Purification of antibodies against N-homocysteinylated proteins by affinity chromatography on Nomega-homocysteinyl-aminohexyl-Agarose.
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.
Perła
Joanna
J
Department of Microbiology & Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, Newark, NJ 07101, USA.
Undas
Anetta
A
Twardowski
Tomasz
T
Jakubowski
Hieronim
H
D000906:Antibodies / Q000302:isolation & purification*; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006710:Homocysteine / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry*
D000906:Antibodies; D011506:Proteins; D006710:Homocysteine; D012685:Sepharose