaction film, adsorptive pads, diaphragm on the described base plate; The end of absorption of sample pad links to each other with the top of reaction film, and the end of reaction film links to each other with the top of adsorptive pads, and the top of absorption of sample pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate; Bonding protective film on the test paper absorption of sample pad that assembles is printed on the MAX mark line on the diaphragm.
2. the assembling of kit
Test paper and micropore reagent set that above-mentioned steps 1 obtains are dressed up kit, in 2～8 ℃ environment, store the term of validity 12 months.
The detection of AMOZ in embodiment 3 animal tissues
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. use the kit test sample
From original packing, take out reagent barrel (if low-temp storage needs pre-balance to room temperature), open rear taking-up requisite number purpose micropore reagent and test strips, and carry out mark; 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.
3. Analysis of test results
Positive: (C) demonstrates band when the Quality Control district, and detection zone (T) does not develop the color, and is judged to the positive, with "+" expression, such as Fig. 4 (a); Negative: as when Quality Control district (C) and detection zone (T) all demonstrate band, to be judged to feminine gender, to represent with "-", such as Fig. 4 (b); Invalid: (C) do not show band when the Quality Control district, and test paper lost efficacy, such as Fig. 4 (c) with (d).
Determining of embodiment 4 kit technical parameters
1. sensitivity test
It is 0.5,1.0,2.0 μ g/kg that AMOZ standard items (available from Sigma company) are diluted; Used diluent is the phosphate buffer of pH7.2,0.2mol/L.
Detect with kit, the result is: when AMOZ standard items concentration is 0.5 μ g/L, demonstrate macroscopic two red lines on the test strips, be negative; When AMOZ standard items concentration was 1.0,2.0 μ g/L, test strips Quality Control district colour developing, but detection zone do not develop the color, and is positive showed that the sensitivity that this kit detects AMOZ is 1.0 μ g/L.
2. false positive rate, false negative rate test
Get known AMOZ content greater than the pork of 1 μ g/kg, chicken, fish, each 20 parts of shrimp positive and AMOZ content less than each 20 parts of pork, chicken, fish, the shrimp negative samples of 1 μ g/kg, kit with 3 batches of productions detects respectively, calculates its yin and yang attribute rate.The results are shown in Table 1, table 2.
Table 1 detects the positive result
Table 2 detects the negative sample result
The result shows: when detecting positive pork, chicken, fish, shrimp sample with the kit of 3 batches of productions, the result is entirely positive, and the positive sample coincidence rate is 100% as can be known, and false negative rate is 0.When detecting negative pork, chicken, fish, shrimp sample, the result is entirely negative, and negative match-rate is 100% as can be known, and false positive rate is 0.Detection AMOZ kit of the present invention can be to the residual fast detecting of carrying out of AMOZ in animal tissue's sample.
3. specific test
Specificity cross reacting rate commonly used represents, refers to the ability of the antigenic determinant generation combination that antibody is different from structure.This kit is 1 μ g/L to the detection sensitivity of AMOZ, the phosphate buffer of the normal other drug (streptomysin, Tetracyclines, quinolones, sulfamido) of examining in AMOZ analog (furaltadone, furazolidone, nitrofurazone, furantoin, Furaxone metabolite, Furacilin metabolite, Cistofuran metabolite) and the animal tissue with pH7.2,0.2mol/L diluted, detect with the kit described in the embodiment 1.The result shows, when detecting furaltadone, furazolidone, nitrofurazone, furantoin, Furaxone metabolite, Furacilin metabolite, Cistofuran metabolite, streptomysin, Tetracyclines, quinolones, sulfa drugs with this kit, test strips Quality Control district and detection zone all develop the color, and illustrate that this kit is to these medicine no cross reactions.
Claims (9)
1. kit that detects AMOZ; it is characterized in that comprising test paper and micropore reagent; described test paper comprises reaction film, absorption of sample pad, adsorptive pads, diaphragm, base plate; detection zone and Quality Control district are arranged on the described reaction film; detection zone is coated with AMOZ hapten-carrier protein conjugate; the Quality Control district is coated with antiantibody, and freeze-drying has AMOZ specific antibody-colloid gold label thing in the described micropore reagent.
2. kit according to claim 1 is characterized in that described test paper is sticked on the base plate successively by absorption of sample pad, reaction film, adsorptive pads, diaphragm to form, and has the micropore plug on the described micropore reagent.
3. kit according to claim 2 is characterized in that described diaphragm sticks on the absorption of sample pad, is the test side, and the above has the MAX mark line.
4. kit according to claim 3 is characterized in that described detection zone is positioned at an end of the diaphragm that is bordering on the MAX mark, and described Quality Control district is positioned at the end away from the diaphragm that the MAX mark is arranged.
5. kit according to claim 1, it is characterized in that described AMOZ hapten-carrier protein conjugate is obtained by AMOZ haptens and carrier protein couplet, described carrier protein can be bovine serum albumin(BSA), oralbumin, hemocyanin, thyroprotein, human serum albumins.
6. according to claim 1 or 5 each described kits, it is characterized in that described AMOZ haptens structural formula is as follows:
7. kit according to claim 1, it is characterized in that the AMOZ specific antibody in described AMOZ specific antibody-colloid gold label thing is to prepare as immunogene with AMOZ hapten-carrier protein conjugate, described AMOZ specific antibody can be AMOZ monoclonal antibody or AMOZ polyclonal antibody.
8. method for preparing each described kit of claim 1-7, it comprises step:
1) the preparation freeze-drying has the micropore reagent of AMOZ specific antibody-colloid gold label thing;
2) preparation has the reaction film in the Quality Control district of the detection zone of coated AMOZ hapten-carrier protein conjugate and coated antiantibody;
3) with 2) reaction film and absorption of sample pad, adsorptive pads, diaphragm, the base plate that prepare be assembled into test paper;
4) with 1) and 3) freeze-drying for preparing has micropore reagent and the test paper of AMOZ specific antibody-colloid gold label thing to be assembled into kit.
9. one kind is detected the residual method of AMOZ in the animal tissue, and it comprises step:
1) sample pre-treatments;
2) detect with each described kit of claim 1-7;
3) analyzing and testing result.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104374910A (en) * | 2014-11-19 | 2015-02-25 | 无锡中德伯尔生物技术有限公司 | Kit and method applied to detection of nitrofurans drug metabolite |
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| WO2007024735A2 (en) * | 2005-08-19 | 2007-03-01 | Charm Sciences, Inc. | Method and antibodies for detecting nitrofuran |
| CN101261274A (en) * | 2008-02-02 | 2008-09-10 | 王学生 | Method for combining colloidal gold immune leaching and immunity-chromatography for rapidly detecting medicament residue |
| CN101407501A (en) * | 2008-11-21 | 2009-04-15 | 华南农业大学 | Preparation of 5-methyl morpholine-3-amino-2-oxazolidinone derivative hapten, antigen and antibody |
| CN101526528A (en) * | 2009-01-12 | 2009-09-09 | 深圳市绿诗源生物技术有限公司 | Furaltadone metabolite detection kit |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104374910A (en) * | 2014-11-19 | 2015-02-25 | 无锡中德伯尔生物技术有限公司 | Kit and method applied to detection of nitrofurans drug metabolite |
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