CN101899109A - Synthesis method of 4-amino azobenzene artificial antigen - Google Patents
Synthesis method of 4-amino azobenzene artificial antigen Download PDFInfo
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- CN101899109A CN101899109A CN 201010215515 CN201010215515A CN101899109A CN 101899109 A CN101899109 A CN 101899109A CN 201010215515 CN201010215515 CN 201010215515 CN 201010215515 A CN201010215515 A CN 201010215515A CN 101899109 A CN101899109 A CN 101899109A
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Abstract
The invention provides a synthesis method of 4-amino azobenzene artificial antigen, belonging to the technical field of biochemical engineering. The method comprises the following steps of: taking 4-amino azobenzene as hapten; coupling the 4-amino azobenzene with carrier protein BSA by means of diazotization; and measuring the coupling ratio of a coupling material by means of spectroscopic luminosity. The synthesis method successfully synthesizes the artificial antigen of the 4-amino azobenzene, is simple and effective in synthesis steps, can be completely used in immunoassay, provides the necessary artificial antigen to the later research of users, and can meet the requirement of the research of the 4-amino azobenzene in China.
Description
Technical field
A kind of synthetic method of general artificial antigen of 4-aminoazobenzene belongs to technical field of biochemical industry.
Background technology
Azoic dyestuff (the azo-group two ends connect a class organic compound of aryl) is fabric clothing most widely used class synthetic dyestuff in dyeing and printing process, under special conditions, it can decompose more than 20 kind of carcinogenic aromatic amine of generation, and the dna structure that changes human body through activation causes pathology and brings out cancer.Azo is one of essential items for inspection of international environmental requirement, and the method for inspection has following three kinds: tlc (TLC), gas-chromatography and mass spectrometry method (GC-MSD) and high performance liquid chromatography (HPLC).Must not contain 23 kinds of azoic dyestuff intermediates in the standard code examined product, if detect wherein a kind of substandard product that is, it is limited the quantity of and is 30ppm.The 4-aminoazobenzene is a kind of in these harmful aromatic amines, and in the azoic dyestuff standard that European Union announces, the 4-aminoazobenzene belongs to a kind of among 2 groups of MAK (III) A in the forbidding aromatic amine.External most of existing relevant criterion rules are all listed the amino diimide benzene of 4-in forbidding aromatic amine ranks, as Germany's " food and daily necessities method " etc., and domesticly all do not have it is listed in the standard that relates to textiles, dyestuff, leatherware, because the restriction of detection technique, the conventional method of inspection can't detect it.In recent years, carried out research both at home and abroad, but still do not had report both at home and abroad,, be necessary to prepare the 4-amino azobenzene artificial antigen in order to remedy this blank at the enzyme linked immunosorbent detection of 4-aminoazobenzene to azo para-immunity analytical procedure.
Summary of the invention
The synthetic method that the purpose of this invention is to provide a kind of 4-amino azobenzene artificial antigen.Prepared product is used for the immune analysis method research of 4-aminoazobenzene.For people's research from now on provides essential artificial antigen.
Technical scheme of the present invention: a kind of synthetic method of 4-amino azobenzene artificial antigen is a haptens with the 4-aminoazobenzene, with the diazotization method with itself and carrier proteins BSA coupling, the coupling ratio of usefulness spectrophotometry conjugate;
(1) preparation of artificial antigen:
A liquid: get 4-aminoazobenzene 0.1mmol and be dissolved in 2mL 1, in the 4-dioxane, ice bath adds 1M hydrochloric acid and transfers pH 2.0, and ice bath adds 0.02M Sodium Nitrite to starch test paper and becomes blue, under 4 ℃, stirs and hatches 30min, is A liquid;
B liquid: bovine serum albumin BSA 0.001mmol is dissolved in the carbonate buffer solution of 2mL, pH 9.6,0.05mol/L, and 4 ℃ of preservations are stand-by, are B liquid;
A liquid is dropwise added in the B liquid, transfer pH 9.0, put 4 ℃ of following temperature then and incubate half an hour, room temperature reaction 4 hours promptly gets 4-amino azobenzene artificial antigen 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;
4-amino azobenzene artificial antigen mixed solution is moved in the dialysis tubing, with 2 * 2L (each 2L, totally 2 times) the phosphate buffer soln of pH 7.4 and 2 * 2L (the each 2L of 0.0lM, totally 2 times) deionized water dialysis 3 days, use lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtain the 4-amino azobenzene artificial antigen;
(2) evaluation of artificial antigen:
The 4-amino azobenzene artificial antigen adopts spectrophotometry to identify its coupling result, utilizes standard protein to obtain typical curve, calculates its coupling ratio.
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.Spectrophotometry is to utilize material that the principle that absorption and its concentration of light is proportionlity is measured respectively by two kinds of molecular conecentrations of link coupled. in macromole and small molecules conjugate, two kinds of molecules all have different separately ultraviolet scanning spectrums, and show the character of spectrogram superposition.
Molar absorptivity ε: preparation 4-aminoazobenzene concentration is 0,10,20,30 μ gmL
-1The PBS solution of 0.01M, by UV scanning as can be known the maximum absorption wavelength of 4-aminoazobenzene be 400nm, survey light absorption value at the 400nm place, each concentration is made parallel sample. molar absorptivity is calculated as: ε=light absorption value/volumetric molar concentration.
The conjugate determination of protein concentration: compound concentration is 0,10,20,40,60,80,100 μ gmL
-1Bovine serum albumin 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 protein 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.
Coupling ratio is measured: prepare 150 μ gmL
-120% ethanolic soln of bovine serum albumin, with coupled product with 20% alcohol dilution to 150 μ gmL
-1, survey light absorption value at the 400nm place, be blank with 20% ethanol, the light absorption value that sample solution, blank solution are measured is respectively A
1, A
2, then coupling ratio γ is: γ=[(A
1-A
2)/ε]/(150 * 10
-3/ 66200).
Wherein ε is molar absorptivity (Lmol
-1), 66200 is the molecular weight of bovine serum albumin, 150 * 10
-3Be bovine serum albumin concentration (μ gmL
-1).
Beneficial effect of the present invention: the present invention has synthesized the artificial antigen of 4-aminoazobenzene, and synthesis step is succinct, effectively, can be used for fully in the middle of the immunoassay, for people's research later on provides approach easily, can satisfy domestic needs to its research.
Description of drawings
Fig. 1: the UV scanning figure before and after the preparation of 4-amino azobenzene artificial antigen.
Fig. 2: 4-amino azobenzene artificial antigen electrophorogram.1, BSA; 2,4-aminoazobenzene-BSA.
Embodiment
(1) preparation of artificial antigen
Get 4-aminoazobenzene 0.1mmol and be dissolved in 2mL 1, in the 4-dioxane, ice bath adds 1M hydrochloric acid and transfers pH 2.0, and ice bath adds 0.02M Sodium Nitrite to starch test paper and becomes blue, under 4 ℃, stirs and hatches 30min, is A liquid.
Bovine serum albumin BSA 0.001mmol is dissolved in 2mL, the 0.05mol/L CBS damping fluid, is B liquid.
A liquid dropwise is added dropwise in the B liquid, and the limit edged is transferred pH 9.0, hatches half an hour under 4 ℃, and room temperature reaction 4 hours promptly gets 4-amino azobenzene artificial antigen 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.
4-amino azobenzene artificial antigen mixed solution is moved in the dialysis tubing, dialysed 3 days with the PBS solution of the 0.01M of 2 * 2L and the deionized water of 2 * 2L.Use lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtain the 4-amino azobenzene artificial antigen.
(2) evaluation of 4-amino azobenzene artificial antigen
Molar absorptivity ε: preparation 4-aminoazobenzene concentration is 0,10,20,30 μ gmL
-1The PBS solution of 0.01M, by UV scanning as can be known the maximum absorption wavelength of 4-aminoazobenzene be 400nm, survey light absorption value at the 400nm place, each concentration is made parallel sample. molar absorptivity is calculated as: ε=light absorption value/volumetric molar concentration.This experimental calculation gets ε=26749.8Lmol
-1
The conjugate determination of protein concentration: compound concentration is 0,10,20,40,60,80,100 μ gmL
-1Bovine serum albumin 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 protein 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.
Coupling ratio is measured: prepare 150 μ gmL
-120% ethanolic soln of bovine serum albumin, with coupled product with 20% alcohol dilution to 150 μ gmL
-1, survey light absorption value at the 400nm place, be blank with 20% ethanol, the light absorption value of measuring is A
1, A
2, then coupling ratio γ is: γ=[(A
1-A
2)/ε]/(150 * 10
-3/ 66200).This experimental calculation gets γ ≈ 23
Wherein ε is molar absorptivity (Lmol
-1), 66200 is the molecular weight of bovine serum albumin, 150 * 10
-3Be bovine serum albumin concentration (μ gmL
-1).
Claims (1)
1. the synthetic method of a 4-amino azobenzene artificial antigen is characterized in that with the 4-aminoazobenzene be haptens, with the diazotization method with itself and carrier proteins BSA coupling, the coupling ratio of usefulness spectrophotometry conjugate;
(1) preparation of artificial antigen:
A liquid: get 4-aminoazobenzene 0.1mmol and be dissolved in 2mL 1, in the 4-dioxane, ice bath adds 1M hydrochloric acid and transfers pH 2.0, and ice bath adds 0.02M Sodium Nitrite to starch test paper and becomes blue, under 4 ℃, stirs and hatches 30min, is A liquid;
B liquid: bovine serum albumin BSA 0.001mmol is dissolved in the carbonate buffer solution of 2mL, pH 9.6,0.05mol/L, and 4 ℃ of preservations are stand-by, are B liquid;
A liquid is dropwise added in the B liquid, transfer pH 9.0, put 4 ℃ of following temperature then and incubate half an hour, room temperature reaction 4 hours promptly gets 4-amino azobenzene artificial antigen 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;
4-amino azobenzene artificial antigen mixed solution is moved in the dialysis tubing, with the deionized water dialysis of the phosphate buffer soln of the pH 7.4 of the 0.01M of 2 * 2L and 2 * 2L 3 days, use lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtain the 4-amino azobenzene artificial antigen;
(2) evaluation of artificial antigen:
The 4-amino azobenzene artificial antigen adopts spectrophotometry to identify its coupling result, utilizes standard protein to obtain typical curve, calculates its coupling ratio.
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| CN2010102155155A CN101899109B (en) | 2010-06-25 | 2010-06-25 | Synthesis method of 4-amino azobenzene artificial antigen |
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| CN101899109B CN101899109B (en) | 2012-06-20 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391373A (en) * | 2011-11-26 | 2012-03-28 | 聊城大学 | 4-aminoazobenzene complete antigen and its preparation method |
| CN102659939A (en) * | 2012-04-26 | 2012-09-12 | 嘉兴九七九生物技术有限公司 | Diphenylhydantoin immunogen, diphenylhydantoin specificity-resistant antibody and diphenylhydantoin detection reagent |
| CN102827271A (en) * | 2012-09-29 | 2012-12-19 | 江南大学 | Synthesis method of artificial antigen suitable for aminoazobenzene |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763103A (en) * | 2005-09-27 | 2006-04-26 | 东华大学 | Dipropyl phthalic acid artificial antigen and its preparation method |
| CN101519364A (en) * | 2009-03-28 | 2009-09-02 | 江苏省原子医学研究所 | Chlorine flucythrinate artificial antigen and method for synthesizing same |
| CN101830981A (en) * | 2010-05-21 | 2010-09-15 | 江南大学 | Preparation method of artificial antigen of 4-aminobenzophenone |
-
2010
- 2010-06-25 CN CN2010102155155A patent/CN101899109B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763103A (en) * | 2005-09-27 | 2006-04-26 | 东华大学 | Dipropyl phthalic acid artificial antigen and its preparation method |
| CN101519364A (en) * | 2009-03-28 | 2009-09-02 | 江苏省原子医学研究所 | Chlorine flucythrinate artificial antigen and method for synthesizing same |
| CN101830981A (en) * | 2010-05-21 | 2010-09-15 | 江南大学 | Preparation method of artificial antigen of 4-aminobenzophenone |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391373A (en) * | 2011-11-26 | 2012-03-28 | 聊城大学 | 4-aminoazobenzene complete antigen and its preparation method |
| CN102659939A (en) * | 2012-04-26 | 2012-09-12 | 嘉兴九七九生物技术有限公司 | Diphenylhydantoin immunogen, diphenylhydantoin specificity-resistant antibody and diphenylhydantoin detection reagent |
| CN102659939B (en) * | 2012-04-26 | 2014-04-23 | 嘉兴九七九生物技术有限公司 | Diphenylhydantoin immunogen, diphenylhydantoin specificity-resistant antibody and diphenylhydantoin detection reagent |
| CN102827271A (en) * | 2012-09-29 | 2012-12-19 | 江南大学 | Synthesis method of artificial antigen suitable for aminoazobenzene |
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| CN101899109B (en) | 2012-06-20 |
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