TY - JOUR
T1 - Immunohistochemical detection of N-homocysteinylated proteins in humans and mice
AU - Perła-Kaján, Joanna
AU - Stanger, Olaf
AU - Łuczak, Michał
AU - Ziółkowska, Agnieszka
AU - Malendowicz, Ludwik K.
AU - Twardowski, Tomasz
AU - Lhotak, Šárka
AU - Austin, Richard C.
AU - Jakubowski, Hieronim
N1 - Funding Information:
This work was supported in part by a grant No. N401 065 32/1504 from the Ministry of Science and Higher Education, Poland.
PY - 2008/9
Y1 - 2008/9
N2 - 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.
AB - 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.
KW - Antibodies
KW - Atherosclerosis
KW - Homocysteine thiolactone
KW - N-homocysteinylated protein
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U2 - 10.1016/j.biopha.2008.04.001
DO - 10.1016/j.biopha.2008.04.001
M3 - Article
C2 - 18501553
AN - SCOPUS:48249093757
SN - 0753-3322
VL - 62
SP - 473
EP - 479
JO - Biomedicine and Pharmacotherapy
JF - Biomedicine and Pharmacotherapy
IS - 7
ER -