Time-resolved fluorescence immunoassay
First Claim
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1. A time-resolved fluorescence immunoassay for multiple analytes, comprising the steps of:
- a. forming an incubation mixture of;
(i) antibodies against each analyte;
(ii) a predetermined amount of fluorescently labeled analytes wherein each fluorescently labeled analyte has a different fluorescene lifetime; and
(iii) a sample to be tested;
b. incubating the mixture under conditions and for a period of time sufficient for antibody and analytes to complex;
c. determining contemporaneously the amount of each fluorescently labeled analyte bound with antibody as an indication of the amount of each corresponding analyte in the sample, by (i) exciting the fluorescently labeled analyte with a light pulse; and
(ii) determining the amplitude of each fluorescence decay curve for the antibody-bound fluorescently labeled analyte by a single amplitude measurement measuring all of the fluorescence reaching the detector from the instant of excitation.
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Abstract
Fluorescence immunoassay for determining single or multiple analytes based upon a single measurement of fluorescence are described.
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Citations
12 Claims
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1. A time-resolved fluorescence immunoassay for multiple analytes, comprising the steps of:
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a. forming an incubation mixture of; (i) antibodies against each analyte; (ii) a predetermined amount of fluorescently labeled analytes wherein each fluorescently labeled analyte has a different fluorescene lifetime; and (iii) a sample to be tested; b. incubating the mixture under conditions and for a period of time sufficient for antibody and analytes to complex; c. determining contemporaneously the amount of each fluorescently labeled analyte bound with antibody as an indication of the amount of each corresponding analyte in the sample, by (i) exciting the fluorescently labeled analyte with a light pulse; and
(ii) determining the amplitude of each fluorescence decay curve for the antibody-bound fluorescently labeled analyte by a single amplitude measurement measuring all of the fluorescence reaching the detector from the instant of excitation. - View Dependent Claims (2, 3, 4)
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5. A time-resolved fluorescence immunoassay for multiple analytes, comprising the steps of:
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a. providing an immunoadsorbent comprising a solid phase having affixed thereto capture antibodies specific for each analyte to be tested; b. incubating the immunoadsorbent with a sample to be tested for a period of time and under conditions sufficient for analytes in the sample to complex with the capture antibodies; c. separating the immunoadsorbent and sample; d. incubating the immunoadsorbent and a solution containing fluorescently labeled antibodies specific for each analyte, each labeled antibody having a different fluorescence lifetime; e. separating the immunoadsorbent and the solution; f. determining contemporaneously the amount of each conjugate associated with the immunoadsorbent as an indication of the amount of each analyte in the sample by (i) exciting the fluorescently labeled antibodies with a light pulse; and
(ii) determining the amplitude of the fluorescence decay curve of each conjugate by measuring all of the fluorescence reaching the detector from the instant of excitation with a single amplitude measurement. - View Dependent Claims (6, 7)
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8. A time-resolved fluorescence immunoassay for multiple analytes, comprising the steps of:
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a. providing an immunoadsorbent comprising a solid phase having affixed thereto capture antibodies specific for each analyte to be tested; b. incubating the immunoadsorbent with a sample to be tested for a period of time and under conditions sufficient for analytes in the sample to complex with the capture antibodies; c. separating the immunoadsorbent and the sample; d. incubating the immunoadsorbent with a solution containing second antibodies against each of the analytes, the incubation being carried out under conditions and for a period of time which permits the second antibodies to complex with analyte bound to the immunoadsorbent wherein each of the second antibodies are antigenically distinct; e. separating the immunoadsorbent and the solution; f. incubating the immunoadsorbent and a solution containing fluorescent antibody conjugates against the second antibodies, the conjugates comprising an antibody against each second antibody labeled with a fluorophore, each conjugate having a different fluorescence lifetime; g. separating the immunoadsorbent and the solution; and h. determining contemporaneously the amount of each conjugate associated with the immunoadsorbent as an indication of the amount of each corresponding analyte in the sample by (i) exciting the fluorophone with a light pulse; and
(ii) determining the amplitude of the fluorescence decay curve of each fluorophore by measuring all of the fluorescence reaching the detector from the instant of excitation with a single amplitude measurement. - View Dependent Claims (9, 10, 11, 12)
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Specification