CN205015348U - Quick test paper of sulfonamides thing jenner rice stick - Google Patents
Quick test paper of sulfonamides thing jenner rice stick Download PDFInfo
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- CN205015348U CN205015348U CN201520752089.7U CN201520752089U CN205015348U CN 205015348 U CN205015348 U CN 205015348U CN 201520752089 U CN201520752089 U CN 201520752089U CN 205015348 U CN205015348 U CN 205015348U
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Abstract
The utility model provides a quick test paper of sulfonamides thing jenner rice stick comprises bottom plate, the board that absorbs water, nitrocellulose membranes, sample imbibition board, MAX group one -tenth, micropore reagent, nitrocellulose membranes stacks on the middle part of bottom plate, absorbing water to pack and putting on the part of the end of the wherein one end of bottom plate, the sample imbibition packs puts in the end of the other end of bottom plate above partly, the nitrocellulose membranes both ends respectively with board and the sample imbibition board looks overlap of absorbing water, nitrocellulose membranes sets gradually test wire and control line, the test wire is by sulfanilamide (SN) - macromolecule albumen conjugate peridium, the control line is by goat -anti mouse polyclonal antibody peridium, micropore reagent is the stick of the jenner's rice after freeze -drying mark sulfanilamide (SN) monoclonal antibody complex. The utility model discloses improve on traditional colloidal gold immunoassay chromatography test paper basis, with the direct freeze -drying of colloidal gold labelled antibody complex in the micropore for the sample that awaits measuring in the testing process fully reacts with gold -labeled antibody, improves detectivity.
Description
Technical field
The utility model relates to animal food field of fast detection, especially relates to a kind of collaurum nanometer rods quick detection test paper of sulfa drugs.
Background technology
Sulfa drugs (Sulfonamides, SAs) is the chemotherapeutic agent that a class has P-aminobenzene-sulfonamide structure, by affecting the synthesis of Nuclear extract matter, and the breeding of anti-bacteria.Sulfa drugs can suppress gram-positive bacteria and some negative bacterium, can treat various bacteria and infect, and veterinary clinic is widely used in treat the various livestock and poultries infected by sensitive bacterial.Due to sulfa drugs effect in vivo and the metabolism time longer, the sulfanilamide (SN) taken in by any approach is all likely at people's body accumulation.When cumulative concentration exceedes certain value, will cause damage to human body.Short time, stimulation that is heavy dose of or long-time low dose can cause acute or slow poisoning respectively, affecting the uropoiesis of body, immune system, destroying the tissues such as muscle, kidney and thyroid gland, as brought out the thyroid cancer etc. of people.In addition, in human body, long-term existence sulfanilamide (SN) can cause many bacteriums to develop immunity to drugs to sulfa drugs.Therefore, Codex Alimentary Commission (CAC) and many national regulations, in animal food, the maximum residue limit (MRL) of sulfamido total amount is 0.1mg/kg.
Existing sulfamido detection method mostly is chromatography, but the sensitivity of these methods is comparatively large by the impact of the purification of sample, the step such as concentrated, moreover these methods need complicated instrument, and process is loaded down with trivial details, is not suitable with the examination of on-the-spot great amount of samples.Sensitive with it, quick, accurate, easy and simple to handle the obtaining of colloidal gold immunity chromatography is applied more and more widely, but current collaurum testing product still exists the low situation of sensitivity.
Utility model content
For the problems referred to above that prior art exists, the applicant provides the colloid gold test paper of sulfa drugs in a kind of quick detection animal food.The utility model improves on traditional colloidal gold immune chromatography test basis, by direct for colloidal gold labeled monoclonal antibody compound freeze-drying in micropore, sample to be tested and golden labeling antibody in testing process is fully reacted, and improves detection sensitivity.
The technical solution of the utility model is as follows:
A kind of sulfa drugs gold nanorods quick detection test paper, is made up of base plate (7), water sucting plate (1), nitrocellulose filter (5), sample imbibition plate (2), MAX line (6) and micropore reagent (8);
Described nitrocellulose filter (5) overlays above the middle part of base plate (7); Described water sucting plate (1) overlays above the end portion of wherein one end of base plate (7), and described sample imbibition plate (2) overlays above the end portion of the other end of base plate (7);
Described nitrocellulose filter (5) two ends are overlapped mutually with water sucting plate (1) and sample imbibition plate (2) respectively, and described nitrocellulose filter (5) is positioned at the below of water sucting plate (1) and sample imbibition plate (2);
From described nitrocellulose filter (5) near that one end of sample imbibition plate (2), set gradually test wire (3) and control line (4); Described test wire (3) is by sulfanilamide (SN)-high molecular weight protein conjugate bag quilt; Described control line (4) is by sheep anti mouse polyclonal antibody bag quilt;
Described micropore reagent (8) is the gold nanorods mark sulfanilamide (SN) monoclonal antibody complex after freeze-drying.
Base plate (7) is PVC backboard, and sample imbibition plate (2) is glass fiber material, and the micropore of micropore reagent (8) is polystyrene material.
The partial-length that described nitrocellulose filter (5) two ends are overlapped mutually with water sucting plate (1) and sample imbibition plate (2) is respectively 1 ~ 2 millimeter.
The using method of this kit is as follows: aqueous phase testing sample is added solid in gold mark micropore reagent (8) to micropore and fully dissolve, if containing sulfa drugs in sample, the sulfa drugs in sample will sulfanilamide (SN) monoclonal antibody-gold nanorods label generation specific binding middle with micropore reagent (8).Insert test strips, because capillarity sample will move along test strips water sucting plate end, when move to be fixed with sulfanilamide (SN)-high molecular weight protein conjugate test wire time, because sulfanilamide (SN) monoclonal antibody-gold nanorods label sulfa drugs in sample is preferentially combined into compound and can not be combined with sulfanilamide (SN)-high molecular weight protein conjugate, therefore the gold nanorods developed the color can not be stranded on test wire, test wire place does not have colour band to show, and namely only has a control line for positive.If there is no sulfa drugs in contrary sample, sulfanilamide (SN) monoclonal antibody-gold nanorods label moves on test wire, sulfanilamide (SN) monoclonal antibody will with sulfanilamide (SN)-high molecular weight protein conjugate generation specific bond, make collaurum be stranded on test wire, namely two colour bands are negative.
When moving to sheep anti mouse polyclonal antibody control line, no matter in sample with or without sulfa drugs, control line all can develop the color.Therefore control line produces then without colour band that representative operation is wrong, when namely detecting sample liquid level more than MAX line or test paper expired.
The technique effect that the utility model is useful is:
This kit changes the method in the past adopting colloid gold label test substance antibody, but adopts the monoclonal antibody of gold nanorods mark test substance.Detection kit provided by the utility model have highly sensitive, easy and simple to handle, be convenient for carrying, the feature such as cost is low, testing result is reliable.
Accompanying drawing explanation
Fig. 1 is the structural drawing of Test paper part in the utility model;
Fig. 2 is the structural drawing of the utility model micropore reagent part;
Fig. 3 is using method figure of the present utility model;
Fig. 4 is negative findings figure of the present utility model;
Fig. 5 is positive findings figure of the present utility model;
Fig. 6 is null result figure of the present utility model;
In figure: 1, water sucting plate, 2, sample imbibition plate, 3, test wire, 4, control line, 5, nitrocellulose filter, 6, MAX line, 7, base plate, 8, micropore reagent.
Embodiment
Below in conjunction with accompanying drawing, the utility model is specifically described.
One, the composition of this kit:
Kit involved by the utility model is made up of Test paper and micropore reagent 8, as shown in Figure 1.Wherein Test paper is made up of base plate 7, water sucting plate 1, nitrocellulose filter 5, sample imbibition plate 2, MAX line 6.Superposition nitrocellulose filter 5 in top in the middle part of base plate 7, on nitrocellulose filter, 5 have a parallel test wire (T line) 3 and a control line (C line) 4; Base plate 7 one end superposes water sucting plate 1 above termination, sample imbibition plate 2 is superposed above other end termination, nitrocellulose filter 5 two ends respectively with water sucting plate 1 with sample imbibition plate 2 is mutual is overlappingly connected (1 ~ 2 millimeter, overlapping coupling part), and are positioned at the below of water sucting plate 1 and sample imbibition plate 2.Wherein test wire 3 is by sulfanilamide (SN)-high molecular weight protein conjugate bag quilt, control line 4 near water sucting plate, by sheep anti mouse polyclonal antibody bag quilt.Micropore reagent 8 for sulfanilamide (SN) monoclonal antibody-direct freeze-drying of gold nanorods label is made in micropore, as shown in Figure 2.
Two, the preparation of sulfa drugs gold-immunochromatographyreagent reagent for assay box:
The synthesis of 1, sulfanilamide (SN)-high molecular weight protein conjugate and sulfonamide hapten-carrier protein couplet thing and qualification
Sulfa drugs is small-molecule substance, only has immunoreactivity, does not have immunogenicity, can not bring out body produce immune response, must with macromolecular carrier albumen coupling after just there is immunogenicity.
(1) synthesis of sulfonamide hapten
2-amino-3-pyridine carboxaldehyde 0.48g and triethylamine 2ml dissolves in 10ml methylene chloride, adds proper catalyst DMAP (DMAP).The 2ml dichloromethane solution of 4-ASC is slowly instilled under room temperature, dropwise rear continuation reaction 5h, steaming desolventizes, after column chromatography purification, add 2mol/LNaOH aqueous solution 80ml, add hot reflux 10h, extraction into ethyl acetate, after column chromatography purification, obtain sulfonamide hapten aldehyde radical sulfapryidine.
(2) immunogenic preparation---sulfonamide hapten and bovine serum albumin(BSA) conjugate synthesize
Get sulfonamide hapten 23mg 3ml dimethyl formamide (DMF) to dissolve completely, make solution I; Get bovine serum albumin(BSA) (BSA) 100mg 7ml0.1mol/LPBS (pH7.0) to dissolve completely, make solution II; Solution I is added in solution II, after room temperature reaction 24h, dialyse three days with 0.01mol/LPBS, change liquid every day three times, obtain immunogene.
(3) preparation of coating antigen---sulfonamide hapten and ovalbumin conjugate synthesize
Get sulfonamide hapten 23mg 3mlDMF to dissolve completely, make solution I; Get ovalbumin (OVA) 100mg 7ml0.1mol/LPBS (pH7.0) to dissolve completely, make solution II; Solution I is added in solution II, after room temperature reaction 24h, dialyse three days with 0.01mol/LPBS, change liquid every day three times, obtain coating antigen.
(4) qualification of sulfanilamide (SN)-high molecular weight protein conjugate and sulfonamide hapten-carrier protein couplet thing
The PBS of sulfonamide hapten, carrier protein, sulfonamide hapten-carrier protein couplet thing pH7.4 is made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PBS, with ultraviolet spectrophotometer in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of sulfonamide hapten, carrier protein, sulfonamide hapten-carrier protein couplet thing.There is different absorption curves in three, shows sulfonamide hapten and carrier protein couplet success.
2, the preparation of sulfanilamide (SN) monoclonal antibody
(1) animal immune
Immunogene step 1 obtained is injected in 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, in 8:1 (quantitative proportion) ratio and SP2/0 myeloma cell fusion, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is utilized to carry out cloning to positive hole, until obtain the hybridoma cell strain of secrete monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma cryopreserving liquid is made the cell suspension of 1 × 106/ml, preserve for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
(4) preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, cultivates under 37 DEG C of conditions, by sad-saturated ammonium sulfate method, the nutrient solution obtained is carried out purifying, obtains monoclonal antibody ,-20 DEG C of preservations.
Described cell culture medium for add calf serum and sodium bicarbonate in RPMI1640 nutrient culture media, and make the massfraction of calf serum in cell culture medium be 20%, the massfraction of sodium bicarbonate in cell culture medium is 0.2%; The pH of described cell culture medium is 7.4.
3, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, for immunogene, immunity is carried out to pathogen-free domestic sheep with mouse source antibody, obtain sheep anti mouse antiantibody.
4, the preparation of gold nanorods mark micropore reagent
(1) gold nanorods is prepared
2.5ml2.5 × 10
4hAuCl
4solution and 2.5ml2.5 × 10
4cTAB solution mixing, then add the freezing 0.01MNaBH of 0.6ml
4solution & stir 10min obtains seed solution.5 × 10 of 0.08M cetyl trimethyl ammonium bromide (CTAB) aqueous solution of 5ml and the 0.25ml of 4ml
4aqueous solution of chloraurate mixing, add the 0.01M silver nitrate (AgNO of 0 ~ 50 microlitre
3) solution, stir and then add 20 microlitre 0.1M ascorbic acid, stir 2min and obtain water white growth solution.1ml seed solution joins in the growth solution of 9ml, stirs 1min, then leaves standstill one week, obtains imperial purple nanometer gold bar solution.
The gold nanorods liquid of sulfa drugs labeling of monoclonal antibody, on absorption micropore, obtain micropore reagent 8 after freeze-drying for subsequent use, as shown in Figure 2.
5, the preparation of test wire and control line
The test wire 3 of test paper and control line 4 are parallel on nitrocellulose filter 5, test wire 3 by sulfanilamide (SN)-high molecular weight protein conjugate bag quilt, control line 4 near water sucting plate by sheep anti mouse polyclonal antibody bag quilt.
6, the combination of Test paper
Test paper is made up of base plate 7, water sucting plate 1, nitrocellulose filter 5, sample imbibition plate 2, MAX line 6, it is nitrocellulose filter 5 in the middle part of base plate, nitrocellulose filter there are a test wire 3 (T line) and a control line 4 (C line), be water sucting plate 1 in termination, base plate 7 one end, other end termination is sample imbibition plate 2, and nitrocellulose filter 5 two ends are respectively with water sucting plate 1 with sample imbibition plate 4 is mutual is overlappingly connected (1 ~ 2 millimeter, overlapping coupling part).
7, using method
As shown in Figure 3, pipette after 100 μ l liquid to be checked adds gold mark hole with liquid-transfering gun and repeatedly blow and beat until bluish violet solid fully dissolves mixing in gold mark hole, hatch 2min; Insert test strips, start timing simultaneously; Within 5 minutes, read result, after 5 minutes, result is invalid.
Three, result interpretation
(1) negative: T line colourity than C line deeply or equally dark, represents that in sample, substrate concentration to be checked is lower than detectability, or not containing thing to be checked, as shown in Figure 4.
(2) positive: T line colourity is more shallow than C line, or T line does not develop the color, then represent that in sample, concentration to be checked is higher than detectability; T line is more shallow than C line, represents that in sample, substrate concentration to be checked is higher, as shown in Figure 5.
(3) invalid: not occur that nature controlling line C line develops the color, show that the incorrect or test card of operating process lost efficacy, when namely detecting sample liquid level more than MAX line or test paper expired.As shown in Figure 6.
Four, test result
The least concentration of the sulfa drugs that this testing cassete can detect is as shown in table 1.The requirement of this kit testing environment is: room temperature.
Table 1
| Medicine name | Detect lower limit ppb (μ g/kg) |
| Sulfamethazine | 6 |
| Sulfamethyldiazine | 4 |
| Sulphadiazine | 3 |
| Daimeton | 6 |
| Sulfanilamide (SN) rope is phonetic | 4 |
| Sulfadimethoxine | 3 |
| Sulfaquinoxaline | 8 |
| Sulfamethoxazole | 20 |
| NU-445 | 15 |
| Sulfamethoxypyridazine | 5 |
| Sulfadoxine | 4 |
| 5-methoxysulfadiazine | 5 |
| Sulfaclozine | 4 |
| Sulfabenzamide | 6 |
| Ayerlucil | 3 |
| Cistosulfa | 5 |
Can see from the data of table 1, the sensitivity of this testing cassete, far above similar products common in the market, has expanded the detection means of sulfa drugs greatly.
Claims (3)
1. a sulfa drugs gold nanorods quick detection test paper, is characterized in that being made up of base plate (7), water sucting plate (1), nitrocellulose filter (5), sample imbibition plate (2), MAX line (6) and micropore reagent (8);
Described nitrocellulose filter (5) overlays above the middle part of base plate (7); Described water sucting plate (1) overlays above the end portion of wherein one end of base plate (7), and described sample imbibition plate (2) overlays above the end portion of the other end of base plate (7);
Described nitrocellulose filter (5) two ends are overlapped mutually with water sucting plate (1) and sample imbibition plate (2) respectively, and described nitrocellulose filter (5) is positioned at the below of water sucting plate (1) and sample imbibition plate (2);
From described nitrocellulose filter (5) near that one end of sample imbibition plate (2), set gradually test wire (3) and control line (4); Described test wire (3) is by sulfanilamide (SN)-high molecular weight protein conjugate bag quilt; Described control line (4) is by sheep anti mouse polyclonal antibody bag quilt;
Described micropore reagent (8) is the gold nanorods mark sulfanilamide (SN) monoclonal antibody complex after freeze-drying.
2. sulfa drugs gold nanorods quick detection test paper according to claim 1, it is characterized in that described base plate (7) is PVC backboard, sample imbibition plate (2) is glass fiber material, and the micropore of micropore reagent (8) is polystyrene material.
3. sulfa drugs gold nanorods quick detection test paper according to claim 1, is characterized in that the partial-length that described nitrocellulose filter (5) two ends and water sucting plate (1) and sample imbibition plate (2) overlap mutually is respectively 1 ~ 2 millimeter.
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| CN201520752089.7U CN205015348U (en) | 2015-09-25 | 2015-09-25 | Quick test paper of sulfonamides thing jenner rice stick |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110818599A (en) * | 2019-10-09 | 2020-02-21 | 广东达元绿洲食品安全科技股份有限公司 | Sulfonamide hapten, artificial antigen and application thereof in immunodetection |
| CN112522368A (en) * | 2020-11-20 | 2021-03-19 | 安徽科技学院 | Immunochromatographic test strip for detecting DNA and preparation method thereof |
-
2015
- 2015-09-25 CN CN201520752089.7U patent/CN205015348U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110818599A (en) * | 2019-10-09 | 2020-02-21 | 广东达元绿洲食品安全科技股份有限公司 | Sulfonamide hapten, artificial antigen and application thereof in immunodetection |
| CN110818599B (en) * | 2019-10-09 | 2022-08-26 | 广东达元绿洲食品安全科技股份有限公司 | Sulfonamide hapten, artificial antigen and application thereof in immunodetection |
| CN112522368A (en) * | 2020-11-20 | 2021-03-19 | 安徽科技学院 | Immunochromatographic test strip for detecting DNA and preparation method thereof |
| CN112522368B (en) * | 2020-11-20 | 2024-04-26 | 安徽科技学院 | Immunochromatography test strip for detecting DNA and preparation method thereof |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of utility model: Quick test paper of sulfonamides thing jenner rice stick Effective date of registration: 20171206 Granted publication date: 20160203 Pledgee: Agricultural Bank of China Limited by Share Ltd. Wuxi science and Technology Branch Pledgor: MEIZHENG BIOTECH Co.,Ltd. Registration number: 2017990001131 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20220804 Granted publication date: 20160203 Pledgee: Agricultural Bank of China Limited by Share Ltd. Wuxi science and Technology Branch Pledgor: MEIZHENG BIOTECH Co.,Ltd. Registration number: 2017990001131 |
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| PC01 | Cancellation of the registration of the contract for pledge of patent right |