Immunochemical crossreactivity of antibodies specific for “advanced glycation endproducts” with “advanced lipoxidation endproducts”  Torsten Richter, Gerald Münch, Hans-Joachim Lüth, Thomas Arendt, Rosemarie Kientsch-Engel, Peter Stahl, Dörte Fengler, Björn Kuhla  Neurobiology of Aging  Volume 26, Issue 4, Pages 465-474 (April 2005) DOI: 10.1016/j.neurobiolaging.2004.04.009

Fig. 1 Chemical structures of AGEs, ALEs and routes to their formation. Formation of AGEs: After the Amadori rearrangement (A), the reaction is irreversible and leads (after subsequent oxidation steps) to fluorescent, often crosslinked AGEs (B), to non-fluorescent AGEs (C), or to FDP-lysine and MP-lysine (derived from the reaction of acrolein with lysine) (D). The reactive carbonyl compounds glyoxal, MDA and acrolein, which can all be derived from lipid peroxidation, are also shown (E). Neurobiology of Aging 2005 26, 465-474DOI: (10.1016/j.neurobiolaging.2004.04.009)

Fig. 2 Set-up of the peptide library containing 20 amino acids. The 20 spots of the peptide library contain one β-alanin-spacer (C-terminus) and one of the 20 amino acids (N-terminus). One peptide strip containing two rows of 20 peptides was used for each experiment. The triple amino acid code is used. Neurobiology of Aging 2005 26, 465-474DOI: (10.1016/j.neurobiolaging.2004.04.009)

Fig. 3 Immunohistochemical staining of human AD brain with the AGE-antibodies 4G9 and K2189. Immunohistochemical staining of Brodmann area 22 of the cerebral cortex of an AD patient with the two AGE-antibodies. (A)–(C) and (D)–(F) shows the immunoreactivity with the antibodies 4G9 and K 2189, respectively. In general, both AGE antibodies demonstrate the same structures in the AD brain, e.g., nerve cells, predominantly pyramidal neurons (A and D), plaques and plaque associated astroglia (B and E), and blood vessels (C and F). Neurobiology of Aging 2005 26, 465-474DOI: (10.1016/j.neurobiolaging.2004.04.009)

Fig. 4 Recognition of glyoxal, MDA or acrolein-modified proteins on dot blots. Unmodified chicken egg albumin, HSA, MAP-tau and β-amyloid and modifications of the proteins with glyoxal, MDA and acrolein in concentrations of 1mM were spotted on nitrocellulose membranes and immunostained with the antibody 4G9 or the antibody K2189. Glyoxal modifications are always strongly immunoreactive, whereas the immunoreactivity of MDA- and acrolein-modifications depend on the protein and the antibody. A typical dot blot is shown in (A), its densitrometric analysis in (B) and (C). The experiments were done in triplicate (asterisk indicates a significant difference compared to the unmodified protein (P < 0.05)). Neurobiology of Aging 2005 26, 465-474DOI: (10.1016/j.neurobiolaging.2004.04.009)

Fig. 5 Reaction of carbonyls with terminal amino acids on peptide membranes. Each strip contains two rows, which were incubated under identical conditions with acrolein, MDA and glyoxal. Immunostaining was performed with antibody 4G9 (A) or antibody K2189 (B). Neurobiology of Aging 2005 26, 465-474DOI: (10.1016/j.neurobiolaging.2004.04.009)

Fig. 6 Quantitative analysis of the immunoreactivity of the peptide spots modified with acrolein, MDA and glyoxal. To evaluate the antibody reactivity shown in Fig. 5, the relative intensity of the signal compared to the background (defined as 1.0) was calculated. Although the signal/noise ratio differs between the two antibodies, a significant immunoreactivity of lysine- and arginine-modifications can be observed for antibody 4G9 (A) and antibody K2198 (B). Neurobiology of Aging 2005 26, 465-474DOI: (10.1016/j.neurobiolaging.2004.04.009)