CN103421108B - Method of synthesizing tetracycline coupled antigen - Google Patents
Method of synthesizing tetracycline coupled antigen Download PDFInfo
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- CN103421108B CN103421108B CN201310350832.1A CN201310350832A CN103421108B CN 103421108 B CN103421108 B CN 103421108B CN 201310350832 A CN201310350832 A CN 201310350832A CN 103421108 B CN103421108 B CN 103421108B
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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 synthesizing tsiklomitsin coupled antigen.
background technology
Since coming out from 1948, tsiklomitsin is applied to clinical successively, and become application at most, the most widely broad-spectrum antibiotics.Existing great mass of data confirms now, and prolonged and repeated use tsiklomitsin, can affect growth and the formation of tooth, not only makes tooth turn yellow, also can cause enamel dysplasia or deformity teeth.Tsiklomitsin can cause liver toxicity, is generally 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, and 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 tetracycline medication remaining in animal food, it is 0.1mg/Kg that European Union, the U.S. and China all define tetracycline medication maximum residue limit in livestock product.
In current rapid detection animal food, residual many employings of tsiklomitsin are highly sensitive, the immunoassay technology that detection time is short, one of core material of this technology is tsiklomitsin coupled antigen, tsiklomitsin coupled antigen synthetic method report a lot, but it is low to there is synthetic ratio at present, to tire low phenomenon, must make the development & production of detection tetracycline residue and cost of goods sold on the high side.
CN1861631 discloses " conjugate of tsiklomitsin and preparation method thereof and application ", the step of the method is: the ratio being 1:1-1:3 in molar ratio by tsiklomitsin and 1-1 '-carbonyl dimidazoles (CDI) is dissolved in anhydrous propanone, pass into nitrogen, under the protection of nitrogen, lucifuge reaction 2-4 hour at 37 DEG C, reaction generates the active intermediate of tsiklomitsin and 1-1 '-carbonyl dimidazoles, for subsequent use; Meanwhile, the amount being 20:1-35:1 with the mole ratio of tsiklomitsin and bovine serum albumin, takes bovine serum albumin for subsequent use; Acetone in above-mentioned reacted solution A is rotated evaporate to dryness, add pH=8.5 ± 0.5, concentration is the tsiklomitsin of borax buffer solution generation and the active intermediate of 1-1 ' carbonyl dimidazoles of 0.5M, and then add above-mentioned bovine serum albumin, the final concentration of bovine serum albumin is made to be 10 ± 5mg/ml, under the protection of nitrogen, 20-30 DEG C of lucifuge reaction 30-48 hour, obtains solution B; With pH7.3-7.5, concentration be the phosphoric acid buffer of 0.01-0.02M at 0-4 DEG C, stir dialysis solution 70-80 hour, then use distill water dialysis 20-30 hour instead, within every 6 hours, change a dialyzate; Solution after freeze-drying dialysis, obtains the conjugate of flaxen tsiklomitsin.The shortcoming of above method is: need in building-up process to pass into nitrogen protection, adds the triviality of synthesis cost and method; Generated time is long, the shortest needs 122 hours.Therefore need to improve for above-mentioned method, explore a kind of low cost, can the method for Fast back-projection algorithm tsiklomitsin coupled antigen.
Summary of the invention
In order to solve above-mentioned technical problem, a kind of method that the invention provides low cost, that synthesize tsiklomitsin coupled antigen fast.
The present invention is realized by following technical scheme:
The method of synthesis tsiklomitsin coupled antigen, comprises following step:
A. the derivatize of tsiklomitsin
Take tsiklomitsin, add DMF and dissolve, obtain solution 1.;
Take N, N-carbonyl dimidazoles, add DMF and dissolve, obtain solution 2.;
1. solution is slowly added drop-wise to solution 2. in, at 34-39 DEG C, stir derivative 1-5 hour;
B. tsiklomitsin and albumen coupling
Take bovine serum albumin, add PBS dissolve, obtain solution 3., tsiklomitsin good for derivatize in step a is slowly added drop-wise to solution 3. in, in 34-39 DEG C of stirring reaction 3-8 hour, then 4 DEG C 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
Tsiklomitsin holoantigen good for coupling in step b is loaded in dialysis tubing, place at 4 DEG C, with distill water dialysis 1-3 days, changed distilled water once every 4-8 hour;
D. the qualification of coupled antigen
Adopt indirect Elisa method, by tsiklomitsin coupled antigen 0.01M PBS gradient dilution good for purifying, described extension rate is 1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 coated elisa plate, measures the activity of coupled antigen and tires.
Preferably, the method for synthesis tsiklomitsin coupled antigen, comprises following step:
A. the derivatize of tsiklomitsin
Take tsiklomitsin 2mg, add 300 μ L DMFs and dissolve, obtain solution 1.;
Take N, N-carbonyl dimidazoles 30mg, add 300 μ L DMFs and dissolve, obtain solution 2.;
1. solution is slowly added drop-wise to solution 2. in, in 37 DEG C, stir derivative 3 hours;
B. tsiklomitsin and albumen coupling
Take bovine serum albumin 10mg, add 600 μ L 0.01M PBS and dissolve, obtain solution 3., tsiklomitsin good for derivatize in step a is slowly added drop-wise to solution 3. in, in 37 DEG C of stirring reactions 5 hours, then 4 DEG C 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
Tsiklomitsin holoantigen good for coupling in step b is loaded in dialysis tubing, 4 DEG C of placements, with 500ml distill water dialysis 3 days, changed distilled water once every 6 hours;
D. the qualification of coupled antigen
By indirect Elisa method, by tsiklomitsin coupled antigen 0.01M PBS gradient dilution good for purifying, described extension rate is 1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 coated elisa plate, measures the activity of coupled antigen and tires.
Reaction principle of the present invention is as follows:
Compared with method in the present invention's disclosed with CN1861631 " conjugate of tsiklomitsin and preparation method thereof and application ", the method disclosed in above-mentioned file, needs in building-up process to pass into nitrogen protection, adds the synthesis cost of nitrogen protection; By changing reaction solvent in method building-up process of the present invention, not needing to pass into nitrogen protection, decreasing synthesis cost;
And method of the present invention is simple, easy handling; Generated time is short, the shortest needs 28 hours, 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, height of tiring, for the development and production of the immunoassay product detecting tetracycline residue improves efficiency, greatly save cost, reduce expense and the price of immunoassay product detection.
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 and dissolve, obtain solution 1.;
Take N, N-carbonyl dimidazoles 30mg, add 300 μ L DMFs and dissolve, obtain solution 2.;
1. solution is slowly added drop-wise to solution 2. in, in 37 DEG C, 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 and dissolve, obtain solution 3., tsiklomitsin good for derivatize is slowly added drop-wise to solution 3. in, in 37 DEG C of stirring reactions 5 hours, then place 12 hours for 4 DEG C, obtain the tsiklomitsin of coupling;
(3) purifying of coupled antigen
Tsiklomitsin holoantigen good for coupling is loaded in dialysis tubing, 4 DEG C of placements, with 500ml distill water dialysis 72 hours, changed distilled water once every 6 hours;
(4) qualification of coupled antigen
By indirect Elisa method, by tsiklomitsin coupled antigen 0.01M PBS gradient dilution (1:2000,1:4000,1:8000,1:16000,1:32000,1:64000) coated elisa plate good for purifying, measure the activity 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 time of synthesis is 28 hours; Generated time in CN1861631 is 142 hours.
In the present embodiment, need not nitrogen protection be added, in CN1861631, add nitrogen protection, thus add cost;
The quality preservation of product: the protein concentration of coupled antigen is 8.1mg/mL, and tiring is 1:32000, namely wrapping by concentration is 0.25 μ g/ml, and the disclosed bag in background technology in CN1861631 is 10 μ g/ml by concentration.
The present embodiment, compared with CN1861631, can save envelope antigen use cost 40 times.
Embodiment 2
(1) derivatize of tsiklomitsin
Take tsiklomitsin 5mg, add 600 μ L DMFs and dissolve, obtain solution 1.; Take N, N-carbonyl dimidazoles 60mg, add 600 μ L DMFs and dissolve, obtain solution 2.; 1. solution is slowly added drop-wise to solution 2. in, in 37 DEG C, stir derivative 3 hours;
(2) tsiklomitsin and albumen coupling
Take bovine serum albumin 20mg, add 1200 μ L 0.01M PBS and dissolve, obtain solution 3., tsiklomitsin good for derivatize is slowly added drop-wise to solution 3. in, in 37 DEG C of stirring reactions 5 hours, then place 12 hours for 4 DEG C;
(3) purifying of coupled antigen
Tsiklomitsin holoantigen good for coupling is loaded in dialysis tubing, 4 DEG C of placements, with 1000ml distill water dialysis 60 hours, changed distilled water once every 6 hours;
(4) qualification of coupled antigen
By indirect Elisa method, by tsiklomitsin coupled antigen 0.01M PBS gradient dilution (1:2000,1:4000,1:8000,1:16000,1:32000,1:64000) coated elisa plate good for purifying, measure the activity 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 and dissolve, obtain solution 1.; Take N, N-carbonyl dimidazoles 15mg, add 150 μ L DMFs and dissolve, obtain solution 2.; 1. solution is slowly added drop-wise to solution 2. in, in 37 DEG C, stir derivative 3 hours;
(2) tsiklomitsin and albumen coupling
Take bovine serum albumin (BSA) 5mg, add 300 μ L 0.01M PBS and dissolve, obtain solution 3., tsiklomitsin good for derivatize is slowly added drop-wise to solution 3. in, in 37 DEG C of stirring reactions 5 hours, then place 13 little for 4 DEG C.
(3) purifying of coupled antigen
Tsiklomitsin holoantigen good for coupling is loaded in dialysis tubing, 4 DEG C of placements, with 500ml distill water dialysis 60 hours, changed distilled water once every 6 hours.
(4) qualification of coupled antigen
By indirect Elisa method, by tsiklomitsin coupled antigen 0.01M PBS gradient dilution (1:2000,1:4000,1:8000,1:16000,1:32000,1:64000) coated elisa plate good for purifying, measure the activity 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 and dissolve, obtain solution 1.;
Take N, N-carbonyl dimidazoles, add DMF and dissolve, obtain solution 2.;
1. solution is slowly added drop-wise to solution 2. in, at 34-39 DEG C, stir derivative 1-5 hour, obtain the tsiklomitsin of derivatize;
B. tsiklomitsin and albumen coupling
Take bovine serum albumin, add PBS and dissolve, obtain solution 3.;
Tsiklomitsin good for derivatize in step a is slowly added drop-wise to solution 3. in, in 34-39 DEG C of stirring reaction 3-8 hour, then 4 DEG C of placements are spent the night, and obtain the tsiklomitsin of coupling;
C. the purifying of coupled antigen
Tsiklomitsin holoantigen good for coupling in step b is loaded in dialysis tubing, place at 4 DEG C, with distill water dialysis 1-3 days, changed distilled water once every 4-8 hour;
D. the qualification of coupled antigen
Adopt indirect Elisa method, by tsiklomitsin coupled antigen 0.01M PBS gradient dilution good for purifying, described extension rate is 1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 coated elisa plate, measures the activity of coupled antigen and tires.
2. the method for synthesis tsiklomitsin coupled antigen as claimed in claim 1, comprises following step:
A. the derivatize of tsiklomitsin
Take tsiklomitsin 2mg, add 300 μ L DMFs and dissolve, obtain solution 1.;
Take N, N-carbonyl dimidazoles 30mg, add 300 μ L DMFs and dissolve, obtain solution 2.;
1. solution is slowly added drop-wise to solution 2. in, in 37 DEG C, stir derivative 3 hours;
B. tsiklomitsin and albumen coupling
Take bovine serum albumin 10mg, add 600 μ L 0.01M PBS and dissolve, obtain solution 3., tsiklomitsin good for derivatize is slowly added drop-wise to solution 3. in, in 37 DEG C of stirring reactions 5 hours, then 4 DEG C of placements were spent the night;
C. the purifying of coupled antigen
Tsiklomitsin holoantigen good for coupling is loaded in dialysis tubing, 4 DEG C of placements, with 500ml distill water dialysis 3 days, changed distilled water once every 6 hours;
D. the qualification of coupled antigen
By indirect Elisa method, by tsiklomitsin coupled antigen 0.01M PBS gradient dilution good for purifying, described extension rate is 1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 coated elisa plate, measures the activity of coupled antigen and tires.
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Citations (4)
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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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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 |
Non-Patent Citations (3)
| Title |
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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》.2002,第10卷(第1期),第46-49页. * |
| 蔡宏道.抗原效价之测定.《使用临床检验学》.上海卫生出版社,1957,第1549页. * |
| 贾万钧等.酶标抗体(单克隆或多克隆)效价参考表.《抗原抗体反应动力学》.军事医学科学出版社,2004,第164-167页. * |
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