Organic compounds -- part of the class 532-570 series – Organic compounds – Sulfur halides
Reexamination Certificate
1999-04-28
2002-01-15
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Sulfur halides
C562S833000, C549S043000, C549S057000, C549S064000, C558S013000, C435S005000, C435S006120, C435S091100, C435S091200, C536S022100, C536S023100
Reexamination Certificate
active
06339172
ABSTRACT:
FIELD OF THE INVENTION
The present invention reates to a novel in situ assay method for an objective substance in a biological sample, comprising assaying on said biological sample, a reagent therefor, particularly a novel labeled nucleic acid probe and a fluorescent complex comprsing said probe and a heavy metal ion, a labeled nucleotide for preparing said labeled nudeic acid probe and a process for preparing said labeled nucleic acid probe. More particularly the present invention relates to a novel method which can be preferably used for analyzing the function and behavior of a certain substance (e.g., nucleic acid) on a biological sample (e.g., a biological tissue and a cell), by assaying the localization or concentration thereof on the biological sample, as well as a labeled probe and a reagent for analysis which contains said probe to be used for said method.
BACKGROUND OF THE INVENTION
In the research field of life science and the field of clinical diagnostic and clinical tests, fluoresct substances have been widel used as a label substance, besides radioactive substances, enzmes and the like. With the progress of the image analyzing technique systems in recent years they have been more increasingly used in a broad range of applications, thereby providing new findings in the function and behavior of biological substances in a living body.
Such fluorescent substances typically indude compounds comprising fluorescein, dansyl group, anthraniloyl group, pyrene, rhodamine, nitrobenzoxadiazol and the like.
The fluorescent substances, which are intercalated in between double strands of nucleic acid (DNA) and enable fluorescent staining of the DNA, include Hoechst 33342 manufactured by Molecular Probe, 4′,6′-diamino-2-phenylindol dihydrochioride (DAPI), propidium iodite (PI), acridium orange and the like. Besides these, commercially available products such as SYTO (TM), BOBO (TM), POPO (TM), TOTO (TM), YOYO (TM) and the like are used similarly.
To label lipids, fluorescent substances, such as 4-nitrobenzene-2-oxa-1,3-diazol (NBD) and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), are used.
In recent years, fluorescent substances capable of labeling various ions or other low molecular substances (e.g., fura-2, indo-1, fluo-3, etc. for calcium ion, SBFI, etc. for sodium ion, mag-fura-2, mag-indo-1, etc. for magnesium ion, TSQ, etc. for zinc ion, SPQ, etc. for chloride ion and FICRhR for cyclic AMP) have been developed, and the behavior of ions in a living body has been studied using these fluorescent substances.
In an assay of a substance in a biological sample, it is desired of a fluorescent substance, theoretically and practically, that (1) it does not deactivate nucleic acid, peptide, low molecular ligand and the like after binding, (2) it has a high fluorescence quantum yield and high photostability, (3) its fluorescence lifetime is long, (4) it is free of the effect of other endogenous fluorescent substances in the biological sample, (5) it does not react non-specifically with an endogenous molecule in the biological sample, (6) it easily dissolves in water and (7) its determination is convenient. Particularly, in an in situ assay on a tissue or cell, a fluorescent substance is further required to not react non-specifically with a biomolecule present in the tissue or cell or on the surface thereof.
However, some of the above-mentioned fluorescent substances are unstable to light and/or heat, some have low quantum yield, and others have short excited fluorescent lifetime and are subject to the effect of autofluorescence of other endogenous substances. The conventionall known fluorescent substances are not ideal fluorescent compounds, but rather, are insufficient, since they are more or less problematic in one or more aspects, such as low S/N ratio, short fluorescence wavelength and the like.
The influence of endogenous fluorescence in the assay of substances in a liquid sample such as body fluid and cell extract can be removed by using, as a lanthanoide metal-containing fluorescent complex, a complex labeled with a novel fluorescent substance and consisting of a substance having affinity for the objective substance and an europium ion. A method has been developed which is free of an influence of the background fluorescence derived from a fluorescent substance or non-fluorescent substance in a biological sample, particularly a serum, during assay of a physiologically active substance in the sample, and which comprises subjecting the complex to a time-resolved fluorescence assay.
For example, use of a diazaophenyl-EDTA-europium complex or isothiocyanatephenyl-EDTA-europium complex for an immnoassay has been known (
Anal. Biochem
. 137, 335-343, 1984). In this immnoassay, &bgr;-naphthoyltrifluoroacetone (&bgr;-NTA) is added to the assay system in the co-existence of &bgr;-diketone and tri-n-octyl-phosphine oxide (TOPO) to achieve the highest sensitivity.
This assay system has been known as a DELFIA system (Dissociation Enhanced Lanthanide Fluoroimmunoassay). A method utilizing a europium complex represented by this system is advantageous in that an assay target in a biological sample can be detected without the influence of fluorescence having a short lifetime which is derived from a contaminating substance in the body, due to the fluorescent property of this complex that its life is long.
On the other hand, the DELFIA system is associated with the defect caused by a reaction between &bgr;-NTA or TOPO used for the assay and europium in a sample or in the environment, thereby producing strong fluorescence, which may prevent detection of the assay target
In addition to the inherent defect this system has in that it is susceptible to the influence of the contaminated europium, the need to add a fluorescence intensifier such as &bgr;-NTA makes the assay on a solid phase unattainable. There is also a problem of manipulative complexity due to the step of adding a fluorescence intensifier. In conclusion, in situ asssy of a physiologicaly active substance (e.g, nucleic acid, receptor, sugar chain, ganglioside and the like) fixed on a tissue or cell (surface) by this system is extremely difficult.
To resolve the above-mentioned defects of the DELFIA system, a Cyber Fluor system that uses a complex of 4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) and europium is known (
Anal. Chem
., 61, 48-53, 1989).
The use of BCPDA has made a great advancement in that nany europium fluorescent complexes can be introduced without a quenching phenomenon (quenching phenomenon strikingly deceases the fluorescence quantum yield) caused when one probe is labeled with many fluoresceins and that it is highly stable and can resolve the defects of the DELFIA system.
However, the Cyber Fluor system has a fatal defect in that its sensitivity is lower than that of the DELFIA system by the order of two digits or more. To compensate for the defect, synthesis of a number of europium complexes was tried, and, for example, trisbipyridine cryptate (TBP) europium complex and the like are known (
Clin. Chem
, 196-201, 1993, U.S. Pat. No. 5,262,526, JP 07-10819 A and the like). These newly developed europium fluorescent complexes have defects in that they have short excitation wavelengths and weak fluorescence, and they require many synthetic steps. Thus, they do not have particulary superior property as compared to the above-mentioned two europium fluorescent complexes.
Many studies have been made so far with respect to europium fluorescent complex and it has been found that &bgr;-diketone-europium fluorescent complex has greater fluorescence intensity than aromatic amine-europium complex, and of the &bgr;-diketone ligands, a europium fluorescent complex of 2-naphthoyltrifluoroacetone (&bgr;-NTA) and 2-thenoytrifluoroacetone (TTA) particurlaly has the greatest fluorescence intensity.
The present inventors snthesized various &bgr;-diketone-europium TOPO complexes to study the effect of &bgr;-diketone as a substituent on the fluorescence property of the &bgr;-diketone-europum
Ikeda Katsunori
Kawamura Yoshihisa
Matsui Kazuhiro
Matsumoto Kazuko
Teshima Shin'ichi
Leydig , Voit & Mayer, Ltd.
Matsumoto Kazuko
Riley Jezia
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