CN202794184U - Colloidal gold test paper card for detecting nitroimidazole drug residue - Google Patents
Colloidal gold test paper card for detecting nitroimidazole drug residue Download PDFInfo
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- CN202794184U CN202794184U CN 201220229497 CN201220229497U CN202794184U CN 202794184 U CN202794184 U CN 202794184U CN 201220229497 CN201220229497 CN 201220229497 CN 201220229497 U CN201220229497 U CN 201220229497U CN 202794184 U CN202794184 U CN 202794184U
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- nitroimidazole
- pad
- test paper
- test card
- paper card
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Abstract
The utility model provides a test paper card. The test paper card comprises a bottom plate, sample absorption pads, a conjugate release pad, a reaction membrane and a water absorption pad, wherein the sample absorption pads are adhered to the bottom plate and are sequentially and closely connected, and the partial region of the conjugate release pad is arranged under the sample absorption pads. The reaction membrane layer is provided with a detection line coated with a nitroimidazole drug hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse IgG; and the conjugate release pad is coated with a colloidal gold marker of a nitroimidazole drug monoclonal antibody. The test paper card provided by the utility model has the advantages that the observation time of testing results can be prolonged, the sample absorption pad can fully absorb detection liquid which is in complete contact with a gold-labeled antibody for full reaction, then the detection liquid transfers to the reaction membrane for reaction through chromatography, and errors can be effectively reduced. Due to the adoption of the test paper card, certain interference components in a detection sample are prevented from deactivating the proteins in the gold-labeled antibody and affecting the combination of the gold-labeled antibody and a coating antigen.
Description
Technical field
The utility model belongs to the immunochemistry speed survey technology field of residue of veterinary drug, is specifically related to a kind of Test paper card.
Background technology
The nitroimidazole medicine is that antiprotozoan commonly used in the animal and fowl fodder infects and antimicrobial, be mainly used in preventing and treating poultry organization trichomonad and parasite, the trichomonad of ox, the hemorrhagic enteritis of pig, the parasitic infection of fish, the microsporidiosis of honeybee etc., and has a growth promoting function, but such medicine and some metabolin thereof have carcinogenic to mammal, teratogenesis, mutagenesis and genetoxic, therefore the unreasonable use of nitroimidazole medicine causes edibility animal tissue drug residue problem to jeopardize human food security, China has forbidden metronidazole, Dimetridazole, Lip river nitre azoles uses in carnivorous food, Tinidazole is forbidden using in veterinary drug, therefore, need a kind of not only sensitivity but also accurate method mensuration nitroimidazole medicine.
The method that detects at present the nitroimidazole medicine mainly contains: high performance liquid chromatography-ultraviolet chromatogram, high performance liquid chromatography diode display detecting device method, gas chromatography nitrogen phosphorous detector method, High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry method, vapor-phase chromatography etc., instrument detection method complex operation and expense are higher, apply being restricted.
The utility model content
The purpose of this utility model is to provide the test card that a Species sensitivity is high, cost is low, simple to operate, detection time is short for above-mentioned deficiency.
Test card of the present utility model comprises base plate, is attached to the absorption of sample pad, bond release pad, reaction film and the adsorptive pads that closely link to each other successively on the base plate, different from conventional test card is, it is not to cover fully under the absorption of sample pad that bond wherein discharges pad, but the subregion is covered under the absorption of sample pad, and 1/2nd to 1/3rd zones that the preferred combination thing discharges pad are covered under the absorption of sample pad.
The benefit of doing like this is to prolong testing result observing time, and the absorption of sample pad also can fully absorb tracer liquid with golden labeling antibody and contact abundant reaction fully, and chromatography reacts to reaction film again, can effectively reduce error.Can also prevent from detecting that some interfering components lose activity the albumen in the golden labeling antibody in the sample, affect the combination of golden labeling antibody and coating antigen.
Be coated with respectively the detection line trace " ︱ " of nitroimidazole medicine hapten-carrier protein conjugate formation and the nature controlling line trace " ︱ " that coated sheep anti-mouse igg consists of on the described reaction film, detection line is parallel with nature controlling line, and detection line is positioned at apart from bond and discharges pad one side 0.4cm place.Thereby can be used for the detection of nitroimidazole medicament residue.
But the material that the base plate of the utility model test card does not absorb water for PVC plate or other hard; Reaction film can be nitrocellulose filter or cellulose acetate membrane; The absorption of sample pad can be made by glass wool or polyester material; Bond discharges bed course to be made by fiber, and bond discharges bed course and is coated with nitroimidazoles medicine monoclonal antibody-collaurum bond.Discharge pad and water accepting layer is coated with diaphragm at absorption of sample pad and bond, at glass wool and the about 0.4cm of fibrage one side place's spray sample mark line.
Coupling nitroimidazole pharmaceutical carrier albumen can be used ovalbumin, human serum albumins, bovine serum albumin(BSA), hemocyanin etc., and the contrast trace assembled arrangement of the detection line trace on reaction film and nature controlling line can be " ‖ "
The utility model test card has following beneficial effect:
1. high specificity, susceptibility is high.Nitroimidazole medicine quick detection test paper card is prepared from as the basis take the monoclonal 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 specificity and affinity impact, and has higher mark rate.Therefore, the quick detection test paper jig has higher specificity and susceptibility.
2. easy and simple to handle quick.Need not any other reagent when using the quick detection test paper card, as long as sample is splashed in the reagent hole clipping with dropper, add rear timing, observations in the 10min.
3. show testing result image, accurately directly perceived.The quick detection test paper card reaches " ‖ " trace as the positive and the negative marker of detection line to show red line look " ︱ ", namely be illustrated in the detected sample liquid at a nitrocellulose filter demonstration red line " ︱ " trace and contain the nitroimidazole medicine, article two, red line " ‖ " trace is illustrated in the detected sample and does not contain the nitroimidazole medicine, the result judges image, directly perceived, accurate, simple and clear, is not easy to occur negative and the false positive erroneous judgement.
4. cost is low, small investment.Use Rapid detection test strip, 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.
5. be easy to apply on a large scale.The quick detection test paper card is simple to operate, everybody operates, can better satisfy different levels personnel's needs, arrive individual cultivation etc. such as specialty chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, animal product processing, collectionization cultivation, have wide market outlook and larger economical, societal benefits.
Description of drawings
Fig. 1 is nitroimidazole medicine quick detection test paper card structure synoptic diagram, wherein, and 1, the absorption of sample pad; 2, bond discharges pad; 3, reaction film; 4, adsorptive pads; 5, detection line; 6, nature controlling line; 7, base plate;
Fig. 2 is nitroimidazole medicine quick detection test paper card plan structure synoptic diagram, and wherein 8-1 and 8-2 are the diaphragms that covers the test card two ends, and 9 is mark line;
Fig. 3 is nitroimidazole medicine quick detection test paper card negative (Fig. 3 .a), positive (Fig. 3 .b), invalid (Fig. 3 .c) synoptic diagram that develops the color, C Quality Control district wherein, and T is detection zone.
Embodiment
The assembling of embodiment 1 test card
Referring to Fig. 1; Fig. 2: absorption of sample pad 1 usefulness glass wool is made; bond discharges bed course 2 has nitroimidazole anti-drug monoclonal antibody-colloid gold label thing for absorption fibrage; reaction rete 3 is nitrocellulose filter; adsorptive pads 4 is made for absorbent material layer; 5 are coated with nitroimidazole medicine-carrier protein couplet thing for detection zone; 6 consist of nature controlling line for the Quality Control district is coated with sheep anti-mouse igg; base plate 7 is the PVC backing; make with the plastic tab bar; on the PVC backing, adhere in order nitrocellulose filter; adsorptive pads; bond discharges pad and sample pad; sample pad is covered bond discharge pad 1/3rd places; 8-1 is white diaphragm; covering absorption of sample pad and bond discharges on the pad; discharging the corresponding diaphragm 8-1 of pad intersection position at absorption of sample pad and bond is partial to absorption of sample pad one side 0.5cm place and is printed on mark line 9; 9 right-hand member is printed on arrow and max printed words, and filter paper layer 4 is and is coated with light blue diaphragm 8-2 on the water accepting layer (handle end).
Make nitroimidazole medicine quick detection test paper card and at first need prepare coupling nitroimidazole pharmaceutical carrier albumen, the preparation detection line, the preparation collaurum for the preparation of golden labeling antibody fiber, secondly must prepare sheep anti-mouse igg antibody, is used for making nature controlling line.Below be the preparation method of associated materials:
1. the nitroimidazole medicine is haptenic synthetic
0.85g metronidazole and 1.0g succinic anhydride add the 10ml pyridine, stir 12-20 hour solvent evaporated under the room temperature, acetic acid ethyl dissolution, saturated ammonium chloride solution washing, washing, drying, after the desolventizing in cyclohexane-ethyl acetate recrystallization obtain white solid, be the nitroimidazole haptens.
2. the Bei – nitroimidazole medicine haptens processed of coating antigen and ovalbumin conjugate is synthetic
1) gets 15mg metronidazole haptens with 1.5ml DMF dissolving, be cooled to 10 ℃;
2) get isobutyl chlorocarbonate 10 μ l and add 1) in, 10 ℃ of stirring reaction 30min;
3) get 25mg ovalbumin 2.2ml 50mmol/L Na
2CO
3Dissolve 10 ℃ of reaction 4h, then 4 ℃ are spent the night;
4) with 4 ℃ of dialysis of 0.01mol/lPBS 3 days, change dislysate every day 3 times, to remove unreacted small-molecule substance, packing saves backup in-20 ℃.
3. immunogenic preparation
1) gets 12mg metronidazole haptens, be dissolved among the 1mL DMF;
2) getting 15mg EDC fully dissolves rear in adding 1 with 0.2ml water) in, 24h stirred under the room temperature;
3) taking by weighing BSA 40mg, make it fully to be dissolved in the 3ml water, with step 2) reactant liquor that obtains dropwise slowly is added drop-wise in the bovine serum albumin solution, and stirs 24 h under room temperature;
4) change dislysate 3 times every day with 4 ℃ of dialysis of 0.01mol/lPBS 3d, to remove unreacted small-molecule substance, packing saves backup in-20 ℃.
5. the evaluation of nitroimidazole medicine-carrier protein couplet thing
Carrier protein, nitroimidazole medicine haptens, nitroimidazole medicine-carrier protein couplet thing 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 200-800 nm scope, obtain the absorption curve of carrier protein, nitroimidazole medicine haptens, nitroimidazole medicine-carrier protein couplet thing with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of nitroimidazole medicine and carrier protein couplet.
6. the preparation of nitroimidazole anti-drug monoclonal antibody
6.1 animal immune
Immunogene is injected in the Balb/c Mice Body, and immunizing dose is 80 μ g/, makes it produce polyclonal antibody.Immune Balb/c mouse boosting cell is got in Fusion of Cells and cloning, merges in 5:1 ratio and SP2/0 myeloma cell, adopts the indirect competitive ELISA method to measure cell conditioned medium liquid, screens positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, directly to the hybridoma cell strain that obtains the stably excreting monoclonal antibody.
6.2 cell cryopreservation and recovery
The strain of nitroimidazole medicine monoclonal hybridoma is made 1 * 10 with cryopreserving liquid
8The 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.
6.3 the preparation and purification of monoclonal antibody
The Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.4ml/, 7 days pneumoretroperitoneum injection nitroimidazole medicine monoclonal hybridomas strain 5 * 10
5Individual/as only, to gather ascites after 7 days.Carry out purifying with sad-saturated ammonium sulfate method, the ascites behind the purifying is put into-20 ℃ of environment and is preserved.
7. the preparation of nitroimidazole anti-drug monoclonal antibody and colloid gold label thing
7.1 the preparation of collaurum
With two ionized waters that boil off 1% gold chloride is diluted to 0.01%, puts to stir on the magnetic force heating rod stirrer and boil, every 100ml 0.01% gold chloride adds 2ml 1% trisodium citrate, continues to boil, and stopped heating when taking on a red color to liquid is supplied dehydration after being cooled to room temperature.The collaurum outward appearance for preparing should be pure, bright, without precipitation and floating thing, the term of validity is one month.
The preparation of nitroimidazole anti-drug monoclonal antibody-colloid gold label thing
Under magnetic agitation, the pH value to 8.2 with 0.1mol/L sal tartari is transferred collaurum adds 50-100 μ g antibody by every milliliter of colloidal gold solution and adds the nitroimidazole drug antibody, continues to stir and evenly mix 30min, adding 10%BSA to final concentration be 1%, leave standstill 30min.12000rpm, 4 ℃ of centrifugal 30min abandon supernatant, and precipitation is with redissolving the damping fluid washed twice, will precipitate with the redissolution damping fluid of initial collaurum volume 1/20 resuspended, put 4 ℃ for subsequent use, preserved 60 days.
Redissolution damping fluid: with containing bovine serum albumin(BSA) (BSA) 0.02 ~ 0.1%, Tween-20 0.05 ~ 0.2%, PVP-300 pyrrolidone 0.3%, the boric acid redissolution damping fluid of 0.02M pH9.0.
8. the preparation of sheep anti-mouse igg antibody
The preparation of sheep anti mouse antiantibody: as immune animal, as immunogene the pathogen-free domestic goat is carried out immunity take mouse source antibody with goat, obtain the sheep anti mouse antiantibody.
9. nitroimidazole medicine quick detection test paper card examinations principle
After nitroimidazole drug test test card sample pad end drips testing sample solution, solution to be checked spreads to reaction film together by the golden labeling antibody in the golden labeling antibody cotton, and finally infiltrate in the filter paper, detecting the nitroimidazole medicine in the diffusion process can combine with golden labeling antibody, and then seal the antigen-combining site of nitroimidazole medicine on the golden antibody, stop the detection trace of golden labeling antibody coupling nitroimidazole medicine hapten-carrier albumen on reaction film to be combined, can not show the detection trace, sheep anti-mouse igg antibody then can be combined with golden labeling antibody, the colour developing of Quality Control district, form a red contrast trace " ︱ ", be a trace positive (Fig. 3 b), then can not stop the carrier protein of golden labeling antibody coupling nitroimidazole medicine on cellulose membrane to detect trace without the nitroimidazole medicine in the opposite sample solution is combined, detection zone shows red trace " ︱ ", same sheep anti-mouse igg antibody also is combined with golden labeling antibody, the Quality Control district also shows red contrast trace " ︱ ", (Fig. 3 a) to form two red lines " ‖ " negative marker, if the Quality Control district does not have red-label to show on the cellulose membrane, then test card invalid (Fig. 3 c).
Claims (8)
1. colloidal gold test paper card that detects the nitroimidazole medicament residue, comprise base plate, be attached to the absorption of sample pad, bond release pad, reaction film and the adsorptive pads that closely link to each other successively on the base plate, it is characterized in that 1/2nd to 1/3rd zones that bond discharges pad are covered under the absorption of sample pad.
2. test card as claimed in claim 1 is characterized in that, has on the described reaction film to be coated with detection line trace " ︱ " that nitroimidazole medicine hapten-carrier protein conjugate consists of and the nature controlling line trace " ︱ " of coated sheep anti-mouse igg formation.
3. test card as claimed in claim 2 is characterized in that, described detection line is parallel with nature controlling line.
4. test card as claimed in claim 3 is characterized in that, detection line is positioned at apart from bond and discharges pad one side 0.4cm place.
5. test card as claimed in claim 1 is characterized in that, is coated with respectively diaphragm at the two ends of test card.
6. test card as claimed in claim 1 is characterized in that, described base plate is the PVC plate, and described reaction film is nitrocellulose filter.
7. test card as claimed in claim 1 is characterized in that, described absorption of sample pad is made by glass wool or polyester material.
8. test card as claimed in claim 1 is characterized in that, described bond is released bed course and made by fiber.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220229497 CN202794184U (en) | 2012-05-21 | 2012-05-21 | Colloidal gold test paper card for detecting nitroimidazole drug residue |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220229497 CN202794184U (en) | 2012-05-21 | 2012-05-21 | Colloidal gold test paper card for detecting nitroimidazole drug residue |
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| Publication Number | Publication Date |
|---|---|
| CN202794184U true CN202794184U (en) | 2013-03-13 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201220229497 Expired - Lifetime CN202794184U (en) | 2012-05-21 | 2012-05-21 | Colloidal gold test paper card for detecting nitroimidazole drug residue |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792349A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Quick detection test strip for nitroimidazoles drug residues |
-
2012
- 2012-05-21 CN CN 201220229497 patent/CN202794184U/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792349A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Quick detection test strip for nitroimidazoles drug residues |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20130313 |
|
| CX01 | Expiry of patent term |