Modulated homogeneous fluorescence biospecific affinity assay employing fluorescing lanthanide chelates as covalent labels
First Claim
1. In the method for a homogenous biospecific affinity assay for determining the content of an analyte in a biological sample, said assay being carried out in an aqueous reaction medium by means of a biospecific affinity reactant labelled with a lanthanide chelate in which a lanthanide ion capable of exhibiting ionic fluorescence is chelated by a ligand bound covalently to the reactant, and said fluorescence being measured by means of time-resolved fluorescence spectroscopy, the improvement comprising that(i) the lanthanide chelate formed by the lanthanide ion and the covalently bound ligand exhibits fluorescence, and that(ii) a fluorescence modulator is added to the reaction medium in an amount so that the lanthanide fluorescence as measured from the medium becomes correlated to the analyte concentration therein, and effects on the lanthanide fluorescence from sample constituents other than analyte become negligible.
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Abstract
Method for a homogeneous biospecific affinity assay for determining the content of a substance (analyte) in a biological sample. The assay is carried out in an aqueous reaction medium by means of time-resolved fluorescence spectroscopy and with a biospecific affinity reactant labeled with a lanthanide chelate in which a lanthanide ion exhibiting ionic fluorescence is chelated by a ligand bound covalently to the reactant. The characteristic feature is
(i) that the lanthanide chelate formed by the lanthanide ion together with the covalently bound ligand forms a fluorescent chelate, and
(ii) that a modulator is added which stabilizes the lanthanide chelate so that the lanthanide fluorescence as measured from the medium becomes a practically pure function of the analyte concentration therein.
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Citations
20 Claims
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1. In the method for a homogenous biospecific affinity assay for determining the content of an analyte in a biological sample, said assay being carried out in an aqueous reaction medium by means of a biospecific affinity reactant labelled with a lanthanide chelate in which a lanthanide ion capable of exhibiting ionic fluorescence is chelated by a ligand bound covalently to the reactant, and said fluorescence being measured by means of time-resolved fluorescence spectroscopy, the improvement comprising that
(i) the lanthanide chelate formed by the lanthanide ion and the covalently bound ligand exhibits fluorescence, and that (ii) a fluorescence modulator is added to the reaction medium in an amount so that the lanthanide fluorescence as measured from the medium becomes correlated to the analyte concentration therein, and effects on the lanthanide fluorescence from sample constituents other than analyte become negligible.
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3. In the method for homogenous biospecific affinity assay for determining the content of an analyte in a biological sample, said assay being carried out in an aqueous reaction medium by means of a biospecific affinity reactant labelled with a lanthanide chelate containing a lanthanide ion capable of exhibiting ionic fluorescence and selected from the group consisting of Eu3+, Tb3+, Sm3+ and Dy3+, said ion being chelated by a ligand covalently bound to the reactant and said fluorescence being measured by means of time-resolved fluorescence spectroscopy, the improvement comprising that
(i) the chelate formed between the covalently bound ligand and the lanthanide ion is fluorescent with a ligand structure which contains a chelating heteroatom having a free electron pair, said heteroatom being selected from the group consisting of nitrogen, oxygen, phosphorus and sulphur and being bound within the ligand in a manner such that the free electron pair is capable of delocalizing to a conjugated system of pi-bonds, and (ii) a fluorescence modulator is added to the reaction medium in an amount so that the fluorescence from the lanthanide chelate as measured from the medium becomes correlated to the analyte concentration therein and effects on lanthanide fluorescence from sample constituents other than analyte become negligible.
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13. In the method for homogenous biospecific affinity assay for determining the content of an analyte in a biological sample, said assay being carried out in an aqueous reaction medium by means of a biospecific affinity reactant labelled with a lanthanide chelate containing a lanthanide ion capable of exhibiting ionic fluorescence and selected from the group consisting of Eu3+, Tb3+, Sm3+ and Dy3+, said ion being chelated by a ligand covalently bound to the reactant and said fluorescence being measured by means of time-resolved fluorescence spectroscopy, the improvement comprising that for a biological sample selected from the group consisting of plasma and serum samples,
(i) the chelate formed between the covalently bound ligand and the lanthanide ion is fluorescent with a ligand structure which contains a chelating heteroatom having a free electron pair, said heteroatom being selected from the group consisting of nitrogen, oxygen, phosphorus and sulphur and being bound within the ligand in a manner such that the free electron pair is capable of delocalizing to a conjugated system of pi-bonds, and (ii) a fluorescence modulator is added to the reaction medium in an amount so that the fluorescence from the lanthanide chelate as measured from the medium becomes correlated to the analyte concentration therein and effects on lanthanide fluorescence from sample constituents other than analyte become negligible.
Specification