A monoclonal antibody has been developed that recognizes only protein-acetaldehyde (AA) adducts prepared under reducing conditions: 5 mM AA with 30 mM sodium cyanoborohydride overnight at 37°. This monoclonal antibody is a mouse IgG2b that has been designated RT1.1. The primary adduct formed when proteins are exposed to acetaldehyde under reducing conditions is N-ethyl lysine (NEL). To examine the epitope specificity of RT1.1, inhibition ELISAs were developed using NEL and other possible inhibitors, such as arginine, ethylamine, lysine and proteins modified with AA under non-reducing conditions. RT1.1 (at half-maximum optical density, 50 ng mL) was inhibited only by NEL and was independent of the carrier or the pH of the buffer used in the ELISA. Further evidence indicating that NEL is the epitope recognized by RT1.1 was obtained using mouse and human epidermal growth factor (EGF). Both proteins contain one alpha amino group but only the human-EGF contains lysine residues with epsilon amino groups. In experiments where these two proteins were modified with AA under reducing conditions, RT1.1 reacted only with human-EGF. These studies demonstrate that RT1.1 is specific for NEL that is formed by the ethylation of proteins with acetaldehyde under reducing conditions. Additionally, these studies demonstrate that the procedures and methods used herein may be useful for characterizing other antibodies prepared to AA-modified proteins under a variety of defined in vitro chemical conditions.
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