Characterization of antibody affinities using an AGE-modified dipeptide spot library.
Recent immunological approaches have greatly helped understanding the significance of advanced glycation end products (AGEs) in age-related diseases. However, immunohistochemical localization of AGEs in tissues or their quantitative determination in biological fluids sometimes yields inconsistent results among different investigators. Since these differences might be caused by the heterogeneity of the AGE antibodies, a systematic mapping of their epitope recognition pattern would help in the evaluation of these different results. For this purpose, an N-terminally acetylated combinatorial dipeptide library with 400 positionally defined dipeptides was modified by glucose to yield AGEs, which are thought to be present on the protein side chains. Using this library, we have characterized six different AGE antibodies in respect to their immunoreactivity towards AGE-modified dipeptides. All antibodies predominantly recognized only one AGE-modified amino acid side chain (and not a particular dipeptide). In most cases, arginine- and lysine- derived AGEs, but also some epitopes on asparagine and on heterocyclic amino acids were recognized. Very interestingly, the immunization of different animals with the same antigen produced AGE antibodies with a totally different epitope recognition pattern. Defining the antigen recognition pattern with this method might help in the quality control of polyclonal AGE antibodies for immunodetection. In addition, defined AGE antibodies (as neutralizing antibodies) could also help to define "signal-active" AGEs in terms of specific AGE-mediated cellular responses.
IZKF Immunobiological Cell Biology Unit, University of Leipzig, Johannisallee 30a 04103 Leipzig, Germany.
Sladjana
S
Dukic-Stefanovic
Dorothee
D
Schicktanz
Amanda
A
Wong
Dieter
D
Palm
Peter
P
Riederer
Toshimitsu
T
Niwa
Reinhard
R
Schinzel
Gerald
G
Münch
D000915:Antibody Affinity; D000918:Antibody Specificity; D004151:Dipeptides / Q000138:chemical synthesis / Q000276:immunology; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D017127:Glycation End Products, Advanced / Q000737:chemistry* / Q000276:immunology*; D006020:Glycopeptides / Q000276:immunology; D019151:Peptide Library
D004151:Dipeptides; D000939:Epitopes; D017127:Glycation End Products, Advanced; D006020:Glycopeptides; D019151:Peptide Library