CN101318990B - Synthesis of multi-antigenic determinant artificial antigen with aminoglycoside medicaments - Google Patents
Synthesis of multi-antigenic determinant artificial antigen with aminoglycoside medicaments Download PDFInfo
- Publication number
- CN101318990B CN101318990B CN2008101230166A CN200810123016A CN101318990B CN 101318990 B CN101318990 B CN 101318990B CN 2008101230166 A CN2008101230166 A CN 2008101230166A CN 200810123016 A CN200810123016 A CN 200810123016A CN 101318990 B CN101318990 B CN 101318990B
- Authority
- CN
- China
- Prior art keywords
- bsa
- artificial antigen
- dialysis tubing
- conjugate
- dialysis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for synthesizing artificial antigen having aminoglycoside medicine multi-antigen determinant, belonging to the biochemical engineering technical field. The method uses neomycin, kanamycin and gentamicin as hapten, uses a carbodiimide method to couple the hapten with protein bovine serum protein BSA as a carrier, and uses a gel electrophoresis method to measure the coupling ratio of coupled matter. The method successfully synthesizes the artificial antigen having the aminoglycoside medicine multi-antigen determinant, is simple and effective in synthesis method, can be used in immune analysis completely, provides the necessary artificial antigen for after research of people, and can meet the needs of the research on the aminoglycoside medicine in China.
Description
Technical field
A kind of synthetic method with aminoglycoside medicaments multi-antigenic determinant artificial antigen belongs to technical field of biochemical industry.
Background technology
Aminoglycoside medicaments is the microbiotic of a class by the glycogen be combined into of aminosugar molecule and aglucone branch, is a kind of extensive pedigree antibiotic.Aminoglycoside antibiotics is mainly used to treat intestinal tract infections, bacillary dysentery, mucocutaneous infections and the ocular infection etc. that intestinal bacteria cause clinically, but has serious ototoxicity and renal toxicity.The degraded of such medicine in environment is relatively more difficult and can pass to the people by food chain, and the medium-term and long-term a large amount of aminoglycoside medicaments that use of animal daily ration can cause serious pollution to environment.In order to ensure animal food safety, answer the interpolation of such medicine in keeping under strict supervision close inspection and the management feed.Yet such drug testing technology is also relatively backward, becomes the principal element of restriction government department to such medicine supervision. existing microorganism detection method and instrument detecting method are difficult to realize aminoglycoside antibiotics is carried out quick, accurate, how residual detection.At home and abroad there is no the report of ideal at present at the how residual immunologic detection method of aminoglycoside medicaments.In order to remedy this blank, having designed with Xin Meisu (neomycin), kantlex (kanamycin), gentamicin (gentamycin) is the synthetic artificial antigen with aminoglycoside medicaments multi-antigenic determinant of haptens.
Summary of the invention
The purpose of this invention is to provide a kind of synthetic method with aminoglycoside medicaments multi-antigenic determinant artificial antigen.Prepared product is used for the immune analysis method research of aminoglycoside antibiotics, for people's research from now on provides essential artificial antigen.
Technical scheme of the present invention: a kind of synthetic method with aminoglycoside medicaments multi-antigenic determinant artificial antigen, with Xin Meisu, kantlex and gentamicin is haptens, with carbodlimide method with itself and the coupling of carrier proteins bovine serum albumin BSA, with the coupling ratio of gel electrophoresis therapy determining conjugate; Step is:
(1) conjugate (NM)
LThe preparation of-BSA
20-50mg Xin Meisu NM, 40mg bovine serum albumin BSA are dissolved in the PBS damping fluid of 3mL 0.01mol/L, dropwise add the methanol solution of the water-soluble carbodiimide EDC that now joins, and room temperature softly stirred 3 hours, promptly got conjugate (NM)
L-BSA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
With conjugate (NM)
L-BSA mixed solution is put into dialysis tubing in the PBS of 0.01mol/L dialysis 3 days, changes liquid three times every day, and it is standby to be concentrated into 3mL with Macrogol 2000 0;
(2) conjugate (NM)
L-(KM)
mThe preparation of-BSA
KM joins (NM) with the 20-50mg kantlex
LIn the PBS solution of-BSA, dropwise add the methanol solution of the water-soluble carbodiimide of now joining, room temperature softly stirred 3 hours, promptly got conjugate (NM)
L-(KM) m-BSA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
With conjugate (NM)
L-(KM)
m-BSA mixed solution is put into dialysis tubing in the PBS of 0.01mol/L dialysis 3 days, changes liquid three times every day, and it is standby to be concentrated into 3mL with Macrogol 2000 0;
(3) artificial antigen (NM)
L-(KM)
m-(GM)
nThe preparation of-BSA
GM joins (NM) with the 20-50mg gentamicin
L-(KM)
mIn the PBS solution of-BSA, dropwise add the methanol solution of the water-soluble carbodiimide of now joining, room temperature softly stirred 3 hours, promptly got artificial antigen (NM)
L-(KM)
m-(GM)
nThe mixed solution of-BSA;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
With (NM)
L-(KM)
m-(GM)
nThe mixed solution of-BSA is put into dialysis tubing in the PBS of 0.01mol/L dialysis 3 days, changes liquid three times every day, uses lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtains having the artificial antigen (NM) of aminoglycoside medicaments multi-antigenic determinant
L-(KM)
m-(GM)
n-BSA;
(4) has the evaluation of the artificial antigen of aminoglycoside medicaments multi-antigenic determinant
Artificial antigen adopts gel electrophoresis to identify its coupling result, utilizes standard protein to obtain typical curve, calculates its coupling ratio.
The methanol solution of used water-soluble carbodiimide of now joining is that 75-100 μ L carbodiimide is dissolved in 75-50 μ L methyl alcohol.Evaluation with artificial antigen of aminoglycoside medicaments multi-antigenic determinant
Coupling ratio is measured: be by the method for the ratio of two kinds of molecules of link coupled (coupling ratio) in the estimation conjugate, though the measuring method kind is a lot, all be to be set up by the principle of two kinds of molecule contents of link coupled (or relative content) according to detecting in the conjugate.Gel electrophoresis is to determine coupling ratio according to the molecular weight increase of the molecular weight ratio carrier proteins of synthetic artificial antigen.
The conjugate determination of protein concentration: compound concentration is 0,40,60,80,100,120,160,200 μ gmL
-1Bovine serum albumen solution 1.5mL, add 5mL coomassie brilliant blue staining liquid, mixing immediately, warm 5 minutes of 30 ℃ of water-baths, each concentration is made parallel sample. survey light absorption value, the relation curve of drafting bovine serum albumin concentration and light absorption value at 595nm place.Antigenic solution is diluted by a certain percentage, measure the light absorption value of antigenic solution at the 595nm place, obtain the corresponding proteins concentration of antigenic solution from curve.
Beneficial effect of the present invention: the present invention successfully synthesizes the artificial antigen with aminoglycoside medicaments multi-antigenic determinant, synthesis step is succinct, effectively, can be used in the immunoassay fully, for people's research later on provides approach easily, can satisfy domestic needs to aminoglycoside medicaments research.
Description of drawings
Fig. 1 lower molecular weight standard protein Mark, bovine serum albumin BSA and conjugate (NM)
L-BSA, (NM)
L-(KM)
m-BSA, (NM)
L-(KM)
m-(GM)
nThe electrophorogram of-BSA.
A: lower molecular weight standard protein Mark, B:BSA, C:(NM)
8.1-BSA, D:(NM)
8.1-(KM)
6.9-BSA, E:(NM)
8.1-(KM)
6.9-(GM)
9.8-BSA.
Fig. 2 electrophoresis canonical plotting (lgMW-Rf figure).
Embodiment
Embodiment 1
(1) conjugate (NM)
LThe preparation of-BSA
Xin Meisu (NM) 25mg, bovine serum albumin BSA 40mg are dissolved in the PBS damping fluid of 3mL 0.01mol/L, dropwise add the methanol solution (75 μ L EDC are dissolved in 75 μ L methyl alcohol) of the water-soluble carbodiimide of now joining, and room temperature softly stirred 3 hours.Promptly get conjugate (NM)
L-BSA mixed solution.
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby.
Reaction solution is put into dialysis tubing in the PBS of 0.01mol/L dialysis 3 days, change liquid three times every day, dialysis tubing is placed in the Macrogol 2000 0, and it is standby that reaction solution is concentrated into 3mL.
(2) conjugate (NM)
L-(KM) preparation of m-BSA
35mg joins (NM) with kantlex (KM)
LIn the PBS concentrated solution of-BSA, dropwise add the methanol solution (75 μ L EDC are dissolved in 75 μ L methyl alcohol) of the water-soluble carbodiimide of now joining, room temperature softly stirred 3 hours.Promptly get conjugate (NM)
L-(KM) m-BSA mixed solution.
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby.
Reaction solution is put into dialysis tubing, and dialysis is 3 days in the PBS of 0.01mol/L, changes liquid three times every day, and it is standby to be concentrated into 3mL with Macrogol 2000 0.
(3) artificial antigen (NM)
L-(KM) preparation of m-(GM) n-BSA
45mg joins (NM) with gentamicin (GM)
L-(KM) in the concentrated solution of m-BSA, dropwise adding the methanol solution (100 μ L EDC are dissolved in 50 μ L methyl alcohol) of the water-soluble carbodiimide of now joining, room temperature softly stirred 3 hours.Promptly get artificial antigen (NM)
L-(KM) mixed solution of m-(GM) n-BSA.
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby.
Reaction solution is put into dialysis tubing in the PBS of 0.01mol/L dialysis 3 days, change liquid three times every day, use lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtain having the artificial antigen (NM) of aminoglycoside medicaments multi-antigenic determinant
L-(KM)
m-(GM)
n-BSA.
(4) has the evaluation of the artificial antigen of aminoglycoside medicaments multi-antigenic determinant
Coupling ratio is measured: be by the method for the ratio of two kinds of molecules of link coupled (coupling ratio) in the estimation conjugate, though the measuring method kind is a lot, all be to be set up by the principle of two kinds of molecule contents of link coupled (or relative content) according to detecting in the conjugate.Gel electrophoresis is to identify the synthetic artificial antigen according to the molecular weight of synthetic artificial antigen.The SDS-polyacrylamide gel electrophoresis utilizes mobility only relevant with molecular weight, and electrically charged with institute, shape of molecule irrelevant, thereby can utilize the SDS-polyacrylamide gel electrophoresis to determine the definite coupling ratio of molecular weight of artificial antigen.10% separation gel is adopted in this experiment, and 5% compression glue is to lower molecular weight standard protein Mark, bovine serum albumin BSA, conjugate (NM)
L-BSA, (NM)
L-(KM)
m-BSA, (NM)
L-(KM)
m-(GM)
n-BSA carries out electrophoresis to be identified.Utilize the biological electrophoresis gel imaging system of FR980 software to draw coupling ratio (NM: KM: GM: BSA) be respectively 8.1: 6.9: 9.8: 1.The form that is artificial antigen is (NM)
8.1-(KM)
6.9-(GM)
9.8-BSA meets immune requirement fully.
The conjugate determination of protein concentration: compound concentration is 0,40,60,80,100,120,160,200 μ gmL
-1Bovine serum albumen solution 1.5mL, add 5mL coomassie brilliant blue staining liquid, mixing immediately, warm 5 minutes of 30 ℃ of water-baths, each concentration is made parallel sample. survey light absorption value, the relation curve of drafting bovine serum albumin concentration and light absorption value at 595nm place.Antigenic solution is diluted by a certain percentage, measure the light absorption value of antigenic solution at the 595nm place, obtain the corresponding proteins concentration of antigenic solution from curve.The protein concentration that this experimental calculation gets antigenic solution is 10.5968mgmL
-1
Claims (2)
1. synthetic method with aminoglycoside medicaments multi-antigenic determinant artificial antigen, it is characterized in that with Xin Meisu, kantlex and gentamicin be haptens, with carbodlimide method with itself and the coupling of carrier proteins bovine serum albumin BSA, with the coupling ratio of gel electrophoresis therapy determining conjugate; Step is:
(1) conjugate (NM)
LThe preparation of-BSA
20-50mg Xin Meisu NM, 40mg bovine serum albumin BSA are dissolved in the PBS damping fluid of 3mL 0.01mol/L, dropwise add the methanol solution of the water-soluble carbodiimide EDC that now joins, and room temperature softly stirred 3 hours, promptly got conjugate (NM)
L-BSA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
With conjugate (NM)
L-BSA mixed solution is put into dialysis tubing in the PBS of 0.01mol/L dialysis 3 days, changes liquid three times every day, and it is standby to be concentrated into 3mL with Macrogol 2000 0;
(2) conjugate (NM)
L-(KM)
mThe preparation of-BSA
KM joins (NM) with the 20-50mg kantlex
LIn the PBS solution of-BSA, dropwise add the methanol solution of the water-soluble carbodiimide of now joining, room temperature softly stirred 3 hours, promptly got conjugate (NM)
L-(KM)
m-BSA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
With conjugate (NM)
L-(KM)
m-BSA mixed solution is put into dialysis tubing in the PBS of 0.01mol/L dialysis 3 days, changes liquid three times every day, and it is standby to be concentrated into 3mL with Macrogol 2000 0;
(3) artificial antigen (NM)
L-(KM)
m-(GM)
nThe preparation of-BSA
GM joins (NM) with the 20-50mg gentamicin
L-(KM)
mIn the PBS solution of-BSA, dropwise add the methanol solution of the water-soluble carbodiimide of now joining, room temperature softly stirred 3 hours, promptly got artificial antigen (NM)
L-(KM)
m-(GM)
nThe mixed solution of-BSA;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
With (NM)
L-(KM)
m-(GM)
nThe mixed solution of-BSA is put into dialysis tubing in the PBS of 0.01mol/L dialysis 3 days, changes liquid three times every day, uses lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtains having the artificial antigen (NM) of aminoglycoside medicaments multi-antigenic determinant
L-(KM)
m-(GM)
n-BSA;
(4) has the evaluation of the artificial antigen of aminoglycoside medicaments multi-antigenic determinant
Artificial antigen adopts gel electrophoresis to identify its coupling result, utilizes standard protein to obtain typical curve, calculates its coupling ratio.
2. synthetic method according to claim 1, the methanol solution that it is characterized in that used water-soluble carbodiimide of now joining are that 75-100 μ L carbodiimide is dissolved in 75-50 μ L methyl alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101230166A CN101318990B (en) | 2008-06-18 | 2008-06-18 | Synthesis of multi-antigenic determinant artificial antigen with aminoglycoside medicaments |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101230166A CN101318990B (en) | 2008-06-18 | 2008-06-18 | Synthesis of multi-antigenic determinant artificial antigen with aminoglycoside medicaments |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101318990A CN101318990A (en) | 2008-12-10 |
| CN101318990B true CN101318990B (en) | 2011-01-26 |
Family
ID=40179287
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008101230166A Expired - Fee Related CN101318990B (en) | 2008-06-18 | 2008-06-18 | Synthesis of multi-antigenic determinant artificial antigen with aminoglycoside medicaments |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101318990B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103575888B (en) * | 2012-08-02 | 2016-04-20 | 北京勤邦生物技术有限公司 | A kind of test strips and method detecting neomycin |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161679A (en) * | 2007-11-01 | 2008-04-16 | 江南大学 | Method for preparing dexamethasone artificial antigen |
-
2008
- 2008-06-18 CN CN2008101230166A patent/CN101318990B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161679A (en) * | 2007-11-01 | 2008-04-16 | 江南大学 | Method for preparing dexamethasone artificial antigen |
Non-Patent Citations (6)
| Title |
|---|
| Haasnoot W et al..Immunochemical detection of aminoglycosides in milk and kidney.《Analyst》.1999,第124卷(第3期),301-305. * |
| JIN Y et al..Development of ELISA and Immunochromatographic Assay for the Detection of Gentamicin.《J. Agric. Food Chem.》.2005,第53卷(第20期),7639-7643. * |
| 刘沙洲.新霉素人工抗原的构建、多克隆抗体的制备及检测方法的初步研究.《中国优秀硕士学位论文全文数据库（农业科技辑）》.2008,(第05期),第11-13页，第24页，第52-53页，图3.7. * |
| 徐亭.庆大霉素单克隆抗体的制备及初步应用.《中国优秀硕士学位论文全文数据库(农业科技辑)》.2005,(第05期),14-15. * |
| 胥传来等.动物性食品中残留甲羟孕酮免疫原的合成与表征.《食品科技》.2004,(第8期),70-73. * |
| 胥传来等.动物源食品中磺胺二甲嘧啶人工抗原的合成研究.《食品科学》.2005,第26卷(第7期),118-121. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101318990A (en) | 2008-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102735851A (en) | Mycoplasma hyopneumoniae multi-recombination antigen ELISA (enzyme-linked immunosorbent assay) detection kit | |
| CN102827076B (en) | Universal hapten, artificial antigen and broad-spectrum monoclonal antibody for fluoroquinolone medicines as well as preparation method and application of universal hapten | |
| CN110616195B (en) | Metformin monoclonal antibody hybridoma cell strain and application thereof | |
| CN103323593B (en) | A kind of test paper and application thereof detecting fluoroquinolones | |
| CN102128923B (en) | One-step ELISA (Enzyme Linked Immunosorbent Assay) method for neomycin (NEO) residues in milk | |
| CN105842464B (en) | Joint based on up-converting phosphor technology quantitatively detects uNGAL and uCr device and preparation method thereof | |
| CN100478357C (en) | Ofloxacin couple and its preparing method and use | |
| CN102608316B (en) | Kit or test strip for detecting quinoxaline compound | |
| CN101318990B (en) | Synthesis of multi-antigenic determinant artificial antigen with aminoglycoside medicaments | |
| CN102120767B (en) | Synthesis method of universal artificial antigen of alkylphenol medicines | |
| CN102321171B (en) | Synthetic method of artificial antigen suitable for alkylphenol medicament | |
| CN101307092B (en) | Method for synthesizing general artificial antigen of beta-lactam drugs | |
| CN101318989B (en) | General use artificial antigen synthesis of aminoglycoside medicaments | |
| CN102382189B (en) | Synthesis method of 5-hydroxymethylfurfural complete antigen | |
| Fahey et al. | Quantitation by ELISA of pili and sheep antibodies to the pili of Bacteroides nodosus | |
| CN106929479B (en) | Vitamin B2 monoclonal antibody hybridoma cell strain GZ-4 and application thereof | |
| CN102675454B (en) | Synthetic method of 4-methylimidazole complete antigen | |
| CN101153871B (en) | Synthesis method for generally used artificial antigen of Beta-lactam medicine | |
| CN101398427A (en) | Aminoside antibiotics ELISA detection method in Animal derived food | |
| CN101899109B (en) | Synthesis method of 4-amino azobenzene artificial antigen | |
| CN103319626A (en) | Method for covalent linkage of hyaluronic acid and isothiocyano-containing organic molecule | |
| CN104004718A (en) | Universal monoclonal antibody hybridoma cell strain capable of resisting pirlimycin and application thereof | |
| CN101585877B (en) | Method for synthesizing rhodamine B complete antigen | |
| CN103509068A (en) | Amikacin semi-antigen, and preparation method and application thereof | |
| Svenungsson et al. | Diagnosis of Salmonella Bacteria: Antibodies Against Synthetic Salmonella O‐Antigen 8 for Immunofluorescence and co‐Agglutination using Sensitized Protein A‐Containing Staphylococci |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110126 Termination date: 20120618 |