CN102288761A - Chloramphenicol testing kit and preparation method of same - Google Patents
Chloramphenicol testing kit and preparation method of same Download PDFInfo
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Abstract
The invention discloses a chloramphenicol testing kit and a preparation method of the same. The kit mainly comprises an enzyme-labeled plate coated with a chloramphenicol-ovalbumin coupled antigen, chloramphenicol monoclonal antibody solution and enzyme-labeled material solution. The ratio of the chloramphenicol monoclonal antibody solution and the enzyme-labeled material solution is 1: 2. Residual veterinary drug chloramphenicol can be rapidly, qualitatively and quantitatively tested through specific antigen-antibody reaction and high-efficiency catalysis of enzyme to substrates. Fish and honey samples can be tested, quality control requirements of food production enterprises for raw materials and finished products can be satisfied, and the kit can be applied in conventional screening for food safety supervision and testing agencies and can also be used in pharmacological and toxicological studies of experiments of animals and microorganisms.
Description
Technical field
The invention belongs to the detection of veterinary drugs in food field, specifically, the present invention relates to a kind of chloromycetin detection kit and preparation method thereof.
Background technology
Chloromycetin (CAP) is a kind of broad-spectrum antibiotic, and, tire height low because of its price once was used widely in China's animal husbandry and aquaculture, and becomes the important drugs of livestock and poultry and aquatic products disease prevention and cure.Along with the widespread use and the research of chloromycetin, it is found that it can cause toxic and side effect, especially very big to the toxicity of human body hemopoietic system.Nineteen fifty finds that it can cause serious lethal hypoplastic anemia.The Ministry of Agriculture in 2002 issues No. 193 bulletin and lists chloromycetin and salt thereof, ester in food animal forbidding medicine list, and regulation chloromycetin must be examined index as the outlet meat products.
The method of chlorine detection mycin mainly contains at present:
Microbial method: be that chloromycetin detects the most frequently used method, this method can be divided into cotton swab method (STOP), cylinder plate method (CPM), paper disk method, agar diffusion method etc. again.The microbial method advantage is simply, fast, in large-scale qualitative screening operation, have certain application value.But its also exist sensitivity low, be subject to disturb, easily omission, result be prone to false positive, the more high shortcoming of detection limit.
Vapor-phase chromatography (GC): can separate for the component that partition factor differs very near, thereby isolate very complicated potpourri.The strong detecting device of many highly sensitive, versatilities or selectivity is arranged for selecting for use, as hydrogen flame ionization detector (FID), electron capture detector (ECD), nitrogen phosphorous detector (NPD) etc., detection limit is generally the ppb level.Vapor-phase chromatography has advantages such as separation efficiency height, selectivity is strong, highly sensitive, detection limit is low, but owing to be subjected to the restriction of chloromycetin volatility and thermal stability, sample pretreatment process complexity, analysis speed are slow, the instrumentation degree is high and cost an arm and a leg, and vapor-phase chromatography is promoted the use of still difficulty relatively in basic unit.
Residual chloromycetin detects the chromatograph-mass spectrometer coupling technology and is mainly LC-MS instrument (LC-MS) and gas chromatograph-mass spectrometer (GC-MS).LC-MS enters the practical stage now, and its sensitivity can be carried out analyzing and testing and structural confirmation to the medicament residue component of ng/kg level easily than high 1 order of magnitude of fluorescence detector, and the LC-MS method also is the chloromycetin conclusive evidence method that U.S. FDA is recommended use at present.But instrument analytical method is because need expensive instrument, and complicated operation detects the high reason of cost and generally uses in scientific research and testing agency.
Enzyme linked immunosorbent assay (ELISA): the ELISA method of chloromycetin detects has now developed the commercial reagents box, but present most of kit testing process needs 2～3h.It is that chloromycetin standard items or sample, enzyme mark thing are added micropore that the ELISA method detects principle, and when temperature was bathed, chloromycetin competition free and enzyme labeling was in conjunction with the binding site of chloramphenicol antibody.All unreacted enzymes mark things are eliminated in cleaning step, the enzyme that combines can come out by chromogenic reagent (enzyme can make colourless developer change blueness into), and the adding of reaction terminating liquid can make blue yellowing.Absorption value is measured at the 450nm place.The content of chloromycetin in the degree reflection sample of change color.The shortcoming of euzymelinked immunosorbent assay (ELISA) is that influence factor is more, wherein the selection of envelope antigen CAP-OVA, anti-CAP monoclonal antibody and use are great to the product quality influence, and the pairing of antigen-antibody is used of crucial importance, its preparation and screening and kit performance are closely related, be prone to false positive results, antibody batch difference, measurement result also difference etc. can occur, but this method is as a kind of rapid screening means, and monitoring and extensive screening have broad application prospects in detecting at the scene.
Summary of the invention
The objective of the invention is to overcome the defective of above-mentioned prior art, a kind of with low cost, easy and simple to handle, chloromycetin detection kit that sensitivity is high is provided.
For achieving the above object, the present invention has taked following technical scheme:
A kind of chloromycetin detection kit mainly includes:
(1) be coated with the ELISA Plate of chloromycetin-ovalbumin coupled antigen, the concentration of chloromycetin in each micropore of described ELISA Plate-ovalbumin coupled antigen is 1 μ g/ml～3 μ g/ml;
(2) chloromycetin monoclonal anti liquid solution, the working concentration of described chloromycetin monoclonal antibody are 0.02 μ g/ml～0.2 μ g/ml;
(3) enzyme labeling thing solution, described enzyme labeling thing are the sheep anti-mouse igg of horseradish peroxidase protein protective agent mark, and the working concentration of described enzyme labeling thing solution is 0.5 μ g/ml～1 μ g/ml;
The amount ratio of described chloromycetin monoclonal anti liquid solution and enzyme labeling thing solution is 1: 2.
Preferably, the concentration of chloromycetin in each micropore-ovalbumin coupled antigen is 2 μ g/ml; The working concentration of described chloromycetin monoclonal antibody is 0.05 μ g/ml～0.1 μ g/ml; The working concentration of described enzyme labeling thing solution is 0.5 μ g/ml～0.8 μ g/ml.
The present invention also aims to provide the preparation method of above-mentioned chloromycetin detection kit, taked following technical scheme:
A kind of preparation method of chloromycetin detection kit may further comprise the steps:
(1) the preparation bag is by the ELISA Plate of chloromycetin-ovalbumin coupled antigen
Adopt active ester method to carry out coupling chloromycetin and ovalbumin and obtain chloromycetin-ovalbumin coupled antigen, the concentration of described chloromycetin-ovalbumin coupled antigen is 6～8mg/ml; With the carbonate buffer solution of 0.05mol/L, pH9.6 with described chloromycetin-ovalbumin coupled antigen dilution after, every hole adds 100 μ l coated elisa plates, 4 ℃ are spent the night, lavation buffer solution is washed plate, every hole adds 200～300 μ l confining liquids in 37 ℃ of sealings, throws away confining liquid behind the sealing 2h, pats dry; With wrap by the ELISA Plate of CAP-OVA coupled antigen dry, that is, the concentration of chloromycetin in each micropore of described ELISA Plate-ovalbumin coupled antigen is 1 μ g/ml～3 μ g/ml;
(2) preparation chloromycetin monoclonal anti liquid solution
Preparation purity more than 90%, concentration is the chloromycetin monoclonal antibody of 10mg/ml～20mg/ml, with 0.2%BSA-PBST described chloromycetin monoclonal antibody is diluted; The working concentration of described chloromycetin monoclonal antibody is 0.02 μ g/ml～0.2 μ g/ml;
(3) preparation enzyme labeling thing solution
The sheep anti-mouse igg of preparation horseradish peroxidase protein protective agent mark, with the sheep anti-mouse igg dilution of 1% skimmed milk power-PBST with described horseradish peroxidase protein protective agent mark, the working concentration of described enzyme labeling thing solution is 0.5 μ g/ml～1 μ g/ml.
Described chloromycetin MONOCLONAL ANTIBODIES SPECIFIC FOR method is: get all big or small Balb/c mouse of 8-10 as immune animal, with chloromycetin-bovine serum albumin(BSA) conjugate is immunogene, with chloromycetin-bovine serum albumin(BSA) conjugate and isopyknic Freund's complete adjuvant emulsification, 100 μ g/ dosage only is to mouse subcutaneous injection, later on once every two all booster immunizations, use incomplete Freund instead, lumbar injection; Merge first three day reinforced immunological once, without adjuvant, but dosage changes 200 μ g/ into only; Fusion of Cells is operated according to conventional method, cell after merging grows into four of culture hole area/for the moment, get the cells and supernatant indirect elisa method and carry out the positive hybridoma cell screening, select positive strong, the eugonic cell of cell, clone with limiting dilution assay, then enlarged culture, frozen and identify; MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying adopt in the body and induce method, with Balb/c mouse peritoneal injection sterilization paraffin oil 0.5ml/ only, pneumoretroperitoneum injection hybridoma was 5-106/in 7-14 days, gathered ascites after 7-10 days, carry out the ascites purifying through sad-saturated ammonium sulfate method, promptly.
Damping fluid: PBS (0.1M pH7.6,0.15M NaCl); The concentration of BSA is 15mg/ml.
The preparation method of the sheep anti-mouse igg of described horseradish peroxidase protein protective agent mark is: be the conventional method preparation; with mouse IgG as immunogene; preparation goat-anti serum; antibody carries out purifying through the affinity chromatography method; remove other non-differential proteins in the goat-anti serum, obtain only to discern the high affinity antibody of mouse IgG, IgM, IgA.Sheep anti-mouse igg adopts conventional method to carry out horseradish peroxidase protein protective agent mark, in order to lower the cross reactivity with other species haemocyanins, need be further purified.
The principle of this kit is the chloromycetin that adopts in indirect competitive ELISA method detection meat (beef, pork, chicken), aquatic products (flesh of fish, shrimp), milk (solid-state, liquid state), honey, the poultry egg equal samples.Kit ELISA Plate micropore endoperidium has the coupled antigen of chloromycetin-ovalbumin, after adding sample or titer, the coupled antigen competition binding antibody solution of chloromycetin in sample or the titer and micropore endoperidium, after adding enzyme labeling thing solution, develop the color with tmb substrate, chloromycetin content and absorbance in sample and the titer are inverse ratio, relatively can draw chloromycetin content with typical curve.
Compared with prior art, the present invention has following beneficial effect:
Specific reaction and the enzyme of this kit by antigen-antibody realized fast qualitative, detection by quantitative to veterinary drug chloromycetin (CAP) residual in the sample to the efficient catalytic effect of substrate; Detectable sample comprises the flesh of fish and honey, can satisfy food production enterprise from the former Quality Control requirement of expecting finished product, also can be used as the conventional examination means of food safety supervision and testing agency, can also be used for the pharmacology and the toxicologic study of animal and Experiment on Microbiology.
Embodiment
Below describe the present invention in detail by specific embodiment.
1 one kinds of chloromycetin detection kit of embodiment
The main constituent of the described chloromycetin detection kit of present embodiment is as follows:
(1) bag is by 1 of the ELISA Plate of chloromycetin-ovalbumin coupled antigen
Cooperate different microplate reader to select, as: 48 holes, 96 holes (12 capillary strip * 8 holes) etc., the concentration of chloromycetin in every hole-ovalbumin coupled antigen is 2 μ g/ml
(2) chloromycetin monoclonal anti liquid solution is 1 bottle: 7ml/ bottle, working concentration are 0.1 μ g/ml
(3) enzyme labeling thing solution is 1 bottle: the 15ml/ bottle
The sheep anti-mouse igg of horseradish peroxidase protein protective agent mark, working concentration are 0.8 μ g/ml
(4) colour developing liquid A is 1 bottle: the 7ml/ bottle
With phosphate-citrate buffer solution preparation of 0.1mol/L, pH5.0, colour developing liquid A contains 0.045% H
2O
2The preparation of phosphate-citrate buffer solution: take by weighing citric acid (C
6H
8O
7H
2O) 21.01g uses deionized water dissolving, is settled to 1000ml, measures the Na of 243ml and 0.2mol/L
2HPO
4Solution 257ml mixes.The H that in phosphate-citrate buffer solution of 100ml, pH5.0, adds 45 μ l
2O
2, mixing, promptly.
(5) colour developing liquid B is 1 bottle: the 7ml/ bottle
Colour developing liquid B contains 0.04% TMB and contains the 0.1mol/L of the tetramethyl benzidine (TMB) of 0.04wt%, phosphate-citrate buffer solution of pH5.0; The TMB that takes by weighing 40mg is dissolved in phosphate-citrate buffer solution of 100ml, pH5.0, promptly.
(6) stop buffer is 1 bottle: the 7ml/ bottle
Be the sulfuric acid of 2mol/L, measure concentrated sulphuric acid 4ml and add in the 32ml deionized water that mixing promptly.
1 bottle of (7) 20 * lavation buffer solution: the 20ml/ bottle, dilution is 20 times during use
Be 0.05%, the PBST damping fluid of pH7.4, in 1 * PBS, add 0.05% Tween-20 and obtain the PBST damping fluid.10 * PBS (phosphate buffer, 0.01mol/L, pH7.4) wherein: take by weighing NaCl80g, KCl 2g, Na
2HPO
414.4g, K
2HPO
42.4g be dissolved in the 800ml deionized water, behind the adjusting pH to 7.4, be settled to 1L.Get 1 * PBS for 10 times with the deionized water dilution during use.
(8) titer is 6 bottles: the 1ml/ bottle
Chloramphenicol concentration is respectively 0ng/ml, 0.05ng/ml, 0.15ng/ml, 0.45ng/ml, 1.35ng/ml, 4.05ng/ml.
(9) valve bag: 1
The preparation method of the described chloromycetin detection kit of present embodiment is as follows:
(1) the preparation bag is by the ELISA Plate of chloromycetin-ovalbumin coupled antigen
Adopt active ester method to carry out coupling chloromycetin and ovalbumin and obtain chloromycetin-ovalbumin coupled antigen, the concentration of described chloromycetin-ovalbumin coupled antigen is 6～8mg/ml; Carbonate buffer solution with 0.05mol/L, pH9.6 dilutes described chloromycetin-ovalbumin coupled antigen; 100 μ l/ hole coated elisa plates, bag is spent the night, and bag is 4 ℃ by temperature, and 1 * lavation buffer solution is washed plate 5 times, and in 37 ℃ of sealings, the amount of confining liquid is 200～300 μ l/ holes to add confining liquid (adding 1%BSA and 0.2% gelatin in the ELISA Plate protective agent).Throw away confining liquid behind the sealing 2h, on thieving paper, pat dry.The concentration of chloromycetin in every hole-ovalbumin coupled antigen is 2 μ g/ml.With wrap by the ELISA Plate of CAP-OVA coupled antigen dry, envelope be stored in 4 ℃ standby.
(2) preparation chloromycetin monoclonal anti liquid solution
Get all big or small Balb/c mouse of 8-10 as immune animal, with chloromycetin-bovine serum albumin(BSA) conjugate is immunogene, with chloromycetin-bovine serum albumin(BSA) conjugate (adopt active ester method to carry out coupling chloromycetin and bovine serum albumin(BSA) (BSA) and obtain immunogene) and isopyknic Freund's complete adjuvant emulsification, 100 μ g/ dosage only is to mouse subcutaneous injection, later on once every two all booster immunizations, use incomplete Freund instead, lumbar injection; Merge first three day reinforced immunological once, without adjuvant, but dosage changes 200 μ g/ into only; Fusion of Cells is operated according to conventional method, cell after merging grows into four of culture hole area/for the moment, get the cells and supernatant indirect elisa method and carry out the positive hybridoma cell screening, select positive strong, the eugonic cell of cell, clone with limiting dilution assay, then enlarged culture, frozen and identify; MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying adopt in the body and induce method, and the Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.5ml/, 7-14 days pneumoretroperitoneum injection hybridoma 5-10
6Individual/as only, to gather ascites after 7-10 days, carry out the ascites purifying through sad-saturated ammonium sulfate method ,-20 ℃ of preservations promptly get the chloromycetin monoclonal antibody.
The Tween-20 of adding 0.05% obtains the PBST damping fluid in 1 * phosphate buffer, and with 0.2%BSA-PBST described chloromycetin monoclonal antibody dilution promptly being got working concentration is 0.1 μ g/ml;
(3) preparation enzyme labeling thing solution
The sheep anti-mouse igg of preparation horseradish peroxidase protein protective agent mark is with the sheep anti-mouse igg dilution of 1% skimmed milk power-PBST with described horseradish peroxidase protein protective agent mark; Obtaining working concentration is the enzyme labeling thing solution of 0.8 μ g/ml.
The performance index of the kit of embodiment 1 are as follows:
(1) sensitivity: 0.1ppb
(2) detect lower limit
Meat (beef, pork, chicken): 0.02ng/g; Aquatic products (flesh of fish, shrimp): 0.025ng/g; Milk (solid-state, liquid state): 0.25ng/g; Honey: 0.05ng/g; Poultry egg: 0.05ng/g;
(3) cross reacting rate
Chloromycetin (Chloramphenicol): 100%; Chloramphenicol sodium succinate (Chloramphenicolsuccinate sodium): 152.63%; Florfenicol (Florfenicol):＜0.01%; Tetracycline (Tetracycline):＜0.01%; Neomycin (Neomycin):＜0.01%; Aureomycin (Chloroteracycline):＜0.01%; Sulfadimidine (Sulphadimidine):＜0.01%; Norfloxacin (Norfloxacin):＜0.01%; Clenobuterol hydrochloride (Clenbuterol Hydrochloride):＜0.01%; Hygromycin B (Hygromycin B):＜0.01%.
(4) recovery
Meat (beef, pork, chicken): 100% ± 5%; Aquatic products (flesh of fish, shrimp): 80%; Milk (solid-state, liquid state): 90% ± 10%; Honey, poultry egg: 80% ± 5%.
2 one kinds of chloromycetin detection kit of embodiment
The concentration of chloromycetin in described each micropore of chloromycetin detection kit of present embodiment-ovalbumin coupled antigen is 1 μ g/ml; The working concentration of described chloromycetin monoclonal antibody is 0.02 μ g/ml; The working concentration of described enzyme labeling thing solution is 0.5 μ g/ml.Other components are all identical with embodiment 1, and the preparation method is also identical with embodiment 1.
3 one kinds of chloromycetin detection kit of embodiment
The concentration of chloromycetin in described each micropore of chloromycetin detection kit of present embodiment-ovalbumin coupled antigen is 3 μ g/ml; The working concentration of described chloromycetin monoclonal antibody is 0.18 μ g/ml; The working concentration of described enzyme labeling thing solution is 1 μ g/ml.Other components are all identical with embodiment 1, and the preparation method is also identical with embodiment 1.
The detection kit of embodiment 1 that adopts embodiment 4 detects the chloromycetin content in the flesh of fish
Place room temperature (20～25 ℃) more than the balance 30min kit before using, must shake up before every kind of liquid reagent uses.Taking-up needs the ELISA Plate capillary strip of quantity to insert in the micropore frame, and sample and the corresponding micropore of titer are numbered according to the order of sequence.Titer is cooked two parallel laboratory tests, notes the position of titer and sample.No capillary strip is put into the aluminium foil bag sealing, be stored in 2～8 ℃.
(1) preparation of sample liquid
A gets the structure of fish muscle sample that 3g rubs, and adds 6ml ethyl acetate, vibration 10min, the centrifugal 15min of 3000g;
B gets the 4ml supernatant, adds the 4ml chloroform, adds 4ml water again, vibration 10min, and the centrifugal 15min of 3000g, the absorption 4ml of lower floor dries up instrument with Rotary Evaporators or nitrogen and dries up (50 degree～60 degree);
C adds 0.5ml PBS and the 0.5ml normal hexane redissolves, and the centrifugal 15min of 3000g behind the vibration 10min removes the upper strata normal hexane, takes off layer 50ul and promptly can be used for analyzing, and extension rate is 0.5;
(2) add 100 μ l titers or the sample liquid handled well in micropore separately, note changing the suction nozzle of pipettor when each hole adds sample liquid or titer.
(3) add 50 μ l chloromycetin monoclonal anti liquid solutions in each micropore, beat ELISA Plate gently, note not running out, in 37 ℃ of incubation 40min with abundant mixing.
(4) pour out liquid in the hole, the micropore frame is upside down in pats on the thieving paper to guarantee to remove fully the liquid in the hole.Add 170 μ l, 1 * lavation buffer solution in every hole, outwell liquid in the micropore once more, on thieving paper, pat dry, repeat aforesaid operations 3 times.
(5) add 100 μ l enzyme labeling thing solution to each micropore, beat ELISA Plate gently, note not running out, in 37 ℃ of incubation 40min with abundant mixing.Take out ELISA Plate, wash 3 times.
(6) every hole adds 50 μ l colour developing liquid A and 50 μ l colour developing liquid B, fully mixes to be incorporated in 37 ℃ of dark places and to hatch 15～30min.After also colour developing liquid A, B equal proportion fully can being mixed, add 100 μ l to every hole, catalytic reaction must not surpass 30min.
(7) every hole adds 50 μ l stop buffers, and the mixing that vibrates is gently located to measure absorbance (OD value) with microplate reader in 450nm (suggestion detects with dual wavelength 450/630nm) at once.Get OD450 or OD450-OD630 (recommendation) and be used for interpretation of result.
(8) result judges
Each the concentration standard liquid that is obtained or the mean value B (diplopore) of sample absorbance are divided by the absorbance B of first standard (0 standard)
0Multiply by 100% again, i.e. the percentage absorbance.
Percentage absorbance (%)=B/B
0* 100%
The mean light absorbency value of B-titer or sample
B
0The mean light absorbency value of-0ng/ml titer
Percentage absorbance with titer is an ordinate, and the semilog value of concentration of standard solution (ng/ml) is a horizontal ordinate, the drawing standard curve map.In the percentage absorbance substitution typical curve with sample, read the pairing concentration of sample, multiply by the actual concentrations that its corresponding extension rate is chloromycetin in the sample from typical curve.If utilize kit specialty analysis software to calculate, be convenient to accurate, the express-analysis of a large amount of samples.
(9) test findings
ELISA kit with embodiment 1 detects 32 parts through the negative flesh of fish of GC-MS method conclusive evidence, and the measured value of 32 duplicate samples is all less than 0.1 μ g/kg, and is negative, conforms to the instrumental method testing result.ELISA kit with embodiment 1 detects 20 parts through the positive flesh of fish of GC-MS method conclusive evidence, and the measured value of 20 duplicate samples is all greater than 0.2 μ g/kg, and is positive, conforms to the instrumental method testing result.
The detection kit of embodiment 1 that adopts embodiment 5 detects the chloromycetin content in the honey
Place room temperature (20～25 ℃) more than the balance 30min kit before using, must shake up before every kind of liquid reagent uses.Taking-up needs the ELISA Plate capillary strip of quantity to insert in the micropore frame, and sample and the corresponding micropore of titer are numbered according to the order of sequence.Titer is cooked two parallel laboratory tests, notes the position of titer and sample.No capillary strip is put into the aluminium foil bag sealing, be stored in 2～8 ℃.
The preparation method of sample liquid is:
A gets 4g honey, adds 4ml water, 4ml ethyl acetate, the centrifugal 15min of 3000g behind the vibration 10min;
B gets upper strata ethyl acetate 2ml, dries up instrument with Rotary Evaporators or nitrogen and dries up (50 degree～60 degree);
C redissolves with lmlPBS, draws 50ul and is used for analyzing.Extension rate is 0.5.
Other steps are all identical with embodiment 4 steps.Test findings is as follows:
ELISA kit with embodiment 1 detects 40 parts through the negative honey of GC-MS method conclusive evidence, and the measured value of 40 duplicate samples is all less than 0.1 μ g/kg, and is negative, conforms to the instrumental method testing result.ELISA kit with embodiment 1 detects 22 parts through the positive honey of GC-MS method conclusive evidence, and the measured value of 22 duplicate samples is all greater than 0.2 μ g/kg, and is positive, conforms to the instrumental method testing result.The detection kit of embodiment 1 that adopts embodiment 6 detects the chloromycetin content in the milk
Before using to the processing of kit with embodiment 4 and 5.
The preparation method of sample liquid is:
A gets 4ml liquid milk (if solid-state milk powder is then got 1g milk powder and added 6ml water, get 1ml behind the mixing and be used for handling), adds the 5.5ml acetonitrile, vibration mixing 5min;
The centrifugal 15min of b4000g, reject upper strata liquid, lower floor's organic phase changes Rotary Evaporators evaporate to dryness or nitrogen over to and dries up instrument and dry up (50～60 ℃);
C redissolves with 4ml PBS, adds 4ml ethyl acetate, vibration mixing 5min, centrifugal after, shift the upper strata organic layer and dry up instrument to Rotary Evaporators evaporate to dryness or nitrogen and dry up (50～60 ℃);
D redissolves with 1ml PBS, draws 50ul and is used for analyzing.As limpid inclusion-free, can further concentrate.The sample extension rate is 0.5.
Other steps are all identical with embodiment 4 steps.Test findings conforms to the instrumental method testing result.
The detection kit of embodiment 1 that adopts embodiment 7 detects the chloromycetin content in the poultry egg
Before using to the processing of kit with embodiment 4 and 5.
The preparation method of sample liquid is:
A is with the egg white of whole egg sample and yellowish, mixing.Get 4g or 4ml compound sample, add the 8ml acetonitrile, vibration mixing 5min;
The centrifugal 15min of b 4000g draws 4ml upper strata liquid, changes Rotary Evaporators evaporate to dryness or nitrogen over to and dries up instrument and dry up (50～60 ℃);
C redissolves with 4ml PBS, adds 4ml ethyl acetate, vibration mixing 5min, centrifugal after, shift the upper strata organic layer and dry up instrument to Rotary Evaporators evaporate to dryness or nitrogen and dry up (50～60 ℃);
D adds 1ml PBS and the 1ml normal hexane redissolves, and the centrifugal 15min of 3000g behind the vibration 10min removes the upper strata normal hexane.Taking off layer 50ul promptly can be used for analyzing.The sample extension rate is 0.5.
Other steps are all identical with embodiment 4 steps.Test findings conforms to the instrumental method testing result.
More than be at the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from equivalence of the present invention and implement or change, all should be contained in the claim of the present invention.
Claims (5)
1. a chloromycetin detection kit is characterized in that, mainly includes:
(1) be coated with the ELISA Plate of chloromycetin-ovalbumin coupled antigen, the concentration of chloromycetin in each micropore of described ELISA Plate-ovalbumin coupled antigen is 1～3 μ g/ml;
(2) chloromycetin monoclonal anti liquid solution, the working concentration of described chloromycetin monoclonal antibody are 0.02～0.2 μ g/ml;
(3) enzyme labeling thing solution, described enzyme labeling thing are the sheep anti-mouse igg of horseradish peroxidase protein protective agent mark, and the working concentration of described enzyme labeling thing solution is 0.5～1 μ g/ml;
The amount ratio of described chloromycetin monoclonal anti liquid solution and enzyme labeling thing solution is 1: 2.
2. chloromycetin detection kit according to claim 1 is characterized in that, the concentration of chloromycetin in each micropore of described ELISA Plate-ovalbumin coupled antigen is 2 μ g/ml; The working concentration of described chloromycetin monoclonal antibody is 0.05～0.1 μ g/ml; The working concentration of described enzyme labeling thing solution is 0.5 μ g/ml～0.8 μ g/ml.
3. the preparation method of claim 1 or 2 described chloromycetin detection kit is characterized in that, may further comprise the steps:
(1) the preparation bag is by the ELISA Plate of chloromycetin-ovalbumin coupled antigen
Adopt active ester method to carry out coupling chloromycetin and ovalbumin and obtain chloromycetin-ovalbumin coupled antigen, the concentration of described chloromycetin-ovalbumin coupled antigen is 6～8mg/ml; With the carbonate buffer solution of 0.05mol/L, pH9.6 with described chloromycetin-ovalbumin coupled antigen dilution after, every hole adds 100 μ l coated elisa plates, 4 ℃ are spent the night, lavation buffer solution is washed plate, every hole adds 200～300 μ l confining liquids in 37 ℃ of sealings, throws away confining liquid behind the sealing 2h, pats dry; With wrap by the ELISA Plate of CAP-OVA coupled antigen dry, that is, the concentration of chloromycetin in each micropore of described ELISA Plate-ovalbumin coupled antigen is 1～～3 μ g/ml;
(2) preparation chloromycetin monoclonal anti liquid solution
Preparation purity more than 90%, concentration is the chloromycetin monoclonal antibody of 10～20mg/ml, with 0.2%BSA-PBST described chloromycetin monoclonal antibody is diluted; The working concentration of described chloromycetin monoclonal antibody is 0.02～～0.2 μ g/ml;
(3) preparation enzyme labeling thing solution
The sheep anti-mouse igg of preparation horseradish peroxidase protein protective agent mark, with the sheep anti-mouse igg dilution of 1% skimmed milk power-PBST with described horseradish peroxidase protein protective agent mark, the working concentration of described enzyme labeling thing solution is 0.5～1 μ g/ml.
4. the preparation method of chloromycetin detection kit according to claim 3, it is characterized in that, described chloromycetin MONOCLONAL ANTIBODIES SPECIFIC FOR method is: get all big or small Balb/c mouse of 8-10 as immune animal, with chloromycetin-bovine serum albumin(BSA) conjugate is immunogene, with chloromycetin-bovine serum albumin(BSA) conjugate and isopyknic Freund's complete adjuvant emulsification, 100 μ g/ dosage only is to mouse subcutaneous injection, later on once every two all booster immunizations, use incomplete Freund instead, lumbar injection; Merge first three day reinforced immunological once, without adjuvant, but dosage changes 200 μ g/ into only; Fusion of Cells is operated according to conventional method, cell after merging grows into four of culture hole area/for the moment, get the cells and supernatant indirect elisa method and carry out the positive hybridoma cell screening, select positive strong, the eugonic cell of cell, clone with limiting dilution assay, then enlarged culture, frozen and identify; MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying adopt in the body and induce method, with Balb/c mouse peritoneal injection sterilization paraffin oil 0.5ml/ only, pneumoretroperitoneum injection hybridoma was 5-106/in 7-14 days, gathered ascites after 7-10 days, carry out the ascites purifying through sad-saturated ammonium sulfate method, promptly.
5. according to the preparation method of claim 3 or 4 described chloromycetin detection kit, it is characterized in that the concentration of chloromycetin in each micropore of ELISA Plate described in the step (1)-ovalbumin coupled antigen is 2 μ g/ml; The working concentration of chloromycetin monoclonal antibody described in the step (2) is 0.05～0.1 μ g/ml; The working concentration of enzyme labeling thing solution described in the step (3) is 0.5 μ g/ml～0.8 μ g/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101186589A CN102288761A (en) | 2011-05-09 | 2011-05-09 | Chloramphenicol testing kit and preparation method of same |
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| CN102585009A (en) * | 2012-02-27 | 2012-07-18 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting chloramphenicol residues |
| CN102914653A (en) * | 2012-10-22 | 2013-02-06 | 大连海洋大学 | Chloramphenicol chemiluminiscence ELISA (Enzyme-Linked Immunosorbent Assay) kit |
| CN106749653A (en) * | 2016-12-07 | 2017-05-31 | 普菲特益斯生物科技（北京）有限公司 | The preparation method of anti-citrinin monoclonal antibody, citrinin kit and its detection method |
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| CN102585009A (en) * | 2012-02-27 | 2012-07-18 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting chloramphenicol residues |
| CN102914653A (en) * | 2012-10-22 | 2013-02-06 | 大连海洋大学 | Chloramphenicol chemiluminiscence ELISA (Enzyme-Linked Immunosorbent Assay) kit |
| CN106749653A (en) * | 2016-12-07 | 2017-05-31 | 普菲特益斯生物科技（北京）有限公司 | The preparation method of anti-citrinin monoclonal antibody, citrinin kit and its detection method |
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