Immunological detection of fructated proteins in vitro and in vivo.

An antibody has been raised against fructated lysine in proteins by immunizing fructated lysine-conjugated ovalbumin in rabbits. The affinity-purified antibody specifically recognized proteins incubated with fructose but not with other reducing sugars such as glucose, galactose or ribose, as judged by immunoblotting and ELISA techniques. Competitive binding to this antibody was observed specifically by fructated lysine but not by glucated lysine, glucose, fructose or lysine. The antibody binds specifically to fructated lysine residues in the protein but not to borohydride-reduced material or advanced glycation end products, indicating that the antibody recognizes only the reducing, carbonyl-containing forms produced in the early stage of the fructation reaction. When BSA was incubated with various concentrations of fructose, the reactivity of the antibody increased in a dose- and time-dependent manner. When soluble proteins prepared from either normal or streptozotocin-induced diabetic rat eyes were analysed by ELISA with this antibody, an increase in the reactive components was observed as a function of aging as well as under diabetic conditions. Western blotting analysis showed that lens crystallin reacted highly with this antibody. Because fructose is biosynthesized largely through the polyol pathway, which is enhanced under diabetic conditions, and lens is known to have a high activity of enzymes in this pathway, this antibody is capable of recognizing fructated proteins in vivo. Thus it is a potentially useful tool for investigating two major issues that seem to be involved in diabetic complications, namely the glycation reaction and the polyol pathway.