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Purification of Antibodies Against N-homocysteinylated Proteins by Affinity Chromatography on Nomega-homocysteinyl-aminohexyl-Agarose

Purification of Antibodies Against N-homocysteinylated Proteins by Affinity Chromatography on Nomega-homocysteinyl-aminohexyl-Agarose

Joanna Perła, Anetta Undas, Tomasz Twardowski, Hieronim Jakubowski

J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Aug 5;807(2):257-61.

PMID: 15203038

Abstract:

Modification with homocysteine (Hcy)-thiolactone leads to the formation of N-Hcy-Lys-protein. Although N-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nomega-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-N-Hcy-Lys-protein antibodies. Nomega-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of omega-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with N-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-N-Hcy-Lys-protein IgG was adsorbed on Nomega-homocysteinyl-aminohexyl-Agarose column at pH 8.6 and eluted with a pH 2.3 buffer. Enzyme-linked immunosorbent assays demonstrate that the antibody recognizes specifically N-homocysteinylated variants of hemoglobin, albumin, transferrin, and antitrypsin.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP58856738	ω-Aminohexyl-Agarose		58856-73-8	Price

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