CN103421108A - Method of synthesizing tetracycline coupled antigen - Google Patents
Method of synthesizing tetracycline coupled antigen Download PDFInfo
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- CN103421108A CN103421108A CN2013103508321A CN201310350832A CN103421108A CN 103421108 A CN103421108 A CN 103421108A CN 2013103508321 A CN2013103508321 A CN 2013103508321A CN 201310350832 A CN201310350832 A CN 201310350832A CN 103421108 A CN103421108 A CN 103421108A
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Abstract
The invention belongs to the technical field of biological detection and particularly relates to a method of synthesizing tetracycline coupled antigen. The method includes the steps of a, derivatizing tetracycline; b, coupling the tetracycline to protein; c, purifying coupled antigen; d, verifying the coupled antigen. The method of efficiently synthesizing tetracycline coupled antigen has the advantages that the method is simple in process, easy to operate, free of pollution, high in synthetic rate and high in valence; the development and production of immune products for detecting tetracycline residue can be more efficient, the cost is saved greatly, and the cost and price in the detection of immune products can be reduced.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of method of synthetic tsiklomitsin coupled antigen.
Background technology
Since 1948 come out, tsiklomitsin is applied to clinical successively, and become application at most, the most widely broad-spectrum antibiotics.Now existing great mass of data confirmation, prolonged and repeated use tsiklomitsin, can affect growth and the formation of tooth, not only makes the tooth flavescence, also can cause enamel dysplasia or deformity teeth.Tsiklomitsin can cause liver toxicity, is generally the fatty liver sex change, the patient Yi Fasheng liver toxicity of pregnancy women, original kidney function damage.Tsiklomitsin can cause transformation reactions: mostly be maculopapule and erythema, small number of patients can occur that urticaria, vasodilation, anaphylactoid purpura, pericarditis and systemic lupus erythematous fash increase the weight of.In order to control residual in animal food of tetracycline medication, European Union, the U.S. and China have all stipulated that in livestock product, the tetracycline medication maximum residue limit is 0.1mg/Kg.
In the rapid detection animal food, residual many employings of tsiklomitsin are highly sensitive at present, the immunoassay technology that detection time is short, one of core material of this technology is the tsiklomitsin coupled antigen, it is a lot of that the synthetic method of tsiklomitsin coupled antigen is reported, but exist at present synthetic ratio low, the low phenomenon of tiring, must make to detect the residual research and development production and selling cost value of tsiklomitsin higher.
CN1861631 discloses " conjugate of tsiklomitsin and preparation method thereof and application ", the step of the method is: tsiklomitsin and 1-1 '-carbonyl dimidazoles (CDI) are dissolved in anhydrous propanone for the ratio of 1:1-1:3 in molar ratio, pass into nitrogen, under the protection of nitrogen, 37 ℃ of lower lucifuge reaction 2-4 hour, reaction generates the active intermediate of tsiklomitsin and 1-1 '-carbonyl dimidazoles, standby; Simultaneously, the amount that the mole ratio of tsiklomitsin and bovine serum albumin of take is 20:1-35:1, take bovine serum albumin standby; By the rotation of the acetone in above-mentioned reacted solution A evaporate to dryness, add pH=8.5 ± 0.5, the tsiklomitsin that the borax buffer solution that concentration is 0.5M generates and the active intermediate of 1-1 ' carbonyl dimidazoles, and then add above-mentioned bovine serum albumin, the final concentration that makes bovine serum albumin is 10 ± 5mg/ml, under the protection of nitrogen, 20-30 ℃ of lucifuge reaction 30-48 hour, obtain solution B; Use pH7.3-7.5, the phosphoric acid buffer that concentration is 0.01-0.02M, under 0-4 ℃, stirs dialysis solution 70-80 hour, then uses distill water dialysis 20-30 hour instead, within every 6 hours, changes a dialyzate; Solution after freeze-drying dialysis, obtain the conjugate of flaxen tsiklomitsin.The shortcoming of above method is: need to pass into nitrogen protection in building-up process, increased the triviality of synthetic cost and method; Generated time is long, the shortest needs 122 hours.Therefore need to be improved for above-mentioned method, explore a kind of cheaply, can synthesize fast the method for tsiklomitsin coupled antigen.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of cheaply, the method for synthetic tsiklomitsin coupled antigen fast.
The present invention realizes by following technical scheme:
The method of synthetic tsiklomitsin coupled antigen comprises following step:
A. the derivatize of tsiklomitsin
Take tsiklomitsin, add DMF to dissolve, obtain solution 1.;
Take N, the N-carbonyl dimidazoles, add DMF to dissolve, and obtains solution 2.;
By solution 1. slowly be added drop-wise to solution 2. in, stir derivative 1-5 hour under 34-39 ℃;
B. tsiklomitsin and albumen coupling
Take bovine serum albumin, add PBS to dissolve, obtain solution 3., by the tsiklomitsin that in step a, derivatize is good slowly be added drop-wise to solution 3. in, in 34-39 ℃ of stirring reaction 3-8 hour, then 4 ℃ of placements are spent the night; Described placement is spent the night and is referred to placement 8-14 hour;
C. the purifying of coupled antigen
The tsiklomitsin holoantigen that in step b, coupling is good is packed in dialysis tubing, place under 4 ℃, with distill water dialysis 1-3 days, every 4-8 hour, change distilled water once;
D. the evaluation of coupled antigen
Adopt indirect Elisa method, the good tsiklomitsin coupled antigen 0.01M PBS gradient dilution by purifying, described extension rate is 1:2000,1:4000,1:8000,1:16000,1:32000, the 1:64000 coated elisa plate, measure the active of coupled antigen and tire.
Preferably, the method for synthetic tsiklomitsin coupled antigen comprises following step:
A. the derivatize of tsiklomitsin
Take tsiklomitsin 2mg, add 300 μ L DMFs to dissolve, obtain solution 1.;
Take N, N-carbonyl dimidazoles 30mg, add 300 μ L DMFs to dissolve, and obtains solution 2.;
By solution 1. slowly be added drop-wise to solution 2. in, in 37 ℃, stir derivative 3 hours;
B. tsiklomitsin and albumen coupling
Take bovine serum albumin 10mg, add 600 μ L 0.01M PBS to dissolve, obtain solution 3., by the tsiklomitsin that in step a, derivatize is good slowly be added drop-wise to solution 3. in, in 37 ℃ of stirring reactions 5 hours, then 4 ℃ of placements were spent the night; Described placement is spent the night and is referred to placement 8-14 hour;
C. the purifying of coupled antigen
The tsiklomitsin holoantigen that in step b, coupling is good is packed in dialysis tubing, and 4 ℃ of placements, with 500ml distill water dialysis 3 days, changed distilled water once every 6 hours;
D. the evaluation of coupled antigen
By indirect Elisa method, the good tsiklomitsin coupled antigen 0.01M PBS gradient dilution by purifying, described extension rate is 1:2000,1:4000,1:8000,1:16000,1:32000, the 1:64000 coated elisa plate, measure the active of coupled antigen and tire.
Reaction principle of the present invention is as follows:
Method in the present invention's disclosed with CN1861631 " conjugate of tsiklomitsin and preparation method thereof and application " is compared, and the method disclosed in above-mentioned file, need to pass into nitrogen protection in building-up process, increased the synthetic cost of nitrogen protection; By changing reaction solvent, do not need to pass into nitrogen protection in method building-up process of the present invention, reduced synthetic cost;
And method of the present invention is simple, easy handling; Generated time is short, the shortest needs 28 hours, and the longest needs 76 hours, and the method disclosed in above-mentioned file is comparatively loaded down with trivial details; And its generated time is long, the shortest needs 122 hours, the longest needs 162 hours.
Beneficial effect provided by the invention is, synthetic method is simple, easy handling, pollution-free, synthetic ratio is high, the height of tiring, for the development and production that detects the residual immunoassay product of tsiklomitsin has improved efficiency, greatly saved cost, reduced expense and price that immunoassay product detects.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described, so that those skilled in the art more understands the present invention, but does not therefore limit the present invention.
Embodiment 1
(1) derivatize of tsiklomitsin
Take tsiklomitsin 2mg, add 300 μ L DMFs to dissolve, obtain solution 1.;
Take N, N-carbonyl dimidazoles 30mg, add 300 μ L DMFs to dissolve, and obtains solution 2.;
By solution 1. slowly be added drop-wise to solution 2. in, in 37 ℃, stir derivative 3 hours, obtain the tsiklomitsin of derivatize;
(2) tsiklomitsin and albumen coupling
Take bovine serum albumin 10mg, add 600 μ L 0.01M PBS to dissolve, obtain solution 3., by derivatize good tsiklomitsin slowly be added drop-wise to solution 3. in, in 37 ℃ of stirring reactions 5 hours, then place 12 hours for 4 ℃, obtain the tsiklomitsin of coupling;
(3) purifying of coupled antigen
During by coupling, good tsiklomitsin holoantigen is packed dialysis tubing into, 4 ℃ of placements, use 500ml distill water dialysis 72 hours, every 6 hours, changes distilled water once;
(4) evaluation of coupled antigen
By indirect Elisa method, 0.01M PBS gradient dilution (1:2000,1:4000,1:8000,1:16000,1:32000, the 1:64000) coated elisa plate for good tsiklomitsin coupled antigen by purifying, measure the active of coupled antigen and tire.
After measured, the protein concentration of coupled antigen is 8.1mg/mL, and tiring is 1:32000.
In the present embodiment, the synthetic time is 28 hours; Generated time in CN1861631 is 142 hours.
In the present embodiment, need not add nitrogen protection, in CN1861631, add nitrogen protection, thereby increase cost;
The quality of product compares: the protein concentration of coupled antigen is 8.1mg/mL, and tiring is 1:32000, and coated concentration is 0.25 μ g/ml, and the disclosed coated concentration in background technology in CN1861631 is 10 μ g/ml.
The present embodiment is compared with CN1861631, can save 40 times of envelope antigen use costs.
Embodiment 2
(1) derivatize of tsiklomitsin
Take tsiklomitsin 5mg, add 600 μ L DMFs to dissolve, obtain solution 1.; Take N, N-carbonyl dimidazoles 60mg, add 600 μ L DMFs to dissolve, and obtains solution 2.; By solution 1. slowly be added drop-wise to solution 2. in, in 37 ℃, stir derivative 3 hours;
(2) tsiklomitsin and albumen coupling
Take bovine serum albumin 20mg, add 1200 μ L 0.01M PBS to dissolve, obtain solution 3., by derivatize good tsiklomitsin slowly be added drop-wise to solution 3. in, in 37 ℃ of stirring reactions 5 hours, then place 12 hours for 4 ℃;
(3) purifying of coupled antigen
During by coupling, good tsiklomitsin holoantigen is packed dialysis tubing into, 4 ℃ of placements, use 1000ml distill water dialysis 60 hours, every 6 hours, changes distilled water once;
(4) evaluation of coupled antigen
By indirect Elisa method, 0.01M PBS gradient dilution (1:2000,1:4000,1:8000,1:16000,1:32000, the 1:64000) coated elisa plate for good tsiklomitsin coupled antigen by purifying, measure the active of coupled antigen and tire.
After measured, the protein concentration of coupled antigen is 7.1mg/mL, and tiring is 1:32000.
Embodiment 3
(1) derivatize of tsiklomitsin
Take tsiklomitsin 1mg, add 150 μ L DMFs to dissolve, obtain solution 1.; Take N, N-carbonyl dimidazoles 15mg, add 150 μ L DMFs to dissolve, and obtains solution 2.; By solution 1. slowly be added drop-wise to solution 2. in, in 37 ℃, stir derivative 3 hours;
(2) tsiklomitsin and albumen coupling
Take bovine serum albumin (BSA) 5mg, add 300 μ L 0.01M PBS to dissolve, obtain solution 3., by derivatize good tsiklomitsin slowly be added drop-wise to solution 3. in, in 37 ℃ of stirring reactions 5 hours, then place 13 little for 4 ℃.
(3) purifying of coupled antigen
During by coupling, good tsiklomitsin holoantigen is packed dialysis tubing into, 4 ℃ of placements, use 500ml distill water dialysis 60 hours, every 6 hours, changes distilled water once.
(4) evaluation of coupled antigen
By indirect Elisa method, 0.01M PBS gradient dilution (1:2000,1:4000,1:8000,1:16000,1:32000, the 1:64000) coated elisa plate for good tsiklomitsin coupled antigen by purifying, measure the active of coupled antigen and tire.
After measured, the protein concentration of coupled antigen is 7.6mg/mL, and tiring is 1:32000.
Claims (2)
1. synthesize the method for tsiklomitsin coupled antigen, comprise following step:
A. the derivatize of tsiklomitsin
Take tsiklomitsin, add DMF to dissolve, obtain solution 1.;
Take N, the N-carbonyl dimidazoles, add DMF to dissolve, and obtains solution 2.;
By solution 1. slowly be added drop-wise to solution 2. in, stir derivative 1-5 hour under 34-39 ℃, obtain the tsiklomitsin of derivatize;
B. tsiklomitsin and albumen coupling
Take bovine serum albumin, add PBS to dissolve, obtain solution 3.;
By the tsiklomitsin that in step a, derivatize is good slowly be added drop-wise to solution 3. in, in 34-39 ℃ of stirring reaction 3-8 hour, then 4 ℃ of placements are spent the night, and obtain the tsiklomitsin of coupling;
C. the purifying of coupled antigen
The tsiklomitsin holoantigen that in step b, coupling is good is packed in dialysis tubing, place under 4 ℃, with distill water dialysis 1-3 days, every 4-8 hour, change distilled water once;
D. the evaluation of coupled antigen
Adopt indirect Elisa method, the good tsiklomitsin coupled antigen 0.01M PBS gradient dilution by purifying, described extension rate is 1:2000,1:4000,1:8000,1:16000,1:32000, the 1:64000 coated elisa plate, measure the active of coupled antigen and tire.
2. the method for synthetic tsiklomitsin coupled antigen as claimed in claim 1 comprises following step:
A. the derivatize of tsiklomitsin
Take tsiklomitsin 2mg, add 300 μ L DMFs to dissolve, obtain solution 1.;
Take N, N-carbonyl dimidazoles 30mg, add 300 μ L DMFs to dissolve, and obtains solution 2.;
By solution 1. slowly be added drop-wise to solution 2. in, in 37 ℃, stir derivative 3 hours;
B. tsiklomitsin and albumen coupling
Take bovine serum albumin 10mg, add 600 μ L 0.01M PBS to dissolve, obtain solution 3., by derivatize good tsiklomitsin slowly be added drop-wise to solution 3. in, in 37 ℃ of stirring reactions 5 hours, then 4 ℃ of placements were spent the night;
C. the purifying of coupled antigen
During by coupling, good tsiklomitsin holoantigen is packed dialysis tubing into, 4 ℃ of placements, use 500ml distill water dialysis 3 days, every 6 hours, changes distilled water once;
D. the evaluation of coupled antigen
By indirect Elisa method, the good tsiklomitsin coupled antigen 0.01M PBS gradient dilution by purifying, described extension rate is 1:2000,1:4000,1:8000,1:16000,1:32000, the 1:64000 coated elisa plate, measure the active of coupled antigen and tire.
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| CN201310350832.1A CN103421108B (en) | 2013-08-13 | 2013-08-13 | Method of synthesizing tetracycline coupled antigen |
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| CN201310350832.1A CN103421108B (en) | 2013-08-13 | 2013-08-13 | Method of synthesizing tetracycline coupled antigen |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104725505A (en) * | 2015-01-08 | 2015-06-24 | 山东绿都生物科技有限公司 | High-affinity anti-vomitoxin monoclonal antibody and preparation method thereof |
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| CN1811454A (en) * | 2006-01-19 | 2006-08-02 | 镇江出入境检验检疫局检验检疫综合技术中心 | Cyclomycin family antibiotic enzyme-linked immunoassay reagent kit |
| CN1861631A (en) * | 2006-03-14 | 2006-11-15 | 山东大学 | Coupling compound of tetracycline, preparation process and application thereof |
| CN101429244A (en) * | 2008-12-04 | 2009-05-13 | 浙江大学 | Tetracycline and carrier protein couplet product, method for producing tetracycline antibiotic antibody uses thereof |
| CN103102300A (en) * | 2011-11-11 | 2013-05-15 | 北京勤邦生物技术有限公司 | Tetracycline hapten and its preparation method and use |
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2013
- 2013-08-13 CN CN201310350832.1A patent/CN103421108B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1811454A (en) * | 2006-01-19 | 2006-08-02 | 镇江出入境检验检疫局检验检疫综合技术中心 | Cyclomycin family antibiotic enzyme-linked immunoassay reagent kit |
| CN1861631A (en) * | 2006-03-14 | 2006-11-15 | 山东大学 | Coupling compound of tetracycline, preparation process and application thereof |
| CN101429244A (en) * | 2008-12-04 | 2009-05-13 | 浙江大学 | Tetracycline and carrier protein couplet product, method for producing tetracycline antibiotic antibody uses thereof |
| CN103102300A (en) * | 2011-11-11 | 2013-05-15 | 北京勤邦生物技术有限公司 | Tetracycline hapten and its preparation method and use |
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| 刘兴泉等: "N，N-二甲基甲酰胺的生产与应用", 《化工科技》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104725505A (en) * | 2015-01-08 | 2015-06-24 | 山东绿都生物科技有限公司 | High-affinity anti-vomitoxin monoclonal antibody and preparation method thereof |
| CN104725505B (en) * | 2015-01-08 | 2018-01-19 | 山东绿都生物科技有限公司 | A kind of high-affinity emesis toxin monoclone antibody and preparation method thereof |
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