Immunohistochemical detection of N-homocysteinylated proteins in humans and mice

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

Research output: Contribution to journalArticlepeer-review

47 Scopus citations


N-homocysteinylation of ε-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 Nε-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 Nε-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.

Original languageEnglish (US)
Pages (from-to)473-479
Number of pages7
JournalBiomedicine and Pharmacotherapy
Issue number7
StatePublished - Sep 2008
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Pharmacology


  • Antibodies
  • Atherosclerosis
  • Homocysteine thiolactone
  • N-homocysteinylated protein


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