Novel probe and its use in bioaffinity assays
First Claim
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1. A homogeneous bioaffinity assay comprising the steps of a) providing a labelled probe comprising (i) a TR-FRET label pair comprising a long excited state lifetime luminescent energy donor and a short excited state lifetime luminescent energy acceptor;
- and (ii) a reactive region, capable of recognizing a bioaffinity component to be determined;
b) contacting said labelled probe with a sample, expected to comprise said bioaffinity component;
c) allowing said probe and said bioaffinity component to undergo a recognition reaction;
said probe having different energy transfer efficiency between the donor and the acceptor when reacted to said bioaffinity component as compared to non-reacted probe;
d) measuring an energy transfer based signal from the acceptor of said reacted sample;
e) measuring an energy transfer based signal from the acceptor of a non-reacted probe in a reference assay;
f) comparing the signals obtained in steps d) and e); and
g) determining the presence or absence of the bioaffinity component in the sample based on a difference in acceptor signals obtained in step (f).
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Abstract
The invention concerns a labeled probe for use in bioaffinity assays based on time-resolved fluorescence resonance energy transfer, wherein said probe comprises an energy donor as well as an energy acceptor, and the energy transfer signal of the acceptor is to be measured. The invention concerns also a homogeneous time-resolved fluorescence resonance energy transfer bioaffinity assay based on the use of said probe.
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18 Claims
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1. A homogeneous bioaffinity assay comprising the steps of
a) providing a labelled probe comprising (i) a TR-FRET label pair comprising a long excited state lifetime luminescent energy donor and a short excited state lifetime luminescent energy acceptor; - and
(ii) a reactive region, capable of recognizing a bioaffinity component to be determined;
b) contacting said labelled probe with a sample, expected to comprise said bioaffinity component;
c) allowing said probe and said bioaffinity component to undergo a recognition reaction;
said probe having different energy transfer efficiency between the donor and the acceptor when reacted to said bioaffinity component as compared to non-reacted probe;
d) measuring an energy transfer based signal from the acceptor of said reacted sample;
e) measuring an energy transfer based signal from the acceptor of a non-reacted probe in a reference assay;
f) comparing the signals obtained in steps d) and e); and
g) determining the presence or absence of the bioaffinity component in the sample based on a difference in acceptor signals obtained in step (f). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A labelled probe for use in a bioaffinity assay, said probe comprising
a) a time-resolved fluorescence resonance energy transfer (TR-FRET) label pair comprising an energy donor and an energy acceptor; - and
b) a reactive region, capable of recognizing a bioaffinity component to be determined, said probe having different energy transfer efficiency between the donor and the acceptor when reacted to said bioaffinity component as compared to non-reacted probe, wherein said donor is a long excited state lifetime luminescent label and said acceptor is a short excited lifetime luminescent label;
said probe being selected from the group consisting of a nucleic acid, a polypeptide comprising 2 to 70 amino acids, an antibody and an antibody fragment; and
said donor is a luminescent label having an excited state lifetime of at least 1 microsecond. - View Dependent Claims (12, 13, 14, 15, 16, 17)
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18. The probe according to claim 19, wherein said probe is an antibody or antibody fragment, capable of binding to an epitope of an antigen.
Specification