CN101955537A - Anti-ractopamine antibody and application thereof - Google Patents
Anti-ractopamine antibody and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-ractopamine antibody and application thereof. The antibody consists of a heavy chain variable region, a short peptide for connecting the heavy chain variable region with a light chain variable region, and the light chain variable region connected in turn, wherein an amino acid sequence of the heavy chain variable region is shown as No.9-No.130 amino acid residues from an N terminal in a sequence 1; and an amino acid sequence of the light chain variable region is shown as No.146-No.250 amino acid residues from the N terminal in the sequence 1. A kit and a colloidal gold test paper card have the advantages of high sensitivity, high accuracy, high precision, low cost, simple operation, short detection time, suitability for various units, simple storage, long quality guarantee period, and can rapidly detect mass samples simultaneously and realize on-site high-throughput rapid detection. Therefore, the antibody, the kit, the colloidal gold test paper and the detection method have important significance for detecting ractopamine.
Description
Technical field
The present invention relates to a kind of Ractopamine hydrochloride antibody and application thereof.
Background technology
(Ractopamine RCT) is a kind of phenyl ethyl amine material to Ractopamine hydrochloride.Be used for the herding fishery, it be a kind of " nutrition reallocation agent ", belong to beta-agonist, Ractopamine hydrochloride is effective as the substitute of " clenbuterol hydrochloride ", the economy return height uses general; Be used for medicine, it is a kind of cardiac tonic, can be used for treatment of obesity and muscular dystrophy.But people's accumulative total is taken in dosage and is surpassed certain value, or edible Ractopamine hydrochloride high residue is when viscera tissue, toxic reaction appears, symptom shows as Skeletal Muscle Contraction to be increased, destroy the fusion between quick muscle fiber and the slow switch fibers, cause muscular tremor, the muscle of four limbs and face is particularly evident, other toxicity symptom comprises tachycardia, heart disorder, stomachache, myalgia, feel sick and dizzy etc., therefore China bans use of, and China Ministry of Agriculture No. 176 bulletin regulation Ractopamine hydrochloride is the medicine of forbidding using in feed and animal drinking water.Therefore, detecting the residual quantity of Ractopamine hydrochloride in animal food is very important.
The method that now is usually used in the Rct opamine residue detection mainly contains microbial method, instrumental method and enzyme-linked immunosorbent assay (ELISA).Though the microorganism detection method is economical, easy and simple to handle, when having other microbial inhibitors to exist in sample, its sensitivity and specificity are restricted; Simple instrument analytical methods such as high efficiency liquid phase chromatographic analysis method, gas spectrum, gas chromatograph mass spectrometer method, though highly sensitive, sample pre-treatment and measurement operation are loaded down with trivial details, the expense height is unwell to the great amount of samples examination.ELISA organically combines high degree of specificity, susceptibility that antibody antigen reacts with the height catalytic of enzyme, in different loadings, carry out the method for toxic substance retention analysis, have easy and simple to handle fast, cost is low, sensitivity is higher, the analyzing samples amount is big advantage.
At present, the antibody that is used for immunodetection all is monoclonal antibody or polyclonal antibody.Monoclonal antibody or Polyclonal Antibody Preparation must obtain by cell cultures, the whole process of production complexity, and elapsed time is long, the expense height, and be difficult for operating.Single-chain antibody is that antibody heavy chain variable region and chain variable region gene are connected the antibody fragment that the back amalgamation and expression comes out by a short peptide chain, has that molecular weight is little, specificity is high, bonding force is strong, is easy to utilize advantage such as genetic engineering technique operation.
Summary of the invention
An object of the present invention is to provide a kind of single-chain antibody and encoding gene thereof that detects Ractopamine hydrochloride.
Single-chain antibody provided by the present invention, small peptide, variable region of light chain by variable region of heavy chain, connection variable region of heavy chain and variable region of light chain connect to form successively, the aminoacid sequence of described variable region of heavy chain as sequence 1 from shown in the N-terminal 9-130 amino acids residue, described variable region of light chain aminoacid sequence as sequence 1 from shown in the N-terminal 146-250 amino acids residue.
In the above-mentioned single-chain antibody, the aminoacid sequence of the small peptide of described connection variable region of heavy chain and variable region of light chain as sequence 1 from shown in the N-terminal 131-145 amino acids residue.
Described encoding gene is following 1), 2), 3), 4) or 5) shown in:
1) encoding gene of described variable region of heavy chain is the dna molecular shown in the 5 ' terminal 25-390 position Nucleotide of sequence 2 in sequence table;
2) encoding gene of described variable region of light chain is the dna molecular shown in the 5 ' terminal 436-750 position Nucleotide of sequence 2 in sequence table;
3) encoding gene of the small peptide of described connection variable region of heavy chain and variable region of light chain is the dna molecular shown in the 5 ' terminal 391-435 position Nucleotide of sequence 2 in sequence table;
4) dna molecular shown in 5 of the sequence 2 ' terminal 25-750 position Nucleotide in the sequence table;
5) under stringent condition with 1), 2) or 3) or 4) dna sequence dna hybridization that limits and dna molecular with identical function.
The recombinant vectors, reorganization bacterium, transgenic cell line and the expression cassette that contain above-mentioned arbitrary described encoding gene also belong to protection scope of the present invention.
The application of above-mentioned arbitrary described single-chain antibody in detecting Ractopamine hydrochloride also belongs to protection scope of the present invention.
Another object of the present invention provides a kind of immunoassay kit that detects Ractopamine hydrochloride.
The immunoassay kit of detection Ractopamine hydrochloride provided by the present invention is following 1), 2), 3) or 4) in arbitrary described test kit:
1) comprises the conjugate of Ractopamine hydrochloride haptens and carrier proteins, above-mentioned arbitrary described single-chain antibody and enzyme labelling anti-antibody in the test kit; Wherein, described conjugate is as coating antigen;
2) comprise the haptenic enzyme labelling thing of Ractopamine hydrochloride, above-mentioned arbitrary described single-chain antibody and anti-antibody in the test kit; Wherein, described anti-antibody is as coating antigen;
3) comprise the haptenic enzyme labelling thing of Ractopamine hydrochloride, above-mentioned arbitrary described single-chain antibody in the test kit; Wherein, described single-chain antibody is as coating antigen;
4) comprise the enzyme labelling thing of the conjugate of Ractopamine hydrochloride haptens and carrier proteins, above-mentioned arbitrary described single-chain antibody in the test kit; Wherein, described conjugate is as coating antigen.
In above-mentioned arbitrary described immunoassay kit, comprise Ractopamine hydrochloride standard solution, washings and sample concentration liquid in the described test kit; Described Ractopamine hydrochloride standard substance are the hydrochloric acid Ractopamine hydrochloride;
Described Ractopamine hydrochloride standard solution is the solution of following each concentration: 0 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35 μ g/L, 4.05 μ g/L and 12.15 μ g/L;
Per 1 liter of described washings is prepared as follows and obtained: with the 10ml polysorbas20,5g sodiumazide and 990ml phosphate buffered saline buffer mix, and obtain described washings; The concentration of described phosphate buffered saline buffer is 0.005M-0.015M, is specially 0.01M, and the pH value is 7.2-7.6, is specially 7.4;
Described sample concentration liquid is that concentration is the phosphate buffered saline buffer of 0.03mol/L-0.05mol/L, is specially the phosphate buffered saline buffer of 0.04mol/L;
The conjugate of described Ractopamine hydrochloride haptens and carrier proteins is to prepare according to the method that comprises the steps: every 68mg Ractopamine hydrochloride haptens is joined in the pyridine solution of 4ml Pyroglutaric acid, 25 ℃ are stirred 22h, nitrogen dries up then, and the material note that obtains is made the material I;
With 8ml DMF and 1, the mixing solutions of 4-diox dissolves described material I, adds the positive Tributylamine of 52.4 μ l again, and 0 ℃ is stirred 10min, adds 28.8 μ L isobutyl chlorocarbonates again, and 25 ℃ are stirred 1h, obtain the solution I; DMF and 1, the mixing solutions of 4-diox are DMF and 1, and the 4-diox mixes the solution that obtains by 1: 1 volume ratio;
Described solution I is dropwise added in the solution of 10ml carrier proteins, and 25 ℃ of shaking tables stir 12h, obtain the conjugate of described Ractopamine hydrochloride haptens and carrier proteins; The solution of carrier proteins is prepared as follows: with concentration be 0.1mol/L, pH value be 8.5 dobell's solution with the dissolving of 100mg carrier proteins, and be settled to 10ml, obtain the solution of described carrier proteins;
Described Ractopamine hydrochloride haptens is the hydrochloric acid Ractopamine hydrochloride; Described carrier proteins is bovine serum albumin, human serum albumin, mouse serum protein, thyroprotein, albumin rabbit serum, hemocyanin or oralbumin;
Described anti-antibody is a mouse-anti HIS tag monoclonal antibody.
Another object of the present invention provides a kind of colloid gold test paper that detects Streptomycin sulphate or Vibriomycin.
The colloid gold test paper of detection Ractopamine hydrochloride provided by the present invention comprises absorption of sample pad, Radioactive colloidal gold pad, reaction film and absorbent pad, and it connects successively; Described glue gold pad is coated with the above-mentioned arbitrary described single-chain antibody of stating of colloid gold label; Contain on the described reacting pad and detect band and quality control band, detect the conjugate that the band position is coated with above-mentioned arbitrary described Ractopamine hydrochloride haptens and carrier proteins, the quality control band position is coated with mouse-anti HIS tag monoclonal antibody; Described Ractopamine hydrochloride haptens is the hydrochloric acid Ractopamine hydrochloride.
Another object of the present invention provides the method for Ractopamine hydrochloride in a kind of test sample.
The method of Ractopamine hydrochloride in the test sample provided by the present invention is following I) or II) shown in:
I) method of Ractopamine hydrochloride comprises the steps: in the test sample
1) testing sample is carried out pre-treatment, obtain sample to be tested solution;
2) with above-mentioned arbitrary described immunoassay kit described sample to be tested solution is detected;
The method of described pre-treatment be following a) or b):
A) described testing sample is pig urine or serum; With described testing sample directly as sample to be tested solution; Or with described testing sample with the centrifugal 5min of the speed of 2000r/min, get supernatant liquor as sample to be tested solution; Or, get filtrate as sample to be tested solution with described testing sample filtration;
B) described testing sample is a feed; The equal pledge of the described testing sample of per 3.0 ± 0.05g is mixed 5min with the vibration of 9mL acetonitrile, and 15 ℃, the centrifugal 5min of 3000r/min get the 1mL supernatant liquid; Described 1mL supernatant liquid nitrogen is dried up, use the 1mL n-hexane dissolution, obtain lysate; With described lysate and 1mL sample diluting liquid mixing, 3000r/min, 15 ℃ of centrifugal 5min take off layer, promptly obtain sample to be tested solution;
C) described testing sample is pork or pork liver; The equal pledge of the described testing sample of per 5.0 ± 0.05g is mixed 10min with the vibration of 10mL acetonitrile, and 3000r/min, 5 ℃ of centrifugal 5min get the 3mL supernatant liquid; Described 3mL supernatant liquid nitrogen is dried up, use the 1mL n-hexane dissolution, obtain lysate; With described lysate and 1mL sample diluting liquid mixing, 3000r/min, 15 ℃ of centrifugal 5min take off layer, promptly obtain sample to be tested solution;
II) method of Ractopamine hydrochloride comprises the steps: in the test sample
1) testing sample is carried out pre-treatment, obtain sample to be tested solution;
2) with above-mentioned arbitrary described colloidal gold test paper card described sample to be tested solution is detected;
The method of described pre-treatment be following a) or b):
A) described testing sample is pig urine or serum; With described testing sample directly as sample to be tested solution; Or with described testing sample with the centrifugal 5min of the speed of 2000r/min, get supernatant liquor as sample to be tested solution; Or, get filtrate as sample to be tested solution with described testing sample filtration;
B) described testing sample is feed, pork or pork liver; The homogenate of the described testing sample of every 4g is mixed with the 0.5mL deionized water, heat 10min in 80 ℃ of-90 ℃ of water-baths, take out, be cooled to room temperature, get supernatant liquor, be sample to be tested solution;
Described sample diluting liquid is that described sample concentration liquid dilution is obtained for 20 times.
The mentioned reagent box both can be enzyme linked immunological kit, can be the electrochemiluminescent immunoassay test kit again, when being enzyme linked immunological kit, comprised substrate colour developing liquid in the described test kit; Described substrate colour developing liquid is made up of A liquid and B liquid, and described A liquid is that concentration is the aqueous solution of the urea peroxide of 1.5%-2.5%, and B liquid is that concentration is the aqueous solution of the tetramethyl benzidine of 0.5%-1.5%;
Described A liquid is preferably the aqueous solution that concentration is 2% urea peroxide, and B liquid is preferably the aqueous solution that concentration is 1% tetramethyl benzidine.
Single-chain antibody of the present invention (scFv) is the recombinant antibodies that antibody heavy chain variable region (VH) and variable region of light chain (VL) is formed by connecting by one section connection peptides (Linker) with gene engineering method, be affine activity of antigen and the functional antibody fragment of specific minimum that has kept parental antibody, can obtain by the genetic engineering technique vivoexpression, can be in bacterium very economical ground scale operation, thereby make the production of immunodetection antibody become very easy, easy and economical, and then significantly reducing the expense of detection reagent, the method that obtains monoclonal antibody than Hybridoma Cell Culture is much simple.The affinity costant of antibody of the present invention is 4.37 * 10
9L/mol, half amount of suppression (IC
50) be 0.26ng/mL.Height is tired, the antibody sources of high specific for the foundation of Rct opamine residue detection method in the food provides in the present invention.
That test kit of the present invention and colloidal gold test paper card have is highly sensitive, accuracy is high, precision is high, cost is low, simple to operate, detection time short, be fit to various units uses, stores advantage simple, long quality-guarantee period; The rapid detection batch samples can realize on-the-spot high-throughput rapid detection simultaneously.Therefore, antibody of the present invention, test kit, colloid gold test paper and detection method will be brought into play significant role in the detection of Ractopamine hydrochloride.
Description of drawings
Fig. 1 is the test kit typical curve.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The preparation of embodiment 1, antibody and Function detection
One, the preparation of Ractopamine hydrochloride single-chain antibody
(1) screening of antibody
Get 6 months male Balb/c mouse, the Trizol single stage method is extracted the total RNA of splenocyte, and purifying obtains mRNA, and reverse transcription obtains cDNA again.Use a complete set of primer, be that template increases respectively and obtains variable region of heavy chain (VH), variable region of light chain (VL) gene with cDNA, extend polymerase chain reaction (Overlap-PCR) through stack VH, VL gene are spliced into single-chain antibody gene (ScFv) at random, then ScFv is connected with carrier pCANTAB5E pCANTAB5E/ScFv, and transformed into escherichia coli TG1, promptly obtain the non-immune single-chain antibody library in mouse source.Obtain having the specific phage particle that resists the single-chain antibody of multiple Ractopamine hydrochloride with the screening of ELISA method, obtain its corresponding nucleotide sequence by order-checking.
This single-chain antibody is connected to form in turn by small peptide, the variable region of light chain of variable region of heavy chain, the described variable region of heavy chain of connection and variable region of light chain.The aminoacid sequence of this antibody is shown in sequence in the sequence table 1, from the N of sequence 1 end 9-130 amino acids residue is the aminoacid sequence of variable region of heavy chain, from the N of sequence 1 end 146-250 amino acids residue is the aminoacid sequence of variable region of light chain, is short peptide sequence from the N of sequence 1 end 131-145 amino acids residue.
The coding gene sequence of this single-chain antibody is shown in sequence in the sequence table 2, from 5 of sequence 2 ' end 25-390 position nucleotide coding variable region of heavy chain, from 5 of sequence 2 ' end 436-750 position nucleotide coding variable region of light chain, from 5 of sequence 2 ' end 391-435 position nucleotide coding small peptide.
(2) preparation of antibody
Expression vector pET20b is available from German NOVAGEN company; E. coli bl21 is available from German NOVAGEN company; Protein purification HisLink
TMProtein Purification Resin is available from U.S. Promega company, and catalog number is V8823.
In the composition sequence table sequence 2 from 5 ' terminal 25-750 position Nucleotide shown in gene, and introduce restriction enzyme site XbaI and Not I at two ends, cut the recovery target gene fragment with restriction enzyme Xba I and Not I enzyme; Cut expression vector pET20b with restriction enzyme Xba I and Not I enzyme, reclaim the big fragment of carrier; Connect, connect the product transformed into escherichia coli, screening and culturing, picking mono-clonal; Mono-clonal is inserted liquid nutrient medium further to be cultivated, extract plasmid, enzyme is cut and sequence verification, the sequence that the result records as sequence 2 from 5 ' terminal 25-750 position Nucleotide shown in, show that gene direction of insertion and sequence are all correct in the recombinant vectors, positive recombinant vectors note is made recombinant expression vector pET20b/ScFv.
Adopt Calcium Chloride Method with recombinant expression vector pET20b/ScFv transformed into escherichia coli BL21, resistance screening, through bacterium liquid PCR and plasmid enzyme restriction checking, obtain containing the recombination bacillus coli of recombinant expression vector pET20b/ScFv, note is made recombination bacillus coli BL21/pET20b/ScFv.
The composition of 2 * TY nutrient solution: be made up of Tryptones, yeast extract, NaCl and water, the concentration of Tryptones is 1.6% in per 1 liter of 2 * TY nutrient solution, the concentration of yeast extract is 1%, the concentration of NaCl is 0.5%; Each percentage composition is the quality percentage composition.
2 * TY the nutrient solution that contains penbritin, paraxin and glucose obtains as follows: add penbritin, paraxin and glucose in 2 * TY nutrient solution, making the final concentration of penbritin in solution is 100 μ g/ml, making the final concentration of paraxin in solution is 34 μ g/ml, and making the final concentration of glucose in solution is 1% (quality percentage composition).
Fermentation reorganization bacterium: the single positive bacterium colony of recombination bacillus coli BL21/pET20b/ScFv is seeded in the 2 * TY nutrient solution that contains penbritin, paraxin and glucose, and 37 ℃ of joltings are to the A of culture system
600Be 0.6 o'clock, collect bacterium; Bacterium is resuspended in the 2ml LB liquid nutrient medium, bacterial suspension inoculation is contained in the antibiotic LB liquid nutrient medium of same concentrations to 50ml that (composition that contains the antibiotic LB liquid nutrient medium of same concentrations: penbritin, paraxin, yeast extract, peptone, NaCl and water are formed by 1: 20 volume ratio; The concentration of each composition is in the solution: yeast extract 0.5%, and peptone 1%, NaCl 1%, penbritin 100 μ g/ml, paraxin 34 μ g/ml), 37 ℃ of joltings are to the A of culture system
600Be 0.6 o'clock, add IPTG (0.7mmol/L) and induce, 30 ℃ of jolting 2.5h, at 4 ℃ of centrifugal 10min of following 5000r/min, the results bacterium.With washings (with 20mmol Tris and 0.15mol NaCl water dissolution, transfer pH value to 8.0 with HCL, water is settled to 1L again, obtains 1 liter of washings) wash 1 time, add lysate (with 20mmol Tris, 10ml Triton X-100,250 μ mol PMSF, 62.5 * 10
4U N,O-Diacetylmuramidase water dissolution is transferred pH value to 8.0 with HCL, and water is settled to 1L again, obtains 1 liter of lysate), place 15min for 30 ℃, at supersound process (output rating 80%) 10s on ice, stop 10s then, 3 times repeatedly, to cell thickness no longer.At 4 ℃ of centrifugal 20min of 2000 * g, collect supernatant and precipitation respectively.To precipitate with binding buffer liquid I (with 20mmol Tris, 0.5mol Nacl and 5mmol imidazoles water dissolution, transfer pH value to 8.0 with HCL, water is settled to 1L again, obtaining 1 liter of binding buffer liquid I) washing is once, then, be suspended from binding buffer liquid II (with 20mmol Tris, 0.5mol Nacl, 5mmol imidazoles and 6mol urea water dissolution, transfer pH value to 8.0 with HCL, water is settled to 1L again, obtains 1 liter of binding buffer liquid II) in, 4 ℃, the centrifugal 20min of 12000 * g collects supernatant, through the 0.45mm membrane filtration, collect filtrate, obtain the rough liquid of antibody.
Purifying: utilize histidine-tagged (His-tag) mark that has on the expression vector by the affinitive layer purification single chain antibody protein.With HisLink
TMProtein Purification Resin adorns post, with the binding buffer of 10 times of column volumes (with 100mmolHEPES, 10mmol imidazoles and 500mmol NaCl water dissolution, re-adjustment pH value to 7.5, water is settled to 1 liter then, obtain 1 liter of binding buffer) the balance purification column, get sample on the rough liquid of antibody, the wash buffer that uses 5 times of column volumes then is (with 100mmolHEPES, 100mmol imidazoles and use water dissolution, re-adjustment pH value to 7.5, water is settled to 1 liter then, obtains 1 liter of wash buffer) the wash-out foreign protein, the elution buffer that uses 10 times of column volumes at last is (with 100mmolHEPES, 250mmol imidazoles water dissolution, re-adjustment pH value to 7.5, water is settled to 1 liter then, obtains 1 liter of elution buffer) the wash-out target protein, collect elutriant, dialysis obtains antibody purified.
Albumen checking Western blot: collect above-mentioned each stage product, carry out the 12%SDS-PAGE electrophoresis; The electrophoretic band trace to the NC film, is hybridized with the hydrochloric acid Ractopamine hydrochloride of HRP mark again, detect luminous band.The hydrochloric acid Ractopamine hydrochloride is available from U.S. Sigma-Aldrich company, and catalog number is 34198,
Simultaneously in contrast, and express according to the method described above and purifying and Western blot detect with the e. coli bl21 that changes empty carrier pET20b over to.
Western blot detected result shows: 1) albumen that obtains of experimental group has and rotten hydrochloric acid Ractopamine hydrochloride bonded function, proteic molecular weight is 30kD, consistent with the molecular weight of albumen of expection, show that target protein is and hydrochloric acid Ractopamine hydrochloride bonded antibody.2) control group does not detect any and hydrochloric acid Ractopamine hydrochloride bonded protein band.
(3) Function detection of antibody
1, uses the ELISA method, detect half inhibiting rate (IC of antibody
50):
A, the conjugate (RCT-BSA) for preparing among the embodiment 2 is cushioned the liquid dissolving with bag, obtains the solution (concentration of RCT-BSA is 1 μ g/ml in this solution) of RCT-BSA.Bag is cushioned the sodium carbonate buffer of liquid: pH9.6,0.1mol/L.
The solution that in the hole of 96 orifice plates, adds RCT-BSA, the every hole of 100 μ L, 37 ℃ of incubation 2h; The coating buffer that inclines, with PBST solution (PBS+0.05%Tween20) washing 3 times, the every hole of 250 μ L, each 30s dries liquid in the hole;
B, in every hole, add 200 μ L2%BSA confining liquids, 37 ℃ of incubation 2h, liquid in the hole of inclining then; With PBST solution (PBS+0.05%Tween20) washing 3 times, the every hole of 250 μ L, each 30s dries liquid in the hole.
C, Xiang Kongzhong add the hydrochloric acid Ractopamine hydrochloride standard solution of single-chain antibody solution and different concns, and hatched 1 hour for 37 ℃ in each every hole of 50 μ L.Make positive control only to add the hole that single-chain antibody solution do not add hydrochloric acid Ractopamine hydrochloride standard solution.
The preparation of single-chain antibody solution: obtain solution with the antibody purification in the sample diluting liquid dilution experiment (two), the concentration of antibody in solution is 5ng/mL; Sample diluting liquid is the phosphate buffered saline buffer of 0.002mol/L.
The preparation of the hydrochloric acid Ractopamine hydrochloride solution of different concns: obtain solution with sample diluting liquid dilute hydrochloric acid Ractopamine hydrochloride.
(PBS 0.05%Tween20) washs 3 times, and the every hole of 250 μ L dries liquid in the hole, adds the mouse-anti His tag monoclonal antibody of HRP mark, hatches 1 hour for 37 ℃ for d, usefulness PBST solution;
E, with PBST solution (PBS, 0.05%Tween20) washing is 3 times, the every hole of 250 μ L; Add the TMB colour developing, 37 ℃ were reacted 10 minutes, added the reaction of 2M sulfuric acid color development stopping, and every hole 50 μ L use microplate reader to carry out reading.
3 repetitions are established in experiment.
The result is as follows:
1) concentration of hydrochloric acid Ractopamine hydrochloride is inversely proportional in the standard solution that added of absorbance and every hole; Proof, the single-chain antibody that expressed purifying obtains has the binding specificity at the hydrochloric acid Ractopamine hydrochloride, and presents linear relationship, illustrates that this antibody can be used for the immunodetection to the hydrochloric acid Ractopamine hydrochloride.
2) half inhibiting rate (IC
50): the absorbance of positive control hole (promptly not adding the hole of hydrochloric acid Ractopamine hydrochloride standard solution) is B
0, the absorbance of each experimental port is B, works as B/B
0The concentration that is 50% o'clock pairing hydrochloric acid Ractopamine hydrochloride standard solution is half inhibiting rate (IC
50).Half inhibiting rate (IC of this monoclonal antibody
50) be 0.26ng/mL.
2, the affinity costant of antibody is measured
Method: get quantitative certain dilution antibody, add in the antigen that increases gradually respectively, the combination of antibody is reached capacity, with bound fraction (B) is ordinate zou, antigen concentration (mol/L) is drawn saturation curve for X-coordinate, obtain the antibody degree of saturation and be 50% o'clock free antigen concentration, its inverse is the affinity costant of this antibody under this extent of dilution.
The result: the affinity costant of antibody is 4.37 * 10
9L/mol.
The enzyme linked immunological kit and the preparation thereof of embodiment 2, detection Ractopamine hydrochloride
One, enzyme linked immunological kit is made up of following substances:
1, bag is by the enzyme plate of hydrochloric acid Ractopamine hydrochloride and carrier protein couplet thing (RCT-BSA);
2, single-chain antibody described in Ractopamine hydrochloride antibody: the embodiment 1.The concentration of antibody working fluid is 5.0ng/mL, and the antibody working fluid is with arriving that the antibody purification among the sample diluting liquid dilution embodiment 1 obtains;
3, Ractopamine hydrochloride standard substance: the Ractopamine hydrochloride standard substance are the hydrochloric acid Ractopamine hydrochloride, and standard solution concentration is respectively 0 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35 μ g/L, 4.05 μ g/L and 12.15 μ g/L; The hydrochloric acid Ractopamine hydrochloride is available from U.S. Sigma-Aldrich company; Catalog number is 34198; Be diluted to above-mentioned each concentration with sample diluting liquid;
4, ELIAS secondary antibody: the mouse-anti HIS tag monoclonal antibody of horseradish peroxidase (HRP) mark; Available from U.S. Sigma-Aldrich company, catalog number is A7058.
5, substrate colour developing liquid: be made up of A liquid and B liquid, A liquid is the aqueous solution of 2% urea peroxide, and B liquid is the aqueous solution of 1% tetramethyl benzidine;
6, stop buffer: 0.2M aqueous sulfuric acid;
7, washings: per 1 liter of described washings is prepared as follows and is obtained: 10ml polysorbas20,5g sodiumazide and 990ml phosphate buffered saline buffer are mixed, obtain described washings; The concentration of described phosphate buffered saline buffer is 0.01M, and the pH value is 7.4;
8, the phosphate buffered saline buffer of sample concentration liquid: 0.04mol/L; It is carried out 20 times of dilutions, use after becoming sample diluting liquid.
Two, the preparation of reagent constituents
(1) preparation of coating antigen
The full name of DMF is N, dinethylformamide;
68mg hydrochloric acid Ractopamine hydrochloride is joined in the pyridine solution of 4ml Pyroglutaric acid, and 25 ℃ are stirred 22h, and nitrogen dries up then, and the material note that obtains is made material I (being RCT one Pyroglutaric acid semialdehyde);
With 8ml DMF and 1, the mixing solutions of 4-diox dissolves described material I, adds the positive Tributylamine of 52.4 μ l again, and 0 ℃ is stirred 10min, adds 28.8 μ L isobutyl chlorocarbonates again, and 25 ℃ are stirred 1h, obtain solution I (being activatory hydrochloric acid Ractopamine hydrochloride solution); DMF and 1, the mixing solutions of 4-diox are DMF and 1, and the 4-diox mixes the solution that obtains by the 1:1 volume ratio;
Described solution I is dropwise added in the solution of 10ml BSA, and 25 ℃ of shaking tables stir 12h, obtain the conjugate (RCT-BSA) of described Ractopamine hydrochloride haptens and carrier proteins; The solution of carrier proteins is prepared as follows: with concentration be 0.1mol/L, pH value be 8.5 dobell's solution with 100mg BSA dissolving, and be settled to 10ml;
Conjugate is purified through Sephadex G 25M chromatography column, measure carrier concn as conjugate concentration with ultraviolet absorption method.The conjugate of purifying adds equivalent glycerine ,-20 ℃ of preservations.
(2) be coated with the enzyme plate and the preparation thereof of coating antigen
Be coated with the polystyrene enzyme plate of synthetic antigen RCT-BSA: with the carbonate solution of 10mM antigen is made 1:50000 and doubly dilute (6.0 μ g/mL), bag is by 96 hole polystyrene enzyme plates, every hole 100 μ L, 37 ℃ of incubation 2h, coating buffer inclines, dilute 20 times of after scouring 3 times with washings, each 30s pats dry, in every hole, add 200 μ L confining liquids then, 37 ℃ of incubation 2h, liquid in the hole of inclining, preserve with the vacuum-sealing of aluminium film dry back.
Bag is cushioned the sodium carbonate buffer of liquid: pH9.6,0.05mol/L;
Confining liquid: per 1 liter of confining liquid is prepared as follows: 5ml horse serum, 1g sodiumazide, 30g casein are mixed, with the phosphate buffered saline buffer dissolving and be settled to 1000ml, obtain confining liquid; Wherein, the concentration of phosphate buffered saline buffer is 0.02M, and the pH value is 7.2.
Three, kit test method
Wash plate liquid in this experiment: be the washings in a kind of described test kit of experiment.
(1) sample pre-treatments
Each sample is made sample to be tested solution, carry out check and analysis then.
1, detecting sample is pig urine or serum: can directly carry out check and analysis as sample to be tested solution; When sample is relatively more muddy, sample with centrifugal 5min of the speed of 2000r/min or filtration, is got supernatant liquor or filtrate as sample to be tested solution.
2, detecting sample is feed: homogenizer homogeneous feed sample; Take by weighing 3.0 ± 0.05g homogeneous sample in the 50mL centrifuge tube, add the 9mL acetonitrile, with vibrator thermal agitation 5min, the centrifugal 5min of 3000r/min, 15 ℃ of centrifugal 5min; Get the limpid organic phase in 1mL upper strata to the clean glass test tube of 10mL, under 50～60 ℃ of nitrogen gas stream, dry up; Add the 1mL normal hexane, with vortex instrument whirling motion 30s; Add the 1mL sample diluting liquid again and mix, with vortex instrument whirling motion 1min, 3000r/min, 15 ℃ of centrifugal 5min remove upper strata normal hexane phase; Taking off layer 50 μ L is used for analyzing.
3, detecting sample is pork or pork liver: with homogenizer homogeneous pork, pork liver sample; Take by weighing the equal pledge of 5.0 ± 0.05g to 50mL polystyrene centrifuge tube, add the 10mL acetonitrile, with vibrator thermal agitation 10min, 3000r/min, 5 ℃ of centrifugal 5min; Get the limpid organic phase in 3mL upper strata to the clean glass test tube of 10mL, under 50～60 ℃ of nitrogen gas stream, dry up; Add the 1mL normal hexane, with vortex instrument whirling motion 30s; Add the 1mL sample diluting liquid again and mix, with vortex instrument whirling motion 1min, more than the 3000r/min, 15 ℃ of centrifugal 5min remove upper strata normal hexane phase; Taking off layer 50 μ L is used for analyzing.
(2) detect with test kit
1, the making of typical curve
In the enzyme plate micropore that is coated with coating antigen, add Ractopamine hydrochloride standard solution 50 μ L, add Ractopamine hydrochloride antibody working fluid 50 μ L again, with cover plate film shrouding, react 30min in 37 ℃ of thermostat containers, pour out liquid in the hole, every hole adds 250 μ L washingss, pours out liquid in the hole behind the 30s, so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Add ELIAS secondary antibody working fluid 100 μ L, react 30min in 37 ℃ of thermostat containers, pour out liquid in the hole, the repeated washing step, every hole adds substrate colour developing liquid, mixing gently vibrates, 37 ℃ of thermostat container lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, and mixing gently vibrates, use microplate reader, measure every hole absorbance.
With the absorbancy mean value (B) of the standard solution of each concentration absorbance (B divided by first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.Semilog value with Ractopamine hydrochloride standard substance concentration (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard graphic representation.The typical curve that obtains as shown in Figure 1.
Percentage absorbance (%)=(B/B
0) * 100%
2, the mensuration of Ractopamine hydrochloride concentration in the sample
In the enzyme plate micropore that is coated with coating antigen, add test sample solution 50 μ L, add Ractopamine hydrochloride single-chain antibody working fluid 50 μ L again, with cover plate film shrouding, react 30min in 37 ℃ of thermostat containers, pour out liquid in the hole, every hole adds 250 μ L washingss, pours out liquid in the hole behind the 30s, so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.The antibody working fluid 100 μ L that add horseradish peroxidase-labeled, react 30min in 37 ℃ of thermostat containers, pour out liquid in the hole, the repeated washing step, every hole adds mixes substrate colour developing liquid 100 μ L, mixing gently vibrates, 37 ℃ of thermostat container lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, and mixing gently vibrates, use microplate reader, measure every hole absorbance.
The result judges: with the absorbancy mean value (B) of each test sample solution absorbance (B divided by first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.The percentage absorbance of corresponding each test sample solution then can be read the absorbance of test sample solution from typical curve, converses the residual quantity of Ractopamine hydrochloride in the sample solution again according to the concentration value of standard solution.
Four, test kit detects effect assessment
(1) accuracy and precision test
Accuracy test: in the sample that does not contain Ractopamine hydrochloride (pig urine, pork, pork liver sample), add the Ractopamine hydrochloride standard substance, make the final concentration of Ractopamine hydrochloride standard substance in sample be respectively 1.0 and 2.0 μ g/kg (L); In the feed sample that does not contain Ractopamine hydrochloride, add the Ractopamine hydrochloride standard substance, make the final concentration of Ractopamine hydrochloride standard substance in sample be respectively 3.0 μ g/kg and 6.0 μ g/kg; Sample after adding is carried out pre-treatment according to method described in the experiment three respectively, obtain test sample solution.
Tolerance range test: in the sample that does not contain Ractopamine hydrochloride (pig urine, pork liver, pork sample), add the Ractopamine hydrochloride standard substance, make the final concentration of Ractopamine hydrochloride standard substance in sample be respectively 4.0 μ g/kg (or μ g/L); In the feed that does not contain Ractopamine hydrochloride, add the Ractopamine hydrochloride standard substance, make the final concentration of Ractopamine hydrochloride standard substance in sample be respectively 10 μ g/kg;
Respectively extract 3 test kits and detect from the test kit of three different batches, each experiment repeats 5 times, calculates the variation coefficient respectively.The result sees Table 1 respectively.
Table 1 Precision test result
The method of calculation of plate within variance coefficient:
Certain sample (being generally medium level) replication number of times gained result's the variation coefficient in plate within variance coefficient=same same block of plate of once measuring.
The method of calculation of variation within batch coefficient:
Variation within batch coefficient=the variation coefficient of each parallel samples in once measuring together.
The method of calculation of interassay coefficient of variation:
Interassay coefficient of variation=same sample is got its mean value in the variation coefficient of different batches measurement result.
The result: the interpolation rate of recovery of all samples is 83.1%～98.5%, and the variation within batch coefficient is 4.7%～11.6%, and interassay coefficient of variation is 7.5%～16.1%.
(2) test kit preservation period
The test kit preservation condition is 2-8 ℃, and through 12 months mensuration, the maximum absorbance value (zero standard) of test kit, 50% inhibition concentration, Ractopamine hydrochloride added the practical measurement value all within normal range.Consider in transportation and the use, have improper preservation condition and occur, test kit was placed 8 days that carry out the accelerated deterioration experiment, the result shows that every index of this test kit meets the requirements fully under the condition of 37 ℃ of preservations.Consider that the freezing situation of test kit takes place, test kit was put into-20 ℃ of refrigerators freezing 8 days, measurement result shows that also the every index of test kit is normal fully.Can draw test kit from above result can preserve more than 12 months at least at 2-8 ℃.
(3) cross reacting rate test:
The specificity of Ractopamine hydrochloride ELISA test kit is determined by carrying out the cross reaction test with respective substance, is obtained its 50% inhibition concentration respectively by various typical curves.Calculate the cross reacting rate of test kit with following formula to other homologue:
The specificity of table 1 test kit
Experiment shows that test kit of the present invention is good to the specificity of Ractopamine hydrochloride, and test kit promptly of the present invention can detect Ractopamine hydrochloride.
Embodiment 3, the test paper that detects Ractopamine hydrochloride and preparation and application
One, the structure of test paper
Described test paper is made up of absorption of sample pad, Radioactive colloidal gold pad, reaction film and absorbent pad;
Described absorption of sample pad, Radioactive colloidal gold pad, reaction film and absorbent pad are linked in sequence successively, and the end of absorption of sample pad links to each other with the top of Radioactive colloidal gold pad, and the end of Radioactive colloidal gold pad links to each other with the top of reaction film, and the end of reaction film links to each other with the top of absorbent pad;
Be coated with the Ractopamine hydrochloride antibody (albumen shown in the sequence 1 in the sequence table) of colloid gold label on the described Radioactive colloidal gold pad;
Detection zone and Quality Control district are arranged on the described reaction film, and detection zone (C line) and Quality Control district (T line) are and the vertical ribbon of the appearance of described test paper; Detection zone is positioned at a side that is bordering on Radioactive colloidal gold pad end; The Quality Control district is positioned at the side away from Radioactive colloidal gold pad end; Detection zone is coated with coating antigen, and the Quality Control district is coated with mouse-anti HIS tag monoclonal antibody;
The absorption of sample pad is a cellulose filter membrane, and reaction film is nitrocellulose filter (a NC film); Absorbent pad is a thieving paper; The Radioactive colloidal gold pad is the glass fibre membrane that is coated with the Ractopamine hydrochloride antibody of colloid gold label; Sample well is positioned at the test paper top.
Two, the preparation of test paper
(1) colloid gold label antibody:
The preparation of colloidal gold solution: get 0.01% aqueous solution of chloraurate 100mL and be heated to boiling with the constant temperature magnetic stirrer, continue to add 1% trisodium citrate aqueous solution 2.5mL under the condition of stirring, continue stirring heating 20min, solution is bright redness.The room temperature cooling returns to original volume with deionized water, 2-8 ℃ of preservation.
0.1mol/L K
2CO
3Regulate colloidal gold solution pH 8.2.To add in the 10mL colloidal gold solution in the 50mL beaker, magnetic stirrer 250r/min stirs, and dropwise adds 1mL and contains 0.35mg single-chain antibody solution.Dropwise add 3mL 5%BSA, continue to stir 10min.The centrifugal 20min of gold labeling antibody solution normal temperature low speed (1500r/min) discards the precipitation that is formed by the gold grain that condenses.2-8 ℃ of red supernatant solution, the centrifugal 40min of 11000r/min.Solution is divided into three layers, transparent supernatant, the gold grain layer of black densification on flowable garnet precipitation in the pipe end and the pipe diapire.Flowable garnet precipitation is transferred in the another one centrifuge tube, be suspended to commercial weight with the 0.01mol/L phosphate buffered saline buffer that contains 1%BSA.Equilibrate overnight, the same repeated centrifugation 2 times.Using the 0.01mol/L PB (containing 0.02% NaN3) of 1%BSA will precipitate suspendible at last is 1/40 of original volume, 2-8 ℃ of preservation.
(2) metal spraying: the antibody of colloid gold label is sprayed onto on the glass fibre, makes the Radioactive colloidal gold pad;
(3) spray film: T line on reaction film and C line position are sprayed coating antigen and mouse-anti HIS tag monoclonal antibody respectively;
(4) assembling: sample pad, Radioactive colloidal gold pad, nitrocellulose filter, thieving paper are assembled according to a conventional method, and slitting is then packed test card in the plastics fabrication into, forms test card.
Three, detect with test paper
(1) sample pre-treatments and detection
Test sample is pig urine, serum, feed, pork or pork liver etc.;
Described testing sample is pig urine or serum; With described testing sample directly as sample to be tested solution; Or with described testing sample with the centrifugal 5min of the speed of 2000r/min, get supernatant liquor as sample to be tested solution; Or, get filtrate as sample to be tested solution with described testing sample filtration;
Get 4g homogenate tissue sample (muddy flesh/degrease) in centrifuge tube, add the 0.5mL deionized water, the tight pipe lid of lid; The centrifuge tube that sample is housed is put into 85 ± 5 ℃ of water-baths heat 10min, take out centrifuge tube and be cooled to room temperature; Take out test card, lie against desktop behind the Kaifeng, the supernatant liquor of drawing in the centrifuge tube (or urine sample supernatant liquor to be checked) dropwise adds 4 in sample well; The 10min judged result, the result behind the 20min is invalid.
(2) result judges
Negative: the colour developing of C line, T line naked eyes as seen, no matter shade all is judged to feminine gender.
Positive: the colour developing of C line, the T line does not develop the color, and is judged to the positive.
Invalid: the C line does not develop the color, and no matter whether the T line develops the color, and it is invalid that this test card all is judged to.
Four, the effect of test paper
(1) false positive rate and false negative rate
Each 50 parts of the negative pig urine samples (containing Ractopamine hydrochloride＜3.0 μ g/L) of the GC-MS that learns from else's experience conclusive evidence and negative porks (containing Ractopamine hydrochloride＜5.0 μ g/kg); Each 50 parts of the positive pig urine samples (containing Ractopamine hydrochloride 〉=3.0 μ g/L) of the GC-MS that learns from else's experience conclusive evidence and positive porks (containing Ractopamine hydrochloride 〉=5.0 μ g/kg).After sample handled according to method described in the experiment three respectively the test card with three batches detect calculating false positive rate and false positive rate.
The result: in 50 parts of negative pork sample determinations, test card detects 2 parts of positive, and false positive rate is 4%.In 50 parts of negative pig urine samples were measured, test card detected 1 part of positive, and false positive rate is 2%.In 50 parts of positive pork sample determinations, test card detects 0 part of negative sample, and false negative rate is 0%.In 50 parts of positive pig urine samples were measured, test card detected 0 part of negative sample, and false negative rate is 0%.
(2) test card preservation period
Stability test is the result show, this test card can be preserved 1 year at shady and cool dry place under 2-8 ℃ or room temperature condition.
Sequence table
＜110〉Beijing Wdwkbio Biotechnology Co., Ltd
＜120〉a kind of Ractopamine hydrochloride antibody and application thereof
<160>2
<210>1
<211>250
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
Met?Asp?Gly?Arg?Gln?Ala?Ala?Ala?Val?Arg?Arg?Asp?Ala?Ala?Tyr?Arg
1 5 10 15
Asp?Trp?Ser?Phe?Ser?Glu?Ala?Val?Leu?Gln?Gly?Phe?Trp?Leu?Leu?Leu
20 25 30
His?Gln?Leu?Leu?Asp?Glu?Leu?Gly?Glu?Ala?Glu?Ala?Trp?Thr?Arg?Pro
35 40 45
Trp?Val?Asp?Trp?His?Asp?Ser?Ser?Phe?Arg?Trp?Cys?Asn?Ser?Val?Lys
50 55 60
Ser?Glu?Val?Gln?Gly?Gln?Gly?His?I1e?Asp?Cys?Arg?Gln?Ile?Leu?Gln
65 70 75 80
His?Ser?Leu?His?Ala?Thr?Gln?Gln?Pro?Asp?Ile?Ser?Gly?Leu?Cys?Gly
85 90 95
Leu?Leu?Leu?Cys?Lys?Ile?Trp?Ser?Arg?Arg?Cys?Tyr?Gly?Leu?Leu?Gly
100 105 110
Pro?Arg?Asp?Leu?Ser?His?Arg?Leu?Leu?Gly?Trp?Arg?Arg?Gln?Arg?Arg
115 120 125
Trp?Arg?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly
130 135 140
Ser?Ser?A1a?Ser?Val?Gly?Glu?Thr?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser
145 150 155 160
Gly?Asn?Ile?His?Asn?Tyr?Leu?Ala?Gly?Ile?Ser?Arg?Asn?Arg?Glu?Asn
165 170 175
Leu?Leu?Arg?Thr?Trp?Ser?Ile?Met?Gln?Glu?Leu?Pro?Gln?Met?Val?Cys
180 185 190
His?Gln?Gly?Ser?Val?Ala?Val?Ala?Gln?Glu?His?Asn?Ile?Leu?Ser?Arg
195 200 205
Ser?Thr?Thr?Cys?Ser?Leu?Lys?Ile?Leu?Val?Val?Ile?Thr?Val?Asn?Ile
210 215 220
Phe?Gly?Ile?Leu?Arg?Thr?Arg?Ser?Glu?Gly?Gly?Pro?Ser?Trp?Lys?Ser
225 230 235 240
Asn?Val?Met?Leu?His?Gln?Leu?Tyr?Pro?Ser
245 250
<210>2
<211>750
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
atggatggcc?gtcaagctgc?agcagtcagg?agagatgcag?cttatagaga?ctggagcttc 60
agtgaagctg?tcctgcaagg?cttctggcta?ctccttcacc?agctactgga?tgaactgggt 120
gaagcagagg?cctggacaag?gccttgggtg?gattggcatg?attcatcctt?ccgatggtgc 180
aactcggtta?aatcagaagt?tcaaggacaa?ggccacattg?actgtagaca?aatcctccag 240
cacagcctac?atgcaactca?gcagcccgac?atctctggac?tctgcggtct?attactgtgc 300
aagatatggt?caaggcgatg?ctatggacta?ctggggccga?gggacctcag?tcaccgtctc 360
ctcgggtggc?ggcggcagcg?gcggtggcgg?ggtggcggcg?gtagcggcgg?tggcggttct 420
ggaggcggcg?gttcttctgc?atctgtggga?gaaactgtca?ccatcacatg?tcgagcaagt 480
gggaatattc?acaattattt?agctggtatc?agcagaaaca?gggaaaatct?cctcagaacc 540
tggtctataa?tgcaagaact?tccgcagatg?gtgtgccatc?aaggttcagt?ggcagtggct 600
caggaacaca?atattctctc?aagatcaaca?acctgcagcc?tgaagatttt?ggtagttatt 660
actgtcaaca?tttttggaat?actccgtaca?cgttcggagg?ggggaccaag?ctggaaatcc 720
aacgtgatgc?tgcaccaact?gtatccaagc 750
Claims (10)
1. single-chain antibody, small peptide, variable region of light chain by variable region of heavy chain, connection variable region of heavy chain and variable region of light chain connect to form successively, the aminoacid sequence of described variable region of heavy chain as sequence 1 from shown in the N-terminal 9-130 amino acids residue, described variable region of light chain aminoacid sequence as sequence 1 from shown in the N-terminal 146-250 amino acids residue.
2. single-chain antibody according to claim 1 is characterized in that: the aminoacid sequence of the small peptide of described connection variable region of heavy chain and variable region of light chain as sequence 1 from shown in the N-terminal 131-145 amino acids residue.
3. the encoding gene of claim 1 or 2 described single-chain antibodies.
4. encoding gene according to claim 3 is characterized in that: described encoding gene is following 1), 2), 3), 4) or 5) shown in:
1) encoding gene of described variable region of heavy chain is the dna molecular shown in the 5 ' terminal 25-390 position Nucleotide of sequence 2 in sequence table;
2) encoding gene of described variable region of light chain is the dna molecular shown in the 5 ' terminal 436-750 position Nucleotide of sequence 2 in sequence table;
3) encoding gene of the small peptide of described connection variable region of heavy chain and variable region of light chain is the dna molecular shown in the 5 ' terminal 391-435 position Nucleotide of sequence 2 in sequence table;
4) dna molecular shown in 5 of the sequence 2 ' terminal 25-750 position Nucleotide in the sequence table;
5) under stringent condition with 1), 2) or 3) or 4) dna sequence dna hybridization that limits and dna molecular with identical function.
5. the recombinant vectors, reorganization bacterium, transgenic cell line and the expression cassette that contain claim 3 or 4 described encoding genes.
6. claim 1 or the 2 described single-chain antibodies application in detecting Ractopamine hydrochloride.
7. an immunoassay kit that detects Ractopamine hydrochloride is following 1), 2), 3) or 4) in arbitrary described test kit:
1) comprises conjugate, claim 1 or the 2 described single-chain antibodies and the enzyme labelling anti-antibody of Ractopamine hydrochloride haptens and carrier proteins in the test kit; Wherein, described conjugate is as coating antigen;
2) comprise the haptenic enzyme labelling thing of Ractopamine hydrochloride, claim 1 or 2 described single-chain antibody and anti-antibodys in the test kit; Wherein, described anti-antibody is as coating antigen;
3) comprise the haptenic enzyme labelling thing of Ractopamine hydrochloride, claim 1 or 2 described single-chain antibodies in the test kit; Wherein, claim 1 or 2 described single-chain antibodies are as coating antigen;
4) comprise the enzyme labelling thing of conjugate, claim 1 or the 2 described single-chain antibodies of Ractopamine hydrochloride haptens and carrier proteins in the test kit; Wherein, described conjugate is as coating antigen.
8. immunoassay kit according to claim 7 is characterized in that:
Comprise Ractopamine hydrochloride standard solution, washings and sample concentration liquid in the described test kit; Described Ractopamine hydrochloride standard substance are the hydrochloric acid Ractopamine hydrochloride;
Described Ractopamine hydrochloride standard solution is the solution of following each concentration: 0 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35 μ g/L, 4.05 μ g/L and 12.15 μ g/L;
Per 1 liter of described washings is prepared as follows and obtained: with the 10ml polysorbas20,5g sodiumazide and 990ml phosphate buffered saline buffer mix, and obtain described washings; The concentration of described phosphate buffered saline buffer is 0.005M-0.015M, is specially 0.01M, and the pH value is 7.2-7.6, is specially 7.4;
Described sample concentration liquid is that concentration is the phosphate buffered saline buffer of 0.03mol/L-0.05mol/L, is specially the phosphate buffered saline buffer of 0.04mol/L;
The conjugate of described Ractopamine hydrochloride haptens and carrier proteins is to prepare according to the method that comprises the steps: every 68mg Ractopamine hydrochloride haptens is joined in the pyridine solution of 4ml Pyroglutaric acid, 25 ℃ are stirred 22h, nitrogen dries up then, and the material note that obtains is made the material I;
With 8ml DMF and 1, the mixing solutions of 4-diox dissolves described material I, adds the positive Tributylamine of 52.4 μ l again, and 0 ℃ is stirred 10min, adds 28.8 μ L isobutyl chlorocarbonates again, and 25 ℃ are stirred 1h, obtain the solution I; DMF and 1, the mixing solutions of 4-diox are DMF and 1, and the 4-diox mixes the solution that obtains by 1: 1 volume ratio;
Described solution I is dropwise added in the solution of 10ml carrier proteins, and 25 ℃ of shaking tables stir 12h, obtain the conjugate of described Ractopamine hydrochloride haptens and carrier proteins; The solution of carrier proteins is prepared as follows: with concentration be 0.1mol/L, pH value be 8.5 dobell's solution with the dissolving of 100mg carrier proteins, and be settled to 10ml, obtain the solution of described carrier proteins;
Described Ractopamine hydrochloride haptens is the hydrochloric acid Ractopamine hydrochloride; Described carrier proteins is bovine serum albumin, human serum albumin, mouse serum protein, thyroprotein, albumin rabbit serum, hemocyanin or oralbumin;
Described anti-antibody is a mouse-anti HIS tag monoclonal antibody.
9. a colloid gold test paper that detects Ractopamine hydrochloride comprises absorption of sample pad, Radioactive colloidal gold pad, reaction film and absorbent pad, and it connects successively; Described Radioactive colloidal gold pad is coated with the claim 1 or the 2 described single-chain antibodies of colloid gold label; Contain on the described reaction film and detect band and quality control band, detect the conjugate that the band position is coated with haptens of Ractopamine hydrochloride described in the claim 8 and carrier proteins, the quality control band position is coated with mouse-anti HIS tag monoclonal antibody; Described Ractopamine hydrochloride haptens is the hydrochloric acid Ractopamine hydrochloride.
10. the method for Ractopamine hydrochloride in the test sample is following I) or II) shown in:
I) method of Ractopamine hydrochloride comprises the steps: in the test sample
1) testing sample is carried out pre-treatment, obtain sample to be tested solution;
2) with claim 7 or 8 described immunoassay kits described sample to be tested solution is detected;
The method of described pre-treatment be following a) or b):
A) described testing sample is pig urine or serum; With described testing sample directly as sample to be tested solution; Or with described testing sample with the centrifugal 5min of the speed of 2000r/min, get supernatant liquor as sample to be tested solution; Or, get filtrate as sample to be tested solution with described testing sample filtration;
B) described testing sample is a feed; The equal pledge of the described testing sample of per 3.0 ± 0.05g is mixed 5min with the vibration of 9mL acetonitrile, and 15 ℃, the centrifugal 5min of 3000r/min get the 1mL supernatant liquid; Described 1mL supernatant liquid nitrogen is dried up, use the 1mL n-hexane dissolution, obtain lysate; With described lysate and 1mL sample diluting liquid mixing, 3000r/min, 15 ℃ of centrifugal 5min take off layer, promptly obtain sample to be tested solution;
C) described testing sample is pork or pork liver; The equal pledge of the described testing sample of per 5.0 ± 0.05g is mixed 10min with the vibration of 10mL acetonitrile, and 3000r/min, 5 ℃ of centrifugal 5min get the 3mL supernatant liquid; Described 3mL supernatant liquid nitrogen is dried up, use the 1mL n-hexane dissolution, obtain lysate; With described lysate and 1mL sample diluting liquid mixing, 3000r/min, 15 ℃ of centrifugal 5min take off layer, promptly obtain sample to be tested solution;
II) method of Ractopamine hydrochloride comprises the steps: in the test sample
1) testing sample is carried out pre-treatment, obtain sample to be tested solution;
2) with the described colloid gold test paper of claim 9 described sample to be tested solution is detected;
The method of described pre-treatment be following a) or b):
A) described testing sample is pig urine or serum; With described testing sample directly as sample to be tested solution; Or with described testing sample with the centrifugal 5min of the speed of 2000r/min, get supernatant liquor as sample to be tested solution; Or, get filtrate as sample to be tested solution with described testing sample filtration;
B) described testing sample is feed, pork or pork liver; The homogenate of the described testing sample of every 4g is mixed with the 0.5mL deionized water, heat 10min in 80 ℃ of-90 ℃ of water-baths, take out, be cooled to room temperature, get supernatant liquor, be sample to be tested solution;
Described sample diluting liquid is that described sample concentration liquid dilution is obtained for 20 times.
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| CN102230936B (en) * | 2011-06-13 | 2014-03-19 | 清华大学深圳研究生院 | Immunochromatography test paper for detecting ractopamine and preparation method thereof |
| CN102653561A (en) * | 2012-05-22 | 2012-09-05 | 中国农业大学 | Single-chain antibody and application thereof in detecting beta-stimulant |
| CN102653561B (en) * | 2012-05-22 | 2013-11-06 | 中国农业大学 | Single-chain antibody and application thereof in detecting beta-stimulant |
| CN105319372A (en) * | 2015-01-29 | 2016-02-10 | 江苏众红生物工程创药研究院有限公司 | Ractopamine detection test paper card and kit |
| CN105319372B (en) * | 2015-01-29 | 2017-03-29 | 江苏晶红生物医药科技股份有限公司 | Ractopamine Test paper card and test kit |
| CN107238701A (en) * | 2016-03-29 | 2017-10-10 | 中国人民解放军军事医学科学院微生物流行病研究所 | Dengue fever Specific IgA antibody in urine as dengue fever diagnosis marker |
| CN107238701B (en) * | 2016-03-29 | 2019-08-16 | 中国人民解放军军事医学科学院微生物流行病研究所 | Diagnosis marker of the dengue fever Specific IgA antibody as dengue fever in urine |
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