CN102093476A - Preparation method of AI (Angiotensin I) immunogen - Google Patents
Preparation method of AI (Angiotensin I) immunogen Download PDFInfo
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- CN102093476A CN102093476A CN2009102504755A CN200910250475A CN102093476A CN 102093476 A CN102093476 A CN 102093476A CN 2009102504755 A CN2009102504755 A CN 2009102504755A CN 200910250475 A CN200910250475 A CN 200910250475A CN 102093476 A CN102093476 A CN 102093476A
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Abstract
The invention relates to a preparation method of AI (angiotensin I) immunogen and a method for preparing a polyclonal antibody. Immunogen prepared from a boric acid buffer solution has higher AI activity and higher antiserum tilter than immunogen prepared from PB (Phosphate Buffer); and immunogen prepared in connection with the AI in a boric acid buffer system by taking hemacyanine as a carrier protein has the highest AI activity and the highest AI antiserum tilter.
Description
Technical field
The present invention relates to a kind of method for preparing the immunogenic method of angiotensin I and this polyclonal antibody of preparation.
Background technology
In the research pathogenesis of hypertension, now confirmed renin-angiotensin system (be called for short RAS) to regulating human blood-pressure and water, electrolyte balance, that keeps the human internal environment stablely plays crucial effect.
The proangiotensin molecular weight is greatly between 66000-110000, no direct physiological action own, its N-terminal 14 peptides are the feritin substrate, the peptide bond between two leucines interrupts on the 10th and the 11st on can be with its peptide chain under the feritin effect, sloughs 4 amino acid of inactive thereafter and forms a kind of angiotensin I (abbreviating AI as) that contains 10 peptide structures.AI sloughs 2 amino acid of carboxyl terminal on the peptide chain by after the effect of AI saccharase, converts the material that boosts with strong vasoconstriction effect to---the Angiotensin II (abbreviating AII as) of 8 peptide structures.
Do not have physiologically active under the AI normal physiological concentration, if when dense, can stimulate adrenal medulla to discharge catecholamine, act on the mesencephalic centre nerve in blood plasma, cause that surrounding blood vessel shrinks, make elevation of blood pressure, also the heavily absorption to sodium, water exerts an influence.
Angiotensin I and Angiotensin II all belong to haptens, must be connected with macromolecular carrier albumen to make the generation of immunogen ability immune animal induce antibody.Shanghai Research Institute of Hypertension selects for use different carrier (albumin rabbit serum, bovine serum albumin, poly-lysine, polyethylene Pyrrolizidine ketone) to prepare five kinds of different Angiotensin II immunogens through carbodiimide (EDC) or glutaraldehyde (GDA) for connecting agent.The immune animal experiment comparison shows that Angiotensin II-glutaraldehyde-bovine serum albumin white effect wherein is satisfied.
The angiotensin I molecular weight is 1295.69, can prepare the angiotensin I immunogen with reference to the immunogenic preparation method of above-mentioned Angiotensin II.But the antibody titer that produces behind the AII immunogen immune animal of this method preparation is not high enough, is used for radio immunoassay and still can.On-radiation solid-phase immunoassay method is used more extensively now, and the tiring of antagonist, avidity require higher, thereby the immunogen and the immunization method that need induce antibody more effectively to produce.
Summary of the invention
The invention provides a kind of method for preparing the immunogenic method of angiotensin I and this polyclonal antibody of preparation.The hemocyanin that the present invention adopts derives from unicellular lower eukaryote, and different originality is stronger, can more effectively stimulate the immunity system of rabbit.Adopt the borate buffer liquor ratio to adopt the immunogen AI activity of PB buffer preparation higher, the antiserum(antisera) titre of generation is also higher.With the hemocyanin is carrier proteins, and the immunogen AI activity that is connected preparation in the borate buffer system with AI is the highest, and the AI antiserum(antisera) work titre of generation is also the highest.
Embodiment
Embodiment:
1) amino-acid sequence of AI is: aspartic acid (Asp)-arginine (Arg)-a word used in person's names propylhomoserin (Val)-tyrosine (Tyr)-Isoleucine (Ile)-Histidine (His)-proline(Pro) (Pro)-phenylalanine (Phe)-Histidine (His)-leucine (Leu) homotype bifunctional reagent glutaraldehyde is as linking agent.Two aldehyde radicals of glutaraldehyde respectively with AI and carrier proteins on amino form schiff alkali, middle bridging with five carbon bonds connects.Reaction formula is as follows:
R-NH
2+HC(CH
2)
3CH+H
2N-P→R-N＝CH(CH
2)
3CH＝N-P
2) being connected of AI and carrier proteins:
Select for use BSA and hemocyanin as carrier proteins, glutaraldehyde is connected with AI by following A, B, three kinds of methods of C as linking agent:
(A) take by weighing 10mg BSA and 5mg AI, be dissolved in 2mL 0.1M pH7.4PB damping fluid.Add 1% glutaraldehyde 0.5mL, stirring at room 1.5h adds 0.25mL 1M glycine, and stirring at room 30min fully dialyses with 0.1M pH 7.4PB damping fluid;
(B) take by weighing 10mg BSA and 5mg AI, be dissolved in 2mL 0.1M pH9.0 borate buffer.Following steps are fully dialysed with 0.05M pH 8.4 borate buffers with (A);
(C) take by weighing 10mg hemocyanin and 12mg AI, be dissolved in 2ml 0.1M pH 9.0 borate buffers.Add 0.31% glutaraldehyde 1mL, stirring at room 2h, following steps are fully dialysed with 0.05M pH 8.4 borate buffers with (A);
3) AI Antiserum Preparation:
New Zealand white rabbit is divided into three groups, three every group, carries out immunity with subcutaneous multiple spot behind A, B, three kinds of AI immunogens of C and the abundant mixing of Fu Shi Freund's complete adjuvant respectively.AI immunogen and the abundant mixing booster immunization of freund 's incomplete adjuvant after two weeks, after this 3-4 week immunity once is total to immunity 6 times at interval, uses AI RIA test kit check antibody titers.
Claims (3)
1. an angiotensin I (AI, Angiotensin I) immunogen preparation method:
Step is as follows:
1) amino-acid sequence of AI is: aspartic acid (Asp)-arginine (Arg)-a word used in person's names propylhomoserin (Val)-tyrosine (Tyr)-Isoleucine (Ile)-Histidine (His)-proline(Pro) (Pro)-phenylalanine (Phe)-Histidine (His)-leucine (Leu) homotype bifunctional reagent glutaraldehyde is as linking agent, two aldehyde radicals of glutaraldehyde respectively with AI and carrier proteins on amino form schiff alkali, middle bridging with five carbon bonds connects, and reaction formula is as follows:
R-NH
2+HC(CH
2)
3CH+H
2N-P→R-N＝CH(CH
2)
3CH＝N-P
2) being connected of AI and carrier proteins:
Select for use BSA and hemocyanin as carrier proteins, glutaraldehyde is connected with AI by following method as linking agent:
Take by weighing 10mg BSA and 5mg AI, be dissolved in the PB damping fluid of 2mL 0.1M pH7.4, add 1% glutaraldehyde 0.5mL, stirring at room 1.5 hours adds 0.25mL 1M glycine, and stirring at room 30 minutes is fully dialysed with the PB damping fluid of 0.1M pH 7.4.
2. a kind of angiotensin I (AI as claimed in claim 1, Angiotensin I) immunogen preparation method: the above-mentioned method that is connected with AI can replace with: take by weighing 10mg BSA and 5mg AI, be dissolved in the borate buffer of 2mL 0.1M pH 9.0, add 1% glutaraldehyde 0.5mL, stirring at room 1.5 hours, add 0.25mL 1M glycine, stirring at room 30 minutes is fully dialysed with 0.05M pH 8.4 borate buffers.
3. a kind of angiotensin I (AI as claimed in claim 1, Angiotensin I) immunogen preparation method: the above-mentioned method that is connected with AI can replace with: take by weighing 10mg hemocyanin and 12mg AI, be dissolved in 2ml 0.1M pH 9.0 borate buffers, add 0.31% glutaraldehyde 1mL, stirring at room 2 hours, add 0.25mL 1M glycine, stirring at room 30 minutes is fully dialysed with 0.05M pH 8.4 borate buffers.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009102504755A CN102093476A (en) | 2009-12-10 | 2009-12-10 | Preparation method of AI (Angiotensin I) immunogen |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103613660A (en) * | 2013-11-25 | 2014-03-05 | 博奥赛斯（天津）生物科技有限公司 | Preparation method of angiotensin complete antigen |
| CN104634965A (en) * | 2015-02-10 | 2015-05-20 | 深圳市新产业生物医学工程股份有限公司 | Angiotensin I detection reagent kit as well as preparation method and application thereof |
| CN105622745A (en) * | 2016-01-22 | 2016-06-01 | 深圳市新产业生物医学工程股份有限公司 | Derivative of amino acid fragment of angiotensin II, angiotensin II antigen and preparation method and application of angiotensin II antigen |
| CN105646668A (en) * | 2016-01-22 | 2016-06-08 | 深圳市新产业生物医学工程股份有限公司 | Derivative of angiotensin I amino acid segment, angiotensin I antigen and preparation and application of angiotensin I antigen |
| CN108181460A (en) * | 2017-12-22 | 2018-06-19 | 苏州博源医疗科技有限公司 | Dipeptidase derivant for angiotensinⅠ detection and its preparation method and application |
-
2009
- 2009-12-10 CN CN2009102504755A patent/CN102093476A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103613660A (en) * | 2013-11-25 | 2014-03-05 | 博奥赛斯（天津）生物科技有限公司 | Preparation method of angiotensin complete antigen |
| CN104634965A (en) * | 2015-02-10 | 2015-05-20 | 深圳市新产业生物医学工程股份有限公司 | Angiotensin I detection reagent kit as well as preparation method and application thereof |
| CN105622745A (en) * | 2016-01-22 | 2016-06-01 | 深圳市新产业生物医学工程股份有限公司 | Derivative of amino acid fragment of angiotensin II, angiotensin II antigen and preparation method and application of angiotensin II antigen |
| CN105646668A (en) * | 2016-01-22 | 2016-06-08 | 深圳市新产业生物医学工程股份有限公司 | Derivative of angiotensin I amino acid segment, angiotensin I antigen and preparation and application of angiotensin I antigen |
| CN108181460A (en) * | 2017-12-22 | 2018-06-19 | 苏州博源医疗科技有限公司 | Dipeptidase derivant for angiotensinⅠ detection and its preparation method and application |
| CN108181460B (en) * | 2017-12-22 | 2020-08-07 | 苏州博源医疗科技有限公司 | Dipeptide derivative for detecting angiotensin I and preparation method and application thereof |
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Application publication date: 20110615 |