CN105004860B - Florfenicol quick detection kit and preparation, using method - Google Patents
Florfenicol quick detection kit and preparation, using method Download PDFInfo
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Abstract
The present invention relates to the improvement of antibiotic detection technique, be specifically related to florfenicol quick detection kit and preparation, using method, belong to field of immunology.Florfenicol quick detection kit, including the supporter arranged from bottom to top, water accepting layer, detection layers, the cover plate of band through hole, preserving number is: secreted by CCTCC C201575 hybridoma FFA C, anti-florfenicol amine monoclonal antibody C of colloid gold label；A liquid: containing the Polyethylene Glycol that 1 5% molecular weight is 20000, the 0.01mol/L phosphate buffer of 0.01 0.25% polysorbas20；B liquid: containing the 0.01mol/L phosphate buffer of 0.01 0.25% polysorbas20.This test kit has simplicity, quick, accurate, sensitive feature, and minimal detectable concentration is 10ug/ml, can be used for the on-the-spot scalping to florfenicol drug residue and detects.
Description
Technical field
The present invention relates to the improvement of antibiotic detection technique, be specifically related to florfenicol quick detection kit and system thereof
Standby, using method, belongs to field of immunology.
Background technology
Florfenicol (Florfenico, FF) Chinese: fluprofen, Florfenicol, chemical name: d (+)-Soviet Union-1-
P-methylphenyl-2-dichloro acetamino-3-fluorine propanol, is that the later stage 1980s is by U.S. Schering-
The broad spectrum antibiotic of the special chloromycetin of a kind of beasts that Plough succeeds in developing, is third generation chloromycetin series antibiotics, this medicine
Nineteen ninety lists in Japan first, as the succedaneum of new chloromycetin, mainly suppresses the combination of 50s subunit thus suppresses bacterial peptide
The activity of acyl group transaminase, is the wide spectrum antibiosis of novel resisting gram-positive bacteria, gram negative bacteria and thiamphenicol fastbacteria
Element, can be used for treating pig, cattle, fowl and the bacterial disease of aquatic animal.
Chloromycetin (Chloramphenicol, CAP) is as first generation chloromycetin series antibiotics ,-NO2 base in its structure
Group is relevant with suppression hemopoietic function, and therefore CAP has spinal cord hemopoietic function suppression toxicity, and reversible hemocyte can be caused to subtract
Few, the probability causing irreversible aplastic anemia is 1:30000.FF-CH3SO2 group instead of-NO2 group, without latent
Dangerous in aplastic anemia.Meanwhile, FF is to resistance to chloromycetin and the Resistant strain of thiamphenicol (Thiamphenicol, TAP)
Still there is antibacterial action.Florfenicol plays biological effect as targeted drug, and dosage is little, instant effect, Increased Plasma Half-life,
Blood drug level is high, can maintain blood medicine for a long time, be widely used in prevention and the control of Animal diseases.China approval fluorobenzene Buddhist nun in 2000
Examine as national two class novel chiral synthon, aquaculture can be used for treat that anguilla japonica Ai Dehuashi is sick and red fin fish disease.
FF metabolism is very fast in animal body, and main metabolites is florfenicol oxamidic acid., florfenicol amine
(Florenicol amine, FFA), florfenicol alcohol etc., but FFA is topmost metabolism in major part edible animal tissue
Product, the mark residue that therefore FFA was calculated as the off-drug period.Although Guo Guifang etc. think that FF injection is without the most acute
Toxicity and subchronic toxicity, but there are some researches show that the FF of high dose can be with mouse thymus, the weight reduction of spleen, and external grinds
Studying carefully humoral immunization and the cellular immunization also indicating that FF can suppress mice, the conclusion that this Guo Gui virtue draws mutually is confirmed, and shows FF
There is stronger immunotoxicity.FF extensive application in aquaculture can lead its residual in animal food, in order to ensure
Public health, the maximum residue limit of florfenicol in animal tissue is specified by China, it is desirable to muscle residue limits be 200
μ g/Kg, liver residue limits are 3000 μ g/Kg, kidney residue limits is 300 μ g/Kg.
Summary of the invention
The present invention is directed to the present situation of fluoride protector, and its harm to human body, design invention offer one can
Quickly, easy, accurately, Site Detection florfenicol and the detection kit of its metabolite florfenicol amine and preparation thereof, make
Use method.
Technical scheme realizes as follows
Florfenicol quick detection kit, it is characterized in that and includes the supporter 5 arranged from bottom to top, for soon
The speed water accepting layer 3 of absorption detecting reagent, the detection layers 2 being made up of nitrocellulose filter, the cover plate 4 of band through hole 1；Preserving number is:
Anti-florfenicol amine monoclonal antibody C secreted by CCTCC C201575 hybridoma FFA-C, colloid gold label (letter
Claim: gold mark monoclonal antibody C)；A liquid: the 0.01mol/L phosphate buffer containing 1-5%PEG20000,0.05-0.25% tween 20
(PBS)；B liquid: the 0.01mol/L phosphate buffer (PBS) containing 0.01-0.25% tween 20；
Described gold mark monoclonal antibody C is to be prepared florfenicol amine (FFA) hapten and bovine serum albumin (BSA) by alkali hydrolysis method
Florfenicol amine-bovine serum albumin (FFA-BSA) complete antigen is synthesized, as immunogen by glutaraldehyde method coupling
The anti-florfenicol amine monoclonal antibody of preparation, causes after colloid gold label；
The preparation process of monoclonal antibody C of described colloid gold label is as follows:
1, preparing colloidal gold solution with the preparation technology of known gold colloidal, the granular size of prepared gold colloidal is 10-
20nm；
2, by monoclonal antibody C (0.5-1g/L antibody protein), above-mentioned 100ml gold colloidal is joined by 150-500 μ l
In solution, mix even slowly；
3, in mixed solution, add PEG20000 so that it is final concentration of 1-5%, stand overnight；
4, by its centrifugal 8000-10000 rev/min, 1-1.5 hour, supernatant is abandoned；
5, centrifugation is taken, with containing 1-5%PEG20000,0.05-0.25% tween 20,0.02% Hydrazoic acid,sodium salt
0.01mol/L phosphate buffer hangs, and makes gold mark monoclonal antibody C；
The pH value of described phosphate buffer is 7.4, wherein contains KCI 0.2g, NaCI 8.0g, KH2PO40.2g、
Na2HPO412H2O 2.9g, distilled water 1000ml；
In described step 2, monoclonal antibody C and colloidal gold solution mix even 30-60min slowly.
The using method of florfenicol quick detection kit, it is characterized in that and comprises the following steps:
1, take solution 5 μ l to be measured, on some nitrocellulose filter in the through hole 1 of cover plate 4, dry；
2, the A liquid 100 μ l of 0.01mol/LPBS containing 1-5%PEG20000,0.05-0.25% tween 20 is added thereon
Penetrate into, close；
3, add 100 μ l gold mark monoclonal antibody C, penetrate into；
4, the B liquid 100 μ l adding the 0.01mol/LPBS containing 0.05-0.25% tween 20 penetrates into；
5, result shows: occur on nitrocellulose filter that punctation is positive, represent detected florfenicol or
Florfenicol amine remains；Redfree speckle is negative, represents and can't detect florfenicol or florfenicol amine, or both
Concentration is less than 10ug/ml.
Florfenicol quick detection kit Cleaning Principle of the present invention:
Be added drop-wise on nitrocellulose filter to dry fixing by testing sample, adding confining liquid A fluid-tight, to close dropping gold mark afterwards single
Anti-C, gold mark monoclonal antibody C specificity and florfenicol or florfenicol amine are combined, and after adding the cleaning of washing liquid B liquid, wash away and do not tie
Close golden labeling antibody, utilize gold colloidal red granules colour developing principle present testing result, on detection layers nitrocellulose filter in
Existing punctation, indicates florfenicol or florfenicol amine residual；Redfree speckle, indicates without florfenicol or fluorobenzene
Buddhist nun examines amine or residual quantity is extremely low.
The invention have the advantages that
1, the florfenicol quick detection kit of the present invention, from actual cultivation needs, by the inspection of drug residue
Survey the cultivation site moved to, without specialized facilities and operating technology during use, be suitable for the detection of common raiser's cultivation site, application
Simply.2, there is the detection feature such as quick, easy, accurate, sensitive, and there is higher stability, just may be used within 5-10min
Display testing result, once detection exceeds standard and drug metabolism can be allowed until residual is less than state how foster for cultivated animals a period of time
Family's standard, can list safely, and this is a kind of method solving the residual problem of medicine of most convenient, decreases because drug residue is to human body
Injury and raiser exceed standard because medicine is residual and caused huge loss by the return of goods, and improve because of medicine residual censorship trouble, the cycle is long, receives
Taking high reason causes more than 99% raiser to be the situation that not censorship medicine remains before cultivated animals lists.3, at whole colloid
Gold labelling and test kit play the bovine serum albumin preventing non-specific binding He playing sealing process during using, all with suitable
When concentration, PEG that molecular weight is 20000 replaced, so can stop the cross reaction caused because of bovine serum albumin, enter one
Step ensure that the specificity of test kit is (because immunogen is that to have bovine serum albumin be carrier protein in prepared by monoclonal antibody
).
Accompanying drawing explanation
Fig. 1: the structural representation of florfenicol quick detection kit of the present invention；
The ultraviolet characteristic spectrum of Fig. 2: FFA-BSA；
The ultraviolet characteristic spectrum of Fig. 3: FFA；
The ultraviolet characteristic spectrum of Fig. 4: BSA；
Fig. 5: FFA-BSA, the ultraviolet characteristic spectrum after tri-groups of collection of illustrative plates matchings of BSA, FFA；
The ultraviolet characteristic spectrum of Fig. 6: OVA；
The ultraviolet characteristic spectrum of Fig. 7: FFA-OVA；
The ultraviolet characteristic spectrum of Fig. 8: FFA；
Ultraviolet characteristic spectrum after tri-groups of collection of illustrative plates matchings of Fig. 9: FFA-OVA, OVA, FFA.
Hybridoma cell strain FFA-C, its deposit number is: CCTCC C201575；Depositary institution's full name and abbreviation: in
State's Type Tissue Collection (CCTCC)；Depositary institution address: in the preservation of Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University
The heart；Preservation date: on May 21st, 2015.
Detailed description of the invention
Embodiments of the invention combine accompanying drawing and are further described below:
Embodiment 1
The synthesis of florfenicol amine: be 1:1 according to mol ratio, weighs FF10.74g, sodium hydroxide 2.56g, adds 35ml
Distilled water dissolves, and heated and stirred is to being completely dissolved, and now solution is claret clarification, adds 6.0g sodium chloride, and stirring is to complete
CL, with thin layer chromatography plate layer chromatography mixed liquor after cooling, with the stifling colour developing of iodine, records the Rf (distance of initial point to spot centers
Ratio from the distance of initial point to solvent front) value is different with FF, it was demonstrated that there is FFA to generate, by solution left at room temperature over night, with filter
Paper filter, distilled water wash to trickle be colourless, be dried after FFA hapten, synthetic route is as follows:
Embodiment 2
The preparation of florfenicol amine complete antigen: weigh 25mg florfenicol amine and be dissolved in 2mLN, dinethylformamide
(DMF) in；Weigh 66mg BSA to be dissolved in the PBS of 10mlPH7.4；Florfenicol amine is added dropwise to above-mentioned
In BSA solution, stir under the conditions of 4 DEG C, be then added dropwise over 100 μ L glutaraldehydes in reactant liquor, be slowly stirred reaction 24h.Will
The product PBS 72h, every 8h of synthesis change liquid once, obtain florfenicol amine-bovine serum albumin (FFA-BSA) immunogen,
Florfenicol amine-oralbumin (FFA-OVA) coating antigen is prepared with method.Synthetic route is as follows:
Embodiment 3
Ultraviolet characteristic spectrum
The characteristic absorption peak of complete antigen FFA-BSA is 222nm, 267nm, and the characteristic absorption peak of FFA be 220nm,
The characteristic absorption peak of 269nm, BSA is 278nm, and the ultraviolet characteristic peak of complete antigen there occurs obvious displacement, shows that coupling becomes
Merit.Shown in scanning result as accompanying drawing 2-5.
The characteristic absorption peak of complete antigen FFA-OVA is 224nm, 267.5nm, and the characteristic absorption peak of FFA be 220nm,
The characteristic absorption peak of 269nm, OVA is 280nm, and the ultraviolet characteristic peak of complete antigen there occurs obvious displacement, shows that coupling becomes
Merit.Scanning result is as shown in accompanying drawing 6-9.
Embodiment 4
The preparation of the monoclonal antibody of anti-florfenicol amine: the monoclonal antibody secreting anti-florfenicol amine of the present invention
Hybridoma, its preparation is as follows with the method selected:
(1) alkali hydrolysis method prepares florfenicol amine (FFA) hapten
(2) florfenicol amine complete antigen is synthesized, including immunogen FFA-BSA, coating antigen FFA-OVA with glutaraldehyde method；
(3) with immunogen FFA-BSA immunity Balb/c white mice；
(4) by the fusion of the splenocyte of immune mouse Yu myeloma cell, 120 strain of hybridoma are cultivated to obtain；
(5) positive hybridoma cell strain is screened with coating antigen FFA-OVA wrapper sheet indirect elisa method；
(6) use limiting dilution assay that wherein 4 strain positive hybridoma cells are cloned；
(7) monoclonal antibody 2D6-1C4 bis-time cloning of the highest anti-florfenicol amine of titer is filtered out by indirect elisa method
Antibody prepared by cell strain, preparation gold mark monoclonal antibody.It is shown in Table 1
Table 1: monoclonal antibody the selection result
Embodiment 5
The specificity identification of the florfenicol amine monoclonal antibody C of the present invention: different from 4 kinds by anti-florfenicol amine monoclonal antibody
Antibiotic be at war with ELLIA detection, use FFA-OVA wrapper sheet.Negative control PBS wrapper sheet；Sun contrasts, and replaces using with PBS
Antibiotic in detection cross reaction.Testing result, florfenicol amine monoclonal antibody can be reacted with florfenicol amine and also can
React with florfenicol, both zero differences.And florfenicol amine monoclonal antibody and similar antibiotic thiamphenicol and other
The oxytetracycline of class such as Macrolide, the norfloxacin hydrochloride of quinolones and the equal no cross reaction of the sulfamethoxazole of sulfonamides.Say
This antibody specificity bright is strong, and can not only detect the former medicine of florfenicol and also can detect its internal metabolite florfenicol
Amine.It is shown in Table 2
Table 2: the experiment of anti-florfenicol amine monoclonal antibody specificity (N=6)
Note: *. comparing with positive controls, OD value is substantially reduced, (P ＜ 0.01)
Embodiment 6
The preparation process of monoclonal antibody C of colloid gold label is as follows:
1, the preparation of gold colloidal: prepared by 10-20nm colloid gold particle, by 0.01% chlorauric acid solution 250ml and 1% Fructus Citri Limoniae
Acid sodium 6.65ml mixing, is heated to 100 degrees Celsius, makes colloidal gold solution, the pH value of this solution: be adjusted to 0.2M potassium carbonate
8.2, standby；
2, the preparation of gold mark monoclonal antibody C:
Anti-fluorobenzene Buddhist nun's amine examines amine monoclonal antibody concentration when being 1g/L, and the monoclonal antibody amount of 100ml colloid gold label is 200 μ l, compares by this
Anti-florfenicol monoclonal antibody amine is joined in colloidal gold solution by example, stirs 50min slowly, adds 3%PEG20000, and 4 DEG C overnight；Then
By it at 4 DEG C, 8000r is centrifuged 60min, centrifugation, abandons supernatant, with containing 3%PEG20000 and 0.25% tween 20
0.01mol/L phosphate buffer (KCL 0.2g, NaCL 8.0g, KH2PO40.2g,Na2HPO412H2O 2.9g, distilled water
1000ml, pH 7.4) hang, then add Hydrazoic acid,sodium salt concentration is adjusted to 0.02%, i.e. make gold mark monoclonal antibody C.
Embodiment 7
The minimum residual quantity of the florfenicol quick detection kit detection of the present invention is 10ug/ml.The results are shown in Table 3
Table 3: gold mark monoclonal antibody C best effort concentration
Note: ++ expression spot colors is aubergine, and testing result is strong positive；+ represent that spot colors is red, detection knot
Fruit is positive；Representing that speckle is colourless, testing result is negative.
Embodiment 8
The using method of the florfenicol quick detection kit of the present invention, the i.e. detecting step of florfenicol: take to be measured
Sample 5 μ l point dries on nitrocellulose filter, adds A liquid (confining liquid) 100 μ l and penetrates into, and then adds gold mark monoclonal antibody C 100 μ l and oozes
Enter, add B liquid (washing liquid) 100 μ l and penetrate into, within 5 minutes, read result.
Positive findings: present punctation on detection layers nitrocellulose filter, indicates florfenicol or florfenicol
Amine remains；
Negative findings: redfree speckle on detection layers nitrocellulose filter, represents and can't detect florfenicol, florfenicol
Amine or both concentration are less than 10ug/ml.
Claims (7)
1. florfenicol quick detection kit, it is characterised in that include the supporter arranged from bottom to top, for quickly absorbing
The water accepting layer (3) of detectable, the detection layers (2) being made up of nitrocellulose filter, the cover plate (4) of band through hole (1)；Preserving number
For: secreted by CCTCC C201575 hybridoma FFA-C, anti-florfenicol amine monoclonal antibody C of colloid gold label
(being called for short: gold mark monoclonal antibody C), A liquid: tell containing the Polyethylene Glycol (PEG20000) that 1-5% molecular weight is 20000,0.05-0.25%
The 0.01mol/L phosphate buffer (PBS) of temperature-20, B liquid: the 0.01mol/L phosphate containing 0.01-0.25% tween 20 delays
Rush liquid.
Florfenicol quick detection kit the most according to claim 1, it is characterised in that: described gold mark monoclonal antibody C be by
Alkali hydrolysis method prepares the florfenicol that florfenicol amine hapten is synthesized by glutaraldehyde method coupling with bovine serum albumin (BSA)
Amine-bovine serum albumin complete antigen, the anti-florfenicol amine monoclonal antibody prepared as immunogen, through gold colloidal
Cause after labelling.
Florfenicol quick detection kit the most according to claim 1, it is characterised in that: described gold mark monoclonal antibody C, its system
Preparation Method is as follows:
(1), preparing colloidal gold solution, the granular size of prepared gold colloidal is 10-20nm；
(2), by monoclonal antibody C, join in above-mentioned 100ml colloidal gold solution by 150-500 μ l, mix even slowly；
(3), in mixed solution, add PEG20000 so that it is final concentration of 1-5%, stand overnight；
(4), it is centrifuged 8000-10000 rev/min, 1-1.5 hour, abandons supernatant；
(5), centrifugation is taken, with containing 1-5%PEG20000,0.05-0.25% tween 20,0.02% Hydrazoic acid,sodium salt
0.01mol/L phosphate buffer hangs, and makes gold mark monoclonal antibody C.
4. according to the florfenicol quick detection kit described in claim 3, it is characterised in that gold colloidal described in step (1)
The preparation method of solution is to be mixed and heated gold chloride and trisodium citrate by certain proportioning to be prepared as colloidal gold solution.
5. according to the florfenicol quick detection kit described in claim 3, it is characterised in that monoclonal antibody in step (2)
C and colloidal gold solution mix even 30-60min slowly.
6. the using method of florfenicol quick detection kit, it is characterised in that comprise the following steps: testing sample is dripped
On nitrocellulose filter, dry, then add A liquid and penetrate into, add gold mark monoclonal antibody C and penetrate into, add B liquid and penetrate into, 5-10 minute, just may be used
Nitrocellulose filter shows testing result.
7. according to the using method of the florfenicol quick detection kit described in claim 6, it is characterised in that include following
Step:
(1) take solution 5 μ l to be measured, on some nitrocellulose filter in the through hole (1) of cover plate (4), dry；
(2) the A liquid 100 μ l adding the 0.01mol/LPBS containing 1-5%PEG20000,0.05-0.25% tween 20 thereon oozes
Enter, close；
(3) add 100 μ l gold mark monoclonal antibody C, penetrate into；
(4) the B liquid 100 μ l adding the 0.01mol/LPBS containing 0.01-0.25% tween 20 penetrates into；
(5) result shows: occurs on nitrocellulose filter that punctation is positive, represents and florfenicol or fluorobenzene detected
Buddhist nun examines amine residual, and redfree speckle is negative, represents and can't detect florfenicol or florfenicol amine, or both concentration
Less than 10 μ g/ml.
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| CN106290927B (en) * | 2016-07-26 | 2018-12-14 | 鲁东大学 | Fortimicin quick detection kit and its preparation, application method |
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Effective date of registration: 20191204 Address after: No.3, unit 1, building 55, No.184, Shixue Road, Zhifu District, Yantai City, Shandong Province Patentee after: Song Kaiwen Address before: 264000 No. 186 Hongqi Middle Road, Zhifu District, Shandong, Yantai Patentee before: Ludong University |
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| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220728 Address after: 255100 1000 meters west of the village committee of zhaowa village, Shuangyang Town, Zichuan District, Zibo City, Shandong Province Patentee after: Shandong Dehao Biotechnology Co.,Ltd. Address before: 264000 No.3, unit 1, building 55, 184 Shixue Road, Zhifu District, Yantai City, Shandong Province Patentee before: Song Kaiwen |