The present invention relates generally to association assays and more specifically to association assays whereby the presence of analytes in samples is determined by methods employing non-isotopic labels or probes.
Since their introduction in the early 1960's, immunoassay techniques employing radioactive labels have found widespread use in the clinical laboratory. Although exhibiting high sensitivities, making possible determination of important biological compositions such as hormones, proteins, drugs and drug metabolites which often exist at very low concentrations in serum samples, the radioimmunoassays possess several problems inherent in the labels employed. Use of radio isotopes requires a special permit and a special laboratory. Radiation can cause health hazards particularly for those working with the commonly used isotopes of iodine. In addition, the useful lifetime of the radio-labelled reagents employed is limited by the half-life of the isotopes and the destructive processes that occur during isotopic decay. The equipment used to determine the amount of radioactivity in the samples is expensive and the counting of a series of samples is relatively time-consuming. Use of radio-labelled reagents in immunoassay techniques necessitates a separation of associated and unassociated radio-labelled material before counting since it is not possible to distinguish between the radio-labelled reagent which is bound to the antibody or which exists unbound in the sample.
The above problems associated with assays involving radio isooopic labels have lead to the development of immunoassay techniques employing non-isotopic labels such as luminescent molecules. See, generally, Smith et al. Ann. Clin. Biochem 18: 253-74 (1981). Luminescent labels emit light upon excitation by an external energy source, and may be grouped into catagories dependent upon the source of the exciting energy, including: radioluminescent labels deriving energy from high energy particles; chemiluminescent labels which obtain energy from chemical reactions; bioluminescent labels wherein the exciting energy is supplied in a biological system; and photoluminescent labels which are excitable by units of electromagnetic radiation (photons) of infra red, visible or ultraviolet light. Id. at 255.
Luminescent assay techniques employing labels excitable by non-radioactive energy sources avoid the health hazards and licensing problems encountered with radio isotopic label assay techniques. Additionally, the use of luminescent labels allows for the development of "homogeneous" assay techniques wherein the labelled probe employed exhibits different luminescent characteristics when associated with an assay reagent than when unassociated, obviating the need fo separation of the associated and unassociated labelled probe.
Several heterogeneous and homogeneous luminescent immunoassay techniques have been reported. Early heterogeneous luminescent immunoassays included fluorescent assays which followed the same basic protocol as a radioimmunoassay except for the substitution of the fluorescent label and the required changes in the equipment employed to detect the fluorescence. However, when these assays are used to assay for the presence of analytes in biological samples, they are frequently reported to exhibit decreased sensitivity (relative to radioimmunoassays) due to interference from the sample components. See, Soni et al., Clin. Chem. 29/1, 65-68 (1983). Serum proteins exhibit fairly strong fluorescence in the emission region of most fluorescent labels (approximately 340-470 nm) resulting in significant levels of background fluorescence. See, Soni et al., Clin. Chem. 25/3, 353-81 (1979). The presence of proteins and other molecules in biological samples may cause the scattering of the exciting light ("Rayleigh scattering") resulting in interference with those luminescent labels which emit light at wavelengths within about 50 nm of the wavelength of the exciting light. The endogenous compounds may also absorb the exciting light and scatter it at longer wavelength characteristic of the absorbing molecules ("Raman scattering"), or may absorb light in the spectrum of emission of the luminescent label, resulting in a quenching of the luminescent probe.
Attempts to improve the sensitivity of heterogeneous luminescent assays have included the development of so-called "time resolved" assays. See, Soni et al., Clin. Chem. 29/1, 65-68 (1983); U.S. Pat. No. 4,176,007. Time resolved assays generally involve employing luminescent labels having emissive lifetimes significantly different from (usually much longer than) the 1-20 nsec emissive lifetime of the natural fluorescence of materials present in the sample. The assay association step is performed and the separated associated or unassociated labelled material is excited by a series of energy pulses provided by a xenon flash tube or other pulsed energy source. Luminescent emission of the label resulting from each pulse is measured at a time greater than the time of the natural fluorescence of background materials in the sample. Interference from the background scattering and short-lived sample fluorescence is thus eliminated from the measured luminescence.
The heterogeneous luminescent assays described above by definition require a separation of associated luminescent labelled material from the unassociated luminescent labelled reagent, resulting in a slower, more laborious assay protocol and increasing the possibility of experimental error.
Homogeneous luminescent association assays have been reported wherein the need for a separation step is avoided by utilizing luminescent labels which exhibit different emissive characteristics when associated with one or more of the assay reagents than when unassociated. See, Ullman, et al., J. Biol. Chem. 251/14: 4172-78 (1976); Smith et al., Ann. Biochem. 18: 253, 262-65, 267-68 (1981). Such reported homogeneous luminescent assays include direct enhancement/quenching assays employing labelled immunological reagents which exhibit enhanced or decreased emission when associated with other reagents, such as antibodies for the ligand or antibodies for the label itself. See, e.g., U.S. Pat. No. 3,998,943; U.S. Pat. No. 4,160,016; U.S. Pat. No. 4,161,515; Smith et al., supra at pp. 262-63.
Of interest to the present invention are homogeneous luminescent assays which utilize energy transfer from the luminescent label excitable by the external energy source to a compound capable of absorbing the transferred energy as means for differentiating between unassociated and associated labelled reagent. See, e.g., U.S. Pat. No. 3,996,345; U.S. Pat. No. 4,174,384; U.S. Pat. No. 4,199,559; Smith et al., supra at p. 264; Ullman et al., supra. Energy transfer assays avoid the problems involved in selecting appropriate labels for direct enhancement/quenching assays in that the label excitable by the external energy source may have its emission modulated by other molecules whose absorbance spectrum overlap with the emission spectrum of the label when the label and modulator are brought in close proximity to one another. U.S. Pat. No. 3,996,345 discloses fluorescent energy transfer immunoassays employing antibodies and a fluorescer/quencher label pair wherein one or both of the label pair are bound to antibodies. The quenching compound is disclosed as a fluorescent compound having its absorption at an emission wavelength of the fluorescer excited by the external energy source. Depending on the protocol utilized, different immunological reagents are used, comprising either a ligand analog bound to one of the fluorescer/quencher label pair and antibodies for the ligand bound to the other of the label components, or a combination of ligands bonded to a hub molecule and one of the fluorescer/quencher labels, and an antibody bound to the other of the fluorescer/quencher label pair. The labelled fluorescer and quencher reagents are added to the sample containing the ligand and compete with the ligand for association with one another, the amount of fluorescer-labelled reagent associated with quencher-labelled reagent being inversely related to the amount of free ligand present in the sample. The association of fluorescer-labelled reagent with the quencher-labelled reagent brings the two labels in close proximity (100 angstroms or less), resulting in absorption by the quencher of energy which would otherwise be emitted as fluorescence from the excited fluorescer. U.S. Pat. 4,174,384 discloses a similar fluorescer/quencher assay wherein the fluorescer and quencher are each covalently bound to an antibody for the ligand, or one of the label pair is bonded to ligand antibody and the other attached to an antibody for the ligand antibody. U.S. Pat. No. 4,199,599 discloses an energy transfer luminescent immunoassay for detection of an antibody in a sample wherein one of the fluorescer/quencher pair is bound to antibody for the sample antibody ligand and the other is bound to a second antibody for the ligand or to a ligand analog.
Other reported homogeneous luminescent assays of interest include modified reagent fluorescent assays wherein a fluorescent label portion of a ligand analog is inhibited from interacting with a modifying substance such as a quencher molecule when the labelled analog is associated with an immunological reagent. See, U.S. Pat. No. 4,208,479. The inhibition results from steric interference caused by the immunological reagent associated with the labelled reagent which prevents the quencher from coming in close proximity to the label. Also reported are luminescent "release" assays utilizing fluorescent-labelled conjugates which include the fluorescent compound bound to a quenching molecule by an enzymaticallycleavable bond. See, U.S. Pat. No. 4,318,981. When the conjugate is associated with an immmunological reagent, the enzyme is inhibited from cleaving the fluorescer-quencher bond and energy transfer quenching or fluorescing by the quencher molecule occurs. If, however, the conjugate is unassociated, ezymatic cleavage occurs, releasing the fluorophore from its close association with the quencher and a resulting increase in fluorescer emission is observed. In both assays the degree of quenching is related to the amount of ligand present due to the competition of the immunological reagents and ligand for association with one another.
Herman et al. have reported an energy transfer association technique utilizing photophore-labelled proteins in the study of muscle differentiation. See, Herman et al., Biochem. 21: 3275-3283 (1982). Two luminescent labels, pyrene and fluorescein isothiocyanate (FITC) were bound to concanavalin A (Conn A) protein and utilized to determine the changes in topography and lateral translational mobility of Conn A receptors on chick muscle cells during the period of myoblast fusion. Labelled Conn A was allowed to associate with its receptors, bringing the pyrene donor and FITC acceptor in sufficient proximity to allow non-radiative energy transfer to occur. Receptor migration was determined by monitoring the decrease in the ratio of acceptor emission relative to donor emission as the acceptor-labelled receptor sites migrated apart from donor-labelled receptors, since the energy transfer process occurs over only short distances and its efficiency decreases as the inverse sixth power of the distance between donor and acceptor. Alternative methods suggested for monitoring migration include monitoring the decrease in donor emission lifetime due to energy transfer or construction of time-resolved emission spectra from fluorescence decay curves collected at various wavelengths by determining unconvoluted donor and acceptor emission. Measurement of unconvoluted donor and acceptor emission decay data was reported to be a very difficult problem. Id. at 3279-80.
Applicant and his coworkers disclose in published EPO Application No. 0070 685 a homogeneous luminescent association assay utilizing a chemiluminescent energy source. The assay employs two labels, a chemiluminescent catalyst or fluorophore and a luminescent acceptor/emitter compound attached to terminal positions on separate polynucleotide strands complementary to contiguous regions of a target polynucleotide. When associated with target polynucleotide, the catalyst and acceptor/emitter are brought in close proximity to one another, allowing the energy released from the provided chemiluminescent reagents when they undergo catalysis to be non-radiatively transferred to the acceptor/emitter. Filters are utilized to block all light except the light emitted by the acceptor/emitter which is measured by a photodetector monitoring device.
By definition, the homogeneous luminescent assays described above are performed in the presence of endogenous sample constituents including proteins and other light absorbing or scattering compounds. The energy transfer assays therefore experience the same potential for background interference due to Rayleigh scattering, Raman scattering, and endogenous luminescence as described above for heterogeneous luminescent assays carried out in the presence of sample. Further, the quenching type energy transfer assays may encounter interference from endogenous quenching compounds which absorb at the wavelengths of emission of the fluorescence label. Chemiluminescent reactions, especially those involving luminol, are also sensitive to trace amounts of catalytic substances such as metal ions. Suggestions that the energy transfer be measured by monitoring the emission of the compound accepting the energy donated by the label excited by the external energy source are also problematic. See, e.g., U.S. Pat. No. 4,318,981; Lim et al., Ann. Biochem. 108: 176-184 (1980). The "acceptor" compound must absorb light in the region of the "donor" emission in an energy transfer scheme. Emission of the donor occurs at a wavelength longer than the wavelength of the exciting light energy source. Therefore the acceptor will necessarily absorb some of the exciting light. This is true since a molecule in solution always absorbs to some extent at wavelengths of higher energy than its lowest energy absorption due to the existence of higher electronic states. The "acceptor" compound will therefore be directly excited by the external energy source to a degree proportional to the intensity of the energy source. Such direct excitation will result in emission by the acceptor whether associated or unassociated, thus increasing the background interference. See, Lim et al., supra at pages 182-183. Color filtering will not eliminate such interference since the direct excitation will cause emission at the wavelength being monitored for energy transfer. Id. The suggested construction of time resolved emission spectra by monitoring donor and acceptor emission decay spectra will not avoid the direct excitation problem unless the spectra are constructed to cover emission of the acceptor due to energy transfer at times greater than the emissive lifetime of the acceptor relative to the exciting energy pulse. Further, construction of the time resolved emission spectra reported requires deconvolution of donor and acceptor emission decay data. Such deconvolution is acknowledged to be a very difficult problem, requiring multiple measurements over a range of emissive wavelengths, (see Herman et al. supra at 3280), and computer matrix analysis of the exponential decay data for each measurement before the curve may be costructed. See, generally, Lakowicz, Principles of Fluorescence Spectroscopy (Plenum Press, N.Y., 1983) pp. 65-75.
In addition to the above-stated problems, another problem results from the inability to distinguish acceptor photophore emission from donor photophore emission. This is particularly a problem if the Strokes' shift of donor and acceptor emissions are both small and spectral overlap is desired between donor photophore emission and the acceptor photophore absorbance transition corresponding to production of the lowest excited singlet state of the acceptor. In general, the donor emission spectrum will extend to some extent through the emission spectrum of the acceptor making complete separation of the two spectra impossible using color filtering alone. In addition, it may be desirable to use excess donor labelled reagent to drive the binding equilibrium, especially at low antigen concentrations. In this event, small overlap between donor and acceptor emission may lead to large backgrounds when measuring acceptor emission due to the "trailing" portion of the donor emission spectrum.
There continues to exist, therefore, a need in the art for simple homogeneous luminescent assays which more rigorously avoid background and other intereference problems present in the variety of assay systems heretofore proposed.