Development of polyclonal antibodies for the detection of styrene oxide modified proteins

Styrene is widely used as one of the most important industrial materials for the production of synthetic rubbers, plastic, insulation, fiberglass, and automobile parts. Inhaled styrene has been reported to produce respiratory toxicity in humans and animals. Styrene oxide, a reactive metabolite of styrene formed via cytochrome P450 enzymes, has been reported to form covalent bonds with proteins, such as albumin and hemoglobin. Among all of the amino acids, cysteine is the most reactive amino acid to be modified by electrophilic species. The purpose of this study is to develop polyclonal antibodies for the detection of styrene oxide cysteinyl protein adducts. Two immunogens were designed, synthesized, and used to induce polyclonal antibodies in rabbits. Immune responses were observed from the raised antibodies by antiserum dilution tests. Competitive ELISA demonstrated that the resulting antibodies specifically recognized the styrene oxide-derived N-acetylcysteine adduct. Western blot results showed that the antibodies recognize styrene oxide-modified albumin. The binding was found to depend on the amount of protein adducts blotted and hapten loading in protein adducts. No cross reaction was observed from the native protein. Competitive Western blots further indicated that these antibodies specifically recognized styrene oxide cysteinyl-protein adducts. Immunoblots revealed the presence of several bands at a molecular weight ranging from 50 to 80 kDa in rat nasal mucosa treated with styrene. In conclusion, we successfully raised polyclonal antibodies to detect styrene oxide-derived protein/cysteine adducts.