Immunological detection of glycated proteins in normal and streptozotocin-induced diabetic rats using anti hexitol-lysine IgG

Abstract

A polyclonal antibody specific for the Amadori compound, a product of an early stage of the Maillard reaction, was raised in rabbits by immunization with hexitol-lysine (1-glucitol-lysine or 1-mannitol-lysine) coupled with various carrier proteins. The affinity purified antibody has a high titre and preferentially recognizes the glucose adduct, in the presence of sodium borohydride, as judged on enzyme-linked immunosorbent assay as well as immunoblot analysis. The glycated proteins (Amadori products) in various tissues of normal and streptozotocin-induced diabetic rats were examined by immunoblot analysis. In diabetic conditions, kidney, liver, lens, brain and lung proteins are more susceptible to glycation than other tissue proteins. Heart, spleen, adrenal gland and muscle proteins exhibit similar extents of glycation in both normal and diabetic conditions. This is the first demonstration of a specific antibody against the Amadori compound being raised with a synthetic compound, and of the tissue distribution of glycated proteins in normal and diabetic conditions. The antibody was very useful for in vitro and in vivo experiments on the Maillard reaction.

Specific detections of the early process of the glycation reaction by fructose and glucose in diabetic rat lens

“…Quite recently we developed a speci¢c antibody against fructated proteins [9]. This, together with our anti-hexitol lysine antibody [10], enabled us to analyze precisely and independently the role of glucation and fructation in vivo.…”

“…For the quanti¢cation of glucation, samples were reduced by adding 10 Wl of 0.1 M NaBH R solution as described previously [10]. The plate was washed four times with PBS containing 0.05% Tween-20 to remove unbound proteins and blocked with 1% bovine serum albumin (BSA) in PBS by incubation at room temperature for 1 h. The a¤nity puri¢ed polyclonal antibody raised against 1-hexitol lysine [10] diluted to 1:500 or that against fructated lysine [9] diluted to 1:250 with PBS was added and reacted at room temperature for 2 h. After washing four times, horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000 dilution in PBS) was added and allowed to react for 2 h. Peroxidase activity was detected by adding 50 Wl of an O-phenylenediamine dihydrochloride solution containing hydrogen peroxide. The reaction was stopped by the addition of 50 Wl of 1 N H P SO R to each well.…”

“…immunoblotting For the detection of fructated proteins by immunoblotting, we biotinylated anti-fructated lysine IgG using a protein biotinylation system (Amersham, Uppsala, Sweden), since the goat anti-rabbit IgG (the second antibody) bound non-speci¢cally to rat Q-crystallins, although it did not cross-react with Q-crystallins in a native conformation [10]. Whole lens proteins were subjected to 15% SDS-PAGE and were transferred onto PVDF membranes under semi-dry conditions using a Trans-blot (Bio-Rad, Hercules, CA, USA).…”

“…Whole lens proteins were subjected to 15% SDS-PAGE and were transferred onto PVDF membranes under semi-dry conditions using a Trans-blot (Bio-Rad, Hercules, CA, USA). For Immunoblot analysis, by anti-hexitol Lys IgG, the blot was reduced with 0.1 M NaBH R for 2 h at room temperature prior to incubation with the IgG [10,11]. The membranes were blocked by incubation with 4% skimmed milk in Tris-bu¡ered saline (TBS; 10 mM TrisHCl, pH 7.4, and 0.15 M NaCl) at room temperature for 1 h with gentle agitation, washed four times with TBS containing 0.05% Tween 20 for 15 min each time, and then incubated with the diluted (1:500) biotinylated anti-fructated lysine IgG at 4³C overnight.…”

“…The use of this antibody, together with antihexitol lysine antibody which speci¢cally recognizes the reduced form of an Amadori product formed in glucated proteins [10], enables the analysis of the early events in the glycation reaction by glucose and fructose to be carried out.…”