TY - JOUR
T1 - An enzyme-linked immunosorbent assay for the detection of diacetyl (2,3-butanedione)
AU - Marri, Lucia
AU - Jansson, Anita M.
AU - Christensen, Caspar Elo
AU - Hindsgaul, Ole
N1 - Copyright © 2017 Elsevier Inc. All rights reserved.
PY - 2017/10/15
Y1 - 2017/10/15
N2 - Diacetyl (2,3-butanedione) is an important metabolic marker of several cancers, as well as an important off-flavour component produced during fermentation. As a small molecule in a complex mixture with many other analytes, existing methods for identification and quantitation of diacetyl invariably involves a chromatographic separation step followed by signal integration with an appropriate stoichiometric detector. Here we demonstrate that the chemical reaction of diacetyl with a 1,2-phenylenediamine derivative yields a chemical adduct, 1,4-quinoxaline which can be conjugated on BSA. The BSA-diacetyl adduct can be used to select an adduct-specific monoclonal antibody in a Fab-format from a 45-billion member phage-display library. The availability of this antibody allowed the development of an enzyme-linked immunosorbent assay for diacetyl, based on the 1,4-quinoxaline competition for the antibodies with the diacetyl adduct immobilized on the plate. The described ELISA assay can detect the captured diacetyl in micromolar concentrations, both in water samples and in cell culture medium.
AB - Diacetyl (2,3-butanedione) is an important metabolic marker of several cancers, as well as an important off-flavour component produced during fermentation. As a small molecule in a complex mixture with many other analytes, existing methods for identification and quantitation of diacetyl invariably involves a chromatographic separation step followed by signal integration with an appropriate stoichiometric detector. Here we demonstrate that the chemical reaction of diacetyl with a 1,2-phenylenediamine derivative yields a chemical adduct, 1,4-quinoxaline which can be conjugated on BSA. The BSA-diacetyl adduct can be used to select an adduct-specific monoclonal antibody in a Fab-format from a 45-billion member phage-display library. The availability of this antibody allowed the development of an enzyme-linked immunosorbent assay for diacetyl, based on the 1,4-quinoxaline competition for the antibodies with the diacetyl adduct immobilized on the plate. The described ELISA assay can detect the captured diacetyl in micromolar concentrations, both in water samples and in cell culture medium.
KW - Animals
KW - Cattle
KW - Diacetyl/analysis
KW - Enzyme-Linked Immunosorbent Assay
KW - Molecular Structure
KW - Serum Albumin, Bovine/chemistry
U2 - 10.1016/j.ab.2017.07.021
DO - 10.1016/j.ab.2017.07.021
M3 - Journal article
C2 - 28739133
SN - 0003-2697
VL - 535
SP - 12
EP - 18
JO - Analytical Biochemistry
JF - Analytical Biochemistry
ER -
