DETECTION OF REDUCED ACETALDEHYDE-PROTEIN ADDUCTS USING A UNIQUE MONOCLONAL-ANTIBODY

Acetaldehyde (AA), the major product of alcohol metabolism, has been shown to bind to proteins in vivo and form chemical adducts. These AA-protein adducts have been shown to alter protein structure and function and may result in tissue damage. Recent reports have shown that polyclonal antibodies can be produced that recognize proteins modified in vitro with AA in the presence of sodium cyanoborohydride (NaCNBH3), a strong reducing (R) agent. Antibodies prepared in this way have been shown to recognize proteins in the livers of rats fed alcohol chronically. Because multiple AA-protein adducts can be recognized by polyclonal antisera, and a variety of adducts may be formed in vitro or in vive, this study was designed to develop monoclonal antibodies specific for proteins modified by AA. In addition, adducts formed under R conditions are probably chemically different than those formed under nonreducing (NR) conditions, and monoclonal antibodies may provide the specificity required to distinguish these chemical differences. Balb/c mice were immunized with bovine brain tubulin that was modified by treatment with 5 mM AA for 7 days under NR conditions. Sera from immunized animals were tested for antibody activity to the immunogen (protein-NR) and for cross-reactivity to protein-R and unmodified protein. Although the highest serum antibody titers were seen toward the NR adduct, antibodies to the R adduct were also detected. This activity difference was independent of the carrier protein, because NR end R bovine serum albumin, keyhole limpet hemocyanin, and actin also gave similar results when used as the adducted protein. Surprisingly, all the monoclonal antibodies (RT1.1, RT1.2, RT1.3, and RT1.4) produced by hybridomas generated from spleen cells from NR-tubulin immunized mice recognized the R and not the NR adduct. One of these hybridomas (RT1.1) produces an IgG2b antibody that reacts with all tested proteins that have been modified with AA under chemical R conditions. Because of its monoclonal specificity, this antibody may be useful in probing for the presence of R AA-protein adducts made both in vitro and in vivo.