Immunological detection of fructated proteins in vitro and in vivo

An antibody has been raised against fructated lysine in proteins by immuniz ing 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 antibod y 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 on ly the reducing, carbonyl-containing forms produced in the early stage of t he fructation reaction. When BSA was incubated with various concentrations of fructose, the reactivity of the antibody increased in a dose- and time-d ependent manner. When soluble proteins prepared from either normal or strep tozotocin-induced diabetic rat eyes were analysed by ELISA with this antibo dy, an increase in the reactive components was observed as a function of ag ing 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 glyca tion reaction and the polyol pathway.