CN106483300A - A kind of colloidal gold immuno-chromatography test paper strip of detection Furaxone metabolite and preparation method and application - Google Patents
A kind of colloidal gold immuno-chromatography test paper strip of detection Furaxone metabolite and preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of colloid gold chromatographic test paper strip of detection Furaxone metabolite and preparation method and application, belong to immunological technique field and wild animal resources technical field.This test strips structure includes base plate, sample pad, colloidal gold pad, coated film and adsorptive pads, and test strips two ends are provided with protecting film, and described sample pad, colloidal gold pad, coated film, adsorptive pads are pasted on base plate successively；The monoclonal antibody that Furaxone metabolite can be identified of colloid gold label is coated with colloidal gold pad；Coated film is provided with and is coupled the stealthy detection line that the carrier protein solution of Furaxone metabolite is printed, and the stealthy nature controlling line printed with goat anti-mouse igg solution.The features such as colloidal gold chromatographic test strip of the present invention has convenient, quick, directly perceived, ageing strong, applied widely, low cost is easy to utilize.
Description
Technical field
The present invention relates to a kind of colloidal gold immuno-chromatography test paper strip and preparation method thereof of detection Furaxone metabolite with
Application, it is particularly suitable for the detection of Furaxone metabolite residual in animal tissue, belongs to immunological technique field and veterinary drug is residual
Stay analysis technical field.
Background technology
Furazolidone (Furazolidone, FZD), also known as furazolidone, are a kind of Nitrofuran metabolites, to common leather
Lan Shi negative bacterium and positive bacteria have inhibitory action, act on microbial enzyme system, suppress S-acetyl-coenzyme-A, interference microorganism sugar generation
Thank, thus playing bacteriostasis.It is widely used in Aquatic product and birdss, in order to treat bacillary dysentery, enteritis, coccidiosiss etc., and cultivate fish
Scabies, red fin fish disease, ulcer etc..Because its sterilizing ability is strong, has a broad antifungal spectrum, it is not likely to produce drug resistance, oral absorption is rapid, with other
Antibiotic no cross resistance the advantages of, be once widely used in clinic.Furazolidone has very strong side effect, and in animal
Internal metabolism soon is 3- amino -2- oxazolidone (AOZ), and AOZ is directly metabolized into having under the conditions of gastric acid and strongly causes to dash forward
The beta-hydroxyethyl hydrazine of degeneration and carcinogenecity, has potential threat to health and ecological balance.At present, European Union, the U.S., day
Basis, China etc. provide against using Nitrofuran antibiotics in edible animal, and the file agriculture and animal husbandry of the Ministry of Agriculture of China sends out [2002] 1
Number file specifies that the detection of furazolidone in food animal must not be limited to and detects.
Detection technique about furazolidone residual mainly has high performance liquid chromatography (HPLC), liquid chromatograph-matter at present
Spectrum combination method (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS/MS) and immuno analytical method, first three methods are sensitive, accurate
Really, but complex operation, higher to experimental facilitiess and technical requirements, be not suitable for the quick detection of batch samples.Immunoassay side
Method includes enzyme linked immunosorbent assay (ELISA) and colloidal gold immunity chromatography (GICA), has that sensitivity is high, specificity is good, becomes
The features such as this is low, easy to operate, is suitable to batch samples screening.GICA, as a kind of new immunological detection method, existing exempts from
The specificity of epidemiology method, has the characteristics that simplicity is quick again, is not required to instrument, it is many in medical science, the animal and plant quarantine, food inspection etc.
Field is widely applied.Exploitation furazolidone colloidal gold strip contributes to AOZ residual and fast and accurately detects have
Effect contains its Misuse in aquatic products and residual excessive problem, improves people's aquatic products dining table quality safety water
Flat.
Content of the invention
The technical problem to be solved is to overcome the defect of prior art to provide a kind of detection furazolidone metabolite
The colloidal gold immuno-chromatography test paper strip of thing, this colloidal gold immuno-chromatography test paper strip have simple to operate, quickly, suitable high-volume sample
The advantage of product primary dcreening operation.Additionally, the present invention further provides the colloidal gold immuno-chromatography test paper strip of this detection Furaxone metabolite
Preparation method and application.
Technical problem of the present invention is realized by technical scheme below.
A kind of colloidal gold immuno-chromatography test paper strip of detection Furaxone metabolite, this test strip is by base plate, sample
Pad, colloidal gold pad, coated film and adsorptive pads composition, reagent paper two ends are provided with protecting film, described sample pad, colloidal gold pad, coated film
It is pasted on successively on base plate with adsorptive pads；Wherein colloidal gold pad is to be adsorbed with the Furaxone metabolite monoclonal of colloid gold label
Antibody, coated film contains the pre-coated detection line (T line) of useful AOZ metabolite coupling carrier albumen, with goat-anti or rabbit anti-mouse
The pre-coated nature controlling line of IgG solution (C line), two lines are vertically arranged in parallel.
Above-mentioned colloidal gold immuno-chromatography test paper strip, Furaxone metabolite monoclonal antibody is Mus resource monoclonal antibody, by
CCTCC NO：The hybridoma cell strain AOZ-4G3 of C2016140 produces.This hybridoma cell strain is named as hybridoma cell strain
AOZ-4G3, delivers China typical culture collection center (CCTCC) preservation on July 31st, 2016, and preserving number is CCTCC
NO:C2016140.
Above-mentioned colloidal gold immuno-chromatography test paper strip, described Furaxone metabolite monoclonal antibody is by Furaxone metabolite
Antigen produces, and it is the conjugate being adopted carbodlimide method synthesis by Furaxone metabolite and carrier protein, including immunogen
Former with detecting；Described carrier protein is bovine serum albumin, mouse serum albumin, gpv protein, thyroprotein, egg white
Albumen, hemocyanin or human serum albumin；Immunogen preferred vector is bovine serum albumin, and the former preferred vector of detection is egg white
Albumen；
Above-mentioned colloidal gold immuno-chromatography test paper strip, described base plate is the hard material that PVC bar or other do not absorb water；Described sample
Product pad and colloidal gold pad are made up of glass fibre cotton；Described adsorptive pads are made up of absorbent filter；Described coated film can be fine for nitric acid
The plain film of dimension or cellulose acetate membrane；Described protecting film is opaque glued membrane.
Colloid gold chromatographic test paper strip preparation method of the present invention, comprises the steps：
(1) prepare Furaxone metabolite antigen working solution：
(a) Furaxone metabolite derivatization：Take 0.25g AOZ, with 2mL about methanol stir at room temperature and be allowed to molten
Solution；Take 0.15g 3- carboxyl benzaldehyde, be allowed to dissolve with 2mL methanol, be slowly dropped under agitation in above-mentioned AOZ solution, room temperature
Lower stirring reaction 5h, filters, and dries derivant CPAOZ that filtering residue obtains final product Furaxone metabolite；
(b) immunogenic synthesis：The bovine serum albumin (BSA) weighing 50mg is dissolved in the 1mL 0.1mol/L of pH value 7.4
PBS in, add N, N- dimethylformamide (DMF) 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in
In 1mLDMF solution, stirring is lower to add dicyclohexyl imines (DCC) 14mg and N-hydroxy-succinamide (NHS) 6mg, at 4 DEG C
Sealing is stirred overnight, and 8000r/min is centrifuged 15 minutes, collects supernatant and loads in bag filter, uses pH value 7.4 under the conditions of 4 DEG C
0.01mol/L PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-BSA solution；
The former synthesis of (c) detection：The ovalbumin (OVA) weighing 40mg is dissolved in the 1mL 0.1mol/L PBS of pH value 7.4
In, add N, N- dimethylformamide (DMF) 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in 1mLDMF molten
In liquid, stirring is lower to add dicyclohexyl imines (DCC) 14mg and N-hydroxy-succinamide (NHS) 6mg, sealing stirring at 4 DEG C
Overnight, 8000r/min is centrifuged 15 minutes, collects supernatant and loads the 0.01mol/L using pH value 7.4 in bag filter under the conditions of 4 DEG C
PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-OVA solution；
(2) prepare Furaxone metabolite monoclonal antibody working solution：
(a) animal immune：Select the immunogen that carrier protein is bovine serum albumin, the female Blab/ of immune 6-8 week old
C mice, interval immunity in 2 weeks 1 time, docking after three immunity takes hematometry potency and suppression ratio, and the optimal mice of selection result is accurate
Standby fusion；
(b) cell fusion：The splenocyte of the selected mice of step (a) and mouse myeloma SP2/O cell is taken to be merged,
Indirect elisa method measures supernatant and chooses positive high hole, carries out sub-clone by limiting dilution assay to positive hole, until setting up
Produce the hybridoma cell strain of the monoclonal antibody of single anti-Furaxone metabolite；
A large amount of preparations of (c) monoclonal antibody：Choose individual larger female Blab/c mice, using inducing ascites in vivo
Method, prepares ascites in a large number, and passes through octanoic acid-ammonium sulfate precipitation purification ascites, is divided into tubule, -20 DEG C of preservations, obtains furazolidone generation
Thank to thing monoclonal antibody；
(3) prepare the Furaxone metabolite monoclonal antibody of colloid gold label：
Furaxone metabolite monoclonal antibody solution 10000r/min to be marked is centrifuged 30min, takes gold colloidal molten
Liquid 10mL, uses 0.1mol/L K2CO3Solution adjusts the pH value of colloidal gold solution to 8.2；Take equivalent Furaxone metabolite Dan Ke
Grand antibody-solutions, are mixed with colloidal gold solution under magnetic agitation, incubated at room 15min, and 10000r/min is centrifuged 30min, abandons
Clearly, Na gained precipitation being 0.02mol/L with every liter of BSA containing 10g, 0.5g sodium azide, concentration2B4O7Solution dilutes, that is, obtain
Obtain the Furaxone metabolite monoclonal antibody of colloid gold label, 4 DEG C save backup；
(4) colloidal gold pad preparation：Cut glass fibre cotton by specification, by the Furaxone metabolite Dan Ke of colloid gold label
Grand antibody gold spraying instrument is uniformly sprayed on glass fibre cotton, and 37 DEG C are dried 1h, sealing, and 4 DEG C save backup；
(5) preparation of detection line and nature controlling line：Furaxone metabolite antigen is put in gold spraying instrument storage pool A, goat-anti is little
Mus IgG solution is put in storage pool B, and fixed fire, in film central authorities, forms the orthoscopic stealth detection line of spacing 0.5cm and hidden respectively for start
Shape nature controlling line, spontaneously dries, sealing, and 4 DEG C save backup；
(6) test strips assembling：Sample pad, colloidal gold pad, coated film and adsorptive pads are pasted on base plate in order successively,
And the surface mount protecting film at test strips two ends, test strips are cut on slot-cutting machine.
Above-mentioned colloid gold chromatographic test paper strip is for detecting in Furaxone metabolite in animal tissue, urine and milk
Application.
Above-mentioned in the application for detecting Furaxone metabolite, comprise the steps：
(1) sample pre-treatments
The pre-treatment of (a) meat, liver or aquatic products sample tissue
After tissue homogenate, weigh 2g in 50ml centrifuge tube, add 4mL deionized water, 1mL 1mol/L hydrochloric acid solution, shake
Swing 1min, add 0.5mL p -carboxybenzaldehyde, vibrate 1min；0.5mol/L disodium phosphate soln 1mL is added after taking-up,
Adjust PH to 7.5 about with 1mol/L sodium hydroxide solution, vibrate 1min, add 5mL ethyl acetate, vibrate 1min, 5000rpm
Centrifugation 10min；Pipette upper strata ethyl acetate layer solution in another centrifuge tube, aqueous phase repeats to extract two with 5mL ethyl acetate again
Secondary, after merging, nitrogen dries up；Add 1ml normal hexane, add a cover vibration 1min, be subsequently adding 0.5mL PBST buffer, vibration
1min, 5000rpm are centrifuged 10min；Draw lower floor's aqueous phase solution to be used for analyzing；
The pre-treatment of (b) urine sample
By urine sample 5000rpm centrifugation 5min to limpid, supernatant 50ul is taken to be detected；
The pre-treatment of (c) milk sample
Take 5000rpm centrifugation 10min under milk sample 2mL room temperature condition, take off layer solution and add 4mL deionized water, 1mL
1mol/L hydrochloric acid solution, vibrates 1min, adds 0.5mL p -carboxybenzaldehyde, vibrates 1min；0.5mol/L phosphorus is added after taking-up
Sour disodium hydrogen solution 1mL, adjusts PH to 7.5 about with 1mol/L sodium hydroxide solution, vibrates 1min, adds 5mL ethyl acetate,
Vibration 1min, 5000rpm are centrifuged 10min；Pipette upper strata ethyl acetate layer solution in another centrifuge tube, aqueous phase uses 5mL second again
Acetoacetic ester repeats to be extracted twice, and after merging, nitrogen dries up；Add 1ml normal hexane, add a cover vibration 1min, be subsequently adding 0.5mL
PBST buffer, vibrates 1min, and 5000rpm is centrifuged 10min；Draw lower floor's aqueous phase solution to be used for analyzing；
(2) detected with colloid gold chromatographic test paper strip
Take out the colloidal gold immuno-chromatography test paper strip of described detection Furaxone metabolite from the package, sample end is inserted
In analyte sample fluid, insertion depth is less than mark line, takes out test strip, horizontal positioned, 3-5 minute within about 10-20 second
Observe result after judging testing result, 10 minutes invalid；
(3) result judgement
If a on () positive coated film, corresponding quality control region location of C shows a reddish brown colo(u)r streak, detection zone T location does not show
Show reddish brown colo(u)r streak, represent that testing result is the positive, illustrate to contain AOZ in testing sample；
If b () feminine gender T, location of C on coated film show two days reddish brown colo(u)r streaks, represent that result is feminine gender, illustrate
AOZ is not contained in testing sample；
C () was lost efficacy and was not shown brownish red band when quality control region C, then no matter whether detection zone T shows brownish red band
It is invalid that this reagent paper is all judged to.
The detection of the Furaxone metabolite colloid gold chromatographic test paper strip of the present invention is limited to 4 μ g/L, with other 3 kinds of nitro furans
All there is not cross reaction, the detection to sample and high performance liquid chromatography in mutter derivant CPAHD, CPSEM of metabolite, CPAMOZ
Result is consistent.With colloid gold chromatographic test paper strip of the present invention detect the method for Furaxone metabolite easy, quick, directly perceived, accurately,
Applied widely, low cost, easily promote the use of.
Brief description
Fig. 1 Furaxone metabolite of the present invention monoclonal antibody affinity measures figure
Fig. 2 Furaxone metabolite of the present invention monoclonal antibody hypotype measures figure
Fig. 3 Furaxone metabolite of the present invention colloid gold chromatographic test paper strip reagent paper cross-sectional view
Fig. 4 Furaxone metabolite of the present invention colloid gold chromatographic test paper strip reagent paper overlooking the structure diagram
Fig. 5 Furaxone metabolite of the present invention colloid gold chromatographic test paper strip test paper result process decision chart
The hybridoma cell strain AOZ-4G3 producing Furaxone metabolite monoclonal antibody of the present invention, in 2016
On July 31, in delivers China typical culture collection center (abbreviation CCTCC, address：Wuhan University, Chinese Typical Representative culture is protected
Tibetan center, postcode：430072) preservation, preserving number is CCTCC NO:C2016140.
Specific embodiment
With reference to specific embodiment, the present invention is described in further detail, cited embodiment is not to the present invention
Content and protection domain constitute any restriction.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
The preparation of embodiment one Furaxone metabolite antigen of the present invention
(1) Furaxone metabolite derivatization：Take 0.25g AOZ, with 2mL about methanol stir at room temperature and be allowed to molten
Solution；Take 0.15g 3- carboxyl benzaldehyde, be allowed to dissolve with 2mL methanol, be slowly dropped under agitation in above-mentioned AOZ solution, room temperature
Lower stirring reaction 5h, filters, and dries filtering residue and obtains final product furazolidone metabolite derivative CPAOZ；
(2) immunogenic synthesis：The bovine serum albumin (BSA) weighing 50mg is dissolved in the 1mL 0.1mol/L of pH value 7.4
PBS in, add N, N- dimethylformamide (DMF) 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in
In 1mLDMF solution, stirring is lower to add dicyclohexyl imines (DCC) 14mg and N-hydroxy-succinamide (NHS) 6mg, at 4 DEG C
Sealing is stirred overnight, and 8000r/min is centrifuged 15 minutes, collects supernatant and loads in bag filter, uses pH value 7.4 under the conditions of 4 DEG C
0.01mol/L PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-BSA solution；
(3) detect former synthesis：The ovalbumin (OVA) weighing 40mg is dissolved in the 1mL 0.1mol/L PBS of pH value 7.4
In, add N, N- dimethylformamide (DMF) 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in 1mLDMF molten
In liquid, stirring is lower to add dicyclohexyl imines (DCC) 14mg and N-hydroxy-succinamide (NHS) 6mg, sealing stirring at 4 DEG C
Overnight, 8000r/min is centrifuged 15 minutes, collects supernatant and loads the 0.01mol/L using pH value 7.4 in bag filter under the conditions of 4 DEG C
PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-OVA solution.
The preparation of embodiment two Furaxone metabolite monoclonal antibody of the present invention
(1) animal immune：Carrier protein is that the female Blab/c of the immunogen immune 6-8 week old of bovine serum albumin is little
Mus, interval immunity in 2 weeks 1 time, three exempt from after docking take hematometry potency and suppression ratio, the optimal mice of selection result prepares fusion；
(2) cell fusion：Take splenocyte and the mouse myeloma SP2/O cell of mice, merged, indirect elisa method
Measure supernatant and choose positive high hole, sub-clone is carried out to positive hole by limiting dilution assay, until set up producing single anti-furan
Mutter oxazolone metabolite monoclonal antibody hybridoma cell strain；
(3) a large amount of preparations of monoclonal antibody：Choose individual larger female Blab/c mice, using inducing ascites in vivo
Method, prepares ascites in a large number, and passes through octanoic acid-ammonium sulfate precipitation purification ascites, is divided into tubule, -20 DEG C of preservations, obtains furazolidone generation
Thank to thing monoclonal antibody.
Embodiment three Furaxone metabolite monoclonal antibody CHARACTERISTICS IDENTIFICATION of the present invention
1st, affinity measures
Measure the affinity costant of Furaxone metabolite monoclonal antibody, result such as Fig. 1 using non-competing euzymelinked immunosorbent assay (ELISA)
Shown, affinity costant Ka=1.6 × 109L/mol.
2nd, hypotype measures
Hypotype mensure is carried out using the Mus source monoclonal antibody hypotype identification kit purchased from Sigma company, result as shown in Figure 2,
Furaxone metabolite monoclonal antibody hypotype is IgG1.
3rd, titration
With 1：40000 dilutions are coated primordial covering detection plate, and monoclonal antibody after purification is carried out 1:200,1:400,1:
800 ... ... 1:800000 dilutions, are added to ELISA Plate in the hole, add the sheep anti mouse two of HRP labelling to resist, finally use TMB after reaction
Colour developing, result shows that Furaxone metabolite MAb concentration after purification reaches 1 for potency during 1mg/ml：1024000.
The colloid gold chromatographic test paper strip of example IV detection of the present invention Furaxone metabolite
1st, gold colloidal liquid preparation：Take 100mL distilled water, boil 5min, add the gold chloride 1mL that mass concentration is 1% and
Mass concentration is 1% citric acid three sodium solution 1.5mL, continues agitating heating, and solution eventually passes through Lycoperdon polymorphum Vitt, claret in thoroughly
Bright redness, is cooled to room temperature, carries out Quality Identification by ocular estimate, ultraviolet spectrophotometry respectively, and qualified gold colloidal is molten
4 DEG C of liquid keeps in Dark Place；
2nd, the preparation of the Furaxone metabolite monoclonal antibody of colloid gold label：By Furaxone metabolite to be marked
Monoclonal antibody solution 10000r/min is centrifuged 30min, takes colloidal gold solution 10mL, uses 0.1mol/L K2CO3Solution adjusts glue
The pH value of body gold solution is to 8.2；Take equivalent Furaxone metabolite monoclonal antibody solution, with colloidal gold solution under magnetic agitation
Mixing, incubated at room 15min, 10000r/min is centrifuged 30min, abandons supernatant, by gained precipitation every liter of BSA containing 10g, 0.5g
Sodium azide, concentration are the Na of 0.02mol/L2B4O7Solution dilutes, that is, obtain the Furaxone metabolite monoclonal of colloid gold label
Antibody, 4 DEG C save backup；
3rd, colloidal gold pad preparation：Cut glass fibre cotton by specification, gold labeling antibody gold spraying instrument is uniformly sprayed on glass
On cellucotton, 37 DEG C are dried 1h, sealing, and 4 DEG C save backup；
4th, the preparation of detection line and nature controlling line：Furaxone metabolite antigen is put in gold spraying instrument storage pool A, sheep anti-Mouse
IgG solution is put in storage pool B, start respectively fixed fire in film central authorities, formed spacing 0.5cm orthoscopic stealth detection line T line and
Stealthy nature controlling line C line, spontaneously dries, sealing, and 4 DEG C save backup；
5th, test strips assembling：Sample pad, colloidal gold pad, coated film and adsorptive pads are pasted on base plate in order successively,
And the surface mount protecting film at test strips two ends, test strips are cut on slot-cutting machine.
6th, the structure of Furaxone metabolite colloid gold chromatographic test paper strip, referring to Fig. 3, Fig. 4.In figure base plate 1 uses PVC board system
Become, sample pad 2 is made with glass fibre cotton, and colloidal gold pad 3 is adsorbed with anti-Furaxone metabolite monoclonal antibody, coated film
4 adopt nitrocellulose filter, and adsorptive pads 7 are made with absorbent filter, and each layer of numbering 2,3,4,7 is pasted fixation from right to left successively
On base plate 1, intersection fiber crosses one another infiltration each other.Stealthy detection line 5 and stealthy nature controlling line 6 are provided with coated film 4,
Stealthy detection line is printed with chicken ovalbumin (OVA) solution being coupled Furaxone metabolite, stealthy nature controlling line sheep anti-Mouse
IgG antibody solution is printed, two traces one-tenth " " arranged in parallel.8 is the sample covering on sample pad 2 and colloidal gold pad 3
End protecting film, 9 is the handle end protecting film covering on adsorptive pads 7, is printed on arrow on the protecting film on the right side of sample mark line
Head and MAX printed words.
Furaxone metabolite in embodiment five colloid gold chromatographic test paper strip detection sample of the present invention
(1) sample pre-treatments
The pre-treatment of (a) meat, liver or aquatic products sample tissue
After tissue homogenate, weigh 2g in 50ml centrifuge tube, add 4mL deionized water, 1mL 1mol/L hydrochloric acid solution, shake
Swing 1min, add 0.5mL p -carboxybenzaldehyde, vibrate 1min；0.5mol/L disodium phosphate soln 1mL is added after taking-up,
Adjust PH to 7.5 about with 1mol/L sodium hydroxide solution, vibrate 1min, add 5mL ethyl acetate, vibrate 1min, 5000rpm
Centrifugation 10min；Pipette upper strata ethyl acetate layer solution in another centrifuge tube, aqueous phase repeats to extract two with 5mL ethyl acetate again
Secondary, after merging, nitrogen dries up；Add 1ml normal hexane, add a cover vibration 1min, be subsequently adding 0.5mL PBST buffer, vibration
1min, 5000rpm are centrifuged 10min；Draw lower floor's aqueous phase solution to be used for analyzing；
The pre-treatment of (b) urine sample
By urine sample 5000rpm centrifugation 5min to limpid, supernatant 50ul is taken to be detected；
The pre-treatment of (c) milk sample
Take 5000rpm centrifugation 10min under milk sample 2mL room temperature condition, take off layer solution and add 4mL deionized water, 1mL
1mol/L hydrochloric acid solution, vibrates 1min, adds 0.5mL p -carboxybenzaldehyde, vibrates 1min；0.5mol/L phosphorus is added after taking-up
Sour disodium hydrogen solution 1mL, adjusts PH to 7.5 about with 1mol/L sodium hydroxide solution, vibrates 1min, adds 5mL ethyl acetate,
Vibration 1min, 5000rpm are centrifuged 10min；Pipette upper strata ethyl acetate layer solution in another centrifuge tube, aqueous phase uses 5mL second again
Acetoacetic ester repeats to be extracted twice, and after merging, nitrogen dries up；Add 1ml normal hexane, add a cover vibration 1min, be subsequently adding 0.5mL
PBST buffer, vibrates 1min, and 5000rpm is centrifuged 10min；Draw lower floor's aqueous phase solution to be used for analyzing；
(2) detected with colloid gold chromatographic test paper strip
Take out the colloidal gold immuno-chromatography test paper strip of described detection Furaxone metabolite from the package, sample end is inserted
In analyte sample fluid, insertion depth is less than mark line, takes out test strip, horizontal positioned, 3-5 minute within about 10-20 second
Observe result after judging testing result, 10 minutes invalid.
(3) result judgement
As shown in figure 5, a is the positive, on coated film, corresponding quality control region location of C shows a reddish brown colo(u)r streak, detection zone T position
Put and do not show reddish brown colo(u)r streak, represent that testing result is the positive, illustrate to contain AOZ in testing sample；B is feminine gender, in coated film
Upper T, location of C show two days reddish brown colo(u)r streaks, represent that result is feminine gender, illustrate not containing AOZ in testing sample；C, d are to lose efficacy,
When quality control region C does not show brownish red band, then no matter detection zone T shows whether this reagent paper is all judged to no brownish red band
Effect.
Embodiment six Furaxone metabolite colloid gold chromatographic test paper strip performance detection of the present invention
1st, sensitivity
1mg/mL furazolidone metabolite derivative (1mg CPAOZ is dissolved in 1mL methanol) is diluted to 0 respectively with PBS, 1,
2nd, the series of standards solution of 3,4,5,10 μ g/L, then take 90 μ L above-mentioned standard solution to drop in the sample pad of test strips respectively,
The colour developing situation of test strips is observed after 3min.
When CPAOZ adds concentration for 3 μ g/L, T line is with respect to the lighter of T line during 0 μ g/L, and works as CPAOZ's
When to add concentration be 4 μ g/L, T line disappears line, illustrates that the detection of colloidal gold strip is limited to 4 μ g/L.
2nd, specificity
Choose 4 kinds of metabolites of nitrofuran derivatization reagent (CPAOZ, CPAHD, CPSEM, CPAMOZ) tests, observe examination
Paper slip color developing effect.
CPAOZ, CPAHD, CPSEM, CPAMOZ mother solution is diluted to 50 μ g/L with the PBS of 0.01mol/L, PH7.4 respectively,
It is added drop-wise to the sample well of test strips, testing result shows, the Furaxone metabolite colloidal gold strip of preparation is several with other
There is not cross reaction in Nitrofuran metatolites derivatization reagent.
3rd, accuracy
Randomly select 8 parts of Aquatic product samples, as sample of having a try.Every part of sample uses HPLC method and this research preparation respectively
CPAOZ colloidal gold colloidal gold detection test paper strip is detected, compares testing result.
Through high performance liquid chromatography detection, wherein 6 parts samples do not detect Furaxone metabolite and remain 8 parts of Aquatic product samples, No. 3
Sample detection goes out AOZ content 1.73 μ g/kg, and No. 7 sample detection go out AOZ content 2.35 μ g/kg.Furan azoles used by these samples simultaneously
Bupropion metabolite thing colloidal gold strip detects, result is complied fully with HPLC method, is shown in Table 1.
Table 1 Furaxone metabolite colloidal gold strip is compared with high performance liquid chromatography testing result
Note："+", represents positive, and " " represents negative.
Above-described embodiment only technology design to illustrate the invention and advantage, the present invention can also have other forms and become
Change, as well known to the skilled person, above-described embodiment functions only as to the exemplary role in foregoing invention protection domain, right
For those of ordinary skill in the art, also has a lot of conventional deformation and other enforcement in the protection domain that the present invention is limited
Example, these deformation and embodiment are all by within the protection domain pending in the present invention.
Claims (8)
1. a kind of detection Furaxone metabolite colloidal gold immuno-chromatography test paper strip it is characterised in that this test strip by
Base plate, sample pad, colloidal gold pad, coated film and adsorptive pads composition, reagent paper two ends are provided with protecting film；Described sample pad, gold colloidal
Pad, coated film and adsorptive pads are pasted on base plate successively；Wherein colloidal gold pad is to be adsorbed with the furazolidone generation of colloid gold label
Thank to thing monoclonal antibody, coated film contains the pre-coated detection line T line of useful Furaxone metabolite coupling carrier albumen, uses sheep
Nature controlling line C line anti-or that rabbit anti-mouse IgG solution is pre-coated, two lines are vertically arranged in parallel.
2. colloid gold chromatographic test paper strip according to claim 1 it is characterised in that described base plate be PVC bar or other not
The hard material of water suction；Described sample pad and colloidal gold pad are made up of glass fibre cotton；Described adsorptive pads are made up of absorbent filter；
Described coated film can be nitrocellulose filter or cellulose acetate membrane；Described protecting film is opaque glued membrane.
3. colloid gold chromatographic test paper strip according to claim 1 is it is characterised in that described Furaxone metabolite monoclonal
Antibody is Mus resource monoclonal antibody, by CCTCC NO:The hybridoma cell strain AOZ-4G3 of C2016140 produces；This hybridoma is thin
Born of the same parents' strain is named as hybridoma cell strain AOZ-4G3, delivers China typical culture collection center on July 31st, 2016 and protects
Hide, preserving number is CCTCC NO:C2016140.
4. colloid gold chromatographic test paper strip according to claim 1 is it is characterised in that described Furaxone metabolite monoclonal
Antibody is produced by Furaxone metabolite antigen, and this Furaxone metabolite antigen is by Furaxone metabolite and carrier protein
Using the conjugate of carbodiimide method synthesis, former with detection including immunogen；Described carrier protein is bovine serum albumin, Mus
Serum albumin, gpv protein, thyroprotein, ovalbumin, hemocyanin or human serum albumin.
5. colloid gold chromatographic test paper strip according to claim 4 is it is characterised in that described Furaxone metabolite immunogen
The use of bovine serum albumin is carrier；Described Furaxone metabolite detects that former use ovalbumin is carrier.
6. prepare the preparation method detecting the colloidal gold immuno-chromatography test paper strip of Furaxone metabolite as claimed in claim 1,
It is characterized in that, comprise the steps：
(1) prepare Furaxone metabolite antigen working solution：
(a) Furaxone metabolite derivatization：Take 0.25g AOZ, with 2mL about methanol stir at room temperature and be allowed to dissolve；
Take 0.15g 3- carboxyl benzaldehyde, be allowed to dissolve with 2mL methanol, be slowly dropped under agitation in above-mentioned AOZ solution, stir under room temperature
Mix reaction 5h, filter, dry derivant CPAOZ that filtering residue obtains final product Furaxone metabolite；
(b) immunogenic synthesis：Weigh 50mg bovine serum albumin BSA be dissolved in pH value 7.4 1mL 0.1mol/L PBS
In, add N, N- dimethylformamide DMF 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in 1mLDMF molten
In liquid, stirring is lower to add dicyclohexyl imines DCC 14mg and N-hydroxy-succinamide NHS 6mg, seals stirred at 4 DEG C
At night, 8000r/min is centrifuged 15 minutes, collects supernatant and loads the 0.01mol/L using pH value 7.4 in bag filter under the conditions of 4 DEG C
PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-BSA solution；
The former synthesis of (c) detection：The ovalbumin OVA weighing 40mg is dissolved in the 1mL 0.1mol/L PBS of pH value 7.4, adds
N, N- dimethylformamide DMF 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in 1mLDMF solution, stirs
Mix lower addition dicyclohexyl imines DCC 14mg and N-hydroxy-succinamide NHS 6mg, sealing at 4 DEG C is stirred overnight,
8000r/min is centrifuged 15 minutes, collects supernatant and loads in bag filter, saturating with the 0.01mol/LPBS of pH value 7.4 under the conditions of 4 DEG C
Analysis 3d, changes liquid 3-4 time daily, obtains CPAOZ-OVA solution；
(2) prepare Furaxone metabolite monoclonal antibody working solution：
(a) animal immune：Select the immunogen that carrier protein is bovine serum albumin, the female Blab/c of immune 6-8 week old is little
Mus, interval immunity in 2 weeks 1 time, docking after three immunity takes hematometry potency and suppression ratio, and the optimal mice of selection result prepares to melt
Close；
(b) cell fusion：The splenocyte of the selected mice of step (a) and mouse myeloma SP2/O cell is taken to be merged, indirectly
ELISA method measures supernatant and chooses positive high hole, carries out sub-clone by limiting dilution assay to positive hole, until set up producing
The hybridoma cell strain of the monoclonal antibody of single anti-Furaxone metabolite；
A large amount of preparations of (c) monoclonal antibody：Choose individual larger female Blab/c mice, using inducing ascites method in vivo,
A large amount of preparation ascites, and pass through octanoic acid-ammonium sulfate precipitation purification ascites, it is divided into tubule, -20 DEG C of preservations, obtain furazolidone metabolite
Thing monoclonal antibody；
(3) prepare the Furaxone metabolite monoclonal antibody of colloid gold label：
Furaxone metabolite monoclonal antibody solution 10000r/min to be marked is centrifuged 30min, takes colloidal gold solution
10mL, uses 0.1mol/L K2CO3Solution adjusts the pH value of colloidal gold solution to 8.2；Take equivalent Furaxone metabolite monoclonal
Antibody-solutions, are mixed with colloidal gold solution under magnetic agitation, incubated at room 15min, and 10000r/min is centrifuged 30min, abandons supernatant,
The Na that gained precipitation is 0.02mol/L with every liter of BSA containing 10g, 0.5g sodium azide, concentration2B4O7Solution dilutes, that is, obtain glue
The Furaxone metabolite monoclonal antibody of body gold labelling, 4 DEG C save backup；
(4) colloidal gold pad preparation：Cut glass fibre cotton by specification, by the Furaxone metabolite monoclonal anti of colloid gold label
Body gold spraying instrument is uniformly sprayed on glass fibre cotton, and 37 DEG C are dried 1h, sealing, and 4 DEG C save backup；
(5) preparation of detection line and nature controlling line：Furaxone metabolite antigen is put in gold spraying instrument storage pool A, goat anti-mouse igg
Solution is put in storage pool B, and fixed fire, in film central authorities, forms the orthoscopic stealth detection line of spacing 0.5cm and stealthy matter respectively for start
Control line, spontaneously dries, sealing, and 4 DEG C save backup；
(6) test strips assembling：Sample pad, colloidal gold pad, coated film and adsorptive pads are pasted on base plate in order successively, and
The surface mount protecting film at test strips two ends, cuts into test strips on slot-cutting machine.
7. colloid gold chromatographic test paper strip as claimed in claim 1 is for detecting furan azoles in animal tissue, urine and milk
Application in bupropion metabolite thing.
8. application according to claim 7 is it is characterised in that comprise the steps：
(1) sample pre-treatments
The pre-treatment of (a) meat, liver or aquatic products sample tissue
After tissue homogenate, weigh 2g in 50ml centrifuge tube, add 4mL deionized water, 1mL 1mol/L hydrochloric acid solution, vibration
1min, adds 0.5mL p -carboxybenzaldehyde, vibrates 1min；Add 0.5mol/L disodium phosphate soln 1mL after taking-up, use
1mol/L sodium hydroxide solution adjust PH to 7.5 about, vibrate 1min, add 5mL ethyl acetate, vibrate 1min, 5000rpm from
Heart 10min；Pipette upper strata ethyl acetate layer solution in another centrifuge tube, aqueous phase repeats to be extracted twice with 5mL ethyl acetate again,
After merging, nitrogen dries up；Add 1ml normal hexane, add a cover vibration 1min, be subsequently adding 0.5mL PBST buffer, vibrate 1min,
5000rpm is centrifuged 10min；Draw lower floor's aqueous phase solution to be used for analyzing；
The pre-treatment of (b) urine sample
By urine sample 5000rpm centrifugation 5min to limpid, supernatant 50ul is taken to be detected；
The pre-treatment of (c) milk sample
Take 5000rpm centrifugation 10min under milk sample 2mL room temperature condition, take off layer solution and add 4mL deionized water, 1mL 1mol/L
Hydrochloric acid solution, vibrates 1min, adds 0.5mL p -carboxybenzaldehyde, vibrates 1min；0.5mol/L phosphoric acid hydrogen two is added after taking-up
Sodium solution 1mL, adjusts PH to 7.5 about with 1mol/L sodium hydroxide solution, vibrates 1min, adds 5mL ethyl acetate, vibration
1min, 5000rpm are centrifuged 10min；Pipette upper strata ethyl acetate layer solution in another centrifuge tube, aqueous phase uses 5mL acetic acid second again
Ester repeats to be extracted twice, and after merging, nitrogen dries up；Add 1ml normal hexane, add a cover vibration 1min, be subsequently adding 0.5mL PBST and delay
Rush liquid, vibrate 1min, 5000rpm is centrifuged 10min；Draw lower floor's aqueous phase solution to be used for analyzing；
(2) detected with colloid gold chromatographic test paper strip
Take out the colloidal gold immuno-chromatography test paper strip of described detection Furaxone metabolite from the package, sample end is inserted to be measured
In sample liquid, insertion depth is less than mark line, takes out within about 10-20 second test strip, horizontal positioned, 3-5 minute is observed
After judging testing result, 10 minutes, result is invalid；
(3) result judgement
A () is positive：If corresponding quality control region location of C shows a reddish brown colo(u)r streak on coated film, detection zone T location does not show palm fibre
Red line, represents that testing result is the positive, illustrates to contain AOZ in testing sample；
B () is negative：If T, location of C show two days reddish brown colo(u)r streaks on coated film, represent that result is feminine gender, illustrate to be measured
AOZ is not contained in sample；
C () was lost efficacy：When quality control region C does not show brownish red band, then no matter detection zone T shows whether this examination of brownish red band
It is invalid that paper is all judged to.
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| CN108530373A (en) * | 2018-05-14 | 2018-09-14 | 广东达元绿洲食品安全科技股份有限公司 | A kind of furans metabolite hapten, artificial antigen and its application in fluorescent quantitation immunochromatography |
| CN108845131A (en) * | 2018-06-20 | 2018-11-20 | 广州质量监督检测研究院 | Detect the colloidal gold immunochromatographimethod detection card and its preparation method and application of bisphenol b |
| CN110441519A (en) * | 2019-07-29 | 2019-11-12 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | A method of identifying the detection card of aurantiamarin and quickly identifies aurantiamarin in Chinese medicine |
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