Homogeneous indirect fluorescence quenching immunoassay for the determination of low molecular weight substances |
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| Authors: | F Sellrie A Warsinke B Micheel |
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| Institution: | (1) Department of Biotechnology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str., 14476 Potsdam-Golm, Germany;(2) Department of Analytical Biochemistry, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str., 14476 Potsdam-Golm, Germany |
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| Abstract: | This paper describes the principle of a homogeneous indirect fluorescence quenching immunoassay that uses monoclonal antibodies.
It is a carrier-free assay system that is performed completely in solution. The assay system was established for the determination
of a low molecular weight substance (hapten), the herbicide diuron, used as a model analyte. A fluorescein–monuron conjugate
together with a fluorescence-quenching monoclonal anti-fluorescein antibody and an anti-analyte antibody (here an anti-diuron/monuron
monoclonal antibody) were used as central components of the assay. The fluorescein–monuron conjugate can be bound either by
the anti-fluorescein monoclonal antibody or by the anti-diuron/monuron monoclonal antibody. Due to steric hindrance, binding
of both antibodies to the conjugate was not possible at the same time. By selecting the antibody concentrations appropriately,
a dynamic equilibrium can be established that permits the preferential binding of the anti-diuron/monuron antibody to the
conjugate, which allows the fluorescein in the conjugate to fluoresce. This equilibrium can be easily altered by adding free
analyte (diuron), which competes with the conjugate to bind to the anti-diuron/monuron antibody. A reduction of anti-diuron/monuron
antibody binding to the conjugate results in an increase in the binding of the anti-fluorescein antibody, which leads to a
decrease in the fluorescence of the conjugate. The fluorescence is therefore a direct indicator of the state of equilibrium
of the system and thus also the presence of free unconjugated analyte. The determination of an analyte based on this test
principle does not require any washing steps. After the test components are mixed, the dynamic equilibrium is rapidly reached
and the results can be obtained in less than 5 min by measuring the fluorescence of the fluorescein. We used this test principle
for the determination of diuron, which was demonstrated for concentrations of ∼5 nM. |
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| Keywords: | Homogeneous immunoassay Fluorescence quenching Indirect fluorescence quenching immunoassay Fluorescein Diuron |
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