PROTEIN-ACETALDEHYDE ADDUCTS IN SERUM OF ALCOHOLIC PATIENTS

Enzyme‐linked immunosorbent assay (ELISA) was used to detect the presence of protein‐acetaldehyde adducts (‐AAs) in human serum samples. Two methods were compared: (1) direct ELISA: samples, rabbit anti‐hemocyanin‐AA IgG, and β‐galactosidase (β‐gal) conjugated goat anti‐rabbit serum IgG added to a 96‐well ELISA plate in a stepwise manner; and (2) two‐site or sandwich ELISA: serum samples added to an ELISA plate that had been precoated with anti‐hemocyanin‐AA IgG (the capture antibody) and incubated stepwise with biotinated anti‐hemocyanin‐AA IgG (the signal anti‐body) and avidin‐β‐gal conjugates. Serum protein‐AA levels were then assayed by bound β‐gal activities at OD405. When human hemoglobin (Hgb)‐AA was used as a model protein‐AA for the sandwich ELISA, the EC60 (estimated concentration that corresponds to 50% of the OD405 response range) was 7 ng/ml. Direct ELISA was less sensitive (EC50 of 120 ng/ml). Adding control human serum to Hgb‐AA increased the EC50 of the direct ELISA more than the sandwich ELISA. Intra‐ and interassay coefficients of variance for sandwich ELISA were both about 8%. Detection of Hgb‐AA by sandwich ELISA was highly specific. The above results with anti‐hemocyanin‐AA IgG were also obtained when anti‐myoglobin‐AA IgG was used in sandwich ELISA. Using sandwich ELISA and anti‐hemocyanin‐AA IgG, OD405 for sera of control subjects and alcoholic patients were 0.036 ± 0.033 (±SEM, n= 28) and 0.150 ± 0.088 (n= 28), respectively. Serum protein‐AAs reacted more strongly with anti‐myoglobin‐AA IgG than anti‐hemocyanin‐AA IgG. The data indicate that high titers of serum protein‐AAs are present in most alcoholic patients. Copyright © 1990, Wiley Blackwell. All rights reserved