title: ReactELISA: Monitoring a Carbon Nucleophilic Metabolite by ELISA-a Study of Lipid Metabolism.
abstract: We here present a conceptually novel reaction-based ELISA principle (ReactELISA) for quantitation of the carbon nucleophilic lipid metabolite acetoacetate. Key to the assay is the utilization of a highly chemoselective Friedländer reaction that captures and simultaneously stabilizes the nucleophilic metabolite directly in the biological matrix. By developing a bifunctional biotinylated capture probe, the Friedländer-acetoacetate adduct can be trapped and purified directly in streptavidin coated wells. Finally, we outline the selection and refinement of a highly selective recombinant antibody for specific adduct quantitation. The setup is very robust and, as we demonstrate via miniaturization for microplate format, amenable for screening of compounds or interventions that alter lipid metabolism in liver cell cultures. The assay-principle should be extendable to quantitation of other nucleophilic or electrophilic and perhaps even more reactive metabolites provided suitable capture probes and antibodies.
authors.affiliation: Department of Forensic Medicine, Aarhus University , Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark.|Department of Chemistry, Aarhus University , Langelandsgade 140, 8000 Aarhus C, Denmark.
authors.forename: Emil F
authors.initials: EF
authors.lastname: Holmquist
authors.affiliation: Department of Forensic Medicine, Aarhus University , Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark.|Department of Chemistry, Aarhus University , Langelandsgade 140, 8000 Aarhus C, Denmark.
authors.forename: Ulrik
authors.initials: U
authors.lastname: B Keiding
authors.affiliation: Department of Forensic Medicine, Aarhus University , Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark.|Department of Chemistry, Aarhus University , Langelandsgade 140, 8000 Aarhus C, Denmark.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Kold-Christensen
authors.affiliation: Department of Forensic Medicine, Aarhus University , Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark.
authors.forename: Trine
authors.initials: T
authors.lastname: Salomón
authors.affiliation: Department of Chemistry, Aarhus University , Langelandsgade 140, 8000 Aarhus C, Denmark.
authors.forename: Karl Anker
authors.identifier: 0000-0002-3482-6236
authors.initials: KA
authors.lastname: Jørgensen
authors.affiliation: Department of Engineering, Aarhus University , Gustav Wieds Vej 10, 8000 Aarhus C, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Kristensen
authors.affiliation: Department of Chemistry, Aarhus University , Langelandsgade 140, 8000 Aarhus C, Denmark.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Poulsen
authors.affiliation: Department of Forensic Medicine, Aarhus University , Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark.
authors.forename: Mogens
authors.identifier: 0000-0002-2548-7025
authors.initials: M
authors.lastname: Johannsen
mesh_terms: D000090:Acetoacetates / Q000097:blood* / Q000737:chemistry; D000098:Acetophenones / Q000138:chemical synthesis / Q000737:chemistry; D000814:Aniline Compounds / Q000138:chemical synthesis / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001710:Biotin / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D051379:Mice
chemical_list: D000090:Acetoacetates; D000098:Acetophenones; D000814:Aniline Compounds; D000911:Antibodies, Monoclonal; C016635:acetoacetic acid; D001710:Biotin