The trapping of phenanthrene-9,10-oxide (PhO) present in environmental mixtures and the stereoselectivity and the mapping of the binding sites of monoclonal antibodies against thiol addition products of PhO

The objective of this research was to develop a simple, sensitive, and useful method for the trapping and detection of environmental electrophiles using specific monoclonal antibodies (MAbs) in Enzyme-Linked Immunosorbent Assays (ELISAs). The MAbs were raised against thiol adducts of phenanthrene-9,10-oxide (PhO). A thiolated polymer on a solid support was used to trap PhO by covalent bonding. Ten picomole level of the PhO was detected with a direct ELISA assay. Some of the antibodies were inhibited in a competitive assay when different thiol compounds, including a purine riboside, were used to trap PhO in solution as a soluble competitor. Picomole and sub-picomole levels of PhO were detected in these assays and in a competitive assay using PhO, without thiolation, as the soluble competitor. No interference with the electrophile trapping or the ELISA assay by tap or distilled water was observed. Turbidity due to bacteria (about 107 million Salmonella per milliliter) and suspended clay particles (up to 2 mg/mL) also did not interfere with the assay procedures. pH values between 5 and 9 did not affect the assay. However, 0.088 mM of chlorine and 1.25 mM humic acid total organic carbon (TOC) did affect the assay. Because chirality is an important determinant of the toxicity of electrophilic metabolites, chiral specificities of the antibodies were determined. Antibody 9PC3 recognized thiol adducts of the K-region epoxides of benzo(a)pyrene (B(a)P), pyrene, benz(a)anthracene (BA), benzo(c)phenanthrene (B(c)Ph) and 1-azaphenanthrene (1-APh) at picomole and sub-picomole levels. Antibody 1PD2 recognized only the thiol adducts of BA and B(c)Ph. It did not recognize 1-APh-oxide thiol adducts despite the recognition of femtomole levels of PhO and its thiol adducts. The antibodies showed between 10- to 50-fold preference for the S,S isomers of the thiol adducts over the R,R isomers. While 9PC3 generally prefered S,S adducts, it also showed an unusual 10-fold preference for one R,R BA-oxide-glutathione adduct over its S,S stereoisomer and a 133-fold preference for an R,R isomer of 1-APh-oxide-glutathione over its S,S isomer. This information was used to propose a model for the binding sites of antibodies 9PC3 and 1PD2.