Method of measuring the luminescence emitted in a luminescent assay

• US 5,527,684 A
• Filed: 07/28/1993
• Issued: 06/18/1996
• Est. Priority Date: 01/28/1991
• Status: Expired due to Term

• Current Assignee: CIS Bio International (Institut National Radio-Elements et D'Util Pub)
• Original Assignee: CIS Bio International (Institut National Radio-Elements et D'Util Pub)
• Inventors: Jolu, Etienne J.-P., Pouyat, Dominique, Mathis, Gerard, Dumont, Christophe, Mabile, Michel
• Primary Examiner(s): Spiegel, Carol A.
• Application Number:
  US08/094,085
• Time in Patent Office: 1,056 Days
• Field of Search: 250/366, 250/459.1, 435/7.1, 435/7.72, 435/7.91-7.95, 435/8, 435/28, 435/962, 435/968, 435/973, 436/518, 436/527, 436/528, 436/530, 436/531, 436/537, 436/546, 436/172, 436/825, 436/56, 436/8, 534/613, 534/15
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

The present invention relates to a method employing at least one luminescent tracer compound and a luminescent compound used as an internal reference, which, when exposed to the same excitation wavelength, are capable of emitting at difference wavelengths, λ₂ and λ₁ respectively, either by direct luminescence or by the induction of a luminescent emission, and correcting the measurement of the luminescence emitted by the tracer compound at wavelength λ₂ on the basis of the measurement of the luminescence emitted by the reference compound at wavelength λ1.

Claims

1. A method for detecting or determining a target analyte in a sample in which it may be present by measurement of emitted luminescence in a chemiluminescent immunoassay, said method comprising the steps of:

(a) forming a reaction mixture by contacting (i) the sample with (ii) a tracer compound comprising a receptor capable of specifically binding to the target analyte conjugated to a luminescent label capable of generating emitted luminescence at a measuring wavelength λ

₂ in the presence of (iii) an internal reference compound capable of emitting luminescence at a correcting wavelength λ

₁, in order to form a specific binding complex between the target analyte and the tracer, wherein the tracer compound and the internal reference compound are the same or different;

(b) irradiating the reaction mixture at a single excitation wavelength by an external radiation source;

(c) simultaneously measuring both the emitted luminescence at λ

₂ due to the tracer bound in the specific binding complex and the emitted luminescence at λ

₁ due to the internal reference compound which takes account of interference parameters of the reaction mixture;

(d) calculating a corrected luminescence for the luminescence emitted by the tracer compound at λ

2 based on the luminescence emitted by the internal reference compound at λ

₁ ; and

(e) correlating the corrected luminescence reading to the presence or amount of the target analyte in the sample.

2. A method for detecting or determining a target analyte in a sample in which it may be present by measurement of emitted luminescence in a luminescent assay, said method comprising the steps of:

(a) forming a reaction mixture by contacting (i) the sample with (ii) a tracer compound comprising a receptor capable of specifically binding to the target analyte conjugated to a luminescent label, said tracer compound capable of generating emitted luminescence at a measuring wavelength λ

₂ in the presence of (iii) an internal reference compound capable of generating emitted luminescence at a correcting wavelength λ

₁, in order to form a specific binding complex between the target analyte and the tracer, wherein the emitted luminescences may be generated by direct luminescence or by induction of a luminescent emission by non-radiative energy transfer, and wherein the tracer compound and the internal reference compound are the same or different;

(b) simultaneously irradiating the trace compound and the internal reference compound at a single excitation wavelength by an external radiation source;

(c) measuring both the emitted luminescence at λ

₂ due to the tracer bound in the specific binding complex and the emitted luminescence at λ

₁ due to the internal reference compound which takes account of interference parameters of the reaction mixture;

(d) calculating a corrected luminescence for the luminescence emitted by the tracer compound at λ

2 based on the luminescence emitted by the internal reference compound at λ

₁ ; and

(e) correlating the corrected luminescence reading to the presence or amount of the target analyte in the sample.

3. The method according to claim 2 wherein the luminescence emitted by the internal reference compound and by the tracer compound, at wavelengths λ

₁ and λ

₂ respectively, are measured simultaneously.

4. The method according to claim 2 wherein the step of calculating a corrected luminescence includes dividing the luminescence at λ

2 by the luminescence at λ

₁ to obtain the corrected luminescence.

5. The method according to claim 2 wherein the luminescence emitted by the internal reference compound at wavelength λ

₁ and the luminescence emitted by the tracer compound at wavelength λ

₂ are measured by a multi-channel luminescent measuring device, and wherein the correction of the measurement of the luminescence emitted by the tracer compound at wavelength λ

₂ includes fixing a counting rate on a channel measuring the luminescent emission of the internal reference compound at wavelength λ

₁, and then, when this rate is reached, triggering the end of the measurement on a channel measuring the luminescent emission at wavelength λ

₂.

6. The method according to claim 2 wherein the tracer compound and the internal reference compound are the same.

7. The method according to claim 2 wherein the tracer compound and the internal reference compound are different.

8. The method according to claim 2 wherein the tracer compound and/or the internal reference compound are fluorescent compounds.

9. The method according to claim 2 wherein the tracer compound and/or the internal reference compound are rare earth chelates or cryprates.

10. The method according to claim 2 wherein the emission lifetime of the tracer compound and/or the emission lifetime of the internal reference compound are of more than one microsecond.

11. The method according to claim 2 wherein the corrected luminescence is measured with a picomol/liter measuring sensitivity.

12. A method for detecting or determining a target analyte in a serum sample in which it may be present by measurement of emitted luminescence in a luminescent immunoassay, said method comprising the steps of:

(a) forming a reaction mixture by contacting (i) the sample with (ii) a tracer compound comprising a receptor capable of specifically binding to the target analyte conjugated to a luminescent label, said tracer compound capable of generating emitted luminescence at a measuring wavelength λ

₂, wherein the emitted luminescence may be generated by direct luminescence or by induction of a luminescent emission by non-radiative energy transfer to the receptor of said analyte conjugated with a luminescent acceptor compound (iii) in the presence of an internal reference compound capable of generating emitted luminescence at a correcting wavelength λ

₁, in order to form a specific binding complex between the target analyte and the tracer, and wherein the tracer compound and the internal reference compound are the same or different;

(b) simultaneously irradiating the tracer compound and the internal reference compound at a single excitation wavelength by an external radiation source;

(c) measuring both the emitted luminescence at λ

₂ due to the tracer bound in the specific binding complex and the emitted luminescence at λ

₁ due to the internal reference compound which takes account of interference parameters of the reaction mixture;

(d) calculating a corrected luminescence for the luminescence emitted by the tracer compound at λ

2 based on the luminescence emitted by the internal reference compound at λ

₁ ; and

(e) correlating the corrected luminescence reading to the presence or amount of the target analyte in the sample.

13. The method according to claim 12 wherein the method is a homogeneous method.

14. The luminescent immunoassay according to claim 12 wherein the luminescence emitted by the internal reference compound and by the tracer compound, at wavelength λ

₁ and λ

₂ respectively, are measured simultaneously.

15. The method according to claim 12 wherein the tracer compound comprising a receptor capable of specifically binding to the target analyte conjugated to a luminescent acceptor compound and the internal reference compound comprises the analyte conjugated to a luminescent donor compound;

step (a) further comprises incubating said reaction mixture after either sequential or simultaneous addition of the tracer compound and the internal reference compound;

step (b) occurs at the excitation wavelength of the luminescent donor compound; and

step (c) comprises measuring the luminescent emitted by the luminescent donor compound at λ

₁ and the luminescence emitted by the accepter compound at λ

₂.

16. An excess method for detecting or determining a target analyte in a serum sample in which it may be present by measurement of emitted luminescence in a luminescent immunoassay, said method comprising the steps of:

(a) forming a reaction mixture by contacting (i) the sample with (ii) a first reagent comprising a first receptor for the target analyte, coupled with a luminescent donor compound, said first receptor capable of specifically binding to the target analyte and (iii) a second reagent comprising a second receptor capable of specifically binding to the target analyte, said second reagent being coupled with a luminescent acceptor compound, said donor compound capable of transferring energy to said acceptor compound, said donor compound and said acceptor compound capable of generating emitted luminescence at measuring wavelengths λ

₁ and λ

₂ respectively;

(b) incubating said mixture after addition of each reagent or after addition of both reagents;

(c) irradiating the resulting mixture at an excitation wavelength of the donor compound;

(d) measuring the emitted luminescence of the donor compound at wavelength λ

₁, this measurement serving as a reference, and the emitted luminescence of the acceptor compound at wavelength λ

₂ resulting from any energy transfer;

(e) calculating a corrected luminescence for the luminescence emitted by the acceptor compound at λ

₂ based on the luminescence emitted by the donor compound at λ

₁ ; and

(f) correlating the corrected luminescence reading the presence or amount of the target analyte in the sample.

17. A competitive method for detecting or determining a target analyte in a serum sample in which it may be present by measurement of emitted luminescence in a luminescent immunoassay, said method comprising the steps of:

(a) forming a reaction mixture by contacting (i) the sample with (ii) a first reagent comprising a first receptor for said target analyte, coupled with a luminescent donor compound, said first receptor capable of specifically binding to the target analyte and (iii) a second reagent comprising the target analyte coupled with a luminescent acceptor compound, said donor compound capable of transferring energy to said acceptor compound, said donor compound and said acceptor compound capable of generating emitted luminescence at measuring wavelength λ

₁ and λ

₂ respectively;

(b) incubating said mixture after addition of each reagent or after addition of both reagents;

(c) irradiating the resulting mixture at an excitation wavelength of the donor compound;

(d) measuring the emitted luminescence of the donor compound at wavelength λ

₁, this measurement serving as a reference, and the emitted luminescence of the acceptor compound at wavelength λ

₂ resulting from any energy transfer;

(e) calculating a corrected luminescence for the luminescence emitted by the acceptor compound at λ

₂ based on the emitted luminescence by the donor compound at λ

₁ ; and

(f) correlating the corrected luminescence reading to the presence or amount of the target analyte in the sample.

18. A method for detecting or determining a target analyte in a serum sample in which it may be present by measurement of emitted luminescence in a luminescent immunoassay, said method comprising the steps of:

(a) forming a reaction mixture by contacting (i) the sample with (ii) a first reagent comprising a first receptor capable of specifically binding to the target analyte, (iii) a second reagent comprising either the target analyte or a second receptor capable of specifically binding to the target analyte, and (iv) a luminescent compound capable of emitting luminescence at wavelength λ

₁ as an internal reference compound, wherein one of (ii) or (iii) is coupled to a fluorescent tracer compound capable of generating emitted luminescence at wavelength λ

₂ and the other is coupled to a heavy atom or moieties containing a heavy atom such that the heavy atom induces an increase in spin-orbit coupling of the fluorescent tracer compound thereby modulating emitted luminescence generated by the fluorescent tracer compound;

(b) incubating the resulting medium either after addition of each reagent or after addition of all reagents;

(c) simultaneously irradiating the mixture at a single excitation wavelength common to both the fluorescent tracer compound and the internal reference compound by an external radiation source;

(d) measuring the emitted luminescence of the fluorescent tracer compound at wavelength λ

₂, and the emitted luminescence of the internal reference compound at wavelength λ

₁, said emitted luminescence at a wavelength λ

₂ being modulated by the heavy atom effect;

(e) calculating a corrected luminescence for the luminescence emitted by the fluorescent tracer compound at λ

₂ based on the luminescence emitted by the internal reference compound at λ

₁ ; and

(f) correlating the corrected luminescence reading to the presence or amount of the target analyte in the sample.