TY - JOUR
T1 - ReactELISA
T2 - Monitoring a Carbon Nucleophilic Metabolite by ELISA - A Study of Lipid Metabolism
AU - Holmquist, Emil F.
AU - Keiding, B. Ulrik
AU - Kold-Christensen, Rasmus
AU - Salomón, Trine
AU - Jørgensen, Karl Anker
AU - Kristensen, Peter
AU - Poulsen, Thomas B.
AU - Johannsen, Mogens
PY - 2017/5/2
Y1 - 2017/5/2
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85020398559&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.7b00507
DO - 10.1021/acs.analchem.7b00507
M3 - Journal article
C2 - 28376300
AN - SCOPUS:85020398559
SN - 0003-2700
VL - 89
SP - 5066
EP - 5071
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 9
ER -