Immunochemical detection of N2-[1-(1-carboxy)ethyl]guanosine, an advanced glycation end product formed by the reaction of DNA and reducing sugars or L-ascorbic acid in vitro.

In the Maillard reaction, free amino groups of proteins and nucleic acids react with reducing sugars to form advanced glycation end products (AGE). A major product found in reaction mixtures of guanosine and glucose is N2-[1-(1-carboxy)ethyl]guanosine (CEG), which, therefore, can be used as a marker of advanced glycation of DNA. An enzyme-linked immunosorbent assay (ELISA) was developed to detect and to semi-quantitate nonenzymatic glycosylation of DNA. A polyclonal antiserum was raised against CEG linked to keyhole limpet hemocyanin. A protocol for a competitive ELISA was developed, and the antiserum was tested for crossreactivity. Several unmodified nucleotides and N2-modified guanosine derivatives showed no or negligible crossreactivity. Only very similar structures like N2-(carboxymethyl)guanosine and N2-(1-carboxy-3-hydroxypropyl)guanosine, which have been identified as reaction products of glucose or l-ascorbic acid and guanosine, display significant binding activity. The signal can be totally repressed by free CEG, yet protein-bound CEG is a stronger inhibitor. DNA incubated with d-glucose, dihydroxyacetone, l-ascorbic or l-dehydroascorbic acid shows a signal inhibition indicating the formation of CEG in vitro. The competitive ELISA procedure proved to be a sensitive method which can be used to detect glycation of DNA in vivo.