CN1766632A - ELISA kit for detecting quinolones in animal derived food - Google Patents
ELISA kit for detecting quinolones in animal derived food Download PDFInfo
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- CN1766632A CN1766632A CNA2005100867794A CN200510086779A CN1766632A CN 1766632 A CN1766632 A CN 1766632A CN A2005100867794 A CNA2005100867794 A CN A2005100867794A CN 200510086779 A CN200510086779 A CN 200510086779A CN 1766632 A CN1766632 A CN 1766632A
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Abstract
The invention provides an agent box for detecting fluorine drugs of animal foodstuff, which uses enzyme immune method to detect the preprocessed animal organization, shrimp, fish and blood product. The enzyme immune agent box comprises: enzyme mark plate which coats fluorine drugs antigen, enzyme mark antibody, ciproloxacin standard solution, base material color developing solution, fluorine drugs antibody, compression cleaning liquid, ending solution and compression twin solution. The invention also discloses a method for applying the detecting method, which comprises: first doing sample front process, then using the agent box to detect, at last analyzing the detected result.
Description
Technical field
The present invention relates to enzyme linked immunological kit and detection method thereof that a kind of detection animal derived food comprises afloqualone class medicine in animal tissue's (muscle, liver), shrimp, fish and the blood product.
Background technology
(be called Comprecin again, QNS), its has a broad antifungal spectrum, antibacterial action are strong, have now become one of most important microbiotic in poultry industry and the culture fishery for afloqualone class antimicrobial.But because afloqualone class medicament residue causes drug resistance and potential carcinogenicity, European Union, Japan and China have all formulated its high residue amount in food.The residue detection of afloqualone class medicine has become the essential items for inspection that China eel exports to Japan.
The residual marker of afloqualone class medicine is Enrofloxacin and Ciprofloxacin in the tissue, and is the highest with residual concentration in liver organization and the renal tissue, secondly is the skin histology that muscle and fat adhere to.Enrofloxacin and Ciprofloxacin metabolic product (CIF) biologically active still wherein; Ofloxacin (OFL) mainly is present in the tissue with the form of former medicine; Pefloxacin (PEF) metabolic rate in vivo is quite high, and near 100%, its metabolic product is Norfloxacin (NOR).
The chemical method that detects afloqualone class drug residue mainly contains thin-layered chromatography (TLC), vapor-phase chromatography (GC), high performance liquid chromatography (HPLC), gas-matter online (GC/MS), liquid-matter online (HPLC/MS), Capillary Electrophoresis (CE) etc., because complicated instrument and equipment and loaded down with trivial details process are not suitable for on-site supervision and great amount of samples examination.
Summary of the invention
(1) technical matters that will solve
The object of the invention is to provide the enzyme linked immunological kit that is used for the drug test of animal derived food afloqualone class of a kind of simple in structure, easy to use, low price, carrying convenience, and a kind of efficient, accurate, easy, qualitative and quantitative analysis method of being suitable for the batch samples examination is provided.
(2) technical scheme
Enzyme linked immunological kit provided by the present invention comprises following ingredients:
(1) bag is by the ELISA Plate of afloqualone class medicine antigen;
(2) enzyme mark antiantibody;
(3) Ciprofloxacin standard solution;
(4) substrate colour developing liquid;
(5) afloqualone class drug antibody;
(6) concentrated cleaning solution;
(7) stop buffer;
(8) concentrate redissolution liquid.
The afloqualone class medicine of indication comprises that Enrofloxacin, Ciprofloxacin, Ofloxacin, Norfloxacin, Pefloxacin etc. have the medicine of common molecular skeleton pharmacologically active among the present invention.
Wherein said bag by the ELISA Plate of afloqualone class medicine antigen in the process of preparation, used coating antigen adopts mixed anhydride method that afloqualone class medicine haptens and carrier protein are carried out coupling and obtains; It is pH9.6 that used bag is cushioned liquid, the carbonate buffer solution of 0.05mol/L; Used confining liquid is pH7.4,0.01mol/L, contains the phosphate buffer of 20% NBCS and 0.05% polysorbas20 in the process of bag quilt; Used afloqualone class medicine haptens is that intermediate adopts the succinic anhydride method synthetic with afloqualone class medicine Ciprofloxacin; The used protein carrier of coating antigen can be bovine serum albumin(BSA) (BSA), human serum albumins (HSA), oralbumin (OVA), thyroprotein (BCG), and wherein preferred oralbumin is the coating antigen carrier protein.
The marker enzyme of wherein said enzyme mark antiantibody can be horseradish peroxidase or bacterium is extracted alkaline phosphatase, and the preferred horseradish peroxidase of the present invention can adopt glutaraldehyde method or sodium periodate method that horseradish peroxidase and antiantibody are carried out coupling.Described antiantibody be with sheep as immune animal, be that immunogene is carried out immunity to the pathogen-free domestic sheep and obtained with mouse source antibody.The sodium periodate method that the present invention preferably improves is with horseradish peroxidase and antiantibody coupling, and its advantage is: need to adopt in traditional sodium periodate method α residual on the dinitrofluorobenzene sealing horseradish peroxidase-and epsilon-amino to avoid crosslinked between the enzyme molecule.The present invention uses instead under low pH and makes NaIO
4The oxidation horseradish peroxidase, thus dinitrofluorobenzene sealing horseradish peroxidase step saved.Horseradish peroxidase is through NaIO
4The hydroformylation enzyme that forms after the oxidation can link to each other with the amino of antibody molecule, forms this Fu Shi alkali, and the latter can further use NaBH
4(or monoethanolamine) reduction generates stable enzyme labeling antiantibody.
Wherein said afloqualone class drug antibody is in preparation process, and used immunogene adopts mixed anhydride method that afloqualone class medicine haptens and carrier protein are carried out coupling and obtains; The immunogen protein carrier can be oralbumin (OVA), albumin rabbit serum (RSA), mouse serum albumin (MSA) or thyroprotein (BCG), wherein be preferably thyroprotein, its advantage is: the albumen relation is far away between thyroprotein and the immunized animal, and complex structure, so immunogenicity is good, can induce stronger immune response.
The afloqualone class drug antibody protein concentration that uses is 0.5～5.0 μ g/L.
The concentration of wherein said Ciprofloxacin pharmaceutical standards product solution is 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L and 81 μ g/L.
Wherein said developer: when marker enzyme was horseradish peroxidase, substrate colour developing liquid A liquid was that hydrogen peroxide or urea peroxide, substrate colour developing liquid B liquid are o-phenylenediamine or tetramethyl benzidine; When marker enzyme was bacterium extraction alkaline phosphatase, substrate colour developing liquid was to the nitro phosphate buffer.
Wherein said concentrated cleaning solution is the phosphate buffer that contains 0.8～1.2% polysorbas20.
Wherein said stop buffer is sulfuric acid, hydrochloric acid or the 2mol/L NaOH of 1～2mol/L.
Wherein said redissolution liquid is 0.01～0.05mol/L, contain the phosphate buffer of 1% gelatin.To add its advantage of a certain amount of gelatin in the phosphate buffer be: reduce the non-specific adsorption of antigen, antibody, thereby increased the combination rate of antigen, antibody.
The carrier mass that can be used as fixedly afloqualone class medicine and carrier protein couplet thing is a lot, as polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.Wherein be preferably the micro-reaction plate shrinkage pool that polystyrene is made, its advantage is: polystyrene has the performance of stronger adsorbed proteins, still keeps original immunocompetence after antigen adsorbs on it, and blank value is low, transparency height at the bottom of the hole is between each plate, performance is close between each hole of same plate.With the similar polyvinyl chloride of polystyrene, as solid phase carrier, the matter soft board is thin, can cut and cut, and is inexpensive, but glossiness is smooth not as polystyrene at the bottom of the hole not as polystyrene, and blank value is also high, so solid phase carrier is preferably polystyrene.
The present invention also provides the method for afloqualone class drug residue in the samples such as use mentioned reagent box qualitative and quantitative analysis animal tissue (muscle, liver), fish, shrimp and serum, and it may further comprise the steps:
(1) sample pre-treatments;
(2) detect with kit;
(3) analyzing and testing result.
Preferably, sample treatment is:
The processing of a, animal tissue's (muscle, liver), fish, shrimp etc.:
Taking by weighing the sample that homogeneous crosses fully mixes with acetonitrile-NaOH solution; Centrifugal, get supernatant liquid and add redissolution liquid and normal hexane, fully mix the back standing demix; Discard the upper strata and add methylene chloride and fully mix, take off a layer organic phase after centrifugal, be evaporated to and do or dry up with nitrogen; Adding normal hexane behind the residue with redissolution liquid dissolving drying fully mixes; Centrifugal, take off layer liquid adding equal-volume redissolution liquid and can analyze.
The processing of b, blood sample:
Blood sampling, room temperature leaves standstill, and waits to separate out behind the blood plasma centrifugal; Get blood plasma and add anhydrous second eyeball and fully mix, get supernatant after centrifugal and add and redissolve liquid and mixing; Add methylene chloride, fully centrifugal behind the mixing; Take off a layer organic phase, be evaporated to and do or dry up with nitrogen; Adding normal hexane behind the residue with redissolution liquid dissolving drying fully mixes; Centrifugal, remove upper organic phase and middle white impurity; Taking off layer liquid adding equal-volume redissolution liquid can analyze.
Preferably, kit test method is: add standard solution or sample solution in the ELISA Plate micropore, add afloqualone class drug antibody again, washing pats dry behind the incubation, adds enzyme mark antiantibody again, and washing pats dry behind the incubation, colour developing, termination are measured absorbance with microplate reader.
Preferably, the testing result analytic process is: the absorbance mean value (B) of standard solution of using each concentration that is obtained is divided by the absorbance (B of first standard solution (0 standard)
0) multiply by 100% again, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
B is the mean light absorbency value of standard solution in the formula, B
0It is the mean light absorbency value of 0 μ g/L standard solution.Natural logarithm value with the Ciprofloxacin drug concentration is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.The percentage absorbance of calculation sample solution uses the same method, the concentration of Ciprofloxacin then can be read from typical curve in corresponding each sample, the depth according to the color sample on the ELISA Plate, but with the concentration range of Ciprofloxacin in the comparison judgement sample of the standard solution color of series concentration, per sample in the residual quantity of Ciprofloxacin utilize cross reacting rate to calculate the residual quantity of other afloqualone class medicine in sample.
Preferably, the analysis of testing result also can be adopted regression equation method, calculates sample solution concentration.
Preferably, the analysis of testing result can also utilize computer professional software, the be more convenient for express-analysis of a large amount of samples of this method, and whole testing process only needed to finish in 1.5 hours, and it is 0.5 μ g/L that lowest detection is limited the quantity of.
Wherein, the preparation method of antigen and antibody is:
(1) antigen is synthetic
Haptens is synthetic: with afloqualone class pharmaceutical intermediate Ciprofloxacin and the synthetic afloqualone haptens of succinic anhydride.Its preparation principle: since afloqualone class medicine in contain ketone group, make its with O-(ethyloic) hydroxyl reaction, the generation have the hapten derivant of carboxyl, thereby in molecule, introduce carboxyl.
Can be as its preparation process:
1, in 70% ethanol, add the haptens of O-(ethyloic) hydroxyl and ketone group, make its concentration be respectively 10mmol/L and 4mmol/L.
2, backflow heating 90min.
3, rotary evaporation reduces volume, extracts with methylene chloride after adding water then.
4, wash dichloromethane extract with water, use Na
2SO
4Be dried to white powder.
Its advantage: the terminal imines succinic anhydride acidylate with the Ciprofloxacin intermediate, pick out a spacerarm that contains 4 carbon, given prominence to the feature structure of afloqualone class medicine haptenic group.This helps to make at afloqualone class drug specificity monoclonal antibody.
Immunogene: adopt mixed anhydride method (isobutyl chlorocarbonate) to carry out coupling afloqualone class medicine haptens and oralbumin, bovine serum albumin(BSA), human serum albumins or thyroprotein and obtain immunogene.
Coating antigen: adopt mixed anhydride method (isobutyl chlorocarbonate) to carry out coupling afloqualone class medicine haptens and hemocyanin, thyroprotein, human serum albumins or mouse serum albumin and obtain coating antigen.
(2) afloqualone class medicine mouse monoclonal antibody preparation
The animal immune program: with afloqualone class medicine haptens and carrier protein couplet thing is that immunogene is carried out immunity to the Balb/c mouse, immunizing dose is 80～100 μ g/, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 2～3 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, booster immunization 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, in 5～10: 1 ratio and SP2/0 myeloma cell are merged, and adopt the indirect competitive ELISA method to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, until 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 1～5 * 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 liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is only injected sterilization paraffin oil 0.5ml/, 7～14 days pneumoretroperitoneum injection hybridomas 5 * 10
5～10
6Individual/as only, to gather ascites after 7～10 days.Carry out the ascites purifying through sad-saturated ammonium sulfate method, bottle packing ,-20 ℃ of preservations.
(3) preparation of antiantibody
As immune animal, is that immunogene to pathogen-free domestic sheep carry out immunity with mouse source antibody with sheep, obtains the sheep anti mouse antiantibody.
(4) enzyme mark antiantibody preparation
Horseradish peroxidase or bacterium are extracted alkaline phosphatase employing sodium periodate method or glutaraldehyde method and coupling of sheep anti mouse antiantibody and purifying extraction, obtain enzyme mark antiantibody.
Wherein the compound method of agents useful for same is:
A. Ciprofloxacin standard solution: concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L;
B. bag is cushioned the carbonate buffer solution of liquid: pH9.6,0.05mol/L;
C. confining liquid: pH7.4,0.01mol/L, contain the phosphate buffer of 20% NBCS, 0.05% polysorbas20;
D. concentrated cleaning solution: contain the phosphate buffer (pH7.4,0.01M) of 0.8～1.2% polysorbas20, be 15～25 times of normal working concentration;
E. afloqualone class drug antibody working fluid: with containing 5 ‰ N, it is 0.5～5.0 μ g/L that N, the phosphate buffer of N '-dimethyl formamide (DMF) are diluted to protein concentration with antibody;
F. enzyme labeling thing working fluid: the concentration of enzyme labeling antiantibody working fluid is 0.1～2.0 μ g/L;
G. substrate colour developing liquid A liquid: hydrogen peroxide or urea peroxide;
H. substrate colour developing liquid B liquid: o-phenylenediamine (OPD) or tetramethyl benzidine (TMB);
I. substrate colour developing liquid to nitro phosphate buffer: pH 8.1, contain MgCl
20.01%100mmolTris-HCl;
J. stop buffer: 1～2mol/L sulfuric acid, hydrochloric acid or 2mol/L sodium hydrate buffer solution;
K. the phosphate buffer that concentrate to redissolve liquid: 0.01～0.05M, contains 1% gelatin is 2～3 times of normal working concentration.
Wherein, bag by the ELISA Plate preparation method of afloqualone class medicine haptens and carrier protein couplet thing is:
Be cushioned liquid with bag afloqualone class medicine haptens and carrier protein couplet thing are diluted to 0.1～1 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h and 4 ℃ spend the night, and the coating buffer that inclines is with cleansing solution washing 3 times, each 30s, pat dry, in every hole, add 150～200 μ l confining liquids then, 37 ℃ of incubation 1～2h, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
The detection principle of kit of the present invention is: the method that adopts indirect competitive ELISA, on capillary strip, wrap in advance by coupling afloqualone class medicine haptens and carrier protein couplet thing, add Ciprofloxacin series standard product or sample solution and add afloqualone class drug antibody again, after hatching washing, Ciprofloxacin residual in the sample will be competed the antibody of anti-afloqualone class medicine with the coupled antigen of pre-bag quilt on the capillary strip, adding enzyme labeling antiantibody carries out the amplification of enzymatic activity, colour developing stops the back and measures every hole absorbance with microplate reader, sample absorbance and residue of ciprofloxacin amount are negative correlation, relatively can draw the content of corresponding residue Ciprofloxacin with typical curve.Simultaneously according to the depth of the color sample on the ELISA Plate, but with the concentration range of the comparison judgement sample of the titer color of the Ciprofloxacin of series concentration.Utilize cross reacting rate to calculate the concentration that contains other afloqualone class medicament residue in the sample according to the residue of ciprofloxacin amount again.
(3) beneficial effect
The enzyme linked immunological kit of detection afloqualone class medicine of the present invention can detect the residual quantity of afloqualone class medicine in the samples such as animal tissue's (muscle, liver), fish, shrimp and serum, pre-treatment to sample requires low, sample pretreatment process is simple, simultaneously the fast detecting gross sample.
The present invention adopts the afloqualone class anti-drug monoclonal antibody of high specific, main agents provides with forms such as working fluid, concentrate or freeze-dried powders, the method of inspection is convenient and easy, have specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height, play a significant role in will detecting at the afloqualone class drug residue of food and feed.
Description of drawings
Fig. 1 is an afloqualone class drug test canonical plotting.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 detects the component preparation of the enzyme linked immunological kit of afloqualone class drug residue
1. antigen is synthetic
A. haptenic synthetic: with afloqualone class medicine Ciprofloxacin is that intermediate adopts the synthetic afloqualone class medicine haptens of succinic anhydride method, and preparation process is:
(1) in 70%200mL ethanol, add the Ciprofloxacin haptens that has O-(ethyloic) hydroxyl and ketone group, make its concentration be respectively 10mmol/L and 4mmol/L;
(2) backflow heating 90min;
(3) rotary evaporation reduces volume, adds water to 50mL then, and methylene chloride extracts;
(4) wash dichloromethane extract with water, use NaSO
4Be dried to white powder.
B. immunogene is synthetic: adopt mixed anhydride method to carry out coupling respectively afloqualone class medicine haptens and thyroprotein and obtain immunogene, preparation process is:
(1) get afloqualone class medicine haptens 2g and be dissolved in 30ml, 50% N is in N '-dimethyl formamide solution;
(2) getting the 0.5ml isobutyl chlorocarbonate again is dissolved in the no Shui diox of 5ml, join in the haptens solution stirring at room reaction 4 hours, get thyroprotein 32g and be dissolved in the 70ml pH9.6 carbonate buffer solution, again thyroprotein is added drop-wise in the haptens 4 ℃ of stirrings and spends the night;
(3) synthetic artificial antigen was dialysed 7 days with the phosphate buffer of 0.2M, change liquid every day 3～4 times, at last concentrated preservation of antigen or freeze-drying are preserved.
C. coating antigen is synthetic: with afloqualone class medicine haptens and oralbumin, adopt mixed anhydride method to carry out coupling and obtain coating antigen, preparation process is:
(1) gets afloqualone class medicine haptens 2g and be dissolved in the sodium hydroxide solution of 20ml, 0.5M, get 2g hydroxyl succinimide (NHS) active ester again and be dissolved in the 8ml pure water, join stirring at room reaction 2.5h in the haptens solution;
(2) get in the carbonate buffer solution that carrier protein 22g is dissolved in 75ml pH9.0, again oralbumin is added drop-wise in the haptens 4 ℃ of stirrings and spends the night;
(3) synthetic artificial antigen was dialysed 7 days with the phosphate buffer of 0.2M, change liquid every day 3～4 times.At last antigen is concentrated and preserve or the freeze-drying preservation.
2. afloqualone class medicine mouse monoclonal antibody preparation
Animal immune program: adopt the Balb/c mouse as immune animal, with afloqualone class medicine haptens and thyroprotein conjugate is immunogene, immunizing dose is 100 μ g/, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 2 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 indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole in 8: 1 ratios and SP2/0 myeloma cell.Utilize limiting dilution assay 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 4 * 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 liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is only injected sterilization paraffin oil 0.5ml/, 10 days pneumoretroperitoneum injection hybridomas 8 * 10
5Individual/as only, to gather ascites after 8 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method, bottle packing ,-20 ℃ of preservations.
3. the preparation of sheep anti mouse antiantibody
With the sheep is immune animal, is that immunogene is carried out immunity to the pathogen-free domestic sheep with the mouse endogenous antibody, obtains the sheep anti mouse antiantibody.
4. enzyme mark antiantibody preparation
Adopt the sodium periodate method with horseradish peroxidase and coupling of sheep anti mouse antiantibody and purifying extraction, obtain enzyme mark antiantibody.Labeling process is:
(1) taking by weighing 5mg horseradish peroxidase (HRP) is dissolved in the 1ml distilled water.
(2) add the 0.1M NaIO that 0.2ml newly joins
4Solution, lucifuge stirs 20min under the room temperature.
(3) solution is packed in the bag filter, with the sodium-acetate buffer dialysis of 1mM pH4.4,4 ℃ are spent the night.
(4) add 20 μ l 0.2M pH9.5 carbonate buffer solutions, make the pH of solution be elevated to 9.0～9.5, add 10mg IgG (antibody) or 5mg staphylococcal protein A then immediately, in the carbonate buffer solution of 1ml 0.01M, the room temperature lucifuge stirs 2h gently.
(5) add the 4mg/ml NaBH that 0.1ml newly joins
4Liquid, mixing, 4 ℃ left standstill 2 hours.
(6) above-mentioned liquid is packed in the bag filter, with 0.15M, the dialysis of pH7.4 phosphate buffer, 4 ℃ are spent the night.
(7) put 4 ℃ of environment and slowly stir, dropwise add the equal-volume saturated ammonium sulfate.
(8) 3000rpm centrifugal half an hour, abandon supernatant.Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved in the phosphate buffer of a small amount of pH7.4,0.15M.
(9) above-mentioned solution is packed in the bag filter,, remove (detecting) behind the ammonium ion with Nai Shi reagent with the phosphate buffered solution dialysis of pH7.4,0.15M, 10, the centrifugal 30min of 000rpm removes precipitation, and supernatant is enzyme conjugates, after the packing, stored frozen.
5. the preparation of ELISA Plate
Be cushioned liquid with bag afloqualone class medicine haptens and oralbumin conjugate are diluted to 0.6 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h, and 4 ℃ spent the night, coating buffer inclines, with cleansing solution washing 3 times, each 30s pats dry, in every hole, add 150 μ l confining liquids then, 37 ℃ of incubation 1.5h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of afloqualone class medicine
Set up the enzyme linked immunological kit that detects afloqualone class medicine, make it comprise following component:
(1) bag is by the ELISA Plate of afloqualone class medicine antigen;
(2) the sheep anti mouse antiantibody of usefulness horseradish peroxidase-labeled;
(3) the Ciprofloxacin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L and 81 μ g/L;
(4) colour developing liquid A liquid is hydrogen peroxide, and colour developing liquid B liquid is o-phenylenediamine;
(5) protein concentration is the afloqualone class drug antibody working fluid of 0.5 μ g/L;
(6) concentrated cleaning solution is the phosphate buffer that contains 0.8% polysorbas20;
(7) stop buffer is the sulfuric acid solution of 1mol/L;
(8) concentrate redissolving liquid is 0.01M, contains the phosphate buffer of 1% gelatin.
Embodiment 3 detects the establishment of the enzyme linked immunological kit of afloqualone class medicine
Set up the enzyme linked immunological kit that detects afloqualone class medicine, make it comprise following component:
(1) bag is by the ELISA Plate of afloqualone class medicine antigen;
(2) the sheep anti mouse antiantibody of usefulness alkali phosphatase enzyme mark;
(3) the Ciprofloxacin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L and 81 μ g/L;
(4) substrate solution is to the nitro phosphate buffer;
(5) protein concentration is the afloqualone class drug antibody working fluid of 5.0 μ g/L;
(6) concentrated cleaning solution is the phosphate buffer that contains 1.2% polysorbas20;
(7) stop buffer is the sodium hydroxide solution of 2mol/L;
(8) concentrate redissolving liquid is 0.01M, contains the phosphate buffer of 1% gelatin.
The detection of afloqualone class medicament residue in embodiment 4 samples
1. sample pre-treatments
(1) muscle or liver
The chicken sample that a, title 2.0g homogeneous are crossed adds acetonitrile-NaOH solution 10ml in the 50ml centrifuge tube.Mix 10min fully up and down, 10000rpm, 15 ℃ of centrifugal 10min get upper phase 5ml.
The damping fluid of b, adding 5ml adds normal hexane 4ml again, mixes 2min, behind the standing demix, removes the upper strata.
C, adding methylene chloride 8ml, abundant mixing 10min, 5000rpm, 15 ℃ of centrifugal 10min.
D, go to the upper strata, take off layer organic phase in drying bottle, 50 ℃ of rotary evaporations are to doing or nitrogen dry up.
E, dilute the good dry residue of redissolution liquid dissolving, add normal hexane 1ml mixings 2min, taking-up, 10000rpm, 15 ℃ of centrifugal 5min with 0.6ml.
F, gently sop up upper organic phase and middle partly liquid, take off layer 50 μ l and drip the good redissolution liquid 50 μ l mixings of dilution, get 50 μ l and analyze.
(2) extraction of shrimp sample is equal to the chicken extracting method.
In above-mentioned f step, if too many foam or jelly between two-phase, occur, be difficult to take out enough lower floor's extracts, sample bottle should be placed in 80 ℃ of water-baths centrifugal again behind the 5min.
(3) processing of chicken plasma sample
A, with the centrifuge tube that is added with liquaemin (25 units/ml blood) gather the chicken blood sample this, the blood sampling syringe is also used the liquaemin rinse, this room temperature of blood sample leaves standstill 1h, after waiting to separate out blood plasma, 8000rpm, 15 ℃ of centrifugal 10min take out blood plasma 1ml.
B, anhydrous acetonitrile 4ml mix 10min fully up and down, 10000rpm, 15 ℃ of centrifugal 10min.
C, move supernatant to another centrifuge tube, add the 2ml damping fluid, mixing.
D, adding methylene chloride 5ml, abundant mixing 10min, 5000rpm, 15 ℃ of centrifugal 10min.Go to the upper strata, take off layer organic phase in drying bottle, 50 ℃ of rotary evaporation to dried or nitrogen dry up.
E, with the dry residue of 0.6ml damping fluid dissolving, add normal hexane 1ml mixings 2min, taking-up 10000rpm, 15 ℃ of centrifugal 5min.
F, gently sop up upper organic phase and middle white impurity, take off layer 50 μ l and 50 μ l and dilute good redissolution liquid mixing, get 50 μ l and can analyze.
2. detect with kit
(1) in afloqualone class medicine haptens and oralbumin conjugate coated elisa plate micropore, adds series standard solution or sample solution 50 μ l, add afloqualone monoclonal antibody working fluid 50 μ l then,, react 30min in 37 ℃ of constant temperature ovens with cover plate film shrouding.
(2) pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pours out liquid in the hole behind the 30s, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.The sheep anti mouse antiantibody working fluid 100 μ l that add horseradish peroxidase-labeled with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens.
(3) take out ELISA Plate, wash plate as described above 5 times.Every hole adds substrate colour developing liquid A liquid 50 μ l, adds B liquid 50 μ l again, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min.
(4) every hole adds stop buffer 50 μ l, and the mixing that vibrates is gently measured every hole absorbance (OD value) with microplate reader.
Interpretation of result: calculate percentage absorbance and drawing standard curve, the concentration of Ciprofloxacin can be read from typical curve in corresponding each sample.Also can use regression equation method, calculate the concentration of Ciprofloxacin in the sample.Utilize computer professional software, the express-analysis of a large amount of samples of being more convenient for.
The depth according to the color sample on the ELISA Plate, but, utilize cross reacting rate to calculate the concentration that contains other afloqualone class medicament residue in the sample according to the residue of ciprofloxacin amount again with the concentration range of Ciprofloxacin in the comparison judgement sample of the standard solution color of series concentration.
The test of experimental example 1 kit precision
The standard items repeatability:
From three batches of kits, respectively get 10 kits, extract 20 micropores in the every elisa plate out, measure the absorbance (OD value) of 9 μ g/L standard solution, calculate the coefficient of variation.Measurement result sees Table 1.
The repeatable test of table 1 standard items
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | ||
| CV％ | 01 batch | 5.6 | 6.7 | 4.5 | 6.7 | 7.5 | 5.2 | 7.3 | 6.2 | 5.5 | 6.1 |
| 03 batch | 8.2 | 8.1 | 6.6 | 7.2 | 5.5 | 7.2 | 6.7 | 7.8 | 6.2 | 5.9 | |
| 06 batch | 7.7 | 7.1 | 7.9 | 8.3 | 6.3 | 6.6 | 9.5 | 5.9 | 7.1 | 8.2 | |
Measurement range determines that the coefficient of variation scope of precision is≤10% between 5.2～9.5% as a result.
The sample repeatability:
Get the Ciprofloxacin standard specimen, add in the sample, get each three of the kits of three different batches respectively, each concentration repeats 5 times, calculates the coefficient of variation respectively, the results are shown in Table 2～5.
The repeatable test of table 2 chicken meat sample
| Lot number | Measured value (μ g/kg) | Coefficient of variation CV% | ||||
| 0506012 | 10.5 | 11.2 | 14.5 | 12.8 | 13.5 | 13.1 |
| 13.5 | 11.9 | 15.6 | 14.2 | 13.9 | 9.6 | |
| 12.7 | 12.6 | 13.4 | 12.9 | 13.6 | 3.4 | |
| 0507004 | 14.6 | 12.8 | 12.0 | 13.6 | 11.2 | 10.4 |
| 13.4 | 12.9 | 14.7 | 13.6 | 12.8 | 5.6 | |
| 13.4 | 12.6 | 13.8 | 12.6 | 14.6 | 6.3 | |
| 0507010 | 12.6 | 13.5 | 14.2 | 13.8 | 12.6 | 5.4 |
| 12.8 | 13.6 | 12.3 | 13.2 | 11.5 | 6.4 | |
| 13.7 | 12.8 | 14.8 | 13.4 | 12.2 | 7.3 | |
The repeatable test of table 3 chicken gizzard sample
| Lot number | Measured value (μ g/kg) | Coefficient of variation CV% | ||||
| 0506012 | 12.6 | 14.5 | 13.5 | 13.8 | 12.9 | 5.6 |
| 12.6 | 13.9 | 10.7 | 14.6 | 11.4 | 13.0 | |
| 13.6 | 12.4 | 14.6 | 12.3 | 12.7 | 7.4 | |
| 0507004 | 13.4 | 12.6 | 13.8 | 14.0 | 12.9 | 4.4 |
| 13.7 | 14.2 | 13.8 | 12.6 | 13.5 | 4.4 | |
| 13.8 | 15.4 | 12.9 | 13.7 | 12.4 | 8.4 | |
| 0507010 | 13.4 | 14.6 | 12.8 | 13.2 | 15.0 | 6.9 |
| 13.5 | 14.5 | 12.7 | 13.8 | 11.8 | 7.8 | |
| 13.4 | 14.6 | 12.6 | 13.8 | 12.7 | 6.2 | |
The repeatable test of sample in table 4 shrimp
| Lot number | Measured value (μ g/kg) | Coefficient of variation CV% | ||||
| 0506012 | 11.6 | 13.5 | 14.2 | 12.8 | 13.5 | 7.5 |
| 12.6 | 12.0 | 13.4 | 13.8 | 14.3 | 7.0 | |
| 13.6 | 12.7 | 13.8 | 12.9 | 14.3 | 4.9 | |
| 0507004 | 14.2 | 13.6 | 12.8 | 11.3 | 13.6 | 8.6 |
| 11.4 | 13.9 | 14.3 | 13.5 | 12.7 | 8.7 | |
| 13.5 | 14.0 | 12.6 | 12.7 | 13.8 | 4.8 | |
| 0507010 | 13.4 | 12.6 | 13.8 | 10.8 | 14.4 | 12.4 |
| 13.8 | 14.4 | 11.5 | 12.7 | 13.2 | 8.4 | |
| 13.4 | 14.7 | 12.9 | 11.0 | 13.9 | 10.5 | |
The repeatable test of table 5 plasma sample
| Lot number | Measured value (μ g/kg) | Coefficient of variation CV% | ||||
| 0506012 | 13.5 | 12.0 | 11.3 | 13.0 | 14.7 | 10.2 |
| 13.5 | 12.4 | 12.9 | 10.3 | 14.8 | 12.6 | |
| 13.4 | 12.8 | 13.6 | 13.5 | 14.0 | 3.2 | |
| 0507004 | 13.7 | 12.6 | 14.5 | 13.8 | 12.9 | 5.6 |
| 14.6 | 12.3 | 11.2 | 13.2 | 13.8 | 10.1 | |
| 14.6 | 12.6 | 12.8 | 14.7 | 13.2 | 7.3 | |
| 0507010 | 13.7 | 13.4 | 14.6 | 13.5 | 14.9 | 4.8 |
| 12.4 | 12.8 | 14.8 | 13.2 | 13.9 | 7.0 | |
| 13.7 | 12.9 | 13.4 | 12.6 | 13.8 | 3.8 | |
The result shows the Variation Lines number average of chicken meat sample less than 15%, and the Variation Lines number average of chicken gizzard sample is less than 15%, and the Variation Lines number average of shrimp sample is less than 15%, and the Variation Lines number average of plasma sample is less than 15%.
The accuracy test of experimental example 2 kits
Get the Ciprofloxacin standard specimen of two concentration, sample added recovery test, each concentration do 4 parallel, calculate recovery rate respectively.
Table 6 accuracy test
| Sample | Chicken | Chicken gizzard | |||
| Add concentration (μ g/kg) | 10 | 50 | 10 | 50 | |
| Recovery % | 1 | 86.4 | 112.3 | 87.4 | 82.4 |
| 2 | 92.5 | 84.2 | 95.2 | 89.5 | |
| 3 | 102.7 | 82.4 | 79.5 | 93.4 | |
| 4 | 89.4 | 97.2 | 102.3 | 112.7 | |
| Mean value | 92.7 | 94.0 | 91.1 | 94.5 | |
| Sample | Shrimp | Blood plasma | |||
| Add concentration (μ g/kg) | 10 | 50 | 10 | 50 | |
| Recovery % | 1 | 82.1 | 100.9 | 97.3 | 114.2 |
| 2 | 79.5 | 86.7 | 120.7 | 87.4 | |
| 3 | 69.5 | 79.5 | 87.4 | 76.5 | |
| 4 | 96.5 | 93.2 | 102.7 | 94.5 | |
| Mean value | 81.9 | 90.0 | 102.0 | 93.1 | |
The result shows that the interpolation recovery of chicken, chicken gizzard sample is 79.5～112.7%; The shrimp sample adds the recovery 69.5～100.9%; The interpolation recovery of plasma sample is 87.4～114.2%.
The specificity test of experimental example 3 kits
Specificity represents that with cross reacting rate cross reacting rate is meant the ability of the antigenic determinant generation combination that antibody is different with structure.
Select and the five kind medicines of Ciprofloxacin with similar structure and similar functions, with the Ciprofloxacin analog of variable concentrations, the accurate solution of the alternate collar third husky asterisk is measured its typical curve, and calculates IC
50Inhibition concentration is calculated cross reacting rate.
Table 7 cross reaction
| Medicine name | Cross reacting rate (%) |
| Enrofloxacin Ciprofloxacin Norfloxacin Ofloxacin Lomefloxacin | 83.3 100 166.7 125 76.9 |
In learning sample, calculate the residual quantity of other afloqualone class medicine under the situation of residue of ciprofloxacin amount according to top cross reacting rate.
The test of experimental example 4 kit storage lives
The kit preservation condition is 2～8 ℃, and through 6 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, afloqualone class medicine added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed 6 days under the condition of 25 ℃ of preservations, carries out the accelerated deterioration experiment, and the result shows that the every index of this kit meets the requirements fully.
Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 6 months at 2～8 ℃.
Claims (8)
1, a kind of enzyme linked immunoassay kit that is used to detect afloqualone class medicine is characterized in that it comprises following component:
(1) bag is by the ELISA Plate of afloqualone class medicine antigen;
(2) enzyme mark antiantibody;
(3) Ciprofloxacin standard solution;
(4) substrate colour developing liquid;
(5) afloqualone class drug antibody;
(6) concentrated cleaning solution;
(7) stop buffer;
(8) concentrate redissolution liquid.
2, kit as claimed in claim 1, it is characterized in that afloqualone class medicine antigen is with the synthetic afloqualone haptens of afloqualone class pharmaceutical intermediate Ciprofloxacin and succinic anhydride, adopt mixed anhydride method afloqualone class medicine haptens and oralbumin, bovine serum albumin(BSA), human serum albumins or thyroprotein to be carried out coupling obtains again.
3, kit as claimed in claim 1, it is characterized in that enzyme mark antiantibody is the sheep anti mouse antiantibody that horseradish peroxidase or bacterium are extracted the alkaline phosphate ester enzyme labeling, described antiantibody be with sheep as immune animal, be that immunogene is carried out immunity to the pathogen-free domestic sheep and obtained with mouse source antibody.
4, kit as claimed in claim 1 is characterized in that concentrated cleaning solution is the phosphate buffer that contains 0.8～1.2% polysorbas20; The liquid that concentrate to redissolve is 0.01～0.05mol/L, contain the phosphate buffer of 1% gelatin; When marker enzyme was horseradish peroxidase, substrate colour developing liquid A liquid was that hydrogen peroxide or urea peroxide, substrate colour developing liquid B liquid are that o-phenylenediamine or tetramethyl benzidine, stop buffer are sulfuric acid or the hydrochloride buffer of 1～2mol/L; When marker enzyme is a bacterium when extracting alkaline phosphatase, substrate colour developing liquid is for being the NaOH of 2mol/L to nitro phosphate buffer, stop buffer.
5, kit as claimed in claim 1, the protein concentration that it is characterized in that afloqualone class drug antibody are 0.5～5.0 μ g/L.
6, kit as claimed in claim 1 is characterized in that the concentration of Ciprofloxacin standard solution is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L and 81 μ g/L.
7, the method for afloqualone class medicament residue in a kind of test sample comprises step:
(1) sample pre-treatments;
(2) the arbitrary described kit with claim 1～6 detects;
(3) analyzing and testing result.
8, method as claimed in claim 7, wherein kit detects to add standard solution or sample solution in the ELISA Plate micropore, add afloqualone class drug antibody again, washing pats dry behind the incubation, add the enzyme labeling antiantibody, washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader.
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| CN101013129B (en) * | 2007-02-13 | 2011-08-17 | 北京望尔生物技术有限公司 | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof |
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| CN101299045B (en) * | 2008-06-12 | 2013-05-08 | 中国农业大学 | Method for detecting florfenicol and florfenicol amine and special-purpose enzyme-linked immunologic reagent kit thereof |
| CN103323594A (en) * | 2012-03-22 | 2013-09-25 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application |
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| CN101013129B (en) * | 2007-02-13 | 2011-08-17 | 北京望尔生物技术有限公司 | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof |
| CN101299045B (en) * | 2008-06-12 | 2013-05-08 | 中国农业大学 | Method for detecting florfenicol and florfenicol amine and special-purpose enzyme-linked immunologic reagent kit thereof |
| CN101358965B (en) * | 2008-08-22 | 2012-09-05 | 北京望尔生物技术有限公司 | Method for detecting norethindrone and special ELISA kit thereof |
| CN102565401A (en) * | 2010-12-15 | 2012-07-11 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescent kit for testing quinolones and application of the kit |
| CN103323594A (en) * | 2012-03-22 | 2013-09-25 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application |
| CN103323594B (en) * | 2012-03-22 | 2016-03-30 | 北京勤邦生物技术有限公司 | A kind of enzyme linked immunological kit and application thereof detecting QNS in aquatic products |
| CN109633147A (en) * | 2018-12-07 | 2019-04-16 | 杭州康力食品有限公司 | The detection method of fluoquinolone constituents in a kind of fresh royal jelly |
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