U.S. patent application number 09/967361 was filed with the patent office on 2002-08-08 for in situ assay of substance in biological sample using labeled probe.
This patent application is currently assigned to Toyo Boseki Kabushiki Kaisha. Invention is credited to Ikeda, Katsunori, Kawamura, Yoshihisa, Matsui, Kazuhiro, Matsumoto, Kazuko, Teshima, Shinichi.
Application Number | 20020106674 09/967361 |
Document ID | / |
Family ID | 26457444 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020106674 |
Kind Code |
A1 |
Matsui, Kazuhiro ; et
al. |
August 8, 2002 |
In situ assay of substance in biological sample using labeled
probe
Abstract
A method for analyzing an objective substance, comprising
reacting a labeled probe with an objective substance on a
biological sample, said probe comprising a label substance of the
formula (I): 1 wherein A.sup.1 is an aromatic group, R.sup.1is a
hydrogen or --COCH.sub.2COC.sub.nF.sub.2n+1 and n is an integer of
1-6, which is bonded to a probe selected from the group consisting
of nucleic acid, nucleic acid binding protein, low molecular ligand
and receptor for ligand (except antibody) to give a fluorescent
complex, reacting the complex with an objective substance on a
biological sample and assaying fluorescence of the resultant
fluorescent complex, a labeled nucleic acid probe and a labeled
nucleotide. According to the method of the present invention,
defects such as hindrance of fluorescence due to contaminant
substance, low sensitivity and the like can be resolved, thereby
enabling analysis on a tissue.
Inventors: |
Matsui, Kazuhiro;
(Tsuruga-shi, JP) ; Ikeda, Katsunori;
(Tsuruga-shi, JP) ; Teshima, Shinichi; (Osaka-shi,
JP) ; Kawamura, Yoshihisa; (Tsuruga-shi, JP) ;
Matsumoto, Kazuko; (Kawasaki-shi, JP) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
Toyo Boseki Kabushiki
Kaisha
Osaka-shi
JP
J
|
Family ID: |
26457444 |
Appl. No.: |
09/967361 |
Filed: |
September 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09967361 |
Sep 28, 2001 |
|
|
|
09301406 |
Apr 28, 1999 |
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Current U.S.
Class: |
435/6.16 ;
435/23 |
Current CPC
Class: |
G01N 33/582 20130101;
G01N 2458/40 20130101 |
Class at
Publication: |
435/6 ;
435/23 |
International
Class: |
C12Q 001/68; C12Q
001/37 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 28, 1998 |
JP |
119768/1998 |
Jun 30, 1998 |
JP |
184852/1998 |
Claims
What is claimed is:
1. A method for analyzing an objective substance, comprising
reacting a labeled probe with an objective substance on a
biological sample, said probe comprising a label substance of the
formula (I): 17wherein A.sup.1 is an aromatic group, R.sup.1is a
hydrogen or --COCH.sub.2COC.sub.nF.sub.- 2n+1 and n is an integer
of 1-6, or a label substance of the following formula (II):
18wherein A.sup.2 and A.sup.3 are the same or different and each is
an aromatic group, R.sup.2 and R.sup.3 are the same or different
and each is a hydrogen or --COCH.sub.2COC.sub.nF.sub.2n+1 and n is
an integer of 1-6, bonded to a probe selected from the group
consisting of nucleic acid, nucleic acid binding protein, low
molecular ligand and receptor for ligand (except antibody) via a
cross-linking group or a cross-linking group and a conjugating
group, adding a heavy metal ion and assaying fluorescence of the
resultant fluorescent complex.
2. A method for analyzing an objective substance, comprising adding
a heavy metal ion to a labeled probe, said probe comprising a label
substance of the formula (I): 19wherein A.sup.1 is an aromatic
group, R.sup.1 is a hydrogen or --COCH.sub.2COC.sub.nF.sub.2n+1 and
n is an integer of 1-6, or a label substance of the following
formula (II): 20wherein A.sup.2 and A.sup.3 are the same or
different and each is an aromatic group, R.sup.2 and R.sup.3 are
the same or different and each is a hydrogen or
--COCH.sub.2COC.sub.nF.sub.2n+1 and n is an integer of 1-6, bonded
to a probe selected from the group consisting of nucleic acid,
nucleic acid binding protein, low molecular ligand and receptor for
ligand (except antibody) to give a fluorescent complex, reacting
the complex with an objective substance on a biological sample and
assaying fluorescence of the resultant fluorescent complex.
3. The analysis method of claim 1 or claim 2, wherein the objective
substance is a member selected from the group consisting of a
nucleic acid, a nucleic acid-bound protein, a low molecular weight
ligand and a ligand receptor.
4. The analysis method of any of claim 1 to claim 3, wherein the
biological sample is a member selected from the group consisting of
a tissue, a cell or a chromosome.
5. The analysis method of claim 1 or claim 2, wherein the
cross-linking group is a sulfonyl group or a carbonyl group.
6. The analysis method of claim 1 or claim 2, wherein the
conjugating group binds a cross-linking group and a probe.
7. The analysis method of claim 1 or claim 2, wherein the
conjugating group is a divalent aliphatic hydrocarbon having 5 to
25 carbon atoms and optionally having 7 or less amide bonds between
carbons.
8. The analysis method of claim 1 or claim 2, wherein the
conjugating group comprises an affinity-bound biotin and
avidin.
9. The analysis method of claim 8, wherein the biotin is
amide-bound with a divalent aliphatic hydrocarbon having 5 to 25
carbon atoms and optionally having 7 or less amide bonds between
carbons.
10. The analysis method of claim 9, wherein the aliphatic
hydrocarbon is
--CH.dbd.CH--CO--NH--CH.sub.2--CH.sub.2--NH--(CO--CH.sub.2--CH.sub.2--CH.-
sub.2--CH.sub.2CH.sub.2--NH).sub.2- which is bonded to a probe.
11. A labeled nucleic acid probe comprising a label substance of
the formula (I): 21wherein A.sup.1 is an aromatic group, R.sup.1 is
a hydrogen or --COCH.sub.2COC.sub.nF.sub.2n+1 and n is an integer
of 1-6, or a label substance of the following formula (II):
22wherein A.sup.2 and A.sup.3 are the same or different and each is
an aromatic group, R.sup.2 and R.sup.3 are the same or different
and each is a hydrogen or --COCH.sub.2COC.sub.nF.sub.2n+1 and n is
an integer of 1-6, bonded to a nucleic acid probe via a
cross-linking group.
12. The labeled nucleic acid probe of claim 11, wherein the label
substance has the formula (III): 23wherein n is an integer of
1-6.
13. The labeled nucleic acid probe of claim 11 or claim 12, wherein
the cross-linking group is a sulfonyl group or a carbonyl
group.
14. The labeled nucleic acid probe of any of claim 11 to claim 13,
wherein the label substance is bonded to nucleic acid probe via a
conjugating group.
15. The labeled nucleic acid probe of claim 14, wherein the
conjugating group is a divalent aliphatic hydrocarbon having 5 to
25 carbon atoms and optionally having 7 or less amide bonds between
carbons.
16. The labeled nucleic acid probe of claim 15, wherein the
conjugating group has the formula (IV): 24wherein a is an integer
of 0-6 and b is 0 or 1.
17. The labeled nucleic acid probe of claim 14, wherein the
conjugating group comprises an affinity-bound biotin and an
avidin.
18. The labeled nucleic acid probe of claim 17, wherein the biotin
is amide-bound with a divalent aliphatic hydrocarbon having 5 to 25
carbon atoms and optionally having 7 or less amide bonds between
carbons.
19. The labeled nucleic acid probe of claim 18, wherein the
aliphatic hydrocarbon is
--CH.dbd.CH--CO--NH--CH.sub.2--CH.sub.2--NH--(CO--CH.sub.2-
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH).sub.2- which is
bonded to a probe.
20. A fluorescent complex comprising the labeled nucleic acid probe
of any of claim 11 to claim 19 and a heavy metal ion.
21. The fluorescent complex of claim 20, wherein the heavy metal
ion is a lanthanoide metal ion or radium ion.
22. The fluorescent complex of claim 21, wherein the lanthanoide
metal ion is a member selected from the group consisting of ions of
europium, samarium, terbium, dysprosium and a mixture thereof.
23. A reagent for analyzing a nucleic acid, comprising the labeled
nucleic acid probe of any of claim 11 to claim 19.
24. The reagent for analyzing of claim 23, comprising a heavy metal
ion.
25. The reagent for analyzing of claim 24, wherein the heavy metal
ion is a lanthanoide metal ion or radium ion.
26. A labeled nucleotide comprising a label substance of the
formula (I): 25wherein A.sup.1 is an aromatic group, R.sup.1 is a
hydrogen or --COCH.sub.2COC.sub.nF.sub.2n+1 and n is an integer of
1-6, which is bonded to a nucleotide via a cross-linking group.
27. The labeled nucleotide of claim 26, wherein the nucleotide is
dUTP.
28. The labeled nucleotide of claim 26 or claim 27, wherein the
cross-linking group is a sulfonyl group orcarbonyl group.
29. The labeled nucleotide of any of claim 26 to claim 28, wherein
the label substance is bonded to a nucleotide via a conjugating
group.
30. The labeled nucleotide of claim 29, wherein the conjugating
group is a divalent aliphatic hydrocarbon having 5 to 25 carbon
atoms and optionally having 7 or less amide bonds between
carbons.
31. The labeled nucleotide of claim 30, wherein the conjugating
group has the formula (IV): 26wherein a is an integer of 0-6 and b
is 0 or 1.
32. The labeled nucleotide of claim 29, wherein the conjugating
group comprises an affinity-bound biotin and avidin.
33. The labeled nucleotide of claim 32, wherein the biotin is
amide-bound with a divalent aliphatic hydrocarbon having 5 to 25
carbon atoms and optionally having 7 or less amide bonds between
carbons.
34. The labeled nucleotide of claim 32, wherein the aliphatic
hydrocarbon is
--CH.dbd.CH--CO--NH--CH.sub.2--CH.sub.2--NH--(CO--CH.sub.2--CH.sub.2---
CH.sub.2--CH.sub.2--CH.sub.2--NH).sub.2 which is bonded to a
probe.
35. A fluorescent complex comprising the labeled nucleotide of any
of claim 26 to claim 34 and a heavy metal ion.
36. The fluorescent complex of claim 35, wherein the heavy metal
ion is a lanthanoide metal ion or radium ion.
37. The fluorescent complex of claim 36, wherein the lanthanoide
metal ion is a member selected from the group consisting of ions of
europium, samarium, terbium, dysprosium and a mixture thereof.
38. A method for producing a labeled nucleic acid probe comprising
reacting the labeled nucleotide of any of claim 26 to claim 34,
dNTPs and a single strand DNA in the presence of a primer and a DNA
polymerase.
39. A method for producing a labeled nucleic acid probe comprising
reacting the labeled nucleotide of any of claim 26 to claim 34,
dNTPs and a double stranded DNA in the presence of 5'-exonuclease,
DNase and a DNA polymerase.
40. A labeled nucleic acid probe obtained by the production method
of claim 38) or claim 39.
41. A reagent for analyzing nucleic acid comprising a label
substance of the formula (I): 27wherein A.sup.1 is an aromatic
group, R.sup.1is a hydrogen or --COCH.sub.2COC.sub.nF.sub.2n+1 and
n is an integer of 1-6, or a label substance of the following
formula (II): 28wherein A.sup.2 and A.sup.3are the same or
different and each is an aromatic group, R.sup.2and R.sup.3 are the
same or different and each is a hydrogen or
--COCH.sub.2COC.sub.nF.sub.2n+1 and n is an integer of 1-6, to
which avidin is covalently bonded via a cross-linking group, and a
nucleotide to which biotin is bonded via a linkage group.
42. The reagent for analysis of nucleic acid of claim 41,
comprising a dNTP primer, a DNA polymerase and a heavy metal.
43. The reagent for analysis of nucleic acid of claim 41,
comprising dNTPs, 5'-exonuclease, DNase, a DNA polymerase and a
heavy metal.
44. The reagent for analysis of nucleic acid of claim 41, wherein
the cross-linking group is a sulfonyl group or carbonyl group.
45. The reagent for analysis of nucleic acid of claim 41, wherein
the nucleotide is dUTP.
46. A method for analyzing an objective substance comprising
reacting the objective substance with a nucleic acid probe
comprising a nucleotide to which biotin is bonded via a linkage
group as a component on a biological sample and then with a the
label substance of the formula (I): 29wherein A.sup.1is an aromatic
group, R.sup.1 is a hydrogen or --COCH.sub.2COC.sub.nF.sub.2n+1 and
n is an integer of 1-6, or a label substance of the following
formula (II): 30wherein A.sup.2 and A.sup.3 are the same or
different and each is an aromatic group, R.sup.2 and R.sup.3 are
the same or different and each is a hydrogen or
--COCH.sub.2COC.sub.nF.sub.2n+1 and n is an integer of 1-6, to
which avidin is covalently bonded via a crosslinking group, adding
a heavy metal ion and assaying fluorescence of the resultant
fluorescent complex.
47. The method for analysis of claim 46, wherein the nucleic acid
probe is obtained by reacting a nucleotide to which biotin is
bonded via a linkage group, dNTPs and a single strand DNA in the
presence of a primer and a DNA polymerase.
48. The method for analysis of claim 46, wherein the nucleic acid
probe is obtained by reacting a nucleotide to which biotin is
bonded via a linkage group, dNTPs and a double stranded DNA in the
presence of 5'-exonuclease, DNase and a DNA polymerase.
49. The method for analysis of claim 46, wherein the linkage group
is a divalent aliphatic hydrocarbon having 5 to 25 carbon atoms and
optionally having 7 or less amide bonds between carbons.
50. The method for analysis of claim 49, wherein the aliphatic
hydrocarbon is
--CH.dbd.CH--CO--NH--CH.sub.2--CH.sub.2--NH--(CO--CH.sub.2--CH.sub.2---
CH.sub.2--CH.sub.2--CH.sub.2--NH).sub.2--.
51. The method for analysis of claim 46, wherein the cross-linking
group is a sulfonyl group or carbonyl group.
52. The method for analysis of claim 46, wherein the nucleotide is
dUTP.
Description
FIELD OF THE INVENTION
[0001] The present invention relates 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 comprising said probe and a heavy metal ion, a labeled
nucleotide for preparing said labeled nucleic 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
[0002] In the research field of life science and the field of
clinical diagnostic and clinical tests, fluorescent substances have
been widely used as a label substance, besides radioactive
substances, enzymes 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.
[0003] Such fluorescent substances typically include compounds
comprising fluorescein, dansyl group, anthraniloyl group, pyrene,
rhodamine, nitrobenzoxadiazol and the like.
[0004] 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 dihydrochloride
(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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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
conventionally 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.
[0009] 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.
[0010] For example, use of a diazophenyl-EDTA-europium complex or
isothiocyanatephenyl-EDTA-europium complex for an immnoassay has
been known (Anal. Biochem, 137,335-343, 1984). In this immnoassay,
.beta.-naphthoyltrifluoroacetone (.beta.-NTA) is added to the assay
system in the co-existence of .beta.-diketone and
tri-n-octyl-phosphine oxide (TOPO) to achieve the highest
sensitivity.
[0011] 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.
[0012] On the other hand, the DELFIA system is associated with the
defect caused by a reaction between .beta.-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.
[0013] 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 .beta.-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 assay of a physiologically
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.
[0014] To resolve the above-mentioned defects of the DELFIA system,
a Cyber Fluor system that uses a complex of
4,7-bis-(chlorosulfophenyl)-1,1- 0-phenanthroline-2,9-dicarboxylic
acid (BCPDA) and europium is known (Anal. Chem., 61, 48-53,
1989).
[0015] The use of BCPDA has made a great advancement in that many
europium fluorescent complexes can be introduced without a
quenching phenomenon (quenching phenomenon strikingly decreases 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.
[0016] 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 particularly superior
property as compared to the above-mentioned two europium
fluorescent complexes.
[0017] Many studies have been made so far with respect to europium
fluorescent complex and it has been found that
.beta.-diketone-europium fluorescent complex has greater
fluorescence intensity than aromatic amine-europium complex, and of
the .beta.-diketone ligands, a europium fluorescent complex of
2-naphthoyltrifluoroacetone (.beta.-NTA) and
2-thenoyltrifluoroacetone (TTA) particurlaly has the greatest
fluorescence intensity.
[0018] The present inventors synthesized various
.beta.-diketonato-europiu- m TOPO complexes to study the effect of
.beta.-diketone as a substituent on the fluorescence property of
the .beta.-diketone-europium fluorescent complex, and found that
the fluorescence intensity of these complexes is dependent on the
composition and structure of the substituents R.sup.1 and R.sup.2
of the .beta.-diketonato (R.sup.1COCH.sub.2COR.sup.2). In other
words, when R.sup.1 is an aromatic hydrocarbon residue, stronger
electron attractiveness of R.sup.2 results in stronger fluorescence
intensity of the complex, based on which finding an immunoassay
utilizing a .beta.-diketone type europium fluorescent complex
having a dramatically improved fluorescence intensity has been
found (U.S. Pat. No. 5,859,297 and Anal. Chem., 70, 596-601,
1988).
[0019] However, the use of this .beta.-diketone type europium
fluorescent complex for the assay of a substance having various
actions that is on a bioloical tissue or cell, such as a
physiologically active substance, has not been disclosed. Many
difficulties are foreseeable in an assay on a tissue or cell of a
physiologically active substance in the biological sample, for
example, a great influence of contaminating substance, a difficult
high sensitivity assay, an unattainable easy assay and the
like.
[0020] It is therefore an object of the present invention to
provide a means of resolving defects such as hindrance of
fluorescence by a contaminating substance and low sensitivity, so
that a substance in a tissue or cell or on surface thereof, such as
nucleic acid, nucleic acid binding protein, receptor, sugar chain,
ganglioside and the like can be assayed as it is on the tissue or
cell with high precision and high sensitivity.
SUMMARY OF THE INVENTION
[0021] The present invention is based on the finding that a
.beta.-diketone form europium fluorescent complex has superior
characteristics as a label for probe for the high sensitivity assay
of a physiologically active substance such as nucleic acid, nucleic
acid binding protein, receptor, enzyme, sugar chain, ganglioside
and the like on a tissue or cell, since it has a noticeably long
fluorescence lifetime and permits time-resolved fluorescence assay,
assay upon elimination of blank fluorescence, use in one step and
has a, long wavelength fluorescence lifetime.
[0022] Accordingly, the present invention provides a method for
analyzing a biological substance comprising the use of a label
substance of the following formula (I): 2
[0023] wherein A.sup.1 is an aromatic group, R.sup.1 is a hydrogen
or --COCH.sub.2COC.sub.nF.sub.2n+1 and n is an integer of 1-6, or a
label substance of the following formula (II) 3
[0024] wherein A.sup.2 and A.sup.3 are the same or different and
each is an aromatic group, R.sup.2 and R.sup.3 are the same or
different and each is a hydrogen or COCH.sub.2COC.sub.nF.sub.2n+1
and n is an integer of 1-6, reagents therefor and a preparation
method thereof. More particularly, the present invention provides
the following.
[0025] (1) A method for analyzing an objective substance,
comprising reacting a labeled probe with an objective substance on
a biological sample, said probe comprising a label substance of the
formula (I) or a label substance of the formula (II) bonded to a
probe selected from the group consisting of nucleic acid, nucleic
acid binding protein, low molecular ligand and receptor for ligand
(except antibody) via a cross-linking group or a cross-linking
group and a conjugating group, adding a heavy metal ion and
assaying fluorescence of the resultant fluorescent complex.
[0026] (2) The method for analyzing an objective substance,
comprising adding a heavy metal ion to a labeled probe, said probe
comprising a label substance of the formula (I) or a label
substance of the formula (II) bonded to a probe selected from the
group consisting of nucleic acid, nucleic acid binding protein, low
molecular ligand and receptor for ligand (except antibody) via a
cross-linking group or a cross-linking group and a conjugating
group to give a fluorescent complex, reacting the complex with an
objective substance on a biological sample and assaying
fluorescence of the resultant fluorescent complex.
[0027] (3) A labeled nucleic acid probe comprising a label
substance of the formula (I) or a label substance of the formula
(II) bonded to a nucleic acid probe via a cross-linking group.
[0028] (4) A fluorescent complex comprising the labeled nucleic
acid probe of (3) and a heavy metal ion.
[0029] (5) A reagent for analyzing a nucleic acid, comprising the
labeled nucleic acid probe of (3).
[0030] (6) A labeled nucleotide comprising a label substance of the
formula (I) bonded to a nucleotide via a cross-linking group.
[0031] (7) A fluorescent complex comprising the labeled nucleotide
of (6) and a heavy metal ion.
[0032] (8) A method for producing a labeled nucleic acid probe
comprising reacting the labeled nucleotide of (6), dNTPs and a
single strand DNA in the presence of a DNA polymerase.
[0033] (9) A method for producing a labeled nucleic acid probe
comprising reacting the labeled nucleotide of (6), dNTPs and a
double stranded DNA in the presence of 5'-exonuclease, DNase and a
DNA polymerase.
[0034] (10) A labeled nucleic acid probe obtained by the production
method of (8) or (9).
[0035] (11) A reagent for analyzing nucleic acid comprising a label
substance of the formula (I) or the formula (II), to which avidin
is covalently bonded via a cross-linking group (hereinafter to be
referred to as label substance A) and a nucleotide to which biotin
is bonded via a linkage group (hereinafter to be referred to as
nucleotide B).
[0036] (12) A method for analyzing an objective substance
comprising reacting the objective substance with a nucleic acid
probe comprising the nucleotide B as a component on a biological
sample and then with the label substance A, adding a heavy metal
ion and assaying the fluorescence of the resultant fluorescent
complex.
[0037] According to the method of the present invention, defects
such as hindrance of fluorescence by a contaminating substance and
low sensitivity can be resolved in the analysis of nucleic acid,
nucleic acid binding protein, receptor, sugar chain, ganglioside
and the like on a biological tissue, cell or chromosome in a
biological sample. In particular, hindrance due to contamination
with a lanthanoide metal ion in a sample or environment can be
removed, and assay of the objective substance with high sensitivity
and with small steps.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The label substance in the present invention is represented
by the formula (I) or the formula (II). In the formulas, A.sup.1,
A.sup.2 and A.sup.3 are the same or different and each is a
trivalent aromatic group, particularly a conjugated double bond,
wherein when R.sup.1, R.sup.2 or R.sup.3 is hydrogen, A.sup.1,
A.sup.2 or A.sup.3 it binds with is a divalent aromatic group. Such
divalent or trivalent aromatic group is exemplified by 4
[0039] and the like. Those having a substituent to these aromatic
rings, such as methylphenylene and methyldibenzothiopehne, are also
exemplified.
[0040] Particularly preferable aromatic group is the following:
5
[0041] R.sup.1, R.sup.2 and R.sup.3 are each independently hydrogen
or COCH.sub.2COC.sub.nF.sub.2n+.
[0042] In the formulas (I), (II) and (III), n and n at R.sup.1,
R.sup.2 and R.sup.3 are an integer of 1-6, preferably 2-4.
[0043] In the present invention, a particularly preferable label
substance is represent ed by the formula (III): 6
[0044] wherein n is an integer of 1-6.
[0045] In the present invention, the probe is selected from the
group consisting of nucleic acid, nucleic acid binding protein,
ligand and receptor for ligand (except antibody).
[0046] In the present invention, the objective substance is a
component in the biological sample, which is to be the subject of
the analysis. Preferable examples thereof include nucleic acid,
nucleic acid binding protein, ligand, receptor for ligand and the
like.
[0047] The nucleic acid binding protein is a protein that
specifically binds with the nucleic acid having a specific
nucleotide sequence, such as histone, DNA binding protein, Lac I
protein and the like. By the use of a transcription regulating
factor of cytokine (a kind of DNA binding proteins), such as
NF-.kappa.B and the like, as a probe to be labeled, the interaction
between the transcription factor and DNA can be visualized.
[0048] The low molecular ligand here means an organic compound such
as sugar chain, aromatic compound, ganglioside, oligosaccharide,
peptide consisting of 2-10 amino acids and the like. Examples
thereof include myc peptide, thyroxine, triiodothyronine,
ganglioside GM2, cellobiose, sugar chain having a sialic acid at
the end thereof and the like.
[0049] The receptor for ligand means a substance that specifically
binds with a specific ligand that is located on or in a cell or
between cells, such as cellulose binding protein, sialic acid
binding lectin, albumin receptor and the like.
[0050] Further examples of the low molecular ligand or receptor
include hormone or hormone receptor such as insulin, insulin
receptor, EGF, EGF receptor, HGF, HGF receptor, TSH, TSH receptor
and the like, and receptors of low molecular ligands such as
receptor of cytokine (e.g., IL-8 and the like) or chemokine,
acetylcholine receptor, histamine receptor and the like.
[0051] The protein kinase C can bind with the derivative of phorbol
ester and can be assayed by the method of the present invention. In
addition, the enzymes such as cAMP-dependent protein kinase,
cGMP-dependent protein kinase, calmodulin-dependent phosphoenzyme,
tyrosine-phosphorylated enzyme and the like can be assayed by way
of ligand-receptor reaction, wherein the labeled probe of the
present invention can be used as the probe for the substrate
binding site of the enzyme.
[0052] Various lectins against various sugar chain and ganglioside
can be used as the probe of the present invention. Examples of
lectin include concanavalin A against D-mannose bonded with various
proteins on the cell, wheat germ agglutinin against
di-N-acetylchitobiose, sialic acid binding lectin against sialic
acid, which is derived from Limulus polyphemus and the like.
[0053] Examples of nucleic acid and nucleic acid probe include DNAs
having a series of various deoxyribonucleic acids (dATP, dGTP,
dTTP, dCTP, dUTP) and RNAs having a series of various ribonucleic
acids (rATP, rGTP, rTTP, rCTP, rUTP). These nucleic acid probe has
a nucleotide sequence consists of cDNA or antisense
oligonucleotides that specifically hybridizes with mRNA which
expresses in the cell. Alternatively, a nucleic acid probe having a
nucleotide sequence complementary to a part of a specific sequence
of nucleic acid or chromosome in the cell can be used.
[0054] Examples of the nucleic acid or gene on chromosome in the
cell include oncogene (e.g., abl, erb, fos, myb, myc, ras, src and
the like), tumor suppressor gene (e.g., p53 and the like),
rearranged T cell receptor gene, rearranged immunoglobulin gene, a
part of the nucleotide sequence of pathogenic virus gene such as
Epstein-Bar virus (EBV), herpes simplex virus (HSV),
cytomegalovirus (CMV), hepatitis B virus (HBV), rotavirus,
adenovirus and the like, a part of the nucleotide sequence of
infectious pathogenic microorganism gene such as malaria protozoa,
fungus, mycoplasma and the like, and nucleic acid having a
nucleotide sequence complementary thereto.
[0055] A part or the whole of the nucleic acid probe complementary
to these genes or homologous therewith may have a modified group
such as methyl group and the like as long as it does not affect
bonding with a label substance.
[0056] The labeled nucleic acid probe of the present invention is a
compound having affinity for a specific substance particularly on
the tissue or cell, such as nucleic acid, nucleic acid binding
protein and the like containing the above-mentioned genes on
chromosome and the like.
[0057] The labeled probe in the present invention consists of a
probe selected from the group consisting of nucleic acid, nucleic
acid binding protein, low molecular ligand and receptor for ligand
(except antibody) and a label substance bonded thereto. The labeled
nucleic acid probe of the present invention consists of a nucleic
acid and a label substance bonded to each other. The labeled
nucleotide of the present invention consists of a nucleotide and a
label substance bonded thereto. The bond between the label
substance and the probe or nucleotide is a bond via a cross-linking
group. It may be a covalent bond via a conjugating group.
[0058] The cross-linking group is via a bond between a label
substance and a conjugating group, probe, nucleotide or avidin.
That is, in a labeled probe and a labeled nucleotide having a
conjugating group, the conjugating group exists between the
cross-linking group and the probe or nucleotide.
[0059] The label substance A in the present invention consists of
avidin and a label substance bonded via a cross-linking group. The
binding ratio of avidin-label substance is 1-50, preferably 2-30.
The nucleotide B in the present invention consists of biotin and
nucleotide bonded via a linkage group.
[0060] Avidin in the present invention is a glycoprotein that is
contained in the egg white and specifically binds with biotin.
Avidin may be a streptoavidin derived from a microorganism (genus
Streptococcus) or a recombinant protein thereof.
[0061] Biotin in the present invention is a substance called
vitamin H and coenzyme R and binds extremely firmly with avidin or
streptoavidin, wherein the bonding strength is far greater than the
bond of typical immunoconjugate.
[0062] The cross-linking group in the present invention is derived
from a group capable of bonding with both nucleic acid, nucleic
acid binding protein, low molecular ligand, receptor for ligand,
nucleotide or avidin and aromatic group. Alternatively, it is
derived from a group capable of bonding with both linkage group and
aromatic group.
[0063] Examples of the cross-linking group include --NH--CS--,
--NH--CO--, --CO--, --N.sub.2--, --NH--, --SO.sub.2--,
--CH.sub.2--S--, --CH.sub.2--NH--,
--(CH.sub.2).sub.6--NH--CO--CH.sub.2--CH.sub.2--CO--, and S--S- and
the like. Particularly preferable cross-lining groups are sulfonyl
group and carbonyl group.
[0064] The linkage group in the present invention is free of
particular limitation as long as it connects a cross-linking group
and a nucleic acid, nucleic acid binding protein, low molecular
ligand, receptor for ligand or nucleotide. Preferable linkage group
is a divalent aliphatic hydrocarbon group having 5-25 carbon atoms
and 7 or less amide bonds between carbons. Specific examples
include a group of the formula (IV): 7
[0065] wherein a is an integer of 0-6 and b is 0 or 1.
[0066] Another preferable mode of the linkage group is a linkage
group containing biotin and avidin through affinity binding.
[0067] Biotin affinity binding with avidin is preferably further
bonded to a probe via a linkage group. Examples of preferable
linkage group include divalent aliphatic hydrocarbon group having 5
to 25 carbon atoms and optionally having 7 or less amide bonds
between carbons. Specifically, it is
--CH.dbd.CH--CO--NH--CH.sub.2--CH.sub.2--NH--(CO--CH.sub.2--CH.sub.2---
CH.sub.2--CH.sub.2--CH.sub.2--NH).sub.2--, wherein preferable bond
is
(probe)--CH.dbd.CH--CO--NH--CH.sub.2--CH.sub.2--NH--(CO--CH.sub.2--CH.sub-
.2--CH.sub.2--CH.sub.2--CH.sub.2--NH).sub.2-biotin:avidin-(cross-linking
group-label substance).
[0068] The linkage group binding biotin and nucleotide in
nucleotide B is free of limitation as long as it binds biotin and
nucleotide. Preferable linkage group include a divalent aliphatic
hydrocarbon group having 5 to 25 carbon atoms and optionally having
7 or less amide bonds between carbons. Specifically, it is
--CH.dbd.CH--CO--NH--CH.sub.2--CH.sub.2--NH--
-(CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH).sub.2--,
wherein preferable bond is
(nucleotide)--CH.dbd.CH--CO--NH--CH.sub.2--CH.-
sub.2--NH--(CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH).sub.-
2-(biotin).
[0069] When the label substance of the formula (II) is bonded to
probe or avidin via two cross-linking groups, the two cross-linking
groups may be the same or different. Again, when it is bonded to
probe or avidin via two conjugating groups, the two conjugating
group may be the same or different.
[0070] The label substance of the formula (II) can be used upon
binding to two probes or avidin. The two probes may be the same or
different two probes or avidin. By binding to two probes, a
synergistic binding effect can be expected.
[0071] For example, one example of the labeled nucleic acid probe
of the present invention is a label substance bonded to different
nucleic acid probes. These probes are nucleic acid probes having
nucleotide sequences complementary to or homologous with the same
or different genes. In other words, a probe having plural nucleic
acids recognizing the specific sites of the assay target binds with
the nucleic acid in the cell having nucleotide sequences
complementary hereto or nucleotide sequences homologous thereto and
becomes a so-called divalent probe by forming a fluorescent complex
upon addition of a heavy metal ion (e.g., lanthanoide metal ion),
thereby affording a possible synergistic effect.
[0072] Even when the two binding probes are the same, each can bind
with an objective substance having same plural specific sites.
Thus, the effect is not a simple addition but expected to be
synergistic.
[0073] In the analysis method of the present invention, different
kinds of labeled probes may be used simultaneously upon mixing.
[0074] While the binding ratio of the probe-label substance is free
of particular limitation, it is generally 1-100, preferably
1-20.
[0075] The binding ratio of the nucleotide-label substance is free
of particular limitation, it is generally 1-50, preferably
1-20.
[0076] The binding ratio of the avidin-label substance is free of
particular limitation, it is generally 1-50, preferably 2-30.
[0077] The labeled probe, labeled nucleotide or label substance A
can be produced by the use of the following functional groups as
long as it does not exert an adverse influence, for binding a label
substance to the probe, nucleotide or avidin. For example, various
binding groups such as isothiocyanate group reactive with amino
group, sulfonyl halide group (sulfonyl chloride group, sulfonyl
fluoride group and the like), o-phthalaldehyde group in the
presence of 2-mercaptoethanol, N-substituted maleimide group and
the like for carbodiimide group and thiol group, iodoacetamide
group for histidine and the like.
[0078] Specifically, a ligand, nucleic acid and the like and a
labeling compound of the following formula in a molar amount of
1-20 per mol of ligand, nucleic acid and the like are reacted in a
solvent. 8
[0079] The present invention also relates to a fluorescent complex
containing a labeled nucleic acid probe and a heavy metal ion.
Examples of the heavy metal ion include lanthanoide metal ion and
radium ion, with preference given to lanthanoide metal ion. The
lanthanoide metal ion to be used in the present invention includes
ions of europium (Eu), samarium (Sm), terbium (Tb), dysprosium (Dy)
and the like. It is typically used in the form of a chloride, but
may be used in the form of other salts as long as the assay is not
influenced. In the present invention, these lanthanoide metal ions
may be used alone or in combination.
[0080] The reaction between the labeled probe and the objective
substance in the present invention is the reactions between nucleic
acid and nucleic acid, nucleic acid and nucleic acid binding
protein, and ligand and receptor for ligand in a biological sample.
For facilitated reaction, the biological sample may be pre-treated.
For example, nucleic acid extraction by AGPC, protein dissociation
treatment with ethanol and the like can be applied.
[0081] The biological sample is preferably a cell, tissue or
chromosome.
[0082] The analysis method of the present invention analyzes the
objective substance in the cell and on the cell surface, wherein a
labeled probe is reacted with the objective substance at a tissue
section, on a cell surface, on a chromosome and the like, a heavy
metal ion such as lanthanoide metal ion, radium ion and the like is
added and fluorescence of the resultant complex is assayed, or a
heavy metal ion such as lanthanoide metal ion, radium ion and the
like is added to a labeled probe to give a fluorescent complex,
which is reacted with the objective substance on a biological
sample and fluorescence of the complex after reaction is
assayed.
[0083] The analysis method of the present invention may comprise
reacting a nucleic acid probe containing nucleotide B as a
component with the objective substance on a biological sample, then
reacting with label substance A, adding a heavy metal ion, and
assaying fluorescent of the resultant fluorescent complex.
[0084] The nucleic acid probe containing nucleotide B as a
component means that one or more nucleotides in the nucleotide
sequence is(are) nucleotide B. Namely, it is a nucleic acid probe
binding with biotin.
[0085] The nucleic acid probe containing nucleotide B as a
component can be obtained by reacting nucleotide B, dNTPs and
single strand DNA in the presence of a primer and a DNA polymerase
to give a double stranded DNA and denaturing the obtained DNA with
heat to give a single strand DNA.
[0086] The nucleic acid probe containing nucleotide B as a
component can be obtained by reacting nucleotide B, dNTPs and a
double stranded DNA in the presence of 5'-exonuclease, DNase and a
DNA polymerase to give a double stranded DNA and denaturing the
obtained DNA with heat to give a single strand DNA.
[0087] More specific analysis method is exemplified by the method
comprising immersing a biological sample in a buffer containing a
labeled probe, incubating the sample to allow reaction of the
objective substance and the labeled probe, washing off excess
labeled probe with the buffer, immersing the probe in a buffer
containing lanthanoide metal ion to form a complex and assaying the
fluorescence of the resultant complex.
[0088] In addition, a method is exemplified, which comprises
admixing buffer containing lanthanoide metal ion with a buffer
containing a labeled probe to form a complex, immersing a
biological sample in this mixture, incubating the sample to allow
reaction with the objective substance, washing off excess (labeled
probe:lanthanoide metal ion) complex and assaying the fluorescence
of the resultant complex on the biological sample.
[0089] As a different specific method, the following method is
exemplified. That is, a double stranded DNA having a sequence to be
the assay target, nucleotide B and dNTPs are reacted in the
presence of 5'-exonuclease, DNase and a DNA polymerase to give a
biotin-bound nucleic acid probe. A biological sample is immersed in
a buffer containing this biotin-bound nucleic acid probe and
incubated to allow reaction of the objective substance and the
biotin-bound nucleic acid probe, and excess biotin-bound nucleic
acid probe is washed off. Then, the sample is immersed in a buffer
containing the label substance A to bind biotin and avidin, and
excess label substance A is removed. Then, the sample is immersed
in a buffer containing lanthanoide metal ion to form a complex and
the fluorescence of the resultant complex is assayed.
[0090] By these methods, the presence of the objective substance in
a biological sample such as a tissue, cell, chromosome and the like
is visualized and analyzed for localization and concentration. In
addition, abnormalities with respect to the objective substance can
be analyzed.
[0091] The visualized image obtained by the use of the inventive
labeled probe can be retained through a fluorescence microscope,
confocal laser-scanning microscope and the like. The fluorescence
signal itself is assayable with a fluorescence assay device,
time-resolved fluorescence assay device and the like.
[0092] In particular, the inventive labeled nucleic acid probe is
reacted with a biological sample of a tissue, cell, chromosome and
the like and visualize the objective substance therein by colony
hybridization, fluorescence in situ hybridization (FISH) of tissue
and chromosome, nucleic acid sandwich hybridization, comparative
genome hybridization (CGH) and the like.
[0093] The present invention also relates to a labeled nucleotide.
The nucleotide of the present invention itself has affinity with a
specific substance on a tissue or cell. It may be used to produce a
labeled nucleic acid probe by binding with a different nucleotide
or by nick translation method from a double stranded DNA.
[0094] The nucleotide in the labeled nucleotide and nucleotide of
nucleotide B of the present invention is not particularly limited
and is exemplified by ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, dTTP,
dUTP and the like, with particular preference given to dUTP.
[0095] The particularly preferable labeled nucleotide has the
following formula (V): 9
[0096] wherein X is a conjugating group of the formula (IV) and Y
is a sulfonyl group or carbonyl group, R is a group of the formula
10
[0097] and p is 0 or 1.
[0098] The present invention further relates to a fluorescent
complex containing the above-mentioned labeled nucleotide and a
heavy metal ion. Examples of the heavy metal ion include the
above-mentioned lanthanoide metal ion and radium ion, with
preference given to the above-mentioned lanthanoide metal ion.
[0099] A labeled probe can be obtained by incorporating a labeled
nucleotide such as the labeled dUTP of the present invention and
the like, when synthesizing a fragmented probe DNA using DNA
extracted from the tissue or cell, particularly chromosomal DNA. To
be specific, labeled nucleotide, dNTPs and double stranded DNA is
reacted in the presence of 5'-exonuclease, DNase and a DNA
polymerase to give a labeled nucleic acid probe. Alternatively, a
labeled nucleotide, dNTPs and a single strand DNA are reacted in
the presence of a DNA polymerase to give a labeled nucleic acid
probe. Particularly preferably, labeled nucleotide of the present
invention, such as labeled dUTP and the like is incorporated by
nick translation method to give a DNA or a DNA fragment usable as a
labeled nucleic acid probe.
[0100] Moreover, labeled nucleic acid probe, labeled nucleotide or
nucleotide B of the present invention can be incorporated into DNA
or RNA by nucleic acid amplification by PCR (polymerase chain
reaction) method, LCR (ligase chain reaction) method, NASBA method
and the like. The obtained DNA or RNA can be used for the analysis
of the objective substance as a labeled nucleic acid probe or a
biotin-bound nucleic acid probe.
[0101] The nucleic acid probe obtained by incorporating the labeled
nucleotide or nucleotide B of the present invention, that comprises
a DNA complementary to the DNA of a tissue or cell can be
particularly suitably used for the analysis of abnormalities in the
chromosome of the objective tissue or cell.
[0102] The reagent for the analysis of nucleic acid of the present
invention contains the above-mentioned novel labeled nucleic acid
probe or labeled nucleotide. Preferably it contains a heavy metal
ion such as the above-mentioned lanthanoide metal ion, radium ion
and the like.
[0103] The reagent for the analysis of nucleic acid of the present
invention contains label substance A and nucleotide B. The reagent
containing label substance A and nucleotide B preferably further
contains dNTPs, primer, DNA polymerase and heavy metal. As a
different mode, a reagent containing label substance A and
nucleotide B preferably further contains dNTPs, 5'-exonuclease,
DNase, DNA polymerase and heavy metal.
[0104] The present invention is explained in detail by illustrative
reference examples and examples, to which the present invention is
not limited in any way.
REFERENCE EXAMPLE 1
Synthesis of 4,4'-diacetyl-o-terphenyl
[0105] To a solution of CH.sub.2Cl.sub.2 (200 ml), AlCl.sub.3 (210
mmol) and CH.sub.3COCl (205 mmol) was gradually added a solution of
CH.sub.2Cl.sub.2 (100 ml) and o-terphenyl (100 mmol) dropwise with
stirring at 0.degree. C. The mixture was stirred at 0.degree. C.
for 30 min and further stirred at room temperature for 24 hr. The
reaction solution was refluxed for 2 hr, and then poured into conc.
hydrochloric acid with ice. The mixture was sufficiently stirred,
and CH.sub.2Cl.sub.2 was distilled away under reduced pressure. The
precipitate was separated by filtration, and thoroughly washed with
water. The product was recrystallized from 2-butanone (about 250
ml) to give needle crystals, which were separated by filtration and
dried in vacuo. The yield was 22.1 g (70.3%). The results of
elemental analysis were as follows.
[0106] Element analysis: calculated: C %=84.05, H %=5.77 found: C
%-84.96, H %=5.87
REFERENCE EXAMPLE 2
Synthesis of intermediate of labeled compound
[0107] The intermediate having the following structure was
synthesized. 11
[0108] To anhydrous ether (Et.sub.2O, 30 g) were added NaOCH.sub.3
(3.0 g), 4,4'-diacetyl-o-terphenyl (10 mmol) and
C.sub.3F.sub.7COOC.sub.2H.sub- .5 (20 mmol), the mixture was
stirred in a sealed container at room temperature for 24 hr.
Anhydrous ether was distilled away to give a residue, which was
dried in vacuo for 30 min. The product was neutralized with 15%
sulfuric acid (100 ml), and the precipitate was separated by
filtration and washed well with water. The precipitate was
dissolved in ethanol (200 ml) under heating, and the insoluble
substance was removed by filtration. The solution was concentrated
to about 20 ml under reduced pressure. This solution was gradually
added dropwise to petroleum ether (200 ml) under stirring. The
mixture was sufficiently stirred and filtered to remove a small
amount of deposited precipitate. The filtrate was evaporated to
completely remove the organic solvents. The obtained oily substance
was dried in vacuo to give a yellow powder. The product was dried
in vacuo for 24 hr. The yield was 460 g (65.0%).
[0109] Elemental analysis: calculated; C %=51.00, H %=2.28 found: C
%=51.22, H %=2.61
[0110] .sup.1H-NMR confirmed that the product was the objective
compound.
REFERENCE EXAMPLE 3
Synthesis of labeled compound
[0111] The labeled compound having the following structure was
synthesized. 12
[0112] To chlorosulfuric acid (3.5 ml) was gradually added
.beta.-diketone (the intermediate obtained in Reference example 2,
2 mmol) under stirring at room temperature. The reaction mixture
was stirred at room temperature for 7 hr, then carefully added
dropwise to ice water (150 ml, outside cooled with ice water) under
stirring. The resultant precipitate was immediately centrifuged,
washed with cold water (about 5.degree. C.) and centrifuged twice.
The precipitate was suspended in a small amount of cold water and
transferred onto a glass filter, and water was removed by suction
filtration. The resultant chlorosulfonylated .beta.-diketone was
dried in vacuo at room temperature for 48 hr or more. The yield was
77%.
[0113] Elemental analysis: calculated: C %=44.76, H %=1.88 found: C
%=44.50, H %=1.92
[0114] .sup.1H-NMR confirmed that the product was the objective
compound.
EXAMPLE 1
Labeling of p53, Human, Probe (exon 4 translated) a labeled
compound
[0115] The labeled compound obtained in Reference Example 3 and
p53, Human, Probe (exon 4 translated) manufactured by Oncogene
Research Product (Cosmo Bio) were reacted in the following manner
to prepare a labeled DNA wherein said labeled compound is bound
with p53, Human, Probe mediated by a sulfonyl group.
[0116] One hundred pmol of p53, Human, Probe (exon 4 translated)
was dissolved in 0.1 mol/l carbonate buffer solution (pH=9.3, 100
.mu.l). To this DNA solution was gradually added a solution (10
.mu.l) of the labeled compound having a mole number equal to said
nucleic acid (having about 280 amino groups/molecule) dropwise
under stirring at room temperature. The mixture was stirred at room
temperature for 1 hr, extracted with phenol/chloroform, and
subjected to ethanol precipitation. The precipitate was washed with
80% ethanol and dried. The dried precipitate was re-dissolved in
0.05 mol/l carbonate buffer solution (pH=8.0, 100 .mu.l).
[0117] The molarity of the labeled compound contained in this
solution was calculated. In addition, the molar absorption
coefficient of the labeled compound at 330 nm was calculated from
the absorbance at 330 nm. As a result, the absorption coefficient
was 0.97 mol.sup.-1cm.sup.-1+. There was no absorption by DNA at
330 nm. Assuming that molar absorption coefficient does not change
during the process of labeling reaction, the concentration of the
label in the labeled DNA solution and the binding ratio of the
label and DNA were calculated.
[0118] The binding ratio of DNA to the labeled compound obtained by
the above method was about 1.
EXAMPLE 2
Hybridization of the labeled DNA with genes in a liver tissue
[0119] A liver tissue excised from human was fixed with 4%
paraformaldehyde-PBS at 4.degree. C. overnight, and dehydrated with
70%, 80%, 90% and 100% ethanol, successively. All water used in
this experiment was purified water treated with
diethylpyrocarbonate (DEPC). Further dehydration was performed by
exchanging the solution in the liver tissue twice with 100%
ethanol. Then, the liver tissue was transferred into xylene, heated
at 60.degree. C. for 2 hr (3 times), and embedded with
paraffin.
[0120] The section (about 5 .mu.m) of this paraffin-embedded tissue
was prepared using a microtome, placed on a thoroughly washed slide
glass, and dried at 37.degree. C. for 6 hr to give a section
preparation. The section preparation was further dried with a
drier, treated with xylene, 100% ethanol, 90% ethanol, 80% ethanol,
70% ethanol and phosphate buffer (PB, pH 7.4), successively, and
treated with proteinase K solution (10 mM Tris-HCl (pH 8.0), 1 mM
EDTA; 10 .mu.g/ml) for 20 min.
[0121] The section preparation was then immersed in 4%
paraformaldehyde-PB solution for 10 min, washed with PB, and
treated with 0.2N hydrochloric acid for 10 min, with PB for 1
minute, with 0.1 M triethanolamine hydrochloric acid (pH 8.0) for 1
minute, with 0.1 M triethanolamine hydrochloric acid (pH 8.0)
containing 0.25% acetic anhydride for 10 min, and with PB for 1
minute. The section preparation was further treated with 70%
ethanol, 80% ethanol, 90% ethanol and 100% ethanol, successively,
and air-dried to give an air-dried sample. The air-dried sample was
immediately subjected to the following hybridization.
[0122] As a hybridization solution, a solution comprising 50%
formamide, 10 mM Tris-HCl (pH 7.6), 10% dextran sulfate, 600 mM
NaCl, 0.25% SDS, 1 mM EDTA, 200 .mu.g/ml tRNA and
1.times.Denhardt's solution was prepared.
[0123] The labeled DNA obtained in the Example was dissolved in
this hybridization solution to the concentration of 5 ng/ml. The
mixture (about 40 ml) was dropped onto the above air-dried sample.
Prior to hybridization, the sample was prehybridized with the
hybridization buffer without the labeled DNA at 37.degree. C. for 2
hr. Then, the air-dried sample was covered with parafilm (CAN Co.),
and incubated at 37.degree. C. for 16 hr in a moisture chamber, in
which a paper towel moistened with 50% formamide solution was set,
for hybridization with the labeled DNA.
[0124] After hybridization, the parafilm was removed from the slide
glass in 5.times.SSC solution (40.degree. C.), and the slide glass
having the section hybridized with the labeled DNA was heated in
2.times.SSC, 50% formamide at 40.degree. C. for 30 min. The slide
glass was washed with TNE solution (aqueous solution containing
Tris-HCl buffer, NaCl, and EDTA), and reacted with 5 .mu.g/ml RNase
A in TNE solution for 10 min.
[0125] The slide glass was then washed with 2.times.SSC at
40.degree. C. for 20 min (once), and 0.2.times.SSC at 40.degree. C.
for 20 min (twice). Finally, the slide glass was immersed in
0.2.times.SSC solution (pH 8.5) containing 0.1 mM europium chloride
to give a fluorescent complex from the reaction of the labeled DNA
with europium chloride. The section on the slide glass was observed
with a fluorescence microscope.
[0126] As a result, signals derived from the fluorescent complex of
the labeled DNA and europium chloride were detected on the section.
When hybridization was performed using DNA labeled with fluorescein
isothiocyanate (FITC) in the place of the labeled DNA above as a
comparative example, hybridization signals were hardly detected. In
this experiment, europium chloride was not added to detect the
signals.
EXAMPLE 3
Preparation of a labeled PCR product
[0127] Two oligonucleotides (SEO ID: NO: 1 and SEQ ID: NO: 2),
having the nucleotide sequences homologous to the sense and
antisense sequence of hepatitis B virus surface antigen (HBsAg)
gene, respectively, were synthesized by phosphoamidite method using
an automatic DNA/RNA synthesizer. In the final step of the
synthesis, a labeled compound was reacted with the 5' termini of
the elongated oligonucleotides to give two kinds of labeled
oligonucleotides shown in the following formulas (1) and (2).
13
[0128] wherein Q.sup.1 is oligonucleotide (SEQ ID: NO: 1) in which
the amino group of the nucleotide at the 5' terminus binds to the
sulfonyl group of the labeled compound. 14
[0129] wherein Q.sup.2 is oligonucleotide (SEQ ID: NO: 2) in which
the amino group of the nucleotide at the 5' terminus binds to the
sulfonyl group of the labeled compound.
[0130] Using the above two labeled oligonucleotides as a pair of
primers and the nucleic acid fraction extracted with
phenol/chloroform from the serum derived from a patient, who was
strongly positive against HBsAg, as a template, PCR was carried out
under the following conditions.
[0131] A reaction mixture consisting of 10 mM Tris-HCl (pH 9.0), 50
mM KCl, 1.5 mM magnesium chloride, 0.1% Triton X-100, dNTPs (50
.mu.M each), 0.02 U/.mu.l Taq DNA polymerase, and primers (0.4 pM
each) was used for amplification.
[0132] After preheating at 95.degree. C. for 2 min, thermal
denaturation was performed at 95.degree. C. for 30 sec, annealing
was performed at 57.degree. C. for 30 sec, and elongation was
performed at 72.degree. C. for 80 sec. These steps were repeated 30
cycles to give a 600 bp DNA as a PCR product.
[0133] Addition of europium chloride to the DNA product resulted in
the generation of extremely strong fluorescence.
EXAMPLE 4
Analysis of liver tissue using labeled PCR product
[0134] A tissue section was prepared from the liver excised from a
human infected with hepatitis B according to the method described
in Example 2, except the use of a normal sterilized purified water
in the place of DEPC-treated water, to give an air-dried sample on
a slide glass.
[0135] A hybridization solution consisting of 50% formamide, 10 mM
Tris-HCl (pH 7.6), 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1
mM EDTA, 200 .mu.g/ml tRNA and 1.times.Denhardt's solution,was
prepared. Prior to hybridization, the air-dried sample was
prehybridized with this hybridization solution without PCR
amplification product for 2 hr.
[0136] The labeled PCR amplification product obtained in Example 3
was dissolved in this hybridization solution to the concentration
of 70 ng/ml in a DNA content. The mixture was preincubated at
85.degree. C. for 10 min, and diluted 10-fold with the
hybridization solution. This hybridization solution (about 40
.mu.l) was added dropwise onto said air-dried sample on the slide
glass. The slide glass was covered with a parafilm and heated at
95.degree. C. for 2 min on a hotplate.
[0137] This slide glass was incubated at 37.degree. C. for 16 hr in
a moisture chamber, in which a paper towel moistened with 50%
formamide solution was set, to allow the air-dried sample to
hybridize with the labeled PCR amplification product.
[0138] After hybridization, the parafilm was removed from the slide
glass in 5.times.SSC solution (40.degree. C.), and the slide glass
was heated in 2.times.SSC and 50% formamide at 40.degree. C. for 30
min. Then, the slide glass was washed with TNE solution, and
reacted with 5 .mu.g/ml RNase in TNE solution for 10 min. The slide
glass was washed successively with 2.times.SSC at 40.degree. C. for
20 min (once), and with 0.2.times.SSC at 40.degree. C. for 20 min
(twice). The slide glass was immersed in 0.2.times.SSC solution (pH
8.5) containing 0.1 mM europium chloride, and the tissue section on
the slide glass was observed with a fluorescence microscope.
[0139] As a result, a mosaic staining pattern was detected in liver
cells in the lobulus on the slide glass. As a comparative example,
hybridization was tried using FITC in the place of the labeled
compound mentioned above. However, the fluorescence of FITC was
significantly degraded when an FITC-labeled PCR product was
obtained, so that the subsequent hybridization step could not be
performed.
[0140] Thus, as an alternative, a DNA product was obtained by
amplification using unlabeled oligonucleotides having the
nucleotide sequences depicted in SEQ ID: NO: 1 and SEQ ID: NO: 2 as
a pair of primers and a nucleic acid fraction derived from an HBsAg
strongly positive patient-derived serum as a template. Said
amplification product was reacted with FITC to give a
fluorescence-labeled probe, which was subjected to hybridization,
wherein no europium chloride was added. Hybridization using said
FITC-labeled probe gave very weak signals, which showed that the
FITC-labeled prove was obviously inferior to the labeled probe of
the present invention.
EXAMPLE 5
Analysis of human pancreas tissue using labeled human insulin
[0141] A labeled human insulin was prepared using a standard human
insulin (Sigma) in the same manner as in Example 1.
[0142] A pancreas tissue excised from a human was treated in the
same manner as in Example 2 to give a section. This section was
placed on a slide glass and immersed in 4% formamide solution at
room temperature for 10 min. To this section was added TBS solution
[Tris-NaCl buffer (pH 7.6), 50 .mu.l] containing 5% skim milk. The
section was heated at 37.degree. C. for 2 hr, and washed with TBS
solution (pH 7.6) 3 times.
[0143] As a hybridization solution, a solution of 1 mM EDTA and
0.2% BSA in TBS solution (pH 7.6) was prepared.
[0144] The labeled human insulin was dissolved in this
hybridization solution to the concentration of 10 ng/ml, and the
mixture (about 40 .mu.l) was dropped onto the pancreas tissue
section. The slide glass was covered with parafilm, and incubated
at 37.degree. C. for 8 hr in a moisture chamber in which a paper
towel moistened with 50% formamide solution was set, to allow the
section to hybridize with the labeled probe.
[0145] After hybridization, the parafilm was removed from the slide
glass in 5.times.SSC solution (40.degree. C.), and the slide glass
was heated in 2.times.SSC and 50% formamide at 40.degree. C. for 30
min. Then, the slide glass was washed with TNE solution, and
reacted with 5 .mu.g/ml RNase in TNE solution for 10 min. The slide
glass was washed successively with 2.times.SSC at 40.degree. C. for
20 min (once), and with 0.2.times.SSC at 40.degree. C. for 20 min
(twice). The slide glass was immersed in 0.2.times.SSC solution (pH
8.5) containing 0.1 mM europium chloride, and the tissue section on
the slide glass was observed with a fluorescence microscope.
[0146] As a result, signals derived from the fluorescent complex of
the labeled human insulin and europium chloride were detected on
the tissue section.
[0147] When hybridization was performed using an anti-human insulin
receptor antibody (Austral Biologicals (ABI)) labeled with
rhodamine in the place of the labeled human insulin mentioned above
as a comparative example, almost the same level of fluorescence
image was obtained.
[0148] Accordingly, it was concluded that the labeled human insulin
of the invention specifically reacted with a human insulin
receptor.
EXAMPLE 6
Analysis of chromosome preparation derived from peripheral blood
cell using denatured DNA probe
[0149] (Culture of Peripheral Lymphocyte)
[0150] Sterilely obtained peripheral blood supplemented with
heparin (1 ml) and RPMI1640 medium (GIBCO BRL, 9 ml) supplemented
with 15% fetal calf serum were mixed, and transferred into a
culture flask. Phytohemagglutinin (Welcome) was added to the final
concentration of 10 g/ml, and the peripheral blood was cultured in
a CO.sub.2 incubator with 5% CO.sub.2 atmosphere at 37.degree. C.
After 48 hrs of culture, thymidine (Sigma) was added to the final
concentration of 300 .mu.g/ml, and the culture was continued. At 63
hr after the start of the culture, peripheral blood cells
comprising lymphocytes were transferred to a new tube, and
centrifuged at 1,200 rpm for 5 min. The cells were rinsed by adding
RPMI1640 medium (10 ml) to the tube and gently stirring. Said
rinsing step was repeated once. RPMI1640 medium supplemented with
15% fetal calf serum (10 ml) and the peripheral blood cells
comprising lymphocytes were mixed, and cultured. At 63.5 hr after
the start of the culture, bromodeoxyuridine (Sigma) was added to
the final concentration of 50 ng/ml, the mixture was stirred, and
the culture was continued. At 70 hr after the start of the culture,
the peripheral blood cells comprising lymphocytes were harvested by
centrifugation at 1,200 rpm.
[0151] (Preparation of Chromosome)
[0152] To the harvested peripheral blood cells was added 0.075 M
KCl (10 ml), and the suspension was stood at room temperature for
30 min. After hypotonization, the suspension was centrifuged at
1,200 rpm for 5 min. The supernatant (about 3 ml) and the
precipitate were sufficiently stirred using a Pasteur pipette, and
gradually dropped into methanol: acetic acid (3:1, carnoy solution,
10 ml). The mixture was sufficiently stirred, stood for 10 min and
centrifuged at 1,200 rpm for 5 min. The supernatant was removed. To
the precipitate was added a fresh carnoy solution (10 ml), and the
both were mixed with stirring. This step of washing with carnoy
solution was repeated twice. The concentration of the suspension
was adjusted with caroy solution. The suspension was dropped onto
the center of a slide glass, which was followed by steam fixation
using a pot containing boiled water. The fixed sample on the slide
glass was dried at 37.degree. C. overnight, which was followed by
adhesion in a dry heat sterilizer at 65.degree. C. for 4 hr to give
a chromosome preparation. This chromosome preparation was stained
with 2.times.SSC containing 1 .mu.g/ml fluorescent dye, Hoechst
33258 (Molecular probe) for 5 min, gently rinsed with 2.times.SSC
and covered with a cover glass, which was followed by standing on a
hot plate (75.degree. C.) for 3 min. The preparation was exposed to
UV light on the hot plate at a distance of 1 cm from the
preparation with a black light (20 W, Toshiba). The cover glass on
the slide glass was removed and the slide glass was rinsed twice
with distilled water, dried and stored at -20.degree. C. with the
chromosome preparation carried thereon.
[0153] (Preparation of Labeled dUTP)
[0154] A labeled dUTP was prepared using dUTP (deoxy UTP, Toyo
Boseki) in the same manner as in Example 1.
[0155] This labeled dUTP and KRAS ONCOGENE (Lab Logics, large
probe) were subjected to nick translation method to give a labeled
nucleic acid.
[0156] The nick translation followed the protocol using a nick
translation kit manufactured by Boehringer. The labeled dUTP was
used at a final concentration of 0.05 mM.
[0157] After the nick translation, 4 M ammonium acetate (2.5
.mu.l), 10 mg/ml salmon sperm DNA (Sigma, 2.0 .mu.l), 10 mg/ml E.
coli tRNA (Sigma, 2.0 .mu.l) and special grade ethanol (75 .mu.l)
were added to the nick translation reaction mixture (20 .mu.l),
admixed well, stored at -80.degree. C. for 1 hr, and centrifuged at
15,000 rpm to give precipitate, which was stirred in special grade
formamide to dissolution.
[0158] (Hybridization)
[0159] To the labeled nucleic acid (5 .mu.l) prepared according to
the above-mentioned method was added 10 mg/ml Cot-1 DNA
(manufactured by Vysis, 5 .mu.l) and incubated in a heat block at
70.degree. C. for 10 min to denature the labeled probe to give a
denatured DNA probe, which was quickly cooled in ice water.
[0160] Then, the above-mentioned slide glass carrying the
chromosome preparation was immersed in a coupling jar filled with
70% formamide (2.times.SSC) at 70.degree. C. to denature the sample
with heat. The sample was immediately moved into 70% ethanol at
-20.degree. C., rapidly cooled for 2 min, dehydrated with 100%
ethanol and dried. The above-mentioned denatured DNA probe was
mixed in a solution having a final concentration of 50% formamide
and 10% dextran sulfate (in 2.times.SSC) and placed on the
above-mentioned chromosome preparation. The denatured DNA probe
solution was uniformly spread thereon using a parafilm strip to
prevent inclusion of air foams. The chromosome sample on the slide
glass and the denatured DNA probe were hybridized in a sealed
moistened chamber containing a filter paper impregnated with
2.times.SSC on the bottom therein at 37.degree. C. for 18 hr.
[0161] The parafilm was stripped off the slide glass and the slide
glass was immersed in a coupling jar filled with 50% formamide (in
2.times.SSC) at 37.degree. C. and rinsed for 15 min. This slide
glass was stood still in 2.times.SSC (room temperature) for 1 min
and then stood still for 15 min in 1.times.SSC (room temperature)
and 5 min in 4.times.SSC (room temperature). Then, 2.times.SSC (pH
8.5) containing 0.1 mM europium chloride was dropped on a slide
glass and the tissue section strip on the slide glass was observed
with a fluorescence microscope.
[0162] As a result, the 11th chromosome was found to have
fluorescence, which coincided with the localization of nucleic acid
containing KRAS ONCOGENE, thereby confirming possible specific
detection.
EXAMPLE 7
[0163] (Preparation of Fluorescence-labeled Streptoavidin)
[0164] Recombinant streptoavidin (24 mg, Boehringer Mannheim) was
dissolved in 100 mM carbonate buffer (pH 9.3, 2 ml) and dialyzed
against the same buffer. From the absorbance of dialysis solution
at 280 nm, it was confirmed to have a 3.4 mg/ml protein
concentration. To the entire amount of 2 ml thereof was dropwise
added an anhydrous DMF solution (0.4 ml) containing the labeled
compound (7.4 mg) of Reference example 3 and the mixture was
stirred at 25.degree. C. for 1 hr. After stirring, the reaction
mixture was eluted with 50 mM ammonium carbonate using Sephadex
G-50 column (about 30 ml bed) to separate a non-reacted labeled
compound. From the absorption coefficient 3.41.times.10.sup.4
(cm.sup.-1M.sup.-1) at 330 nm of the protein fraction and the
molecular weight of recombinant streptoavidin of about 52,000, the
cross-linked labeled compound was calculated to be about 20
molecules per 1 molecule of streptoavidin. Sodium azide was added
to the protein fraction to the concentration of 0.1%, adjusted to
pH 6.5 with 1N HCl and stored at 4.degree. C.
[0165] (Preparation of DNA Probe Using Nick Translation Kit)
[0166] Using the KRAS ONCOGENE (Lab Logics, large probe) used in
Example 6 and biotin-21-dUTP nick translation kit (Clontech), and
following the protocol attached to the kit, biotin-21-dUTP was
incorporated into the nucleic acid KRAS ONCOGENE to give a
biotinylated KRAS ONCOGENE DNA probe. The biotin-21-dUTP has the
following structure including a linkage group. 15
[0167] wherein R has the formula 16
[0168] To the reaction mixture (20 .mu.l) after nick translation
reaction were added 4 M ammonium acetate (2.5 .mu.l), 10 mg/ml
salmon sperm DNA (Sigma, 2.0 .mu.l), 10 mg/ml E. coli tRNA (Sigama,
2.0 .mu.l) and special grade ethanol (75 .mu.l) and admixed well.
After storing at -80.degree. C. for 1 hr, it was centrifuged at
15,000 rpm and the precipitate was thoroughly stirred in special
grade formamide to dissolution.
[0169] (Hybridization)
[0170] Completely in the same manner as in Example 6, a denatured
DNA probe was prepared and hybridized with a chromosome
preparation.
[0171] After hybridization, a parafilm was stripped off the slide
glass and the slide glass was immersed in a coupling jar filled
with 50% formamide (in 2.times.SSC) at 37.degree. C. and rinsed for
15 min. This slide glass was stood still in 2.times.SSC (room
temperature) for 1 min and then stood still for 15 min in
1.times.SSC (room temperature) and 5 min in 4.times.SSC (room
temperature). The labeled streptoavidin prepared above was diluted
with 2.times.SSC to the concentration of 0.02 mg/ml. The slide
glass was left standing still in the solution at room temperature
for 15 min. Using 1.times.SSC (room temperature), the slide glass
was left standing still for 5 min, which step was repeated three
times. Then, 2.times.SSC (pH 8.5) containing 0.1 mM europium
chloride was dropped on a slide glass and the tissue section strip
on the slide glass was observed with a fluorescence microscope.
[0172] As a result, the 11th chromosome was found to have
fluorescence like Example 6, but apparently had stronger
fluorescence. This fluorescence intensity was considered to reflect
the high incorporation rate of biotinylated-21-UTP into the nucleic
acid and the great number of the labeled compounds bonded to
streptoavidin. The synchronized localization with KRAS ONCOGENE was
confirmed and the method was concluded to be a specific detection
method.
[0173] This application is based on patent application Nos.
119768/1998 and 184852/1998 filed in Japan, the contents of which
are hereby incorporated by reference.
Sequence CWU 1
1
2 1 27 DNA Artificial Sequence Oligonucleotide designed to act as
sense primer for amplifying HBsAg gene. 1 catggagaac atcacacatc
aggattc 27 2 24 DNA Artificial Sequence Oligonucleotide designed to
act as antisense primer for amplifying HBsAg gene. 2 aatgtatacc
cagagacaaa acaa 24
* * * * *