CN101995468A - Time resolution immunoassay test kit and method of ciprofloxacin residual - Google Patents

Abstract

The invention provides a time resolution immunoassay test kit and method of ciprofloxacin residual. The test kit of the invention is coated with an elisa plate of a ciprofloxacin antigen, a lanthanide-labeled goat anti-rabbit or goat anti-mouse antibody, a ciprofloxacin antibody and the like. The invention also discloses the method for testing the ciprofloxacin residual by utilizing the test kit. The test kit for testing the ciprofloxacin provided by the invention has the advantages that an indirect competition time resolution immunoassay technique is adopted, the sensitivity is high, the stability is good, the operation step and the reaction time are greatly simplified, errors caused by the complex operation are reduced, and the cost is lowered, thereby being extremely suitable for screening a large number of samples, and having important realistic significance.

Description

The time resolution immunoassay detection kit and the detection method thereof of residue of ciprofloxacin

Technical field

The invention belongs to technical field of immunoassay, relate to residual detection kit of a kind of Pollution by Chemicals and preparation method thereof, be specifically related to a kind of time resolution immunoassay kits that detects residue of ciprofloxacin and preparation method thereof.

Background technology

Food security is a major issue that is related to the daily life of numerous people.Food-safety problem mainly comprises the Several Factors such as physics harm, chemical hazard and microorganism harm in the food, and wherein chemical hazard mainly comprises residue of veterinary drug, residues of pesticides, heavy metal, environment harmful etc.

Residual associating Legislative Council of FAO (Food and Agriculture Organization of the United Nation) (FAO) and The World Health Organization (WHO) is defined as residue of veterinary drug: the parent compound and the metabolin thereof of the contained veterinary drug of any edible part of animal product, and the relevant impurity of veterinary drug.Residue of veterinary drug has become the difficult problem of puzzlement whole world food security, also is that various countries are in order to be provided with one of topmost means of trade barrier.

Ciprofloxacin (Enrofloxacin) is the disinfecting antibacterials of a class synthetic, and its has a broad antifungal spectrum, antibacterial activity are strong, is used widely on clinical and veterinary clinic.Yet drug resistance, residual toxicity that Ciprofloxacin is produced when veterinary clinic is used have caused extensive concern to the bad reaction of human body, and it is residual to work the mischief to human health and bacterium is developed immunity to drugs by food chain accumulation meeting.European Union, the U.S., Japan, China have all done this type of microbiotic of Ciprofloxacin and have limited the quantity of or forbid.Because the residue of veterinary drug problem in the animal food more and more receives the concern of international community, the detection to animal food veterinary drug residue in the international trade has all been strengthened in countries in the world.Therefore, in order to guarantee the safety of animal food, ensure the consumer health, promote carrying out smoothly of international trade, set up that the examination criteria of Ciprofloxacin has great importance in the animal food.Its structural formula is as follows:

Now, set up many methods both at home and abroad and detected residual Ciprofloxacin in the animal food, comprised microorganism detection method, HPLC method, MS method and methods such as LC-MS, immunoassay.Wherein the detectability of microorganism detection method often is higher than the maximum residue limit of medicine, and is sensitive inadequately, and is difficult to use in the qualitative of sample.Instrumental method is accurate, sensitive, thereby but owing to cost an arm and a leg, reasons such as time length, technical requirement height limit the screening that it is applied to batch samples.Immune analysis method commonly used at present has enzyme linked immunological immunoassay (ELISA), chemiluminescence immune assay (CLIA), time resolution immunoassay (TRFIA) method, wherein time resolution immunoassay (TRFIA) has highly sensitive, quick, high specificity, good stability, easy and simple to handle, advantage such as analysis result is accurate, is one of immune analysis method of at present potentialization.

Time resolution immunoassay (TRFIA) and common common fluorescence analysis principle are basic identical, just TRFIA has adopted a kind of special label labelled protein, polypeptide, hormone, antibody, nucleic acid probe or biologically active cell, after the question response system takes place, with the fluorescence intensity in the time resolution luminoscope mensuration end product, according to fluorescence intensity and relative intensity of fluorescence ratio, judge the concentration of analyte in the reaction system, reach the purpose of quantitative test.

Time resolution immunoassay (TRFIA) is to be label with lanthanide series such as europium (Eu), samarium (Sm), dysprosium (Dy), terbium rare earth ions such as (Te), rare earth ion need be by the bridge linking effect ability labelled antigen or the antibody of bifunctional chelating agent, an end and the Eu of bifunctional chelating agent ³⁺Deng connection, the amino on other end synantigen or the antibody molecule connects.Sequestrant commonly used has N ₂-[p-isothiocyanic acid-phenyl]-diethylenetriamine four acetic acid (DTTA), isothiocyanic acid-phenyl-EDTA and cyclisation diethylene triamine pentaacetic acid acid anhydride (cDPTA) etc., they contain band amino carboxylic acid or band isothiocyanic acid yl carboxylic acid isoreactivity group, and the ability of very strong chelating rare earth element is arranged.

Time resolution immunoassay (TRFIA) can be divided into two kinds of competition law and sandwich methods according to the difference of immune analysis method pattern.

(1) competition law: be mainly used in micromolecule antigen, haptens and some detection of antibodies.Comprise direct competition method or indirect competition method, direct competition method is generally wrapped by second antibody in advance at microwell plate, adds earlier a certain amount of first antibody and sample, adds Eu again after hatching washing ³⁺The micromolecule antigenic competition of mark is in conjunction with first antibody.The indirect competition method generally at microwell plate envelope antigen in advance, adds a certain amount of first antibody and sample, adds Eu again after hatching washing ³⁺The second antibody of mark is carried out spike.And in some detection of antibodies, the antibody in labelled antibody and the sample by competition in conjunction with in a certain amount of and antigen analyze.At last, add enhancing solution by hatching washing, the measured fluorescence intensity and the content of sample are inversely proportional to, and typical curve suppresses curve for competition.

(2) sandwich method: be used for the detection of big molecular biological activity material, coated in microporous plate one strain antibody of this analysis, another strain antibody of rare earth ion mark, form antibody-Ag-Ab-marker complex at last, measured fluorescence intensity is directly proportional with the content of sample, and typical curve is the saturation balance curve.

Time resolution immunoassay (TRFIA) is paid attention to by people gradually with its outstanding advantage in recent years, and at present, this technology has been not limited to the clinical diagnosis field, and is penetrated into the every field of biological study.It is one of at present the sensitiveest ultramicro-analysis technology, and its sensitivity is up to 10 ^-19mol/ L is than 10 of radioimmunoassay (RIA) ^-16mol/ L exceeds 3 orders of magnitude.

But still there is not the correlation technique report that adopts the time resolution immunoassay to detect Ciprofloxacin at present, more there is not a kind of kit that Ciprofloxacin time resolution immunoassay detects that is fit to carry out, so that realize high sensitivity, fast detecting in enormous quantities simple to operate.

Summary of the invention

An object of the present invention is to overcome the deficiencies in the prior art, a kind of time resolution immunoassay detection kit that detects residue of ciprofloxacin is provided.

Another object of the present invention provides the method for described detection residue of ciprofloxacin, and high sensitivity, simple to operate can fast detecting in enormous quantities.

Purpose of the present invention is achieved by the following technical programs:

A kind of time resolution immunoassay detection kit that detects residue of ciprofloxacin is provided, comprises by the ELISA Plate of Ciprofloxacin antigen, Ciprofloxacin antibody, lanthanide series mark goat-anti rabbit or sheep anti-mouse antibody (two is anti-); Described lanthanide series comprises Eu ³⁺, Tb ³⁺, Dy ³⁺Or Sm ³⁺Etc. rare earth element.

Described Ciprofloxacin antigen is with Ciprofloxacin and bovine serum albumin(BSA) (BSA), human serum albumins (HSA), keyhole limpet hemocyanin carrier proteins such as (KLH), carries out coupling and obtains envelope antigen and immunogene by carbodlimide method (EDC), active ester method (DCC, NHS), mixed anhydride method (isobutyl chlorocarbonate).Immunogene and coating antigen carry out purifying by column chromatography, and purity is identified through the SDS-PAGE electrophoresis.

Described Ciprofloxacin antibody comprises monoclonal antibody, rabbit polyclonal antibody or genetic engineering antibody.

Described monoclonal antibody induces method and prepares in animal immune, Fusion of Cells and clone, cell cryopreservation and recovery and body; Described animal immune is, and to be immunogene with Ciprofloxacin and carrier protein couplet thing carry out the interval immunity to the Balb/c mouse, and immunoassay detects and obtain containing in the blood mouse spleen of Ciprofloxacin specific antibody indirectly.Described Fusion of Cells is to get the Balb/c mouse boosting cell and the myeloma cell SP20 that produce specific antibody to merge with the clone, adopts indirect competition time resolution immune analysis method to measure cell conditioned medium liquid, screens positive hole.Utilize limiting dilution assay or microclone method that cloning is carried out in positive hole, obtain and set up the hybridoma cell strain that produces monoclonal antibody.Described cell cryopreservation and recovery are to get the hybridoma that is in exponential phase to make cell suspension with cryopreserving liquid, are sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.Described MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying are to adopt in the body to induce method, and Balb/c mouse (8 age in week) abdominal cavity is injected the sterilization paraffin oil, and 7～14 days pneumoretroperitoneum injection hybridomas were gathered ascites after 7～10 days.Carry out the ascites purifying through sad-saturated sulfuric acid amine method or affinity chromatography, purity is identified through the SDS-PAGE electrophoresis, bottle packing ,-20 ℃ of preservations.

Described Ciprofloxacin rabbit polyclonal antibody is to adopt new zealand white rabbit as immune animal, with Ciprofloxacin and carrier protein couplet thing is that immunogene is carried out immunity to new zealand white rabbit, repeatedly serum antibody titer is measured in the immunity back, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through sulfuric acid amine fractional precipitation.

Described Ciprofloxacin genetic engineering antibody is meant small molecular antibody, comprising: Fab (being made of complete light chain and Fd), Fv is (by V _HAnd V _LConstitute), ScFv (single-chain antibody, V _HAnd V _LBetween be formed by connecting by a connection peptides), single domain antibody is (only by V _HForm) etc. through the improved antibody of technique for gene engineering.

The preparation method is: the RNA that extracts Ciprofloxacin monoclonal cell or the mouse boosting cell after the ciprofloxacin immunogen immunity, reverse transcription is cDNA, designerantibodies weight chain amplimer, utilize round pcr to amplify the weight chain gene of antibody, insert suitable expression plasmid, at expression in escherichia coli, utilize immune affine method to carry out purifying, purity is identified by the SDS-PAGE electrophoresis.

The preparation of described lanthanide series mark goat-anti rabbit or sheep anti-mouse antibody is with Eu ³⁺Mark goat-anti rabbit is an example, gets the 5mg/mL sheep anti-mouse igg 1ml that is dissolved in 0.05mol/L PBS pH7.4, and through the conversion buffered salt condition of PD-10 post, eluent is 50mmol/L pH 9.6NaCO ₃-NaHCO ₃Damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis ₂₈₀-0.74A ₂₆₀), dilute sheep anti mouse to 2mg/mL with above-mentioned eluent.The sheep anti-mouse igg of getting after 500 μ L dilute adds the Eu that contains 0.2mg ³⁺-N ₂-[p-isocyanic acid-benzyl]-oxalic acid alkene triamine tetraacethyl (Eu ³⁺In-DTTA) the brown vial, place 28 ℃ of constant temperature ovens to react 48h, (1 * 30cm) chromatography is measured the photofluorometer numerical value of every pipe to reactant liquor with the SepharoseCL-6B of 50mmol/L Tris-HCl pH 7.8 damping fluid balances, detect first counting peak after diluting, the dilution back is standby.

Mark rate=mark number/IgG molecular number

For convenience of execute-in-place and batch detection, described kit can also comprise the Ciprofloxacin standard solution, strengthen liquid, concentrated cleaning solution, diluted sample concentrate.

Preferably, can also comprise that box body, bag are cushioned liquid, lavation buffer solution, confining liquid and cover plate film.

Described Ciprofloxacin standard solution be with concentration be the Ciprofloxacin of 1000 μ g/L as stock solution, being made into concentration gradient before the use again is the Ciprofloxacin standard solution of 0,0.1,0.3,0.9,2.7,8.1 μ g/L;

Described enhancing liquid comprises beta-diketon body, trioctylphosphine oxide (TOPO), TritonX-100, glacial acetic acid and Potassium Hydrogen Phthalate (the pH value is 2.0～3.2); With the Potassium Hydrogen Phthalate adjust pH to 3.2 of 0.1mol/L, (1mL Triton X-100 adds tri-distilled water and is settled to 1L for β-NTA), 50 μ mol trioctyl phosphine oxides to add 15 μ mol beta-diketon bodies with the 6mL glacial acetic acid.

Described concentrated washing lotion comprises that (pH value 7.4 0.1mol/L), is 15～25 times of conventional working concentration for the phosphate buffer of 0.5～1.5% (volume by volume concentration) polysorbas20.

The phosphate buffer (PBS) of described concentration and dilution liquid pH value 7.4～8.0,0.1～0.25mol/L is 5～15 times of conventional working concentration.

Described ELISA Plate is 96/40 hole ELISA Plate, be coated with can with the Ciprofloxacin antigen of anti-Ciprofloxacin antibody specific bond, and closed porosity surface adsorption site not; Described confining liquid comprises the skimmed milk power solution that is dissolved in PBS (5g skimmed milk power: 100mL PBS) with the calf serum 3mL (deactivation) of diluted to final concentration 3% (volume by volume concentration).

The present invention provides the method that detects residue of ciprofloxacin simultaneously, may further comprise the steps:

(1) sample pre-treatments;

A. honey

Take by weighing sample 3g, add 3mL distilled water, vibration evenly adds 6mL ethyl acetate, vibration 15min.The centrifugal 10min of 5000r/min draws upper strata 2mL rotation evaporate to dryness.Add the dry thing of 1mL n-hexane dissolution, add 1mL PBS solution again and redissolve, remove upper strata normal hexane phase, lower floor's stillnesss of night 50, μ L was to be measured;

B. birds, beasts and eggs

Take by weighing sample 3g, add acetonitrile-aqueous solution (80: 20) 6mL, vibration 10min, the centrifugal 10min of 5000r/min gets upper strata 2mL, adds 2mL ethyl acetate, vibration 15min, the centrifugal 10min of 5000r/min draws whole upper solution, the rotation evaporate to dryness.Add the dry thing of 1mL n-hexane dissolution, add 1mL PBS solution again and redissolve.Remove upper strata normal hexane phase, lower floor's stillnesss of night 50, μ L was to be measured,

C. organize (detection) with muscle, liver or kidney etc.

Take by weighing sample 3g, add PBS 3mL, vibration 10min, the centrifugal 10min of 5000r/min gets upper strata 2mL and adds the equivalent normal hexane, mixing gently, the centrifugal 10min of 5000r/min, it is to be measured to take off layer stillness of night 50 μ L.

(2) utilize kit of the present invention that standard solution and sample are detected.

(3) according to typical curve and sample solution fluorescent value calculation sample concentration.

Main agents of the present invention provides with the form of working fluid, saves the running time, and described detection specifically may further comprise the steps:

(1) kit is taken out from cold storage environment, place 20～24 ℃ of environment balances to be no less than 30min, ELIAS strip is fixed, do two parallel laboratory tests, number in order;

(2) add 50 μ L standard solution in the standard items hole, sample well adds 50 μ L testing samples, and every then hole adds 50 μ L monoclonal antibody against ciprofloxacin working fluids, mixing; Oscillating reactions 45min;

(3) wash plate behind the question response six times, and on thieving paper, pat dry, to guarantee to remove fully the liquid in the hole.Every hole adds 100 μ L europiums and marks two anti-working fluids, oscillating reactions 45min;

(4) wash plate behind the question response six times, and on thieving paper, pat dry, to guarantee to remove fully the liquid in the hole.Every hole adds 200 μ L and strengthens liquid, mixing, and the lucifuge room temperature is patted vibration 5min;

(5) differentiate immunoassay (TRFIA) detector with the time and measure each hole fluorescent value;

(6) do typical curve according to standard solution fluorescent value and concentration semilog, according to typical curve and sample solution fluorescent value calculation sample concentration.

With the mean value calculation percentage fluorescent value of obtained standard model light absorption value, be ordinate with the percentage fluorescent value, the semilog of Ciprofloxacin concentration of standard solution is a horizontal ordinate drawing standard curve, obtains straight-line equation.The use the same method percentage fluorescent value of calculation sample solution is obtained the Ciprofloxacin concentration of counter sample according to equation.The calculating formula of described percentage fluorescent value is:

Percentage fluorescence value (%)=(B/B ₀) * 100

Wherein, B is the mean fluorecence value of standard solution or sample, B ₀It is the mean fluorecence value of 0 μ g/L standard solution.

The analysis of testing result can also utilize computer professional software to calculate and analyze.

Beneficial effect of the present invention:

The present invention adopts the lanthanide series thing of marking, and DTTA is a sequestrant, has set up the quantitative analysis method to Ciprofloxacin, and this method operating process is short, simple, fast, and precision and accuracy are all better, and sample pre-treatments is simple.Especially adopt europium (Eu ³⁺) thing of marking, DTTA is sequestrant, and is effective, cost is low.

The mode that the present invention takes to seal preservation guarantees that the fluorescent value of the blank plate of described ELISA Plate is lower than 1000, guarantees not have the pollution of rare earth element.

Concentrated cleaning solution is that (pH7.4 0.1mol/L), is 15～25 times of normal working concentration for the phosphate buffer that contains 0.5～1.5% polysorbas20; Described diluted sample concentrate is the phosphate buffer of pH7.4～8.0,0.1～0.25mol/L, is 5～15 times of normal working concentration, and the minimizing volume conveniently is equipped with in kit.

The present invention uses the acid liquid that strengthens, and makes europium ion (Eu ³⁺) from chelate, disintegrate down free Eu ³⁺Down collaborative at trioctyl phosphine oxide, form a kind of new chelate that can accept exciting light expeditiously with the beta-diketon body again.Under excitation, the europium ion capacitation, the process electronic transition is also returned ground state, just can launch extremely strong fluorescence signal, reads its fluorescent value on time resolution immunity (TRFIA) analyser, and its sensitivity can reach 16 * 10 ^-18MolEu ³⁺

The present invention is 0.1～8.1 μ g/L to the Ciprofloxacin linear detection range, and detectability can reach 0.1 μ g/L, and whole testing process only needs 2h just can finish.

Description of drawings

Fig. 1 typical curve

Embodiment

Further describe the present invention below in conjunction with the drawings and specific embodiments.Employed test method is conventional method if no special instructions among the following embodiment; Employed material, reagent etc. if no special instructions, are the reagent and the material that can obtain from commercial channels.

The preparation of embodiment 1 antigen

The preparation of Ciprofloxacin antigen:

A. 50 μ mol/L Ciprofloxacins are dissolved among the DMF of 1mL, in this solution, add equimolar DCC and NHS then, be allowed to condition to react under the room temperature and spend the night;

B. centrifugal, get supernatant 800 μ L, slowly join among the BSA or OVA carrier protein carbonate buffer solution of 4mL 15mg/mL, under magnetic agitation, react 4h then;

C. after question response was finished, the bag filter of packing into was used distill water dialysis 2 times earlier, uses 0.8% normal saline dialysis then, gets product;

D. adopt UV scanning to measure in conjunction with than (Chen Xinjian etc., 1998), antigen is concentrated preserve or freeze-drying is preserved and obtained ciprofloxacin immunogen and coating antigen at last, packing is stored in-20 ℃ the refrigerator.

The preparation of embodiment 2 antibody

The preparation of Ciprofloxacin rabbit monoclonal antibodies:

Animal immune: with Ciprofloxacin and carrier protein couplet thing is that immunogene is carried out the interval immunity to the Balb/c mouse, and immunoassay detects and obtain containing in the blood mouse spleen of Ciprofloxacin specific antibody indirectly.

Fusion of Cells and clone: get the Balb/c mouse boosting cell and the myeloma cell SP20 that produce specific antibody and merge, adopt the indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay or microclone method that cloning is carried out in positive hole, obtain and set up the hybridoma cell strain that produces monoclonal antibody.

Cell cryopreservation and recovery: get the hybridoma that is in exponential phase and make cell suspension, be sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen with cryopreserving liquid.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.

MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is injected the sterilization paraffin oil, 7～14 days pneumoretroperitoneum injection hybridomas were gathered ascites after 7～10 days.Carry out the ascites purifying through sad-saturated sulfuric acid amine method or affinity chromatography, purity is identified through the SDS-PAGE electrophoresis, bottle packing ,-20 ℃ of preservations.

(3) preparation of Ciprofloxacin rabbit polyclonal antibody

Adopt new zealand white rabbit as immune animal, with Ciprofloxacin and carrier protein couplet thing is that immunogene is carried out immunity to new zealand white rabbit, repeatedly serum antibody titer is measured in the immunity back, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying through sulfuric acid amine fractional precipitation.

(4) preparation of Ciprofloxacin genetic engineering antibody

Described Enrofloxacin genetic engineering antibody is meant small molecular antibody, comprising: Fab (being made of complete light chain and Fd), Fv is (by V _HAnd V _LConstitute), ScFv (single-chain antibody, V _HAnd V _LBetween be formed by connecting by a connection peptides), single domain antibody is (only by V _HForm) etc. through the improved antibody of technique for gene engineering.

The preparation method is: the RNA that extracts Ciprofloxacin monoclonal cell or the mouse boosting cell after the ciprofloxacin immunogen immunity, reverse transcription is cDNA, designerantibodies weight chain amplimer, utilize round pcr to amplify the weight chain gene of antibody, insert suitable expression plasmid (TG1 bacterial strain, commercial), at expression in escherichia coli, utilize immune affine method to carry out purifying, purity is identified by the SDS-PAGE electrophoresis.

V wherein _H(Back), V _H(For) two primers antibody heavy chain variable region gene V that is used to increase _HV _L(Back), V _L(For) two primers antibody chain variable region gene V that is used to increase _LTwo primers of Linker1, Linker2 are used for overlap extension PCR method splicing V as the joint primer _H, V _LGenetic fragment becomes the scFv genetic fragment, and this joint primer includes coding connection peptides (Gly ₄Ser) ₃Genetic fragment, 5 ' end and V _H3 ' the complementation of fragment end, 3 ' end and V _LFragment 5 ' end is complementary.RS (Back), two primers of RS (For) are used for secondary PCR amplification total length scFv genetic fragment, introduce BgI and Not I restriction enzyme site simultaneously.Two primers of R1, R2 are primer on the carrier, are used for the PCR evaluation that carrier inserts fragment.

V _H(Back) 5 -CAG?GTS?MAR?CTG?CAG?SAG?TCW?GG-3

V _H(For) 5

-TGA?GGA?GAC?GGT?GAC?CGT?GGT?GCC-3

V _L(Back) 5

-GAC?ATC?GAG?CTC?ACT?CAG?TCT?CCA-3

V _L(For) 5

-CCG?TTT?TAT?TTC?CAG?CCT?GGT?CCC-3

Linker1 5

-GGC?ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGA

GGC?GGT?TCA?GGC?GGA?GGT?GGC?TCT?GG-3

Linker2 5

-TGG?AGA?CTG?AGT?GAG?CTC?GAT?GTC?CGATCC?GCC

ACC?GCC?AGA?GCC?ACC?TCC?GCC-3

RS(Back) 5

-GTC?CTC?GCA?ACT?GCG? GCC?CAG?CCG?GCC?ATG

(containing Bgl I site) GCC CAG GTC AAA CTG CAG GAG TCA GG-3

RS(For) 5

-GAG?TCA?TTC?T GC?GGC?CGC?CCG?TTT?TAT?TTC

(containing Not I site) CAG CCT GGT CCC-3

R1 5

-CCA?TGA?TTA?CGC?CAA?GCT?TTG?GAG?CC-3

R2 5 -CGA?TCT?AAA?GTT?TTG?TCG?TCT?TTC?C-3

V _LAmplification condition: reaction solution vortex mixing, of short duration centrifugal after, carry out pcr amplification reaction, course of reaction and condition are: 94 ℃ * 5min sex change, add 1 μ L high-fidelity Pfu enzyme (2.5U), and carry out following circulation: 94 ℃ * 30s, 54 ℃ * 1min, 72 ℃ * 1min, totally 25 circulations, last 72 ℃ are extended 10min.After reaction finishes, V _LReaction product is got 5 μ L and is carried out the detection of 1% agarose gel electrophoresis.

V _HAmplification condition: reaction solution vortex mixing, of short duration centrifugal after, carry out pcr amplification reaction, course of reaction and condition are: 94 ℃ * 5min sex change, add 1 μ L high-fidelity Pfu enzyme (2.5U), and carry out following circulation: 94 ℃ * 30s, 60 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, last 72 ℃ are extended 10min.After reaction finishes, V _HReaction product is got 5 μ L and is carried out the detection of 1% agarose gel electrophoresis.

(1) splicing of scFv genetic fragment and pcr amplification:

The overlap extension reaction

At first carry out the pre-splicing of linker by following reaction system:

Linker1 primer (10 μ mol/L) 0.5 μ L

Linker2 primer (10 μ mol/L) 0.5 μ L

10×PCR?Buffer 5μL

dNTP(2.5mmol/L) 4μL

Deionized water 4.5 μ L

Cumulative volume 44.5 μ L

Reaction solution vortex mixing, of short duration centrifugal.The pre-sex change of 94 ℃ * 5min on the PCR instrument adds 0.5 μ L high-fidelity Pfu enzyme, carries out following circulation: 94 ℃ * 45s, and 50 ℃ * 1min, 72 ℃ * 1min, totally 3 circulations.

Add V in the system in the above _H, V _LCarry out the splicing of scFv:

V _HPurified product (50ng) 3 μ L

V _LPurified product (50ng) 2.5 μ L

Linker splices solution 44.5 μ L in advance

Cumulative volume 50 μ L

Successively with V _H, V _LAfter the adding, with rifle mixing gently.0.5 μ L high-fidelity Pfu enzyme is added in the pre-sex change of 94 ℃ * 5min on the PCR instrument, carries out following circulation: 94 ℃ * 45s, and 50 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, 72 ℃ are extended 10min.

(2) pcr amplification of scFv gene

Adopt following reaction system:

Splicing product 4 μ L

5×PCR?Buffer 10μL

dNTP(2.5mmol/L) 4μL

RS (Back) primer (10 μ mol/L) 1 μ L

RS (For) primer (10 μ mol/L) 1 μ L

Sterile pure water 30 μ L

Cumulative volume 50 μ L

Mix, of short duration centrifugal after, on the PCR instrument, the pre-sex change of 94 ℃ * 2min adds 0.5 μ L high-fidelity Pfu enzyme, carries out following circulation: 94 ℃ * 45s, 60 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, last 72 ℃ are extended 10min.Get 5 μ L reaction product and carry out electrophoresis detection.

Embodiment 3Eu ³⁺The preparation of mark sheep anti mouse and purifying

Get the 5mg/mL sheep anti-mouse igg 1ml that is dissolved in 0.05mol/L PBS pH7.4, through the conversion buffered salt condition of PD-10 post, eluent is 50mmol/L pH 9.6NaCO ₃-NaHCO ₃Damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis ₂₈₀-0.74A ₂₆₀), dilute sheep anti mouse to 1mg/mL with above-mentioned eluent.The sheep anti-mouse igg of getting after 500 μ L dilute adds the Eu that contains 0.2mg ³⁺-N ₂-[p-isocyanic acid-benzyl]-oxalic acid alkene triamine tetraacethyl (Eu ³⁺In-DTTA) the brown vial, place 28 ℃ of constant temperature ovens to react 48h, (1 * 30cm) chromatography is measured the photofluorometer numerical value of every pipe to reactant liquor with the SepharoseCL-6B of 50mmol/L Tris-HCl pH 7.8 damping fluid balances, detect first counting peak after diluting, the dilution back is standby.

Mark rate=mark number/IgG molecular number

The preparation of embodiment 4 time resolution immunoassay kits components

(1) preparation of thickening and washing damping fluid: (pH7.4 0.1mol/L), is 15～25 times of normal working concentration to contain the phosphate buffer of 0.5～1.5% Tween-20.

(2) preparation of diluted sample concentrate: pH7.4～8.0,0.1 ~ 0.25mol/L, the phosphate buffer that contains are 5～15 times of normal working concentration.

(3) preparation of confining liquid: skimmed milk power 1.0～5.0g is dissolved in 100mL distilled water.

(4) preparation of enhancing liquid: the 6mL glacial acetic acid adds 15 μ mol with the Potassium Hydrogen Phthalate adjust pH to 3.2 of 0.1mol/L -two ketoboidies (

-NTA), and 50 μ mol trioctyl phosphine oxides (TOPO), 1mL Triton X-100 adds tri-distilled water and is settled to 1L.

(5) the bag quilt of ELISA Plate microwell plate: envelope antigen pH9.6,0.05mol/L carbonate buffer solution (contain 1～2g sodium carbonate and 2～4g sodium bicarbonate, distilled water 1L) be diluted to 0.1～5ug/mL, add 100uL in every hole of ELISA Plate, 37 ℃ of bags by 1h after 4 ℃ down bag spent the night, coating buffer inclines, with PBST washing 3 times, pat dry, in every hole, add 200uL1.0～5.0% skimmed milk power then, wash 3 times with PBST after putting into 37 ℃ of incubator 1h, 4 ℃ of preservations in the aluminium foil bag are enclosed in dry back.

(6) preparation of Ciprofloxacin standard solution: accurately take by weighing Ciprofloxacin standard specimen 8.1mg, be dissolved in the 0.1L damping fluid, prepare 8.1 μ g/L, 2.7 μ g/L, 0.9 μ g/L, 0.3 μ g/L, 0.1 μ g/L ciprofloxacin solution respectively with the damping fluid dilution then, damping fluid is prepared 0 μ g/L in the same old way in addition, 4 ℃ of preservations.

(8) reagent packing: all ingredients is prepared on request, measure the aseptic subpackaged Ciprofloxacin antibody working fluid 7mL/ bottle in qualified back, Ciprofloxacin standard model 1mL/ bottle, two anti-working fluid 10mL/ bottles, strengthen liquid 20mL/ bottle, 6 bottles of Ciprofloxacin standard solution concentrate washing lotion 50mL/ bottle, concentrating sample dilution 50mL/ bottle.Label after the packing, indicate the lot number and the term of validity, 4 ℃ of preservations.

(9) assembling of kit: will detachably wrap respectively by 1 of the microwell plate of good envelope antigen, Ciprofloxacin antibody working fluid, two anti-working fluids, strengthen liquid, concentrate washing lotion, each 1 bottle of concentrating sample dilution, 6 bottles of Ciprofloxacin standard solution, operation instructions are put assigned address in the kit for 1 part.Kit encapsulates after the assay was approved, 4 ℃ of preservations.

The time resolution immunoassay kits that detects Ciprofloxacin is set up in the establishment of the time resolution immunoassay kits of embodiment 5 detection Ciprofloxacins, comprise that other working fluids can be equipped with at control laboratory by the ELISA Plate of Ciprofloxacin antigen, Ciprofloxacin antibody, lanthanide series mark goat-anti rabbit or sheep anti-mouse antibody.

If for Ciprofloxacin in the test sample fast in enormous quantities, the time resolution immunoassay kits of the detection Enrofloxacin of establishment comprises following component:

(1) wraps by the ELISA Plate of Ciprofloxacin antigen 96 holes;

(2) monoclonal antibody against ciprofloxacin, the 7mL/ bottle;

(3) europium is marked two anti-working fluids, 10mL/ bottle;

(4) the Ciprofloxacin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 1mL/ bottle;

(5) strengthen liquid, 7mL/ bottle;

(6) concentrated cleaning solution, the 50mL/ bottle;

(7) concentrating sample dilution, the 50mL/ bottle;

(8) operation instructions, 1 part;

(9) cover plate film, 2;

(10) valve bag (containing drying agent), 1.

The preparation method of the fluorescence immunoassay kit of detection residue of ciprofloxacin of the present invention may further comprise the steps:

(1) bag is by the preparation method of ELISA Plate of Ciprofloxacin antigen: it is 100 μ g/L that CIP-OVA is cushioned the liquid dilution with bag, in the ELISA Plate micropore, add antigen 1 00 μ L, putting into 4 ℃ of refrigerator bags is spent the night, wash plate behind the equilibrium at room temperature twice, with 37 ℃ of sealings of skimmed milk power confining liquid (250 μ L) 1h, wash plate three times, with patting dry on the dustless thieving paper, preserve with the vacuum seal of aluminium film dry back.Fixedly the Ciprofloxacin antigen vectors is a kind of in the following material, for example polystyrene, cellulose nitrate, tygon, polypropylene, polyacrylamide, cross-linked glucose, glass, silicon rubber, Ago-Gel etc.

(2) preparation method of Ciprofloxacin antibody diluent: the immunogene that Ciprofloxacin is used obtains for adopting active ester method (DCC, NHS) or mixed anhydride method (isobutyl chlorocarbonate) that Ciprofloxacin and carrier protein covalent coupling are synthesized, with immune antigen immune rabbit or mouse, prepare the Ciprofloxacin polyclonal antibody, utilize hybridoma technology to prepare monoclonal antibody against ciprofloxacin or utilize gene engineering method to prepare genetic engineering antibody.Collect antiserum, ascites, fermentation liquor etc., carry out purifying with sad ammonium sulfate precipitation purifying or mistake affinity column.

Antibody with sad-ammonium sulfate method purifying after, it is standby to be diluted to the 1mg/mL packing with PBS.

(3) Eu ³⁺The preparation method of mark sheep anti mouse: get the 6mg/mL sheep anti-mouse igg 0.1ml that is dissolved in 0.05mmol/L PBS pH7.4, through the conversion buffered salt condition of PD-10 post, eluent is 50mmol/L pH 9.6NaCO ₃-NaHCO ₃Damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis ₂₈₀-0.74A ₂₆₀), dilute sheep anti mouse to 1mg/mL with above-mentioned eluent.The sheep anti-mouse igg of getting after 500 μ L dilute adds the Eu that contains 0.4mg ³⁺-N ₂-[p-isocyanic acid-benzyl]-oxalic acid alkene triamine tetraacethyl (Eu ³⁺In-DTTA) the brown vial, place 28 ℃ of constant temperature ovens to react 48h, (1 * 30cm) chromatography is measured the photofluorometer numerical value of every pipe to reactant liquor with the Sepharose CL-6B of 50mmol/L Tris-HCl pH 7.8 damping fluid balances, detect first counting peak after diluting, the dilution back is standby.Mark rate=mark number/IgG molecular number

(4) preparation of Ciprofloxacin standard solution: with concentration be the Ciprofloxacin of 1000 μ g/L as stock solution, being made into concentration gradient before the use again is the Ciprofloxacin standard items working fluid of 0,0.1,0.3,0.9,2.7,8.1 μ g/L;

(5) preparation of enhancing liquid: the 6mL glacial acetic acid adds 15 μ mol with the Potassium Hydrogen Phthalate adjust pH to 3.2 of 0.1mol/L -two ketoboidies (

-NTA), and 50 μ mol trioctyl phosphine oxides (TOPO), 1mL Triton X-100 adds tri-distilled water and is settled to 1L.

(6) bag is cushioned the preparation of liquid: get Na ₂CO ₃0.375g, NaHCO ₃0.732g, NaCl 2.250g, NaN ₃0.100g adding distil water gets the 0.1mol/L carbonate buffer solution to 250mL.

(7) preparation of lavation buffer solution (PBST): get KH ₂PO ₄0.4g, Na ₂HPO ₄12H ₂O5.8g, NaCl 16.0g, KCl 0.4g, Tween-201.0mL, adding distil water is to 2000mL.

(8) confining liquid: 1) skimmed milk power 5g is dissolved in 100mL PBS; 2) calf serum 3mL (deactivation), with diluted to final concentration 3%.

(9) dilution (50mmol/L Tris-HCl, pH 7.85): get Tris-base 6.4g, NaCl 9.0g, NaN ₃0.4g adjusting its pH value with dense HCl (12M) is 7.85, adding distil water is settled to 1L.

Embodiment 6 sample pre-treatments

A. honey

Take by weighing sample 3g, add 3mL distilled water, vibration evenly adds 6mL ethyl acetate, vibration 15min.The centrifugal 10min. of 5000r/min draws upper strata 2mL rotation evaporate to dryness.Add the dry thing of 1mL n-hexane dissolution, add 1mL PBS (pH value 7.4,0.1mol/L phosphate buffer) solution again and redissolve.Remove upper strata normal hexane phase, lower floor's stillnesss of night 50, μ L was to be measured.

B. birds, beasts and eggs

Take by weighing sample 3g, add acetonitrile-aqueous solution (volume ratio 80: 20) 6mL, vibration 10min.The centrifugal 10min of 5000r/min.Get upper strata 2mL, add 2mL ethyl acetate, vibration 15min.The centrifugal 10min of 5000r/min draws whole upper solution, the rotation evaporate to dryness.Add the dry thing of 1mL n-hexane dissolution, add 1mL PBS solution again and redissolve.Remove upper strata normal hexane phase, lower floor's stillnesss of night 50, μ L was to be measured.

C. organize (detection) with muscle, liver or kidney etc.

Take by weighing sample 3g, add PBS 3mL, vibration 10min.The centrifugal 10min of 5000r/min.Get upper strata 2mL and add the equivalent normal hexane, mixing gently, the centrifugal 10min of 5000r/min.It is to be measured to take off layer stillness of night 50 μ L.

The detection method of embodiment 7 kits

(1) kit is taken out from cold storage environment, place 20～24 ℃ of environment balances to be no less than 30min, ELIAS strip is fixed, do two parallel laboratory tests, number in order.

(2) add 50 μ L standard solution in the standard items hole, sample well adds 50 μ L testing samples, and every then hole adds 50 μ L monoclonal antibody against ciprofloxacin working fluids, mixing; Oscillating reactions 45min.

(3) wash plate behind the question response six times, and on thieving paper, pat dry, to guarantee to remove fully the liquid in the hole.Every hole adds 100 μ L europiums and marks two anti-working fluids, oscillating reactions 45min.

(4) wash plate behind the question response six times, and on thieving paper, pat dry, to guarantee to remove fully the liquid in the hole.Every hole adds 200 μ L and strengthens liquid, mixing, and the lucifuge room temperature is patted vibration 5min.

(5) measure each hole fluorescent value with the TRFIA detector.

(6) do typical curve according to standard solution fluorescent value and concentration semilog, according to typical curve and sample solution fluorescent value calculation sample concentration.

With the mean value calculation percentage fluorescent value of obtained standard fluorescent sample value, be ordinate with the percentage fluorescent value, the semilog of Ciprofloxacin concentration of standard solution is a horizontal ordinate drawing standard curve, obtain straight-line equation, see shown in the accompanying drawing 1 straight-line equation y=67.184-56.17x, R ²0.99246.The use the same method percentage fluorescent value of calculation sample solution is obtained the Ciprofloxacin concentration of counter sample according to equation.The calculating formula of described percentage fluorescent value is:

Percentage fluorescent value (%)=(B/B ₀) * 100

Wherein, B is the mean fluorecence value of standard solution or sample, B ₀It is the mean fluorecence value of 0 μ g/L standard solution.

The analysis of testing result can also utilize computer professional software to calculate and analyze, and is 0.1～8.1 μ g/L to the Ciprofloxacin linear detection range, detects and is limited to 0.1 μ g/L, and whole testing process only needs 2h just can finish.

Embodiment 8 kit precision and accuracy tests

1, standard solution replica test

From 3 batches of ELISA Plate according to the preparation of the method the embodiment 4, each extracts 20 micropores out, measures the fluorescent value (CPS value) of 0.9 μ g/L standard solution, repeats 20 times, calculates coefficient of variation CV%, the results are shown in Table 1.

Table 1 standard solution replica test

The result shows the variation within batch coefficient scope of kit standard items detection between 2.9～4.8%, and interassay coefficient of variation is 7.6%.

2, sample repeatability and accuracy test

Accuracy is meant the matching degree of measured value and true value, and in immune analysis determination, accuracy often represents with the recovery that precision is often represented with the coefficient of variation.In blank honey, birds, beasts and eggs, tissue (chicken), it is 10 μ g/L (μ g/kg), 15 μ g/L (μ g/kg) that Ciprofloxacin is added into final concentration, and each 10 of each concentration are parallel, measure 3 batches.Calculating mean value, the interpolation recovery and batch interior and interassay coefficient of variation.The results are shown in Table 2.

Table 2 sample repeatability and accuracy test result

The result shows the interpolation recovery of honey, birds, beasts and eggs, tissue samples between 81.0～110%, and the variation within batch coefficient is between 4.9～9.1%, and interassay coefficient of variation is between 14.2～18.5%.

The test of embodiment 9 storage lives

(1) kit is positioned over 2～8 ℃, get 0,2,4,6,8,9,10,11 and 12 months kit respectively, to fluorescent value, 50% inhibition concentration of Ciprofloxacin standard model (0.1 μ g/L), add the recovery, each parameter of variation within batch coefficient is measured.

(2) kit was placed 12 days under the condition of 37 ℃ of preservations, measure fluorescent value, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of Ciprofloxacin standard model (0.1 μ g/L) every day.

(3) kit was preserved 12 days at-20 ℃ of refrigerators, measure fluorescent value, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of Ciprofloxacin standard model (0.1 μ g/L) every day.

Can find out that from the result preserve test through three kinds of conditions, the fluorescent value of Ciprofloxacin standard model (0.1 μ g/L) descends less than 5%, and CPS is not less than 100000; 50% inhibiting rate is between 0.5～1.0 μ g/L; Add the recovery between 80～110%; The variation within batch coefficient is less than 10%; Every index all conforms to quality requirements, and therefore, kit can be preserved 12 months at 2～8 ℃.

Claims (10)

1. a time resolution immunoassay detection kit that detects residue of ciprofloxacin is characterized in that comprising by the ELISA Plate of Ciprofloxacin antigen, Ciprofloxacin antibody, lanthanide series mark goat-anti rabbit or sheep anti-mouse antibody; Described lanthanide series is Eu ³⁺, Tb ³⁺, Dy ³⁺Or Sm ³⁺

2. kit according to claim 1 is characterized in that described Ciprofloxacin antigen is that Ciprofloxacin and carrier protein couplet are obtained; Described Ciprofloxacin antibody comprises monoclonal antibody, rabbit polyclonal antibody or genetic engineering antibody.

3. kit according to claim 1 is characterized in that also comprising the Ciprofloxacin standard solution, strengthens liquid, concentrated cleaning solution and diluted sample concentrate.

4. kit according to claim 3 is characterized in that described Ciprofloxacin standard solution is is that the concentration gradient that the Ciprofloxacin of 1000 μ g/L is made into is the Ciprofloxacin standard solution of 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L with concentration.

5. kit according to claim 3 is characterized in that described enhancing liquid comprises that beta-diketon body, trioctylphosphine oxide (TOPO), Triton X-100, glacial acetic acid and pH value are 2.0～3.2 Potassium Hydrogen Phthalate.

6. kit according to claim 3 is characterized in that described concentrated washing lotion comprises the phosphate buffer of 0.5～1.5% polysorbas20, and described phosphate buffer pH value is 7.4, and concentration is 0.1mol/L; Described concentration and dilution liquid is the phosphate buffer of pH value 7.4～8.0,0.1～0.25mol/L.

7. time resolution immunoassay kits according to claim 1 it is characterized in that described ELISA Plate is 96/40 hole ELISA Plate, and the fluorescent value of blank plate is lower than 1000.

8. the detection method of a Ciprofloxacin is characterized in that may further comprise the steps:

(1) sample pre-treatments;

(2) detect with each described kit of claim 1～7;

(3) result treatment and analysis.

9. detection method according to claim 8 is characterized in that described sample pre-treatments is:

(1) honey:

Take by weighing sample 3g, add 3mL distilled water, vibration evenly adds 6mL ethyl acetate, vibration 15min, the centrifugal 10min of 5000r/min, draw upper strata 2mL rotation evaporate to dryness, add the dry thing of 1mL n-hexane dissolution, add 1mL PBS solution again and redissolve, remove upper strata normal hexane phase, lower floor's stillnesss of night 50, μ L was to be measured;

Or (2) birds, beasts and eggs:

Take by weighing sample 3g, add acetonitrile-aqueous solution 6mL, vibration 10min, the centrifugal 10min of 5000r/min gets upper strata 2mL, adds 2mL ethyl acetate, vibration 15min, the centrifugal 10min of 5000r/min draws whole upper solution, the rotation evaporate to dryness, add the dry thing of 1mL n-hexane dissolution, add 1mL PBS solution again and redissolve, remove upper strata normal hexane phase, lower floor's stillnesss of night 50, μ L was to be measured;

Or (3) tissue:

Described tissue comprises muscle, liver or kidney; Take by weighing sample 3g, add PBS 3mL, vibration 10min, the centrifugal 10min of 5000r/min gets upper strata 2mL and adds the equivalent normal hexane, mixing gently, the centrifugal 10min of 5000r/min, it is to be measured to take off layer stillness of night 50 μ L.

10. detection method according to claim 5 is characterized in that may further comprise the steps:

(1) kit is taken out from cold storage environment, place 20～24 ℃ of environment balances to be no less than 30min, ELIAS strip is fixed, do parallel laboratory test, number in order;

(2) add 50 μ L standard solution in the standard items hole, sample well adds 50 μ L testing samples, and every then hole adds 50 μ L Ciprofloxacin antibody working fluids, mixing; Oscillating reactions;

(3) plate is washed in the described oscillating reactions of step (2), pats dry, and every hole adds 100 μ L lanthanide series and marks two anti-working fluids, oscillating reactions;

(4) plate is washed in the described oscillating reactions of step (2), pats dry, and every hole adds 200 μ L and strengthens liquid, mixing, and the lucifuge room temperature is patted vibration;

(5) differentiate the immunoassay detector with the time and measure each hole fluorescent value;

(6) do typical curve according to standard solution fluorescent value and concentration semilog, according to typical curve and sample solution fluorescent value calculation sample concentration.

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