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Usefulness of specific antibodies to immobilize pyridylaminated N-glycans for solid-phase interaction analyses
http://hdl.handle.net/10083/49657
http://hdl.handle.net/10083/4965756f6c3b6-396a-4e2e-9d76-bd8a16ffa7b8
名前 / ファイル | ライセンス | アクション |
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Item type | 紀要論文 / Departmental Bulletin Paper(1) | |||||
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公開日 | 2011-03-10 | |||||
タイトル | ||||||
タイトル | Usefulness of specific antibodies to immobilize pyridylaminated N-glycans for solid-phase interaction analyses | |||||
言語 | ||||||
言語 | eng | |||||
資源タイプ | ||||||
資源 | http://purl.org/coar/resource_type/c_6501 | |||||
タイプ | departmental bulletin paper | |||||
著者 |
Le, Na
× Le, Na× Kato, Mari× Ogawa, Haruko |
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抄録 | ||||||
内容記述タイプ | Abstract | |||||
内容記述 | Pyridylamination is a method of fluorescence-tagging oligosaccharides that enables efficient purification of each glycan from mixtures of biological materials and structural analyses of glycans by HPLC mapping techniques. A method to utilize purified pyridylamino (PA)-oligosaccharides immediately in interaction studies with specific binding proteins would be useful to study the biological functions of glycans. To achieve this, we prepared a neoglycoprotein by complexing periodate-oxidated PA-GlcNAc2 and rabbit serum albumin (RSA) by reductive amination. Immunizing a rabbit with the neoglycoprotein, specific polyclonal antibodies, generated anti-PA-GlcNAc2-IgGs that were purified from the rabbit antiserum by using a protein A column and subsequently an RSA-Sepharose column to remove anti-RSA-IgGs. When the wells of a plastic plate were coated with anti-PA-GlcNAc2-IgGs and PA-GlcNAc2 was added to the wells, PA-GlcNAc2 was immobilized and concentration-dependently bound with biotinylated concanavalin A or Psathyrella velutina lectin (PVL) by ELISA. The IgGs were capable of immobilizing PA-N-glycans in the solid phas\ e. Using F(ab’)2 fragments prepared from the IgGs as an immobilizing reagent for PA-GlcNAc2 decreased the background absorbance in the ELISA, suggesting that N-glycans in the Fc region of the IgGs interfered with lectin binding. The effects of de-N-glycosylation of F(ab’)2 suggested that the N-glycans which are reactive to Con A but not involved in the antigen recognition are present in the fragments. Quantitative studies using surface plasmon resonance indicated that the dissociation constant of the interaction between the F(ab’)2 and PA-GlcNAc2 was 6 x 10-6 M. The results demonstrate the utility of the IgGs and F(ab’)2 as immobilizing reagents for PA-N-glycans. Our method provides a new approach to interaction analyses of PA-oligosaccharides. |
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書誌情報 |
お茶の水女子大學自然科學報告 巻 61, 号 2, p. 31-45, 発行日 2011-03 |
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ISSN | ||||||
収録物識別子タイプ | ISSN | |||||
収録物識別子 | 00298190 | |||||
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収録物識別子タイプ | NCID | |||||
収録物識別子 | AN00033958 | |||||
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内容記述タイプ | Other | |||||
内容記述 | application/pdf | |||||
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919229 bytes | ||||||
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値 | publisher | |||||
日本十進分類法 | ||||||
主題Scheme | NDC | |||||
主題 | 400 | |||||
出版者 | ||||||
出版者 | お茶の水女子大学 | |||||
資源タイプ | ||||||
内容記述タイプ | Other | |||||
内容記述 | 紀要論文 | |||||
資源タイプ・ローカル | ||||||
紀要論文 | ||||||
資源タイプ・NII | ||||||
Departmental Bulletin Paper | ||||||
資源タイプ・DCMI | ||||||
text | ||||||
資源タイプ・ローカル表示コード | ||||||
03 | ||||||
所属 | ||||||
Ochanomizu University |