CN1401786A - Process for preparing beta-D-mannuronic acid oligosaccharide monoclonal antibody - Google Patents
Process for preparing beta-D-mannuronic acid oligosaccharide monoclonal antibody Download PDFInfo
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- CN1401786A CN1401786A CN 01135114 CN01135114A CN1401786A CN 1401786 A CN1401786 A CN 1401786A CN 01135114 CN01135114 CN 01135114 CN 01135114 A CN01135114 A CN 01135114A CN 1401786 A CN1401786 A CN 1401786A
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- beta
- mannuronic acid
- acid oligosaccharide
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- monoclonal antibody
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Abstract
A process for preparing beta-D-mannuronate oligose monoclonal antibody includes chemically linking beta-D mannuronate oligose with bovine serum albumin, immunizing mouse, taking its spleen cells and myloma cells, fusing them, selective culture, diluting, cloning, choosing positive cloned strain, amplifying a lot of it, in-vitro cell culture, and separating.
Description
The present invention relates to a kind of beta-D-mannuronic acid oligosaccharide, particularly relate to a kind of preparation method of beta-D-mannuronic acid oligosaccharide monoclonal antibody.
Beta-D-mannuronic acid oligosaccharide (HSH-971) is the acid oligosaccharides class of a kind of lower molecular weight material that separation and Extraction obtains from marine alga, and its weight-average molecular weight is 2000 dalton.The contriver recognizes that its complex structure does not have the characteristic ultra-violet absorption spectrum in the practice for many years of this glucide of development, lack the specific criteria quantitative detecting method.Thereby all have difficulties in all many-sides such as biological effect of the detection of HSH-971, mensuration, quality standard, immune purifying and blocking-up HSH-971.
The preparation method who the purpose of this invention is to provide a kind of beta-D-mannuronic acid oligosaccharide monoclonal antibody, the monoclonal anti physical efficiency that it is prepared solves above-mentioned difficulties of the prior art.
A kind of preparation method of beta-D-mannuronic acid oligosaccharide monoclonal antibody, it is characterized in that beta-D-mannuronic acid oligosaccharide is connected with the bovine serum albumin chemistry, make the immunocomplex immune mouse, getting this mouse boosting cell and myeloma cell merges, carrying out selectivity with selective medium cultivates, carry out cloning through dilution, select positive clone strain, and a large amount of amplifications, injecting the homology mouse peritoneal induces ascites or cultivates separation acquisition beta-D-mannuronic acid oligosaccharide monoclonal antibody by cell in vitro.
The beta-D-mannuronic acid oligosaccharide monoclonal antibody of the present invention's preparation can be used for the biological effect of detection, mensuration, quality standard, purifying and the blocking-up beta-D-mannuronic acid oligosaccharide of beta-D-mannuronic acid oligosaccharide, and is easy to use.
Below by embodiment the present invention is described.
It is in 4 the sodium-acetate salt buffer that 450mg HSH-971 is dissolved in 0.4ml pH, the sodium periodate aqueous solution that adds 2.4% (concentration expressed in percentage by weight), vibration 30min, the aqueous glycol solution that adds 3ml 5% (concentration expressed in percentage by volume), place 30min down for 4 ℃, with above-mentioned acetate buffer solution dialysed overnight; Adding 20ml contains the sodium carbonate salt damping fluid of 270mg bovine serum albumin (BSA), 4 ℃ of following dialysed overnight; Add 40ml again and contain 300mg sodium borohydride (NaHB
4) the aqueous solution, place down 30min for 4 ℃.So far finished chemical connection.The gained reaction solution is concentrated into 5ml, separates with separator column Sephadex G-100, adding pH is 7.4 phosphoric acid buffer wash-out, makes the BSA-HSH-971 immunocomplex.
4mg BSA-HSH-971 immunocomplex is dissolved in 5ml physiological saline, mixes with complete Freund's adjuvant (CFA) 1: 1 (volume ratio); Get female Balb/c mouse in 8 ages in week, make subcutaneous multi-point injection complete Freund's adjuvant in back and BSA-HSH-971 mixture 0.5ml; Abdominal injection CFA and BSA-HSH-971 mixture 0.5ml again after one week, booster immunization, booster immunization is once weekly; Getting blood examination during the 4th weekend and survey antibody titer, after with physiological saline serum being diluted by 1: 10000, carry out enzyme linked immunosorbent assay (ELISA) and detect, is standard with the positive that develops the color; First three sky of cytogamy, the above-mentioned BSA-HSH-971 immunocomplex of intravenous injection 0.25ml.
Put to death mouse, prepare single splenocyte suspension, in the 50ml centrifuge tube, mix 10
8Individual splenocyte and 10
7The murine myeloma cell (NS-1 cell) of individual logarithmic phase growth, and adding the simple substratum of 30ml, centrifugal 8 minutes of 800 rev/mins of room temperatures are removed supernatant liquor, centrifuge tube is put in 37 ℃ of water-baths, slowly add minute adding of 0.8ml 50% (weight percent) polyoxyethylene glycol-4000,2 and finish, shake centrifuge tube while adding, slowly add simple substratum then, add 4ml in 3 minutes altogether, centrifugal 8 minutes of 800 rev/mins of room temperatures are removed supernatant liquor; (xanthoglobulin (H), aminopterin (A), thymidine (T), concentration is respectively H:1 * 10 to the HAT of adding 45ml20% (volume percent) calf serum
-6Mol/L, A:4 * 10
-9Mol/L, T:1.6 * 10
-7Mol/L) selective medium is inoculated in after the piping and druming in 3 96 orifice plates, contains 5% (volume percent) CO in air
2With cultivate under 37 ℃ the condition, after 7 days the nutrient solution in the culture plate is filled it up with, observe after 10 days, detect the antibody titer of hybrid cell nutrient solution with the ELISA method.To potency ratio preferably hybrid cell breed, go down to posterity, and (concentration is respectively H:10 to use HT instead gradually
-6Mol/L, T:1.6 * 10
-7Mol/L) substratum is until the substratum of 20% (volume percent) calf serum.
Take out the antibody positive porocyte, this cell dilution is become 1 cell/ml, be inoculated in 96 orifice plates of inoculating splenocyte with the HT substratum, every hole 0.1ml, making cell content is 2/hole, 1/hole or 0/hole.In air, contain 5% (volume percent) CO
2With cultivate under 37 ℃ the condition.Microscopically is observed, and selects the hole of having only a colony growth, and a large amount of amplification, detects the antibody in the culture supernatant.The positive porocyte of antagonist, a large amount of amplifications obtain clone strain three strains.
Get female Balb/c mouse in 8 ages in week, every mouse peritoneal injection 0.5ml pristane, week back inoculation hybridoma 10
6/ only, collect ascites after 7-10 days, 12000 rev/mins centrifugal 30 minutes, collect supernatant liquor, add 1/10000 (weight percent) NaN
3(sodium azide) aqueous solution is preserved down for-20 ℃.Make HSH-971 monoclonal antibody of the present invention with Sepharose CL-4B Protein A post (Immunopure (A) IgG Purification Kit) separation.
By analysis, the affinity constant of monoclonal antibody of the present invention and HSH-971 is 5 * 10
-7-2 * 10
-9, with the equal no cross reaction of polysaccharide of similar such as polysaccharide and alginic acid in the human bodies such as heparin, heparan, dermatan sulfate, chondroitin sulfate, hyaluronic acid.Through continuous passage in 8 months, frozen 1 year, recovery twice, the secretion capacity of this monoclonal antibody does not have considerable change; This antibody is preserved a week, the no considerable change of tiring at 37 ℃; This antibody subclass is respectively IgG1 (κ), IgG2a (κ), and karyomit(e) bar number is the 92-96 bar.This antibody can be used for detection, mensuration, quality standard, the purifying of HSH-971, and the biological effect of blocking-up HSH-971.
The step that induces ascites from mouse peritoneal in the present embodiment also can be used cell in vitro instead and be cultivated, concrete grammar is to contain the culture medium culturing of 15% calf serum with the clone strain cell, collect culture supernatant, separate preparation HSH-971 monoclonal antibody of the present invention with Sepharose CL-4B Protein A post (Immunopure (A) IgGPurification Kit).
Claims (3)
1, a kind of preparation method of beta-D-mannuronic acid oligosaccharide monoclonal antibody, it is characterized in that beta-D-mannuronic acid oligosaccharide is connected with the bovine serum albumin chemistry, make the immunocomplex immune mouse, get this mouse boosting cell and myeloma cell and merge, carry out selectivity with selective medium and cultivate, carry out cloning through dilution, select positive clone strain, and a large amount of amplifications, inject the homology mouse peritoneal and induce ascites or cultivate separation by cell in vitro.
2, preparation method as claimed in claim 1 is characterized in that described beta-D-mannuronic acid oligosaccharide and bovine serum albumin chemistry is connected and is in the sodium-acetate salt buffer and adds to carry out under the condition of the sodium periodate aqueous solution.
3, preparation method as claimed in claim 1 is characterized in that described immune mouse is bovine serum albumin-beta-D-mannuronic acid oligosaccharide immunocomplex to be dissolved in physiological saline and to mix with complete Freund's adjuvant (CFA) mouse is made the subcutaneous multi-point injection in back.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01135114 CN1401786A (en) | 2001-11-15 | 2001-11-15 | Process for preparing beta-D-mannuronic acid oligosaccharide monoclonal antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01135114 CN1401786A (en) | 2001-11-15 | 2001-11-15 | Process for preparing beta-D-mannuronic acid oligosaccharide monoclonal antibody |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005089776A1 (en) * | 2004-03-24 | 2005-09-29 | Ocean University Of China | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| EP1791967A2 (en) * | 2004-09-21 | 2007-06-06 | The Brigham And Women's Hospital, Inc. | Methods and compositions relating to mannuronic acid specific binding peptides |
| CN106432371A (en) * | 2016-10-31 | 2017-02-22 | 中国人民解放军第二军医大学 | Beta-1,2-D-oligomeric mannoprotein conjugates and preparation method and application thereof |
-
2001
- 2001-11-15 CN CN 01135114 patent/CN1401786A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005089776A1 (en) * | 2004-03-24 | 2005-09-29 | Ocean University Of China | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| KR100844918B1 (en) | 2004-03-24 | 2008-07-09 | 오션 유니벌시티 오브 차이나 | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| AU2005223923B2 (en) * | 2004-03-24 | 2008-10-23 | Ocean University Of China | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| EA011417B1 (en) * | 2004-03-24 | 2009-02-27 | Оушн Юниверсити Оф Чайна | Algin oligosaccharides and the derivatives thereof, the manufacture and the use of the same |
| US8835403B2 (en) | 2004-03-24 | 2014-09-16 | Meiyu Geng | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| US9493496B2 (en) | 2004-03-24 | 2016-11-15 | Ocean University Of China | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| US10213456B2 (en) | 2004-03-24 | 2019-02-26 | Ocean University Of China | Alginate oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| EP1791967A2 (en) * | 2004-09-21 | 2007-06-06 | The Brigham And Women's Hospital, Inc. | Methods and compositions relating to mannuronic acid specific binding peptides |
| EP1791967A4 (en) * | 2004-09-21 | 2009-03-25 | Brigham & Womens Hospital | Methods and compositions relating to mannuronic acid specific binding peptides |
| CN106432371A (en) * | 2016-10-31 | 2017-02-22 | 中国人民解放军第二军医大学 | Beta-1,2-D-oligomeric mannoprotein conjugates and preparation method and application thereof |
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