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 beta-galactosidase (beta-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 antibody) and avidin-beta-gal conjugates. Serum protein-AA levels were then assayed by bound beta-gal activities at OD405. When human hemoglobin (Hgb)-AA was used as a model protein-AA for the sandwich ELISA, the EC50 (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.(ABSTRACT TRUNCATED AT 250 WORDS)
Lin
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Department of Medicine, Indiana University School of Medicine, Indianapolis.
Lumeng
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Shahidi
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S
Kelly
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Pound
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D000079:Acetaldehyde / Q000097:blood*; D000437:Alcoholism / Q000097:blood* / Q000175:diagnosis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D009211:Myoglobin / Q000378:metabolism; D011485:Protein Binding
D006454:Hemoglobins; D009211:Myoglobin; D000079:Acetaldehyde