CN201181297Y - Colloidal gold test paper card for detecting Ractopamine medicine residue - Google Patents
Colloidal gold test paper card for detecting Ractopamine medicine residue Download PDFInfo
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- CN201181297Y CN201181297Y CNU2008200788397U CN200820078839U CN201181297Y CN 201181297 Y CN201181297 Y CN 201181297Y CN U2008200788397 U CNU2008200788397 U CN U2008200788397U CN 200820078839 U CN200820078839 U CN 200820078839U CN 201181297 Y CN201181297 Y CN 201181297Y
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- ractopamine
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Abstract
The utility model provides a detection test paper card, which comprises a bottom plate, and a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad, which are attached on the bottom plate and are tightly connected with each other in sequence, wherein a portion region of the conjugate release pad is covered by the sample absorption pad. The reaction membrane layer has a detection line coated with ractopamine-carrier protein conjugation and a quality control line coated with goat anti-mouse IgG; and the conjugate release pad is coated with colloidal gold mark with ractopamine. The test paper card can prolong the observation time of the detection result, the sample absorption pad can fully absorb the detected liquid to make the detection liquid completely contact and react with a gold-labeled antibody, and then detected liquid is separated by chromatography and reacts with a reaction membrane, thereby effectively reducing the error. The test paper card can also prevent the deactivation of the protein in the gold-labeled antibody due to the presence of interference components in the detected sample from influencing the combination of the gold-labeled antibody with a coatingen.
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 detection test card.
Background technology
Ractopamine (Ractopamine) belongs to beta-stimulants.Beta-stimulants is that the heavy partitioning agent of nutrition is a kind of, is a class formation and the function class phenolethanolamine analog derivative like adrenaline and norepinephrine, and it can accelerate growth of animals or poultry speed, reduces the ketoboidies fat content, improves lean meat percentage.Being under an embargo at European Union's beta-stimulants is used for the growth promoter of poultry, and also there is explicit stipulation in China in " forbid use types of drugs catalogue " in feed and animal drinking water.But because Ractopamine belongs to non-prescribed medicine, and price is far below clenbuterol, so its illegal application person is on the increase.This method is a Ractopamine monoclonal antibody enzyme linked immunological quick detection kit, can be used for the residue detection in urine sample, the animal tissue's (muscle, liver etc.).
The detection method that Rct opamine residue is commonly used has two kinds at present.A kind of is stratographic analysis, as vapor-phase chromatography (GC), because complicated instrument and equipment and loaded down with trivial details process are not suitable for on-site supervision and great amount of samples examination.Another kind is an immunological method, as enzyme linked immunosorbent assay (ELISA).Its shortcoming is that detection time is long, and expense is higher, is unfavorable for promoting the use of in grass-roots unit.And these two kinds of methods all need the professional and technical personnel to operate.
The utility model content
The purpose of this utility model is to provide the test card that a kind of susceptibility height, cost are low, simple to operate, detection time is short at above-mentioned deficiency.
Test card of the present utility model comprises base plate, discharges pad, reaction film and adsorptive pads attached to the absorption of sample pad that closely links to each other successively on the base plate, bond, different with 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 the half territory that the preferred combination thing discharges pad is 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 and the complete full contact reaction of golden labeling antibody, and chromatography reacts to reaction film again, can effectively reduce error.Can also prevent to detect that some interfering components lose activity the albumen in the golden labeling antibody in the sample, influence combining of golden labeling antibody and coating antigen.
Be coated with the detection line trace " | " of Ractopamine-carrier protein couplet thing formation and the nature controlling line trace " | " that bag is constituted by sheep anti-mouse igg on the described reaction film respectively.Thereby can be used for the detection of Rct opamine 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 pad can be made by fiber, and bond discharges bed course and is coated with antibody-collaurum bond.On absorption of sample pad and bond release pad and water accepting layer, be coated with diaphragm, at glass wool and the about 0.4cm of fibrage one side place's spray sample mark line.
Coupling Ractopamine gold mark carrier protein can be with ovoserum albumen or human serum albumins, bovine serum albumin(BSA), hemocyanin etc., and the contrast trace assembled arrangement of detection line trace on reaction film and nature controlling line can be " || ".
The utlity model has useful good effect, it is every a bit that Ractopamine quick detection test paper jig is got row ready:
1. high specificity, the susceptibility height.Ractopamine quick detection test paper card is that the basis is prepared from 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 to monoclonal specificity and affinity influence, and has higher mark rate.Therefore, the quick detection test paper jig has higher specificity and susceptibility.Can detect 5ng/ml.
2. easy and simple to handle quick.Need not any other reagent when using the quick detection test paper card,, drip 3 (about 100 microlitres), add the back timing, observations in 5～8min as long as sample is splashed in the reagent hole clipping with dropper.
3. show testing result image, accurately directly perceived.The quick detection test paper card is to show that red line look " | " and " || " trace are as the positive and the negative marker that detect, showing on nitrocellulose filter promptly that a red line " | " trace is illustrated in the detected sample liquid contains Ractopamine, article two, red line " || " trace is illustrated in and does not contain Ractopamine in the detected sample, 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 the quick detection test paper bar, do not need to join in addition instrument and equipment and other reagent, the on-the-spot detection 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 can operate, can better satisfy different levels personnel's needs, arrive individual breed etc. as specialty chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, animal product processing, collectionization breed, have vast market prospect and bigger economical, societal benefits.
Description of drawings
Fig. 1 is a Ractopamine 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 a Ractopamine 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 Ractopamine quick detection test paper card feminine gender (Fig. 3 .a), positive (Fig. 3 .b), invalid (Fig. 3 .c) synoptic diagram that develops the color, T Quality Control district wherein, and C is a detection zone.
Embodiment
The assembling of embodiment 1 test strips
Referring to Fig. 1; Fig. 2: absorption of sample pad 1 usefulness glass wool is made; bond discharges bed course 2 is marked the carrier protein label for the gold that is adsorbed with Ractopamine fibrage; according to pressing noodle producing method; preparation is coated with Ractopamine gold labeling antibody fibrage; be called for short Ractopamine colloid gold label thing; 3 are the reaction rete; present embodiment adopts nitrocellulose filter; 4 adsorptive pads are that absorbent material layer is made; 5 are coated with Ractopamine-carrier protein couplet thing for detection zone; 6 constitute and to refer to nature controlling line for the Quality Control district is coated with sheep anti-mouse igg; 7 for the PVC backing be supporting layer; make with the plastic tab bar; on the PVC backing, adhere to nitrocellulose filter in order; adsorptive pads; bond discharges pad and sample pad; sample pad is covered bond discharge pad 1/2nd 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 Ractopamine quick detection test paper card and at first need prepare coupling Ractopamine carrier protein, the preparation detection line, the preparation collaurum is used to prepare golden labeling antibody fiber, secondly must prepare sheep anti-mouse igg antibody, is used to make nature controlling line.Below be the preparation method of associated materials:
1. the coupling of Ractopamine carrier protein
1.1 preparation Ractopamine haptens
The Ractopamine haptens: the 6g Ractopamine, 12g succinic anhydride and 8.7ml triethyl ammonia dissolve with the 100ml pyridine.Refluxed one hour.Decompression distillation is desolvated, residue 5%NaHCO
3The ether washed twice is used in the aqueous solution dissolving, uses H then
2SO
4Carry out acidifying (pH to 2.0), wash solid content (being three chloroethyl hemisuccinic acid esters) twice with water, (output is about 75%, fusing point (88 ℃～89 ℃) to make its crystallization with chloroform-ethane.Get 2.5g hemisuccinic acid ester and be dissolved in the 6.5ml thionyl chloride, 65 ℃ were heated 30 minutes.Reduction vaporization, dry 1 hour (under the high vacuum condition).Above-mentioned product is dissolved in 15ml N, in N-dimethyl-acetate acetamide, stirring at room 2 hours.After 65 ℃ of vacuum evaporation, crystallization is separated out,, obtain Ractopamine-hemisuccinic acid ester with being dissolved in zinc in the dimethyl formamide and the acetic acid trichloro ethyl ester that dissociates with isopropyl alcohol.
1.2 the coupling of Ractopamine haptens and carrier protein
Get EDC100mg, make it abundant dissolving (I liquid) with the 10mmol/L PBS liquid 1.5mL of pH8.0.Get Ractopamine hemisuccinic acid ester 25mg, with 0.5mlDMF dissolving (II liquid).Get HSA80mg, be dissolved in 2ml 10mmol/L PBS (PH8.0) liquid (III liquid).II liquid is mixed with III liquid, under magnetic agitation, dropwise add I liquid (remaining 0.5ml).Lucifuge stirred 1 hour under the room temperature, dropwise added remaining I liquid.4 degree stirred 12 hours.Leave standstill 10 hours (4 degree).Make it abundant dialysis (about 48 hours) with distilled water.
1.3 the evaluation of Ractopamine-carrier conjugates
Carrier protein, Ractopamine haptens, Ractopamine-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 pH7.4PBS, in the interscan of wavelength 200-800nm scope, obtain the absorption curve of carrier protein, Ractopamine haptens, Ractopamine-carrier protein couplet thing with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of Ractopamine and carrier protein couplet.
2. the preparation of anti-Ractopamine clonal antibody
2.1 animal immune
Immunogene is injected in the Balb/c mouse 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 ratios 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.
2.2 cell cryopreservation and recovery
The monoclonal hybridoma strain of Ractopamine 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 frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
2.3 MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
The Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.4ml/, monoclonal hybridoma strain c-1-15 * 10 of 7 days pneumoretroperitoneum injection Ractopamines
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.
3. the preparation of Ractopamine gold labeling antibody and gold mark label
3.1 the preparation of collaurum
With two ionized waters that boil off 1% gold chloride is diluted to 0.01%, put 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, when liquid takes on a red color, stop heating, supply dehydration after being cooled to room temperature.The collaurum outward appearance for preparing should be pure, bright, do not have precipitation and floating thing, and the term of validity is one month.
The preparation of Ractopamine monoclonal antibody-colloid gold label thing
Under magnetic agitation, the pH value to 8.2 with 0.1M sal tartari is transferred collaurum adds antibody Ractopamine antibody by 1～2 μ g/ml antibody colloidal gold, continues stirring and evenly mixing 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 ℃ standby, preserved 60 days.
Redissolution damping fluid: with containing bovine serum albumin(BSA) (BSA) 0.02～0.1%, Tween-20 0.05～0.2%, vitamin C (Vit-C) 0.1%, the boric acid redissolution damping fluid of 0.02M pH9.0.
4. the preparation of sheep anti-mouse igg antibody
The preparation of sheep anti mouse antiantibody: as immune animal, is that immunogene to pathogen-free domestic goat carry out immunity with mouse source antibody with goat, obtains the sheep anti mouse antiantibody.
5. Ractopamine quick detection test paper card implements to detect principle
Detect the absorption of sample pad end dropping testing sample solution of test card when Ractopamine after, 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 Ractopamine in the diffusion process can combine with golden labeling antibody, and then seal the antigen-combining site of Ractopamine on the golden antibody, stop the detection trace of coupling Ractopamine look carrier protein on golden labeling antibody and the reaction film to combine, can not show the detection trace, sheep anti-mouse igg antibody then can combine with golden labeling antibody, the colour developing of Quality Control district, form a red contrast trace " | ", it is positive to be a trace, nos Ractopamine then can not stop the carrier protein detection trace of coupling Ractopamine on golden labeling antibody and the cellulose membrane to combine in the opposite sample solution, detection zone shows red trace " | ", same sheep anti-mouse igg antibody also combines with golden labeling antibody, the Quality Control district also shows red contrast trace " | ", form two red lines " || " negative marker, if there is not red-label to show that then test card is invalid on the cellulose membrane.
5. Ractopamine quick detection test paper card detects the implementation column method of operating
5.1 sample pre-treatment:
Animal tissue's (chicken, chicken gizzard, pork, pork liver) sample: take by weighing tissue samples that the even matter of 1.0 ± 0.05g crosses in centrifuge tube, add 1ml PB damping fluid, cover tight bottle cap water-bath 10min in little water-bath that boils, leave standstill after the taking-up be back to room temperature after, get 100 microlitres and carry out point sample and detect.Detection is limited to 5ng/g.
Urine specimen is handled:
Urine sample: generally can directly detect, after urine is muddy state and then adopts filter paper filtering to handle, get 100 microlitres and carry out point sample and detect.
5.2 detect with test card
Sample is splashed in the reagent hole clipping with dropper, drip 3, add the back timing, observations in 5～8min.It is invalid to surpass the interpretation of 10min sample detection.
5.3 testing result analysis
Ractopamine is when concentration is higher than 5ng/ml in sample, and the Ractopamine antibody of colloid gold label combines with Ractopamine is whole, thereby does not occur red stripes in detection zone because competitive reaction can not combine with the Ractopamine conjugate.Negative sample owing to lack the antibody antigen competitive reaction, will red stripes occur in detection zone and Quality Control district in testing process.
Positive; When red stripes of Quality Control district demonstration, and detection zone does not develop the color, and the quick detection test paper card is judged to the positive, shown in Fig. 3 middle graph to show red line look " | ".
Negative: when the Quality Control district shows a red stripes, detection zone also demonstrates a red stripes simultaneously, and the detection zone color near or when being shallower than the Quality Control district, the quick detection test paper card is judged to feminine gender to show that the red line look is " || ".Shown in the left figure of Fig. 3.
Invalid: when the Quality Control district does not demonstrate red stripes, then no matter whether detection zone demonstrates red stripes, and it is invalid that this test card is judged to.Shown in the right figure of Fig. 3.
Claims (10)
1. colloidal gold test paper card that detects the Ractopamine medicament residue, comprise base plate, discharge pad, reaction film and adsorptive pads attached to the absorption of sample pad that closely links to each other successively on the base plate, bond, it is characterized in that the subregion that bond discharges pad is covered under the absorption of sample pad.
2, test card as claimed in claim 1 is characterized in that, the half territory that bond discharges pad is covered under the absorption of sample pad.
3, test card as claimed in claim 1 or 2 is characterized in that, has detection line trace " | " that is coated with Ractopamine-carrier protein couplet thing formation and the nature controlling line trace " | " that bag is constituted by sheep anti-mouse igg on the described reaction film.
4, test card as claimed in claim 3 is characterized in that, described detection line is parallel with nature controlling line.
5, 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.
6, test card as claimed in claim 1 or 2 is characterized in that, is coated with diaphragm respectively at the two ends of test card.
7, test card as claimed in claim 1 or 2 is characterized in that, described base plate is the PVC plate, and described reaction film is a nitrocellulose filter.
8, test card as claimed in claim 1 or 2 is characterized in that, described absorption of sample pad is made by glass wool or polyester material.
9, test card as claimed in claim 1 or 2 is characterized in that, described bond is released bed course and made by fiber.
10, test card as claimed in claim 1 or 2 is characterized in that, described bond discharges bed course and is coated with antibody-collaurum bond.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNU2008200788397U CN201181297Y (en) | 2008-01-30 | 2008-01-30 | Colloidal gold test paper card for detecting Ractopamine medicine residue |
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| CNU2008200788397U CN201181297Y (en) | 2008-01-30 | 2008-01-30 | Colloidal gold test paper card for detecting Ractopamine medicine residue |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993488A (en) * | 2009-08-27 | 2011-03-30 | 深圳市三方圆生物科技有限公司 | Ractopamine immunogen, coating antigen and use thereof in colloid-gold test paper |
| CN102183641A (en) * | 2011-01-28 | 2011-09-14 | 王继华 | Ractopamine immunochromatographic assay test paper strip |
| CN102230936A (en) * | 2011-06-13 | 2011-11-02 | 清华大学深圳研究生院 | Immunochromatography test paper for detecting ractopamine and preparation method thereof |
| CN103100237A (en) * | 2012-12-31 | 2013-05-15 | 南宁市蓝光生物技术有限公司 | Mini-type efficient ractopamine immunoaffinity purification enriching column preparation and application |
| CN103170309A (en) * | 2013-03-12 | 2013-06-26 | 中国农业大学 | Ractopamine antibody immunoaffinity chromatographic column and application thereof |
| CN104090248A (en) * | 2013-12-24 | 2014-10-08 | 上海容晖生物科技有限公司 | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay |
| CN104714018A (en) * | 2015-04-14 | 2015-06-17 | 武汉华美生物工程有限公司 | Colloidal gold micropore type detecting kit and preparation method thereof |
| CN107340398A (en) * | 2017-06-06 | 2017-11-10 | 北京热景生物技术股份有限公司 | Turn the anti-Miao Le Shi pipes hormone quantitative determination reagent and method of luminescence method based on |
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2008
- 2008-01-30 CN CNU2008200788397U patent/CN201181297Y/en not_active Expired - Lifetime
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993488A (en) * | 2009-08-27 | 2011-03-30 | 深圳市三方圆生物科技有限公司 | Ractopamine immunogen, coating antigen and use thereof in colloid-gold test paper |
| CN101993488B (en) * | 2009-08-27 | 2014-05-21 | 深圳市三方圆生物科技有限公司 | Ractopamine immunogen, coating antigen and use thereof in colloid-gold test paper |
| CN102183641A (en) * | 2011-01-28 | 2011-09-14 | 王继华 | Ractopamine immunochromatographic assay test paper strip |
| CN102183641B (en) * | 2011-01-28 | 2013-12-11 | 广州万孚生物技术股份有限公司 | Ractopamine immunochromatographic assay test paper strip |
| CN102230936B (en) * | 2011-06-13 | 2014-03-19 | 清华大学深圳研究生院 | Immunochromatography test paper for detecting ractopamine and preparation method thereof |
| CN102230936A (en) * | 2011-06-13 | 2011-11-02 | 清华大学深圳研究生院 | Immunochromatography test paper for detecting ractopamine and preparation method thereof |
| CN103100237A (en) * | 2012-12-31 | 2013-05-15 | 南宁市蓝光生物技术有限公司 | Mini-type efficient ractopamine immunoaffinity purification enriching column preparation and application |
| CN103100237B (en) * | 2012-12-31 | 2015-12-02 | 南宁市蓝光生物技术有限公司 | Micro high efficiency Ractopamine immunoaffinity purification enriching column preparations and applicatio |
| CN103170309A (en) * | 2013-03-12 | 2013-06-26 | 中国农业大学 | Ractopamine antibody immunoaffinity chromatographic column and application thereof |
| CN104090248A (en) * | 2013-12-24 | 2014-10-08 | 上海容晖生物科技有限公司 | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay |
| CN104714018A (en) * | 2015-04-14 | 2015-06-17 | 武汉华美生物工程有限公司 | Colloidal gold micropore type detecting kit and preparation method thereof |
| CN107340398A (en) * | 2017-06-06 | 2017-11-10 | 北京热景生物技术股份有限公司 | Turn the anti-Miao Le Shi pipes hormone quantitative determination reagent and method of luminescence method based on |
| CN107340398B (en) * | 2017-06-06 | 2019-07-26 | 北京热景生物技术股份有限公司 | Turn the anti-Miao Le Shi pipe hormone quantitative determination reagent and method of luminescence method based on |
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