CN101713778A - Detection kit for ciprofloxacin and detection method thereof - Google Patents
Detection kit for ciprofloxacin and detection method thereof Download PDFInfo
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- CN101713778A CN101713778A CN200910232655A CN200910232655A CN101713778A CN 101713778 A CN101713778 A CN 101713778A CN 200910232655 A CN200910232655 A CN 200910232655A CN 200910232655 A CN200910232655 A CN 200910232655A CN 101713778 A CN101713778 A CN 101713778A
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- cip
- antibody
- goat anti
- ciprofloxacin
- rabbit antibody
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- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 title claims abstract description 62
- 229960003405 ciprofloxacin Drugs 0.000 title claims abstract description 31
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 102100030176 Muscular LMNA-interacting protein Human genes 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 29
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 238000005406 washing Methods 0.000 claims abstract description 8
- 238000005259 measurement Methods 0.000 claims abstract description 5
- 239000012530 fluid Substances 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 25
- 238000013016 damping Methods 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 19
- 239000000427 antigen Substances 0.000 claims description 17
- 102000036639 antigens Human genes 0.000 claims description 17
- 108091007433 antigens Proteins 0.000 claims description 17
- 241000283707 Capra Species 0.000 claims description 15
- 238000010790 dilution Methods 0.000 claims description 13
- 239000012895 dilution Substances 0.000 claims description 13
- 239000012086 standard solution Substances 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 230000036039 immunity Effects 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
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- 229910052693 Europium Inorganic materials 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 230000002708 enhancing effect Effects 0.000 claims description 6
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000000376 reactant Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 238000003018 immunoassay Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 239000002671 adjuvant Substances 0.000 claims description 3
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- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 3
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- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
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- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 1
- WVVLURYIQCXPIV-UHFFFAOYSA-N 4,4,4-trifluoro-1-naphthalen-2-ylbutane-1,3-dione Chemical class C1=CC=CC2=CC(C(=O)CC(=O)C(F)(F)F)=CC=C21 WVVLURYIQCXPIV-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a detection kit for ciprofloxacin and a detection method thereof, which belong to the technical field of TRFIA and are used for detecting CIP in animal derived food, blood, urine and fodder. The prepared kit adopts the TRFIA to detect the CIP, and the measurement base is labeled immune response. A micro-pore plate is clad with CIP-carrier protein; and CIP standard or sample and CIP antibody are added into the micro-pore plate. The free CIP and the CIP-carrier protein on the micro-pore plate compete for the CIP antibody, the unconnected CIP antibody is removed by washing, then Eu3+-goat anti-rabbit antibody is added into the micro-pore plate, and the unconnected Eu3+-goat anti-rabbit antibody is removed by washing after the labeled immune response. After the enhancement liquid is added, the fluorescence intensity cps is measured by using a time-resolved fluorescence instrument, the fluorescence intensity is in inverse proportion to the concentration of the CIP in the sample, and the content of the CIP in the detected sample can be determined by referring to a stand curve. The kit provided by the invention for detecting the CIP has the advantages of simple structure, using convenience, low price and high sensitivity, and can reach over 0.01ng/mL.
Description
Technical field
The present invention relates to a kind of detection Ciprofloxacin (Ciprofloxacin, CIP) detection kit and detection method thereof, belong to time resolved fluoro-immunoassay (Time resolvedfluoroisnmunoassay, TRFIA) technical field is used for the detection of animal derived food, blood, urine and feed Ciprofloxacin drug residue.
Background technology
Ciprofloxacin (ciprofloxacin, CIP), claim Ciprofloxacin again, belong to the antibiotic synthetic drug of third generation quinolones, its has a broad antifungal spectrum, antibacterial activity are strong, be one of medicine that antibacterial activity is the strongest in the fluoroquinolones of present clinical practice, be widely used in human clinical and livestock breeding industry.Because this class medicine easily brings out bacterial drug resistance, accumulate poisoning can appear in prolonged application, thereby can cause articular cartilage disease, photosensitized reaction, liver renal toxicity and cause the genotoxicity of lymphocyte chromosomal variation, and can cause that people's tendon breaks.It is as the shared medicine of a kind of people and animals, and medicament residue is bigger to human health damage by food chain, is unfavorable for the treatment of such medicine to human diseases.Therefore strengthen the residue detection of this medicine in animal food and supervision are just seemed very necessary.The regulation that European Union, the U.S., Japan, China etc. have all made forbidding or limited the use of Ciprofloxacin maximum residue limit(MRL) in the animal derived food.
The main method that detects residue of ciprofloxacin in the animal derived food at present has microbial method, fluorescence method, high performance liquid chromatography (HPLC), LC-MS method (LC-MS), capillary electrophoresis and enzyme linked immunosorbent assay (ELISA) etc.Ciprofloxacin detection method commonly used is chromatographic detection and immunological detection.Chromatographic detection sensitivity, accurate, instrument that need be expensive and special staff, and also sample preparation is more loaded down with trivial details, and its application is subjected to certain restriction, and is especially more outstanding in the extensive screening of basic unit detects with the scene.Immunological detection sensitivity, special, quick, easy has overcome above deficiency, is fit to extensive screening.
The time resolved fluoro-immunoassay detection technique is to develop highly sensitive rapidly detection means recently.Its principle of TRFIA is to utilize the sequestrant with bifunctional group structure, one end and lanthanide series combination, and the other end is connected with free amino group on the antibody (antigen), makes Eu
3+Antigen (antibody) in the labelled antibody (antigen), it and testing sample is combined into immune complex.Ideally, the fluorescence intensity of measuring lanthanide series in the compound just can be determined the content of antigen in the sample, but the fluorescence intensity of in fact this compound quite a little less than, have only and add a kind of enhancing solution (Enhancement solution) again, lanthanide series is disintegrated down from compound, and with strengthen β-Nai formyl trifluoroacetone contained in the liquid (β-NTA) form microcapsules again, the very strong fluorescence of emission under the exciting of ultraviolet light, up to a million times of enhancing effects.Differentiate luminoscope with the time and measure its fluorescence intensity cps, can determine the amount of antigen in the sample.
Summary of the invention
The object of the present invention is to provide a kind of Ciprofloxacin detection kit and detection method, be used for qualitative or detect the residual quantity of animal derived food, blood, urine and feed Ciprofloxacin quantitatively; Detection time is short, average recovery rate is high, batch in, batch between error little, and have easy, characteristics fast and accurately.
Technical scheme of the present invention: this Ciprofloxacin detection kit be by 1, the porous bag is by plate, 2, damping fluid, 3, the Ciprofloxacin standard solution, 4, Ciprofloxacin antibody, 5, europium mark goat anti-rabbit antibody, 6, cleansing solution, 7, strengthen liquid and form.
The present invention mainly adopts time resolved fluoro-immunoassay method (TRFIA) to detect CIP.Adopt the technology of TRFIA to mainly contain two aspects: the first, the preparation of specific polyclonal antibody utilizes the artificial antigen immunizing rabbit, obtains to contain the serum of antibody, through biochemical purification separating immune globulin; The second, Eu
3+The preparation of mark goat anti-rabbit antibody.
Assay method is: the basis of mensuration is the labelled immune reaction.Be coated with the microwell plate of CIP-carrier protein, add CIP standard solution or sample preparation liquid in micropore separately, add CIP antibody again, oscillating reactions, free CIP and the competition of the CIP-carrier protein on microwell plate CIP antibody, the cleansing solution washing, the CIP antibody that does not have to connect is removed in washing step.Add Eu
3+-goat anti-rabbit antibody carries out the labelled immune reaction, and with the cleansing solution washing, the reaction back does not have the Eu of connection again
3+-goat anti-rabbit antibody is removed in washing step.After adding the vibration of enhancing liquid, the very strong fluorescence of emission under the exciting of ultraviolet light is differentiated luminoscope with the time and is measured its fluorescence intensity cps, and the concentration in fluorescence intensity and the sample is inversely proportional to, and the reference standard curve can be determined the amount of CIP in the sample.
Beneficial effect of the present invention: this kit is simple in structure, and is easy to use, cheap, highly sensitive, can reach more than the 0.01ng/mL.
Description of drawings
Fig. 1: Ciprofloxacin detection kit synoptic diagram.1, the porous bag is by plate, and 2, damping fluid, 3, the Ciprofloxacin standard solution, 4, Ciprofloxacin antibody, 5, europium mark goat anti-rabbit antibody, 6, cleansing solution, 7, strengthen liquid.
Fig. 2: CIP-TRFIA canonical plotting.
Embodiment
Embodiment 1: the preparation of kit and animal derived food detect
The preparation of immunizing antigen:
Immunizing antigen of the present invention carries out animal immune with synthetic CIP-BSA as immunizing antigen for Ciprofloxacin is adopted mixed anhydride method and macromolecular carrier protein combination.
Carrier protein adopts bovine serum albumin(BSA) (BSA).Take by weighing 30mg～300mg bovine serum albumin(BSA), be dissolved among 50% dioxane aqueous solution 6mL～10mL, adjust pH is 9～10, is A liquid; Take by weighing 10mg～40mg CIP, be dissolved in the 2mL dioxane, put 4 ℃ of 10min, add tri-n-butylamine 20 μ L and isobutyl chlorocarbonate 10 μ L successively, in 4 ℃ of stirring 30min, as B liquid.A liquid is splashed into B liquid at 4 ℃, and stirring is spent the night.With reactant liquor to distill water dialysis 72 hours, during change distilled water 5 times.Or the purification of dextran gel G50 (Sephadex G50) post, with 0.05mol/L pH8.0 carbonate buffer solution wash-out.Collect the post eluent at dialysis refined solution or the 1st peak, packing is stored in-20 ℃ and makes immunogene usefulness.
The Ciprofloxacin Polyclonal Antibody Preparation:
Above-mentioned synthetic Ciprofloxacin-bovine serum albumin(BSA) bond (CIP-B SA) is made immunizing antigen, adopt intracutaneous or subcutaneous multi-point injection mode, the male new zealand rabbit of inoculation 1kg～1.5kg; Every 4 week immunity 1 time, during immunity for the first time, in immunogene, add equivalent Freund's complete adjuvant mixing and make emulsion, immunity later on all adds the equivalent freund 's incomplete adjuvant; From immunity beginning for the second time, each immunity back blood sampling test in 8～10 days antiserum titre.Observe the antiserum titre growth pattern, reach the satisfied separation of whole blood serum of getting when tiring.
Antiserum is used the chromatographic column separation and purification again through 3 sulphoxylic acid ammonium post precipitations, and phosphate buffered solution is dialysed.Draw antiserum 10mL, balance adds the 0.01mol/LpH7.8 phosphate buffered solution of equivalent to room temperature, fully mixing adds 20mL saturated ammonium sulfate solution (transferring to pH7.8 with strong aqua), shakes up, 4 ℃ after static 1 hour, with 5000 rev/mins centrifugal 5 minutes, remove supernatant.Sediment adds 10mL saturated ammonium sulfate solution (pH7.8) then with the dissolving of 20mL 0.01mol/L pH7.8 phosphate buffered solution, shake up, 4 ℃ after static 30 minutes, with 5000 rev/mins centrifugal 5 minutes, remove supernatant.Repeat to saltout twice.Finally with 2mL0.01mol/L pH7.8 phosphate buffered solution lytic immunity globulin IgG sediment, the bag filter of packing into was to 2000mL 0.01mol/L pH7.2 phosphate buffered solution dialysis 12 hours.With above-mentioned dislysate, join cellulose DEAE-52 (extracting cellulose DEAE-52 dry powder 10 grams of about 25 gram weight in wet bases, phosphate buffered solution soaked overnight with 500mL 0.01mol/L pH8.0, suction filtration) in, 4 ℃ of balances 1 hour, stirred once in per 10 minutes, in the impouring Buchner funnel, with the 0.2mol/L phosphate buffered solution filter wash of pH8.0.Collect filtrate, to 0.01mol/L pH7.4 phosphate buffered solution dialysis 12 hours, add an amount of glycerine-18 ℃ preservation at last again.
Eu
3+The preparation of-goat anti-rabbit antibody:
Get the 5g/L goat anti-rabbit antibody 1mL～2mL that is dissolved in 50mmol/L PBS pH7.0, through the conversion buffered condition of PD-10 post, eluent is the 50mmol/LNa that contains 0.15mol/L NaCl
2CO
3-NaHCO
3The pH8.5 damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis
280-0.74A
260), dilute goat anti-rabbit antibody to 2g/L with above-mentioned eluent.The goat anti-rabbit antibody of getting after 500 μ L～1000 μ L dilute adds the Eu that contains 0.2mg～0.4mg
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu
3+In-DTTA) the bottle, 30 ℃ of magnetic agitation reactions 20 hours.Reactant liquor is through SepharoseCL-6B post (1 * 40cm) chromatography, A with 80mmol/L Tris-HCl pH7.8 damping fluid balance
280Protein peak is collected in monitoring, and the dilution packing is standby.
The solid phase antigen bag is prepared by plate:
Ciprofloxacin-oralbumin bond (CIP-OVA) is used 50mmol/LNa
2CO
3-NaHCO
3The pH9.6 damping fluid is diluted to the coating buffer of 10mg/L, and each hole of microwell plate, 96 (or 48) hole adds 100 μ L, and 4 ℃ of placements are spent the night.Discard coating buffer, wash three times, add the above-mentioned damping fluid sealing that 150 μ L contain 3g/L OVA, 4 ℃ of placements are spent the night.Discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
The preparation of reagent:
(1) Ciprofloxacin standard solution: (0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 60ng/mL 100ng/mL), dilutes from the pure product of CIP and obtains, and dilution is the 0.1mol/LpH7.5 phosphate buffer.
(2) damping fluid: 8mmol/L NaCl, 0.2% OVA, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s (DTPA), 0.1mL/L Tween-80 and 0.1%NaN
3Tris-HCl pH7.8.
(3) cleansing solution: 14.5mmol/L NaCl, 0.2mL/L Tween-80 and 0.2%NaN
350mmol/L Tris-HCl pH7.8.
(4) strengthen liquid: 1LpH3.2 Potassium Hydrogen Phthalate damping fluid contains 15 μ mol β-naphthoyltrifluoroacetones (β-NTA), 50 μ mol trioctyl-phosphine oxide (TOPO), 1mL triton x-100 (Triton X-100).
The reagent that kit provides:
Reagent in each box enough carries out 96 measurements, and the material in the box is as follows:
(1) 1 * 96 orifice plate (8 * 12 hole can be split as single hole) is coated with CIP-OVA.
(2) 6 * CIP standard solution, the 1.0mL/ bottle, concentration of standard solution is: 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 60ng/mL, 100ng/mL.
(3) 1 * CIP antibody, time spent 0.5mL dissolved in distilled water.
(4) 1 * Eu
3+-goat anti-rabbit antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(5) 1 * enhancing liquid: 15mL.
(6) 1 * cleansing solutions: 30mL, the time spent is with distilled water dilution in 1: 25.
(7) 1 * damping fluids: 30mL.
The reagent that the laboratory should be provided for oneself:
(1) distilled water or deionized water;
(2) methylene chloride;
(3) normal hexane;
(4) 0.1mol/L sodium hydroxide solution;
(5) acetonitrile-0.1mol/L sodium hydroxide solution: get anhydrous acetonitrile 84mL adding 0.1mol/L sodium hydroxide solution 16mL and mix;
(6) phosphate-buffered working fluid: pH7.2 gets 1.45g sodium hydrogen phosphate (containing 12 water of crystallization), 0.1g potassium dihydrogen phosphate (anhydrous), 8.0g sodium chloride, is dissolved in the water and fixed molten to 1000mL.
Points for attention before measuring:
(1) uses before all reagent to be gone up to room temperature (18 ℃～30 ℃).
(2) all reagent are put back to 2 ℃～8 ℃ immediately after the use.
(3) if sample size is big, the hyperchannel pipettor is used in suggestion.
(4) hatch in the process at all constant temperature, avoid irradiate light, use the cap covers micropore.
(5) take out microwell plate and the framework that needs with quantity, no microwell plate is put in the former Fresco Bag, and resealed, be stored in 2 ℃～8 ℃ with the drying agent that provides.
Concrete detection step is as follows:
Take by weighing pork sample 3.0g (being accurate to 0.01g), place the 50mL centrifuge tube, add acetonitrile-0.1mol/LNaOH solution 9mL, fully mix 10min, more than the 3800r/min, 15 ℃ of centrifugal 10min get supernatant 3mL, add 0.02mol/L PB damping fluid 3mL, add methylene chloride 8mL, fully mix 10min, more than the 3800r/min, 15 ℃ of centrifugal 10min, remove supernatant, take off layer organic phase 4mL to drying receptacle, 50 ℃ of nitrogen dry up, with the dry residue of buffering working fluid 1mL dissolving, add normal hexane 1mL mixing 2min, more than the 3800r/min, 15 ℃ of centrifugal 5min gently sop up upper strata and center section liquid, get subnatant 50 μ L and are used for analyzing.
Get the CIP-OVA lath, add the CIP standard solution of 50 μ L or sample preparation liquid in micropore separately, each standard and sample solution must use new suction nozzle, CIP antibody 50 μ L with damping fluid dilution in 1: 20,25 ℃ vibrated 1 hour, cleansing solution is given a baby a bath on the third day after its birth inferior, with the Eu of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ L, 25 ℃ vibrated 1 hour, washed six times with cleansing solution, added 200 μ L and strengthened liquid vibration measurement after 5 minutes.CIP content according in the typical curve calculation sample sees Table 1, and CIP concentration is 3.28ng/mL in this routine sample preparation liquid.
Table 1
The preparation of embodiment 2 kits
The CIP-BSA antigen preparation:
Take by weighing 12mg CIP 40mg, N-hydroxyl succinamide (NHS), 35mg carbodiimide (EDC) and be dissolved in N, among the N '-dimethyl formamide (DMF), stirring at room 24h makes A liquid; Take by weighing 50mg BSA and be dissolved among the 3mL 0.01mol/mL pH7.4 PBS, make B liquid.A liquid is dropwise added B liquid, and the limit edged stirs, room temperature reaction 3h, will be in the reactant liquor distilled water dialysis 72 hours, during change distilled water 5 times.Or through the purification of dextran gel G50 (Sephadex G50) post, with 0.05mol/L pH8.0 carbonate buffer solution wash-out.Collect the post eluent at dialysis refined solution or the 1st peak, packing is stored in-20 ℃ and makes immunizing antigen usefulness.
The preparation of polyclone Ciprofloxacin antibody: identical with embodiment 1.
Eu
3+The preparation of-goat anti-rabbit antibody: identical with embodiment 1.
Solid phase antigen preparation: identical with embodiment 1.
The preparation of reagent: identical with embodiment 1.
The reagent that kit provides: identical with embodiment 1.
The reagent that the laboratory should be provided for oneself: identical with embodiment 1.
Points for attention before measuring: with embodiment 1.
The concrete step that detects: with embodiment 1.
Embodiment 3
The reagent that kit provides is identical with embodiment 1, is used to detect honey sample.
The reagent that the laboratory should be provided for oneself:
(1) distilled water or deionized water;
(2) methylene chloride;
(3) the PB damping fluid of 0.02mol/L: take by weighing Na
2HPO
412H
2O 5.16g, NaH
2PO
40.87g to volumetric flask, be dissolved in water and be settled to 1L;
(4) phosphate-buffered working fluid: pH7.2 gets 1.45g sodium hydrogen phosphate (containing 12 water of crystallization), 0.1g potassium dihydrogen phosphate (anhydrous), 8.0g sodium chloride, is dissolved in the water and fixed molten to 1000mL.
Points for attention before measuring: with embodiment 1.
Concrete detection step is as follows:
Accurately take by weighing 1.0g ± 0.01g honey sample in the 50mL centrifuge tube, the PB damping fluid 2mL that adds 0.02mol/L shakes to honey and all dissolves, and adds methylene chloride 8mL, 5min is extracted in concussion, the above centrifugal 5min of 3800r/min gently sops up upper strata liquid, takes off layer organic phase to drying receptacle, 50 ℃ of nitrogen dry up, with the dry residue of buffering working fluid 1mL dissolving, dilute in proportion with the buffering working fluid again, get 50 μ L and be used for analyzing.
Get the CIP-OVA lath, add the CIP standard solution of 50 μ L or sample preparation liquid in micropore separately, each standard and sample solution must use new suction nozzle, CIP antibody 50 μ L with damping fluid dilution in 1: 20,25 ℃ vibrated 1 hour, and cleansing solution is given a baby a bath on the third day after its birth inferior, with the Eu of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ L, 25 ℃ vibrated 1 hour, washed six times with cleansing solution, added 200 μ L and strengthened liquid vibration measurement after 5 minutes.CIP content from the typical curve calculation sample sees Table 2 and Fig. 2, and CIP concentration is 2.42ng/mL in this routine sample solution.
Table 2
Claims (8)
1. time resolved fluoro-immunoassay method kit that detects Ciprofloxacin is characterized in that by the porous bag by plate (1) damping fluid (2), Ciprofloxacin standard solution (3), the antibody of Ciprofloxacin (4), the goat anti-rabbit antibody of europium mark (5), cleansing solution (6) and enhancing liquid (7) are formed.
2. kit according to claim 1, bag wherein by solid phase antigen, is used 50mmol/L pH9.6Na by plate (1) bag
2CO
3-NaHCO
3Damping fluid CIP-OVA is diluted to 10mg/L as coating buffer, 96 or 48 each hole of hole microwell plate add 100 μ L, 4 ℃ of placements are spent the night, discard coating buffer, wash three times, add the above-mentioned damping fluid sealing that 150 μ L contain 3g/L OVA, 4 ℃ of placements are spent the night, discard confining liquid, vacuum is drained, rearmounted-18 ℃ of freezing preservations of lath sealing.
3. kit according to claim 1, Ciprofloxacin standard solution (3) wherein dilutes from the pure product of CIP and obtains, and dilution is a 0.1mol/L pH7.5 phosphate buffer, totally 6 bottles, CIP concentration is respectively: 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 60ng/mL, 100ng/mL.
4. kit according to claim 1, the antibody of Ciprofloxacin wherein (4), with Freund's complete adjuvant or incomplete Freund 1.2mL mixing 2mg CIP-BSA, mixed 2 hours with homogenizer, make Water-In-Oil antigen emulsifying agent, get the Water-In-Oil antigen emulsifying agent that 600 μ L prepare, carry out hypodermic injection on one's body at new zealand white rabbit and BALB/c small white mouse multidigit point, after immunity 3～4 times, can identify that the dilution of the qualified back of serum and ascites, packing, freeze-drying are standby.
5. kit according to claim 1, the goat anti-rabbit antibody (5) of europium mark wherein with the goat anti-rabbit antibody of buying, to pH9.0, is collected protein peak through the conversion buffered condition of PD-10 post, gets switched goat anti-rabbit antibody and adds Eu
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, reaction is spent the night, and reactant liquor is through column chromatography, collects protein peak, and dilution, packing are standby.
6. kit according to claim 5, the goat anti-rabbit antibody (5) of europium mark wherein, get the 5g/L goat anti-rabbit antibody 1mL～2mL that is dissolved in 50mmol/L PBS pH7.0, through the conversion buffered condition of PD-10 post, eluent is the 50mmol/LNa that contains 0.155mol/L NaCl
2CO
3-NaHCO
3PH8.5～9.0 damping fluids collects protein peak, and is quantitative through the uv absorption analysis, and to 2g/L, the goat anti-rabbit antibody of getting after 500 μ L～1000 μ L dilute adds the Eu that contains 0.2mg～0.4mg with above-mentioned eluent dilution goat anti-rabbit antibody
3+-N
2In the bottle of-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, 28 ℃～30 ℃ magnetic agitation were reacted 16 hours, (1 * 40cm) chromatography, collection protein peak dilute reactant liquor, packing is standby through the Sephadex-G50 post with 80mmol/L Tris-HCl pH7.8 damping fluid balance.
7. method that detects Ciprofloxacin with the described kit of claim 1, it is characterized in that getting be coated with CIP-OVA the micropore bag by plate, add CIP standard solution or sample preparation liquid in micropore separately, add CIP antibody again, oscillating reactions, the cleansing solution washing, the goat anti-rabbit antibody that adds the europium mark carries out the labelled immune reaction, the cleansing solution washing, add and strengthen liquid vibration back measurement fluorescence intensity cps, the CIP content in the reference standard curve calculation sample.
8. the method for detection Ciprofloxacin according to claim 8, it is operating as: get be coated with CIP-OVA the micropore bag by plate, the sample that adds the CIP standard of 50 μ L or handle well is in micropore separately, add 50 μ L and make thinning agent with damping fluid (2), 1: 20 dilution CIP antibody, 25 ℃～37 ℃ vibrations 0.5～1 hour, cleansing solution is given a baby a bath on the third day after its birth time, in addition 100 μ L Eu of damping fluid (2) dilutions in 1: 20
3+-goat anti-rabbit antibody, 25 ℃～37 ℃ vibrated 0.5～1 hour, washed six times with cleansing solution, added the vibration of 200 μ L enhancing liquid and measured fluorescence intensity cps, the content of CIP from the typical curve calculation sample after 5 minutes.
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| CN200910232655A CN101713778A (en) | 2009-12-04 | 2009-12-04 | Detection kit for ciprofloxacin and detection method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910232655A CN101713778A (en) | 2009-12-04 | 2009-12-04 | Detection kit for ciprofloxacin and detection method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101995468A (en) * | 2010-08-31 | 2011-03-30 | 华南农业大学 | Time resolution immunoassay test kit and method of ciprofloxacin residual |
| CN102323415A (en) * | 2011-06-13 | 2012-01-18 | 清华大学深圳研究生院 | Immunochromatography test paper for detecting Ciprofloxacin and preparation method thereof |
| CN102455358A (en) * | 2010-10-28 | 2012-05-16 | 深圳出入境检验检疫局食品检验检疫技术中心 | Time-resolved fluoroimmunoassay kit for detecting diazepam and detection method thereof |
| CN102707045A (en) * | 2012-05-04 | 2012-10-03 | 嘉兴博泰生物科技发展有限公司 | Enzyme linked immunosorbent assay kit and method for detecting ciprofloxacin |
| CN106525835A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for gentamycin in food |
| CN106706581A (en) * | 2016-12-07 | 2017-05-24 | 无锡艾科瑞思产品设计与研究有限公司 | Detection method and equipment special for detecting ciprofloxacin |
-
2009
- 2009-12-04 CN CN200910232655A patent/CN101713778A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101995468A (en) * | 2010-08-31 | 2011-03-30 | 华南农业大学 | Time resolution immunoassay test kit and method of ciprofloxacin residual |
| CN102455358A (en) * | 2010-10-28 | 2012-05-16 | 深圳出入境检验检疫局食品检验检疫技术中心 | Time-resolved fluoroimmunoassay kit for detecting diazepam and detection method thereof |
| CN102323415A (en) * | 2011-06-13 | 2012-01-18 | 清华大学深圳研究生院 | Immunochromatography test paper for detecting Ciprofloxacin and preparation method thereof |
| CN102323415B (en) * | 2011-06-13 | 2014-03-19 | 清华大学深圳研究生院 | Immunochromatography test paper for detecting Ciprofloxacin and preparation method thereof |
| CN102707045A (en) * | 2012-05-04 | 2012-10-03 | 嘉兴博泰生物科技发展有限公司 | Enzyme linked immunosorbent assay kit and method for detecting ciprofloxacin |
| CN106525835A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for gentamycin in food |
| CN106706581A (en) * | 2016-12-07 | 2017-05-24 | 无锡艾科瑞思产品设计与研究有限公司 | Detection method and equipment special for detecting ciprofloxacin |
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