CN202956385U - Test paper used for detecting florfenicol and thiamphenicol - Google Patents
Test paper used for detecting florfenicol and thiamphenicol Download PDFInfo
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- CN202956385U CN202956385U CN201220519389.7U CN201220519389U CN202956385U CN 202956385 U CN202956385 U CN 202956385U CN 201220519389 U CN201220519389 U CN 201220519389U CN 202956385 U CN202956385 U CN 202956385U
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Abstract
The utility model provides test paper used for detecting florfenicol and thiamphenicol. The test paper used for detecting the florfenicol and the thiamphenicol comprises a box body of a test paper box.
Description
Technical field
The utility model relates to a kind of test paper that detects Florfenicol and Thiamphenicol, particularly detects the test paper of Florfenicol and Thiamphenicol in milk.
Background technology
Florfenicol, Thiamphenicol and chloromycetin all belong to the chloromycetin medicine, and wherein Thiamphenicol is the derivant of Florfenicol, and their antibacterial action is similar to chloromycetin.Because there is serious side effects in the chloromycetin medicine, in succession forbid in the world or strictly limit and use chloromycetin.Florfenicol and Thiamphenicol all have higher antibacterial activity, the existing trend that substitutes chloromycetin, and the bacteriosis that is mainly used in treating fish, pig, ox and poultry is used on a large scale in aquaculture.But the extensive application on Production of Livestock and Poultry of Florfenicol and Thiamphenicol also causes it residual in animal products, the harm public health.In European Union (EEC) 96/23 instruction, stipulate, Thiamphenicol MRL value is 50 μ g/kg, Florfenicol MRL value is 50 ~ 2000 μ g/kg, the MRL value of China's regulation Thiamphenicol in various animal tissues is 50 μ g/kg, Florfenicol sign residue is florfenicol amine, and its MRL value in muscle is 1000 μ g/kg.
In animal tissue, the residue detection of chloromycetin medicine is mainly physics and chemistry detection method, immunoassay.The physics and chemistry detection method has vapor-phase chromatography, gas chromatography and mass spectromentry method etc., and physics and chemistry detection method degree of accuracy is high, but it exists testing cost high, and the checkout equipment complexity, to having relatively high expectations of testing staff, detect the shortcomings such as length consuming time; Immune analysis method commonly used, be mainly the enzyme linked immunosorbent detection method at present, and its testing cost needed is still higher, is not suitable for enterprise and detects cheaply needs.
The utility model content
The purpose of this utility model provides a kind of highly sensitive, cost is low, simple to operate, detection time is short detection Florfenicol and the test paper of Thiamphenicol.
Test paper of the present utility model comprises the test paper box body, there is the holding appliance in 12 holes and place 12 tool plug reagent barrels wherein, reagent barrel comprises micropore reagent strip and 8 test strips, the micropore reagent strip has 8 reacting holes, there is the micropore plug on reacting hole, test strips is by base plate, be attached on base plate successively closely connected absorption of sample pad, reaction film, the composition such as adsorptive pads and diaphragm, different from conventional test strips is, Thiamphenicol monoclonal antibody wherein-colloid gold label thing gold is not to be coated on bond to discharge on pad, be assembled into test strips, but freeze-drying is in micropore reagent strip plate hole.
By golden labeling antibody freeze-drying, in the micropore reagent strip, the benefit of doing like this is in testing process, can make golden labeling antibody fully contact with sample liquid to be checked, fully reaction, thus reduce error, increase the reaction sensitivity of whole system.
Be coated with respectively the detection line trace " ︱ " of Thiamphenicol hapten-carrier protein conjugate formation and the nature controlling line trace that coated sheep anti mouse antiantibody forms on described reaction film.On reaction film, detection line trace and nature controlling line trace assembled arrangement are " ︱ ︱ ".
Described detection line is positioned at apart from absorption of sample pad one side 5-8mm, preferred 6mm, and described nature controlling line is positioned at apart from detection line 4-7mm, preferably 5mm.
Described test strips covers on the diaphragm on the absorption of sample pad the MAX mark line.
In the utility model test paper, the test strips base plate can be the material that PVC base plate or other hard do not absorb water; 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; Diaphragm is PE material diaphragm; The test paper box body is carton box; Reagent barrel is the plastics reagent barrel; Holding appliance is the rigid support material.
The utility model test paper has following beneficial effect:
1. highly sensitive.Florfenicol and Thiamphenicol Test paper be take the monoclonal antibody of colloid gold label high-affinity and are prepared from as basis, priceless strong formation between gold grain and 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.Therefore, the utility model test paper has higher specificity and sensitivity.This test paper is 5 μ g/L to the detectability of Florfenicol and Thiamphenicol.
2. easy and simple to handle quick.While using detection paper, without any other reagent, as long as sample solution to be checked is dripped in micropore reagent, after mixing, the test strips that test paper is indicated to MAX wire tag end is downward, timing after the micropore reagent after insertion is hatched, observations in 5min.
3. show testing result image, accurately directly perceived.Detection is that test strips is usingd and shown that red line look " ︱ " reaches " ‖ " trace as the positive and negative marker, on nitrocellulose filter, show content that a red line " ︱ " trace is illustrated in Florfenicol in detected sample liquid or Thiamphenicol greater than or equal to test paper the lowest detectable limit to Florfenicol and Thiamphenicol, article two, red line " ‖ " trace be illustrated in detected sample Florfenicol or Thiamphenicol content lower than test paper the lowest detectable limit to Florfenicol and Thiamphenicol, result is judged image, intuitively, accurately, simple and clear, be not easy to occur the result erroneous judgement.
4. cost is low, small investment.Use the utility model test paper, do not need separately to join instrument and equipment and other reagent, Site Detection is settled at one go, with low cost, small investment, instant effect.
5. be easy to apply on a large scale.Test paper is simple to operate, can better meet different levels personnel's needs, as specialty chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, dairy industry enterprise etc., has wide market outlook and larger economical, societal benefits.
The accompanying drawing explanation
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 the utility model ELISA test strip process decision chart as a result.
Fig. 5 is the utility model reagent barrel structural representation;
The vertical view that Fig. 6 is the utility model holding appliance;
The side view that Fig. 7 is the utility model box body and holding appliance.
Embodiment
One, the assembling of Florfenicol and Thiamphenicol Test paper preparation
1), test strips:
Absorption of sample pad 1, reaction film 2, adsorptive pads 3 and diaphragm are pasted successively in order on described base plate 6; the end of absorption of sample pad is connected with reaction film top; the end of reaction film is connected 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; be adhesive with diaphragm 7 on test strips absorption of sample pad; be printed on the MAX mark line on diaphragm; detection line 4 is coated with Thiamphenicol 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 in plate hole, freeze-drying has Thiamphenicol anti-drug monoclonal antibody-colloid gold label thing.
3) by 1) test strips and 2) the micropore reagent strip plastics reagent barrel 10 of packing into, the plastics reagent barrel is positioned in the holding appliance 11 in test paper box body 12.
Two, the use of test paper
Milk sample solution to be checked drips 200 μ l in micropore reagent, and after mixing, the test strips that test paper is indicated to MAX wire tag end is downward, counts observations in 5min after the micropore reagent after insertion is hatched.
Three, Analysis of test results
Florfenicol and the Thiamphenicol content in sample is prescribed a time limit to its lowest detection greater than or equal to test paper, Thiamphenicol medicine monoclonal-colloid gold label thing is combined with Florfenicol or Thiamphenicol, antigen binding site on the gold labeling antibody is closed, thereby because competitive reaction, can not be combined with Thiamphenicol hapten-carrier protein conjugate and red stripes do not occur in detection zone.Negative sample owing to lacking the antigen-antibody competitive reaction, will red stripes occur in testing process on detection line and nature controlling line.As shown in Figure 4.
Positive: when nature controlling line (C) shows a red stripes, and detection line (T) does not develop the color, and test strips, to show red line look " ︱ ", is judged to the positive, as shown in Fig. 4 .a figure.
Negative: when nature controlling line (C) shows a red stripes, detection line (T) simultaneously also demonstrates a red stripes, and detection line (T) color approaches or while being shallower than nature controlling line (C), and test strips be take and shown that the red line look is " ‖ ", be judged to feminine gender, as shown in Fig. 4 .b.
Invalid: when nature controlling line (C) does not demonstrate red stripes, no matter whether detection line (T) demonstrates red stripes, and it is invalid that this test strips is judged to, as shown in Fig. 4 .c, 4.d.
Four, the material preparation method of using in Test paper is as follows:
1, the synthetic and evaluation of Thiamphenicol hapten-carrier protein conjugate
The Thiamphenicol medicine is small-molecule substance, only has immunoreactivity, there is no immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just there is immunogenicity.
1) the haptenic preparation of Thiamphenicol:
0.39g the solution of bromo-acetic acid tert-butyl in 5ml dimethyl sulfoxide (DMSO) (DMSO), slowly be added dropwise in 0.71g Thiamphenicol and the potpourri of 1ml pyridine in 10ml DMSO under 40 ℃.After dropwising, continue reaction after 4 hours, steaming desolventizes.After simple column chromatography for separation, except desolventizing, add 20ml DMSO and 5ml formic acid, room temperature reaction 20 hours, steaming desolventizes, and in ethanol-water system, recrystallization obtains the Thiamphenicol haptens.
2) immunogenic preparation-Thiamphenicol haptens and bovine serum albumin(BSA) conjugate are synthetic
Get Thiamphenicol haptens 10mg and be dissolved in 1ml water, obtain I liquid; Get bovine serum albumin(BSA) 50mg and be dissolved in 5ml water, then add wherein carbodiimides (EDC) 25mg priming reaction 30min, obtain II liquid; After I liquid is added to II liquid, stirring reaction spends the night, and obtains immunogene.
3) preparation of coating antigen-Thiamphenicol haptens and ovalbumin conjugate are synthetic
Get Thiamphenicol haptens 10mg and be dissolved in 1ml water, obtain I liquid; Get ovalbumin 50mg and be dissolved in 5ml water, then add wherein EDC 25mg priming reaction 30min, obtain II liquid; After I liquid is added to II liquid, stirring reaction spends the night, and obtains coating antigen.
4) evaluation of Thiamphenicol hapten-carrier conjugates
Carrier protein, Thiamphenicol haptens, Thiamphenicol hapten-carrier protein conjugate are made into to the solution of 0.5mg/ml with the PBS of pH7.4, with 0.01mol/L pH7.4 PBS, return to zero, in the interscan of wavelength 200-800 nm scope, obtain the absorption curve of carrier protein, Thiamphenicol haptens, Thiamphenicol hapten-carrier protein conjugate with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows Thiamphenicol haptens and carrier protein couplet success.
2, the preparation of Thiamphenicol anti-drug monoclonal antibody
(1) prepare 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, in the 9:1(quantitative proportion) ratio and SP2/0 myeloma cell's fusion, screening obtains the Thiamphenicol medicine monoclonal hybridoma strain of stably excreting Thiamphenicol anti-drug monoclonal antibody.
Obtain the Thiamphenicol monoclonal antibody through screening and hand over tumor cell strain D-4-1.This hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC, China, Beijing) on May 3rd, 2012, and preserving number is CGMCC No. 6067.The antibody of this hybridoma cell strain secretion is good for Florfenicol and Thiamphenicol specificity, and detection sensitivity can reach 5 μ g/L.
C. cell cryopreservation and recovery
Hybridoma is made to 1 * 10 with cryopreserving liquid
9The cell suspension of individual/ml is preserved for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
D. the preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in to cell culture medium, is cultivated under 37 ℃ of conditions, by sad-saturated ammonium sulfate method, the nutrient solution obtained is carried out to 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), 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
Using sheep as immune animal, and the mouse source antibody of take carries out immunity as immunogene to the pathogen-free domestic sheep, obtains the sheep anti mouse antiantibody.
4, the preparation of Thiamphenicol anti-drug monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
With two ionized waters that boil off, 1% gold chloride is diluted to 0.01%(quality percentage composition), put on the magnetic force heating stirrer and stir and boil, every 100ml 0.01% gold chloride adds 2.5 ml 1% trisodium citrates, continue agitating heating and react stop heating when liquid takes on a red color, supply dehydration after being cooled to room temperature.The collaurum outward appearance prepared is pure, bright, nothing precipitates and floating thing.
(2) preparation of Thiamphenicol anti-drug monoclonal antibody-colloid gold label thing
Under magnetic agitation, adjust the pH value to 7.0 of collaurum with 0.2mol/L sal tartari, standard by 50-100 μ g antibody/ml collaurum adds above-mentioned Thiamphenicol anti-drug monoclonal antibody in colloidal gold solution, continue to stir and evenly mix 30min, the final concentration in colloidal gold solution is the 1%(volumn concentration to bovine serum albumin(BSA) to add 10% bovine serum albumin(BSA)), standing 30min.12000rpm, 4 ℃ of centrifugal 30min, abandon supernatant, precipitation is with redissolving the damping fluid washed twice, the redissolution damping fluid that is initial collaurum volume 1/20 with volume will precipitate resuspended, the concentration of the Thiamphenicol anti-drug monoclonal antibody obtained-colloid gold label thing solution is 50 μ g monoclonal antibodies/ml solution, put 4 ℃ standby.
Redissolution damping fluid: containing bovine serum albumin(BSA), the 0.02mol/L of Tween-80, the phosphate solution of pH7.2, wherein the final concentration of bovine serum albumin(BSA) in the redissolution damping fluid is 0.06～0.1%(volumn concentration), the final concentration of Tween-80 in the redissolution damping fluid is 0.05～0.10%(quality percentage composition).
5, by Thiamphenicol anti-drug monoclonal antibody-colloid gold label thing freeze-drying to micropore reagent
Add 50 μ l Thiamphenicol anti-drug monoclonal antibodies-colloid gold label thing in micropore reagent microwell plate, put into freeze drier, condenser temperature is under-70 ℃ of conditions, after pre-freeze 4h, freeze-drying 14h again, can take out, obtain the micropore reagent that freeze-drying has Thiamphenicol anti-drug monoclonal antibody-colloid gold label thing.
6, the preparation of absorption of sample pad
The absorption of sample pad is placed in containing bovine serum albumin(BSA) (bovine serum albumin(BSA) is the 0.5%(volumn concentration at the final concentration of damping fluid)), pH is 7.2, the 0.05mol/L phosphate buffer soaks 2h, 37 ℃ to dry 2h standby.
7, the preparation of reaction film
Coated process: with phosphate buffer, Thiamphenicol haptens-ovalbumin conjugate is diluted to 10mg/mL, with Isoflow point film instrument, it is coated in to the detection zone on nitrocellulose filter, package amount is 1.0 μ g/cm
2With 0.01mol/L, pH 7.4 PBS damping fluids, sheep anti-mouse igg antibody is diluted to 200 μ g/ml, with Isoflow point film instrument, it is coated in to the Quality Control district on nitrocellulose filter, package amount is 1.0 μ g/cm2.The reaction film be coated with is placed in to dry 2h under 37 ℃ of conditions, standby.
Claims (8)
1. a test paper that detects Florfenicol and Thiamphenicol; it is characterized in that test paper comprises the test paper box body, has holding appliance and places tool plug reagent barrel wherein; reagent barrel comprises micropore reagent strip and test strips, test strips by base plate, be attached on base plate successively closely connected absorption of sample pad, reaction film, adsorptive pads and diaphragm and form.
2. test paper as claimed in claim 1, is characterized in that on described reaction film having and be coated with detection line trace " ︱ " that Thiamphenicol hapten-carrier protein conjugate forms and the nature controlling line trace " ︱ " of coated sheep anti mouse antiantibody formation.
3. test paper as claimed in claim 2, is characterized in that described detection line is parallel with nature controlling line.
4. as claim 1,2 or 3 described test paper, it is characterized in that described detection line is positioned at apart from absorption of sample pad one side 5-8mm, described nature controlling line is positioned at apart from detection line 4-7mm.
5. test paper as claimed in claim 1, 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, and diaphragm is PE material diaphragm.
6. test paper as claimed in claim 1, is characterized in that described test paper box body is carton box, and reagent barrel is the plastics reagent barrel, and holding appliance is the rigid support material.
7. test paper as claimed in claim 1, is characterized in that the described micropore reagent strip hole that responds, and has the micropore plug on reacting hole.
8. test paper as claimed in claim 7, in described micropore reagent, freeze-drying has Thiamphenicol anti-drug monoclonal antibody-colloid gold label thing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201220519389.7U CN202956385U (en) | 2012-10-11 | 2012-10-11 | Test paper used for detecting florfenicol and thiamphenicol |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201220519389.7U CN202956385U (en) | 2012-10-11 | 2012-10-11 | Test paper used for detecting florfenicol and thiamphenicol |
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| CN202956385U true CN202956385U (en) | 2013-05-29 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105004860A (en) * | 2015-07-24 | 2015-10-28 | 烟台昊清生物科技有限公司 | Florfenicol rapid detection kit and preparation and use method thereof |
| CN105137068A (en) * | 2015-07-24 | 2015-12-09 | 烟台昊清生物科技有限公司 | Florfenicol on-site test paper, and preparation and use methods thereof |
| CN110508029A (en) * | 2019-09-04 | 2019-11-29 | 武玉香 | It is a kind of for extracting the immune affinity column of Florfenicol, chloramphenicol, Thiamphenicol simultaneously |
-
2012
- 2012-10-11 CN CN201220519389.7U patent/CN202956385U/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105004860A (en) * | 2015-07-24 | 2015-10-28 | 烟台昊清生物科技有限公司 | Florfenicol rapid detection kit and preparation and use method thereof |
| CN105137068A (en) * | 2015-07-24 | 2015-12-09 | 烟台昊清生物科技有限公司 | Florfenicol on-site test paper, and preparation and use methods thereof |
| CN105137068B (en) * | 2015-07-24 | 2017-03-01 | 鲁东大学 | Florfenicol Site Detection reagent paper and its preparation, using method |
| CN110508029A (en) * | 2019-09-04 | 2019-11-29 | 武玉香 | It is a kind of for extracting the immune affinity column of Florfenicol, chloramphenicol, Thiamphenicol simultaneously |
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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: 20130529 Termination date: 20211011 |
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| CF01 | Termination of patent right due to non-payment of annual fee |