Development of an immunoassay to detect hemoglobin adducts formed by benzene exposure

Polyclonal murine antibodies that recognize the adducts formed by benzene metabolites in hemoglobin (Hb) were prepared and used to develop immunoassays. In competitive inhibition assays, the concentration of competitor needed to reduce the signal by 50% (IC50) was less than 10 pmoles for hydroquinone-hemoglobin (HQHb) adducts and less than 1 pmole for 1,2,4 trihydroxybenzene-hemoglobin (TriOH Hb). Hemoglobin (Hb) incubated with either phenol or catechol (CAT) did not elicit antibodies suitable for quantitative immunoassays. The metabolite-directed immunoassays were tested using hemoglobin from mice previously administered [C14] benzene for two to four weeks. The most sensitive assay for hydroquinone measured 0.49 pmoles adduct/40 pmoles Hb (191 pmoles adduct/mg Hb) in mice treated with 200 mg/kg benzene (P < 0.05, Student's t test). TriOH Hb adducts were not detected.