CN101290317A - Salbutamolum ELISA method and reagent kit and method for making same - Google Patents
Salbutamolum ELISA method and reagent kit and method for making same Download PDFInfo
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- CN101290317A CN101290317A CNA2008100286361A CN200810028636A CN101290317A CN 101290317 A CN101290317 A CN 101290317A CN A2008100286361 A CNA2008100286361 A CN A2008100286361A CN 200810028636 A CN200810028636 A CN 200810028636A CN 101290317 A CN101290317 A CN 101290317A
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Abstract
The invention discloses a EUSA detection method for salbutamol and a kit as well as a method for making the same. The detection method comprises the following steps that: the salbutamol antigen is coated on a solid phase carrier, a sample or a sample to be detected is added, a salbutamol double-functional genetic engineering antibody is added, after the reaction, substrate fluid is added to display the color, the percentage absorbency is detected, according to a standard curve of the salbutamol and the percentage absorbency of the sample to be detected, the concentration of the salbutamol in the sample to be detected is calculated, etc. The invention also discloses the kit realizing the detection method and the method for making the kit. The invention adopts the salbutamol double-functional genetic engineering antibody and the direct competition EUSA adsorption analysis technique, has high sensitivity and good stability, greatly simplifies the operation procedure, shortens the reaction time, lowers the cost, is suitable for screening a large quantity of samples and has important realistic significance.
Description
Technical field
The invention belongs to the detection technique field, be specifically related to the bifunctional genetically engineered antibody immunologic detection method of salbutamolum residue in a kind of animal derived food and realize the preparation method of the monitoring of described method with kit and kit.
Background technology
(salbutamol Sal), has another name called methoxyphenamini hydrochloridum to salbutamol, belongs to a kind of of beta 2-adrenergic activator (BAA), is widely used in treatment bronchial astehma clinically.β 2-excitants such as while salbutamol also can be used as the growth accelerator of livestock and poultry such as ox, sheep, fowl, pig, and the researchist has also carried out going deep into extensive studies to its effect and effect, and once it is widely used in Production of Livestock and Poultry.But, and can enter human body by food chain, the serious harm human health in animal viscera because that β 2-excitant easily gathers is residual.Along with the extensive poisoning of the meat products that the edible β of use 2-excitant produces takes place in succession on ground such as Spain, Holland, France and Hong-Kong, Guangdong, Shanghai, countries and regions such as European Union, the U.S., China all legislation successively forbid using in Production of Livestock and Poultry the β 2-excitant of actuating the thing growth as feed addictive.Salbutamol and clenbuterol have same growth promotion effect, clenbuterol (clenbuterol hydrochloride) is owing to the heavily further investigation of distributional effects and the attention that widespread use causes the parties concerned of its nutrition, all having worked out fast and effectively detection method both at home and abroad monitors it, make its illegal use be subjected to more effectively control, and salbutamol relatively lagging behind owing to detection means research, thereby progressively replace clenbuterol, become the new tool that the lawless person seeks economic interests.Therefore, setting up quick, simple, convenient, effective detection means is the urgent task that the control salbutamol illegally uses.
Usually, the method for detection salbutamol mainly contains high performance liquid chromatography (HPLC), gas chromatography-mass spectrography (GC-MS), enzyme-linked immuno assay technology (ELISA) or the like.HPLC method and GC-MS method are the conclusive evidence methods that salbutamolum residue detects, and its advantage is to detect the degree of accuracy height, but because of its instrumentation degree height, detection time length, complicated sample handling procedure, testing cost costliness etc., are not suitable for being used as the detection of gross sample.In various detection methods, what be fit to be applied to large quantities of test sample is EL ISA detection method.It has fast, sensitive, simple to operate and characteristics that the one-time detection sample size is big, and can directly detect with urine, blood sample, with HPLC, GC-MS the higher rate that conforms to is arranged, and is fit to very much the detection of living animal.
The ultimate principle of enzyme-linked immuno assay technology is exactly to utilize the antigen of enzyme labeling or the reaction between the antibody, analyzes with antagonist or antigen by the colour developing of zymolyte.Obviously, when antigen molecule was detected, the quality of antibody itself and enzyme labelled antibody was the key that guarantees that this detection means is implemented.In the immunization of salbutamol being detected in the past, employing be polyclonal antibody or monoclonal antibody.Many anti-easily obtain but specificity is not high has the researcher to report in the desertification butylamine alcohol polyclonal antibody because of there being different I gG hypotype, thereby salbutamol and carrier protein BSA shown different binding abilities.Then there is not this problem in monoclonal antibody, but it should be noted that adopt Hybridoma Cell Culture carry out monoclonal antibody (McAb) cycle long, yield poorly that (output on average has only 10～100mg/L).In addition, no matter be polyclonal antibody or monoclonal antibody, all will carry out the external labeling process of enzyme when adopting enzyme-linked immuno assay to it, will have following shortcoming inevitably: 1) chemical covalent cross-linking process causes certain sex change to enzyme and antibody, thereby influence is active; 2) inhomogeneity of enzyme labelled antibody batch product causes needing before use it is proofreaied and correct; 3) product all needs to carry out purifying, complex steps before and after enzyme and the antibody coupling.At present, the ELISA kit that China produces voluntarily is in sensitivity and quantitatively all can't satisfy actual needs, and only expense of the annual import salbutamol ELISA kit in Shenzhen reaches millions of units.Because kit price height, each sample detection cost is more than dozens of yuan.Therefore, setting up effective, actual antibody and enzyme labelled antibody preparation method is the key problem that realizes that further the salbutamol fast detecting need solve.
The recombinant antibodies technology has caused the revolution of immunoassay technology, and especially phage surface presents the appearance of technology, makes to obtain to have desirable antigen compatibility and specific genetic engineering antibody becomes possibility.Its advantage is the repertoire antibody variable region gene to be assembled into and forms phage antibody library in the expression vector, enrichment process by " absorption-wash-out-amplification ", from phage antibody library, filter out the variable region gene of specific antibody very effectively, be convenient to further genetic manipulation.Based on this bifunctional genetically engineered antibody, just be meant the antibody molecule compound protein that on dna level, the genetic fragment of a kind of gene of antibody fragment and other functional moleculars such as enzyme etc. is carried out amalgamation and expression by reorganization.It is little that this antibody not only has a genetic engineering antibody molecular weight, the characteristics that specificity is high, and kept the activity of enzyme spcificity bound substrates; Even more noteworthy, bifunctional genetically engineered antibody can be expressed in various expression systems, and can produce in a large number by technique for gene engineering, nutrient culture media there is not specific (special) requirements, fermentation density height, fermentation period weak point, easy and simple to handle, whole cost is lower, and this just fundamentally provides guarantee for the practical application of bifunctional genetically engineered antibody in each association area.
The applicant discloses a kind of enzyme linked immunological kit and using method thereof that detects salbutamolum residue in application number is 200810025905.9 patented claim, do not detect but realize utilizing the bifunctional genetically engineered antibody technology to carry out salbutamol, do not see other prior art report correlation techniques yet.The applicant expects to utilize phage surface to present technology acquisition desertification butylamine alcohol antibody gene, by recombination to construct desertification butylamine alcohol bifunctional genetically engineered antibody expression vector, the method that adopts molecular biology, cell biology to combine with bio-separation, realize desertification butylamine alcohol bifunctional genetically engineered antibody efficiently expressing and separation and purification in Escherichia coli, and its alternative vitro enzyme mark polyclone/monoclonal antibody is used for enzyme immunoassay analysis salbutamol.This will provide a kind of new way to the fast detecting that the enzyme immunoassay technology further is applied to micromolecule farming, residue of veterinary drug and obtain independent intellectual property right significance is arranged.
Summary of the invention
An object of the present invention is deficiency, a kind of high specific, high sensitivity, cheap, simple to operate are provided, in enormous quantities the bifunctional genetically engineered antibody immunologic detection method of fast detecting salbutamol at existing salbutamol detection technique existence.
Another object of the present invention provides the kit of the described detection method of realization and the preparation method of kit.
Purpose of the present invention is achieved by the following technical programs:
A kind of enzyme-linked immune detection method of husky butanolamine is provided, may further comprise the steps:
(1) husky butanolamine is antigen coated on solid phase carrier;
(2) add standard specimen or testing sample, add husky butanolamine bifunctional genetically engineered antibody again, the reaction back adds substrate solution develops the color, and measures the percentage absorbance;
(3) according to the semilog between percentage absorbance and husky butanolamine concentration relation map typical curve, according to the typical curve of husky butanolamine and the percentage absorbance of sample to be checked, extrapolate the concentration of husky butanolamine in the testing sample.
Measuring principle of the present invention is as follows: at first that salbutamol is antigen coated in solid phase carrier for example on the ELISA Plate, add standard specimen or testing sample then, add salbutamol bifunctional genetically engineered antibody (horseradish peroxidase HRP or alkaline phosphatase AP genetic engineering antibody) again, salbutamol competition bifunctional genetically engineered antibody in envelope antigen and the testing sample, when testing sample salbutamol content is high, then the bifunctional genetically engineered antibody that combines with solid phase antigen is just few, otherwise the bifunctional genetically engineered antibody that is combined on the solid phase antigen is just many, adding substrate in reaction back develops the color and is measured, when one timing of bifunctional genetically engineered antibody amount, it is many more that the testing sample that adds contains salbutamol, the bifunctional genetically engineered antibody that combines with solid phase antigen is just few more, the color development habituation, the percentage absorbance is low, otherwise, color development increased response then, the percentage absorbance increases, thereby according to the semilog between percentage absorbance and the salbutamol concentration relation mapping promptly gets typical curve, according to the typical curve of salbutamol and the percentage absorbance of sample to be checked, can extrapolate the concentration of salbutamol in the testing sample again.
The material of solid phase carrier that can be used as the described fixedly salbutamol of step (1) antigen is more, for example polystyrene, cellulose nitrate, tygon, polypropylene, polyacrylamide, cross-linked glucose, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be shrinkage pool, the scraps of paper, globule etc.Preferred 96 holes of the present invention or 40 hole ELISA Plate, be coated with can with the salbutamol antigen of salbutamol bifunctional genetically engineered antibody specific bond, and closed porosity surface adsorption site not, used coating buffer is the carbonate buffer solution of pH9.6,0.05mol/L, carbonate buffer solution contains 1～2g sodium carbonate and 2～4g sodium bicarbonate, distilled water 1L envelope, closing liquid is 1～5% skimmed milk power solution.Described husky butanolamine bifunctional genetically engineered antibody utilizes gene engineering method to prepare horseradish peroxidase or alkaline phosphatase salbutamol genetic engineering antibody, antibody can be Fab, Fv, ScFv or single domain antibody, great expression is collected fermentation liquor, carries out purifying with the affinity chromatography method.Fab is made of complete light chain and Fd, and Fv is made of VH and VL, and the ScFv single-chain antibody is formed by connecting by a connection peptides between VH and the VL, and single domain antibody only is made up of VH.
The present invention provides a kind of kit of realizing the bifunctional genetically engineered antibody immunologic detection method of described husky butanolamine simultaneously, comprises following ingredients:
(1) kit box body;
(2) wrap by the ELISA Plate of husky butanolamine antigen 1;
(3) husky butanolamine bifunctional genetically engineered antibody working fluid, 1 bottle;
(4) husky butanolamine standard solution, totally 6 bottles;
(5) substrate solution, 1 bottle;
(6) substrate buffer solution, 1 bottle;
(7) stop buffer, 1 bottle;
(8) concentrated cleaning solution, 1 bottle;
(9) operation instructions, 1 part;
(10) cover plate film, 2;
(11) contain the valve bag of drying agent, 1.
When enzyme is horseradish peroxidase, described substrate solution is for containing 3,3,5, the pH5.0 phosphoric acid-citric acid solution of 5-tetramethyl benzidine or o-phenylenediamine, described substrate buffer solution are that the pH5.0 phosphoric acid-citric acid solution that contains hydrogen peroxide or urea peroxide, described stop buffer are 1～2mol/L sulfuric acid solution; When enzyme was alkaline phosphatase, substrate solution was that 4-nitrophenols sodium ascorbyl phosphate (PNPP) diethanolamine solution, the stop buffer of pH9.8 is the sodium hydroxide solution of 2mol/L.
Described 6 bottles of husky butanolamine standard solution concentration are respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L and 8.1 μ g/L; Described concentrated cleaning solution is the phosphate buffer that contains 0.5～1.5% polysorbas20, and phosphate buffer pH value is 7.4, and concentration is 0.1mol/L, is 15～25 times of existing conventional method working concentration; Described husky butanolamine bifunctional genetically engineered antibody working fluid volume is 7mL; Husky butanolamine standard solution 1mL/ bottle; The substrate solution volume is 7mL; The substrate buffer solution volume is 7mL; The stop buffer volume is 7mL; The concentrated cleaning solution volume is 50mL.
Described box body is a carton box; ELISA Plate is the polystyrene ELISA Plate in 96 holes, is put in the aluminide-coating bag; The cover plate film is the opaque plastics paster; Standard solution, husky butanolamine bifunctional genetically engineered antibody, substrate solution, substrate buffer solution, stop buffer and concentrated cleaning solution adopt the transparent plastic bottle packing of different colours lid; Be provided with interior pad in the box, interior pad is that foams plastic material is made, and is provided with recessed bottle position.
The preparation method of described kit may further comprise the steps:
(1) the husky butanolamine genetic engineering antibody of preparation;
(2) preparation ELISA Plate;
(3) prepare required solution and packing as requested;
(4) assembling kit.
The method of the husky butanolamine genetic engineering antibody of described preparation may further comprise the steps:
(a) RNA of extraction salbutamol monoclonal cell or the mouse boosting cell after the salbutamol immunogen immune, reverse transcription is cDNA, the amplimer of designerantibodies weight chain and connection peptides, utilize round pcr to amplify the weight chain gene of antibody and connect, be inserted into suitable expression plasmid, at expression in escherichia coli, utilize immune affine method to carry out purifying, purity is identified by the SDS-PAGE electrophoresis;
(b) be template with recombinant phage positive colony plasmid, pcr amplification desertification butylamine alcohol gene engineered antibody fragment; Cut through enzyme, purifying, subclone be to the carrier pPhoA (+) that carries horseradish peroxidase HRP or alkaline phosphatase AP gene, transformed into escherichia coli, and resistance screening obtains the recombination bacillus coli positive colony; Induce the expression of difunctional gene antibody; The optimization expression condition, efficiently express the salbutamol bifunctional genetically engineered antibody.
Described amplimer is:
V
H(Back)：5’-TTACTCGCGGCCCAGCCGCCATGGCCCAGGTSMARCTGCAGSAGTCWGG-3’；
V
H(For)：5’-GCCAGAGCCACCTCCGCCTGAACCGTCCACCTGAGGAGACGGTGAC?CGTGGTGCC-3’；
V
L(Back)：5’-TCAGGCGGAGGTGGCTCTGGCGGTGGGGATCGGACATTGAG?CTC?ACC?CAG?TCTCCA-3’；
V
L(For)：5’-GAGTCATTCTGCGGCCGCCCGTTTTATTTCCAGCTTGGTCCC-3’；
R
1：5’-CCATGATTACGCCAAGCTTTGGAGCC-3’；
R
2：5’-CGATCTAAAGTTTTGTCGTCTTTCC-3’；
Primer V wherein
H(Back) contain Sfi I restriction enzyme site, V
H(For) contain (Gly4Ser)
3The connection peptides partial sequence; V
L(For) contain Not I restriction enzyme site, V
L(Back) contain (Gly
4Ser)
3Connection peptides partial sequence, wherein 21 bases and V
H(For) partial sequence overlaid, R
1, R
2Be the carrier specificity primer, be used to insert the PCR evaluation of fragment.
The described method for preparing ELISA Plate of step (2) is to be cushioned liquid with bag salbutamol antigen is diluted on demand, adds antigenic dilution in the elisa plate micropore, puts into 37 ℃ of environment and hatches, and puts into 4 ℃ of environment night incubation again; The coating buffer that inclines washs with cleansing solution; In every hole, add confining liquid, hatch for 37 ℃; The liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
Kit lowest detection of the present invention is limited to 0.1ng/mL, the recovery is 95 ± 15%, the variation within batch coefficient is less than 10%, the cross reaction of analogues such as same Clenbuterol, Ractopamine, salbutamol, Mabuterol, Arubendol, adrenaline, norepinephrine is all less than 0.01%, and kit can be preserved 12 months at 2～8 ℃.
The invention has the beneficial effects as follows:
(1) the invention provides the new method that the enzyme immunoassay technology further is applied to the fast detecting of salbutamolum residue, the sex change of avoiding traditional chemical covalent cross-linking process that enzyme and antibody are caused can both activity of fine maintenance; The differences between batches of having avoided the inhomogeneity of enzyme labelled antibody batch product to cause; Thereby the step that has reduced enzyme and antibody coupling front and back product purification has reduced the detection cost;
(2) bifunctional genetically engineered antibody can be expressed in various expression systems, and can produce in a large number by technique for gene engineering, and nutrient culture media is not had specific (special) requirements, and fermentation density height, fermentation period weak point can reduce manufacturing cost;
(3) kit provided by the invention adopts direct competitive ELISA detecting pattern, has reduced operation steps, has improved the sensitivity, the accuracy that detect; Adopt envelope antigen to carry out the bag quilt of ELISA Plate, with respect to antibody sandwich, more help reaching and wrap preferably, thereby improved precision, accuracy and stability that kit detects by effect and long holding time.
Be highly suitable for the trace analysis and the batch detection of salbutamolum residue based on above this kit of advantage, have important practical significance.
Description of drawings
Fig. 1 is a typical curve
Fig. 2 is the synoptic diagram directly perceived of kit
Fig. 3 is the ELISA Plate schematic appearance
Fig. 4 is an ELISA Plate
Fig. 5 is the reagent bottle synoptic diagram
Fig. 6 is the kit package material
Fig. 7 kit annex
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.
The preparation of embodiment 1 salbutamol bifunctional genetically engineered antibody
The design degenerate primer:
V
H(Back)：5’-TTACTCGCGGCCCAGCCGCCATGGCCCAGGTSMARCTGCAGSAGTCWGG-3’；
V
H(For)：5’-GCCAGAGCCACCTCCGCCTGAACCGTCCACCTGAGGAGACGGTGAC?CGTGGTGCC-3’；
V
L(Back)：5’-TCAGGCGGAGGTGGCTCTGGCGGTGGGGATCGGACATTGAG?CTC?ACC?CAG?TCTCCA-3’；
V
L(For)：5’-GAGTCATTCTGCGGCCGCCCGTTTTATTTCCAGCTTGGTCCC-3’；
R
1：5’-CCATGATTACGCCAAGCTTTGGAGCC-3’；
R
2：5’-CGATCTAAAGTTTTGTCGTCTTTCC-3’；
Primer V wherein
H(Back) contain Sfi I restriction enzyme site, V
H(For) contain (Gly4Ser)
3The connection peptides partial sequence; V
L(For) contain Not I restriction enzyme site, V
L(Back) contain (Gly
4Ser)
3Connection peptides partial sequence, wherein 21 bases and V
H(For) partial sequence overlaid, R
1, R
2Be the carrier specificity primer, be used to insert the PCR evaluation of fragment.
With synthetic salbutamol artificial antigen animal has been carried out immunity, detected the serum titer titre and reach requirement; Get the sensitized animal spleen, extract total RNA, utilize the degenerate primer that designs, heavy chain variable region gene light through the complete set that RT-PCR has successfully amplified immune mouse,, the amplification condition of light chain is: 94 ℃ * 5min sex change, add 1 μ L high-fidelity Pfu enzyme (2.5U), carry out following circulation: 94 ℃ * 30s, 54 ℃ * 1min, 72 ℃ * 1min, totally 25 circulations, last 72 ℃ are extended 10min.The amplification condition of heavy chain is: 94 ℃ * 5min sex change, add 1 μ L high-fidelity Pfu enzyme (2.5U), and carry out following circulation: 94 ℃ * 30s, 60 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, last 72 ℃ are extended 10min.V
HThe genetic fragment size is about 350bp, V
LThe genetic fragment size is 320bp.With the V that amplifies
H, V
LFragment is template each other, adopts to contain (Gly
4Ser)
3The joint primer of connection peptides, overlap extension PCR method synthetic antibody genetic fragment, and adopting two upstream and downstream primer antagonists that contain Bgl I and Not I restriction enzyme site to carry out the secondary PCR amplification, condition is the pre-sex change of 94 ℃ * 5min, adds 0.5 μ L high-fidelity Pfu enzyme, carry out following circulation: 94 ℃ * 45s, 50 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, 72 ℃ are extended 10min, obtain the full length antibody genetic fragment of 760bp.Behind antibody fragment usefulness Bgl I and Not I double digestion, pCANTAB5E is connected with carrier, and transformed into escherichia coli TG1 uses helper phage M
13KO
7Carry out superinfection, make up phage antibody library.Antibody library storage capacity is about 1.6 * 10
4Adopt the solid phase antigen method,, phage antibody library is carried out affine enrichment, filter out 15 positive recombinant phage clones through ELISA with salbutamol envelope antigen coated elisa plate.
The positive colony that will filter out has antibody activity extracts plasmid, and plasmid is connected with the carrier pPhoA (+) that cuts through same enzyme after with Bgl I and Not I double digestion, connects product transformed into escherichia coli resistance screening positive colony, and passes through that enzyme is cut, the PCR evaluation.Be accredited as from above among the positive clone and extract plasmid DNA, transform amber and stop non-inhibition type Escherichia coli HB2151, IPTG induces desertification butylamine alcohol bifunctional antibody to carry out solubility expression.Employing osmotic shock method has been extracted the solubility bifunctional antibody in the somatic cells pericentral siphon chamber, and the antibody of expressing in culture supernatant and the pericentral siphon chamber extract carried out SDS-PAGE, Western-Blotting and ELISA identify, and utilize affinity chromatography that it is carried out purifying.
The preparation of embodiment 2 enzyme linked immunological kit components
(1) preparation of thickening and washing damping fluid: contain the phosphate buffer of 0.5～1.5% polysorbas20, phosphate buffer pH7.4, concentration is 0.1mol/L, is 15～25 times of existing conventional method working concentration.
(2) preparation of confining liquid: skimmed milk power 1.0～5.0g is dissolved in 100mL distilled water.
(3) preparation of substrate buffer solution: 30% hydrogen peroxide, 30 μ L are dissolved in pH5.0 phosphoric acid-citrate buffer solution of 19mL 4 ℃ of preservations.Phosphoric acid-citrate buffer solution adopts 0.2MNa
2HPO
425.7mL, 0.1M citric acid 24.3mL, adding distil water 50mL be formulated.
(4) preparation of substrate solution: substrate solution is with 3,3,5 when enzyme is horseradish peroxidase (HRP), and 5-tetramethyl benzidine (TMB) 80mg is dissolved in 10mL pH5.0 phosphoric acid-citrate buffer solution, 4 ℃ of preservations.Described phosphoric acid-citrate buffer solution adopts 0.2MNa
2HPO
425.7mL, 0.1M citric acid 24.3mL, adding distil water 50mL) formulated.Substrate solution is 4-nitrophenols sodium ascorbyl phosphate (PNPP) 100mg to be dissolved in the diethanolamine buffer of 100mL pH9.8 4 ℃ of preservations when enzyme is alkaline phosphatase (AP).Diethanolamine buffer adopts diethanolamine 97mL, Sodium azide 0.2g to be dissolved in the 1000mL distilled water, regulates pH value to 9.8 with concentrated hydrochloric acid, and is stored in 4 ℃.
(5) the bag quilt of ELISA Plate microwell plate: envelope antigen pH9.6,0.05mol/L carbonate buffer solution be diluted to 0.1～5ug/mL, every hole in ELISA Plate adds 100uL, 37 ℃ of bags by 1h after 4 ℃ down bag spent the night, the coating buffer that inclines is with PBST washing 3 times, pat dry, add 200uL1.0～5.0% skimmed milk power then in every hole, put into behind 37 ℃ of incubator 1h with PBST washing 3 times, 4 ℃ of preservations in the aluminium foil bag are enclosed in dry back.It is formulated that carbonate buffer solution adopts 1～2g sodium carbonate and 2～4g sodium bicarbonate to be dissolved in distilled water 1L.
(6) preparation of salbutamol standard solution: accurately take by weighing salbutamol standard specimen 8.1mg, be dissolved in the 0.1L damping fluid, prepare 8.1 μ g/L, 2.7 μ g/L, 0.9 μ g/L, 0.3 μ g/L, 0.1 μ g/L salbutamol solution respectively with the damping fluid dilution then, damping fluid is prepared 0 μ g/L in the same old way in addition, 4 ℃ of preservations.
(7) reagent packing: all ingredients is prepared on request, and it is aseptic subpackaged to measure qualified back.Salbutamol bifunctional genetically engineered antibody working fluid 7mL/ bottle, salbutamol standard model 1mL/ bottle, substrate solution 7mL/ bottle, substrate buffer solution 7mL/ bottle, stop buffer 7mL/ bottle concentrates washing lotion 50mL/ bottle, concentrating sample dilution 50mL/ bottle.Label after the packing, indicate the lot number and the term of validity, 4 ℃ of preservations.
(8) assembling of kit: will detachably wrap respectively by 1 of the microwell plate of good envelope antigen, respectively 1 bottle of salbutamol bifunctional genetically engineered antibody working fluid, substrate solution, substrate buffer solution, stop buffer, concentrating sample cleansing solution, 6 bottles of salbutamol standard solution, operation instructions are put assigned address in the kit for 1 part.Kit encapsulates after the assay was approved, 4 ℃ of preservations.Concrete assembling example is seen embodiment 3.
The kit of the detection salbutamol of setting up is shown in accompanying drawing 2～accompanying drawing 7, and kit comprises following component:
(1) kit box body; Can adopt carton box;
(2) wrap by the ELISA Plate of salbutamol antigen 96 holes;
(3) salbutamol bifunctional genetically engineered antibody working fluid, 7mL/ bottle, green lid;
(4) the salbutamol standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 1mL/ bottle, red lid;
(5) substrate solution, 7mL/ bottle, black lid;
(6) substrate buffer solution, 7mL/ bottle, white lid;
(7) stop buffer, 7mL/ bottle, yellow lid;
(8) concentrated cleaning solution, 50mL/ bottle, white transparency cover;
(9) operation instructions, 1 part;
(10) cover plate film, 2;
(11) contain the valve bag of drying agent, 1.
Wherein, in the accompanying drawing 21: the salbutamol standard solution; 2: salbutamol bifunctional genetically engineered antibody working fluid; 3: bag is by the ELISA Plate of salbutamol antigen; 4: operation instructions; 5:20 times of concentrated cleaning solution; 6: substrate buffer solution; 7: substrate solution; 8: stop buffer; 9: valve bag; 10: the cover plate film; In the accompanying drawing 3 11: aluminium foil bag; 12:96 hole ELISA Plate sheet frame in the accompanying drawing 4; 13:96 hole removable enzyme mark bar; 14:20 times of concentrated cleaning solution (transparent white lid) in the accompanying drawing 5; 15: enzyme labeling thing (green lid); 16: substrate solution (black lid); 17: substrate buffer solution (white lid); 18: stop buffer (yellow lid); 19: standard solution (6 bottles of red lids); In the accompanying drawing 6 20: the standard items shrinkage pool; 21: the stop buffer shrinkage pool; 22: the substrate solution shrinkage pool; 23: the substrate buffer solution shrinkage pool; 24: enzyme labeling thing shrinkage pool; 25:20 times of concentrate shrinkage pool; 26: outer packing box; In the accompanying drawing 7 27: cover plate film (2); 28: valve bag (band drying agent); 29: instructions.
One, sample pre-treatments
A. urine specimen is handled
Limpid urine sample can directly carry out check and analysis.If urine sample is muddy shape, centrifugal 5min of 2000g or filtration detect with supernatant.
B. feed
Feed is pulverized, and sample thief 2g adds 20mL 0.1mol/L HCl solution, vibration 30min or use Extraction by Ultrasound 15min, every 5min slightly vibrates 1 time, then the centrifugal 10min of 4000r/min, get supernatant and regulate pH value to 10 with 1mol/L NaOH, add the extraction of 20mL isobutyl alcohol, tell organic phase, the water-bath evaporated under reduced pressure, dissolve with the 10mL sample diluting liquid, get 50 μ L and be used for detecting, extension rate is 5, considers when calculating.
C. organize, for example muscle, liver, kidney etc.
Tissue sample is shredded, sample thief 5g adds 20mL 0.1mol/L HCl solution, vibration 30min or use Extraction by Ultrasound 15min, the slight vibration of every 5min 1 time, the centrifugal 10min of 4000r/min gets supernatant and regulates pH value to 10 with 1mol/L NaOH then, adds the extraction of 20mL isobutyl alcohol, tell organic phase, the water-bath evaporated under reduced pressure with the dissolving of 5mL sample diluting liquid, is got 50 μ L and is used for detecting.
Two, detection method
(1) kit is taken out from cold storage environment, place room temperature (20～24 ℃) more than the balance 30min, the batten of enough standards and the used quantity of sample is fixed in support, standard and sample are done two parallel laboratory tests, number in order.
(2) add 50 μ L standard items in the standard items hole, sample well adds 50 μ L testing samples.Every then hole adds 50 μ L bifunctional antibodies, pats mixing.Cover the cover plate film, at incubated at room 20min.
(3) pour out liquid in the hole, the micropore frame is upside down on the thieving paper pats (every take turns wash plate pat 3 times) to guarantee to remove fully the liquid in the hole.Charge in the hole with 250 μ L distilled water, outwell the liquid in the micropore once more, repetitive operation is 3 times again.
(4) every hole adds 100 μ L colour developing liquid, pats mixing, covers the cover plate film, dark place incubated at room 15min.
(5) add 50 μ L reaction terminating liquids in micropore.Mixing at wavelength 450nm, is blank with the air, measures each hole light absorption value, must read light absorption value in the 60min after adding stop buffer.
Three, testing result is calculated and is analyzed:
Mean value calculation percentage absorbance with obtained standard model light absorption value, with the percentage absorbance is ordinate, the semilog of salbutamol concentration of standard solution is a horizontal ordinate drawing standard curve, obtains straight-line equation, and straight-line equation is y=-14.407x+87.317.The use the same method percentage absorbance of calculation sample solution is obtained the salbutamol concentration of counter sample according to equation.The calculating formula of described percentage absorbance is:
Percentage absorbance (%)=(B/B
0) * 100
Wherein, B is the average light absorption value of standard solution or sample, B
0It is the mean light absorbency value of 0 μ g/L standard solution.
The analysis of testing result can also utilize computer professional software to calculate and analyze, and is 0.1～8.1 μ g/L to the salbutamol linear detection range, detects and is limited to 0.1 μ g/L, and whole testing process only needs 35min just can finish.
1, standard solution replica test
From 3 batches of ELISA Plate according to the preparation of the method the embodiment 2 (6), each extracts 20 micropores out, measures the absorbance (OD value) of 0.9 μ g/L standard solution, repeats 20 times, calculates coefficient of variation CV%, the results are shown in Table 1.
Table 1 standard solution replica test
The result shows the variation within batch coefficient scope of kit standard items detection between 3.8～4.9%, and interassay coefficient of variation is 8.8%.
2, sample repeatability and accuracy test
Accuracy is meant the matching degree of measured value and true value, and in ELISA measured, accuracy often represented with the recovery that precision is often represented with the coefficient of variation.In blank pig urine, pork liver, it is 1 μ g/L (μ g/kg), 5 μ g/L (μ g/kg) that salbutamol is added into final concentration, and in blank feed, it is 50 μ g/kg, 100 μ g/kg that salbutamol is added into final concentration, each 10 of each concentration are parallel, measure 3 batches.Calculating mean value, the interpolation recovery and batch interior and interassay coefficient of variation.The results are shown in Table 2.
Table 2 sample repeatability and accuracy test result
The result shows the interpolation recovery of urine sample, pork liver, feed sample between 80.9～106.0%, and the variation within batch coefficient is between 3.2～9.1%, and interassay coefficient of variation is between 11.2～14.8%.
The test of embodiment 6 storage lives
(1) kit is positioned over 2～8 ℃, get 0,2,4,6,8,9,10,11 and 12 months kit respectively, to absorbance, 50% inhibition concentration of salbutamol standard model (0.1 μ g/L), add the recovery, each parameter of variation within batch coefficient is measured.
(2) kit was placed 12 days under the condition of 37 ℃ of preservations, measure absorbance, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of salbutamol standard model (0.1 μ g/L) every day.
(3) kit was preserved 12 days at-20 ℃ of refrigerators, measure absorbance, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of salbutamol standard model (0.1 μ g/L) every day.
Can find out that from the result preserve test through three kinds of conditions, the absorbance of salbutamol standard model (0.1 μ g/L) descends less than 5%, and OD is not less than 1.5; 50% inhibiting rate is between 0.6～1.0 μ g/L; Add the recovery between 80～110%; The variation within batch coefficient is less than 10%; Every index all conforms to quality requirements, and therefore, kit can be preserved 12 months at 2～8 ℃.
Claims (10)
1, a kind of enzyme-linked immune detection method of husky butanolamine is characterized in that may further comprise the steps:
(1) husky butanolamine is antigen coated on solid phase carrier;
(2) add standard specimen or testing sample, add husky butanolamine bifunctional genetically engineered antibody again, the reaction back adds substrate solution develops the color, and measures the percentage absorbance;
(3) according to the semilog between percentage absorbance and husky butanolamine concentration relation map typical curve, according to the typical curve of husky butanolamine and the percentage absorbance of sample to be checked, extrapolate the concentration of husky butanolamine in the testing sample.
2, according to the enzyme-linked immune detection method of the described husky butanolamine of claim 1, it is characterized in that the described solid phase carrier of step (1) is 96 holes or 40 hole ELISA Plate, be coated with can with the salbutamol antigen of salbutamol bifunctional genetically engineered antibody specific bond, and closed porosity surface adsorption site not; Described husky butanolamine bifunctional genetically engineered antibody is horseradish peroxidase or alkaline phosphatase salbutamol single-chain bifunctional genetic engineering antibody, and genetic engineering antibody is Fab, Fv, ScFv or single domain antibody.
3, a kind of kit of realizing the enzyme-linked immune detection method of the described husky butanolamine of claim 1 is characterized in that comprising following ingredients:
(1) kit box body;
(2) wrap by the ELISA Plate of husky butanolamine antigen 1;
(3) husky butanolamine bifunctional genetically engineered antibody working fluid, 1 bottle;
(4) husky butanolamine standard solution, totally 6 bottles;
(5) substrate solution, 1 bottle;
(6) substrate buffer solution, 1 bottle;
(7) stop buffer, 1 bottle;
(8) concentrated cleaning solution, 1 bottle;
(9) operation instructions, 1 part;
(10) cover plate film, 2;
(12) contain the valve bag of drying agent, 1.
4, kit according to claim 3, it is characterized in that when enzyme is horseradish peroxidase, described substrate solution is for containing 3,3,5, the pH5.0 phosphoric acid-citric acid solution of 5-tetramethyl benzidine or o-phenylenediamine, described substrate buffer solution are that the pH5.0 phosphoric acid-citric acid solution that contains hydrogen peroxide or urea peroxide, described stop buffer are 1～2mol/L sulfuric acid solution; When enzyme was alkaline phosphatase, substrate solution was that 4-nitrophenols sodium ascorbyl phosphate diethanolamine solution, the stop buffer of pH9.8 is the sodium hydroxide solution of 2mol/L.
5, kit according to claim 3 is characterized in that described 6 bottles of husky butanolamine standard solution concentration are respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L and 8.1 μ g/L; Described concentrated cleaning solution is the phosphate buffer that contains 0.5～1.5% polysorbas20, and phosphate buffer pH value is 7.4, and concentration is 0.1mol/L; Described husky butanolamine bifunctional genetically engineered antibody working fluid volume is 7mL; Husky butanolamine standard solution 1mL/ bottle; The substrate solution volume is 7mL; The substrate buffer solution volume is 7mL; The stop buffer volume is 7mL; The concentrated cleaning solution volume is 50mL.
6, kit according to claim 3 is characterized in that described box body is a carton box; ELISA Plate is the polystyrene ELISA Plate in 96 holes, is put in the aluminide-coating bag; The cover plate film is the opaque plastics paster; Standard solution, husky butanolamine bifunctional genetically engineered antibody, substrate solution, substrate buffer solution, stop buffer and concentrated cleaning solution adopt the transparent plastic bottle packing of different colours lid; Be provided with interior pad in the box, interior pad is that foams plastic material is made, and is provided with recessed bottle position.
7, the preparation method of the described kit of a kind of claim 3 is characterized in that may further comprise the steps:
(1) the husky butanolamine genetic engineering antibody of preparation;
(2) preparation ELISA Plate;
(3) prepare required solution and packing as requested;
(4) assembling kit.
8,, it is characterized in that the husky butanolamine genetic engineering antibody of the described preparation of step (1) may further comprise the steps according to the described preparation method of claim 7:
(a) RNA of extraction salbutamol monoclonal cell or the mouse boosting cell after the salbutamol immunogen immune, reverse transcription is cDNA, the amplimer of designerantibodies weight chain and connection peptides, utilize round pcr to amplify the weight chain gene of antibody and connect, be inserted into suitable expression plasmid, at expression in escherichia coli, utilize immune affine method to carry out purifying;
(b) be template with recombinant phage positive colony plasmid, pcr amplification desertification butylamine alcohol gene engineered antibody fragment; Cut through enzyme, purifying, subclone be to the carrier pPhoA (+) that carries horseradish peroxidase HRP or alkaline phosphatase AP gene, transformed into escherichia coli, and resistance screening obtains the recombination bacillus coli positive colony; Induce the expression of difunctional gene antibody; The optimization expression condition, efficiently express the salbutamol bifunctional genetically engineered antibody.
9, described according to Claim 8 preparation method is characterized in that described amplimer is:
V
H(Back)：5’-TTACTCGCGGCCCAGCCGCCATGGCCCAGGTSMARCTGCAGSAGTCWGG-3’；
V
H(For)：5’-GCCAGAGCCACCTCCGCCTGAACCGTCCACCTGAGGAGACGGTGAC?CGTGGTGCC-3’；
V
L(Back)：5’-TCAGGCGGAGGTGGCTCTGGCGGTGGGGATCGGACATTGAGCTCACCCAGTCTCCA-3’；
V
L(For)：5’-GAGTCATTCTGCGGCCGCCCGTTTTATTTCCAGCTTGGTCCC-3’；
R
1：5’-CCATGATTACGCCAAGCTTTGGAGCC-3’；
R
2：5’-CGATCTAAAGTTTTGTCGTCTTTCC-3’；
Primer V wherein
H(Back) contain Sfi I restriction enzyme site, V
H(For) contain (Gly4Ser)
3The connection peptides partial sequence; V
L(For) contain Not I restriction enzyme site, V
L(Back) contain (Gly
4Ser)
3Connection peptides partial sequence, wherein 21 bases and V
H(For) partial sequence overlaid, R
1, R
2Be the carrier specificity primer, be used to insert the PCR evaluation of fragment.
10, according to the described preparation method of claim 7, it is characterized in that the described method for preparing ELISA Plate of step (2) is to be cushioned liquid with bag salbutamol antigen is diluted on demand, in the elisa plate micropore, add antigenic dilution, put into 37 ℃ of environment and hatch, put into 4 ℃ of environment night incubation again; The coating buffer that inclines washs with cleansing solution; In every hole, add confining liquid, hatch for 37 ℃; The liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100286361A CN101290317A (en) | 2008-06-06 | 2008-06-06 | Salbutamolum ELISA method and reagent kit and method for making same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100286361A CN101290317A (en) | 2008-06-06 | 2008-06-06 | Salbutamolum ELISA method and reagent kit and method for making same |
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| Publication Number | Publication Date |
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| CN101290317A true CN101290317A (en) | 2008-10-22 |
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|---|---|---|---|
| CNA2008100286361A Pending CN101290317A (en) | 2008-06-06 | 2008-06-06 | Salbutamolum ELISA method and reagent kit and method for making same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608310A (en) * | 2012-02-21 | 2012-07-25 | 天津康利缘生物工程有限公司 | Salbutamol enzyme linked immunoassay kit and use method thereof |
| CN102768278A (en) * | 2012-05-31 | 2012-11-07 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant |
| CN103018447A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for salbutamol detection, and applications thereof |
| CN113651890A (en) * | 2021-07-05 | 2021-11-16 | 新乡学院 | anti-SAL single-chain antibody, and screening method and application thereof |
-
2008
- 2008-06-06 CN CNA2008100286361A patent/CN101290317A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103018447A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for salbutamol detection, and applications thereof |
| CN103018447B (en) * | 2011-09-20 | 2016-01-20 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and the method thereof of salbutamol |
| CN102608310A (en) * | 2012-02-21 | 2012-07-25 | 天津康利缘生物工程有限公司 | Salbutamol enzyme linked immunoassay kit and use method thereof |
| CN102768278A (en) * | 2012-05-31 | 2012-11-07 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant |
| CN113651890A (en) * | 2021-07-05 | 2021-11-16 | 新乡学院 | anti-SAL single-chain antibody, and screening method and application thereof |
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