Production of antibodies that recognize the heterogeneity of immunoreactive sites in human hemoglobin chemically modified by acetaldehyde.
Human hemoglobin (Hgb) was incubated with acetaldehyde under two different conditions: (a) in the presence of 250 mM acetaldehyde for 1 hr then reduced with 100 mM NaCNBH3 for an additional 4 hr at room temperature; and (b) in the presence of 500 mM acetaldehyde for 10 days at room temperature and then reduced with 1 mM NaBH4 for 1 hr. It was found that 44% and 27% of free amino groups in Hgb-acetaldehyde adduct (AA) remained unmodified when Hgb was treated under conditions (a) and (b), respectively. SDS-PAGE analysis revealed that the molecular weight of Hgb-AA(a) [Hgb modified under condition (a)] was slightly greater than that of unmodified Hgb and extensive protein cross-linking had occurred in Hgb-AA(b) [Hgb modified under condition (b)]. Electrophoresis on agarose gel showed the order of negative charge was Hgb-AA(b) > Hgb-AA(a) > unmodified Hgb. Polyclonal antibody raised in rabbits using keyhole limpet hemocyanin as the carrier protein modified by acetaldehyde under condition (a) [i.e., KLH-AA(a)] preferentially recognized Hgb-AA(a), whereas antibody raised using KLH-AA(b) as the immunogen recognized only Hgb-AA(b). In conclusion, antibodies raised with protein-AA antigens produced under different conditions recognize different epitopes.
Lin
R C
RC
Department of Medicine, Indiana University School of Medicine, Indianapolis.
Shahidi
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S
Lumeng
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D000079:Acetaldehyde / Q000276:immunology / Q000633:toxicity*; D000910:Antibodies, Heterophile / Q000096:biosynthesis; D015478:Antigens, Heterophile / Q000276:immunology*; D001323:Autoantibodies / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D006441:Hemoglobin A / Q000187:drug effects* / Q000276:immunology*; D006519:Hepatitis, Alcoholic / Q000276:immunology*; D006801:Humans; D005779:Immunodiffusion; D008970:Molecular Weight
D000910:Antibodies, Heterophile; D015478:Antigens, Heterophile; D001323:Autoantibodies; C028672:hemoglobin AA; D006441:Hemoglobin A; D000079:Acetaldehyde