CN103529209B - The test strips of quick detection oxolinic acid and preparation method - Google Patents
The test strips of quick detection oxolinic acid and preparation method Download PDFInfo
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- CN103529209B CN103529209B CN201310334329.7A CN201310334329A CN103529209B CN 103529209 B CN103529209 B CN 103529209B CN 201310334329 A CN201310334329 A CN 201310334329A CN 103529209 B CN103529209 B CN 103529209B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
Abstract
The invention discloses test strips of a kind of quick detection oxolinic acid and preparation method thereof, this test strips bottom is supporting layer, middle layer is adsorbed layer, protective seam is fixed on adsorbed layer, adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete and handle end from test lead, and the golden labeling antibody in golden labeling antibody fibrage is anti-oxolinic acid polyclonal antibody or the monoclonal antibody of colloid gold label; The detection trace that the carrier protein that cellulose rete is provided with coupling oxolinic acid is printed and the contrast trace printed with goat-anti or rabbit anti-mouse IgG or goat anti-rabbit igg antibody.Test strips structure of the present invention is simple, high specificity, sensitivity and accuracy high, easy and simple to handle, applied widely, cheap, be easy to apply, there is society and economic worth preferably.
Description
Technical field
The invention belongs to the field of immunological detection of residue of veterinary drug, particularly relate to and a kind ofly detect the residual immunity colloidal gold test paper strip of oxolinic acid and preparation method.
Background technology
Oxolinic acid (OxolinicAcid is called for short OA), another name Oxolinic Acid, molecular formula is C
13h
11nO
5, molecular weight is 261.23, and belong to wide spectrum class antibacterials, particularly have very strong antibacterial activity to Gram-negative bacteria, consumption is few, good anti-bacterial effect, is one of desirable agricultural chemicals thing for the treatment of aquatic animal disease.Oxolinic acid is generally white or the crystalline powder of off-white color, odorless, tasteless, and water insoluble and ethanol, can be dissolved in formic acid and sodium hydroxide solution, nonhygroscopic, to light, thermally-stabilised.But it has certain toxic and side effect to human body, common mainly comprises gastrointestinal reaction, liver renal toxicity and muscle skeleton spinoff etc., the U.S. and Japan's forbidding oxolinic acid; European Union and China are defined as 50-300 μ g/kg to its highest limitation in the meats such as ox, pig, chicken and fish.Although China's residue of veterinary drug limitation is with reference to international and developed country's standard formulation, and veterinary drug use has the off-drug period to specify, fundamentally solves animal food residue of veterinary drug excessive problem and still there is larger difficulty.Therefore be badly in need of research and set up the residual detection method of quick, sensitive, effective oxolinic acid.
The method detecting oxolinic acid residual both at home and abroad at present mainly adopts physico-chemical analysis method, comprise high performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry combination analysis method (LC/MS), gas chromatography/mass spectrometry analytic approach (GC/MS) etc., these methods detect all exists certain defect: (1) detected sample preprocessing process is complicated, loaded down with trivial details time-consuming; (2) need expensive instrument and equipment, promote the use of and be subject to a definite limitation; (3) high to the competency profiling of professional, certain correlation experience must be had to obtain reliable results; (4) instrument maintenance requires high, the accuracy of the direct impact analysis result of quality of maintenance; (5) testing cost is high; (6) Site Detection and extensive batch detection cannot be carried out, and ageing poor.Immunological detection has sxemiquantitative and certain quantitation capabilities, can provide the preliminary information of determinand, and this method sensitivity is higher, accuracy is good, and analytic process is relatively simple, and the examination as oxolinic acid has unique advantage, the detection technique needing to first develop, anxious solution to be studied.
Summary of the invention
The technical problem to be solved in the present invention is: overcome the defect that prior art exists, provide a kind of high specificity, highly sensitive, easy and simple to handle, precisely fast for detecting the residual test strips of oxolinic acid and preparation method.
Technical scheme of the present invention:
A kind of test strips of quick detection oxolinic acid, bottom is supporting layer, middle layer is adsorbed layer, protective seam is fixed on adsorbed layer, adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete and handle end from test lead, and the golden labeling antibody in golden labeling antibody fibrage is anti-oxolinic acid polyclonal antibody or the monoclonal antibody of colloid gold label; The detection trace that the carrier protein that cellulose rete is provided with coupling oxolinic acid is printed and the contrast trace printed with goat-anti or rabbit anti-mouse IgG or goat anti-rabbit igg antibody.
The carrier protein of described coupling oxolinic acid obtains by the following method:
Take oxolinic acid 12mg and dissolve in 1mLN, in dinethylformamide solution, add 10 μ L tri-n-butylamine mixings under room temperature condition, then add 10 μ L isobutyl chlorocarbonate magnetic agitation reaction 3h, then add the PBS solution containing 20mg carrier protein, stirring reaction spends the night; Reactant liquor PBS dialyses 3 days, and the centrifugal 5min of 4000r/min, abandons precipitation, obtains the carrier protein couplet thing of oxolinic acid, and packing is also stored in-20 DEG C, for subsequent use.
Described cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made.
Described adsorbing fiber layer PVDF membrane, nylon membrane, glass fibre cotton or polyester film are made; Absorbent material layer absorbent filter is made, and the supporting layer toughness material do not absorbed water is made; The golden labeling antibody glass fibre cotton of gold labeling antibody fibrage absorption oxolinic acid is made.
Described
the carrier protein of coupling oxolinic acid is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin.
Described detection trace and contrast trace be arranged in parallel "
︱ ︱" orthoscopic trace, " 10 " font arrangement trace, " ┬ ┬ " font arrangement trace, " ┴ ┴ " font arrangement trace, " ├ ├ " font arrangement trace or " ┤ ┤ " font arrangement trace.
Described adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer are coated with diaphragm, 0.3-0.6cm place, adsorbing fiber layer side are partial to by the diaphragm that adsorbing fiber layer is corresponding with golden labeling antibody fibrage intersection and are printed with sample mark line.
The preparation method of the test strips of described oxolinic acid, comprises the following steps:
(1) preparation of coupling oxolinic acid carrier protein
Oxolinic acid 12mg is dissolved in 1mLN, in dinethylformamide solution, adds 10 μ L tri-n-butylamine mixings under room temperature condition, then add 10 μ L isobutyl chlorocarbonate magnetic agitation reaction 3h, then add the PBS solution containing 20mg carrier protein, stirring reaction spends the night; Dialyse 3 days with PBS, the centrifugal 5min of 4000r/min, abandons precipitation, obtains the carrier protein couplet thing of oxolinic acid;
(2) preparation of anti-oxolinic acid monoclonal antibody or polyclonal antibody;
(3) preparation of oxolinic acid gold labeling antibody;
(4) preparation of goat-anti or rabbit anti-mouse IgG or goat anti-rabbit igg antibody;
On cellulose rete, make detection trace with the oxolinic acid carrier protein couplet thing of gained, on cellulose rete, prepare contrast trace with goat-anti or rabbit anti-mouse IgG or goat anti-rabbit igg antibody; Supporting layer, adsorbed layer and protective seam, for the preparation of golden labeling antibody fibrage, are then assembled into test strips by oxolinic acid gold labeling antibody successively.
Test strips of the present invention has the following advantages:
high specificity, highly sensitive.Test strips of the present invention is prepared from based on the monoclonal antibody of colloid gold label high-affinity, formed without covalent bond between gold grain and antibody molecule in gold labeling antibody, the two is combined by the Van der Waals force between the charges of different polarity, colloid gold label affects very little on the specificity of monoclonal antibody and affinity, and there is higher mark rate, there is stronger specificity and higher sensitivity simultaneously, the micro-oxolinic acid of 50ng/g can be detected.
easy, quick.Use test strips of the present invention, can execute-in-place.Only test strips need be inserted 10 ~ 20 seconds in test sample, take out in latter 5 minutes and can judge testing result.
result display is vivid, directly perceived, accurate.Test strips with show brownish red "
︱" (or " ten ", " ┬ ", " ┴ ", " ├ ", " ┤ ") trace as testing result positive and negative marker, namely show on cellulose rete a brownish red "
︱" trace, to represent in test sample containing oxolinic acid, show two brownish reds "
︱ ︱" trace, represent in test sample not containing oxolinic acid.It is vivid, directly perceived, accurate, simple and clear that result judges, not easily occurs the artificially erroneous judgement such as false positive and false negative.
cost saving.Use test strip, without the need to separately joining instrument and equipment and other reagent, can detect whenever and wherever possible, low cost, can a large amount of expensive instrument and cost of equipment be saved.
applied widely, easy to utilize.Easy and simple to handle, can meet the needs of different levels personnel, this test strips has wide market outlook and obvious economic benefit and social benefit.
6. convenient storage, long shelf-life.Test strip convenient storage, and it is less demanding to storing temperature, at 4-8 DEG C, the term of validity can keep 1 year, and under room temperature, effective shelf-life can reach six months.
Accompanying drawing explanation
Fig. 1 is the plan structure schematic diagram of test strips of the present invention.
Fig. 2 is the side structure schematic diagram of Fig. 1 test strips.
In figure, 1 is supporting layer, and 2 is adsorbing fiber layer, and 3 is golden labeling antibody fibrage, and 4 is cellulose rete, and 5 is absorbent material layer, and 6 for detecting trace, and 7 is contrast trace, and 8-1 is test lead diaphragm, and 8-2 is handle end diaphragm, and 9 is sample mark line.
Embodiment
Embodiment one: the test strips and the preparation method that detect micro-oxolinic acid fast, see Fig. 1, Fig. 2.In figure, supporting layer 1 plastic slice bar is made, adsorbing fiber layer 2 is made with glass fibre cotton, gold labeling antibody fibrage 3 is adsorbed with the monoclonal antibody of anti-oxolinic acid, cellulose rete 4 adopts nitrocellulose filter to make, absorbent material layer 5 is made with absorbent filter, be pasted and fixed on successively from right to left on supporting layer 1 by each layer of numbering 2,3,4,5, intersection fiber crosses one another infiltration each other.Cellulose rete 4 is provided with and detects trace 6 and contrast trace 7, bovine serum albumin(BSA) (BSA) solution detecting trace coupling oxolinic acid is printed and is formed, and contrast trace rabbit anti-mouse IgG antibody trace is made, two trace formation trace arranged in parallel bands "
︱ ︱".8-1 covers the test lead diaphragm (white) above adsorbing fiber layer 2 and golden labeling antibody fibrage 3; 8-2 is the handle end diaphragm (as yellow) covered above absorbent material layer 5; 9 is sample mark line; namely at adsorbing fiber layer 2 and golden labeling antibody fibrage 3 intersection; corresponding white diaphragm deflection adsorbing fiber layer 2 side is about the mark line printed at 0.5cm place, the diaphragm on the right side of sample mark line is printed on arrow and max printed words.
The preparation of testing sample solution and detecting step:
Detect chicken muscle: sample is cut into block, smashs to pieces, then carry out centrifugal, the centrifugal 5min of 4000r/min, draw upper strata juice as testing sample;
Method of operating: inserted in testing sample by test strips test lead, insertion depth is no more than mark line, and about 10 ~ 20 seconds took out test strips, horizontal positioned, observe and judge testing result in 5 minutes.
Result judges: if (a) positive show on cellulose rete a brownish red trace band "
︱", represent that testing result is positive, illustrate that testing sample contains oxolinic acid; If b () negative show on cellulose rete two brownish red trace bands "
︱ ︱", testing result is negative, illustrates in testing sample not containing oxolinic acid; On cellulose rete, do not have reddish brown colour band to show if c () is lost efficacy, then showed that test strips lost efficacy.
The susceptibility of test strips of the present invention and detectability detect: prepare the oxolinic acid standard solution that 8 concentration are 0,50,100,200,400,800,1600 and 3200 μ g/L altogether, detect according to Test paper operation requirements, each concentration establishes 6 repetitions, judges susceptibility and the detectability of Test paper according to test findings.Test findings shows, the detection of this test strips is limited to 50ng/g.
Make and detect micro-oxolinic acid test strips fast, first prepare coupling oxolinic acid carrier protein and oxolinic acid gold labeling antibody, thus preparation detects trace and golden labeling antibody fibrage; Next prepares goat-anti or rabbit anti-mouse IgG(or goat anti-rabbit igg) antibody, for the preparation of contrast trace.
(1) preparation of the carrier protein couplet thing antigen of oxolinic acid
Adopt mixed anhydride method, oxolinic acid and carrier protein are carried out coupling, prepares artificial conjugated antigen.
Take oxolinic acid 12mg and dissolve in 1mLN, in dinethylformamide (DMF) solution, 10 μ L tri-n-butylamine mixings are added under room temperature condition, add 10 μ L isobutyl chlorocarbonates again, magnetic agitation reaction 3h, then add containing 20mg carrier protein BSA(or carrier protein OVA, KLH) PBS solution, stirring reaction spends the night; Dialyse 3 days with PBS, the centrifugal 5min of 4000r/min, abandons precipitation, obtains the carrier protein couplet thing of oxolinic acid, and packing is also stored in-20 DEG C, for subsequent use.
(2) preparation of anti-oxolinic acid monoclonal antibody or polyclonal antibody
Prepared by monoclonal antibody: with the oxolinic acid carrier protein couplet thing of preparation with 30 μ g ~ 50 μ g/ consumption immunity Balb/C mouse in 6 ~ 8 week age only 3 ~ 4 times, 3 ~ 5 weeks each immunization interval time, determine that antibody titer carries out superpower immunity after meeting the requirements, and its suppression valency was detected before fusion, within 3 ~ 4 days afterwards, by hole blood sampling under immune mouse socket of the eye, be separated positive serum; De-neck is lethal, and the alcohol-pickled mouse 5 ~ 10min with 75% sterilizes body surface, asepticly gets its spleen, is shredded by spleen and grinds, filtering, the centrifugal 10min of 1000rpm through 120 order nylon gauzes, collection splenocyte.By 1 × 10
8splenocyte mix in the ratio of 10:1 with NS0 myeloma cell, 1000rpm is centrifugal, and 10min abandons supernatant, cell precipitation thing is slowly added in 37 DEG C of water-baths the PEG4000 of 50% of 0.7 ~ 1.0mL, add in 1min, within first 30 seconds, add 0.1 ~ 0.3mL, centre adds 0.2 ~ 0.4mL in 15 seconds, within last 15 seconds, adds; Then serum-free 1640 nutrient culture media 15mL is slowly added, to stop the effect of PEG, 37 DEG C of water-bath 5 ~ 10min, 1000rpm is centrifugal, and 10min abandons supernatant, cell precipitation thing is resuspended in HAT Selective agar medium, and add (8 ~ 10 pieces) in 96 porocyte culture plates, 100 μ L/ holes, μ L ~ 200, be placed in 37 DEG C, the CO of 5%
2cultivate in incubator.Cultivate 10 ~ 14 days, positive hole sizer choosing is carried out with indirect elisa method, selection strong positive, the hole that inhibiting rate is high, Growth of Cells is vigorous carry out 3 ~ 6 limited dilution clonings (until cell clone is monoclonal, tire in clone hole, suppression valency is basically identical to detect each), then expand cultivation, set up hybridoma cell strain.The monoclonal antibody of prepared hybridoma secretion can be reacted with oxolinic acid specifically, and affinity constant reaches 10
10-10
12, light chain subtype is κ or λ, and heavy chain subgroup is IgG
1, IgG
2a, IgG
2b, IgG
3, for the monoclonal antibody of oxolinic acid specific epitope, for the preparation of golden labeling antibody glass fibre cotton.
How anti-preparation: with oxolinic acid carrier protein couplet thing immunize New Zealand white rabbits, immunizing dose is 200 μ g ~ 500 μ g/ time, and dorsal sc divides 4 ~ 6 injections.Head exempts from, and the oxolinic acid carrier protein couplet thing dissolving preparation with aseptic PBS, mixes with equivalent Freund's complete adjuvant (FCA), fully emulsified; Booster immunization, dissolves oxolinic acid carrier protein couplet thing with aseptic PBS, mixes with equivalent incomplete Freund's adjuvant (FIA), fully emulsified, within 2 ~ 3 weeks, carry out after head exempts from, continuous immunity 4 ~ 5 times, every minor tick 2 ~ 3 weeks, after last immunity 10 ~ 15 days, surveys it with ELISA method and surely tires and reach 10
5time above, blood sampling is separated and collected hyper-immune serum also.IgG antibody is extracted with saturated ammonium sulfate salting out method, namely get 1 portion of hyper-immune serum and add 2 parts of PBS(pH7.2) mixing, add the mixing of equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerator 12h, 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant, again with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration 33%, put 4 DEG C of refrigerator 2h, 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant, with appropriate PBS(pH7.2) dissolution precipitation, put 4 DEG C of interior PBS(pH7.2 of refrigerator) dialyse 48 ~ 72h, liquid is changed for several times in centre, 4 DEG C, the centrifugal 15min of 12000rpm, collect supernatant, obtain the anti-oxolinic acid polyclonal antibody of purifying,-20 DEG C frozen, for the preparation of golden labeling antibody glass fibre cotton.
(3) preparation of oxolinic acid gold labeling antibody and golden labeling antibody glass fibre cotton
Adopt reduction of sodium citrate legal system for colloidal gold solution.In 200mL0.01 ~ 0.02% chlorauric acid solution of boiling, add freshly prepared 1% sodium citrate 8mL, obtain the colloidal gold solution that diameter is about 15nm, with the K of 0.1mol/L
2cO
3adjust ph to 8.5 ~ 9.5, are placed in 2 ~ 8 DEG C and save backup.With the mark of 1:2000 than oxolinic acid monoclonal antibody to be marked or polyclonal antibody are added in the aurosol of pH8.5 ~ 9.5, after mark 10min, the PEG10000 adding 20% is 0.05% to final concentration, 4 DEG C, the centrifugal 20min of 1500 ~ 3000rpm, remove unconjugated colloid gold particle, 4 DEG C, the centrifugal 1h of 15000rpm, abandon supernatant, obtain the golden labeling antibody protein mixture of preliminary purification, carry out separation and purification with propylene glucosan S-400 column chromatography again, obtain oxolinic acid colloidal gold labeled monoclonal antibody.The colloidal gold labeled monoclonal antibody that 1:100 ~ 1:500 dilutes is adsorbed in processed glass cellucotton, 4 DEG C of low-temperature vacuum dryings, prepares oxolinic acid gold labeling antibody glass fibre cotton.
(4) goat-anti or rabbit anti-mouse IgG(or goat anti-rabbit igg) preparation of antibody
The preparation (method with extraction mice serum IgG identical, no longer repeat) of oxolinic acid negative mice serum IgG (or negative rabbit anteserum IgG) for oxolinic acid Rapid detection test strip contrast trace is extracted with saturated ammonium sulfate.
(5) the detection reaction principle of test strips
After the test lead of oxolinic acid test strips inserts testing sample solution, solution to be measured drives the golden labeling antibody in oxolinic acid to be measured and golden labeling antibody glass fibre cotton to spread to cellulose rete together by syphonic effect, and the final absorbent material layer infiltrating handle end.In diffusion process, oxolinic acid to be measured can combine with the golden labeling antibody in golden labeling antibody fibrage, and then close the antigen-combining site of oxolinic acid on golden labeling antibody, the detection trace of golden labeling antibody coupling oxolinic acid carrier protein on cellulose membrane is stoped to be combined, test strips cannot show detection trace, sheep or rabbit anti-mouse IgG(or goat anti-rabbit igg) antibody then can be combined with golden labeling antibody, formed brownish red contrast trace band "
︱", namely reddish brown colour band "
︱" trace is positive expression.If otherwise without oxolinic acid in sample solution, then the detection trace of golden labeling antibody coupling oxolinic acid carrier protein on cellulose membrane can not be stoped to be combined, test strips display brownish red detection trace band "
︱"; Same goat-anti or rabbit anti-mouse IgG(or goat anti-rabbit igg) antibody also can be combined with golden labeling antibody, display brownish red contrast trace band "
︱", formed two reddish brown colour bands "
︱ ︱" be negative expression.If cellulose membrane does not have reddish brown colour band show, then show that test strips lost efficacy.
Embodiment two: test strips structure is substantially identical with embodiment one; difference is: golden labeling antibody fibrage is adsorbed with the polyclonal antibody of anti-oxolinic acid; adsorbing fiber layer nylon membrane is made; cellulose rete adopts pure cellulose film; detect trace band to be " ten " with stealthy contrast trace band, the handle end diaphragm covered above absorbent material layer is blue look.
Testing sample is chicken gizzard, and sample is cut into block, smashs to pieces, then carries out centrifugal, the centrifugal 5min of 4000r/min, draws upper strata juice as testing sample;
Result judge: if (a) positive show on cellulose membrane a brownish red trace band "
ten", represent that testing result is positive, illustrate in testing sample containing oxolinic acid; If (b) feminine gender show on cellulose membrane two brownish red trace bands "
ten", represent that testing result is negative, illustrate in testing sample not containing oxolinic acid; On cellulose membrane, do not have reddish brown colour band to show if c () is lost efficacy, then showed that test strips lost efficacy.
Operation and preparation method, with embodiment one, do not repeat.
Embodiment three: test strips structure is substantially identical with embodiment one; difference is: adsorbing fiber layer PVDF membrane (PVDF) is made; coupling oxolinic acid carrier protein solution is chicken ovalbumin (OVA); stealthy contrast trace band rabbit anti-mouse IgG antibody solution trace on cellulose membrane is made; cellulose rete adopts carboxylated cellulose film; the handle end diaphragm covered above absorbent material layer is green, detects trace band and is " ┬ " with stealthy contrast trace band.
Result judges: if (a) positive shows a brownish red trace band " ┬ " on cellulose membrane, represents that testing result is positive, illustrates in testing sample containing oxolinic acid; If b () feminine gender shows two brownish red trace bands " ┬ " on cellulose membrane, represent that testing result is negative, illustrate in testing sample not containing oxolinic acid; On cellulose membrane, do not have reddish brown colour band to show if c () is lost efficacy, then showed that test strips lost efficacy.Operation and preparation method, with embodiment one, do not repeat.
Embodiment four: test strips structure is substantially identical with embodiment one, and difference is: adsorbing fiber layer polyester film is made, cellulose rete adopts carboxylated cellulose film, and coupling oxolinic acid carrier protein solution is hemocyanin (KLH).Detect trace band to be " ┴ " with stealthy contrast trace band.Preparation method, operation and result decision method, with embodiment one, do not repeat.
Embodiment five: test strips structure is substantially identical with embodiment one, and difference is: stealthy contrast trace band goat anti-rabbit igg antibody solution trace on cellulose membrane is made, and adsorbing fiber layer nylon membrane is made.Detect trace band to be " ├ " with stealthy contrast trace band.The judgement of preparation method, result and method of operating, with embodiment one, do not repeat.
Embodiment six: substantially identical with embodiment one, difference is: oxolinic acid gold labeling antibody fibrage is adsorbed with anti-oxolinic acid polyclonal antibody, and detection sample is chicken gizzard.Detect trace band to be " ┤ " with stealthy contrast trace band.
Embodiment seven: substantially identical with embodiment one, difference is: oxolinic acid gold labeling antibody fibrage is adsorbed with anti-oxolinic acid polyclonal antibody, and detection sample is chicken muscle.
Embodiment eight: substantially identical with embodiment one, difference is: oxolinic acid gold labeling antibody fibrage is adsorbed with anti-oxolinic acid polyclonal antibody, and detection sample is chicken gizzard.
Embodiment nine: substantially identical with embodiment one, difference is: coupling oxolinic acid carrier protein solution is chicken ovalbumin (OVA), detects sample, result judgement and method of operating with embodiment one.
Embodiment ten: substantially identical with embodiment one, difference is: coupling oxolinic acid carrier protein solution is hemocyanin (KLH), detects sample, result judgement and method of operating with embodiment one.
Claims (9)
1. one kind is detected the test strips of oxolinic acid fast, bottom is supporting layer, middle layer is adsorbed layer, protective seam is fixed on adsorbed layer, it is characterized in that: adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete and handle end from test lead, the golden labeling antibody in golden labeling antibody fibrage is anti-oxolinic acid polyclonal antibody or the monoclonal antibody of colloid gold label; The detection trace that the carrier protein that cellulose rete is provided with coupling oxolinic acid is printed and the contrast trace printed with goat-anti or rabbit anti-mouse IgG or goat anti-rabbit igg antibody;
The carrier protein of described coupling oxolinic acid obtains by the following method:
Take oxolinic acid 12mg and dissolve in 1mLN, in dinethylformamide solution, add 10 μ L tri-n-butylamine mixings under room temperature condition, then add 10 μ L isobutyl chlorocarbonate magnetic agitation reaction 3h, then add the PBS solution containing 20mg carrier protein, stirring reaction spends the night; Reactant liquor PBS dialyses 3 days, and the centrifugal 5min of 4000r/min, abandons precipitation, obtains the carrier protein couplet thing of oxolinic acid, and packing is also stored in-20 DEG C, for subsequent use.
2. test strips according to claim 1, is characterized in that: described cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made.
3. test strips according to claim 1, is characterized in that: described adsorbing fiber layer PVDF membrane, nylon membrane, glass fibre cotton or polyester film are made; Absorbent material layer absorbent filter is made, and the supporting layer toughness material do not absorbed water is made; The golden labeling antibody glass fibre cotton of gold labeling antibody fibrage absorption oxolinic acid is made.
4. the test strips according to any one of claim 1-3, is characterized in that: described in
the carrier protein of coupling oxolinic acid is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin.
5. the test strips according to any one of claim 1-3, is characterized in that: described detection trace and contrast trace be arranged in parallel "
︱ ︱" orthoscopic trace, " 10 " font arrangement trace, " ┬ ┬ " font arrangement trace, " ┴ ┴ " font arrangement trace, " ├ ├ " font arrangement trace or " ┤ ┤ " font arrangement trace.
6. the test strips according to any one of claim 1-3; it is characterized in that: on described adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer, be coated with diaphragm, 0.3-0.6cm place, adsorbing fiber layer side is partial to by the diaphragm that adsorbing fiber layer is corresponding with golden labeling antibody fibrage intersection and is printed with sample mark line.
7. the preparation method of the test strips of oxolinic acid described in claim 1, is characterized in that: the method comprises the following steps:
(1) preparation of coupling oxolinic acid carrier protein
Oxolinic acid 12mg is dissolved in 1mLN, in dinethylformamide solution, adds 10 μ L tri-n-butylamine mixings under room temperature condition, then add 10 μ L isobutyl chlorocarbonate magnetic agitation reaction 3h, then add the PBS solution containing 20mg carrier protein, stirring reaction spends the night; Dialyse 3 days with PBS, the centrifugal 5min of 4000r/min, abandons precipitation, obtains the carrier protein couplet thing of oxolinic acid;
(2) preparation of anti-oxolinic acid monoclonal antibody or polyclonal antibody;
(3) preparation of oxolinic acid gold labeling antibody;
(4) preparation of goat-anti or rabbit anti-mouse IgG or goat anti-rabbit igg antibody;
On cellulose rete, make detection trace with the oxolinic acid carrier protein couplet thing of gained, on cellulose rete, prepare contrast trace with goat-anti or rabbit anti-mouse IgG or goat anti-rabbit igg antibody; Supporting layer, adsorbed layer and protective seam, for the preparation of golden labeling antibody fibrage, are then assembled into test strips by oxolinic acid gold labeling antibody successively.
8. preparation method according to claim 7, is characterized in that: the preparation method of described oxolinic acid gold labeling antibody is as follows:
In 200mL0.01 ~ 0.02% chlorauric acid solution of boiling, add 1% sodium citrate 8mL of new moss preparation, obtain the colloidal gold solution of diameter 15-20nm, with the K of 0.1mol/L
2cO
3adjust ph to 8.5 ~ 9.5, put 2 ~ 8 DEG C of preservations, for subsequent use; With the mark of 1:2000 than oxolinic acid monoclonal antibody to be marked or polyclonal antibody are added in the aurosol of pH8.5 ~ 9.5, after mark 10min, the PEG10000 adding 20% is 0.05% to final concentration, 4 DEG C, the centrifugal 20min of 1500 ~ 3000rpm, remove unconjugated colloid gold particle, 4 DEG C, the centrifugal 1h of 15000rpm, abandon supernatant, obtain the golden labeling antibody protein mixture of preliminary purification, carry out separation and purification with propylene glucosan S-400 column chromatography again, obtain the oxolinic acid antibody of colloid gold label.
9. preparation method according to claim 7, is characterized in that: described cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made; Adsorbing fiber layer PVDF membrane, nylon membrane, glass fibre cotton or polyester film are made; Absorbent material layer absorbent filter is made, and the supporting layer toughness material do not absorbed water is made; The golden labeling antibody glass fibre cotton of gold labeling antibody fibrage absorption oxolinic acid is made; The carrier protein of coupling oxolinic acid is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin.
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| CN1409113A (en) * | 2002-08-06 | 2003-04-09 | 张少恩 | Use of colloidal gold immunological chromatography in detection of aquatic, agricutural and livestock products |
| CN101955540A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Antibody against fluoroquinolones and application thereof |
| CN202814981U (en) * | 2012-08-09 | 2013-03-20 | 河南省农业科学院 | Test strip for quickly detecting trace amount of chlorothalonil |
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| CN1409113A (en) * | 2002-08-06 | 2003-04-09 | 张少恩 | Use of colloidal gold immunological chromatography in detection of aquatic, agricutural and livestock products |
| CN101955540A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Antibody against fluoroquinolones and application thereof |
| CN202814981U (en) * | 2012-08-09 | 2013-03-20 | 河南省农业科学院 | Test strip for quickly detecting trace amount of chlorothalonil |
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