Development of anti-cotinine antibody & its application to EIA and carrier for cotinine-conjugated molecule

Abstract

Cotinine is a major metabolite of nicotine and is widely used tobacco biomarker. Both active smoking and passive exposure to tobacco smoke are major risk factors for cardiovascular, pulmonary, and oncological diseases. The serum level of cotinine reflects both active and passive exposure to tobacco smoke. However, currently available enzyme-linked immunosorbent assays (ELISAs) for cotinine have limited sensitivity, and a high-throughput quantification of the severity of passive exposure to tobacco smoke has not been possible thus far. For this reason, I generated recombinant antibody against cotinine from combinatorial antibody using phage display technique, at first. Then a sensitive ELISA was developed using this antibody and evaluated the ELISA in a clinical setting and an animal model. The lower limit of quantification and cut off value were 1 ng/mL and 5.1 ng/mL cotinine, respectively. The intra- and inter-assay precisions based on three quality control samples were 3.8–13.5% and 14.0–15.0%, respectively. No significant interference from nicotine, trans-3'-hydroxy cotinine, tobacco alkaloids, or from other serum components was found. When this ELISA was applied to serum samples, passive smokers and active smokers were classified from non-smokers. In addition, the ELISA yielded reproducible and accurate results, which were comparable to those of LC/MS in a split assay. In animal studies, I was able to distinguish between rats injected with a nicotine dose equivalent to that of passive exposure to tobacco and rats without exposure. In addition to the development of ELISA, I applied anti-cotinine antibody to in vivo and in vitro carriers for cotinine conjugated molecule such as peptide or aptamer as another application for anti-cotinine antibody. When cotinine is chemically conjugated to a molecule such as protein, peptide, aptamer, and chemical substance with a linker, it can mediate the formation of a cotinine conjugated molecule/anti‐cotinine antibody complex. This complex can retain the original characteristics of both cotinine conjugated molecule, including the specific reactivity and functions of the molecule and the antibody that includes long half life, ADCC, and CDC. To test that this system could function as an in vivo carrier of cotinine conjugated therapeutic molecule, I conjugated WKYMVm-NH2, therapeutic peptide in sepsis, to cotinine in this platform. After conjugation and complex with anti-cotinine antibody, the short elimination half-lives of WKYMVm-NH2 were dramatically increased with retaining its biological activity. And the survival of sepsis mouse administrated with peptide with our platform was improved about 35% in comparison to that of sepsis mouse administrated with WKYMVm-NH2 only. And I also conjugated aptamers to cotinine in this platform for the purpose of this platform to provide another affinity unit in various biological assays and experiments. I developed a method to label the 5' ends of aptamers with cotinine, which allows the formation of a stable complex with anti-cotinine antibodies. To demonstrate the functionality of this affinity unit in biological assays, I utilized two well-known aptamers in this topic: AS1411, which binds to nucleolin, and pegaptanib, which binds to vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. In this study, I generated anti-cotinine antibody using phage display technique and developed competitive ELISA for detection and quantification of the severity of risk of passive smoking and also applied it to in vivo and in vitro carriers for cotinine-conjugated molecule.