Abstract 2733: Development of novel monoclonal antibodies for a cyclic DNA adduct derived from oxidation of ω-6 polyunsaturated fatty acids

Abstract

Abstract Dietary polyunsaturated fatty acids (ω-3 and ω-6 PUFAs) play a role in certain human cancers. The oxidation of PUFAs produces reactive α,β-unsaturated aldehydes (enals) that can modify DNA producing mutagenic lesions. Of particular interest, 7-(1′,2′-dihydroxyheptyl)-1,N6- ethenodeoxyadenosine (DHHϵdA) is a novel DNA adduct in vitro and in vivo derived from lipid peroxidation of ω-6 PUFAs. Despite its recent identification in vivo, the role of DHHϵdA in tumorigenesis is not yet known. Previously, the endogenously formed DHHϵdA was detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). Due to its low levels in vivo (1-30 adducts/109 DNA bases), a relatively high quantity of DNA is needed for its detection and quantification. In this study we developed a monoclonal antibody against DHHϵdA in order to detect and quantify this adduct in cells and tissues. Two antigens were synthesized by conjugating activated hapten: 5′-carboxy-7-(1′,2′-dihydroxyheptyl) adenosine (5′-carboxy DHHϵA) to BSA and KLH carrier proteins. KLH-conjugated hapten was used for mice immunization, whereas BSA-based hapten was used to test for an immune response. After immunization and several screenings, 6 monoclonal cell lines were chosen for further tests. Normal and competitive ELISA was performed to characterize the antibodies from all cell lines. Competitive ELISA revealed no cross-reactivity towards normal nucleosides and α- and γ-OHPdG, the cyclic 1,N2-propano adduct derived from acrolein, and 8-oxo-dG for all of the tested antibodies. However, we observed a weak (< 1:100) specificity for HNE-dG, an adduct derived from (E)-4-hydroxy-2-nonenal produced from oxidized ω-6 PUFAs. Because of the relatively low level of endogenous HNE-dG compared to DHHϵdA, this weak cross-reactivity should not affect the specificity of the developed antibody to detect DHHϵdA. Two cell lines were discarded after antibodies from them showed moderate competitive effects against 1,N6-etheno-2′-deoxyadenosine (ϵdA). From the remaining 4 cell lines the best one was chosen to produce purified antibody. We believe that this antibody will be a useful tool in the studies of the role of DHHϵdA as an endogenous DNA lesion in cancer development associated with ω-6 PUFAs. This work was supported by the NCI grant CA134892. Citation Format: Marcin Dyba, Brandon Da Silva, Jishen Pan, Fung-Lung Chung. Development of novel monoclonal antibodies for a cyclic DNA adduct derived from oxidation of ω-6 polyunsaturated fatty acids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2733. doi:10.1158/1538-7445.AM2015-2733