Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-08-31
2001-05-29
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06239271
ABSTRACT:
BACKGROUND OF THE INVENTION
1) Field of the Invention
The present invention relates to the field of hybridization assay methods, and more particularly, it relates to hybridization assay techniques in which the presence of an analyte is determined by means of energy transfer.
2) Brief Description of the Prior Art
Nucleic acid hybridization is an effective method for detecting and identifying pathogenic organisms and genetic disorders. It is also useful for mapping genes on chromosomes and in general has a wide spectrum of applications in clinical research. But despite the commercial availability of numerous target specific polynucleotide probes, detection or identification of infectious agents by hybridization is often not the preferred method. Instead, infectious agents are still cultured and identified by laborious, subjective and frequently inaccurate procedures.
Some of the commonly cited reasons for not using hybridization assays are the relative complexity of performing the assay procedure and the lack of sufficient sensitivity for detection or quantification when small numbers of the target organism are present. The most sensitive hybridization assays depend upon the use of radioisotopes for labeling the nucleic acid probe. However, the use of radioisotopes necessitates safety precautions and elaborate means for their disposal. In addition, radiolabels often have a short half-life (e.g., the half-life of
32
P is fourteen days), which makes their use expensive. Thus, it is recognized as being highly desirable to develop and improve nonisotopic hybridization detection methods.
At present, alternative methods are available which depend, inter alia, either on the development of color or on the emission of fluorescence. Recently, highly sensitive time-resolved fluorescence (TRF) labeling, based on the long-lived emissions of lanthanide chelates, has been used in immunoassay and immunological detection of hybridization assay probes. Even though lanthanide ions, such as europium (Eu
+3
) and terbium (Tb
+3
) exhibit extremely weak luminescence when they are directly excited by visible light, it has been shown that these ions become highly fluorescent when they are chelated by organic ligands with good energy absorption properties. Absorption of the light by the ligand is followed by an efficient energy transfer from the excited ligand to the energy levels of the lanthanide ions. The fluorescence of lanthanide chelates is characterized by broad excitation in the absorption region of the ligand, a large Stokes' shift (>250 nm), narrow emission lines typical of the metal and an exceptionally long fluorescence lifetime (100-1000 usec). See, Horrocks, W. D. et al., “Laser-Induced Lanthanide Ion Luminescence Lifetime Measurement by Direct Excitation of Metal Ion Levels, A New Class of Structional Probe for Calcium-Binding Proteins and Nucleic Acids,”
Journal of the American Chemical Society
99(7):2378-2380(1977) and Hemmilia, Clin. Chem., 31:359-370(1985).
In TRF assays, signals at the emission wavelengths of the lanthanide chelates are measured after a lapse in time between excitation and emission. This time-lag is sufficiently long to ensure that the specific long-lived fluorescence emissions of the lanthanide chelates are detected, but that the short-lived (<1 usec) background resulting from the intrinsic fluorescence of biological materials and other assay components (a serious problem in fluorometric measurements in biological samples) is not measured. This results in higher signal-to-noise ratios than are commonly observed in conventional fluorescence assays and consequently in an improvement in assay sensitivity.
Typically, TRF immunoassays are performed using Eu
+3
linked to antibodies via covalently bound chelators. After formation of the complex between the antigen and the antibody-chelate Eu
+3
, the Eu
+3
in the complex is released, bound to suitable chelators such as those referred to above, then trapped in micelles. Pulsed light with an appropriate wavelength is applied to this micelle system. The resulting fluorescence is measured using a time resolving fluorometer. Under optimal conditions (i.e., high quality antibodies, stable chelation complexes, and suitable time resolving fluorometers), TRF immunoassays have the potential to exceed sensitivity levels obtainable with radioisotopic labels. See, Gibson, et al., Arch. of Virol., 84:233(1985).
TRF immunoassays with sensitivity levels comparable to radioimmunoassays using
125
I labels have been successfully used to measure levels of peptide hormones, alpha-fetoprotein, thyrotropin, choriogonadotropin and influenza viruses in clinical specimens. See, Hemmilia, supra: Soini and Kojola, Clin. Chem., 29:65(1983); Chun, P. K. et al., “Rapid Detection of Antigens Using Colloidal Gold in Membrane Based Immunoassays,” from International Symposium on Rapid Methods and Automation in Microbiology and Immunology (5th) 1987, Florence, Italy, Published in
Rapid Methods and Automation in Microbiology and Immunology,
Balows, A. et al., Editors, Brixia Academic Press, Brescia, pages 572-577 (1989).
Syvanen, et al., Nuc. Ac. Res. 14:1017(1986) have applied the technique of TRF immunoassays to the detection of DNA hybrids formed in sandwich hybridization assays. DNA probes carrying haptenic sulfone groups were hybridized to nitrocellulose-bound target DNA sequences (adenovirus genomic DNA). The hybrids were detected using a two-step antibody system. The first antibodies specifically recognized and became bound to the sulfone labels on the probe. The second antibodies consisted of sheep anti-rabbit IgG labeled with Eu
+3
by chelation. The Eu
+3
was released from this complex, chelated to diketones, trapped in micelles, and excited with UV light. The resulting emission was detected using a time resolving fluorometer. Syvanen et al state that Eu
+3
can be bound to organic molecules by mediation of EDTA derivatives and that these chelates are unstable at hybridization conditions (data not shown), and that is the reason why the probe DNA cannot be directly labelled with Eu-EDTA chelates.
Two additional solid phase sandwich hybridization assays which employ TRF detection of hybridized probes and which are similar to the method described by Syvanen, et al., have been reported. Dahlen, et al., used streptavidin-Eu+
3
, with the Eu
+3
attached to streptavidin through a diethylenetriamine pentaacetic acid (DTPA) chelator, to detect biotinylated probes in matrix-bound hybridization complexes. Dahlen, et al., Mol. & Cell. Probes 1:159(1987). In the sandwich assay developed by Oser, et al., DTPA was attached to poly-L-lysine groups which were covalently bound to probe DNA via psoralen linkages. The probes were labeled with Eu
+3
following hybridization. Nitrocellulose was used as the matrix in this system. Oser. et al., Nuc. Ac. Res., 16:1181(1988).
In both of these assays, as well as in the Syvanen, et al., assay, Eu
+3
was measured by TRF following its release from the hybridization-detection complex. Notwithstanding the high sensitivity of these reported TRF-DNA hybridization assays, the length and complexity of these procedures make them unattractive for use in clinical laboratory settings. In addition, the lower detection levels of these assays are limited by high backgrounds resulting from the presence of measurable quantities of Eu
+3
in assay reagents, as well as in the environment (i.e., dust).
In another approach Sheldon III, et al., in U.S. Pat. No. 4,582,789, disclose a process for labeling nucleic acids with psoralen derivatives, which are also intercalators. A spacer arm chemically links the alkylating intercalation moiety with the label moiety, thereby allowing the label to react without interference, with detection means, such as antibodies.
Letsinger et al., U.S. Ser. No. 444,438, filed Nov. 24, 1982 now abandoned, is said by Sheldon et al., supra, to disclose bifunctional intercalators containing a phenanthridinium moiety as an agent for intro
Hurley Ian
Rabbani Elazar
Enzo Diagnostics, Inc.
Fedus, Esq. Ronald C.
Horlick Kenneth R.
Rogers, Esq. James L.
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