Usefulness of specific antibodies to immobilize pyridylaminated N-glycans for solid-phase interaction analyses
| Autor: | Le, Na, Kato, Mari, Ogawa, Haruko |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Zdroj: | お茶の水女子大學自然科學報告. 61(2):31-45 |
| ISSN: | 0029-8190 |
| Popis: | application/pdf 紀要論文 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. |
| Databáze: | OpenAIRE |
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