CN101526528A - Furaltadone metabolite detection kit - Google Patents
Furaltadone metabolite detection kit Download PDFInfo
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- CN101526528A CN101526528A CN200910045162A CN200910045162A CN101526528A CN 101526528 A CN101526528 A CN 101526528A CN 200910045162 A CN200910045162 A CN 200910045162A CN 200910045162 A CN200910045162 A CN 200910045162A CN 101526528 A CN101526528 A CN 101526528A
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Abstract
The invention relates to a furaltadone metabolite detection kit and provides an elisa kit for detecting furaltadone metabolite, comprising an enzyme yoke plate with an encrusting furaltadone metabolite derivate antigen, an enzyme yoke plate with an enzyme-labeling furaltadone metabolite derivate specific antibody or an encrusting furaltadone metabolite derivate specific antibody, and an enzyme-labeling furaltadone metabolite derivate antigen, wherein the furaltadone metabolite derivate antigen is obtained by coupling a furaltadone metabolite derivate hapten and carrier protein by an active ester method. The kit is convenient to use and has favorable performance in aspects, such as accuracy, sensitivity, specificity, and the like.
Description
Technical field
The present invention relates to Furaltadone metabolite detection kit.
Background technology
The itrofurans medicine is the broad-spectrum antibiotic of synthetic, and it has extraordinary antibacterial action and the dynamic (dynamical) characteristic of medicine, once is widely used as domestic animal, poultry, propagates the growth-promoting additive of aquatic products artificially.But find by long-term laboratory study, itrofurans medicine and metabolin all can make animal used as test that canceration and gene mutation take place, and infer thus, human long-term use such medicine or long-term edible domestic animal, poultry of adding such growth accelerator, propagate aquatic products artificially, equally also canceration and gene mutation can take place.So this type of drug withdrawal uses in treatment and feed.
China has issued in 2002 and has banned use of the antibiotic ban of itrofurans, in " food animal forbidding veterinary drug and other compound inventories " of No. 193 bulletins of Ministry of Agriculture issue, the use that in all food animals and all purposes, all is under an embargo of itrofurans microbiotic.
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 performance limit value (Minimum Required Performance Limits, MRPL) be 1 μ g/kg, the content of European Union's metabolin of itrofurans medicine from the animal product of third country import must not surpass 1 μ g/kg.U.S. FDA had been announced 11 kinds of list of substance forbidding using in the import animal derived food in 2004, comprising furaltadone.Therefore, 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.
Because furaltadone in vivo soon can be by metabolism, the metabolic product of combination (AMOZ) then can retain long period of time in tissue, so the product often will analyze its metabolism when analyzing this type of medicine residual after, detecting supervision department is the purpose that means reach the detection residue of furaltadone through to detect metabolic product just.
Now developed the enzyme linked immunological kit that detects furaltadone both at home and abroad, still present domestic most of demand is from external import, and the kit of domestic production can't be better than the import reagent box at aspects such as accuracy, sensitivity, specificitys.
Summary of the invention
In order to overcome the above-mentioned defective that exists in the prior art.
The invention provides the enzyme linked immunological kit of a kind of detection AMOZ (AMOZ), include: bag is by the elisa plate of AMOZ derivatives antigens and enzyme labeling AMOZ derivant specific antibody or wrap by the elisa plate of AMOZ derivant specific antibody and enzyme labeling AMOZ derivatives antigens; Described AMOZ derivatives antigens adopts active ester method that AMOZ derivative hapten and carrier protein are carried out coupling and obtains.
Above-mentioned carrier protein is bovine serum albumin(BSA), chicken egg white, rabbit anteserum albumen, human serum albumins, human fibrin or hemocyanin.
Above-mentioned elisa plate is to be carrier with polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber or Ago-Gel, be cushioned liquid with pH value 9.6,0.05mol/L, the carbonate buffer solution that contains 0.5%v/v methyl alcohol as bag, contain that the solution of the skimmed milk of 8%-15%w/v and 1%w/v inert protein is prepared from as confining liquid.
The preparation method of above-mentioned elisa plate is as follows:
(1) is cushioned liquid with bag the AMOZ derivative hapten is become antigenic dilution or antibody diluent with carrier protein couplet thing or antiantibody with 0.02-0.08 μ g/ml concentration dilution;
(2) in every hole of elisa plate, add 100 μ l and diluted good antigenic dilution or antiantibody dilution, 37 ℃ of incubation 2h, the coating buffer that inclines, with cleansing solution washing 4 times, each 15-30s removes moisture;
(3) in every hole of elisa plate, add 150-200 μ l confining liquid, 37 ℃ of incubation 1-2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
As optimization, above-mentioned enzyme is peroxidase or the sweet enzyme of galactose, is preferably peroxidase.
Above-mentioned enzyme labeling AMOZ derivant specific antibody is to adopt glutaraldehyde method to carry out coupling to make.
Above-mentioned enzyme-labelled antigen is to adopt active ester method that marker enzyme and AMOZ haptens are carried out coupling to obtain.
Above-mentioned AMOZ derivant specific antibody is mouse resource monoclonal antibody or rabbit source polyclonal antibody.
As optimization, the mentioned reagent box also comprises AMOZ derivant standard solution, substrate colour developing liquid, stop buffer, concentrated cleaning solution and concentrates redissolution liquid.More preferably substrate colour developing liquid is single substrate solution.
The kit of producing in the kit provided by the invention is easy to use, and all functional at aspects such as accuracy, sensitivity, specificitys.
Description of drawings
Fig. 1 is a furaltadone examination criteria curve map.
Embodiment
Detection principle of the present invention: the present invention adopts the ELISA direct competition method, be that AMOZ derivative hapten and bovine gamma globulin(BGG) conjugate (AMOZ-BGG) are adsorbed on the solid phase carrier when coating antigen is AMOZ derivant coupled antigen, add sample or AMOZ derivant standard items, add enzyme labeling AMOZ specific antibody then, the AMOZ derivatives antigens competition enzyme labeling AMOZ derivant specific antibody of bag quilt on residual AMOZ (after the pre-treatment derivatization) and the ELISA Plate in the testing sample.Colour developing back stops, the absorbance of working sample, and the AMOZ residual quantity is negative correlation in this value and the sample, relatively can draw the concentration of AMOZ with typical curve.
Coating protein is that specific antibody is adsorbed on the solid phase carrier when being AMOZ derivant specific antibody, add enzyme labeling AMOZ derivatives antigens, add sample or AMOZ derivant standard solution again, AMOZ (after the pre-treatment derivatization) and enzyme labeling AMOZ derivatives antigens residual in the sample to be tested are competed AMOZ derivant specific antibody, the colour developing back stops, the absorbance of working sample, the AMOZ residual quantity is negative correlation in this value and the sample, relatively can draw the concentration of AMOZ with typical curve, but with the standard solution color concentration range of AMOZ in the judgement sample more then.
The invention provides a kind of enzyme linked immunological kit that detects AMOZ in the animal derived food, it contains:
(1) bag by the elisa plate of AMOZ derivatives antigens or the bag by the elisa plate of AMOZ derivant specific antibody;
(2) enzyme labeling AMOZ derivant specific antibody or enzyme labeling AMOZ derivatives antigens;
(3) AMOZ derivant standard solution;
(4) substrate colour developing liquid;
(5) stop buffer;
(6) concentrated cleaning solution;
(7) concentrate redissolution liquid;
AMOZ derivant envelope antigen adopts active ester method that AMOZ derivative hapten and carrier protein are carried out coupling to obtain in the kit of the present invention, and this carrier protein can be macromolecular carriers such as bovine serum albumin(BSA), chicken egg white, rabbit anteserum albumen, human serum albumins, human fibrin, hemocyanin.To be cushioned liquid be pH value 9.6,0.05mol/L to used bag, the carbonate buffer solution that contains 0.5% methyl alcohol in the preparation elisa plate process; Bag be can be polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc. by the carrier mass of AMOZ derivatives antigens or antibody; The form of carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.; Used cleansing solution is deionized water or ultrapure water; Used confining liquid is to contain the skimmed milk of 8%-15%w/v and the phosphate solution of 1%w/v inert protein.
Bag by the preparation process of the elisa plate of the elisa plate of AMOZ derivatives antigens or coated antibody is in the kit of the present invention:
(1) is cushioned liquid with bag the AMOZ derivative hapten is become antigenic dilution or antibody diluent with carrier protein couplet thing or antibody with 0.02-0.08 μ g/ml concentration dilution;
(2) in every hole of elisa plate, add 100 μ l and diluted good antigenic dilution or antibody diluent, 37 ℃ of incubation 2h, the coating buffer that inclines, with cleansing solution washing 4 times, each 15-30s pats dry;
(3) in every hole of elisa plate, add 150-200 μ l confining liquid, 37 ℃ of incubation 1-2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
The elisa plate of above method preparation has good stability, and through cold and hot stability test, the correlation technique parameter of elisa plate is all in normal range, and coating antigen has good specificity.
The enzyme labeling thing is enzyme labeling AMOZ derivant specific antibody or enzyme labeling AMOZ derivatives antigens in the kit of the present invention, and used enzyme can be peroxidase or the sweet enzyme of galactose, the preferred peroxidase of the present invention; Enzyme labeling thing form can be freeze-dried powder, concentrate and working fluid; The used dilution of enzyme labeling thing working fluid is for containing the solution of 50% glycerine (can prevent that the enzyme labeling thing of putting into-20 ℃ of environment from freezing, also can keep the biologically active of enzyme labeling thing for a long time), 1% sodium azide antiseptic (being convenient to preserve).
The preparation process of enzyme labeling AMOZ derivant specific antibody is in the kit of the present invention:
(1) preparation of peroxidase labelling AMOZ derivant specific antibody: antibody and peroxidase (HRP) are carried out coupling, the method that adopts is a glutaraldehyde method, adopt glutaraldehyde method to make the combination rate of antibody and horseradish peroxidase raise, tradition GA single stage method coupling reaction is wayward, spontaneous polymerization easily takes place in the fast molecule of reaction velocity, and coupling efficiency is not high.In order to address these problems, we improve single stage method, have overcome the shortcoming of single stage method.At first in by two kinds of molecules of coupling, the molecule more weak with the coupling agent reflection activates with excessive coupling agent earlier, then the unnecessary coupling agent in place to go; Second step was connected an end with certain molecule coupling agent couples together with another kind of molecule by changing reaction conditions.In the glutaraldehyde two step method, enzyme itself does not produce condensation, because a free amine group on the second step enzyme molecule only with glutaraldehyde on an active aldehyde radical connect, another active aldehyde radical then can combine the generation enzyme conjugates with second amino that goes on foot on the antibody globulin molecule that adds on the glutaraldehyde, therefore, the two-step approach advantage is the enzyme conjugates that can generate homogeneous, can make a molecule enzyme and the effect of a molecular ball albumen mostly, the loss of activity of antibody and enzyme is less, and gained enzyme conjugates specific activity single stage method is high about 10 times.Though the two step method operation is more numerous, coupling efficiency improves, and the same Molecularly Imprinted Polymer that forms reduces.
Enzyme-labelled antigen is to adopt glutaraldehyde method that marker enzyme and AMOZ haptens are carried out coupling to obtain in the kit of the present invention.
AMOZ derivant specific antibody is mouse resource monoclonal antibody or rabbit source polyclonal antibody in the kit of the present invention, and immunogene adopts active ester method that AMOZ derivative hapten and key hole maple hemocyanin are carried out coupling and obtains; Antibody formation can be freeze-dried powder, concentrate, working fluid; Antibody diluent is pH value 7.4,0.08mol/L, contain the phosphate buffer of 0.3% gelatin, 5 ‰ Tween-80s and 5 ‰ methyl alcohol.
AMOZ derivant specific antibody can be monoclonal antibody or polyclonal antibody in the kit of the present invention, and its preparation method is as follows:
(1) step of AMOZ derivant Monoclonal Antibody is:
A. animal immune program: the 8-12 Balb/c mouse in age in week of adopting pure lines is as immune animal, immunogene (conjugate of AMOZ derivative hapten and key hole maple hemocyanin) immunizing dose is 100-300 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, At intervals of two to three weeks is got the same dose immunogene and is added equivalent incomplete Freund mixing and emulsifying, booster immunization once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days;
B. Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, in 5-10: 1 ratio and SP2/0 myeloma cell are merged, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole, 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;
C. 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, takes out frozen pipe during recovery, puts into 37 ℃ of water-bath middling speeds immediately and melts, and behind the centrifugal removal cryopreserving liquid, moves in the culture flask and cultivates;
D. 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 with sad-saturated ammonium sulfate method, bottle packing ,-20 ℃ of preservations;
E. the antibody freeze-dried powder can be dried ascites under 37 ℃ of environment, puts into-20 ℃ of preservations;
F. the antibody working fluid is with antibody diluent antibody to be diluted with 0.02-0.08 μ g/ml concentration.
(2) step of AMOZ derivant polyclonal antibody preparation is:
Adopt new zealand white rabbit as immune animal, immunogene (conjugate of AMOZ derivative hapten and key hole maple hemocyanin) immunizing dose is 1mg/kg, 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 3-4 week adds equivalent incomplete Freund mixing and emulsifying at interval, booster immunization once immune 5 times altogether, does not add adjuvant for the last time.Take a blood sample after last immune 7-10 days, measure serum antibody titer, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
In the kit of the present invention when marker enzyme is peroxidase substrate colour developing liquid be that hydrogen peroxide or urea peroxide and tetramethyl benzidine sulfate mixed solution, stop buffer are sulfuric acid or the hydrochloride buffer of 0.1-0.5mol/L; When marker enzyme was the sweet enzyme of galactose, substrate colour developing liquid was the 0.5mol/L kaliumphosphate buffer, and stop buffer is the citrate buffer solution of 2mol/L; Concentrated cleaning solution is deionized water or ultrapure water; Concentrating redissolution liquid is the phosphate buffer that contains 1% bovine serum albumin(BSA).
AMOZ derivant standard solution is the AMOZ derivative solution of six concentration gradients in the kit of the present invention, and AMOZ derivant dilution is the phosphate buffer that contains 5 ‰ methyl alcohol.
The preparation of reagent is specially in the kit of the present invention:
A. AMOZ derivant standard solution: 6 bottles of AMOZ derivant series standard solution, concentration are 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35 μ g/L, 4.05 μ g/L, the 1-3ml/ bottle.
B. bag is cushioned liquid: pH value 9.6,0.05mol/L, contain the carbonate buffer solution of 0.5% methyl alcohol.
C. confining liquid: contain the skimmed milk of 8%-15% and the solution of 1% inert protein.
D. cleansing solution: deionized water or ultrapure water, 30-50ml/ bottle, 1 bottle.
E. enzyme labeling thing: enzyme labeling antiantibody working fluid or enzyme labeling AMOZ derivatives antigens working fluid, 7-12ml/ bottle, 1 bottle.
F. substrate colour developing liquid hydrogen peroxide or urea peroxide and tetramethyl benzidine sulfate mixed solution, 10-15ml/ bottle, 1 bottle.
G. substrate colour developing liquid to nitro phosphate buffer: pH8.1, contain MgCl
20.01% 100mmolTris-HCl;
H. stop buffer: 1-2mol/L sulfuric acid, hydrochloric acid or 2mol/L sodium hydrate buffer solution, 5-8ml/ bottle, 1 bottle.
I. antibody diluent: pH value 7.4,0.08mol/L, contain the phosphate buffer of 0.3% gelatin, 5 ‰ Tween-80s and 5 ‰ methyl alcohol.
J. concentrate to redissolve liquid: contain the phosphate buffer of 5% methyl alcohol, 1% calf serum (BSA), for the 5-10 of normal working concentration doubly, 30-50ml/ bottle, 1 bottle.
K. derivatization reagent solution: 100% methyl alcohol or 100%N, N '-dimethyl formamide (DMF)
The method of AMOZ in the detection animal derived food of the present invention has comprised following steps:
(1) sample pre-treatments;
(2) detect with kit of the present invention;
(3) analyzing and testing result.
(1) sample-pretreating method is among the present invention:
The sample pre-treatment need be prepared:
With deionized water with 2 * concentrate and redissolve liquid by dilution in 1: 1, be used for the redissolution after the sample extraction.
Derivatization reagent: claim the 15.1mg o-nitrobenzaldehyde fixed molten to 10ml with derivatization reagent solution.
C liquid: (using for the milk sample) claims the 12.5g potassium nitroferrocyanide fixed molten to 100ml with deionized water.
D liquid: (using for the milk sample) claims 29.8g zinc sulfate fixed molten to 100ml with deionized water dissolving.
0.1M K
2HPO
4Claim 22.8g K
2HPO
43H
2It is fixed molten to 1L that O adds deionized water dissolving
1M HCl gets the dense HCl of 8.6ml, and to add water fixed molten to 100ml.
1M NaOH takes by weighing 4g NaOH, and to add water fixed molten to 100ml.
A. meat sample or fishes and shrimps
The homogeneous sample (meat sample/fishes and shrimps) of getting 1 ± 0.05g adds the deionized water of 4ml, 0.5ml1M HCl and 100 μ l derivatization reagents, fully vibration; Place 72 ℃ of night incubation (approximately 2-4h); Add 5ml 0.1M K respectively
2HPO
4, the ethyl acetate of 0.4ml 1M NaOH and 5ml, 30s fully vibrates; The above centrifugal 10min of (20-25 ℃) 4000r/min at room temperature; The ethyl acetate that takes out 2.5ml in another clean container in 50 ℃ of nitrogen/air blow drying; With the dry thing of 1ml n-hexane dissolution, fully mix with the good redissolution liquid of oneself dilution of 1ml; The above centrifugal 10min of (20-25 ℃) 4000r/min at room temperature; Getting 50 μ l lower floors is used for analyzing mutually.
Sample extension rate: 2
The advantage of this law has been to improve the temperature of derivatization, and the reaction time is shortened greatly.
B. the sample of suckling
Get 2ml milk, it is put the centrifugal 10min of 5000rpm, remove upper strata fat, take off layer 50 μ l, the redissolution liquid that kit provided is by 1: 40 times of dilution.
Sample extension rate: 40
C. honey
Get 1 ± 0.05g sample in centrifuge tube; Add the deionized water dissolving of 4ml, add 0.5ml 1M HCl and 100 μ l derivatization reagents again, fully vibration; Place 72 ℃ of night incubation (approximately 2-4h); Add 5ml 0.1M K respectively
2HPO
4, the ethyl acetate of 0.4ml 1M NaOH and 5ml, 30s fully vibrates; The above centrifugal 10min of (20-25 ℃) 4000r/min at room temperature; The ethyl acetate that takes out 2.5ml in another clean container in 50 ℃ of nitrogen/air blow drying; With the dry thing of 1ml n-hexane dissolution, fully mix with the good redissolution liquid of oneself dilution of 1ml; The above centrifugal 10min of (20-25 ℃) 4000r/min at room temperature.
Sample extension rate: 2
D. eggs
Take by weighing the egg sample 2g for preparing, join in the 50ml centrifuge tube, add 4ml water respectively, 0.5ml 1M HCl, 200 μ l C liquid, vibration mixing; Add 200 μ l D liquid, the 5min that fully vibrates, the above centrifugal 10min of room temperature (20-25 ℃) 3000g; Get whole supernatants, add 200 μ l derivatization reagents, fully vibration, 50 ℃ of water-bath 2h (middle per half an hour thermal agitation 1-2 minute); Add 5ml 0.1M K respectively
2HPO
4, 0.4ml 1M NaOH5ml ethyl acetate, thermal agitation 30s, the above centrifugal 10min of room temperature (20-25 ℃) 4000r/min; Getting the 2.5ml upper organic phase dries up in 50 ℃ of following nitrogen; Add the dry thing of 1ml n-hexane dissolution; Add the redissolution liquid after 2ml dilutes, vibration 10s, the above centrifugal 10min (, please in 60 ℃ of water-baths, heating 5min, again repeated centrifugation) of 4000r/min as emulsion occurring; Remove upper organic phase; Taking off layer water 50 μ l is used for analyzing.
(2) detect with kit of the present invention:
1) from 4 ℃ of cold storage environment, takes out required reagent, put room temperature (20-25 ℃) more than the balance 30min, note to shake up before every kind of liquid reagent uses.
2) take out micropore and the framework that needs quantity, no micropore is put into valve bag, be stored in 2-8 ℃.
3) dosing: 40ml concentrated cleaning solution (20 times concentrate) is diluted to 800ml standby (or amount dilution on demand) with distilled water or deionized water.
4) numbering: the corresponding micropore of sample and standard items is numbered according to the order of sequence, and it is parallel that each sample and standard items are done 2 holes, and the position at record standard hole and sample aperture place.
5) add standard items/sample 50 μ l/ holes in micropore separately, add enzyme labeling AMOZ derivant specific antibody 50 μ l/ holes then, with cover plate film shrouding, light shaking mixing.React 30min in 37 ℃ of environment.
6) take out liquid drying in the hole, wash plate 4-5 time, each 10 seconds at interval, pat dry (bubble that is not eliminated after patting dry can puncture with clean rifle head) with thieving paper with washing lotion 250 μ l/ holes.
7) colour developing: every hole adds substrate solution 100 μ l, the mixing that vibrates gently, lucifuge colour developing 15min in 37 ℃ of environment.
8) measure: every hole adds stop buffer 50 μ l, and the mixing that vibrates is gently set microplate reader in the 450nm place (suggestion detects with dual wavelength 450/630nm, runs through data in 5min), measures every hole OD value.
(3) analyzing and testing result:
The result judges two kinds of methods, judges available the 1st kind of method roughly, quantitatively judges with the 2nd kind of method.Notice that the sample light absorption value becomes negative correlation with the content of SEM.
1) qualitatively judge:
Mean light absorbency value and standard value with sample relatively can draw the contained AMOZ concentration range of sample (ng/ml).The absorbance of assumes samples 1 is 0.268, and the absorbance of sample 2 is 1.230, and the titer absorbance is respectively: 0ppb is 1.671; 0.05ppb be 1.425; 0.15ppb be 1.103; 0.45ppb be 0.567; 1.35ppb be 0.205; 4.05ppb be 0.104.Then the concentration range of sample 1 is 0.45ppb-1.35pb; The concentration range of sample 2 is 0.05ppb-0.15ppb.(multiply by corresponding extension rate again)
2) quantitatively judge:
Each the concentration standard solution that is obtained and the mean value (B) of sample absorbance are divided by the absorbance (B of first standard (0 standard)
0) multiply by 100% again, i.e. percentage absorbance.
The mean light absorbency value of B-standard solution or sample solution
B
0The mean light absorbency value of-0ng/ml standard solution
With standard items percentage absorptance is ordinate, is horizontal ordinate with the semilog of AMOZ standard items concentration (ng/ml), and the drawing standard curve map is seen Fig. 1.In the percentage absorptance substitution typical curve with sample, read the pairing concentration of sample, multiply by its corresponding extension rate and be AMOZ actual concentrations in the sample from typical curve.
Residual enzyme linked immunological kit and the method for AMOZ in the detection animal derived food provided by the invention, sample pretreatment process is simple, and detection time is disconnected, expense is cheap, and can detect gross sample simultaneously.
Embodiment 1 detects the preparation of the enzyme linked immunological kit component of AMOZ
1. antigen is synthetic
A. coating antigen is synthetic
Adopt the o-nitrobenzaldehyde derivative method to synthesize the AMOZ derivative hapten AMOZ, again haptens is carried out coupling by diazo-reaction and bovine gamma globulin(BGG) carrier protein with active ester method and obtain.
B. immunogenic synthetic
Adopt the o-nitrobenzaldehyde derivative method to synthesize the AMOZ derivative hapten AMOZ, again haptens is carried out coupling by diazo-reaction and key hole maple hemocyanin carrier protein with active ester method and obtain.
2. the preparation of AMOZ derivant mouse monoclonal antibody
A. animal immune
Adopt.The Balb/c mouse is as immune animal, with AMOZ derivative hapten and key hole maple hemocyanin conjugate is immunogene, immunizing dose is 300 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.
B. 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 5: 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.
C. 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 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.
D. MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Adopt in the body and induce method, the Balb/c mouse peritoneal injection in 8 ages in week is only sterilized paraffin oil 0.5ml/, 7 days pneumoretroperitoneum injection hybridomas 5 * 10
6Individual/as only, to gather ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method, bottle packing, 20 ℃ of preservations.
3. the preparation of AMOZ derivant rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with AMOZ derivative hapten and key hole maple hemocyanin conjugate is immunogene, immunizing dose is 3mg/kg, 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 3 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once, immunity is 5 times altogether, does not add adjuvant for the last time.Serum antibody titer is measured in last immunity blood sampling after 7 days, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
4. the preparation of ELISA Plate
Be cushioned liquid with bag Furaxone metabolite derivant specificity rabbit antibody dilution is become 0.06 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 6h, the coating buffer that inclines is with cleansing solution washing 4 times, each 1min, pat dry, in every hole, add 150 μ l confining liquids, 37 ℃ of incubation 1h then, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of AMOZ
Set up the enzyme linked immunological kit that detects AMOZ, make it comprise following component:
(1) bag is by the elisa plate of AMOZ derivatives antigens;
(2) the AMOZ derivant specific murine antibody of usefulness horseradish peroxidase-labeled;
(3) the AMOZ standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35g/L, 4.05 μ g/L;
(4) substrate colour developing liquid hydrogen peroxide and tetramethyl benzidine sulfate mixed solution;
(5) stop buffer is the phosphate buffer of 2mol/L;
(6) concentrated cleaning solution is deionized water or ultrapure water;
(7) antibody diluent is pH value 7.4,0.08mol/L, contains the phosphate buffer of 0.3% gelatin, 5 ‰ Tween-80s and 5 ‰ methyl alcohol.
(8) concentrate the phosphate buffer that redissolution liquid contains 5% methyl alcohol, 1% calf serum (BSA);
Embodiment 3 detect AMOZs enzyme linked immunological kit establishment
Set up the enzyme linked immunological kit that detects AMOZ, make it comprise following component:
(1) bag is by the elisa plate of AMOZ derivant specificity rabbit antibody;
(2) the AMOZ derivatives antigens of usefulness alkaline phosphate ester enzyme labeling;
(3) AMOZ derivant standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35g/L, 4.05 μ g/L;
(4) substrate colour developing liquid is to the nitro phosphate buffer;
(5) stop buffer is the sodium hydrate buffer solution of 2mol/L;
(6) concentrated cleaning solution is PH 7.4, contains 0.8% polysorbas20 and 0.1% sodium azide (NaN
3) phosphate buffer of antiseptic;
(7) concentrate redissolution liquid for containing the phosphate buffer of 50% methyl alcohol, 1% calf serum (BSA).
The residual detection of AMOZ in embodiment 4 samples
1. sample pre-treatments
Get the equal pledge of 1g fishes and shrimps, add the distilled water of 4ml successively, the 2-nitrobenzaldehyde of the HCl of 0.5ml1M and 100 μ l 10mM, fully vibration.Hatched 2 hours for 72 ℃, add 5ml 0.1M K respectively
2HPO
4, the ethyl acetate of 0.4ml 1M NaOH and 5ml, 30 seconds of thermal agitation, at room temperature (20-25 ℃) was centrifugal, and rotating speed is 3000rpm.Take out the ethyl acetate layer evaporate to dryness of 2.5ml, with the dry thing of the n-hexane dissolution of 1ml, with the suitable mixing of redissolution liquid of 1ml.Centrifugal in room temperature (20-25 ℃), 3000rpm.Lower floor's liquid with 50 μ l is analyzed.
2. detect with kit
In 96 hole ELISA Plate micropores of AMOZ derivant coupled antigen bag quilt, add series standard product or sample solution (each 2 hole) 50 μ l, add enzyme labeling AMOZ derivant antibody working fluid 50 μ l again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens.Pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pours out liquid in the hole after 30 seconds, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Add substrate colour developing liquid 100 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min.Every hole adds stop buffer 2mol/L sulfuric acid 50 μ l, and the mixing that vibrates is gently measured every hole absorbance with microplate reader.
3. testing result analysis
With the absorbance mean value (B) of the standard solution of each concentration that is obtained, the absorbance (Bo) divided by first standard solution (0 standard) multiply by 100% again, obtains the percentage absorbance.Semilog with AMOZ concentration is the x axle, and the percentage absorbance is a Y-axis, the drawing standard curve map.Percentage absorbance with same way calculation sample solution, in the percentage absorbance substitution typical curve with sample solution, read the pairing concentration of AMOZ the sample solution from typical curve, multiply by the actual concentrations that its corresponding extension rate is AMOZ in the sample solution.
The residual detection of AMOZ in embodiment 5 samples
1. sample pre-treatments
Get 2ml milk, it is put the centrifugal 10min of 5000rpm, remove upper strata fat, take off layer 50 μ l, the redissolution liquid that kit provided is by 1: 40 times of dilution.
2. detect with kit
In 96 hole ELISA Plate micropores of AMOZ derivant specificity rabbit antibody sandwich, add the AMOZ derivatives antigens 50 μ l that bacterium is extracted the alkaline phosphate ester enzyme labeling, add series standard product or sample solution 50 μ l again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens, the repeated washing process.Adding is to nitro phosphate buffer 1 00 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuges colour developing 15min.Every hole adds stop buffer 2mol/L.NaOH 50 μ l, the mixing that vibrates is gently measured every hole absorbance with microplate reader.
3. testing result analysis
Multiply by 100% with the absorbance mean value (B) of the standard solution of each concentration that is obtained again divided by the absorbance (Bo) of first standard solution (0 standard), obtain the percentage absorbance.Semilog with AMOZ derivatives concentration (μ g/L) is the x axle, and the percentage absorbance is a Y-axis, the drawing standard curve map.Percentage absorbance with same way calculation sample solution, in the percentage absorbance substitution typical curve with sample solution, read the pairing concentration of sample solution from typical curve, multiply by its corresponding extension rate and be AMOZ actual concentrations in the sample solution.
Experimental example 1: standard items precision test
From every batch of elisa plate according to the preparation of the method the embodiment 1 (4), each extracts 10 micropores out, measures 0.45 μ g/L.The absorbance of standard solution (OD value) repeats 3 times, calculates coefficient of variation CV%, the results are shown in Table 1.
The repeatable test of table 1 standard (CV%)
The result shows coefficient of variation scope between 6.1%-12.8%, has met the coefficient of variation less than 20% regulation, illustrates that this kit standard items precision has reached standard.
Experimental example 2: the repeatable test of sample
With 1 μ g/L, the AMOZ of concentration adds in the sample fishes and shrimps, muscle and milk, gets each five of the kits of three different batches respectively, and each concentration repeats 5 times, calculates the coefficient of variation respectively, the results are shown in Table 2, table 3.
Table 2 fishes and shrimps, the repeatable test of muscle samples
But table 3 milk sample repeated experiments
The result shows that fishes and shrimps, muscle sample coefficient of variation all are lower than 20%, the Variation Lines number average of milk sample is lower than 20%, has met the coefficient of variation less than 25% regulation, illustrates that the precision of this kit measurement sample has reached standard.
Experimental example 3: the accuracy test of kit
Get the furaltadone metabolism standard solution of two concentration, be respectively 0.3 μ g/kg (L) and 1 μ g/kg (L), respectively sample is added recovery test, each concentration do 4 parallel, calculate recovery rate respectively.
The accuracy of table 4 kit
The result shows the recovery of fishes and shrimps, muscle interpolation between 82%-95%, and milk adds the recovery between 75%-93%.
Experimental example 4
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, AMOZ 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 37 ℃ 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 (10)
1. Furaltadone metabolite detection kit includes:
Bag by the elisa plate of AMOZ derivatives antigens and enzyme labeling AMOZ derivant specific antibody or bag by the elisa plate of AMOZ derivant specific antibody and enzyme labeling AMOZ derivatives antigens;
Described AMOZ derivatives antigens adopts active ester method that AMOZ derivative hapten and carrier protein are carried out coupling and obtains.
2. Furaltadone metabolite detection kit according to claim 1 is characterized in that, described carrier protein is bovine serum albumin(BSA), chicken egg white, rabbit anteserum albumen, human serum albumins, human fibrin or hemocyanin.
3. Furaltadone metabolite detection kit according to claim 1, it is characterized in that, described elisa plate is to be carrier with polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber or Ago-Gel, be cushioned liquid with pH value 9.6,0.05mol/L, the carbonate buffer solution that contains 0.5%v/v methyl alcohol as bag, contain that the solution of the skimmed milk of 8%-15%w/v and 1%w/v inert protein is prepared from as confining liquid.
4. Furaltadone metabolite detection kit according to claim 1 is characterized in that, the preparation method of described elisa plate is as follows:
(1) is cushioned liquid with bag the AMOZ derivative hapten is become antigenic dilution or antibody diluent with carrier protein couplet thing or antiantibody with 0.02-0.08 μ g/ml concentration dilution;
(2) in every hole of elisa plate, add 100 μ l and diluted good antigenic dilution or antiantibody dilution, 37 ℃ of incubation 2h, the coating buffer that inclines, with cleansing solution washing 4 times, each 15-30s pats dry;
(3) in every hole of elisa plate, add 150-200 μ l confining liquid, 37 ℃ of incubation 1-2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
5. Furaltadone metabolite detection kit according to claim 1 is characterized in that, described enzyme is peroxidase or the sweet enzyme of galactose.
6. Furaltadone metabolite detection kit according to claim 1 is characterized in that, described enzyme labeling AMOZ derivant specific antibody adopts glutaraldehyde method to carry out coupling and makes.
7. Furaltadone metabolite detection kit according to claim 1 is characterized in that, described enzyme labeling AMOZ derivatives antigens is to adopt glutaraldehyde method that marker enzyme and AMOZ haptens are carried out coupling to obtain.
8. Furaltadone metabolite detection kit according to claim 1 is characterized in that, described AMOZ derivant specific antibody is mouse resource monoclonal antibody or rabbit source polyclonal antibody.
9. according to the described Furaltadone metabolite detection kit of claim 1, it is characterized in that described kit also comprises AMOZ derivant standard solution, substrate colour developing liquid, stop buffer, concentrated cleaning solution and concentrates redissolution liquid.
10. according to the described Furaxone metabolite detection kit of claim 9, it is characterized in that described substrate colour developing liquid is single substrate solution.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910045162A CN101526528A (en) | 2009-01-12 | 2009-01-12 | Furaltadone metabolite detection kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910045162A CN101526528A (en) | 2009-01-12 | 2009-01-12 | Furaltadone metabolite detection kit |
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| CN101526528A true CN101526528A (en) | 2009-09-09 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101957375A (en) * | 2010-10-15 | 2011-01-26 | 扬州大学 | Label-free impedance type immunosensor for furaltadone residues, preparation method and application thereof |
| CN102539763A (en) * | 2010-12-15 | 2012-07-04 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescent kit for detecting furaltadone metabolite and application thereof |
| CN102721805A (en) * | 2011-03-29 | 2012-10-10 | 宝瑞源生物技术(北京)有限公司 | 3-amino-5-morpholinomethyl-2-oxazolidinone(AMOZ) assay kit and method for making the same |
| CN102936583A (en) * | 2011-09-23 | 2013-02-20 | 泰州康正生物技术有限公司 | Furaltadone metabolite derivative monoclonal antibody and applications thereof |
| CN103364557A (en) * | 2012-04-07 | 2013-10-23 | 北京勤邦生物技术有限公司 | Kit and method for detecting furaltadone metabolin |
| CN104841381A (en) * | 2015-04-28 | 2015-08-19 | 中国农业科学院上海兽医研究所 | Oriented adsorption carrier, immunoaffinity chromatography column, preparation method and kit |
| CN106645687A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | Kit for detecting furaltadone metabolites in food |
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2009
- 2009-01-12 CN CN200910045162A patent/CN101526528A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101957375A (en) * | 2010-10-15 | 2011-01-26 | 扬州大学 | Label-free impedance type immunosensor for furaltadone residues, preparation method and application thereof |
| CN101957375B (en) * | 2010-10-15 | 2013-03-20 | 扬州大学 | Preparation method of label-free impedance type immunosensor for furaltadone residues and application thereof |
| CN102539763A (en) * | 2010-12-15 | 2012-07-04 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescent kit for detecting furaltadone metabolite and application thereof |
| CN102721805A (en) * | 2011-03-29 | 2012-10-10 | 宝瑞源生物技术(北京)有限公司 | 3-amino-5-morpholinomethyl-2-oxazolidinone(AMOZ) assay kit and method for making the same |
| CN102936583A (en) * | 2011-09-23 | 2013-02-20 | 泰州康正生物技术有限公司 | Furaltadone metabolite derivative monoclonal antibody and applications thereof |
| CN102936583B (en) * | 2011-09-23 | 2014-09-17 | 泰州康正生物技术有限公司 | Furaltadone metabolite derivative monoclonal antibody and applications thereof |
| CN103364557A (en) * | 2012-04-07 | 2013-10-23 | 北京勤邦生物技术有限公司 | Kit and method for detecting furaltadone metabolin |
| CN103364557B (en) * | 2012-04-07 | 2016-02-24 | 北京勤邦生物技术有限公司 | A kind of kit and method detecting AMOZ |
| CN104841381A (en) * | 2015-04-28 | 2015-08-19 | 中国农业科学院上海兽医研究所 | Oriented adsorption carrier, immunoaffinity chromatography column, preparation method and kit |
| CN106645687A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | Kit for detecting furaltadone metabolites in food |
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Application publication date: 20090909 |