Polycyclic aromatic hydrocarbons (PAHs) are organic compounds which show a variety of toxic, mutagenic, and carcinogenic properties. PAHs are ubiquitous due to their production in incomplete combustion processes both natural and anthropogenic causes. These have caused an accumulation in environments and food chains. Compounding these effects is the increased stability of two or more fused aromatic hydrocarbon rings. 16 PAHs are listed as priority environmental pollutants by the United States Environmental Protection Agency \citep{Ghosal_2016}. Benzo[a]pyrene (BAP) is the most well-studied and characterized PAH and has strict concentration limits in the European Union. Antibody design for developing highly sensitive methods for detection for environmental testing and in vitro testing has only recently been applied for benzo[a]pyrene \citep{Eichinger_2017}. Genotoxic studies in vivo of PAHs in mice have shown other highly damaging PAHs beyond benzo[a]pyrene. Particularly, benzo[k]fluoranthene (BKF) show concerning potency for carcinogenic effects in the stomach, small intestine, liver, lung, and bone marrow \citep{Long_2016}. Developing a battery of antibodies for high specificity and affinity for various PAH molecules could allow for improved bioanalytical quantification of potential carcinogenic and mutagenic pollutants. The concern for these molecules has prompted the development of immunoassays for sample monitoring. The realization of this technology is underlined from work by Lux et al. that showed electro-switchable biosurfaces driven by association with the antibodies can detect PAH concentrations exceeding a 40 pM limit within 1 hour \citep{Lux_2015}
PAHs' primary mechanism of cell damage is through the formation of bulky DNA adducts and as P450 inducers through AhR activation. These impacts are sustained through the activity of PAH metabolites as well \citep{Spink_2008}. These reactive states are common variations of additional hydroxyl or epoxide groups, which arise through various degradation pathways catalyzed by Cytochrome P450 1a1 through oxidation or radical cation pathways \citep{Long_2016}. Spink et al. identified five metabolites and the starting molecule, which were shown to be CYP1 inducers. These metabolites include 3-, 8-, and 9-OH BkF, BkF-2,3-diol, and BkF-8,9-diol \citep{Spink_2008}, which align with other known PAH metabolite trends, like BAP-3-ol. Creating specific antibodies for PAH molecules will allow for better quantification of these toxic molecules, especially since PAHs are shown to act synergistically to induce toxicity. By binding BkF and identifying reactive intermediate potency of BkF metabolites, one can create a biosensor to quantify potential BkF toxicity levels. Specific binding with high affinity to BkF is more challenging due to the unreactive polyaromaticity, so BkF-3-ol is chosen as the target ligand. Focusing on increasing hydrophobic interactions far from the hydroxyl group and increasing hydrogen bonding to the hydroxyl group within the synthetic mAb-binding site will be crucial in improving affinity for BkF-3-ol. By mutating mAb 22F12/benzo[a]pyrene-3-ol complex, shown in Figure 1, from Eichinger et al. (2017), a new Fab fragment can be designed for binding and measuring the presence of other PAHs – in this case, benzo[k]fluoranthene-3-ol.