CN202903799U - Macrodantin metabolin detection kit - Google Patents
Macrodantin metabolin detection kit Download PDFInfo
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- CN202903799U CN202903799U CN201220552158.6U CN201220552158U CN202903799U CN 202903799 U CN202903799 U CN 202903799U CN 201220552158 U CN201220552158 U CN 201220552158U CN 202903799 U CN202903799 U CN 202903799U
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- cistofuran metabolite
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Abstract
The utility model relates to a macrodantin metabolin detection kit which comprises a solid phase carrier, a concave bottle position for holding the kit and a reagent bottle positioned above the concave bottle position, wherein a micro-pore strip is arranged on the solid phase carrier; the micro-pore strip comprises micro-pores and a coating layer; the coating layer consists of macrodantin metabolin coupling antigens; and the reagent bottle comprises a reagent bottle with a macrodantin metabolin monoclonal antibody solution and a reagent bottle with a goat-anti-mouse anti-antibody marked with horse radish peroxidase. The kit can be used for detecting residual amount of the macrodantin metabolin; and compared with an instrument analysis technique, the kit has the chrematistics of being convenient to use, high in sensitivity, large in detection sample quantity and the like.
Description
(1) technical field: the utility model relates to a kind of Cistofuran metabolite detection reagent kit, particularly detects the kit of Cistofuran metabolite residual quantity in the animal derived food.
(2) background technology: the itrofurans medicine once was widely used because the dynamic (dynamical) characteristic of extraordinary antibacterial action and medicine is arranged, as the somatotrophic adjuvant of bird, aquatic products and pig.But finding in the process of experimental for a long time, itrofurans medicine and metabolin all can make animal used as test generation canceration and gene mutation, just cause Just because of this this type of drug withdrawal to use in treatment and feed.Furantoin is disabled in nineteen ninety-five.
Because the itrofurans medicine in vivo soon can be by metabolism, the metabolic product of combination then can retain long period of time in tissue, 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.Furantoin metabolic product AHD; Furaltadone metabolic product AMOZ; Furazolidone metabolite product A OZ; Nitrofurazone (nitrofurazone) metabolic product SEM.
The most popular method that is used for detecting Cistofuran metabolite is LC-UV, LC-MS and LC-MS/MS.Enzyme-linked immunoassay method combines chromatographic technique, adopts the specific antibody of AHD derivant, has very high degree of accuracy and sensitivity, and lower operative technique requires and of short duration detection time, detects the characteristics such as sample size is large and well shows in detection.
(3) utility model content: the utility model provides a kind of Cistofuran metabolite detection reagent kit of small volume and less weight, it is easy and simple to handle, detection sensitivity is high, cost is low, can carry out the mensuration that one-time continuous is carried out Cistofuran metabolite residual quantity in a plurality of samples, reduce the needed time of detection sample.
Cistofuran metabolite detection reagent kit, comprise solid phase carrier, put reagent recessed bottle position, be positioned at the reagent bottle on the recessed bottle position, solid phase carrier is provided with capillary strip, capillary strip comprises micropore, coated layer; The reagent bottle of the sheep anti mouse antiantibody that reagent bottle comprises reagent bottle that the Cistofuran metabolite monoclonal antibody solution is housed, be equipped with horseradish peroxidase-labeled.
Described coated layer is comprised of the Cistofuran metabolite coupled antigen.
Described kit also comprises the 6 bottled reagent bottles that the Cistofuran metabolite titer is arranged, the 2 bottled reagent bottles that the substrate nitrite ion is arranged, the 1 bottled reagent bottle that stop buffer is arranged, the 1 bottled reagent bottle that cleansing solution is arranged, the 1 bottled reagent bottle that damping fluid is arranged, the 1 bottled reagent bottle that derivatization reagent is arranged.
Described solid phase carrier is comprised of 96 hole polystyrene agent plate.
Reagent in each kit enough carries out 96 times to be measured, and both can carry out the detection of a plurality of samples of one-time continuous, also can take plate hole apart repeated detection.During detection, Cistofuran metabolite in the sample will be competed the Cistofuran metabolite monoclonal antibody with the Cistofuran metabolite coupled antigen of the coated layer of capillary strip, after adding the sheep anti mouse antiantibody of horseradish peroxidase-labeled, develop the color with the substrate nitrite ion, the residual quantity of Cistofuran metabolite becomes negative correlation in sample absorbance and the sample, multiply by more again its corresponding extension rate with typical curve, can draw Cistofuran metabolite residual quantity in the sample.
Our experiments show that this kit has very high sensitivity: detection sensitivity is: 0.1 μ g/L.With respect to other Cistofuran metabolite detection methods, the required instrument of this kit is less, and generally all there is outfit in similar laboratory, and required cost is lower.
The beneficial effects of the utility model are: can be used for quick, easy, sensitive, exactly the detection of animal derived food Cistofuran metabolite residual quantity.
(4) description of drawings:
The solid phase carrier one row capillary strip figure of Fig. 1 the utility model kit.
Fig. 2 the utility model solid phase carrier figure.
Fig. 3 the utility model box body, recessed bottle bit architecture schematic diagram;
(5) embodiment:
As shown in Figure 1 to Figure 3, Cistofuran metabolite detection reagent kit, has the recessed bottle position 2 that fixed mould forms in the box body 1, recessed bottle position is used for placing the reagent bottle that solution is housed, the solid phase carrier 3 that 96 hole polystyrene agent plate form, solid phase carrier capillary strip 4 comprise micropore 5 and coated layer 6, coated layer is comprised of the Cistofuran metabolite coupled antigen, the reagent bottle of the sheep anti mouse antiantibody that reagent bottle comprises reagent bottle that the Cistofuran metabolite monoclonal antibody solution is housed, be equipped with horseradish peroxidase-labeled.
In order more to be conducive to detect the residual detection of Cistofuran metabolite, kit can also include the 6 bottled reagent bottles that the Cistofuran metabolite titer is arranged, the 2 bottled reagent bottles that the substrate nitrite ion is arranged, the 1 bottled reagent bottle that stop buffer is arranged, the 1 bottled reagent bottle that cleansing solution is arranged, the 1 bottled reagent bottle that damping fluid is arranged, the 1 bottled reagent bottle that derivatization reagent is arranged.
Concrete composition in the reagent bottle of kit is as follows:
(1) Cistofuran metabolite monoclonal antibody working fluid 10ml/bottle;
(2) Cistofuran metabolite standard solution 1ml/ bottle, concentration are respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L;
(3) the sheep anti mouse antiantibody working fluid 12ml/ bottle of horseradish peroxidase-labeled;
(4) substrate nitrite ion is respectively substrate colour developing A liquid, substrate colour developing B liquid, and substrate nitrite ion A liquid is urea peroxide 7ml/ bottle, substrate nitrite ion B liquid is tetramethyl benzidine 7ml/ bottle;
(5) stop buffer 7ml/ bottle;
(6) damping fluid 50ml/ bottle is for the pH value is 7.2, the 0.2-0.5mol/L phosphate buffer.2 times of dilutions during use.
(7) cleansing solution 40ml/ bottle is for containing 0.6%-0.8% Tween-20,0.05% thimerosal, 0.02mol/L phosphate buffer.20 times of dilutions during use.
(5) derivatization reagent 15.1mg/ bottle, derivatization reagent are the 2-nitrobenzaldehyde.
The preparation method of the main material of using in the kit:
The preparation of Cistofuran metabolite coupled antigen
(1) the haptenic preparation of Cistofuran metabolite
Cistofuran metabolite with carboxyl benzaldehyde is obtained by reaction.
(2) preparation of Cistofuran metabolite coupled antigen
Cistofuran metabolite haptens and ovalbumin are obtained the Cistofuran metabolite coupled antigen by the mixed anhydride method coupling.
The preparation of Cistofuran metabolite monoclonal antibody
(1) animal immune
Immunogene (Cistofuran metabolite haptens and bovine serum albumin(BSA) obtain by the mixed anhydride method coupling) is injected in the Balb/c Mice Body, and immunizing dose is 100 μ g/, makes it produce polyclonal antibody.
(2) Fusion of Cells and cloning
After the mice serum measurement result is higher, get its splenocyte, merge in 9:1 ratio and SP2/0 myeloma cell, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, until obtain the hybridoma cell strain of secrete monoclonal antibody.
(3) cell cryopreservation and recovery
The strain of Cistofuran metabolite monoclonal hybridoma is made 1 * 10 with cryopreserving liquid
9The cell suspension of individual/ml is preserved in liquid nitrogen for a long time.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) production of monoclonal antibody and purifying
The Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.5ml/, 7 days pneumoretroperitoneum injection Cistofuran metabolite monoclonal hybridomas strain 5 * 10
7Individual/as only, to gather ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method ,-20 ℃ of preservations.
(5) 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.
(6) preparation of the sheep anti mouse antiantibody of horseradish peroxidase-labeled
Sodium periodate method after sheep anti mouse antiantibody and horseradish peroxidase (HRP) the employing improvement is carried out coupling to be obtained.
(1) use Cistofuran metabolite detection reagent kit to detect the operation steps of Cistofuran metabolite:
1, takes out the microwell plate that needs quantity.
2, add standard items/sample: add standard items/sample 50 μ l in the micropore of correspondence, adding Cistofuran metabolite monoclonal antibody working fluid 50 μ l/ holes, the mixing that vibrates is gently put in 25 ℃ of lucifuge environment and is reacted 30min.
3, wash plate: liquid in the hole is dried, with wash operating solution 250 μ l/ holes, fully wash 4-5 time, every minor tick 10s pats dry with thieving paper.
4, ELIAS secondary antibody: add ELIAS secondary antibody working fluid 100 μ l in the micropore of correspondence, the mixing that vibrates is gently put in 25 ℃ of lucifuge environment and is reacted 30min.
5, colour developing: add each 50 μ l/ hole of substrate solution nitrite ion, mixing is put in 25 ℃ of lucifuge environment and is reacted 15min.
6, measure: add stop buffer 50 μ l/ holes, mixing is set microplate reader and is measured every hole OD value in the 450nm place.
(2) result judges
(1) calculating of percentage absorptance, the percentage absorptance of standard items or sample equal the mean value (diplopore) of percentage absorbance of standard items or sample divided by the absorbance of first standard (0 standard), multiply by 100%, namely again
The mean light absorbency value of B-standard solution or sample solution
B
0The mean light absorbency value of-0 μ g/L standard solution
(2) drafting of typical curve and calculating
Take standard items percentage absorptance as ordinate, take the semilog of Cistofuran metabolite standard items concentration (μ g/L) as horizontal ordinate, the drawing standard curve map.In the percentage absorptance substitution typical curve with sample, read the corresponding concentration of sample from typical curve, multiply by its corresponding extension rate and be Cistofuran metabolite practical residue limit in the sample.
Claims (4)
1. Cistofuran metabolite detection reagent kit, comprise solid phase carrier, put reagent bottle recessed bottle position, be positioned at the reagent bottle on the recessed bottle position, it is characterized in that: solid phase carrier is provided with capillary strip, capillary strip comprises micropore, coated layer; The reagent bottle of the sheep anti mouse antiantibody that reagent bottle comprises reagent bottle that the Cistofuran metabolite monoclonal antibody solution is housed, be equipped with horseradish peroxidase-labeled.
2. Cistofuran metabolite detection reagent kit as claimed in claim 1 is characterized in that: described coated layer is comprised of the Cistofuran metabolite coupled antigen.
3. Cistofuran metabolite detection reagent kit as claimed in claim 1, it is characterized in that: described kit also comprises the 6 bottled reagent bottles that the Cistofuran metabolite titer is arranged, the 2 bottled reagent bottles that nitrite ion is arranged, the 1 bottled reagent bottle that stop buffer is arranged, the 1 bottled reagent bottle that cleansing solution is arranged, the 1 bottled reagent bottle that damping fluid is arranged, the 1 bottled reagent bottle that derivatization reagent is arranged.
4. Cistofuran metabolite detection reagent kit as claimed in claim 1, it is characterized in that: described solid phase carrier is comprised of 96 hole polystyrene agent plate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201220552158.6U CN202903799U (en) | 2012-10-25 | 2012-10-25 | Macrodantin metabolin detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201220552158.6U CN202903799U (en) | 2012-10-25 | 2012-10-25 | Macrodantin metabolin detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN202903799U true CN202903799U (en) | 2013-04-24 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201220552158.6U Expired - Fee Related CN202903799U (en) | 2012-10-25 | 2012-10-25 | Macrodantin metabolin detection kit |
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| CN (1) | CN202903799U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107315091A (en) * | 2017-06-29 | 2017-11-03 | 重庆师范大学 | One kind detection Cistofuran metabolite quantum dot immune chromatographic test paper, preparation method and applications |
-
2012
- 2012-10-25 CN CN201220552158.6U patent/CN202903799U/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107315091A (en) * | 2017-06-29 | 2017-11-03 | 重庆师范大学 | One kind detection Cistofuran metabolite quantum dot immune chromatographic test paper, preparation method and applications |
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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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130424 Termination date: 20211025 |