Purification of antibodies against N-homocysteinylated proteins by affinity chromatography on Nω-homocysteinyl-aminohexyl-Agarose

Modification with homocysteine (Hcy)-thiolactone leads to the formation of Nε-Hcy-Lys-protein. Although Nε-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nω-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-Nε-Hcy-Lys-protein antibodies. Nω-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of ω-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with Nε-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-Nε-Hcy-Lys-protein IgG was adsorbed on Nω-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.