CN103713122A - Quick detection kit for dexamethasone - Google Patents
Quick detection kit for dexamethasone Download PDFInfo
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- CN103713122A CN103713122A CN201410001015.XA CN201410001015A CN103713122A CN 103713122 A CN103713122 A CN 103713122A CN 201410001015 A CN201410001015 A CN 201410001015A CN 103713122 A CN103713122 A CN 103713122A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention relates to a quick detection kit for dexamethasone. The quick detection kit comprises (1) an array plate coated with a dexamethasone antigen or array plate coated with a dexamethasone specific antibody, (2) an enzyme labeled dexamethasone specific antibody or enzyme labeled dexamethasone antigen, (3)a dexamethasone standard substance solution, (4) a substrate color developing solution, (5) a stop solution, (6) a washing concentrate and (7) a concentration compound solution. According to the quick detection kit, sample pretreatment process is simple, detection time is short, cost is low, the sensitivity and detectable limit can be greatly improved, and a large quantity of samples can be simultaneously detected. A detection line of the quick detection kit is capable of reaching the sensitivity of the kit of 0.05 microgram/kilogram, the minimum sample detectable limit tissue of 0.05 microgram/kilogram, and the recycling rate tissue of 85+/-10 percent; a one-step method is adopted for the kit, and the detection time is shortened for 45min.
Description
Technical field
The present invention relates to a kind of dexamethasone quick detection kit, is a kind of enzyme linked immunological kit that detects dexamethasone in animal derived food.
Background technology
Dexamethasone (Dexamethasone, DSMS) has another name called dexamethasone, fluorine first PRD dragon, Dexamethasone, is a kind of artificial synthetic cortex hormone of aadrenaline.Be glucocorticosteroid steroids anti-inflammatory, Claritin, its pharmacological action is mainly anti-inflammatory, antitoxin, antiallergy, antirheumatic, and clinical use is more extensive.In recent years, the normal function of the aspects such as the edible beef that contains the hormones such as female enediol, progesterone, Medroxyprogesterone can be upset human endocrine, grows, immune system, reproductive system is thought by European Union, also may carcinogenic, teratogenesis, thereby forbid the use of this parahormone in animal.In addition, dexamethasone, also as regulant for animal's growth, promotes animal protein to synthesize and metabolism, increases meat yield, is once widely used as domestic animal, poultry, use.But find by long-term laboratory study, dexamethasone can make animal used as test generation canceration and gene mutation, dexamethasone and residual people, animal are all had to obvious toxic and side effect, is used the dexamethasone of high dose can cause the spinoffs such as muscular atrophy, growth inhibition.And infer thus, the mankind use for a long time such medicine or long-term edible domestic animal, poultry of adding such growth accelerator, equally also canceration and gene mutation can occur.So this type of drug withdrawal is used in treatment and feed.
China is in the < < animal food herbal medicine maximum residue limit(MRL) > > of No. 235 bulletins of 2002 Nian Ministry of Agriculture issue, dexamethasone must not surpass in 0.75ug/kg liver and must not surpass 2ug/kg therefore in all food animal muscle, for guaranteeing the safety of animal derived food and the development of export abroad trade, set up accurately and reliably, highly sensitive qualitative-and-quantitative method is very necessary.
Now developed the enzyme linked immunological kit that detects dexamethasone both at home and abroad, but the kit of domestic production now can't reach the requirement of detection completely at aspects such as accuracy, sensitivity, specificitys.The exploitation of this kit improved detect sensitivity and accuracy, can carry out the detection of great amount of samples, the time that shortened is that enterprise has reduced cost.The present invention produces thus.
Summary of the invention
For solving the above-mentioned technical matters of prior art, the object of this invention is to provide a kind of dexamethasone quick detection kit, sample pretreatment process is simple, and detection time is short, expense is cheap, and sensitivity and detectability all increase substantially and can detect gross sample simultaneously.
For achieving the above object, the present invention is achieved by the following technical solutions:
A dexamethasone quick detection kit, described kit includes:
(1) the coated elisa plate of dexamethasone antigen or the elisa plate of coated dexamethasone specific antibody; (2) enzyme labeling dexamethasone specific antibody or enzyme labeling dexamethasone antigen; (3) dexamethasone standard solution; (4) substrate nitrite ion; (5) stop buffer; (6) the concentrated liquid that redissolves in concentrated cleaning solution and (7).
The preparation process of the elisa plate of the elisa plate of described coated dexamethasone antigen or coated dexamethasone specific antibody is:
(1) with coated damping fluid, dexamethasone haptens is become to antigenic dilution or antibody diluent with carrier protein couplet thing or antibody with 0.02-0.08 μ g/ml concentration dilution;
(2) to the antigenic dilution or the antiantibody dilution that add 100 μ l to dilute in every hole of elisa plate, 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, dry rear with aluminium film vacuum seal preservation.
The carbonate buffer solution that described damping fluid is pH value 9.6,0.05mol/L, contain 0.5% methyl alcohol; Described cleansing solution is deionized water or the ultrapure water that contains 8%-15% Tween-20 ℃; Described confining liquid is the skimmed milk that contains 8%-15% and the solution of 1% inert protein; Described carrier protein is bovine serum albumin(BSA), chicken egg white, rabbit anteserum albumen, human serum albumins, human fibrin or hemocyanin.
Described enzyme-labelled antigen obtains for adopting glutaraldehyde method that marker enzyme and dexamethasone haptens are carried out to coupling.
Described dexamethasone specific antibody is monoclonal antibody or polyclonal antibody.
Step prepared by described monoclonal antibody is:
(1) animal immune program: adopt Balb/c mouse as immune animal, the conjugate immunizing dose of dexamethasone haptens and key hole maple hemocyanin is 80-100 μ g/, when head exempts from, the Freund's complete adjuvant of immunogene and equivalent is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, At intervals of two to three weeks is got same dose immunogene and is added equivalent incomplete Freund's adjuvant mixing and emulsifying, once, four exempt from pneumoretroperitoneum booster immunization once to booster immunization, extracting spleen cell after 3 days;
(2) Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, in 5-10:1 ratio and SP2/0 myeloma cell, merge, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole, utilize limiting dilution assay to carry out cloning to positive hole, until obtain the hybridoma cell strain of stably excreting monoclonal antibody;
(3) cell cryopreservation and recovery: get hybridoma in exponential phase and with cryopreserving liquid, make the cell suspension of l-5 * 106/ml, be sub-packed in cryopreservation tube, in liquid nitrogen, preserve for a long time, during recovery, take out cryopreservation tube, putting into immediately 37 ℃ of water-bath middling speeds melts, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate;
(4) preparation and purification of monoclonal antibody: adopt in body and induce method, the Balb/c mouse peritoneal in 8 week age is only injected to sterilizing paraffin oil 0.5ml/, within 7-14 days, pneumoretroperitoneum injection hybridoma is 5 * l05-106/, after 7-10 days, gather ascites, by sad-saturated ammonium sulfate method, carrying out ascites purifies, bottle packing ,-20 ℃ of preservations;
(5) antibody freeze-dried powder can be dried ascites under 37 ℃ of environment, puts into-20 ℃ of preservations;
(6) antibody working fluid is with antibody diluent, antibody to be diluted with 0.02 ~ 0.08 μ g/ml concentration.
Step prepared by described polyclonal antibody is:
Adopt new zealand white rabbit as immune animal, the conjugate immunizing dose of dexamethasone haptens and key hole maple hemocyanin is 1mg/kg, when head exempts from, the Freund's complete adjuvant of immunogene and equivalent is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, get same dose immunogene interval 3-4 week and add equivalent incomplete Freund's adjuvant mixing and emulsifying, once, immunity is 5 times altogether, does not add for the last time adjuvant for booster immunization.After last immune 7-l0 days, take a blood sample, measure serum antibody titer, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
Substrate nitrite ion described in when marker enzyme is peroxidase is hydrogen peroxide or urea peroxide and tetramethyl benzidine sulfate mixed solution, the sulfuric acid that described stop buffer is 0.1-0.5mol/L or hydrochloride buffer; When marker enzyme is the sweet enzyme of galactose, described substrate nitrite ion is 0.5mol/L kaliumphosphate buffer, the citrate buffer solution that described stop buffer is 2mol/L.
Described concentrated cleaning solution is deionized water or ultrapure water.
Described concentrated redissolution liquid is the phosphate buffer containing 1% bovine serum albumin(BSA).
Beneficial effect of the present invention is as follows:
The residual enzyme linked immunological kit of dexamethasone in detection animal derived food provided by the invention, sample pretreatment process is simple, and detection time is short, expense is cheap, and sensitivity and detectability all increase substantially and can detect gross sample simultaneously.This kit method, sensitivity and detectability all increase, and have strengthened the accuracy detecting, and shorten detection time to some extent, and step is easier.In the < < animal food herbal medicine maximum residue limit(MRL) > > of No. 235 bulletins of Ministry of Agriculture's issue, dexamethasone must not surpass 0.75ug/kg in all food animal muscle, the detection line of this method can reach kit sensitivity: 0.05ug/kg, sample lowest detectable limit is organized 0.05ug/kg, recovery tissue 85 ± 10%, kit adopts single stage method, and the time shorten of detection is 45 minutes.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited to this.
Dexamethasone quick detection kit of the present invention includes:
(1) the coated elisa plate of dexamethasone antigen or the elisa plate of coated dexamethasone specific antibody; (2) enzyme labeling dexamethasone specific antibody or enzyme labeling dexamethasone antigen; (3) dexamethasone standard solution; (4) substrate nitrite ion; (5) stop buffer; (6) the concentrated liquid that redissolves in concentrated cleaning solution and (7).
The preparation process of the coated elisa plate of dexamethasone antigen or the elisa plate of coated dexamethasone specific antibody is:
(1) with coated damping fluid, dexamethasone haptens is become to antigenic dilution or antibody diluent with carrier protein couplet thing or antibody with 0.02-0.08 μ g/ml concentration dilution;
(2) to the antigenic dilution or the antiantibody dilution that add 100 μ l to dilute in every hole of elisa plate, 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, dry rear with aluminium film vacuum seal preservation.
In the present invention damping fluid be pH value 9.6,0.05mol/L, the carbonate buffer solution that contains 0.5% methyl alcohol; Cleansing solution is deionized water or the ultrapure water that contains 8%-15% Tween-20 ℃; Confining liquid is the skimmed milk that contains 8%-15% and the solution of 1% inert protein; In kit of the present invention, dexamethasone envelope antigen adopts active ester method that dexamethasone haptens and carrier protein are carried out to coupling to obtain, and this carrier protein is bovine serum albumin(BSA), chicken egg white, rabbit anteserum albumen, human serum albumins, human fibrin or hemocyanin.
Substrate nitrite ion described in when marker enzyme is peroxidase is hydrogen peroxide or urea peroxide and tetramethyl benzidine sulfate mixed solution, the sulfuric acid that described stop buffer is 0.1-0.5mol/L or hydrochloride buffer; When marker enzyme is the sweet enzyme of galactose, described substrate nitrite ion is 0.5mol/L kaliumphosphate buffer, the citrate buffer solution that described stop buffer is 2mol/L.
The carrier mass of coated dexamethasone antigen or antibody can be polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.; The form of carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.; Concentrated cleaning solution is deionized water or ultrapure water; The concentrated liquid that redissolves is for the phosphate buffer containing 1% bovine serum albumin(BSA).
Elisa plate prepared by above method has good stability, and through cold and hot stability test, the correlation technique parameter of elisa plate is all in normal range, and coated original good specificity.
In kit of the present invention, enzyme labeling thing is enzyme labeling dexamethasone specific antibody or enzyme labeling dexamethasone antigen, and enzyme used 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; Enzyme labeling thing working fluid dilution used 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 for a long time the biologically active of enzyme labeling thing), 1% sodium azide antiseptic (being convenient to preserve).
In kit of the present invention, the preparation process of enzyme labeling dexamethasone specific antibody is:
(1) preparation of peroxidase labelling dexamethasone specific antibody: antibody and peroxidase (HRP) are carried out to coupling, the method adopting is glutaraldehyde method, adopt glutaraldehyde method that the combination rate of antibody and horseradish peroxidase is raise, tradition GA single stage method coupling reaction is wayward, easily there is spontaneous polymerization 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.First in two kinds of coupled molecules, the molecule weak with coupling agent reflection first activates with excessive coupling agent, then the unnecessary coupling agent in place to go; Second step, by the coupling agent of one end and certain minute sub-connection, couples together with another kind of molecule by changing reaction conditions.Although two step method operation is more numerous, coupled efficiency improves, and the same Molecularly Imprinted Polymer forming reduces.
Enzyme-labelled antigen obtains for adopting glutaraldehyde method that marker enzyme and dexamethasone haptens are carried out to coupling.
In kit of the present invention, dexamethasone specific antibody is mouse resource monoclonal antibody or rabbit source polyclonal antibody, and immunogene adopts mixed anhydride method that dexamethasone haptens and the coupling of chicken egg white (OVA) row are obtained; Antibody formation can be freeze-dried powder, concentrate, working fluid; The phosphate buffer that antibody diluent is pH value 7.4,0.08mol/L, contain 0.3% gelatin, 5 ‰ twen-80s and 5 ‰ methyl alcohol.
Dexamethasone specific antibody is monoclonal antibody or polyclonal antibody.
Step prepared by monoclonal antibody is:
(1) animal immune program: adopt Balb/c mouse as immune animal, the conjugate immunizing dose of dexamethasone haptens and key hole maple hemocyanin is 80-100 μ g/, when head exempts from, the Freund's complete adjuvant of immunogene and equivalent is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, At intervals of two to three weeks is got same dose immunogene and is added equivalent incomplete Freund's adjuvant mixing and emulsifying, once, four exempt from pneumoretroperitoneum booster immunization once to booster immunization, extracting spleen cell after 3 days;
(2) Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, in 5-10:1 ratio and SP2/0 myeloma cell, merge, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole, utilize limiting dilution assay to carry out cloning to positive hole, until obtain the hybridoma cell strain of stably excreting monoclonal antibody;
(3) cell cryopreservation and recovery: get hybridoma in exponential phase and with cryopreserving liquid, make the cell suspension of l-5 * 106/ml, be sub-packed in cryopreservation tube, in liquid nitrogen, preserve for a long time, during recovery, take out cryopreservation tube, putting into immediately 37 ℃ of water-bath middling speeds melts, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate;
(4) preparation and purification of monoclonal antibody: adopt in body and induce method, the Balb/c mouse peritoneal in 8 week age is only injected to sterilizing paraffin oil 0.5ml/, within 7-14 days, pneumoretroperitoneum injection hybridoma is 5 * l05-106/, after 7-10 days, gather ascites, by sad-saturated ammonium sulfate method, carrying out ascites purifies, bottle packing ,-20 ℃ of preservations;
(5) antibody freeze-dried powder can be dried ascites under 37 ℃ of environment, puts into-20 ℃ of preservations;
(6) antibody working fluid is with antibody diluent, antibody to be diluted with 0.02 ~ 0.08 μ g/ml concentration.
Step prepared by polyclonal antibody is:
Adopt new zealand white rabbit as immune animal, the conjugate immunizing dose of dexamethasone haptens and key hole maple hemocyanin is 1mg/kg, when head exempts from, the Freund's complete adjuvant of immunogene and equivalent is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, get same dose immunogene interval 3-4 week and add equivalent incomplete Freund's adjuvant mixing and emulsifying, once, immunity is 5 times altogether, does not add for the last time adjuvant for booster immunization.After last immune 7-l0 days, take a blood sample, measure serum antibody titer, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
In kit of the present invention, dexamethasone standard solution is the dexamethasone solution of six concentration gradients, the phosphate buffer that dexamethasone dilution is 0.02M.
As preferably, in kit of the present invention, the preparation of reagent is specially:
(1) dexamethasone standard solution: 6 bottles of dexamethasone series standard solution, concentration is 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, 1-3ml/ bottle;
(2) coated damping fluid: pH value 9.6,0.05mol/L, the carbonate buffer solution that contains 0.5% methyl alcohol;
(3) confining liquid: the solution of the skimmed milk that contains 8%-15% and 1% inert protein;
(4) cleansing solution: the deionized water or the ultrapure water that contain 8%-15% Tween-20 ℃, 30-50ml/ bottle, 1 bottle;
(5) enzyme labeling thing: enzyme labeling antiantibody working fluid or enzyme labeling dexamethasone antigen working fluid, 7-12ml/ bottle, l bottle;
(6) substrate nitrite ion hydrogen peroxide or urea peroxide and tetramethyl benzidine sulfate mixed solution, 10-15ml/ bottle, l bottle;
(7) substrate nitrite ion is to nitro phosphate buffer: pH8.1, the 100mmolTris-HCl that contains MgCl2 0.01%;
(8) stop buffer: 1-2mol/L sulfuric acid, hydrochloric acid or 2mol/L sodium hydrate buffer solution, 5-8ml/ bottle, 1 bottle.
(9) antibody diluent: pH value 7.4,0.08mol/L, the phosphate buffer that contains 0.3% gelatin, 5 ‰ twen-80s and 5 ‰ methyl alcohol.
(10) the concentrated liquid that redissolves: the phosphate buffer that contains 5% methyl alcohol, 1% calf serum (BSA), for the 5-10 of normal working concentration doubly, 30-50ml/ bottle, 1 bottle.
The present invention detects the method for dexamethasone in animal derived food, has comprised following steps:
(1) sample pre-treatments; Sample pretreatment needs preparation:
With deionized water, the 2 * concentrated liquid that redissolves is pressed to 1:1 dilution, for the redissolution after sample extraction.
0.1M NaOH takes 0.4g NaOH and adds water and be dissolved to 100ml.
Fowl poultry kind tissue (sample extension rate 2)
A, ± tissue samples that 0.05g homogeneous is crossed are in 50ml centrifuge tube;
B, enter 3ml 0.1M sodium hydroxide solution, fully mix 1 minute;
C, 4 ml ethyl acetate fully vibrate 5 minutes, room temperature 4000r/min, centrifugal 10 minutes;
In the extremely clean glass test tube of the limpid upper organic phase of d, 1ml, 56 ℃ of water-bath nitrogen/air blow drying;
E, l normal hexane vibration 30 seconds, then add 0.5ml to redissolve working fluid fully to vibrate and mix 30 seconds; 4000 revs/min of room temperatures, centrifugal 5 minutes;
F, except upper organic phase, take off layer 50 μ l liquid for analyzing.
(2) with kit of the present invention, detect:
A, required reagent take out from cold storage environment, and at room temperature balance is 30 minutes, before every kind of liquid is used, must shake up; Notice that titer all needs to do 2 parallel experiments;
B and enzyme mark concentrate mix: it (is 1000ml antibody working fluid+100ml enzyme mark concentrate that antibody working fluid and enzyme mark concentrate are mixed by 10:1 volume ratio, this mixed liquor is now with the current, can not preserve) add 50ml standard items or sample, in corresponding micropore, add again 50 μ l antibody working fluids and enzyme mark concentrate mixed liquor, good with membrane cover, lucifuge reaction is 30 minutes at 25 ℃;
C, wash plate: carefully open cover plate film, liquid in hole is dried, add cleansing solution 250ml/ hole, soak 15-30 second at every turn, fully wash 4-5 time, with thieving paper, pat dry;
D, colour developing: every hole first adds substrate solution A 50 μ l, respectively adds substrate solution B 50 μ l, rock and mix gently, and lucifuge is reacted 15 minutes at 25 ℃;
E, reading: every hole adds 50 μ l stop buffers, measure OD value (suggestion detects with 450/630nm dual wavelength, runs through data in 5 minutes) in 450nm place in microplate reader.
(3) analyzing and testing result.
Each the concentration standard solution obtaining and the mean value (B) of sample absorbance are multiplied by 100% again divided by the absorbance (B0) of first standard (0 standard), i.e. percentage absorbance.
The mean light absorbency value of B-standard solution or sample solution
The mean light absorbency value of B0-0ng/ml standard solution
The standard items percentage absorptance of take is ordinate, and the semilog of DSMS standard items concentration (ng/ml) of take is horizontal ordinate, drawing standard curve map.By in the percentage absorptance substitution typical curve of sample, from typical curve, read the corresponding concentration of sample, be multiplied by its corresponding extension rate and be DSMS actual concentrations in sample.
The residual enzyme linked immunological kit of dexamethasone in detection animal derived food provided by the invention, sample pretreatment process is simple, and detection time is short, expense is cheap, and sensitivity and detectability all increase substantially and can detect gross sample simultaneously.This kit method, sensitivity and detectability all increase, and have strengthened the accuracy detecting, and shorten detection time to some extent, and step is easier.In the < < animal food herbal medicine maximum residue limit(MRL) > > of No. 235 bulletins of Ministry of Agriculture's issue, dexamethasone must not surpass 0.75ug/kg in all food animal muscle, the detection line of this method can reach kit sensitivity: 0.05ug/kg, sample lowest detectable limit is organized 0.05ug/kg, recovery tissue 85 ± 10%, kit adopts single stage method, and the time shorten of detection is 45 minutes.
Above-described embodiment is only for the inventive concept of the present invention of explaining, but not restriction to rights protection of the present invention, allly utilizes this design to carry out the change of unsubstantiality to the present invention, all should fall into protection scope of the present invention.
Claims (10)
1. a dexamethasone quick detection kit, is characterized in that, described kit includes:
(1) the coated elisa plate of dexamethasone antigen or the elisa plate of coated dexamethasone specific antibody; (2) enzyme labeling dexamethasone specific antibody or enzyme labeling dexamethasone antigen; (3) dexamethasone standard solution; (4) substrate nitrite ion; (5) stop buffer; (6) the concentrated liquid that redissolves in concentrated cleaning solution and (7).
2. dexamethasone quick detection kit as claimed in claim 1, is characterized in that, the preparation process of the elisa plate of the elisa plate of described coated dexamethasone antigen or coated dexamethasone specific antibody is:
(1) with coated damping fluid, dexamethasone haptens is become to antigenic dilution or antibody diluent with carrier protein couplet thing or antibody with 0.02-0.08 μ g/ml concentration dilution;
(2) to the antigenic dilution or the antiantibody dilution that add 100 μ l to dilute in every hole of elisa plate, 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, dry rear with aluminium film vacuum seal preservation.
3. dexamethasone quick detection kit as claimed in claim 2, is characterized in that, the carbonate buffer solution that described damping fluid is pH value 9.6,0.05mol/L, contain 0.5% methyl alcohol; Described cleansing solution is deionized water or the ultrapure water that contains 8%-15% Tween-20 ℃; Described confining liquid is the skimmed milk that contains 8%-15% and the solution of 1% inert protein; Described carrier protein is bovine serum albumin(BSA), chicken egg white, rabbit anteserum albumen, human serum albumins, human fibrin or hemocyanin.
4. dexamethasone quick detection kit as claimed in claim 1, is characterized in that, described enzyme labeling dexamethasone antigen obtains for adopting glutaraldehyde method that marker enzyme and dexamethasone haptens are carried out to coupling.
5. dexamethasone quick detection kit as claimed in claim 1, is characterized in that, described dexamethasone specific antibody is monoclonal antibody or polyclonal antibody.
6. dexamethasone quick detection kit as claimed in claim 5, is characterized in that step prepared by described monoclonal antibody is:
(1) animal immune program: adopt Balb/c mouse as immune animal, the conjugate immunizing dose of dexamethasone haptens and key hole maple hemocyanin is 80-100 μ g/, when head exempts from, the Freund's complete adjuvant of immunogene and equivalent is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, At intervals of two to three weeks is got same dose immunogene and is added equivalent incomplete Freund's adjuvant mixing and emulsifying, once, four exempt from pneumoretroperitoneum booster immunization once to booster immunization, extracting spleen cell after 3 days;
(2) Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, in 5-10:1 ratio and SP2/0 myeloma cell, merge, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole, utilize limiting dilution assay to carry out cloning to positive hole, until obtain the hybridoma cell strain of stably excreting monoclonal antibody;
(3) cell cryopreservation and recovery: get hybridoma in exponential phase and with cryopreserving liquid, make the cell suspension of l-5 * 106/ml, be sub-packed in cryopreservation tube, in liquid nitrogen, preserve for a long time, during recovery, take out cryopreservation tube, putting into immediately 37 ℃ of water-bath middling speeds melts, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate;
(4) preparation and purification of monoclonal antibody: adopt in body and induce method, the Balb/c mouse peritoneal in 8 week age is only injected to sterilizing paraffin oil 0.5ml/, within 7-14 days, pneumoretroperitoneum injection hybridoma is 5 * l05-106/, after 7-10 days, gather ascites, by sad-saturated ammonium sulfate method, carrying out ascites purifies, bottle packing ,-20 ℃ of preservations;
(5) antibody freeze-dried powder can be dried ascites under 37 ℃ of environment, puts into-20 ℃ of preservations;
(6) antibody working fluid is with antibody diluent, antibody to be diluted with 0.02 ~ 0.08 μ g/ml concentration.
7. dexamethasone quick detection kit as claimed in claim 5, is characterized in that step prepared by described polyclonal antibody is:
Adopt new zealand white rabbit as immune animal, the conjugate immunizing dose of dexamethasone haptens and key hole maple hemocyanin is 1mg/kg, when head exempts from, the Freund's complete adjuvant of immunogene and equivalent is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, get same dose immunogene interval 3-4 week and add equivalent incomplete Freund's adjuvant mixing and emulsifying, once, immunity is 5 times altogether, does not add for the last time adjuvant for booster immunization; After last immune 7-l0 days, take a blood sample, measure serum antibody titer, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
8. dexamethasone quick detection kit as claimed in claim 1, it is characterized in that: the substrate nitrite ion described in when marker enzyme is peroxidase is hydrogen peroxide or urea peroxide and tetramethyl benzidine sulfate mixed solution, the sulfuric acid that described stop buffer is 0.1-0.5mol/L or hydrochloride buffer; When marker enzyme is the sweet enzyme of galactose, described substrate nitrite ion is 0.5mol/L kaliumphosphate buffer, the citrate buffer solution that described stop buffer is 2mol/L.
9. dexamethasone quick detection kit as claimed in claim 1, it is characterized in that: in kit, dexamethasone standard solution is the dexamethasone solution of six concentration gradients, dexamethasone dilution is phosphate buffer, dexamethasone concentration of standard solution is: 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, 1 ~ 3ml/ bottle.
10. dexamethasone quick detection kit as claimed in claim 1, is characterized in that: described concentrated redissolution liquid is the phosphate buffer containing 1% bovine serum albumin(BSA).
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| CN201410001015.XA CN103713122A (en) | 2014-01-02 | 2014-01-02 | Quick detection kit for dexamethasone |
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| CN201410001015.XA CN103713122A (en) | 2014-01-02 | 2014-01-02 | Quick detection kit for dexamethasone |
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| CN103713122A true CN103713122A (en) | 2014-04-09 |
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| CN105842450A (en) * | 2016-05-13 | 2016-08-10 | 云南省畜牧兽医科学院 | Bluetongue antibody competition liquid and corresponding quick ELISA detection method |
| CN105838681A (en) * | 2016-05-30 | 2016-08-10 | 江南大学 | Anti-dexamethasone-specificity monoclonal antibody hybridoma cell strain C3 and application thereof |
| CN105907725A (en) * | 2016-07-11 | 2016-08-31 | 江南大学 | Anti-hydrocortisone specific monoclonal antibody hybridoma cell strain YH7 and application thereof |
| CN106596951A (en) * | 2015-10-15 | 2017-04-26 | 镇江亿特生物科技发展有限公司 | Kit for rapidly detecting amitraz content in crops |
| CN106771007A (en) * | 2016-12-07 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | The detection method and test strips of dexamethasone in a kind of dairy products |
| CN106769960A (en) * | 2016-11-29 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of detection method of dexamethasone content |
| CN109324183A (en) * | 2018-11-23 | 2019-02-12 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | A kind of glucocorticoid immunochromatography wide spectrum detection card and the preparation method and application thereof |
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| CN105842450A (en) * | 2016-05-13 | 2016-08-10 | 云南省畜牧兽医科学院 | Bluetongue antibody competition liquid and corresponding quick ELISA detection method |
| CN105842450B (en) * | 2016-05-13 | 2019-02-19 | 云南省畜牧兽医科学院 | A kind of bluetongue antibody competition liquid and its corresponding Rapid ELISA detection method |
| CN105838681A (en) * | 2016-05-30 | 2016-08-10 | 江南大学 | Anti-dexamethasone-specificity monoclonal antibody hybridoma cell strain C3 and application thereof |
| CN105838681B (en) * | 2016-05-30 | 2019-04-30 | 江南大学 | One plant of anti-dexamethasone monoclonal antibody specific hybridoma cell strain C3 and its application |
| CN105907725A (en) * | 2016-07-11 | 2016-08-31 | 江南大学 | Anti-hydrocortisone specific monoclonal antibody hybridoma cell strain YH7 and application thereof |
| CN105907725B (en) * | 2016-07-11 | 2019-07-16 | 江南大学 | One plant of anti-hydrocortisone monoclonal antibody specific hybridoma cell strain YH7 and its application |
| CN106769960A (en) * | 2016-11-29 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of detection method of dexamethasone content |
| CN106771007A (en) * | 2016-12-07 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | The detection method and test strips of dexamethasone in a kind of dairy products |
| CN109324183A (en) * | 2018-11-23 | 2019-02-12 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | A kind of glucocorticoid immunochromatography wide spectrum detection card and the preparation method and application thereof |
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