Quantitation of the binding of phenanthrene-9,10-oxide to hemoglobin

Phenanthrene-9,10-oxide (PhO) is a moderately potent mutagen in the Ames microbial mutagenesis assay. The purpose of this project was to detect hemoglobin (Hb) adduct formation in the blood caused by PhO. A series of monoclonal antibodies were tested in competition enzyme-linked immunosorbent assays (ELISAs) to see what amounts of PhO attached to Hb could be detected. The ability of the ELISAs to recognize Hb adducts of PhO was poorer than their ability to recognize thiol adducts of PhO, so proteolytic cleavage was done on Hb containing PhO adducts to increase the sensitivity of the ELISAs. ELISA sensitivity did not increase after proteolysis. ELISAs that employed four monoclonal antibodies, 1PD2, 9PC3, 3PD3, and 1PA1, gave a good response to PhO:Hb adducts. The 50% inhibition points in the ELISAs were at 65, 100, 82, and 70 picomole for 1PD2, 9PC3, 3PD3, and 1PA1 respectively.