CN105004860A - Florfenicol rapid detection kit and preparation and use method thereof - Google Patents
Florfenicol rapid detection kit and preparation and use method thereof Download PDFInfo
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Abstract
The invention relates to an improvement of an antibiotic detecting technology, in particular to a florfenicol rapid detection kit and a preparation and use method thereof, and belongs to the field of immunology. The florfenicol rapid detection kit comprises a supporting body, a water absorption layer, a detecting layer and a cover plate with a through hole, the supporting body, the water absorption layer, the detecting layer and the cover plate are arranged from bottom to top. The conservation number is CCTCC-C201575. Anti-florfenicol amine monoclonal antibody C secreted by hybridomas FFA-C and marked by colloidal gold comprises liquid A and liquid B, the liquid A includes 1%-5% of polyethylene glycol with the molecular weight of 20,000 and 0.01%-0.25% oftween-20 0.01 mol/L phosphate buffer, and the liquid B comprises 0.01%-0.25% of tween-20 0.01 mol/L phosphate buffer. The kit has the advantages of being simple, convenient to use, fast, accurate and sensitive, the minimum detecting concentration is 10 microgramme/ml, and the kit can be used for carrying out coarse screening detection on florfenicol drug residues on site.
Description
Technical field
The present invention relates to the improvement of microbiotic detection technique, be specifically related to Florfenicol quick detection kit and preparation thereof, using method, belong to field of immunology.
Background technology
Florfenicol (Florfenico, FF) Chinese: Fluprofen, Florfenicol, chemical name: d (+)-Su-1-p-methylphenyl-2-dichloro acetamino-3-fluorine propyl alcohol, it is the broad spectrum antibiotic of the special chloromycetin of a kind of beasts succeeded in developing by U.S. Schering-Plough in the later stage 1980s, it is third generation chloromycetin series antibiotics, this medicine nineteen ninety goes on the market in Japan first, as the substitute of new chloromycetin, the combination of main suppression 50s subunit thus the activity of anti-bacteria acyltransferase polypeptide transaminase, it is novel resisting gram-positive bacteria, the broad-spectrum antibiotic of Gram-negative bacteria and Thiamphenicol drug-fast bacteria, can be used for treating pig, ox, the bacteriosis of fowl and aquiculture animal.
Chloromycetin (Chloramphenicol, CAP) as first generation chloromycetin series antibiotics, its structural-NO2 group is relevant with suppression hematopoiesis function, therefore CAP has spinal cord hematopoiesis function suppression toxicity, reversible haemocyte can be caused to reduce, and the probability causing irreversible alpastic anemia is 1:30000.FF-CH3SO2 group instead of-NO2 group, dangerous without potential alpastic anemia.Meanwhile, the antibody-resistant bacterium of FF to resistance to chloromycetin and Thiamphenicol (Thiamphenicol, TAP) still has antibacterial action.Florfenicol plays biological effect as targeted drug, and dosage is little, instant effect, Increased Plasma Half-life, and blood concentration is high, can maintain blood medicine for a long time, be widely used in the prevention and corntrol of Animal diseases.Within 2000, China's approval Florfenicol is national two class novel chiral synthon, and aquaculture can be used for treatment common eel Ai Dehuashi disease and red fin fish disease.
FF metabolism is very fast in animal body, main metabolites is Florfenicol oxamic acid, florfenicol amine (Florenicol amine, FFA), Florfenicol alcohol etc., but FFA is topmost metabolic product in most of edible animal tissue, and therefore FFA is by the mark residue calculated as the off-drug period.Although Guo Guifang etc. think that FF parenteral solution is without obvious acute toxicity and subchronic toxicity, but there are some researches show high dose FF can mouse thymus, spleen weight reduce, external research also shows that FF can suppress humoral immunity and the cellular immunity of mouse, the conclusion that this Guo Gui virtue draws is confirmed mutually, shows that FF has stronger immunotoxicity.The extensive application of FF in aquaculture can lead its remaining in animal food, in order to protect public health, China specifies the maximum residue limit of Florfenicol in animal tissue, and the muscle residue limits of requirement is 200 μ g/Kg, liver residue limits is 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 it is to the harm of human body, design invention provide a kind of can the detection kit of quick, easy, accurate, Site Detection Florfenicol and its metabolic product florfenicol amine and preparation thereof, using method.
Technical scheme of the present invention realizes as follows
Florfenicol quick detection kit, its special character be to comprise arrange from bottom to top supporter 5, the water accepting layer 3 for quick absorption detecting reagent, the detection layers 2 be made up of nitrocellulose filter, band through hole 1 cover plate 4; Preserving number is: anti-florfenicol amine monoclonal antibody C secreted by CCTCC-C201575 hybridoma FFA-C, colloid gold label (being called for short: gold mark monoclonal antibody C); A liquid: containing the 0.01mol/L phosphate buffer (PBS) of 1-5%PEG20000,0.05-0.25% Tween-20; B liquid: containing the 0.01mol/L phosphate buffer (PBS) of 0.01-0.25% Tween-20;
Described gold mark monoclonal antibody C prepares florfenicol amine (FFA) haptens by alkali hydrolysis method and bovine serum albumin(BSA) (BSA) synthesizes florfenicol amine-bovine serum albumin(BSA) (FFA-BSA) comlete antigen by glutaraldehyde method coupling, it can be used as immunogene and the anti-florfenicol amine monoclonal antibody prepared, cause after colloid gold label;
The preparation process of the monoclonal antibody C of described colloid gold label is as follows:
1, prepare colloidal gold solution with the preparation technology of known collaurum, the grain size of obtained collaurum is 10-20nm;
2, by monoclonal antibody C (0.5-1g/L antibody protein), join in above-mentioned 100ml colloidal gold solution by 150-500 μ l, mix even slowly;
3, in mixed solution, add PEG20000, make its final concentration be 1-5%, hold over night;
4, by its centrifugal 8000-10000 rev/min, 1-1.5 hour, supernatant is abandoned;
5, get centrifugation, hang with the 0.01mol/L phosphate buffer containing 1-5%PEG20000,0.05-0.25% Tween-20,0.02% sodium azide, make gold mark monoclonal antibody C;
The pH value of described phosphate buffer is 7.4, wherein containing KCI 0.2g, NaCI 8.0g, KH
2pO
40.2g, Na
2hPO
412H
2o 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, its special character is to comprise the following steps:
1, get solution 5 μ l to be measured, put on the nitrocellulose filter in the through hole 1 of cover plate 4, dry;
2, the A liquid 100 μ l added thereon containing the 0.01mol/LPBS of 1-5%PEG20000,0.05-0.25% Tween-20 infiltrates, and closes;
3, add 100 μ l gold mark monoclonal antibody C, infiltrate;
4, the B liquid 100 μ l added containing the 0.01mol/LPBS of 0.05-0.25% Tween-20 infiltrates;
5, result display: nitrocellulose filter occurs punctation is positive, represents and has detected that Florfenicol or florfenicol amine remain; Redfree spot is negative, represent and can't detect Florfenicol or florfenicol amine, or both concentration is lower than 10ug/ml.
Florfenicol quick detection kit Cleaning Principle of the present invention:
Testing sample is added drop-wise to nitrocellulose filter dries fixing, add after confining liquid A fluid-tight is closed and drip gold mark monoclonal antibody C, gold is marked monoclonal antibody C specificity and Florfenicol or florfenicol amine and is combined, after adding the cleaning of washing lotion B liquid, wash away unconjugated golden labeling antibody, utilize the collaurum red granules principle that develops the color to present testing result, detection layers nitrocellulose filter presents punctation, indicates that Florfenicol or florfenicol amine are residual; Redfree spot, indicate without Florfenicol or florfenicol amine or residual quantity extremely low.
The present invention has the following advantages:
1, Florfenicol quick detection kit of the present invention, from the cultivation needs of reality, by the cultivation site that the detection of medicament residue is moved to, without the need to specialized facilities and operative technique during use, be applicable to common raiser's cultivation site and detect, application is simple.2, have and detect fast, easy, accurately, the feature such as sensitive, and there is higher stability, just testing result can be shown within 5-10min, once detect, exceed standard can by how foster for cultivated animals a period of time, allow drug metabolism until residual lower than national standard, can go on the market safely, this is a kind of method solving the residual problem of medicine of most convenient, decrease and caused huge loss because medicament residue to exceed standard because medicine is residual to the injury of human body and raiser by the return of goods, and improve because medicine remains censorship trouble, cycle is long, high reason of charging causes more than 99% raiser to be the situation that not censorship medicine remains before cultivated animals listing.3, in whole colloid gold label and kit use procedure, the bovine serum albumin(BSA) preventing non-specific binding and play sealing process is played, all with debita spissitudo, PEG that molecular weight is 20000 replaced, the cross reaction caused because of bovine serum albumin(BSA) can be stopped like this, further ensure the specificity (because in monoclonal antibody preparation, immunogene has bovine serum albumin(BSA) to be carrier protein) of kit.
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, BSA, FFA tri-picture group spectrum matching after ultraviolet characteristic spectrum;
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;
Fig. 9: FFA-OVA, OVA, FFA tri-picture group spectrum matching after ultraviolet characteristic spectrum.
Hybridoma cell strain FFA-C, its deposit number is: CCTCC-C201575; Depositary institution's full name and abbreviation: China typical culture collection center (CCTCC); Depositary institution address: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center; Preservation date: on May 21st, 2015.
Embodiment
Embodiments of the invention further describe as follows by reference to the accompanying drawings:
Embodiment 1
The synthesis of florfenicol amine: be 1:1 according to mol ratio, take FF10.74g, NaOH 2.56g, add 35ml distilled water to dissolve, heating is stirred to dissolves completely, now solution is claret clarification, add 6.0g sodium chloride again, be stirred to and dissolve completely, with thin-layer chromatography plate layer chromatography mixed liquor after cooling, with the stifling colour developing of iodine, record Rf (initial point is to the distance of spot centers and the ratio of initial point to the distance of solvent front) value different with FF, proof has FFA to generate, by solution left at room temperature over night, with Filter paper filtering, distilled water washing is colourless to trickle, FFA haptens after drying, synthetic route is as follows:
Embodiment 2
The preparation of florfenicol amine comlete antigen: take 25mg florfenicol amine and be dissolved in 2mLN, in dinethylformamide (DMF); Weighing 66mg BSA is dissolved in the PBS damping fluid of 10mlPH7.4; Florfenicol amine is dropwise joined in above-mentioned BSA solution, stir under 4 DEG C of conditions, then dropwise add 100 μ L glutaraldehydes in reactant liquor, slow stirring reaction 24h.Dialyse the product PBS of synthesis 72h, and every 8h changes liquid once, obtains florfenicol amine-bovine serum albumin(BSA) (FFA-BSA) immunogene, with legal system for florfenicol amine-oralbumin (FFA-OVA) coating antigen.Synthetic route is as follows:
Embodiment 3
Ultraviolet characteristic spectrum
The characteristic absorption peak of comlete antigen FFA-BSA is 222nm, 267nm, and the characteristic absorption peak of FFA is 220nm, 269nm, and the characteristic absorption peak of BSA is 278nm, and the ultraviolet characteristic peak of comlete antigen there occurs obvious displacement, shows coupling success.Scanning result is shown in shown in accompanying drawing 2-5.
The characteristic absorption peak of comlete antigen FFA-OVA is 224nm, 267.5nm, and the characteristic absorption peak of FFA is 220nm, 269nm, and the characteristic absorption peak of OVA is 280nm, and the ultraviolet characteristic peak of comlete antigen there occurs obvious displacement, shows coupling success.Scanning result is as shown in accompanying drawing 6-9.
Embodiment 4
The preparation of the monoclonal antibody of anti-florfenicol amine: the hybridoma of the monoclonal antibody of the anti-florfenicol amine of secretion of the present invention, its preparation is as follows with the method selected:
(1) alkali hydrolysis method prepares florfenicol amine (FFA) haptens
(2) with glutaraldehyde method synthesis florfenicol amine comlete antigen, immunogene FFA-BSA is comprised, coating antigen FFA-OVA;
(3) with immunogene FFA-BSA immunity Balb/c small white mouse;
(4) by the splenocyte of immune mouse and the fusion of myeloma cell, 120 strain of hybridoma are cultivated to obtain;
(5) with the strain of coating antigen FFA-OVA wrapper sheet indirect elisa method screening positive hybridoma cell;
(6) limiting dilution assay is adopted to clone wherein 4 strain positive hybridoma cells;
(7) antibody prepared by the cell line being filtered out monoclonal antibody 2D6-1C4 bis-time cloning of the highest anti-florfenicol amine of tiring by indirect elisa method, preparation gold mark monoclonal antibody.In table 1
Table 1: monoclonal antibody the selection result
Embodiment 5
The specificity identification of florfenicol amine monoclonal antibody C of the present invention: detect with anti-florfenicol amine monoclonal antibody microbiotic different from the 4 kinds ELLIA that is at war with, use FFA-OVA wrapper sheet.Negative control PBS wrapper sheet; Sun contrasts, and replaces the microbiotic for detecting cross reaction with PBS.Testing result, florfenicol amine monoclonal antibody can be reacted with florfenicol amine and also can be reacted with Florfenicol, both indifferences.And florfenicol amine monoclonal antibody and similar microbiotic Thiamphenicol and other classes are as the terramycin of macrolides, the norfloxacin hydrochloride of quinolones and the equal no cross reaction of the radonil of sulfamido.This antibody high specificity is described, and the former medicine of Florfenicol can not only be detected also can detects metabolic product florfenicol amine in its body.In table 2
Table 2: anti-florfenicol amine monoclonal antibody specificity experiment (
n=6)
Note: *. compare with positive controls, OD value significantly reduces, (P < 0.01)
Embodiment 6
The preparation process of the monoclonal antibody C of colloid gold label is as follows:
1, the preparation of collaurum: prepared by 10-20nm colloid gold particle, mixed by 0.01% chlorauric acid solution 250ml, be heated to 100 degrees Celsius, make colloidal gold solution, the pH value of this solution: be adjusted to 8.2 with 0.2M sal tartari with 1% sodium citrate 6.65ml, for subsequent use;
2, the preparation of gold mark monoclonal antibody C:
Anti-fluorobenzene Buddhist nun amine examines amine monoclonal antibody concentration when being 1g/L, and the monoclonal antibody amount of 100ml colloid gold label is 200 μ l, and join in colloidal gold solution in this ratio by anti-Florfenicol monoclonal antibody amine, stir 50min slowly, add 3%PEG20000,4 DEG C are spent the night; Then by it at 4 DEG C, the centrifugal 60min of 8000r, centrifugation, abandons supernatant, with 0.01mol/L phosphate buffer (KCL 0.2g, NaCL 8.0g, KH containing 3%PEG20000 and 0.25% Tween-20
2pO
40.2g, Na
2hPO
412H
2o 2.9g, distilled water 1000ml, pH 7.4) hang, then add sodium azide concentration is adjusted to 0.02%, namely make gold mark monoclonal antibody C.
Embodiment 7
The minimum residual quantity that Florfenicol quick detection kit of the present invention detects 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; + representing that spot colors is red, testing result is positive;-representing that spot is colourless, testing result is negative.
Embodiment 8
The using method of Florfenicol quick detection kit of the present invention, the i.e. detecting step of Florfenicol: get testing sample 5 μ l point and dry on nitrocellulose filter, add A liquid (confining liquid) 100 μ l to infiltrate, then add gold mark monoclonal antibody C 100 μ l to infiltrate, add B liquid (washing lotion) 100 μ l to infiltrate, within 5 minutes, read result.
Positive findings: detection layers nitrocellulose filter presents punctation, indicates Florfenicol or florfenicol amine remains;
Negative findings: redfree spot on detection layers nitrocellulose filter, represents and can't detect Florfenicol, florfenicol amine or both concentration lower than 10ug/ml.
Claims (7)
1. Florfenicol quick detection kit, its special character be to comprise arrange from bottom to top supporter, the water accepting layer (3) for quick absorption detecting reagent, the detection layers (2) be made up of nitrocellulose filter, band through hole (1) cover plate (4), preserving number is anti-florfenicol amine monoclonal antibody C secreted by CCTCC-C201575 hybridoma FFA-C, colloid gold label (being called for short: gold mark monoclonal antibody C); A liquid: be the polyglycol (PEG20000) of 20000, the 0.01mol/L phosphate buffer (PBS) of 0.05-0.25% Tween-20 containing 1-5% molecular weight; B liquid: containing the 0.01mol/L phosphate buffer of 0.01-0.25% Tween-20.
2. Florfenicol quick detection kit according to claim 1, it is characterized in that: described gold mark monoclonal antibody C: be prepare by alkali hydrolysis method florfenicol amine-bovine serum albumin(BSA) (FFA-BSA) comlete antigen that florfenicol amine (FFA) haptens and bovine serum albumin(BSA) (BSA) synthesized by glutaraldehyde method coupling, it can be used as immunogene and the anti-florfenicol amine monoclonal antibody prepared, cause after colloid gold label.
3. Florfenicol quick detection kit according to claim 1, it is characterized in that: described gold mark monoclonal antibody C, its preparation method is as follows:
(1), prepare colloidal gold solution, the grain size of obtained collaurum is 10-20nm;
(2), by monoclonal antibody C (0.5-1g/L antibody protein), join in above-mentioned 100ml colloidal gold solution by 150-500 μ l, mix even slowly;
(3), in mixed solution add PEG20000, make its final concentration be 1-5%, hold over night;
(4), centrifugal 8000-10000 rev/min, 1-1.5 hour, abandon supernatant;
(5), get centrifugation, hang with the 0.01mol/L phosphate buffer containing 1-5%PEG20000,0.05-0.25% Tween-20,0.02% sodium azide, make gold mark monoclonal antibody C.
4., according to Florfenicol quick detection kit according to claim 3, it is characterized in that gold chloride mixes with trisodium citrate and heat by certain proportioning to be prepared into colloidal gold solution by the preparation method of colloidal gold solution described in step (1).
5., according to Florfenicol quick detection kit according to claim 3, it is characterized in that step 2) in monoclonal antibody C and colloidal gold solution mix even 30-60min slowly.
6. the using method of Florfenicol quick detection kit, is characterized in that comprising the following steps: be added drop-wise to by testing sample on nitrocellulose filter, dry, then add A liquid to infiltrate, add gold mark monoclonal antibody C and infiltrate, add B liquid and infiltrate, 5-10 minute, just can show testing result on nitrocellulose filter.
7., according to the using method of Florfenicol quick detection kit according to claim 6, it is characterized in that comprising the following steps:
(1) get solution 5 μ l to be measured, put on the nitrocellulose filter in the through hole 1 of cover plate 4, dry;
(2) the A liquid 100 μ l added thereon containing the 0.01mol/LPBS of 1-5%PEG20000,0.05-0.25% Tween-20 infiltrates, and closes;
(3) add 100 μ l gold mark monoclonal antibody C, infiltrate;
(4) the B liquid 100 μ l added containing the 0.01mol/LPBS of 0.01-0.25% Tween-20 infiltrates;
(5) result display: nitrocellulose filter occurs punctation is positive, represent and detected that Florfenicol or florfenicol amine remain, redfree spot is negative, represent and can't detect Florfenicol or florfenicol amine, or both concentration is lower than 10ug/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 |
| CN117402254A (en) * | 2023-10-19 | 2024-01-16 | 河北农业大学 | Genetic engineering antibody for identifying florfenicol and application thereof |
| CN117402254B (en) * | 2023-10-19 | 2024-04-16 | 河北农业大学 | Genetic engineering antibody for identifying florfenicol and application thereof |
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