Immunohistochemical detection of N-homocysteinylated proteins in humans and mice Journal Articles uri icon

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  • N-homocysteinylation of epsilon-amino group of protein lysine residues by homocysteine (Hcy) thiolactone has been implicated in vascular disease in humans. We have previously generated polyclonal rabbit anti-N-Hcy-protein IgG antibodies that specifically recognize the Nepsilon-Hcy-Lys epitope on N-homocysteinylated proteins. The present work was undertaken to examine the utility of these antibodies for the immunohistochemical detection of N-homocysteinylated proteins in biological samples. We found that the rabbit antibody specifically detected N-Hcy-protein in a dot-blot assay, that the signal resulting from the reaction of the antibody with N-Hcy-protein depended on the amount of the antigen, and that the sensitivity of the assay was protein-dependent. The rabbit anti-N-Hcy-protein IgG also specifically detected Nepsilon-Hcy-Lys epitopes in human tissues, as shown by positive immunohistochemical staining of myocardium and aorta samples from cardiac surgery patients, and a lack of staining when the antibody was pre-adsorbed with N-Hcy-albumin. We also observed increased immunohistochemical staining for N-Hcy-proteins in aortic lesions from ApoE-/- mice with hyperhomocysteinemia induced by a high methionine diet, relative to ApoE-/- mice fed a control chow diet. In conclusion, polyclonal rabbit anti-N-Hcy-protein antibody can detect and monitor N-homocysteinylated proteins in human and mouse tissues with good sensitivity and specificity.


  • Perła-Kaján, Joanna
  • Stanger, Olaf
  • Łuczak, Michał
  • Ziółkowska, Agnieszka
  • Malendowicz, Ludwik K
  • Twardowski, Tomasz
  • Lhotak, Šárka
  • Austin, Richard C
  • Jakubowski, Hieronim

publication date

  • September 2008

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