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Production of Antibodies That Recognize the Heterogeneity of Immunoreactive Sites in Human Hemoglobin Chemically Modified by Acetaldehyde
Author(s) -
Lin Renee C.,
Shahidi Samin,
Lumeng Lawrence
Publication year - 1993
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/j.1530-0277.1993.tb00858.x
Subject(s) - hemoglobin , acetaldehyde , antibody , chemistry , biochemistry , biology , immunology , ethanol
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 NaCNBH 3 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 NaBH 4 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.