Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye

PURPOSE: To develop an antibody that recognizes a variety of advanced glyca tion endproduct (AGE) epitopes. METHODS: Glycolaldehyde was used to modify bovine serum albumin and HPLC an alysis was used to measure pentosidine formation as an indicator of AGE for mation. A polyclonal anti-AGE antibody was synthesized by injecting glycola ldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin colum n, and characterized by immunoblot analysis. RESULTS: HPLC analysis of glycolaldehyde treated bovine serum albumin detec ted high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody r ecognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evide nce of AGEs in Bruch's membrane (including basal laminar deposits and druse n), choroidal extracellular matrix, and vessel walls in an 82 year old nond iabetic globe. A similar staining pattern was observed in an age-matched di abetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. CONCLUSIONS: A unique anti-AGE antibody was synthesized that recognizes a v ariety of AGE epitopes including carboxymethyllysine and pentosidine. Its b est use might be in broad surveys of the age-dependent accumulation of a la rge number of AGE epitopes that might not be revealed by antibodies to pent osidine or CML.