Analyte detection by means of energy transfer

We claim:

1. An assay for detecting the presence of an analyte comprising the steps of:

a. forming a complex comprising said analyte and binding entity comprising an analyte recognition segment and a first partner of a fluorescent energy transfer system, wherein said first partner is selected from the group consisting of an energy donor and an energy acceptor, wherein said energy donor is a fluorescent aromatic agent or a lanthanide metal and said energy acceptor is a fluorescent, aromatic agent or a lanthanide metal, with the proviso that when said energy donor is a fluorescent, aromatic agent, then said energy acceptor is a fluorescent, aromatic agent or a lanthanide metal and when said energy donor is a lanthanide metal, then said energy acceptor is a fluorescent, aromatic agent;

b. contacting said complex with a reporting entity composition comprising a second partner of said fluorescent energy transfer system and at least one component for rendering said analyte detectable which binds to said second partner to form a unit, wherein: (i) said second partner is selected from the group consisting of an energy donor and an energy acceptor, with the proviso that when said first partner is an energy donor then said second partner is an energy acceptor and when said first partner is an energy acceptor then said second partner is an energy donor, and (ii) the distance between said first partner and said second partner is 30 Angstroms or less;

c. irradiating said unit with energy that can be absorbed by said energy donor and not by said energy acceptor with the proviso that said energy donor emits fluorescent energy which can excite said energy acceptor; and

d. detecting the fluorescence emitted by said energy acceptor.

2. The method of claim 1 wherein said first partner is attached covalently to said recognition segment of said binding entity by means of a linker arm.

3. The method of claim 1 wherein said binding entity comprises an analyte-specific binding substance, said first partner is selected from the group consisting of europium and terbium, and said second partner is a fluorescent aromatic intercalating agent.

4. The method of claim 1 wherein said lanthanide metal is chelated.

5. The method of claim 1 wherein said assay is carried out in a one phase system.

6. The method of claim 1 wherein said analyte is selected from the group consisting of antigens, haptens, antibodies and target polynucleotides.

7. The method of claim 6 wherein said analyte is an antigen.

8. The method of claim 7 wherein said antigen is selected from the group consisting of proteins, polysaccharides, viruses, phages, and bacteria.

9. The method of claim 1 wherein said first partner is an energy acceptor and said second partner is an energy donor.

10. The method of claim 1 wherein said energy donor is a fluorescent aromatic agent and said energy acceptor is selected from the group consisting of fluorescent aromatic agents and lanthanide metals.

11. The method of claim 10 wherein said energy donor is a fluorescent aromatic agent and said energy acceptor is a lanthanide metal.

12. The method of claim 1 wherein aromatic agents are selected from the group consisting of auromine O, lumichrome, and 9-aminoacridine.

13. The method of claim 10 wherein said metal is selected from the group consisting of europium and terbium.

14. The method of claim 1 wherein said second partner is attached covalently or non-covalently to said component of said reporting entity by means of a linker arm.

15. The method of claim 6 wherein said analyte is selected from the group consisting of antigens and antibodies and wherein said component of said reporting entity is selected from the group consisting of Clq, antibodies, and solid supports.

16. The method of claim 15 wherein said analyte is selected from the group consisting of antigens and antibodies and wherein said component of said reporting entity is selected from the group consisting of Clq and solid supports.

17. The method of claim 6 wherein said analyte is a target polynucleotide and wherein said component of said reporting entity is selected from the group consisting of intercalating agents and solid supports.

18. The method of claim 3 wherein said solid support is selected from the group consisting of glass, plastic, cellulose, and gel polymers.

19. The method of claim 1 wherein said assay is carried out in a two phase system, wherein said two phases comprise a solid support and a liquid.

20. The method of claim 1 wherein said analyte is an antigen, said first partner is an energy acceptor, said second partner is an energy donor, and said component of said reporting entity selected from Clq and a solid support.

21. The method of claim 20 wherein said energy acceptor is a lanthanide metal.

22. The method of claim 5 wherein said analyte is an antigen, said binding entity comprises an antibody said first partner is an energy acceptor, said second partner is an energy donor, and said reporting component entity comprises Clq.

23. The method of claim 22 wherein said energy acceptor is a lanthanide metal.

24. The method of claim 1 wherein said analyte is a target polynucleotide, said recognition segment is a complementary polynucleotide, said first partner is a lanthanide metal, and said second partner is a fluorescent aromatic intercalating agent.

25. The method of claim 5 wherein said analyte is a target polynucleotide, said binding entity comprises a complementary polynucleotide, said first partner is a lanthanide metal, and said second partner is a fluorescent aromatic intercalating agent.

26. The method of claim 19 wherein said analyte is a target polynucleotide, said binding entity comprises a complementary polynucleotide, said first partner is a lanthanide metal, said second partner is a fluorescent aromatic intercalating agent.

27. The method of claim 1 wherein said analyte and said binding entity form a complex selected from the group consisting of antigen/antibody, lectin/sugar, hormone/receptor, inhibitor/enzyme, cofactor/enzyme, and ligand/substrate.

28. The method of claim 5 wherein said analyte is an antigen and wherein said binding entity comprises a specific antibody.

29. An assay for detecting the presence of an analyte comprising the steps of:

a. forming a complex comprising said analyte and a binding entity comprising an analyte recognition segment and a first partner of a fluorescent energy transfer system, wherein said first partner is selected from the group consisting of an energy donor and an energy acceptor, wherein said energy donor is a fluorescent, aromatic agent and said energy acceptor is selected from the group consisting of fluorescent, aromatic agents, europium and terbium;

b. contacting said complex with a reporting entity comprising a second partner of said fluorescent energy transfer system and a component which binds to said second partner to form a unit, wherein: (i) said second partner is selected from the group consisting of an energy donor and an energy acceptor, with the proviso that when said first partner is an energy donor then said second partner is an energy acceptor and when said first partner is an energy acceptor then said second partner is an energy donor, and (ii) the distance between said first partner and said second partner is 30 Angstroms or less;

c. irradiating said unit with energy that can be absorbed by said energy donor and not by said energy acceptor with the proviso that said energy donor emits fluorescent energy which can excite said energy acceptor and wherein said irradiation is performed without prior separation of said binding entity which has not formed said complex with said analyte; and

d. detecting the fluorescence emitted by said energy acceptor.

30. The method of claim 29 wherein said binding entity comprises an analyte-specific binding substance, said first partner is selected from the group consisting of europium and terbium, and said second partner is a fluorescent aromatic intercalating agent.

31. The method of claim 29 wherein said assay is carried out in a two phase system, wherein said two phases comprise a solid support and a liquid.

32. The method of claim 29 wherein said europium is chelated.

33. The method of claim 29 wherein said terbium is chelated.

34. The method of claim 29 wherein said assay is carried out in a one phase system.

Description Submit all comments and votes

BACKGROUND OF THE INVENTION

The present invention relates to a method for determining the presence of an analyte by means of an energy transfer that results in the generation of bathochromic and/or delayed fluorescence emission. Fluorescence radiation, emitted from a first energy emitter (E.sub.1), is absorbed by a second energy emitter (E.sub.2). This second energy emitter emits fluorescence radiation of a longer wavelength than the first energy emitter. The second energy emitter may in addition emit fluorescence for a substantially longer period than the first energy emitter (in a delayed manner). The detection of either the bathochromic fluorescence or of any fluorescence after a time period during which fluorescence radiation from background sources has decayed verifies the presence of the analyte.

Methods for the in-vitro detection of analytes are well known in the art. The methods include the formation of antibody-antigen complexes (immunodetection), and the formation of nucleic acid complexes (polynucleotide hybridization). The analyte can be an intact cell or a component of the cell. Examples of analytes are bacteria, viruses, antigens, antibodies, and polynucleotides.

The immunoassay for detecting antigen (or antibody) analytes is well established in the art. The assay involves the formation of antigen-antibody complexes. In radioimmunoassay (RIA), a radioactive isotope is used to report the presence of the analyte. In enzyme immunoassay, chromogen or fluorescence generated by means of an enzyme is used to report the presence of the analyte. Several enzyme immunoassays are currently in use. They include the enzyme multiplied immunoassay technique (EMIT) and the enzyme-linked immunosorbent assay (ELISA). The ELISA method comprises the "sandwich" technique for antigen, the antibody assay, and the competitive assay for antigen.

A typical ELISA assay using the sandwich technique is carried out by adsorbing an antibody to the surface of a support. The test specimen is added to the support and the antigen allowed to complex to the antibody. Unbound antigen is washed away. An enzyme-conjugated antibody is added and allowed to react with a different set of determinants on the bound antigen which are not blocked by the support-absorbed antibody. After the reaction, the excess of unbound enzyme-linked antibody is washed away and a substrate of the enzyme is added to the support. The generation of a colored product indicates the presence of the antigen in the test specimen. See Enzyme Immunoassays by S. Bakerman in Laboratory Management, August 1980, p. 21.

A drawback of these methods is that they cannot be carried out in one-step, to achieve detection, i.e., by adding the antibody to the antigen or the antigen to the antibody. One or more washing steps are required to remove antibody unbound to antigen (or vice versa). Also, a number of these methods involves competition kinetics which in some instances can provide ambiguous results.

Polynucleotide hybridization assays using a polynucleotide probe for verifying the presence of a target polynucleotide analyte is a well known method. Hybridization is based on complementary base-pairing.

When single-stranded polynucleotide probes are incubated in solution with single-stranded target polynucleotides that are immobilized on a support, complementary base sequences pair to form double-stranded hybrid molecules. The double-stranded hybrid molecules remain immobilized on the support while unbound polynucleotide probe molecules are washed off. See M. Grunstein and J. Wallis, METHODS IN ENZYMOLOGY, volume 68, R.W.U (Ed) (1979) pp. 379-469; A. R. Dunn, and J. Sambrook, METHODS IN ENZYMOLOGY, volume 65; part 1, (1980) pp. 468-478; Modified Nucleotides And Methods Of Preparing And Using The Same by D. C. Ward, A. A. Waldrop, and P. R. Langer, European Patent Publication No. 0,063,879 published Nov. 3, 1982; DNA Probes for Infectious Disease by A. J. Berry and J. B. Peter, Diagnostic Medicine (March, 1984) pp. 1-8; and Recombinant DNA Technology: Some Applications In Clinical Microbiology by Wie-Shing Lee and James L. Bennington, Laboratory Management (April, 1985) pp. 21-26.

The polynucleotide probes generally comprise a polynucleotide segment and a signalling segment which is attached to the polynucleotide. The polynucleotide segment of the probe has the ability to base-pair, i.e. hybridize to a sequence of interest, namely the analyte or target polynucleotide. The signalling segment of the probe has or produces the means by which the presence of the analyte moiety can be verified. The means can be, for example, fluorescence, phosphorescence, radioactivity, chromogen, or electron density.

The method of detecting the presence of a target polynucleotide generally involves several steps, one of which is the separation of hybridized polynucleotide probe from unhybridized probe. The separation can be facilitated by immobilizing either the probe or the target onto a solid support. Typically, double-stranded polynucleotides are isolated from a sample suspected of containing a target polynucleotide. The double-stranded polynucleotides are cut into smaller segments by means of restriction endonuclease enzyme digestion, the segments are separated by gel electrophoresis, and the segments are transferred from the gel onto a support, for example, nitrocellulose paper. Alternatively, the double-stranded polynucleotides are fixed directly onto the support without any prior enzyme digestion. The fixed polynucleotides are contacted with a solution containing the polynucleotide probe, and the support is heated to about 80.degree.-90.degree. C. to denature the polynucleotide double-strands. (The double-strands can alternatively be denatured by means of alkali). The system, which now contains the denatured target polynucleotide and the polynucleotide probe, is allowed to cool to an appropriate temperature to allow hybridization to take place. After sufficient time has elapsed for hybridization to be complete, which can be for ten minutes to several hours, the fixed target polynucleotide is washed to remove all unbound polynucleotide probes. The signalling moiety of the polynucleotide probe is now detected, either directly, for example, by means of radioactivity or fluorescence, or indirectly, for example, by means of a chromogen formed through an enzymatic reaction.

A drawback of this method is that it requires several steps before the presence of the target polynucleotide can be verified. Namely, it requires the fixation of the target polynucleotide to a support, the contacting of the target polynucleotide with a polynucleotide probe, and the removal of all unhybridized polynucleotide probes from the support. Besides being time consuming, the method is not readily amenable to automation and requires some expertise for obtaining reproducible results. In addition, hybridization and detection of the target polynucleotide in a one phase system is not possible.

One method seeking to overcome the above drawbacks by detecting the presence of a target polynucleotide with a homogenous (one-step or one phase) nucleic acid hybridization assay has been reported. The method comprises hybridizing first and second single-stranded polynucleotides, both of which contain light-sensitive labels, with a complementary single-stranded polynucleotide target from a sample such that non-radiative energy transfer occurs between the light-sensitive labels of the first and second polynucleotides. At least one of the light-sensitive labels is of the absorber/emitter type such that energy absorbed by this label from the emission of the other light-sensitive label is reemitted at a different wavelength. These secondary emissions can only occur if hybridization of both the first and second single-stranded polynucleotides to the target polynucleotide has taken place. The quantity of the target polynucleotides in the sample is related to the amount of secondary light emitted. See European Patent Publication No. 0,070,685 by Michael James Heller, published Jan. 26, 1983.

A drawback of this method is that it requires two separate polynucleotide strands to detect the presence of a target polynucleotide. In addition, the method requires the presence of a chemiluminescent catalyst, an absorber/emitter moiety, and chemiluminescent reagents effective for causing light emission in the presence of the chemiluminescent catalyst. Furthermore, only one label can be attached per polynucleotide probe because the light-sensitive label is attached to the sugar moiety of a terminal nucleoside. Also, the bulky labels may prevent hybridization of the bases adjacent to the labels.

Another method for detecting the presence of a target polynucleotide by means of a homogeneous assay has been recently reported. The method involves forming a hybrid between the target polynucleotide and the polynucleotide probe, wherein the hybrid has binding sites for two specific binding reagents, one of which comprises a first label and the other a second label. The interaction of the first and second labels provide a detectable response which is measurably different when the two labeled reagents are both bound to the same hybrid, as compared to when the two labeled reagents are not so bound. The formation of the hybrid assay product brings the two labels within approximate interaction distance of one another, e.g., as in the cases of sequential catalyst (enzyme) interaction and energy transfer. Since the labels provide a response which is distinguishable when the labels are associated with a hybridized probe, no separation step is required. See European Patent Application No. 0,144,914 by James P. Albarella et al., published Nov. 29, 1984.

The method has two main embodiments. The first embodiment involves the generation of a component which subsequently produces a color. This embodiment has a drawback in that it requires the use of two distinct chemical reactions, namely, the reaction of the first label to produce a diffusible mediator product, and the reaction of the mediator product with the second label to yield a detectable product. In addition, detection depends on the formation and maintenance of a higher localized concentration of the mediator product in the vicinity of the first label as compared to elsewhere in the solution. Furthermore, both reactions require the use of bulky enzyme molecules attached to the polynucleotide probe. These bulky molecules may sterically "clash" with each other.

A second embodiment involves that of energy transfer, namely the emission of photons from a first label, for example, fluorescence, followed by absorption of the photons by a second label, to either quench the emission, or to provide a second emission. This has a drawback in that when an intercalator is the first label, it is attached to the polynucleotide probe covalently. In addition, the method requires the formation of two complexes, namely the formation of a polynucleotide/polynucleotide complex, and the formation of an antigen/antibody complex. Furthermore, one aspect involves the quenching of emitted photons, and since hybridization of probe to target is usually no more than a few percent, such minute quenching would produce ambiguous results.

Fluorescence detection is widely used in hybridization assays. In fluorescence spectroscopy the substance to be determined which is present in a liquid or a solid phase is subjected to a radiation with a known spectral distribution, for instance light with a limited band width. The fluorescent radiation thereby emitted has a longer wavelength than the exciting radiation and this radiation is specific for the substance to be determined. The measurement of the intensity of the fluorescent radiation constitutes a quantification of the substance to be determined. Fluorescent moieties attached to polynucleotide probes are most efficient when they have a high intensity, a relatively long emission wavelength (more than 500 nm), a high Stoke's shift, and the ability to be bound covalently to a polynucleotide probe without negatively affecting its hybridization capabilities. Aromatic agents used in biological systems that give a rather strong fluorescence and are relatively stable include, for example, fluorescenisothiocyanate (FITC), rhodamines (RBITC, TRITC, RB-200-SC), dansil chloride (DNS-Cl), and fluorescamine (FL).

Fluorescence is generally measured with a spectrofluorimeter. A disadvantage of current methods for detecting signalling moieties with spectrofluorimeters is that the detection sensitivity is limited because of interfering fluorescence or noise in the exciting and detecting systems that increases the background. Interfering fluorescence is generated from substances such as substrate molecules, non-specifically bound compounds, sample holders, air particles, and the intrinsic fluorescence of the biological systems. The background is also affected by a heavy scattering which gives rise to an interference, especially when aromatic organic agents with a small Stoke's shift (less than 50 nm) are used.

Several approaches have been described that attempt to overcome the background problem with fluorescence detection. One approach, described in U.S. Pat. No. 4,058,732, measures delayed fluorescence using a signalling moiety comprising a substance with a fluorescence emission having a duration that considerably exceeds the duration of the fluorescence of the noise sources. A laser pulse is used to excite a sample, and the detection of the fluorescence from the signalling moiety takes place only when a sufficiently long time has passed for the fluorescence from the noise sources to have decayed. This method has drawbacks in that it is not readily adaptable to commercial use, and is not amenable for a homogenous assay.

A second approach, described in U.S. Pat. No. 4,374,120, by E. Soini and I. Hemmilia, discloses a method for determining the presence of an antigen by attaching a first ligand to an antibody, complexing a lanthanide metal to the first ligand, and complexing a second ligand to the lanthanide metal. The antigen-containing sample is fixed to a support, antibodies are then contacted with the sample, and unbound antibodies are washed away. A radiation pulse of short duration is used to excite the second ligand. Energy is transfered from the triplet state of this ligand to the chelated metal which emits radiation at a longer wavelength and for a longer time period than the noise sources. Detection of this delayed fluorescence verifies the presence of the antigen. This method has a drawback in that it cannot be carried out in one step; all unbound antibodies must be washed away from the support.

BRIEF SUMMARY OF THE INVENTION

It is an object of this invention to provide a method for detecting an analyte by complexing it to a binding entity comprising a first partner of an energy transfer system, wherein the formation of the complex induces or allows for the localization of a reporting entity comprising a second partner of the energy transfer system within a proximate distance of the first partner so that energy emitted by one partner, the energy donor or E.sub.1, can be absorbed by the other partner, the energy acceptor or E.sub.2, and wherein, the fluorescent energy emitted by the second partner is of longer wavelength than that emitted by the first partner and in addition may have fluorescence of substantially greater duration than the first partner or of the background fluorescence.

It is another object of this invention to provide a method for detecting an analyte by complexing it to a binding entity comprising a first energy emitter (E.sub.1), wherein the formation of the complex induces or allows for the localization of a reporting entity comprising a second energy emitter (E.sub.2) within a proximate distance of E.sub.1 so that energy emitted by E.sub.1 can be absorbed by E.sub.2, and wherein, the fluorescent energy emitted by E.sub.2 is of longer wavelength than that emitted by the E.sub.1 and in addition may have fluorescence of substantially greater duration than E.sub.1 or background fluorescence.

It is an additional object of this invention to provide a method for detecting an analyte by complexing it to a binding entity comprising a second energy emitter (E.sub.2), wherein the formation of the complex induces or allows for the localization of a reporting entity comprising a first energy emitter (E.sub.1) within a proximate distance of E.sub.2 so that energy emitted by E.sub.1 can be absorbed by E.sub.2, and wherein, the fluorescent energy emitted by E.sub.2 is of longer wavelength than that emitted by the E.sub.1 and in addition may have fluorescence of substantially greater duration than E.sub.1 or background fluorescence.

It is another object of this invention to provide a method for detecting the presence of an antigen in solution by complexing it to a specific antibody comprising an E.sub.2 (or E.sub.1), contacting the formed complex with Clq (of complement) comprising an E.sub.1 (or E.sub.2) or a second antibody comprising an E.sub.1 (or E.sub.2) to form a unit, irradiating the E.sub.1 with appropriate energy, and measuring the fluorescence emission.

It is a further object of this invention to provide a method for detecting the presence of an antigen by fixing the antigen to a support, contacting the antigen with a solution containing a specific antibody comprising an E.sub.2 (or E.sub.1) to form an antigen/antibody complex, contacting said complex with Clq comprising an E.sub.1 (or E.sub.2) or a second antibody comprising an E.sub.1 (or E.sub.2) to form an entity, irradiating the E.sub.1 with appropriate energy, and measuring the fluorescence emission.

It is an additional object of this invention to provide a method for detecting the presence of an antigen in solution by fixing a specific antibody comprising an E.sub.2 (or E.sub.1) to a support, contacting the antibody with a solution containing the antigen to form an antigen/antibody complex, contacting said complex with Clq comprising an E.sub.1 (or E.sub.2) or a second antibody comprising an E.sub.1 (or E.sub.2) to form an entity, and measuring the fluorescence emission.

It is also an object of this invention to provide a method for detecting the presence of an antigen by fixing the antigen to a support which has attached to it the E.sub.1 (or E.sub.2), contacting the support with a solution containing an antibody comprising an E.sub.2 (or E.sub.1), allowing the antibody to complex with the antigen, irradiating the E.sub.1 with appropriate energy, and measuring the fluorescence emission.

It is a further object of this invention to provide a method for detecting the presence of a target polynucleotide in solution by hybridizing it to a polynucleotide probe comprising an E.sub.2, permitting an E.sub.1 to intercalate into the formed hybrid, irradiating the E.sub.1 with appropriate energy, and measuring the fluorescence emission.

It is also an object of this invention to provide a method for detecting the presence of a target polynucleotide by fixing the target polynucleotide to a support, contacting the target polynucleotide with a solution containing a polynucleotide probe comprising an E.sub.2 to form a hybrid, permitting an E.sub.1 to intercalate into the formed hybrid, irradiating the E.sub.1 with appropriate energy, and measuring the fluorescence emission.

It is another object of this invention to provide a method for detecting the presence of a target polynucleotide by fixing a polynucleotide probe comprising an E.sub.2 to a support, contacting the polynucleotide probe with a solution containing the target polynucleotide to form a hybrid, permitting an E.sub.1 to intercalate into the formed hybrid, irradiating the E.sub.1 with appropriate energy, and measuring the fluorescence emission.

It is yet another object of this invention to provide a method for detecting the presence of a target polynucleotide by fixing the target polynucleotide to a support which has attached to it the E.sub.1 (or E.sub.2), contacting the support with a solution containing a polynucleotide probe comprising an E.sub.2 (or E.sub.1), allowing the target polynucleotide to hybridize to the polynucleotide probe, irradiating the E.sub.1 with appropriate energy, and measuring the fluorescence emission.

It is an additional object of this invention to provide a method for detecting the presence of a target polynucleotide in solution by hybridizing it to a polynucleotide probe comprising a hapten, binding an antibody specific for the hapten or for a specific double-stranded polynucleotide comprising an E.sub.1 (or E.sub.2) to said hybrid to form a complex, contacting said complex with Clq comprising an E.sub.2 (or E.sub.1) to form an entity, irradiating the E.sub.1 with appropriate energy, and measuring the fluorescence emission.

A method is disclosed herein for detecting the presence of an analyte in a homogeneous or one-step assay. The assay can be carried out either in one phase (liquid) or in two phases (liquid and solid). The method comprises first complexing an analyte with a binding entity. The binding entity and the analyte can both be dissolved in the liquid phase or one of them can be dissolved in the liquid phase and one of them can be fixed to a solid support. A reporting entity which is dissolved in the liquid phase or comprises the solid support, is then brought into contact with the complex to form a unit.

The analyte comprises an antigen, antibody, or polynucleotide. The binding entity comprises a recognition segment and a signalling segment. The recognition segment comprises an antibody, antigen, or polynucleotide. The signalling segment comprises either an E.sub.1 (an energy donor) or an E.sub.2 (an energy acceptor). The reporting entity comprises an E.sub.1 or an E.sub.2 depending on what the signalling entity does not comprise. The actual composition of the binding entity and the reporting entity depend on the composition of the analyte and the embodiment used for carrying out the detection.

The E.sub.1 and E.sub.2 constitute the two partners in the energy transfer system. The E.sub.1 or E.sub.2 can be either a fluorescent aromatic agent or a lanthanide metal. When the E.sub.1 is a fluorescent aromatic agent, then the E.sub.2 can be a fluorescent aromatic agent or a lanthanide metal. When the E.sub.1 is a lanthanide metal, then the E.sub.2 must be a fluorescent aromatic agent.

The E.sub.1 always absorbs the initial energy and then emits some of this energy at a wavelength which is absorbed by the E.sub.2. The E.sub.2 then emits some of this energy as fluorescence of a longer wavelength than the E.sub.1 and in addition may emit fluorescence whose duration considerably exceeds the duration of the E.sub.1 and of the background fluorescence. The presence of this bathochromic and/or delayed fluorescence emission indicates the presence of the analyte.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a depicts the detection of an analyte antigen in solution with a binding entity comprising an antibody and the E.sub.2 and a reporting entity comprising Clq and the E.sub.1.

FIG. 1b depicts the detection of an analyte antigen fixed to a solid support with a binding entity comprising an antibody and E.sub.1 and a reporting entity comprising Clq and the E.sub.2.

FIG. 1c depicts the detection of an analyte antigen fixed to a solid support with a binding entity comprising an antibody and the E.sub.2 and a reporting entity comprising the solid support and the E.sub.1.

FIG. 1d depicts the detection of an analyte target polynucleotide in solution with a binding entity comprising a complementary polynucleotide and the E.sub.2 and a reporting entity comprising an intercalating agent as the E.sub.1.

FIG. 1e depicts the detection of an analyte target polynucleotide fixed to a solid support with a binding entity comprising a complementary polynucleotide and the E.sub.2 and a reporting entity comprising an intercalating agent and the E.sub.1.

FIG. 1f depicts the detection of an analyte target polynucleotide fixed to a solid support with a binding entity comprising a complementary polynucleotide and the E.sub.2 and a reporting entity comprising the solid support and the E.sub.1.

FIG. 2 shows a schematic diagram of a fluorimeter which can be used to carry out the detection of an analyte with a probe comprising a lanthanide metal.

DETAILED DESCRIPTION OF THE INVENTION

1. GENERAL DESCRIPTION OF THE INVENTION This invention discloses homogeneous assay for determining the presence of an analyte. A homogeneous assay, also known as a one-step assay, permits the detection of an analyte upon the contacting of the analyte with a binding entity and a reporting entity (and other components) in an assay medium. There is no need to remove unbound binding entities from the assay medium before detection can be achieved.

The method comprises the use of a first energy emitter, the E.sub.1 (energy donor), and a second energy emitter, the E.sub.2 (energy acceptor). The E.sub.2 is capable of absorbing some of the energy emitted by the E.sub.1. The complexing of the binding entity to the analyte causes or allows the reporting entity to contact the formed complex to form a unit. The formation of this unit places the E.sub.1 sufficiently proximate to the E.sub.2 such that energy emitted by the E.sub.1 can be absorbed by the E.sub.2. The E.sub.2 emits its absorbed energy as fluorescence of a longer wavelength (bathochromic) than the fluorescence of the E.sub.1, and in addition, may emit fluorescence of substantially greater duration (delayed) than the E.sub.1 (or other background fluorescence). The presence of this bathochromic and/or delayed fluorescence indicates the presence of the analyte.

The method is applicable to the detection of analytes which include, for example, antigens haptens, antibodies, hormones, enzymes, or polynucleotides, and can be carried out in a one phase system i.e. in a solution, or in a two phase system, i.e. in a solution over a solid support. The detection is carried out by forming a complex between the analyte and a binding entity.

The binding entity contains a recognition segment and a signalling segment. The recognition segment is the part of the binding entity that complexes to a part of the analyte. The signalling segment is the part that is involved in the formation of an energy-transfer system to produce a signal indicating that recognition of the analyte by the binding entity has occurred. If the analyte is an antigen, then the binding entity comprises an antibody. If the analyte is an antibody, then the binding entity comprises a antigen. If the analyte is a target polynucleotide, then the binding entity comprises a complementary polynucleotide. The signalling segment comprises either the E.sub.1 or the E.sub.2. The E.sub.1 can be a fluorescent aromatic agent; the E.sub.2 can be a fluorescent aromatic agent or a lanthanide metal. The reporting entity comprises either the E.sub.1 or the E.sub.2. When the signalling segment comprises the E.sub.1, then the reporting entity comprises the E.sub.2. When the signalling segment comprises the E.sub.2, then the reporting entity comprises the E.sub.1.

In some embodiments of the assay, all of the components are dissolved in a solution (liquid phase). In other embodiments, one or more of the components are fixed to a solid support while the remaining components are dissolved in a solution. A number of various embodiments are described below. These embodiments are not meant for limitation.

1. The analyte is an antibody and the binding entity comprises an antigen and the E.sub.1. The E.sub.2 is attached to Clq (of complement) or to an antibody. All the components are dissolved in the liquid phase.

2. The analyte is an antibody and the binding entity comprises an antigen and the E.sub.2. The E.sub.1 is attached to the Clq or to an antibody. All the components are dissolved in the liquid phase.

3. The analyte is an antigen and the binding entity comprises an antibody and the E.sub.1. The E.sub.2 is attached to Clq or to an antibody. All the components are dissolved in the liquid phase.

4. The analyte is an antigen and the binding entity comprises an antibody and the E.sub.2. The E.sub.1 is attached to the Clq or to an antibody. All the components are dissolved in the liquid phase.

5. The analyte is an antibody and is fixed onto a sold support. The binding entity comprises an antigen and the E.sub.1. The E.sub.2 is attached to Clq or to an antibody. Both the binding entity and the Clq or antibody are dissolved in the liquid phase.

6. The binding entity comprising an antigen and the E.sub.1 is fixed onto a solid support. The analyte is an antibody. The E.sub.2 is attached to Clq. Both the analyte and the Clq or antibody are dissolved in the liquid phase.

7. The analyte is an antibody and is fixed onto a solid support. The binding entity comprises an antigen and the E.sub.2. The E.sub.1 is attached to Clq or to an antibody. Both the binding entity and the Clq are dissolved in the liquid phase.

8. The binding entity comprising an antigen and the E.sub.2 is fixed onto a solid support. The analyte is an antibody. The E.sub.1 is attached to Clq or to an antibody. Both the analyte and the Clq or antibody are dissolved in the liquid phase.

9. The analyte is an antigen and is fixed onto a solid support. The binding entity comprises an antibody and the E.sub.1. The E.sub.2 is attached to Clq or to an antibody. Both the binding entity and the Clq or antibody are dissolved in the liquid phase.

10. The binding entity comprising an antibody and the E.sub.1 is fixed onto a solid support. The analyte is an antigen. The E.sub.2 is attached to Clq or to an antibody. Both the analyte and the Clq or antibody are dissolved in the liquid phase.

11. The analyte is an antigen and is fixed onto a solid support. The binding entity comprises an antibody and the E.sub.2. The E.sub.1 is attached to Clq or to an antibody. Both the binding entity and the Clq or antibody are dissolved in the liquid phase.

12. The binding entity comprising an antibody and the E.sub.2 is fixed onto a solid support. The analyte is an antigen. The E.sub.1 is attached to Clq or to an antibody Both the analyte and the Clq or antibody are dissolved in the liquid phase.

13. The analyte is an antibody and is fixed onto a solid support. The binding entity comprises an antigen and the E.sub.1. The E.sub.2 is attached onto the solid support. The binding entity is dissolved in the liquid phase.

14. The analyte is an antibody and is fixed onto a solid support. The binding entity comprises an antigen and the E.sub.2. The E.sub.1 is attached onto the solid support. The binding entity is dissolved in the liquid phase.

15. The analyte is an antigen and is fixed onto a solid support. The binding entity comprises an antibody and the E.sub.1. The E.sub.2 is attached onto the solid support. The binding entity is dissolved in the liquid phase.

16. The analyte is an antigen and is fixed onto a solid support. The binding entity comprises an antibody and the E.sub.2. The E.sub.1 is attached onto the solid support. The binding entity is dissolved in the liquid phase.

17. The analyte is a target polynucleotide and the binding entity comprises a complementary polynucleotide and the E.sub.2. The E.sub.1 is either an intercalating agent or attached to an intercalating agent. All the components are dissolved in the liquid phase.

18. The analyte is a target polynucleotide and the binding entity comprises a complementary polynucleotide, a hapten attached to the polynucleotide, and an E.sub.2 which is attached to an antibody bound to the hapten. The E.sub.1 is an intercalating agent or attached to an intercalating agent. All the components are dissolved in the liquid phase.

19. The analyte is a target polynucleotide and is fixed onto a solid support. The binding entity comprises a complementary polynucleotide and the E.sub.2. The E.sub.1 is an intercalating agent or attached to an intercalating agent. Both the binding entity and the E.sub.1 are dissolved in the liquid phase.

20. The binding entity comprising a complementary polynucleotide and the E.sub.2 is fixed onto a solid support. The analyte is a target polynucleotide. The E.sub.1 is an intercalating agent or attached to an intercalating agent. Both the analyte and the E.sub.1 are dissolved in the liquid phase.

21. The analyte is a target polynucleotide and is fixed onto a solid support. The binding entity comprises a complementary polynucleotide, a hapten attached to the polynucleotide, and an E.sub.2 which is attached to an antibody bound to the hapten. The E.sub.1 is an intercalating agent or attached to an intercalating agent. The binding entity, the antibody, and the E.sub.1 are dissolved in the liquid phase.

22. The binding entity comprising a polynucleotide, a hapten attached to the polynucleotide, and a E.sub.2 which is attached to an antibody bound to the hapten is fixed onto a solid supp