CN101995460B - Ractopamine residual time resolution immunoassay kit and detection method thereof - Google Patents
Ractopamine residual time resolution immunoassay kit and detection method thereof Download PDFInfo
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- CN101995460B CN101995460B CN 201010268992 CN201010268992A CN101995460B CN 101995460 B CN101995460 B CN 101995460B CN 201010268992 CN201010268992 CN 201010268992 CN 201010268992 A CN201010268992 A CN 201010268992A CN 101995460 B CN101995460 B CN 101995460B
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Abstract
The invention provides a ractopamine residual time resolution immunoassay kit and a detection method thereof. The kit of the invention contains an ELISA plate of ractopamine antigen, lanthanide labelled goat anti rabbit or goat anti mouse antibody and ractopamine. The invention also discloses a method for detecting ractopamine residue by applying the kit. The kit for detecting ractopamine provided by the invention adopts indirect competitive time resolution immunoassay technology, sensitivity is high, stability is good, operation steps are greatly simplified and reaction time is reduced, error caused by complex operation is reduced, and cost is reduced, thus being applicable to screening of massive samples and having important reality significance.
Description
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 Timed-resolved fluoroimmunoassay kit that detects Rct opamine residue 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 harms of microbe in food, and wherein chemical hazard mainly comprises residue of veterinary drug, residues of pesticides, heavy metal, environment harmful etc.
In the past few decades, along with the development of intensive culture industry, veterinary drug is extensively used as adjuvant and medicine in animal husbandry, and the residue problem of the animal food herbal medicine caused thus becomes increasingly conspicuous.It mainly refers to that animal is when being used chemoprophylaxis, treatment disease, and after use growth-promoting feed medicated premix, medicine is accumulated in the histoorgan or edible products (as milk, egg) of animal with original shape or its metabolic product.Same animal different organs Residual Veterinary Medicines difference is generally from high to low by residual quantity: liver>bile>kidney>muscle, the residual quantity of injection site and fat is generally also more.The illegal interpolation and the increasing dose, do not observe the off-drug period regulation, enlarge arbitrarily the residue of veterinary drug event that the behaviors such as usable range and drug abuse cause, serious harm is to the people's health, severe patient even can cause death, these left drugs also can exert an influence to environment simultaneously, and surrounding food is produced again and pollutes.
Ractopamine (Ractopamine is called for short RAC), have another name called Ractopamine, chemistry 1-(4-hydroxy phenyl) by name-2[1-methyl-3-(4-hydroxy phenyl) the-third amino]-the ethylate hydrochlorate, belong to
2-adrenoreceptor agonists class medicine is the most often added by abuse after Clenbuterol
2-adrenoreceptor agonists class medicine, its structural formula is suc as formula shown in (I):
It is a kind of medical material, a kind ofly can be used for treatment and rushes the cardiotonic drug of courageous and upright disease in heart failure.Can also be used for the treatment of muscular dystrophy, increase muscle, reduce lipopexia, and useful to the fetus an d neonate growth.U.S. FDA, approval in 2000, can, for Animal nutrition ingredients again, be widely used for animal husbandry and aquaculture.Can improve the daily gain of animal simultaneously, improve efficiency of feed utilization, improve the protein content of animal.But due to
the 2-excitant easily gathers residual in animal viscera, and can enter human body by food chain, the serious harm human health.At present, Ractopamine is one of the most frequently used adjuvant, and in China, abuse is especially serious, and it progressively replaces clenbuterol, becomes the new tool that the lawless person seeks economic interests.Setting up quick, simple, convenient, effective detection means is to control at present the urgent task of the illegal use of Ractopamine.
At present, detecting the residual method of RAC mainly contains following several: high performance liquid chromatography (HPLC), gas chromatography-mass spectrography (GC-MS), liquid chromatography-mass spectrography (LC-MS), By Capillary Zone Electrophoresis (CE) and immune analysis method (IA).Although instrumental method is sensitive accurately, need expensive professional instrument, to analyze time-consumingly, cost is higher.Simple, quick, sensitive and characteristics that cost is low that immune analysis method has, can reach trace (μ g kg
-1) level, it is a kind of high flux prescreening method that current countries in the world are carried out and implemented, the Timed-resolved fluoroimmunoassay detection method belongs to a kind of of immune analysis method, and it is actually and grows up on the basis of fluorescence analysis (FIA), is a kind of special fluorescence analysis.
The ultimate principle of Timed-resolved fluoroimmunoassay (TRFIA) be its to have adopted special lanthanide series metal be trivalent rare earth ions (Eu
3+, Tb
3+, Dy
3+, Sm
3+) and sequestrant (replacing fluorescent material, isotope or enzyme).Labelled protein, polypeptide, hormone, antibody, nucleic acid probe or biologically active cell, after question response system (as antigen-antibody reaction, nucleic acid probe reaction, the reaction of biotin Avidin, target cell react with killing and wounding of effector cell etc.) occurs, measure the fluorescence intensity in end product with time resolved analysis instrument, power according to fluorescent value, the concentration of analyte in the judgement system, reach the purpose of quantitative test.
The Timed-resolved fluoroimmunoassay detection method has been widely used in clinical detection abroad, in recent years in many frontiers, is applied, and as immunohistochemistry, microarray, and can be used for the multiple labeling immunoassays such as double-tagging, three marks and four marks.The fluorescence immunoassay detection method, as an emerging biology techniques with wide development potentiality, is learned and is brought a revolution to labelled immune.The fast detecting that the present invention further is applied to the food veterinary drug residue to immunoassay technology provides a kind of new way.
But there is no at present the correlation technique report that adopts Timed-resolved fluoroimmunoassay to detect Ractopamine, more there is no a kind of kit that is applicable to carrying out the detection of Ractopamine Timed-resolved fluoroimmunoassay, 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 that detects Rct opamine residue is provided.
Another object of the present invention is to provide the method for described detection Rct opamine residue, 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 that detects Rct opamine residue is provided, comprises ELISA Plate, Anti-ractopamine antibody, lanthanide series mark goat-anti rabbit or the sheep anti-mouse antibody (two is anti-) that have been coated with Ractopamine antigen; Described lanthanide series comprises Eu
3+, Tb
3+, Dy
3+, Sm
3+etc. rare earth element.
To be carbodlimide method (EDC), active ester method (DCC, NHS), mixed anhydride method (isobutyl chlorocarbonate) carry out coupling by Ractopamine haptens and carrier protein to described Ractopamine antigen obtains.Described carrier protein comprises the carrier proteins such as bovine serum albumin(BSA) (BSA), human serum albumins (HSA), keyhole limpet hemocyanin (KLH).
Ractopamine and sodium chloroacetate are carried out to halogenating reaction, replace the hydroxyl on the Ractopamine molecule, the Ractopamine haptens of the synthetic carboxyl spacerarm that contains 2 carbon; Or the amino in maleic anhydride and Ractopamine is carried out to acylation reaction, the Ractopamine haptens of the synthetic carboxyl spacerarm that contains 4 carbon.
To contain the haptens of carboxyl spacerarm of 2 carbon for immunogenic preparation, will contain the haptens of carboxyl spacerarm of 4 carbon for the preparation of coating antigen, immunogene and coating antigen carry out purifying by column chromatography, and purity is identified through the SDS-PAGE electrophoresis.Immunogene of the present invention adopts different haptens structures will more be conducive to improve the sensitivity detected from coating antigen.
Described Anti-ractopamine antibody comprises monoclonal antibody, rabbit polyclonal antibody or genetic engineering antibody.
The preparation of monoclonal antibody:
Animal immune: haptens and the carrier protein couplet thing of the carboxyl spacerarm that contains 2 carbon of take carries out Immunity at intervals as immunogene to the Balb/c mouse, and immunoassay detects and obtain in blood the mouse spleen that contains the Ractopamine 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 competition Time-resolved Fluoroimmunoassay for Human to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay or microclone method to carry out cloning to positive hole, obtain and set up the hybridoma cell strain that produces monoclonal antibody.
Cell cryopreservation and recovery: get in the hybridoma of exponential phase and make cell suspension with cryopreserving liquid, be sub-packed in cryopreservation tube, preserve for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
The preparation and purification of monoclonal antibody: adopt in body and induce method, by Balb/c mouse (8 week age) Intraperitoneal injection sterilizing paraffin oil, 7～14 days pneumoretroperitoneum injection hybridomas, 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.
The preparation of Ractopamine rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, take Ractopamine and carrier protein couplet thing carries out immunity to new zealand white rabbit as immunogene, repeatedly after the immunity, measure serum antibody titer, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through sulfuric acid amine fractional precipitation.
The preparation of Ractopamine genetic engineering antibody
Genetic engineering antibody mainly refers to small molecular antibody, comprising: Fab (consisting of complete light chain and Fd), Fv is (by V
hand V
lform), ScFv (single-chain antibody, V
hand V
lbetween connect peptide by one and be formed by connecting), 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 Ractopamine monoclonal cell or the mouse boosting cell after the Ractopamine immunogen immune, 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, with Eu
3+mark goat-anti rabbit is example, gets the 5mg/mL sheep anti-mouse igg 1ml that is dissolved in 0.05mol/L PBS pH7.0, 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 containing 0.2mg
3+-N
2-[p-isocyanic acid-benzyl]-oxalic acid alkene triamine tetraacethyl (Eu
3+-DTTA) in brown vial, be placed in 28 ℃ of constant temperature ovens and react 48h, SepharoseCL-6B (1 * 30cm) chromatography of 50mmol/L Tris-HCl pH 7.8 damping fluid balances for reactant liquor, measure the photofluorometer numerical value of every pipe, detect first counting peak after diluting, standby after dilution.
Mark rate=mark/IgG molecular number
In order to facilitate execute-in-place and batch detection, described kit can also comprise the Ractopamine standard solution, strengthen liquid, concentrated cleaning solution, Sample Dilution concentrate.
Preferably, can also comprise box body, coated damping fluid, lavation buffer solution, confining liquid and cover plate film.
Described Ractopamine standard solution is Ractopamine (RAC) the standard items stock solution that concentration is 1000 μ g/L, and being made into concentration gradient before use is Ractopamine (RAC) the standard items stock solution of 8 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ g/L, 0.5 μ g/L, 0 μ g/L again.
Described enhancing liquid comprises beta-diketon body, trioctylphosphine oxide (TOP0), TritonX-100, glacial acetic acid and Potassium Hydrogen Phthalate (the pH value is 2.0～3.2); Potassium Hydrogen Phthalate adjust pH to 3.2 by the 6mL glacial acetic acid with 0.1mol/L, add 15 μ mol beta-diketon bodies (β-NTA), 50 μ mol trioctyl phosphine oxides, and 1mL Triton X-100, add tri-distilled water and be settled to 1L.
Described concentrated washing lotion comprises 0.5～1.5% (volume by volume concentration).The phosphate of polysorbas20
(pH value 7.4 0.1mol/L), is 15～25 times of conventional working concentration to damping fluid.
The Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) 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.
ELISA Plate is 96 hole ELISA Plate, adopts polystyrene micropore plate, and it is the anti-Ractopamine antigen of 75 μ g/L that this coated in microporous plate has concentration, and closed porosity surface adsorption site not; With the sealing of confining liquid room temperature, described confining liquid comprises skimmed milk power solution (the 5g skimmed milk power: 100mL PBS) that is dissolved in PBS.Calf serum 3mL (deactivation) with diluted to final concentration 3% (volume by volume concentration).
Main agents of the present invention provides with the form of working fluid, saves the running time, and the preparation method of kit is as follows:
(1) be coated with the preparation method of the ELISA Plate of Ractopamine antigen: RAC-OVA be take to be coated with damping fluid dilution be 75 μ g/L, add antigen 1 00 μ L in the ELISA Plate micropore, put into that 4 ℃ of refrigerators are coated to spend the night, wash plate after equilibrium at room temperature twice, with 37 ℃ of sealing 1h of skimmed milk power confining liquid (250 μ L), wash plate three times, with patting dry on dustless thieving paper, dry rear with aluminium film vacuum seal preservation.Fixedly the Ractopamine antigen vectors is a kind of in following material, such as polystyrene, cellulose nitrate, tygon, polypropylene, polyacrylamide, cross-linked glucose, glass, silicon rubber or Ago-Gel etc.
(2) preparation method of RAC antibody diluent: after the RAC monoclonal antibody is purified with caprylic acid-ammonium, be diluted to the 1mg/mL packing with PBS standby.
(3) Eu
3+mark sheep anti mouse: with 200 times of the dilutions of the pH 7.85Tris-HCl containing 0.2%
(4) preparation of Ractopamine standard solution: the RAC that the concentration of usining is 1000 μ g/L is as stock solution, and being made into concentration gradient before use is the RAC standard items working fluid of 0,0.1,0.3,0.9,2.7,8.1 μ g/L again.
(5) strengthen the preparation of liquid: the Potassium Hydrogen Phthalate adjust pH to 3.2 of 0.1mol/L for the 6mL glacial acetic acid adds 15 μ mol
-bis-ketoboidies (
-NTA), 50 μ mol trioctyl phosphine oxides (TOPO), 1mL Triton X-100, add tri-distilled water and be settled to 1L.
(6) preparation of coated damping fluid: get Na
2cO
30.375g, NaHCO
30.732g, NaCl 2.250g, NaN
30.100g adding distil water, to 250mL, obtains the 0.1mol/L carbonate buffer solution.
(7) preparation of lavation buffer solution (PBST): get KH
2pO40.4g, 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 it is 7.85 that the dense HCl (12M) of take adjusts its pH value, adding distil water is settled to 1L.
The present invention provides the method that detects Rct opamine residue simultaneously, comprises the following steps:
(1) sample pre-treatments;
A. urine specimen is processed
Limpid urine sample can directly detect analysis.If urine sample is muddy shape, the centrifugal 5min of 2000r/min or filtration, detect with supernatant.
B. feed sample process
By fodder crushing, take 2g and be placed in the 50mL test tube, add 12mL10% ammonification methyl alcohol (1 part of the 9 parts+methyl alcohol of ammonia spirit that the pH value is 9～10), fully mix vibration 1 ~ 2min.Add 9mL ethyl acetate, vibration 20min.The centrifugal 10min of 5000r/min (4000g).Get organic phase 500 μ L in another test tube, dry up with nitrogen.Add the rear direct-detection of sample diluting liquid 500 μ L dilution.Extension rate 10 should be considered in result is calculated.
C. organize (muscle, liver, kidney etc.) sample process
By historrhexis, with Ultrasound Instrument or analogous instrument, tissue is homogenized, the tissue samples after taking 2g and homogenizing is placed in sealable test tube, adds the pure methyl alcohol of 10mL, and vortex mixes 5min.The centrifugal 5min of 10000r/min or the centrifugal 20min of 3000r/min, get supernatant.Add the pure methyl alcohol of 10mL in precipitation, vortex mixes rear centrifugal (method is the same), gets supernatant again.Mix centrifugal supernatant twice, take out 10mL and dry up with nitrogen.After getting the 1mL sample diluting liquid and again fully dissolving for detection of (noting: please carry out immediately analyzing and testing after dissolving).Extension rate is 1.
(2) utilize kit of the present invention to be detected standard solution and sample.
(3) according to typical curve and sample solution fluorescent value calculation sample concentration.
Applying described kit is detected specifically and is comprised the following steps:
(1) kit is taken out from cold storage environment, be placed in 20～24 ℃ of environment balances and 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/enters 50 μ L testing samples, and then every hole adds 50 μ L Ractopamine monoclonal antibody working fluids, mixes; Oscillating reactions 45min.
(3) wash plate after question response six times, and pat dry on thieving paper, to guarantee to remove fully the liquid in hole.Every hole adds 100 μ L europium mark two anti-working fluids, oscillating reactions 45min.
(4) wash plate after question response six times, and pat dry on thieving paper, to guarantee to remove fully the liquid in hole.Every hole adds 200 μ L to strengthen liquid, mixes, and the lucifuge room temperature is patted vibration 5min.
(5) measure each hole fluorescent value with Timed-resolved fluoroimmunoassay (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.
Testing result is processed and is analyzed:
With the mean value calculation percentage fluorescent value of obtained standard model light absorption value, take the percentage fluorescent value as ordinate, the semilog of Ractopamine concentration of standard solution is horizontal ordinate drawing standard curve, obtains straight-line equation.The use the same method percentage fluorescent value of calculation sample solution, obtain the Ractopamine concentration of counter sample according to equation.The calculating formula of described percentage fluorescent value is:
Percentage fluorescent value (%)=(B/B
0) * 100
Wherein, the mean fluorecence value that B is 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 to the Ractopamine linear detection range, is 0.1～8.1 μ g/L, and detectability can reach 0.01 μ g/L, and whole testing process needs 2h just can complete.
Beneficial effect of the present invention:
The present invention adopts the lanthanide series thing of marking, and DTTA is sequestrant, has set up the quantitative analysis method to Ractopamine, and this method operating process is short, simple, quick, and preci-sion and accuracy is all better, and sample pre-treatments is simple, especially adopts europium (Eu
3+) thing of marking, DTTA is sequestrant, effective, cost is low.
Concentrated cleaning solution is for containing the phosphate buffer (pH7.4,0.1mol/L) of 0.5～1.5% polysorbas20, is 15～25 times of normal working concentration; The phosphate buffer that described Sample Dilution concentrate is pH7.4～8.0,0.1～0.25mol/L is 5～15 times of normal working concentration.So that the minimizing volume conveniently is equipped with in kit.
The mode that the present invention adopts sealing to preserve guarantees that the fluorescent value of blank plate of described ELISA Plate, lower than 1000, guarantees not have the pollution of rare earth element.
The present invention uses the acid liquid that strengthens, and makes europium ion (Eu
3+) disintegrate down free Eu from chelate
3+under trioctyl phosphine oxide is collaborative, again with the beta-diketon body, form a kind of new chelate that can accept expeditiously exciting light.Under excitation, the europium ion capacitation, the process electronic transition is also returned to ground state, just can launch extremely strong fluorescence signal, on time resolution immunity (TRFIA) analyser, reads its fluorescent value, and its sensitivity can reach 16 * 10
-18molEu
3+.
The accompanying drawing explanation
The haptens one-level ESI-MS collection of illustrative plates of Fig. 1 Ractopamine (RAC)
The haptens secondary ESI-MS collection of illustrative plates of Fig. 2 Ractopamine (RAC)
Fig. 3 typical curve
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.The test method of using in following embodiment if no special instructions, is conventional method; The material used, reagent etc. if no special instructions, are reagent and the material that can obtain from commercial channels.
The haptenic preparation of embodiment 1
The haptenic synthetic route of Ractopamine is as follows:
The hydrochloric acid Ractopamine is dissolved in anhydrous pyridine and reacts in stirring at room with succinic anhydride.Reaction finishes, through extraction, to remove pyridine and unreacted raw material, and evaporated under reduced pressure on vacuum rotary evaporator, obtain haptens Ractopamine-succinate crude product.Above post-reaction treatment process is monitored in real time with TLC.Crude product obtains purer haptens through column chromatography purification, with ESI-MS, is identified.
Ractopamine haptenic (+) ESI-MS full scan mass spectrum as shown in Figure 1, the molecular ion peak of m/z 400 occurs in figure, corresponding with the hapten molecule quality.In order further to determine its structure, molion to m/z 400 has carried out (+) ESI-MS2 analysis, the fragment ion peaks such as m/z 384.2,284.2,236.2,164.3 appear after the molion cracking of m/z400, can rationally belong to, see shown in accompanying drawing 2, so m/z 400 molions can be attributed to haptens Ractopamine succinate.
The preparation of embodiment 2 antigens
The preparation of Ractopamine antigen:
A. 50 μ mol/L Ractopamine haptens are dissolved in the dimethyl formamide (DMF) of 1mL, then add equimolar dicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (NHS) in this solution, be allowed to condition at reaction under room temperature and spend the night;
B. centrifugal, get supernatant 800 μ L, slowly join in the BSA or OVA carrier protein carbonate buffer solution of 4mL 15mg/mL, then under magnetic agitation, react 4h;
C., after question response completes, the bag filter of packing into, first use distill water dialysis 2 times, then uses 0.8% normal saline dialysis, obtains product;
D. adopt UV scanning to measure in conjunction with than (Chen Xinjian etc., 1998), finally preserve or freeze-drying is preserved and obtained Ractopamine immunogene and coating antigen antigen is concentrated, packing is stored in the refrigerator of-20 ℃.
The preparation of embodiment 3 antibody
The preparation of monoclonal antibody:
Animal immune program: adopt the Balb/c mouse as immune animal, take Ractopamine haptens and bovine serum albumin(BSA) conjugate is immunogene, immunizing dose is 60 μ g/, when head exempts from, the Freund's complete adjuvant of immunogene and equivalent is mixed and made into to emulsifying agent, lumbar injection, interval is got the same dose immunogene in 3 weeks and is added equivalent incomplete Freunds adjuvant mixing and emulsifying, and booster immunization is once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days.
Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, merge in 4: 1 ratios and SP2/0 myeloma cell, adopt the indirect competition Time-resolved Fluoroimmunoassay for Human to measure cell conditioned medium liquid, screen positive hole.Utilize the microclone method to carry out cloning to positive hole, until obtain the hybridoma cell strain of stably excreting monoclonal antibody.
Cell cryopreservation and recovery: get in the hybridoma of exponential phase and make 5 * 10 with cryopreserving liquid
6the cell suspension of individual/mL, be sub-packed in cryopreservation tube, in-70 ℃ of ultra low temperature freezers, preserves for a long time.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
The preparation and purification of monoclonal antibody: adopt in body and induce method, by Balb/c mouse (8 week age) lumbar injection hybridoma 5 * 10
6individual/as only, after 14 days, to gather ascites.Carry out ascites with immunochromatographic method and purify, bottle packing ,-20 ℃ of preservations.
(2) preparation of Ractopamine rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, take Ractopamine and carrier protein couplet thing carries out immunity to new zealand white rabbit as immunogene, first immunisation is with 100 μ L antigens and the fully emulsified rear immunity of 100 μ L Freund's complete adjuvant, second and third, four immunity are with 100 μ L antigens and the fully emulsified immunity of 100 μ L Freunds incomplete adjuvant, rear mensuration serum antibody titer and inhibition, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through sulfuric acid amine fractional precipitation.
(3) preparation of Ractopamine genetic engineering antibody
Genetic engineering antibody mainly refers to small molecular antibody, comprise: Fab (being formed by complete light chain and Fd), Fv (being formed by VH and VL), ScFv (single-chain antibody, connecting peptide by one between VH and VL is formed by connecting), single domain antibodies (only being comprised of VH) etc. are through the improved antibody of technique for gene engineering.
The preparation method is: the RNA that extracts Ractopamine monoclonal cell or the mouse boosting cell after the Ractopamine immunogen immune, 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 are for increasing antibody heavy chain variable region gene V
h; V
l(Back), V
l(For) two primers are for increasing antibody chain variable region gene V
l; Linker1, two primers of Linker2 splice V as the joint primer for overlapping extension PCR method
h, V
lgenetic fragment becomes the scFv genetic fragment, and this joint primer includes coding and connects peptide (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), for secondary PCR amplification total length scFv genetic fragment, are introduced BgI and Not I restriction enzyme site simultaneously.R1, two primers of R2 are primer on carrier, for the PCR evaluation of carrier Insert 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 CGA TCC 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: the reaction solution vortex mixes, 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 completion of the reaction, V
lreaction product is got 5 μ L and is carried out 1% agarose gel electrophoresis detection.
V
hamplification condition: the reaction solution vortex mixes, 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 completion of the reaction, V
hreaction product is got 5 μ L and is carried out 1% agarose gel electrophoresis detection.
(1) splicing of scFv genetic fragment and pcr amplification:
Overlapping extension
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 34.5 μ L
Cumulative volume 44.5 μ L
The reaction solution vortex mixes, of short duration centrifugal.94 ℃ * 5min denaturation on the PCR instrument, add 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 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
The pre-splicing solution 44.5 μ L of Linker
Cumulative volume 50 μ L
Successively by V
h, V
lafter adding, with rifle, mix gently.94 ℃ * 5min denaturation on the PCR instrument, add 0.5 μ L high-fidelity Pfu enzyme, 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:
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, 94 ℃ * 2min denaturation, add 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 4Eu
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 2mg/mL with above-mentioned eluent.The sheep anti-mouse igg of getting after 500 μ L dilute adds the Eu containing 0.2mg
3+-N
2-[p-isocyanic acid-benzyl]-oxalic acid alkene triamine tetraacethyl (Eu
3+-DTTA) in brown vial, be placed in 28 ℃ of constant temperature ovens and react 48h, SepharoseCL-6B (1 * 30cm) chromatography of 50mmol/L Tris-HCl pH 7.8 damping fluid balances for reactant liquor, measure the photofluorometer numerical value of every pipe, detect first counting peak after diluting, standby after dilution.
Mark rate=mark/IgG molecular number
The preparation of embodiment 5 Timed-resolved fluoroimmunoassay reagent constituents
(1) preparation of thickening and washing damping fluid: containing the phosphate buffer (pH7.4,0.1mol/L) of 0.5～1.5% polysorbas20, be 15～25 times of normal working concentration.
(2) preparation of Sample Dilution 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) strengthen the preparation of liquid: the Potassium Hydrogen Phthalate adjust pH to 3.2 of 0.1mol/L for the 6mL glacial acetic acid adds 15 μ mol-
two ketoboidies (
-NTA), 50 μ mol trioctyl phosphine oxides (TOPO), 1mL Triton X-100, add tri-distilled water and be settled to 1L.
(5) the ELISA Plate microwell plate is coated: envelope antigen pH9.6,0.05mol/L carbonate buffer solution (containing 1～2g sodium carbonate and 2～4g sodium bicarbonate, distilled water 1L) be diluted to 0.1～5ug/mL, every hole in ELISA Plate adds 100uL, coated spending the night under 4 ℃ after 37 ℃ of coated 1h, coating buffer inclines, with PBST washing 3 times, pat dry, then add 200uL1.0～5.0% skimmed milk power in every hole, wash 3 times the dry rear 4 ℃ of preservations in aluminium foil bag of enclosing after putting into 37 ℃ of incubator 1h with PBST.
(6) preparation of Ractopamine standard solution: accurately take Ractopamine standard specimen 8.1mg, be dissolved in the 0.1L damping fluid, then prepare respectively 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 Ractopamine solution with the damping fluid dilution, damping fluid is prepared 0 μ g/L control sample, 4 ℃ of preservations in addition.
(8) reagent packing: various reagent is prepared on request, measures qualified rear aseptic subpackaged Anti-ractopamine antibody working fluid 7mL/ bottle, Ractopamine standard model 1mL/ bottle, two anti-working fluid 10mL/ bottles, strengthen liquid 20mL/ bottle, concentrated washing lotion 50mL/ bottle, concentrating sample dilution 50mL/ bottle.Label after packing, indicate lot number and the term of validity, 4 ℃ of preservations.
(9) assembling of kit: 1 of the microwell plate that will detachably be coated with respectively good envelope antigen, Anti-ractopamine antibody working fluid, two anti-working fluids, strengthen each 1 bottle of liquid, concentrated washing lotion, Ractopamine standard solution, concentrating sample dilution, 6 bottles of Ractopamine standard solution, 1 part of operation instructions are put assigned address in kit.Kit encapsulates after the assay was approved, 4 ℃ of preservations.
Embodiment 6 detects the establishment of the Timed-resolved fluoroimmunoassay kit of Ractopamine
Set up the Timed-resolved fluoroimmunoassay kit that detects Ractopamine, comprise the ELISA Plate, Anti-ractopamine antibody, lanthanide series mark goat-anti rabbit or the sheep anti-mouse antibody that have been coated with Ractopamine antigen, other working fluids can be equipped with at control laboratory.
If, in order to detect fast in enormous quantities Ractopamine in sample, the Timed-resolved fluoroimmunoassay kit of the detection Ractopamine of establishment comprises following component:
(1) ELISA Plate of coated Ractopamine antigen, 96 holes;
ELISA Plate is 96 hole ELISA Plate, adopts polystyrene micropore plate, and it is the anti-Ractopamine antigen of 75 μ g/L that this coated in microporous plate has concentration, and closed porosity surface adsorption site not; Described sealing refers to take PBS (with weight ratio) the confining liquid room temperature sealing that concentration is 5% skimmed milk power.
(2) Anti-ractopamine antibody working fluid, the 7mL/ bottle; Concentration is 2mg/mL;
(3) the anti-working fluid of europium mark two, the 10mL/ bottle; Mark rate is 11.2, and concentration is 0.15mg/mL;
(4) the Ractopamine 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 Rct opamine residue of the present invention comprises the following steps:
(1) be coated with the preparation method of the ELISA Plate of Ractopamine antigen: RAC-OVA be take to be coated with damping fluid dilution be 75 μ g/L, add antigen 1 00 μ L in the ELISA Plate micropore, put into that 4 ℃ of refrigerators are coated to spend the night, wash plate after equilibrium at room temperature twice, with 37 ℃ of sealing 1h of skimmed milk power confining liquid (250 μ L), wash plate three times, with patting dry on dustless thieving paper, dry rear with aluminium film vacuum seal preservation.Fixedly the Ractopamine antigen vectors is a kind of in following material, such as polystyrene, cellulose nitrate, tygon, polypropylene, polyacrylamide, cross-linked glucose, glass, silicon rubber or Ago-Gel etc.
(2) preparation method of RAC antibody diluent: after the RAC monoclonal antibody is purified with caprylic acid-ammonium, be diluted to the 1mg/mL packing with PBS standby.
(3) Eu
3+mark sheep anti mouse: with 200 times of the dilutions of the pH 7.85Tris-HCl containing 0.2%
(4) preparation of Ractopamine standard solution: the RAC that the concentration of usining is 1000 μ g/L is as stock solution, and being made into concentration gradient before use is the RAC standard items working fluid of 0,0.1,0.3,0.9,2.7,8.1 μ g/L again.
(5) strengthen the preparation of liquid: the Potassium Hydrogen Phthalate adjust pH to 3.2 of 0.1mol/L for the 6mL glacial acetic acid adds 15 μ mol
-bis-ketoboidies (
-NTA), 50 μ mol trioctyl phosphine oxides (TOPO), 1mL Triton X-100, add tri-distilled water and be settled to 1L.
(6) preparation of coated damping fluid: get Na
2cO
30.375g, NaHCO
30.732g, NaCl 2.250g, NaN
30.100g adding distil water, to 250mL, obtains the 0.1mol/L carbonate buffer solution.
(7) preparation of lavation buffer solution (PBST): get KH
2pO40.4g, 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/LTris-HCl, pH 7.85): get Tris-base 6.4g, NaCl 9.0g, NaN
30.4g it is 7.85 that the dense HCl (12M) of take adjusts its pH value, adding distil water is settled to 1L.
Embodiment 7 sample pre-treatments
A. urine specimen is processed
Limpid urine sample can directly detect analysis.If urine sample is muddy shape, the centrifugal 5min of 2000r/min or filtration, supernatant for detection of.
B. feed sample process
By fodder crushing, take 2g and be placed in the 50mL test tube, add 12mL10% ammonification methyl alcohol (1 part of the 9 parts+methyl alcohol of ammonia spirit of pH9-10), fully mix vibration 1～2min.Add 9mL ethyl acetate, vibration 20min.The centrifugal 10min of 5000r/min (4000g).Get organic phase 500 μ L in another test tube, dry up with nitrogen.Add the rear direct-detection of sample diluting liquid 500 μ L dilution.Extension rate 10 should be considered in result is calculated.
C. organize (detecting with muscle, liver or kidney etc.) sample process
By historrhexis, with Ultrasound Instrument or analogous instrument, tissue is homogenized, the tissue samples after taking 2g and homogenizing is placed in sealable test tube, adds the pure methyl alcohol of 10mL, and vortex mixes 5min.The centrifugal 5min of 10000r/min or the centrifugal 20min of 3000r/min, get supernatant.Add the pure methyl alcohol of 10mL in precipitation, vortex mixes rear centrifugal (method is the same), gets supernatant again.Mix centrifugal supernatant twice, take out 10mL and dry up with nitrogen.After getting the 1mL sample diluting liquid and again fully dissolving for detection of (noting: please carry out immediately analyzing and testing after dissolving).Extension rate is 1.
The detection method of embodiment 8 kits
(1) kit is taken out from cold storage environment, be placed in 20～24 ℃ of environment balances and 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 then every hole adds 50 μ L Ractopamine monoclonal antibody working fluids, mixes; Oscillating reactions 45min.
(3) wash plate after question response six times, and pat dry on thieving paper, to guarantee to remove fully the liquid in hole.Every hole adds 100 μ L europium mark two anti-working fluids, oscillating reactions 45min.
(4) wash plate after question response six times, and pat dry on thieving paper, to guarantee to remove fully the liquid in hole.Every hole adds 200 μ L to strengthen liquid, mixes, 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.
Testing result is processed and is analyzed:
With the mean value calculation percentage fluorescent value of obtained standard model light absorption value, take the percentage fluorescent value as ordinate, the semilog of Ractopamine concentration of standard solution is horizontal ordinate drawing standard curve, obtains straight-line equation, straight-line equation is y=48.17142-36.52x, R
2=0.99486.The use the same method percentage fluorescent value of calculation sample solution, obtain the Ractopamine concentration of counter sample according to equation.The calculating formula of described percentage fluorescent value is:
Percentage fluorescent value (%)=(B/B
0) * 100
Wherein, the mean fluorecence value that B is 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 to the Ractopamine linear detection range, is 0.1～8.1 μ g/L, and detectability can reach 0.01 μ g/L, and whole testing process needs 2h just can complete.
Embodiment 9 kit precision and accuracy tests
1, standard solution replica test
From 3 batches of ELISA Plate that prepare according to the method embodiment 4 (6), 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
Result shows that the variation within batch coefficient range of kit standard items detection is between 3.9～5.9%, and interassay coefficient of variation is 8.7%.
2, sample repeatability and accuracy test
Accuracy refers to the matching degree of measured value and true value, and in immune analysis determination, accuracy often means with the recovery, and precision often means with the coefficient of variation.In blank pig urine, pork liver, it is 1 μ g/L (μ g/kg), 5 μ g/L (μ g/kg) that Ractopamine is added into to final concentration, and in blank feed, it is 50 μ g/kg, 100 μ g/kg that Ractopamine is added into to final concentration, 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
Result shows that the interpolation recovery of urine sample, pork liver, feed sample is between 81.0～105%, and the variation within batch coefficient is between 5.9～9.1%, and interassay coefficient of variation is between 14.2～18.3%.
(1) kit is positioned over to 2～8 ℃, get respectively 0,2,4,6,8,9,10,11 and the kit of 12 months, to fluorescent value, 50% inhibition concentration of Ractopamine standard model (0.1 μ g/L), add the recovery, each parameter of variation within batch coefficient is measured.
(2) kit is placed 12 days under the condition of 37 ℃ of preservations, measured every day to fluorescent value, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of Ractopamine standard model (0.1 μ g/L).
(3) by kit-20 ℃ of Refrigerator stores 12 days, every day to fluorescent value, 50% inhibition concentration of Ractopamine standard model (0.1 μ g/L), add the recovery, each parameter of variation within batch coefficient is measured.
Can find out from result, through three kinds of conditions, preserve test, the fluorescent value of Ractopamine standard model (0.1 μ g/L) descends and is less than 5%, and CPS is not less than 100000; 50% inhibiting rate is between 0.5～1.0 μ g/L; Its detectability can reach 0.01 μ g/mL, IC
50can reach 0.2ng/mL; Add the recovery between 80～110%; The variation within batch coefficient, between 5.9～9.1%, is less than 10%; Interassay coefficient of variation is between 14.2～18.3%.Indices all conforms to quality requirements, and therefore, kit can be preserved 12 months at 2～8 ℃.
Claims (2)
1. a time resolution immunoassay that detects Rct opamine residue, is characterized in that comprising the ELISA Plate, Anti-ractopamine antibody, lanthanide series mark goat-anti rabbit or the sheep anti-mouse antibody that have been coated with Ractopamine antigen; Described lanthanide series is Eu
3+, Tb
3+, Dy
3+or Sm
3+;
Described kit also comprises the Ractopamine standard solution, strengthens liquid, concentrated cleaning solution and Sample Dilution concentrate; Described Ractopamine standard solution is to take the Ractopamine standard items working fluid that concentration gradient that Ractopamine standard items that concentration is 1000 μ g/L are made into is 8 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ g/L, 0.5 μ g/L, 0 μ g/L;
Described Ractopamine antigen is that haptens Ractopamine succinate and carrier protein couplet are obtained;
Described enhancing liquid comprises the Potassium Hydrogen Phthalate that beta-diketon body, trioctylphosphine oxide, Triton X-100, glacial acetic acid and pH value are 2.0～3.2.
2. kit according to claim 1, is characterized in that described Anti-ractopamine antibody comprises rabbit polyclonal antibody or genetic engineering antibody.
3. kit according to claim 1, is characterized in that described concentrated cleaning solution 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; The Tris-HCl that described Sample Dilution concentrate is pH value 7.4～8.0,0.1～0.25mol/L.
4. kit according to claim 1, is characterized in that described ELISA Plate is 96 hole ELISA Plate, and being coated with concentration is the anti-Ractopamine antigen of 75 μ g/L, uses not adsorption site of confining liquid closed porosity surface; Described confining liquid comprises the skimmed milk power solution that is dissolved in PBS.
5. the detection method of a Ractopamine is characterized in that comprising the following steps:
(1) sample pre-treatments;
(2) with the described kit of claim 1～4 any one, detected;
(3) result treatment and analysis.
6. detection method according to claim 5 is characterized in that described sample pre-treatments is:
(1) urine specimen is processed:
Limpid urine sample can directly detect analysis; If urine sample is muddy shape, the centrifugal 5min of 2000r/min or filtration, detect with supernatant;
Or (2) feed sample process:
By fodder crushing, take 2g and be placed in the 50mL test tube, add 12mL10% ammonification methyl alcohol, fully mix vibration 1～2min, add 9mL ethyl acetate, vibration 20min, the centrifugal 10min of 5000r/min, get organic phase 500 μ L in another test tube, dry up with nitrogen, add the rear direct-detection of sample diluting liquid 500 μ L dilution;
Or (3) tissue samples is processed:
Described tissue comprises muscle, liver or kidney; By historrhexis and homogenizing, tissue samples after taking 2g and homogenizing is placed in sealable test tube, adds the pure methyl alcohol of 10mL, and vortex mixes 5min, the centrifugal 5min of 10000r/min or the centrifugal 20min of 3000r/min, get supernatant, add the pure methyl alcohol of 10mL in precipitation again, vortex mixes rear centrifugal, get supernatant, mix centrifugal supernatant twice, take out 10mL and dry up with nitrogen, after getting the 1mL sample diluting liquid and again fully dissolving for detection of.
7. detection method according to claim 5 is characterized in that comprising the following steps:
(1) kit is taken out from cold storage environment, be placed in 20～24 ℃ of environment balances and 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 then every hole adds 50 μ L Ractopamine monoclonal antibody working fluids, mixes; Oscillating reactions;
(3) wash plate after the described oscillating reactions of step (2), pat dry, every hole adds 100 μ L europium mark two anti-working fluids, oscillating reactions;
(4) wash plate after the described oscillating reactions of step (3), pat dry, every hole adds 200 μ L to strengthen liquid, mixes, and the lucifuge room temperature is patted vibration;
(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.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569404A (en) * | 2014-12-17 | 2015-04-29 | 浙江工商大学 | Method for detecting olaquindox by directly-competing TRFIA (Time Resolved Fluorescence Immunoassay) |
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|---|---|---|---|---|
| CN102539750A (en) * | 2011-11-25 | 2012-07-04 | 佛山市中南农业科技有限公司 | Time-resolved immunoassay kit for detecting chlorpromazine residues and detection method thereof |
| CN102778564B (en) * | 2012-05-31 | 2014-06-04 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting ractopamine |
| CN104090248A (en) * | 2013-12-24 | 2014-10-08 | 上海容晖生物科技有限公司 | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1456894A (en) * | 2003-06-09 | 2003-11-19 | 江南大学 | Diethylstilbestrol enzyme linked immunoassay reagent box and determination |
| CN101482562A (en) * | 2008-12-30 | 2009-07-15 | 江苏省微生物研究所有限责任公司 | Detection reagent kit and detection method for diethyl stilbestrol |
| CN101799472A (en) * | 2010-03-25 | 2010-08-11 | 中国农业科学院上海兽医研究所 | Diethylstilbestrol detection kit and detection method |
-
2010
- 2010-08-31 CN CN 201010268992 patent/CN101995460B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1456894A (en) * | 2003-06-09 | 2003-11-19 | 江南大学 | Diethylstilbestrol enzyme linked immunoassay reagent box and determination |
| CN101482562A (en) * | 2008-12-30 | 2009-07-15 | 江苏省微生物研究所有限责任公司 | Detection reagent kit and detection method for diethyl stilbestrol |
| CN101799472A (en) * | 2010-03-25 | 2010-08-11 | 中国农业科学院上海兽医研究所 | Diethylstilbestrol detection kit and detection method |
Non-Patent Citations (2)
| Title |
|---|
| 动物组织中氯霉素和莱克多巴胺残留的时间分辨荧光免疫检测方法的研究;张祯;《万方学位论文数据库》;20070723;第30页第3.1.1节、第33页第3.2.4.1、3.2.4.3节，第33-34页第3.2.5节 * |
| 张祯.动物组织中氯霉素和莱克多巴胺残留的时间分辨荧光免疫检测方法的研究.《万方学位论文数据库》.2007, |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569404A (en) * | 2014-12-17 | 2015-04-29 | 浙江工商大学 | Method for detecting olaquindox by directly-competing TRFIA (Time Resolved Fluorescence Immunoassay) |
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