CN101241132A - Nitrofurans medicament metabolite residue ELISA kit and use method - Google Patents
Nitrofurans medicament metabolite residue ELISA kit and use method Download PDFInfo
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- CN101241132A CN101241132A CNA2008100259044A CN200810025904A CN101241132A CN 101241132 A CN101241132 A CN 101241132A CN A2008100259044 A CNA2008100259044 A CN A2008100259044A CN 200810025904 A CN200810025904 A CN 200810025904A CN 101241132 A CN101241132 A CN 101241132A
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- metabolin
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- nitrofuran
- enzyme
- antibody
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Abstract
The present invention discloses enzyme-linked immune kits for detecting nitrofurans metabolite residue including antigenic enzyme label plate coated with nitrofuran meatbolites, enzyme labeled antibody working fluid, standard solution, substrate solution, substrate buffer solution, reaction termination liquid, condense washing liquid and sample diluted concentrated liquid. The present invention also discloses using method for detecting by said kits including steps of pretreatment of sample, detecting with kits, result process and analysis and so on. The kits provided by present invention employ directly competing enzyme-linked immunoadsorption analysis to detect not only multiple nitrofurans metabolite residues at the same time, but also singleness nitrofurans metabolite residue, avoid many times detection of the same sample and reduce miss ratio, has merits of high sensitivity, good stability, less steps and low cost, and suit for screening of a large mount of samples, and has importance practical significance.
Description
Technical field
The present invention relates to the enzyme linked immunosorbent detection technical field, be specifically related to a kind of enzyme-linked immunologic detecting kit and method thereof that detects nitrofurans medicament metabolite residue in the animal derived food.
Background technology
Itrofurans medicine (Nitrofurans) is the extensive pedigree antibiotic with 5-nitrofuran basic structure of synthetic, the itrofurans microbiotic mainly comprises furazolidone (Furazolidone), furaltadone (Furaltadone), nitrofurazone (Nitrofurazone), furantoin (Nitrofurantoin) etc., this class microbiotic has biocidal property because of it and bactericidal properties is widely used in zoonotic prevention and treatments such as poultry, domestic animal, aquatic products, honeybee, the part kind has somatotrophic effect, can be used as feed addictive.
It is potential carcinogenic and induce organism to produce the material of sudden change that the itrofurans medicine is that a class has.Because metabolism is rapid in vivo for itrofurans microbiotic such as furazolidone, furaltadone, nitrofurazone, furantoin, its metabolic product 3-amino-2-oxazolidone (AOZ), 3-amino-5-beautiful jade form the protein conjugates more stable than former drug compound for methyl-2-oxazolidone (AMOZ), semicarbazides (SC) and 1-amido-glycolyurea (AH) energy and protein bound.These metabolins can (as the acid condition of human gastric juice) discharge from protein under solutions of weak acidity, therefore when the human food that contains the nitrofuran antibiotics residue of having eaten, these metabolins just can discharge from protein under the acid condition of human gastric juice and be absorbed by the body and human health is worked the mischief.
EU Committee has passed through the resolution of the 2003/181/EC council in 2003, the minimum of having set up the whole bag of tricks of the metabolin that is used for detecting poultry product and aquatic products itrofurans medicine requires the performance limit value, and (Minimum Required Performance Limits MRPL) is 1 μ gkg
-1, that is to say that the European Union laboratory detects the sensitivity of the whole bag of tricks of the metabolin of itrofurans medicine and all will reach 1 μ gkg
-1, the content of European Union's metabolin of itrofurans medicine from the animal product of third country import must not surpass 1 μ gkg
-1Therefore, be the safety of guaranteeing animal derived food and the development of export abroad trade, set up accurately and reliably that highly sensitive qualitative-and-quantitative method is very necessary.
At home and abroad delivered in the document of relevant itrofurans residues of antibiotics detection method, early stage bibliographical information great majority are to detect former drug compound.Yet because the itrofurans microbiotic is to photaesthesia, have metabolism characteristics fast, half life period in animal body is a few hours only, usually unlikely detect the residual of former medicine, for example furazolidone disappears from tissue in the 12h after drug withdrawal, its metabolite 3-amino-2-oxazolidone (AOZ) then exists with the form of histone bond in animal body, in vivo can residual several weeks, AOZ exists in the pig muscle tissue at least 6 weeks after drug withdrawal, therefore reduce to detectability when following when former concentration, it is possible detecting its metabolite concentration.Other itrofurans microbiotic also have similar characteristics.
At present, detect AMOZ, residual method such as AOZ, SC and AH mainly contains electron spray-tandem mass spectrum (ESI-MS-MS), liquid chromatograph mass spectrography method (LS-MS) and liquid chromatography-tandem mass spectrometry method (LS-MS-MS) etc.Although instrument analytical method sensitivity, accurately need be carried out loaded down with trivial details pre-service to sample usually, consuming time, cost is high, and also used instrument is relatively more expensive and huge heaviness, needs the professional and technical personnel to safeguard, is unsuitable for on-the-spot the detection.The application of immunoassay has remedied the defective of above-mentioned technology, has simple, quick and sensitive characteristics, can reach trace (μ g kg
-1) level, be a kind of high flux prescreening method that present countries in the world are carried out and implemented.
Existing at present immunoassay and the kit that is applied to the detection of itrofurans microbiotic metabolin, but all can only carry out the detection of single medicine metabolite residue, for example commercially available domestic and international kit is single medicine residue detection kit at present, and search domestic relevant patent and also be single medicine metabolite residue detection kit and method, as patent " detecting the enzyme linked immunological kit and the application thereof of Furacilin metabolite ", application number: 200710063871.8; Patent " Furantoin metabolite enzyme linked immunosorbent analytical reagent casing and application thereof ", application number: 200710063837.7; Patent " detecting the enzyme linked immunological kit and the application thereof of AMOZ ", application number: 200710063872.2; Patent " a kind of enzyme-linked immunologic detecting kit and application that is used for the furazolidone residual analysis ", application number: 200510086346.9 etc.Yet, immunoassay is as a kind of method of high flux primary dcreening operation, if can only detect single metabolin, especially just little to the meaning of itrofurans microbiotic metabolite residue detection, because it is several that the itrofurans microbiotic comprises, as furazolidone, furaltadone, nitrofurazone, furantoin etc., its metabolic product is respectively 3-amino-2-oxazolidone (AOZ) again, 3-amino-5-beautiful jade is for methyl-2-oxazolidone (AMOZ), semicarbazides (SC) and 1-amido-glycolyurea (AH), so detecting unit needs these several metabolites are all detected in actual detected, if immune reagent kit can only detect a kind of metabolin, because 4 kinds of metabolins are arranged, a sample just must detect 4 times, detection time will have been prolonged like this, increased the testing amount, and increased the detection cost, lost the meaning of prescreening method.
In a word, existing nitrofuran immunologic detection method detected object is single both at home and abroad, be difficult to be applied to practice, and existing product is because ubiquity poor stability, sample pre-treatments and detect step complexity, appointed condition deficiency such as have relatively high expectations, cost an arm and a leg, therefore have a strong impact on itrofurans detection of antibiotics and monitoring, developed many residue detection, the how residual ELISA kit of itrofurans microbiotic metabolin simple to operate, that equipment requirements is low, cheap has very important economy and social effect.
Summary of the invention
The objective of the invention is deficiency at existing nitrofuran detection technique, provide a kind of and can detect multiple itrofurans microbiotic metabolin total residual simultaneously, can detect single nitrofuran microbiotic metabolite residue again, high sensitivity, cheap, simple to operate, the enzyme-linked immunologic detecting kit of fast detecting itrofurans microbiotic metabolin in enormous quantities.
Another object of the present invention provides the using method that described enzyme linked immunological kit detects itrofurans microbiotic metabolite residue.
To achieve these goals, the present invention adopts following measuring principle: at first with multiple metabolin coupled antigen bag by in solid phase carrier for example on the ELISA Plate, add standard specimen or testing sample then, the mixed liquor that adds plurality of enzymes mark metabolin antibody again, metabolin competition enzyme labelled antibody in envelope antigen and the testing sample, when the testing sample metabolite content is high, then the enzyme labelled antibody that combines with solid phase antigen is just few, otherwise the enzyme labelled antibody that is combined on the solid phase antigen is just many, adding substrate in reaction back develops the color and is measured, when one timing of enzyme labelled antibody amount, it is many more that the testing sample of adding contains metabolin, just few more with solid phase antigen desmoenzyme labeling antibody, the color development habituation, the percentage absorbance is low, otherwise, color development increased response then, the percentage absorbance increases, thereby according to the semilog between percentage absorbance and the metabolite concentration relation mapping promptly gets typical curve, again according to the percentage absorbance of typical curve and sample to be checked, can extrapolate the concentration of metabolin in the testing sample.
Technical scheme of the present invention provides a kind of enzyme linked immunological kit that detects nitrofurans medicament metabolite residue, comprising:
(1) bag is by the ELISA Plate of multiple nitrofuran metabolin coupled antigen;
(2) enzymic-labelled antibody working fluid;
(3) nitrofuran metabolin standard solution;
(4) substrate solution;
(5) substrate buffer solution;
(6) reaction terminating liquid;
(7) concentrated cleaning solution;
(8) diluted sample concentrate.
Described ELISA Plate is 96 holes or 40 hole ELISA Plate, ELISA Plate hole endoperidium have can with the multiple nitrofuran metabolin coupled antigen of anti-nitrofuran metabolin antibody specific bond, it is to use carbodlimide method (EDC), active ester method (DCC, NHS), mixed anhydride method (isobutyl chlorocarbonate) is with AOZ, AMOZ, metabolin such as SC and AH haptens and carrier protein couplet obtain, used coating buffer is pH9.6,0.05mol/L carbonate buffer solution, carbonate buffer solution contains 1～2g sodium carbonate and 2～4g sodium bicarbonate and distilled water 1L, and confining liquid is 1～5% skimmed milk power solution.
In the preparation process of described nitrofuran metabolin antibody, used immunogene obtains for adopting active ester method (DCC, NHS) or mixed anhydride method (isobutyl chlorocarbonate) that metabolin haptens and carrier protein covalent coupling are synthesized, with immune antigen immune rabbit or mouse, prepare nitrofuran metabolin polyclonal antibody, utilize hybridoma technology to prepare nitrofuran metabolin monoclonal antibody 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.
Described enzyme labeling nitrofuran metabolin antibody working fluid obtains enzyme with mixing by a certain percentage after metabolin (AOZ, AMOZ, SC and AH etc.) antibody carries out coupling respectively for adopting glutaraldehyde method or periodates oxidizing process.Used marker enzyme can be horseradish peroxidase or bacterium is extracted alkaline phosphatase, the present invention is preferably horseradish peroxidase, and adopt the periodates oxidizing process after the improvement to carry out mark, improved labeling effciency, saved the consumption of enzyme and antibody, enzyme and antibody have good activity behind the assurance mark.
Described nitrofuran metabolin standard solution is respectively AOZ standard solution, AMOZ standard solution, SC standard solution and AH standard solution etc., and the concentration of different metabolic thing standard solution is: 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 and 0 μ g/L.
Described substrate colour developing liquid is when marker enzyme is horseradish peroxidase, substrate solution is for containing 3,3,5, pH5.0 phosphoric acid-the citric acid solution of 5-tetramethyl benzidine (TMB) or o-phenylenediamine (OPD), substrate buffer solution is the pH5.0 phosphoric acid-citric acid solution that contains hydrogen peroxide or urea peroxide, and described stop buffer is the sodium hydroxide solution of 1～2mol/L sulfuric acid solution or 2mol/L;
When marker enzyme is the alkaline phosphatase of bacterium extraction, described substrate solution is to the nitro phosphate buffer, described substrate buffer solution is the pH5.0 phosphoric acid-citric acid solution that contains hydrogen peroxide or urea peroxide, and described stop buffer is the sodium hydroxide solution of 1～2mol/L sulfuric acid solution or 2mol/L.
Described concentrated cleaning solution is the phosphate buffer that contains 0.5～1.5% polysorbas20, phosphate buffer pH7.4, and concentration is 0.1mol/L, is 15～25 times of normal working concentration.
Described diluted sample concentrate is the phosphate buffer of pH7.4,0.1～0.25mol/L, is 5～15 times of normal working concentration.
The material that can be used as the fixing carrier of nitrofuran metabolin antigen is more, for example polystyrene, cellulose nitrate, tygon, polypropylene, polyacrylamide, cross-linked glucose, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be shrinkage pool, the scraps of paper, globule etc.
The preparation method of antigen-antibody of the present invention specifically is presented below:
(1) antigen is synthetic
Haptenic synthetic:
A. metabolin AOZ is haptenic synthetic
3-amino-2 oxazolidone (AOZ) generates the haptens that has carboxyl with the terephthalaldehydic acid reaction.
B. metabolin AMOZ is haptenic synthetic
3-amino-5-beautiful jade reacts in water for methyl-2-oxazolidone (AMOZ) and terephthalaldehydic acid and generates the haptens that has carboxyl.
C. metabolin SC is haptenic synthetic
With the nitroreduction of semicarbazides (SC) is amino, generates to have amino haptens.
D. metabolin AH is haptenic synthetic
A 1-amido-glycolyurea (AH) and an aldehyde benzoic acid react in water and generate the haptens that has carboxyl.
Coating antigen and immunogenic synthetic:
With nitrofuran metabolin haptens and bovine serum albumin(BSA) (BSA), human serum albumins (HSA), keyhole limpet hemocyanin carrier proteins such as (KLH), carry out coupling and obtain 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.
(2) nitrofuran metabolin MONOCLONAL ANTIBODIES SPECIFIC FOR
Animal immune: with metabolin haptens and carrier protein couplet thing is that immunogene is carried out the interval immunity to the Balb/c mouse, and indirect ELISA detects and obtain containing in the blood mouse spleen of metabolin specific antibody.
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 nitrofuran metabolin rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with nitrofuran metabolin haptens 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 nitrofuran genetic engineering antibody
Genetic engineering antibody mainly refers to small molecular antibody, comprise: Fab (constituting) by complete light chain and Fd, Fv (constituting) by VH and VL, ScFv (single-chain antibody, be formed by connecting by a connection peptides between VH and the VL), single domain antibody (only forming) by VH, or multi-specificity antibody (not homospecific single-chain antibody is linked together by connection peptides, have the specificity of multiple single-chain antibody simultaneously) etc. is through the improved antibody of technique for gene engineering.
The preparation method is: the RNA that extracts nitrofuran metabolin monoclonal cell or the mouse boosting cell after nitrofuran metabolin immunogen immune, reverse transcription is cDNA, designerantibodies weight chain amplimer, utilize round pcr to amplify the weight chain gene of antibody, the weight chain gene is connected the preparation single-chain antibody, or more different single-chain antibodies are connected preparation polyspecific single-chain antibody, insert suitable expression plasmid then, at expression in escherichia coli, utilize immune affine method to carry out purifying, purity is identified by the SDS-PAGE electrophoresis.
Wherein, the preparation of enzyme labeling nitrofuran metabolin antibody:
Adopt the sodium periodate method to carry out coupling nitrofuran metabolin antibody and horseradish peroxidase (HRP).Concrete grammar is:
1. dissolve 5mg HRP in the 1mL ultrapure water, the 0.1mol/L sodium periodate 75 μ L of the new configuration of adding put room temperature or 4 ℃ of refrigerators react 20min or 30min.
2. the bag filter of packing into after having reacted, 4 ℃ of dialysed overnight of 0.001mol/L pH4.0 hac buffer, during need to change dislysate several times.
3. antibody is diluted to 10mg/mL with the 0.1mol/L carbonic acid buffer, the pH value of solution that will activate good HRP with the 0.1mol/L carbonic acid buffer transfers to 9.5 in addition.0.5mL antibody is added in the HRP solution, put room temperature or 4 ℃ of refrigerator reaction 2h.
4. add 100 μ L 4mg/mL sodium borohydrides, 4 ℃ of refrigerator reaction 2h.
5. to 0.01mol/L PBS dialysed overnight, it is standby that liquid-20 ℃ preservation is preserved in adding.
Different enzyme labeling nitrofuran metabolin antibody is mixed according to a certain percentage, promptly get enzyme labeling thing solution.
Wherein, the preparation method of ELISA Plate is:
Being cushioned liquid with bag dilutes different nitrofuran metabolin antigen on demand, in the elisa plate micropore, add antigenic dilution, put into 37 ℃ of environment and hatch, put into 4 ℃ of environment night incubation again, the good stability of the elisa plate that obtains, coating buffer inclines, with the cleansing solution washing, in every hole, add confining liquid then, hatch for 37 ℃, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
The present invention provides simultaneously and has utilized above-mentioned enzyme linked immunological kit to carry out the method that nitrofuran detects in the animal derived food, may further comprise the steps:
(1) sample pre-treatments;
(2) use kit to detect;
(3) result treatment and analysis.
The invention provides the testing sample pre-treating method is:
With tissue sample tissue refiner's high-speed homogenization; The centrifugal removal fat deposit of milk sample; The dissolving of honey sample adding distil water evenly; Prevent that with taking out the yellow and white after the broken shell of egg sample, stirring evenly gently foam from producing.
(1) for example chicken, pork or fishes and shrimps of animal tissue
Get the equal pledge of the tissue sample of 2 ± 0.05g, add the distilled water of 8mL, the 2-nitrobenzaldehyde of 1mL 1M HCL and 200 μ L 10mM, fully vibration; At 37 ℃ of about 12h of night incubation; Add 10mL 0.1M K
2HPO
4, 0.8mL 1M NaOH and 10mL ethyl acetate, thermal agitation 1min; Centrifugal 10min more than 20～25 ℃ of following 3000g of room temperature; Taking out 5mL ethyl acetate 50 ℃ of following nitrogen in another container dries up; With the dry thing of 2mL n-hexane dissolution, diluted good redissolution liquid with 2mL and fully mixed; Centrifugal 10min more than 20～25 ℃ of following 3000g of room temperature; Getting 50 μ L subnatant bodies is used for analyzing.
(2) milk
Take out 10mL milk sample in the glass centrifuge tube; Add each 200 μ L of 0.36M sodium nitroprusside damping fluid and 1M zinc sulfate damping fluid respectively; Fully behind the mixing sample, 4～10 ℃ with constant temperature hydro-extractor 3000g more than centrifugal 10min.Get the centrifuged supernatant 2mL of milk, add the distilled water of 16mL, the 2-nitrobenzaldehyde of 2mL 1M HCL and 200 μ L 10mM, fully vibration; At 37 ℃ of about 12h of night incubation; Add 10mL 0.1M K
2HPO
4, 0.8mL1M NaOH and 10mL ethyl acetate, thermal agitation 1min; Centrifugal 10min more than 20～25 ℃ of following 3000g of room temperature; Taking out 5mL ethyl acetate 50 ℃ of following nitrogen in another container dries up; With the dry thing of 2mL n-hexane dissolution, diluted good redissolution liquid with 2mL and fully mixed; Centrifugal 10min more than 20～25 ℃ of following 3000g of room temperature; Getting 50 μ L subnatant bodies is used for analyzing.
(3) honey
Take out 2g honey sample in centrifuge tube; The distilled water vibration dissolving that adds 16mL, the 2-nitrobenzaldehyde of 2mL1M HCL and 200 μ L 10mM, fully vibration; At 37 ℃ of about 12h of night incubation; Add 10mL 0.1M K
2HPO
4, 2mL 1M NaOH and 10mL ethyl acetate, thermal agitation 1min; Centrifugal 10min more than 20～25 ℃ of following 3000g; Taking out 12mL ethyl acetate 50 ℃ of following nitrogen in another container dries up; With the dry thing of 2mL n-hexane dissolution, diluted good redissolution liquid with 4mL and fully mixed; Centrifugal 10min more than 20～25 ℃ of following 3000g; Getting 50 μ L subnatant bodies is used for analyzing.
(4) egg
The egg sample that taking-up 4g has prepared is in the 100mL centrifuge tube; Add 16mL water respectively, 2mL1M HCL, the sodium nitroprusside damping fluid of 400 μ L 0.36M, vibration mixing; Add 400 μ L 1M zinc sulfate damping fluids, the 8min that fully vibrates, the above centrifugal 10min of room temperature (20～25 ℃) 3000g, take out whole supernatants, add the 2-nitrobenzaldehyde of 400 μ L10mM, fully vibration, 50 ℃ of water-bath 2h, per half an hour thermal agitation 1～2 minute; Add 10mL0.1M K respectively
2HPO
4, 1mL 1M NaOH and 8mL ethyl acetate, thermal agitation 1min, the at room temperature above centrifugal 10min of (20～25 ℃) 3000g; Taking out the 6mL upper organic phase dries up in 50 ℃ of following nitrogen; With the dry thing of 2mL n-hexane dissolution, adding 4mL has diluted good redissolution liquid fully to be mixed; The above centrifugal 10min of (20～25 ℃) 3000g at room temperature; Remove upper organic phase; Taking off layer water 50 μ L is used for analyzing.
The step of using kit to detect is:
(1) kit is taken out from cold storage environment, place equilibrium at room temperature;
(2) standard items or testing sample adding have been coated with in the ELISA Plate hole of nitrofuran metabolin antigen, every then hole adds the enzyme labeling thing, pats mixing, hatches;
(3) washing;
(4) every hole adds the substrate buffer solution and the substrate solution of equivalent, pats mixing, and lucifuge is hatched;
(5) every hole adds reaction terminating liquid, mix, and under wavelength 450nm or 492nm, be blank with the air, microplate reader is measured each hole light absorption value;
Testing result provided by the invention is handled with analytical approach:
With the mean value calculation percentage absorbance of obtained standard model light absorption value, be ordinate with the percentage absorbance, the semilog of nitrofuran metabolin concentration of standard solution is a horizontal ordinate drawing standard curve, obtains straight-line equation.The use the same method percentage absorbance of calculation sample solution is obtained the nitrofuran metabolite concentration of counter sample according to equation.The calculating formula of described percentage absorbance is:
Percentage absorbance (%)=(B/B
0) * 100
Wherein, B is the average light absorption value of standard solution or sample, B
0It is the mean light absorbency 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 nitrofuran metabolin linear detection range, detects and is limited to 0.1 μ g/L, and whole testing process only needs 35min just can finish.
Compared with prior art, the present invention has following beneficial effect:
(1) kit of the present invention both can detect multiple nitrofuran microbiotic metabolin total residual simultaneously, can detect single nitrofuran metabolite residue again, avoid same sample need detect repeatedly problem, saved detection time and cost, reduced the probability of omission simultaneously;
(2) kit of the present invention adopts direct competitive ELISA detecting pattern, has reduced operation steps, has improved the sensitivity, the accuracy that detect;
(3) kit of the present invention adopts different envelope antigens to mix the bag quilt that carries out ELISA Plate, with respect to antibody sandwich, more helps reaching and wraps preferably by effect and long holding time, thereby improved precision and stability that kit detects;
(4) kit of the present invention utilizes enzyme-labelled antibody technique, enzyme directly is marked on the different nitrofuran metabolin specific antibodies, nitrofuran metabolin specific antibody and two kinds of most important reactants of enzyme are united two into one, not only simplified greatly operation steps and reaction time, reduced the error that causes because of complicated operation, and need not in kit, to dispose again antiantibody, simultaneously also save the consumption of nitrofuran metabolin specific antibody and enzyme, thereby greatly reduced the cost of kit.
Be highly suitable for the trace analysis and the batch detection of nitrofuran metabolite residue based on above this kit of advantage, have important practical significance.
Description of drawings
Fig. 1 is a typical curve.
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.
The preparation of embodiment 1 antigen
(1) the haptenic preparation of nitrofuran metabolin:
A. metabolin AOZ is haptenic synthetic
3-amino-2-oxazolidone (AOZ) generates the haptens that has carboxyl with the terephthalaldehydic acid reaction.
B. metabolin AMOZ is haptenic synthetic
3-amino-5-beautiful jade reacts in water for methyl-2-oxazolidone (AMOZ) and terephthalaldehydic acid and generates the haptens that has carboxyl.
C. metabolin SC is haptenic synthetic
With the nitroreduction of semicarbazides (SC) is amino, generates to have amino haptens.
D. metabolin AH is haptenic synthetic
An amido-glycolyurea (AH) and an aldehyde benzoic acid react in water and generate the haptens that has carboxyl.
(2) preparation of nitrofuran metabolin antigen:
A. 50 μ mol/L nitrofuran metabolin haptens 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 disclosed UV scanning such as Chen Xinjian in 1998 measure in conjunction with ratio method measure in conjunction with than, antigen is concentrated preserve or freeze-drying is preserved and obtained nitrofuran immunogene and coating antigen at last, packing is stored in-20 ℃ the refrigerator.
The preparation of embodiment 2 antibody
The preparation of nitrofuran metabolin mouse monoclonal antibody:
Animal immune program: adopt the Balb/c mouse as immune animal, with nitrofuran metabolin haptens and bovine serum albumin(BSA) conjugate is immunogene, immunizing dose is 60 μ g/, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, lumbar injection, getting the same dose immunogene 3 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, 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, adopt the indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole in 4: 1 ratios and SP2/0 myeloma cell.Utilize the microclone method that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains the stably excreting monoclonal antibody.
Cell cryopreservation and recovery: get the hybridoma that is in exponential phase and make 5 * 10 with cryopreserving liquid
6The cell suspension of individual/mL is sub-packed in frozen pipe, in the medium-term and long-term preservation of-70 ℃ of ultra low temperature freezers.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, with Balb/c mouse (8 age in week) lumbar injection hybridoma 5 * 10
6Individual/as only, to gather ascites after 14 days.Carry out the ascites purifying with immunochromatographic method, bottle packing ,-20 ℃ of preservations.
The extraction of embodiment 3 horseradish peroxidases or alkaline phosphatase
1, the extraction of horseradish peroxidase
A. water extracts: take by weighing 20 kilograms of bright horseradish or horseradish skins of wash clean, be cut into small pieces, rub in comminutor.Disintegrating slag slurry adds 10 kg water stirring and leaching 8 hours at low temperatures, with centrifugal 10 minutes of 3000 rev/mins of speed, collects supernatant.
B. ammonium sulfate fractionated: every liter of filtrate adds 226 gram ammonium sulfate powder, and the limit edged stirs, and puts under the room temperature and spends the night.Draw supernatant next day, add 258 gram ammonium sulfate powder by whenever going up clear liquid again, with adding with stirring, treat that ammonium sulfate dissolves fully after, put the cold house and spend the night.Inhale next day and remove supernatant, the precipitation part in refrigerated centrifuge with 13000 rev/mins centrifugal 20 minutes, abandoning supernatant, collecting precipitation.Precipitation is dissolved in 200～300 ml distilled waters, is sub-packed in the bag filter, in circulating water, dialysed 1～2 day, till the water adding barium chloride solution that appears does not have the precipitation generation.And then in distilled water, dialysed 8 hours.Merge dislysate, in refrigerated centrifuge with 4000 rev/mins centrifugal 15 minutes, collect supernatant.
C. acetone fractionated: pour supernatant into beaker and put during cryosel bathes, under constantly stirring, add equal-volume with dropper along wall of cup and be chilled to-15 ℃ acetone in advance, centrifugal 15 minutes with 4000 rev/mins in refrigerated centrifuge, collect supernatant,-15 ℃ of acetone that add 0.8 times of former supernatant volume again, centrifugal (condition is the same) collecting precipitation.Precipitation is dissolved in a small amount of distilled water, and acetone is removed in dialysis (method is the same), promptly gets thick HRP.
D. refining: every liter of crude enzyme liquid adds 1 milliliter of 1 mol sulfuric acid zinc solution, in refrigerated centrifuge with 5000 rev/mins centrifugal 10 minutes, collect supernatant, be sub-packed in the bag filter, dialysis removes zinc sulfate in flowing water, needs 1 day approximately, dialysis 8 hours in distilled water then.Dislysate is merged, carry out vacuum drying and promptly get and make with extra care HRP.Product is the fibrous fluffer of ecru.
2, alkaline phosphatase
E.Coli 1,317 strain fermentation that utilize to produce alkaline phosphatase is cultivated, fermentation liquor through centrifugal (8000r/min, 10min) after, the thalline of precipitation is through 5 * 10
-4Back lysozyme broken wall is handled in the high sepage of M EDTA-0.03MpH8.0 Tris-0.5M sucrose, and last clear enzyme solution is through DEAE cellulose stirring and adsorbing and wash-out, thermal treatment and Sephadex G-100 sieve chromatography purifying.
The preparation of embodiment 4 enzyme labeling nitrofuran metabolin antibody
The periodates oxidizing process is adopted in the preparation of horseradish peroxidase HRP marker nitro furans metabolin antibody, and concrete grammar is:
A. dissolve 5mg HRP in the 1mL ultrapure water, the 0.1mol/L sodium periodate 75 μ L of the new configuration of adding put room temperature or 4 ℃ of refrigerators react 20min or 30min.
B. the bag filter of packing into after having reacted, 4 ℃ of dialysed overnight of 0.001mol/L pH4.0 hac buffer, during need to change dislysate several times.
C. nitrofuran metabolin antibody is diluted to 10mg/mL with the 0.1mol/L carbonic acid buffer, the pH value of solution that will activate good HRP with the 0.1mol/L carbonic acid buffer transfers to 9.5 in addition.0.5mL antibody is added in the HRP solution, put room temperature or 4 ℃ of refrigerator reaction 2h.
D. add 100 μ L 4mg/mL sodium borohydrides, 4 ℃ of refrigerator reaction 2h.
E. to 0.01mol/L PBS dialysed overnight, it is standby that liquid-20 ℃ preservation is preserved in adding.
The preparation of embodiment 5 enzyme linked immunological kit components
(1) preparation of thickening and washing damping fluid: contain the phosphate buffer of 0.5～1.5% polysorbas20, phosphate buffer pH7.4, concentration is 0.1mol/L, is 15～25 times of normal working concentration.
(2) phosphate buffer of the preparation of diluted sample concentrate: pH7.4,0.1～0.25mol/L is 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 substrate buffer solution: 30% hydrogen peroxide, 30 μ L are dissolved in pH5.0 phosphoric acid-citrate buffer solution of 19mL 4 ℃ of preservations.The preparation of phosphoric acid-citrate buffer solution: 0.2MNa
2HPO
425.7mL, 0.1M citric acid 24.3mL, adding distil water 50mL.
(5) preparation of substrate solution: with 3,3,5,5-tetramethyl benzidine (TMB) 80mg is dissolved in 10mL pH5.0 phosphoric acid-citrate buffer solution, 4 ℃ of preservations.The preparation of phosphoric acid-citrate buffer solution: 0.2M Na
2HPO
425.7mL, 0.1M citric acid 24.3mL, adding distil water 50mL.
(6) the bag quilt of ELISA Plate microwell plate: the coupled antigen pH9.6 of 4 kinds of nitrofuran metabolic product AOZ, AMOZ, SC and AH, 0.05mol/L carbonate buffer solution be diluted to 0.1～5ug/mL then in 4: 4: 1: 1 ratio is mixed, wherein carbonate buffer solution contains 1～2g sodium carbonate and 2～4g sodium bicarbonate, distilled water 1L.Every hole in ELISA Plate adds 100uL, 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.
(7) the different enzyme labelled antibodies of 4 kinds of metabolic product AOZ, AMOZ, SC and AH that mark is good were according to 4: 4: 1: 1 ratio mixed preparing enzymic-labelled antibody working fluid.
(8) preparation of nitrofuran metabolin standard solution: accurately take by weighing nitrofuran metabolin AOZ standard specimen 8.1 μ g, be dissolved in the 0.01L 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 nitrofuran metabolin AOZ standard 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; The preparation of the standard solution of other 3 kinds of metabolic product AMOZ, SC and AH is the same.
(9) reagent packing: all ingredients is prepared on request, and it is aseptic subpackaged to measure qualified back.Enzymic-labelled antibody working fluid 7mL/ bottle, nitrofuran metabolin standard model 1mL/ bottle, substrate solution 7mL/ bottle, substrate buffer solution 7mL/ bottle, stop buffer 7mL/ bottle concentrates 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.
(10) assembling of kit: will detachably wrap respectively by 1 of the microwell plate of good multiple nitrofuran metabolin coupled antigen, enzymic-labelled antibody working fluid, substrate solution, substrate buffer solution, stop buffer, concentrate each 1 bottle of washing lotion, concentrating sample dilution, 24 bottles of nitrofuran metabolin standard solution, each 6 of 4 kinds of different metabolic things, operation instructions are put assigned address in the kit for 1 part.Kit encapsulates after the assay was approved, 4 ℃ of preservations.
The preparation of embodiment 6 enzyme linked immunological kit components
Experimental procedure is with embodiment 5, different is that the alkaline phosphatase that adopts bacterium to extract is a marker enzyme, described substrate solution is to the nitro phosphate buffer, described substrate buffer solution is the pH5.0 phosphoric acid-citric acid solution that contains hydrogen peroxide or urea peroxide, described stop buffer is the sodium hydroxide solution of 1～2mol/L sulfuric acid solution or 2mol/L, prepares according to the laboratory conventional method.
Embodiment 7 is set up the enzyme linked immunological kit that detects nitrofuran, comprises following component:
(1) bag is by 96 hole ELISA Plate of multiple nitrofuran metabolin coupled antigen; Perhaps select 40 hole ELISA Plate as required;
(2) enzymic-labelled antibody working fluid, the 7mL/ bottle;
(3) nitrofuran metabolin standard solution is 24 bottles, and the concentration of each metabolin standard solution of AOZ, AMOZ, SC and AH is 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;
(4) substrate buffer solution, the 7mL/ bottle;
(5) substrate solution, the 7mL/ bottle;
(6) stop buffer, the 7mL/ bottle;
(7) concentrated cleaning solution, the 50mL/ bottle;
(8) concentrating sample dilution, the 50mL/ bottle;
(9) operation instructions, 1 part;
(10) cover plate film, 2;
(11) valve bag (containing drying agent), 1.
Embodiment 8 testing sample pre-treatments
With tissue sample tissue refiner's high-speed homogenization; The centrifugal removal fat deposit of milk sample; The dissolving of honey sample adding distil water evenly; Prevent that with taking out the yellow and white after the broken shell of egg sample, stirring evenly gently foam from producing.
(1) for example chicken, pork or fishes and shrimps of animal tissue
Get the equal pledge of the tissue sample of 2 ± 0.05g, add the distilled water of 8mL, the 2-nitrobenzaldehyde of 1mL1MHCL and 200 μ L10mM, fully vibration; At 37 ℃ of about 12h of night incubation; Add 10mL 0.1M K
2HPO
4, 0.8mL 1M NaOH and 10mL ethyl acetate, thermal agitation 1min; The above centrifugal 10min of (20～25 ℃) 3000g at room temperature; Taking out 5mL ethyl acetate 50 ℃ of following nitrogen in another container dries up; With the dry thing of 2mL n-hexane dissolution, diluted good redissolution liquid with 2mL and fully mixed; The above centrifugal 10min of (20～25 ℃) 3000g at room temperature; Getting 50 μ L subnatant bodies is used for analyzing.
(2) milk
Take out 10mL milk sample in the glass centrifuge tube; Add each 200 μ L of 0.36M sodium nitroprusside damping fluid and 1M zinc sulfate damping fluid respectively; Fully behind the mixing sample, 4～10 ℃ with constant temperature hydro-extractor 3000g more than centrifugal 10min.Get the centrifuged supernatant 2mL of milk, add the distilled water of 16mL, the 2-nitrobenzaldehyde of 2mL 1M HCL and 200 μ L 10mM, fully vibration; At 37 ℃ of about 12h of night incubation; Add 10mL 0.1M K
2HPO
4, 0.8mL1M NaOH and 10mL ethyl acetate, thermal agitation 1min; The above centrifugal 10min of (20～25 ℃) 3000g at room temperature; Taking out 5mL ethyl acetate 50 ℃ of following nitrogen in another container dries up; With the dry thing of 2mL n-hexane dissolution, diluted good redissolution liquid with 2mL and fully mixed; The above centrifugal 10min of (20～25 ℃) 3000g at room temperature; Getting 50 μ L subnatant bodies is used for analyzing.
(3) honey
Take out 2g honey sample in centrifuge tube; The distilled water vibration dissolving that adds 16mL, the 2-nitrobenzaldehyde of 2mL1M HCL and 200 μ L 10mM, fully vibration; At 37 ℃ of about 12h of night incubation; Add 10mL 0.1M K
2HPO
4, 2mL 1M NaOH and 10mL ethyl acetate, thermal agitation 1min; The above centrifugal 10min of (20～25 ℃) 3000g at room temperature; Taking out 12mL ethyl acetate 50 ℃ of following nitrogen in another container dries up; With the dry thing of 2mL n-hexane dissolution, diluted good redissolution liquid with 4mL and fully mixed; The above centrifugal 10min of (20～25 ℃) 3000g at room temperature; Getting 50 μ L subnatant bodies is used for analyzing.
(4) egg
The egg sample that taking-up 4g has prepared is in the 100mL centrifuge tube; Add 16mL water respectively, 2mL1M HCL, the sodium nitroprusside damping fluid of 400 μ L 0.36M, vibration mixing; Add 400 μ L 1M zinc sulfate damping fluids, the 8min that fully vibrates, the above centrifugal 10min of room temperature (20～25 ℃) 3000g, take out whole supernatants, add the 2-nitrobenzaldehyde of 400 μ L 10mM, fully vibration, 50 ℃ of water-bath 2h, per half an hour thermal agitation 1～2 minute; Add 10mL0.1M K respectively
2HPO
4, 1mL 1M NaOH and 8mL ethyl acetate, thermal agitation 1min, the at room temperature above centrifugal 10min of (20～25 ℃) 3000g; Taking out the 6mL upper organic phase dries up in 50 ℃ of following nitrogen; With the dry thing of 2mL n-hexane dissolution, adding 4mL has diluted good redissolution liquid fully to be mixed; The above centrifugal 10min of (20～25 ℃) 3000g at room temperature; Remove upper organic phase; Taking off layer water 50 μ L is used for analyzing.
The detection method of embodiment 9 kits
(1) kit is taken out from cold storage environment, place room temperature (20～24 ℃) more than the balance 30min, the batten of enough standards and the used quantity of sample is fixed in support, standard and sample are done two parallel laboratory tests, number in order;
(2) add 50 μ L standard items in the standard items hole, sample well adds 50 μ L testing samples.Every then hole adds 50 μ L enzyme labeling things, pats mixing.Cover the cover plate film, at incubated at room 20min;
(3) pour out liquid in the hole, the micropore frame is upside down on the thieving paper pats, the every wheel washed plate and patted 3 times, to guarantee to remove fully the liquid in the hole.Charge in the hole with 250 μ L distilled water, outwell the liquid in the micropore once more, repetitive operation is 3 times again;
(4) every hole adds 100 μ L colour developing liquid (substrate buffer solution is mixed with the substrate solution equal-volume), pats mixing, covers the cover plate film, dark place incubated at room 15min;
(5) add 50 μ L reaction terminating liquids in micropore.Mixing at wavelength 450nm or 492nm, is blank with the air, measures each hole light absorption value, must read light absorption value in the 60min after adding stop buffer.
Testing result is calculated and is analyzed:
With the mean value calculation percentage absorbance of obtained standard model light absorption value, be ordinate with the percentage absorbance, the semilog of nitrofuran concentration of standard solution is a horizontal ordinate drawing standard curve, obtains straight-line equation.See accompanying drawing 1.Y＝-15.201X+87.907；R
2＝0.994。The use the same method percentage absorbance of calculation sample solution is obtained the nitrofuran metabolite concentration of counter sample according to equation.The calculating formula of described percentage absorbance is:
Percentage absorbance (%)=(B/B
0) * 100
Wherein, B is the average light absorption value of standard solution or sample, B
0It is the mean light absorbency 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 nitrofuran metabolin linear detection range, detects and is limited to 0.1 μ g/L, and whole testing process only needs 35min just can finish.
Embodiment 10 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 (6), each extracts 20 micropores out, measures the absorbance (OD 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
| Sample | Concentration μ g/L | The 1st batch | The 2nd batch | The 3rd batch | CV% between crowd |
| CV％ | CV％ | CV％ | |||
| Standard items | 0.9 | 4.7 | 4.9 | 4.3 | 8.1 |
The result shows the variation within batch coefficient scope of kit standard items detection between 4.3～4.9%, and interassay coefficient of variation is 8.1%.
2, sample repeatability and accuracy test
Accuracy is meant the matching degree of measured value and true value, and in ELISA measured, accuracy often represented with the recovery that precision is often represented with the coefficient of variation.In blank fishes and shrimps, milk, honey, it is 1 μ g/L (μ g/kg), 5 μ g/L (μ g/kg) that nitrofuran 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
| Sample | Add concentration μ g/L | Content | The 1st crowd of recovery % | CV％ | Content | The 2nd crowd of recovery % | CV％ | Content | The 3rd crowd of recovery % | CV％ | CV% between crowd |
| Fishes and shrimps | 1 | 0.83 | 83.0 | 6.7 | 0.85 | 85.0 | 8.4 | 0.81 | 81.0 | 9.3 | 14.9 |
| Milk | 5 1 5 | 4.205 0.913 4.565 | 84.1 91.3 91.3 | 5.7 4.4 7.1 | 4.09 0.835 4.435 | 81.8 83.5 88.7 | 7.9 5.1 6.1 | 4.18 0.889 4.18 | 83.6 88.9 83.6 | 8.9 4.3 5.8 | 13.4 16.8 13.1 |
| Honey | 1 5 | 0.813 4.76 | 81.3 95.2 | 8.2 8.1 | 0.853 4.155 | 85.3 83.1 | 5.2 9.8 | 0.813 4.06 | 81.3 81.2 | 6.3 8.9 | 15.6 13.9 |
The result shows the interpolation recovery of fishes and shrimps, milk, honey sample between 81.0～95.2%, and the variation within batch coefficient is between 4.3～9.8%, and interassay coefficient of variation is between 13.1～16.8%.
The test of embodiment 11 storage lives
(1) kit is positioned over 2～8 ℃, get 0,2,4,6,8,9,10,11 and 12 months kit respectively, to absorbance, 50% inhibition concentration of nitrofuran metabolin 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 absorbance, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of nitrofuran metabolin standard model (0.1 μ g/L) every day.
(3) kit was preserved 12 days at-20 ℃ of refrigerators, measure absorbance, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of nitrofuran metabolin standard model (0.1 μ g/L) every day.
Can find out that from the result preserve test through three kinds of conditions, the absorbance of nitrofuran metabolin standard model (0.1 μ g/L) descends less than 5%, and OD is not less than 1.6; 50% inhibiting rate is between 0.5～1.0 μ g/L; Add the recovery between 75～105%; 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 kind of enzyme-linked immunologic detecting kit of nitrofurans medicament metabolite residue is characterized in that comprising following ingredients:
(1) bag is by the ELISA Plate of multiple nitrofuran metabolin coupled antigen;
(2) enzymic-labelled antibody working fluid;
(3) nitrofuran metabolin standard solution;
(4) substrate solution;
(5) substrate buffer solution;
(6) reaction terminating liquid;
(7) concentrated cleaning solution;
(8) diluted sample concentrate.
2, enzyme linked immunological kit according to claim 1, it is characterized in that described ELISA Plate adopts 96 holes or 40 hole ELISA Plate, be coated with can with the nitrofuran metabolin coupled antigen of nitrofuran metabolin antibody specific bond, and the site of nitrofuran metabolin coupled antigen is not adsorbed on the closed porosity surface.
3, enzyme linked immunological kit according to claim 1 is characterized in that described nitrofuran metabolin antigen is to adopt carbodlimide method, active ester method or mixed anhydride method that nitrofuran metabolin haptens and carrier protein are carried out coupling to obtain.
4,, it is characterized in that described enzyme labeling nitrofuran metabolin antibody working fluid obtains marker enzyme with mixing by a certain percentage after metabolin antibody carries out coupling respectively for adopting glutaraldehyde method or periodates oxidizing process according to the described enzyme-linked immuno assay kit of claim 1; Described marker enzyme is the alkaline phosphatase that horseradish peroxidase or bacterium are extracted; Described nitrofuran metabolin antibody be mouse monoclonal antibody, rabbit polyclonal antibody or genetic engineering antibody any one.
5, enzyme linked immunological kit according to claim 1, it is characterized in that when marker enzyme is horseradish peroxidase, described substrate solution is for containing 3,3,5, pH5.0 phosphoric acid-the citric acid solution of 5-tetramethyl benzidine or o-phenylenediamine, described substrate buffer solution are the pH5.0 phosphoric acid-citric acid solution that contains hydrogen peroxide or urea peroxide, and described stop buffer is the sodium hydroxide solution of 1～2mol/L sulfuric acid solution or 2mol/L;
When marker enzyme is the alkaline phosphatase of bacterium extraction, described substrate solution is to the nitro phosphate buffer, described substrate buffer solution is the pH5.0 phosphoric acid-citric acid solution that contains hydrogen peroxide or urea peroxide, and described stop buffer is the sodium hydroxide solution of 1～2mol/L sulfuric acid solution or 2mol/L.
6, enzyme linked immunological kit according to claim 1 is characterized in that described concentrated cleaning solution is the phosphate buffer that contains 0.5～1.5% polysorbas20, phosphate buffer pH7.4, and concentration is 0.1mol/L.
7, enzyme linked immunological kit according to claim 1 is characterized in that described diluted sample concentrate is the phosphate buffer of pH7.4,0.1～0.25mol/L.
8, enzyme linked immunological kit according to claim 1, it is characterized in that described nitrofuran metabolin standard solution is respectively AOZ standard solution, AMOZ standard solution, SC standard solution or AH standard solution, the concentration of described metabolin standard solution is: 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 and 0 μ g/L.
9, the using method of the described enzyme linked immunological kit of a kind of claim 1 is characterized in that may further comprise the steps:
(1) sample pre-treatments;
(2) use kit to detect;
(3) result treatment and analysis.
10, method according to claim 9 is characterized in that step (2) may further comprise the steps:
(1) kit is taken out from cold storage environment, place equilibrium at room temperature;
(2) standard items or testing sample adding have been coated with in the ELISA Plate hole of nitrofuran metabolin hybrid antigen, every then hole adds the enzyme labelled antibody working fluid, pats mixing, hatches;
(3) washing;
(4) every hole adds the substrate buffer solution and the substrate solution of equivalent, pats mixing, and lucifuge is hatched;
(5) every hole adds reaction terminating liquid, mix, and under wavelength 450nm or 492nm, be blank with the air, microplate reader is measured each hole light absorption value.
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| CN113189348A (en) * | 2021-06-08 | 2021-07-30 | 中山生物工程有限公司 | Hepatitis C virus antibody detection kit and application thereof |
| CN113884680A (en) * | 2021-09-16 | 2022-01-04 | 天津温阳生物技术有限公司 | Carbon quantum dot fluorescence immunoassay rapid detection kit and detection method for six veterinary drug antibiotics in poultry tissues |
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