
* feat: add PATENT_USPTO as input format Signed-off-by: Cesar Berrospi Ramis <ceb@zurich.ibm.com> * feat: add USPTO backend parser Add a backend implementation to parse patent applications and grants from the United States Patent Office (USPTO). Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com> * refactor: change the name of the USPTO input format Change the name of the patent USPTO input format to show the typical format (XML). Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com> * refactor: address several input formats with same mime type Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com> * refactor: group XML backend parsers in a subfolder Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com> * chore: add safe initialization of PatentUsptoDocumentBackend Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com> --------- Signed-off-by: Cesar Berrospi Ramis <ceb@zurich.ibm.com> Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com>
1381 lines
74 KiB
Plaintext
Vendored
1381 lines
74 KiB
Plaintext
Vendored
PATN
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WKU 057006474
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SRC 8
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APN 5686806
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APT 1
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ART 189
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APD 19951207
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TTL Carbocation containing cyanine-type dye
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ISD 19971223
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NCL 20
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ECL 1
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EXP Houtteman; Scott W.
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NDR 1
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NFG 1
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INVT
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NAM Miyazaki; Takeshi
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CTY Ebina
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CNT JPX
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INVT
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NAM Tanaka; Kazumi
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CTY Yokohama
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CNT JPX
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INVT
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NAM Santo; Tsuyoshi
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CTY Yokohama
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CNT JPX
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INVT
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NAM Ohnishi; Toshikazu
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CTY Machida
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CNT JPX
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INVT
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NAM Fukui; Tetsuro
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CTY Kawasaki
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CNT JPX
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INVT
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NAM Okamoto; Tadashi
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CTY Yokohama
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CNT JPX
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ASSG
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NAM Canon Kabushiki Kaisha
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CTY Tokyo
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CNT JPX
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COD 03
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PRIR
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CNT JPX
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APD 19910621
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APN 3-150428
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PRIR
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CNT JPX
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APD 19911028
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APN 3-281645
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PRIR
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CNT JPX
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APD 19920610
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APN 4-150665
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RLAP
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COD 74
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APN 900302
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APD 19920618
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PSC 01
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PNO 5512446
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CLAS
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OCL 435 6
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XCL 436139
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XCL 430 93
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XCL 585 16
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XCL 585406
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XCL 260350
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EDF 6
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ICL C12Q 168
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FSC 435
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FSS 6
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FSC 436
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FSS 139;63;56
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FSC 430
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FSS 93
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FSC 585
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FSS 16;406
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FSC 260
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FSS 350
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UREF
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PNO 3770383
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ISD 19731100
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NAM Price
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OCL 436509
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UREF
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PNO 3789116
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ISD 19740100
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NAM Kay
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OCL 436800
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UREF
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PNO 4738908
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ISD 19880400
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NAM Oguchi et al.
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OCL 430 20
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UREF
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PNO 5112960
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ISD 19920500
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NAM Bronstein et al.
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OCL 536 18.1
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FREF
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PNO 2191674
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ISD 19900700
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CNT JPX
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OREF
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PAL K. Sauda et al., "Determination of Protein in Human Serum by
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High-Performance Liquid Chromatography with Semiconductor Laser
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Fluorometric Detection," Analytical Chemistry, vol. 58, No. 13, Nov. 1986,
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pp. 2649-2653.
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PAL Derwent Abstract Accession No. 91-068399/10 (1991).
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PAL Smith, et al., Nature, vol. 321 (1986), pp. 674-679.
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PAL Wingrove, Organic Chemistry, pub. by Harper & Row, New York, pp. 163-166,
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1981.
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PAL Mujumdar et al., Cytometry 10:11-19, 1989.
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LREP
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FRM Fitzpatrick, Cella, Harper & Scinto
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ABST
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PAL To provide a reagent with excellent stability under storage, which can
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detect a subject compound to be measured with higher specificity and
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sensitibity.
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PAL Complexes of a compound represented by the general formula (IV):
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##STR1##
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PARN
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PAR This application is a division of application Ser. No. 07/900,302 filed
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Jun. 18, 1992, now U.S. Pat. No. 5,512,446.
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BSUM
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PAC BACKGROUND OF THE INVENTION
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PAR 1. Field of the Invention
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PAR The present invention relates to a labeled complex for microassay using
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near-infrared radiation. More specifically, the present invention relates
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to a labeled complex capable of specifically detecting a certain
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particular component in a complex mixture with a higher sensitivity.
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PAR 2. Related Background Art
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PAR On irradiating a laser beam on a trace substance labeled with dyes and the
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like, information due to the substance is generated such as scattered
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light, absorption light, fluorescent light and furthermore light
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acoustics. It is widely known in the field of analysis using lasers, to
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detect such information so as to practice microassays rapidly with a
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higher precision.
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PAR A gas laser represented by an argon laser and a helium laser has
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conventionally been used exclusively as a laser source. In recent years,
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however, a semi-conductor laser has been developed, and based on the
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characteristic features thereof such as inexpensive cost, small scale and
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easy output control, it is now desired to use the semiconductor laser as a
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light source.
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PAR If diagnostically useful substances from living organisms are assayed by
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means of the wave-length in ultraviolet and visible regions as has
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conventionally been used, the background (blank) via the intrinsic
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fluorescence of naturally occurring products, such as flavin, pyridine
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coenzyme and serum proteins, which are generally contained in samples, is
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likely to increase. Only if a light source in a near-infrared region can
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be used, such background from naturally occurring products can be
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eliminated so that the sensitivity to substances to be measured might be
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enhanced, consequently.
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PAR However, the oscillation wavelength of a semiconductor laser is generally
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in red and near-infrared regions (670 to 830 nm), where not too many dyes
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generate fluorescence via absorption or excitation. A representative
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example of such dyes is polymethine-type dye having a longer conjugated
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chain. Examples of labeling substances from living organisms with a
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polymethine-type dye and using the labeled substances for microanalysis
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are reported by K. Sauda, T. Imasaka, et al. in the report in Anal. Chem.,
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58, 2649-2653 (1986), such that plasma protein is labeled with a cyanine
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dye having a sulfonate group (for example, Indocyanine Green) for the
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analysis by high-performance liquid chromatography.
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PAR Japanese Patent Application Laid-open No. 2-191674 discloses that various
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cyanine dyes having sulfonic acid groups or sulfonate groups are used for
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labeling substances from living organisms and for detecting the
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fluorescence.
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PAR However, these known cyanine dyes emitting fluorescence via absorption or
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excitation in the near-infrared region are generally not particularly
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stable under light or heat.
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PAR If the dyes are used as labeling agents and bonded to substances from
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living organisms such as antibodies for preparing complexes, the complexes
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are likely to be oxidized easily by environmental factors such as light,
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heat, moisture, atmospheric oxygen and the like or to be subjected to
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modification such as generating cross-links. Particularly in water, a
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modification such as hydrolysis is further accelerated, disadvantageously.
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Therefore, the practical use of these complexes as detecting reagents in
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carrying out the microassay of the components of living organisms has
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encountered difficulties because of their poor stability under storage.
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PAC SUMMARY OF THE INVENTION
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PAR The present inventors have made various investigations so as to solve the
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above problems, and have found that a dye of a particular structure, more
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specifically a particular polymethine dye, and among others, a dye having
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an azulene skelton, are extremely stable even after the immobilization
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thereof as a labeling agent onto substances from living organisms. Thus,
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the inventors have achieved the present invention. It is an object of the
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present invention to provide a labeled complex with excellent storage
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stability which can overcome the above problems.
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PAR According to an aspect of the present invention, there is provided a
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labeled complex for detecting a subject compound to be analyzed by means
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of optical means using near-infrared radiation which complex comprises a
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substance from a living organism and a labeling agent fixed onto the
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substance and is bonded to the subject compound to be analyzed, wherein
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the labeling agent comprises a compound represented by the general formula
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(I), (II) or (III):
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##STR2##
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wherein R.sub.1 through R.sub.7 are independently selected from the group
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consisting of hydrogen atom, halogen atom, alkyl group, aryl group,
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aralkyl group, sulfonate group, amino group, styryl group, nitro group,
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hydroxyl group, carboxyl group, cyano group, or arylazo group; R.sub.1
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through R.sub.7 may be bonded to each other to form a substituted or an
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unsubstituted condensed ring; R.sub.1 represents a divalent organic
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residue; and X.sub.1.sup..crclbar. represents an anion;
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##STR3##
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wherein R.sub.8 through R14 are independently selected from the group
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consisting of hydrogen atom, halogen atom, alkyl group, aryl group,
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aralkyl group, sulfonate group, amino group, styryl group, nitro group,
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hydroxyl group, carboxyl group, cyano group, or arylazo group; R.sub.8
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through R14 may be bonded to each other to form a substituted or an
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unsubstituted condensed ring; and R.sub.A represents a divalent organic
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residue;
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##STR4##
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wherein R.sub.15 through R.sub.21 are independently selected from the
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group consisting of hydrogen atom, halogen atom, alkyl group, aryl group,
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a substituted or an unsubstituted aralkyl group, a substituted or an
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unsubstituted amino group, a substituted or an unsubstituted styryl group,
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nitro group, sulfonate group, hydroxyl group, carboxyl group, cyano group,
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or arylazo group; R.sub.15 through R.sub.21 may or may not be bonded to
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each other to form a substituted or an unsubstituted condensed ring;
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R.sub.B represents a divalent organic residue; and X.sub.1.sup..crclbar.
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represents an anion.
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PAR According to another aspect of the present invention, there is provided a
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labeled complex for detecting a subject compound to be analyzed by means
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of optical means using near-infrared radiation which complex comprises a
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substance from a living organism and a labeling agent fixed onto the
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substance and is bonded to the subject compound to be analyzed, wherein
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the labeling agent comprises a compound represented by the general formula
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(IV):
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##STR5##
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wherein A, B, D and E are independently selected from the group consisting
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of hydrogen atom, a substituted or an unsubstituted alkyl group having two
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or more carbon atoms, alkenyl group, aralkyl group, aryl group, styryl
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group and heterocyclic group; r.sub.1 ' and r.sub.2 ' are individually
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selected from the group consisting of hydrogen atom, a substituted or an
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unsubstituted alkyl group, cyclic alkyl group, alkenyl group, aralkyl
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group and aryl group; k is 0 or 1; 1 is 0, 1 or 2; and
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X.sub.2.sup..crclbar. represents an anion.
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PAR According to another aspect of the present invention, there is provided a
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method of detecting a subject compound to be analyzed by means of optical
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means which method comprises using a labeled complex comprised of a
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substance from a living organism and a labeling agent fixed onto the
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substance and bonding the complex to the subject compound to be analyzed,
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wherein the labeling agent comprises a compound represented by the general
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formula (I), (II) or (III).
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PAR According to still another aspect of the present invention, there is
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provided a method of detecting a subject compound to be analyzed by means
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of optical means which method comprises using a labeled complex comprised
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of a substance from a living organism and a labeling agent fixed onto the
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substance and bonding the complex to the subject compound to be analyzed,
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wherein the labeling agent comprises a compound represented by the general
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formula (iv).
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DRWD
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PAC BRIEF DESCRIPTION OF THE DRAWINGS
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PAR FIG. 1 depicts one example of fluorescence emitting wave form of a labeling
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agent.
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DETD
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PAC DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
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PAR The present invention will now be explained in detail hereinbelow.
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PAR In accordance with the present invention, the compound of the general
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formula (I), (II) or (III) is employed as a labeling agent, wherein
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R.sub.1 to R.sub.21 individually represent hydrogen atom, halogen atom
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(chlorine atom, bromine atom, and iodine atom) or a monovalent organic
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residue, and other such functional groups described above. The monovalent
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organic residue can be selected from a wide variety of such residues.
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PAR The alkyl group is preferably in straight chain or branched chain, having a
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carbon number of 1 to 12, such as for example methyl group, ethyl group,
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n-propyl group, iso-propyl group, n-butyl group, sec-butyl group,
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iso-butyl group, t-butyl group, n-amyl group, t-amyl group, n-hexyl group,
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n-octyl group, t-octyl group and the like.
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PAR The aryl group preferably has a carbon number of 6 to 20, such as for
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example phenyl group, naphthyl group, methoxyphenyl group,
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diethylaminophenyl group, dimethylaminophenyl group and the like.
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PAR The substituted aralkyl group preferably has a carbon number of 7 to 19,
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such as for example carboxybenzyl group, sulfobenzyl group, hydroxybenzyl
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group and the like.
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PAR The unsubstituted aralkyl group preferably has a carbon number of 7 to 19,
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such as for example benzyl group, phenethyl group, .alpha.-naphthylmethyl
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group, .beta.-naphthylmethyl group and the like.
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PAR The substituted or unsubstituted amino group preferably has a carbon number
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of 10 or less, such as for example amino group, dimethylamino group,
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diethylamino group, dipropylamino group, acetylamino group, benzoylamino
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group and the like.
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PAR The substituted or unsubstituted styryl group preferably has a carbon
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number of 8 to 14, such as for example styryl group, dimethylaminostyryl
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group, diethylaminostyryl group, dipropylaminostyryl group, methoxystyryl
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group, ethoxystyryl group, methylstyryl group and the like.
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PAR The aryl azo group preferably has a carbon number of 6 to 14, such as for
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example phenylazo group, .alpha.-naphthylazo group, .beta.-naphthylazo
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group, dimethylaminophenylazo group, chlorophenylazo group, nitrophenylazo
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group, methoxyphenylazo group and the like.
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PAR Of the combinations of R.sub.1 and R.sub.2, R.sub.2 and R.sub.3, R.sub.3
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and R.sub.4, R.sub.4 and R.sub.5, R.sub.5 and R.sub.6, and R.sub.6 and
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R.sub.7 of the general formula (I), at least one combination may form a
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substituted or an unsubstituted condensed ring. The condensed ring may be
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five, six or seven membered, including aromatic ring (benzene,
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naphthalene, chlorobenzene, bromobenzene, methyl benzene, ethyl benzene,
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methoxybenzene, ethoxybenzene and the like); heterocyclic ring (furan
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ring, benzofuran ring, pyrrole ring, thiophene ring, pyridine ring,
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quinoline ring, thiazole ring and the like); and aliphatic ring
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(dimethylene, trimethylene, tetramethylene and the like). This is the case
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with the general formulas (II) and (III).
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PAR For the general formula (II), at least one combination among the
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combinations of R.sub.8 and R.sub.9, R.sub.9 and R.sub.10, R.sub.10 and
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R.sub.11, R.sub.11 and R.sub.12, R.sub.12 and R.sub.13, and R.sub.13 and
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R.sub.14, may form a substituted or an unsubstituted condensed ring.
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PAR Also for the general formula (III), at least one combination of the
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combinations of R.sub.15 and R.sub.16, R.sub.16 and R.sub.17, R.sub.17 and
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R.sub.18, R.sub.18 and R.sub.19, R.sub.19 and R.sub.20, and R.sub.20 and
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R.sub.21, may form a substituted or an unsubstituted condensed ring.
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PAR In the general formulas (I) to (IV) described above, the general formula
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(I) is specifically preferable; preference is also given individually to
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hydrogen atom, alkyl group and sulfonate group in the case of R.sub.1 to
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R.sub.7 ; hydrogen atom, alkyl group and sulfonate group in the case of
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R.sub.8 to R.sub.14 ; hydrogen atom, alkyl group and sulfonate group in
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the case of R.sub.15 to R.sub.21 ; alkyl group and aryl group in the case
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of A, B, D and E; hydrogen atom and alkyl group in the case Of r.sub.1 '
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to r.sub.2 '.
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PAR In the general formula (I), R represents a divalent organic residue bonded
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via a double bond. Specific examples of a compound containing such R to be
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used in the present invention, include those represented by the following
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general formulas (1) to (12), wherein Q.sup..sym. represents the following
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azulenium salt nucleus and the right side excluding Q.sup..sym.
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represents R.
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##STR6##
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wherein the relation between the azulenium salt nucleus represented by
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Q.sup..crclbar. and the azulene salt nucleus on the right side in the
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formula (3) may be symmetric or asymmetric.
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##STR7##
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In the above formulas (1) to (12) as in the case of R.sub.1 to R.sub.7,
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R.sub.1 ' to R.sub.7 ' and R.sub.1 " to R.sub.7 " independently represent
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hydrogen atom, halogen atom, alkyl group, aryl group, aralkyl group, amino
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group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano
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group or aryl azo group, while R.sub.1 ' to R.sub.7 ' and R.sub.1 " to
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R.sub.7 " independently may form a substituted or an unsubstituted
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condensed ring; n is 0, 1 or 2; r is an integer of 1 to 8; S represents 0
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or 1; and t represents 1 or 2.
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PAR M.sub.2 represents a non-metallic atom group required for the completion of
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a nitrogen-containing heterocyclic ring.
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PAR Specific examples of M.sub.2 are atom groups required for the completion of
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a nitrogen-containing heterocyclic ring, including pyridine, thiazole,
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benzothiazole, naphthothiazole, oxazole, benzoxazole, naphthoxazole,
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imidazole, benzimidazole, naphthoimidazole, 2-quinoline, 4-quinoline,
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isoquinoline or indole, and may be substituted by halogen atom (chlorine
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atom, bromine atom, iodine atom and the like), alkyl group (methyl, ethyl,
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propyl, butyl and the like), aryl group (phenyl, tolyl, xylyl and the
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like), and aralkyl (benzene, p-trimethyl, and the like).
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PAR R.sub.22 represents hydrogen atom, nitro group, sulfonate group, cyano
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group, alkyl group (methyl, ethyl, propyl, butyl and the like), or aryl
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group (phenyl, tolyl, xylyl and the like). R.sub.23 represents alkyl group
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(methyl, ethyl, propyl, butyl and the like), a substituted alkyl group
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(2-hydroxyethyl, 2-methoxyethyl, 2-ethoxyethyl, 3-hydroxypropyl,
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3-methoxypropyl, 3-ethoxypropyl, 3-chloropropyl, 3-bromopropyl,
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3-carboxylpropyl and the like ), a cyclic alkyl group (cyclohexyl,
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cyclopropyl), aryl aralkyl group (benzene, 2-phenylethyl, 3-phenylpropyl,
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3-phenylbutyl, 4-phenylbutyl, .alpha.-naphthylmethyl,
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.beta.-naphthylmethyl), a substituted aralkyl group (methylbenzyl,
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ethylbenzyl, dimethylbenzyl, trimethylbenzyl, chlorobenzyl, bromobenzyl
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and the like), aryl group (phenyl, tolyl, xylyl, .alpha.-naphtyl,
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.beta.-naphthyl) or a substituted aryl group (chlorophenyl,
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dichlorophenyl, trichlorophenyl, ethylphenyl, methoxydiphenyl,
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dimethoxyphenyl, aminophenyl, sulfonate phenyl, nitrophenyl, hydroxyphenyl
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and the like).
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PAR R.sub.24 represents a substituted or an unsubstituted aryl group or the
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cation group thereof, specifically including a substituted or an
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unsubstituted aryl group (phenyl, tolyl, xylyl, biphenyl, aminophenyl,
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.alpha.-naphthyl, .beta.-napthyl, anthranyl, pyrenyl, methoxyphenyl,
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dimethoxyphenyl, trimethoxyphenyl, ethoxyphenyl, diethoxyphenyl,
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chlorophenyl, dichlorophenyl, trichlorophenyl, bromophenyl, dibromophenyl,
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tribromophenyl, ethylphenyl, diethylphenyl, nitrophenyl, aminophenyl,
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dimethylaminophenyl, diethylaminophenyl, dibenzylaminophenyl,
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dipropylaminophenyl, morpholinophenyl, piperidinylphenyl,
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piperidinophenyl, diphenylaminophenyl, acetylaminophenyl,
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benzoylaminophenyl, acetylphenyl, benzoylphenyl, cyanophenyl, sulfonate
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phenyl, carboxylate phenyl and the like).
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PAR R.sub.25 represents a heterocyclic ring or the cation group thereof,
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specifically including a monovalent heterocyclic ring derived from cyclic
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rings, such as furan, thiophene, benzofuran, thionaphthene, dibenzofuran,
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carbazole, phenothiazine phenoxazine, pyridine and the like.
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PAR R.sub.26 represents hydrogen atom, alkyl group (methyl, ethyl, propyl,
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butyl and the like), or a substituted or an unsubstituted aryl group
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(phenyl, tolyl, xylyl, biphenyl, ethylphenyl, chlorophenyl, methoxyphenyl,
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ethoxyphenyl, nitrophenyl, aminophenyl, dimethylaminophenyl,
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diethylaminophenyl, acetylaminophenyl, .alpha.-naphthyl, .beta.-naphthyl,
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anthraryl, pyrenyl, sulfonate phenyl, carboxylate phenyl and the like. In
|
|
the formula, Z.sub.7 represents an atom group required for the completion
|
|
of pyran, thiapyran, selenapyran, telluropyran, benzopyran,
|
|
benzothiapyran, benzoselenapyran, benzotelluropyran, naphthopyran,
|
|
naphthothiapyran, or naphthoselenapyran, or naphthotelluropyran.
|
|
PAR L.sub.7 represents sulfur atom, oxygen atom or selenium atom or tellurium
|
|
atom.
|
|
PAR R.sub.27 and R.sub.28 individually represent hydrogen atom, alkoxy group, a
|
|
substituted or an unsubstituted aryl group, alkenyl group and a
|
|
heterocyclic group,
|
|
PAR More specifically, R.sub.27 and R.sub.28 individually represent hydrogen
|
|
atom, alkyl group (methyl, ethyl, propyl, butyl and the like), alkyl
|
|
sulfonate group, alkoxyl group (methoxy, ethoxy, propoxy, ethoxyethyl,
|
|
methoxyethyl and the like), aryl group (phenyl, tolyl, xylyl, sulfonate
|
|
phenyl, chlorophenyl, biphenyl, methoxyphenyl and the like), a substituted
|
|
or an unsubstituted styryl group (styryl, p-methylstyryl, o-chlorostyryl,
|
|
p-methoxystyryl and the like), a substituted or an unsubstituted 4-phenyl,
|
|
1,3-butadienyl group (r-phenyl, 1,3-butadienyl, 4-(p-methylphenyl),
|
|
1,3-butadienyl and the like), or a substituted or an unsubstituted
|
|
heterocyclic group (quinolyl, pyridyl, carbazoyl, furyl and the like).
|
|
PAR As in the case of R, the same is true with R.sub.A and R.sub.B of the
|
|
general formulas (II) and (III), respectively.
|
|
PAR Then, in R, the symbols R.sub.8 ' to R.sub.14 ' individually correspond to
|
|
R.sub.1 ' to R.sub.7 '; R.sub.8 " to R.sub.14 " individually correspond to
|
|
R.sub.1 " to R.sub.7 "; in R.sub.B, R.sub.14 ' to R.sub.21 " individually
|
|
correspond to R.sub.1 ' to R.sub.7 '; R.sub.14 " to R.sub.21 "
|
|
individually correspond to R.sub.1 " to R.sub.7 ".
|
|
PAR In the azulenium nucleus of the (1) to (12), described above, those
|
|
represented by the formulas (3), (9) and (10) are more preferably used;
|
|
and particularly, the formula (3) is preferable.
|
|
PAR R.sub.1 to R.sub.28, R.sub.1 ' to R.sub.21 ' and R.sub.1 " to R.sub.21 "
|
|
preferably contain one or more well-known polar groups in order to impart
|
|
water solubility to a compound (labeling agent) represented by the general
|
|
formula (I), (II) or (III). The polar groups include, for example,
|
|
hydroxyl group, alkylhydroxyl group, sulfonate group, alkylsulfonate
|
|
group, carboxylate group, alkylcarboxylate group, tetra-ammonium base and
|
|
the like. R.sub.1 to R.sub.28, R.sub.1 ' to R.sub.21 ', and R.sub.1 " to
|
|
R.sub.21 " preferably contain one or more well-known reactive groups in
|
|
order that the compound of the general formula (I) can form a covalent
|
|
bond with a substance from a living organism.
|
|
PAR The reactive groups include the reactive sites of isocyanate,
|
|
isothiocyanate, succinimide ester, sulfosuccinimide ester, imide ester,
|
|
hydrazine, nitroaryl halide, piperidine disulfide, maleimide,
|
|
thiophthalimide, acid halide, sulfonyl halide, aziridine, azide
|
|
nitrophenyl, azide amino, 3-(2-pyridyldithio) propionamide and the like.
|
|
In these reactive sites, the following spacer groups
|
|
##STR8##
|
|
(n=0, 1 to 6) may be interposed in order to prevent steric hindrance
|
|
during on the bonding of a labeling agent and a substance from a living
|
|
organism.
|
|
PAR Preferable such reactive groups include isothiocyanate, sulfosuccinimide
|
|
ester, succinimide ester maleimide and the like X.sub.1.sup..sym.
|
|
represents an anion, including chloride ion, bromide ion, iodide ion,
|
|
perchlorate ion, benzenesulfonate ion, p-toluene sulfonate ion,
|
|
methylsulfate ion, ethylsulfate ion, propylsulfate ion, tetrafluoroborate
|
|
ion, tetraphenylborate ion, hexafluorophosphate ion, benzenesulfinic acid
|
|
salt ion, acetate ion, trifluoroacetate ion, propionate ion, benzoate ion,
|
|
oxalate ion, succinate ion, malonate ion, oleate ion, stearate ion,
|
|
citrate ion, monohydrogen diphosphate ion, dihydrogen monophosphate ion,
|
|
pentachlorostannate ion, chlorosulfonate ion, fluorosulfonate ion,
|
|
trifluoromethane sulfonate ion, hexafluoroantimonate ion, molybdate ion,
|
|
tungstate ion, titanate ion, zirconate ion and the like.
|
|
PAR Specific examples of these labeling agents are illustrated in Tables 1, 2
|
|
and 3, but are not limited thereto.
|
|
PAR The synthetic method of these azulene dyes is described in U.S. Pat. No.
|
|
4,738,908.
|
|
TBL3 TABLE 1
|
|
- No. G R X.sub.1 R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 R.sub.6
|
|
R.sub.7
|
|
1 (I) (3) R'.sub.1 = R'.sub.3 = R'.sub.5 = R'.sub.6 = HR'.sub.2 =
|
|
R'.sub.7 = CH.sub.3R'.sub.4 = CH(CH.sub.3).sub.2, R'.sub.22 = H, n = 2
|
|
BF.sub.4 H H H
|
|
##STR9##
|
|
H H CH.sub.3
|
|
2 (I) (3) R'.sub.1 = R'.sub.2 = R'.sub.4 = R'.sub.6 = H ClO.sub.4 H H
|
|
CH.sub.3 H OCH.sub.3 H CH.sub.3
|
|
R'.sub.3 = R'.sub.7 =
|
|
CH.sub.3 R'.sub.5 =
|
|
OCH.sub.3, R'.sub.22 = H, n =
|
|
2 3 (I) (3) R'.sub.1 =
|
|
R'.sub.2 = R'.sub.3 = R'.sub.7 = R'.sub.4 = R'.sub.6 = H I H H CH.sub.3
|
|
H CH.sub.2
|
|
CH.sub.2 H CH.sub.3
|
|
R'.sub.5 = CH.sub.2 CH.sub.2 CH.sub.2 COONa CH.sub.2 CH.sub.3
|
|
R'.sub.22 = H n =
|
|
2 4 (I) (3) R'.sub.1 =
|
|
R'.sub.2 = R'.sub.3 = R'.sub.4 = R'.sub.6 = R'.sub.7 = H ClO.sub.4 H H'
|
|
H H C(CH.sub.2).sub.3 H H
|
|
R'.sub.5 =
|
|
C(CH.sub.2).sub.3 R'.sub.22
|
|
= H, n =
|
|
2
|
|
5 (I) (3) R'.sub.1 = R'.sub.5 = R'.sub.6 = HR'.sub.2 and R'.sub.3
|
|
are cyclizedwith (CH.sub.2).sub.2R'.sub.4 = R'.sub.7 = CH.sub.3,
|
|
R'.sub.22 = H, n = 2 BF.sub.4 H SO.sub.3.sup..crclbar. Na.sup..sym. H
|
|
##STR10##
|
|
H H CH.sub.3
|
|
6 (I) (3)
|
|
##STR11##
|
|
BF.sub.4 H H H H C(CH.sub.2).sub.3 H H
|
|
7 (I) (3) R'.sub.1 = R'.sub.2 = R'.sub.4 = R'.sub.6 = HR'.sub.3 =
|
|
R'.sub.5 = R'.sub.7 = CH.sub.3R'.sub.22 = H, n =
|
|
2
|
|
##STR12##
|
|
H H CH.sub.3 H CH.sub.3 H CH.sub.3
|
|
8 (I) (9)
|
|
##STR13##
|
|
BF.sub.4 H CH.sub.3 H R.sub.4 and R.sub.5 arecombined to formSCHC(CH.sub
|
|
.3) H CH.sub.3
|
|
9 (I) (10)
|
|
##STR14##
|
|
ClO.sub.4 H SO.sub.3.sup..crclbar. Na.sup..sym. H CH(CH.sub.3).sub.2
|
|
##STR15##
|
|
H CH.sub.3
|
|
10 (I) (11)
|
|
##STR16##
|
|
BF.sub.4 H CH.sub.3 H CH(CH.sub.3).sub.2 H H CH.sub.3
|
|
11 (I) (12)
|
|
##STR17##
|
|
##STR18##
|
|
H CH.sub.3 H CH(CH.sub.3).sub.2 H H CH.sub.3
|
|
*G: General Formula
|
|
TBL TABLE 2
|
|
__________________________________________________________________________
|
|
No.
|
|
G R.sub.A R.sub.8
|
|
R.sub.9
|
|
R.sub.10
|
|
R.sub.11
|
|
R.sub.12 R.sub.13
|
|
R.sub.14
|
|
__________________________________________________________________________
|
|
12 (II)
|
|
(1)
|
|
R'.sub.8 = R'.sub.10 = R'.sub.12 = R'.sub.13 = H
|
|
H SO.sub.3.sup..crclbar. Na.sup..sym.
|
|
H CH(CH.sub.3).sub.2
|
|
H H CH.sub.3
|
|
R'.sub.9 = R'.sub.14 = CH.sub.3
|
|
R'.sub.11 = CH(CH.sub.3).sub.2
|
|
13 (II)
|
|
(1)
|
|
R'.sub.8 = R'.sub.12 = R'.sub.13 = R'.sub.14 = H
|
|
H CH.sub.3
|
|
SCHC(CH.sub.3)
|
|
H H H
|
|
R'.sub.10 < R'.sub.11CSCHC(CH.sub.3)
|
|
14 (II)
|
|
(2)
|
|
R'.sub.8 = R'.sub.10 = R'.sub.12 = R'.sub.13 = H
|
|
H SO.sub.3.sup..crclbar. Na.sup..sym.
|
|
H CH(CH.sub.3).sub.2
|
|
H H CH.sub.3
|
|
R'.sub.9 = R'.sub.14 = CH.sub.3
|
|
R'.sub.11 = CH(CH.sub.3).sub.2
|
|
15 (II)
|
|
(2)
|
|
R'.sub.8 = R'.sub.9 = R'.sub.11 = R'.sub.13 = H R'.sub.10 =
|
|
R'.sub.14 = CH.sub.3 R'.sub.12 = OC.sub.2 H.sub.5
|
|
H H CH.sub.3
|
|
H
|
|
##STR19## H CH.sub.3
|
|
__________________________________________________________________________
|
|
*G: General Formula
|
|
TBL3 TABLE 3
|
|
- No. G R.sub.B X.sub.1 R.sub.15 R.sub.16 R.sub.17 R.sub.18 R.sub.19
|
|
R.sub.20 R.sub.21
|
|
16 (III) (10)
|
|
##STR20##
|
|
BF.sub.4 H CH.sub.3 H formation ofSCHC H CH.sub.3
|
|
1
|
|
7 (III) (4) R'.sub.15 = R'.sub.16 = R'.sub.18 = R'.sub.20 = H I H
|
|
SO.sub.3.sup..crclbar.
|
|
Na.sup..sym. H H CH.sub.3 H CH.sub.3 R'.sub.17 =
|
|
R'.sub.19 = R'.sub.21 =
|
|
CH.sub.3 r =
|
|
1
|
|
18 (III) (10)
|
|
R'.sub.15 = R'.sub.18 = R'.sub.20 = HR'.sub.16 = NO.sub.2R'.sub.17 =
|
|
R'.sub.19 = R'.sub.21 = CH.sub.3r =
|
|
3
|
|
##STR21##
|
|
H NO.sub.2 CH.sub.3 H CH.sub.3 H CH.sub.3
|
|
19 (III) (5) R'.sub.15 = R'.sub.16 = R'.sub.17 = R'.sub.18 = HR'.sub.19
|
|
= R'.sub.20 = R'.sub.21 = HR".sub.15 = R".sub.17 = R".sub.18 = R".sub.19
|
|
=R".sub.20 = R".sub.21 = H ClO.sub.4 H SO.sub.3.sup..crclbar.
|
|
Na.sup..sym. H H
|
|
##STR22##
|
|
H H
|
|
20 (III) (8)
|
|
##STR23##
|
|
##STR24##
|
|
H CH.sub.3 H CH(CH.sub.3).sub.2 H H CH.sub.3
|
|
21 (III) (9)
|
|
##STR25##
|
|
BF.sub.4 H SO.sub.3.sup..crclbar. Na.sup..sym. H H n-C.sub.8 H.sub.17 H
|
|
H
|
|
22 (III) (10)
|
|
##STR26##
|
|
##STR27##
|
|
H SO.sub.3.sup..crclbar. Na.sup..sym. H CH(CH.sub.3).sub.2 H H CH.sub.3
|
|
23 (III) (12)
|
|
##STR28##
|
|
I H CH.sub.3 H CH(CH.sub.3).sub.2
|
|
##STR29##
|
|
H CH.sub.3
|
|
No. G R X.sub.1 R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 R.sub.6
|
|
R.sub.7
|
|
24 I (6)
|
|
##STR30##
|
|
I H H H
|
|
##STR31##
|
|
H H CH.sub.3
|
|
25 I (7)
|
|
##STR32##
|
|
BF.sub.4 H H H CH(CH.sub.3).sub.2 H H CH.sub.3
|
|
26 I (3) R'.sub.1 = R'.sub.3 = R'.sub.5 = R'.sub.6 = HR'.sub.2 =
|
|
SO.sub.3.sup..crclbar. Na.sup..sym.R'.sub.7 = CH.sub.3 R'.sub.22 =
|
|
HR'.sub.4 = CH(CH.sub.3).sub.2n =
|
|
2 I H H CH.sub.3
|
|
##STR33##
|
|
H H CH.sub.3
|
|
27 I (3) R'.sub.1 = R'.sub.3 = R'.sub.4 = R'.sub.6 = R'.sub.7 =
|
|
HR'.sub.2 = SO.sub.3.sup..crclbar. Na.sup..sym.R'.sub.5 =
|
|
(CH.sub.2).sub.3COO.sup..crclbar.
|
|
Na.sup..sym. BF.sub.4 H SO.sub.3.sup..crclbar.
|
|
Na.sym. H H
|
|
##STR34##
|
|
H H
|
|
*G: General Formula
|
|
PAR These illustrated labeling agents absorb light in a near-infrared
|
|
wavelength region of 670 to 900 nm, and the molar absorption coefficient
|
|
.epsilon. is in the region of 50,000 to 300,000 1/mol.cm. The illustrated
|
|
labeling agents include those generating strong fluorescence.
|
|
PAR Table 4 shows the maximum absorption wavelength (.lambda.max) and maximum
|
|
fluorescence wavelength of (.lambda.em) each of the labeling agents
|
|
generating fluorescence in the region of the semiconductor laser
|
|
wavelength (medium: ethanol/dichloromethane=1/4).
|
|
TBL TABLE 4
|
|
______________________________________
|
|
Maximum absorption
|
|
Maximum fluorescence
|
|
No. wavelength (.lambda. max)
|
|
wavelength (.lambda. em)
|
|
______________________________________
|
|
2 828 863
|
|
3 833 871
|
|
4 825 857
|
|
6 825 851
|
|
7 830 871
|
|
16 790 828
|
|
27 826 870
|
|
______________________________________
|
|
PAR FIG. 1 shows the fluorescence emitting wave form on the incidence of
|
|
semiconductor laser beam (10 mW) of 830 nm into a labeling agent No. 3.
|
|
The apparatus for measurement is IMUC-7000 manufactured by Otsuka Electron
|
|
Co., Ltd.
|
|
PAR In FIG. 1, curve A shows incident wave form of semiconductor laser beam.
|
|
Curve B shows A fluorescence emitting wave form of a labeling agent No. 3.
|
|
PAR Alternatively, the labeling agent to be used in the present invention is a
|
|
compound of the general formula (IV), wherein A, B, D and E individually
|
|
represent hydrogen atom or alkyl group (for example, ethyl group, n-propyl
|
|
group, iso-propyl group, n-butyl group, sec-butyl group, iso-butyl group,
|
|
t-butyl group, n-amyl group, t-amyl group, n-hexyl group, n-octyl group,
|
|
t-octyl group and the like); and additionally, other alkyl groups such as
|
|
for example a substituted alkyl group (for example, 2-hydroxyethyl group,
|
|
3-hydroxypropyl group, 4-hydroxybutyl group, 2-acetoxyethyl group,
|
|
carboxymethyl group, 2-carboxyethyl group, 3-carboxypropyl group,
|
|
2-sulfoethyl group, 3-sulfopropyl group, 4-sulfobutyl group, 3-sulfate
|
|
propyl group, 4-sulfate butyl group, N-(methylsulfonyl)-carbamylmethyl
|
|
group, 3-(acetyl-sulfamyl)propyl group, 4-(acetylsulfamyl)butyl group and
|
|
the like); cyclic alkyl groups (for example cyclohexyl group), allyl group
|
|
(CH.sub.2 .dbd.CH--CH.sub.2 --), alkenyl group (vinyl group, propenyl
|
|
group, butenyl group, pentenyl group, hexenyl group, heptenyl group,
|
|
octenyl group, dodecyl group, prenyl group and the like), aralkyl group
|
|
(for example, benzyl group, phenethyl group, .alpha.-naphthylmethyl group,
|
|
.beta.-naphthylmethyl group and the like), a substituted aralkyl group
|
|
(for example, carboxybenzyl group, sulfobenzyl group, hydroxybenzyl group
|
|
and the like), a substituted or an unsubstituted aryl group (for example,
|
|
phenyl group, aminophenyl group, naphthyl group, tolyl group, xylyl group,
|
|
methoxyphenyl group, dimethoxyphenyl group, trimethoxyphenyl group,
|
|
ethoxyphenyl group, dimethylaminophenyl group, diethylaminophenyl group,
|
|
dipropylaminophenyl group, dibenzylaminophenyl group, diphenylaminophenyl
|
|
group, sulfonate phenyl group, carboxylate phenyl group and the like), a
|
|
substituted or an unsubstituted heterocyclic group (for example, pyridyl
|
|
group, quinolyl group, lepidyl group, methylpyridyl group, furyl group,
|
|
phenyl group, indolyl group, pyrrolle group, carbazolyl group,
|
|
N-ethylcarbazolyl group and the like), or a substituted or an
|
|
unsubstituted styryl group (for example, styryl group, methoxystyryl
|
|
group, dimethoxystyryl group, trimethoxystyryl group, ethoxystyryl group,
|
|
aminostyryl group, dimethylaminostyryl group, diethylaminostyryl group,
|
|
dipropylaminostyryl group, dibenzylaminostyryl group, diphenylaminostyryl
|
|
group, 2,2-diphenylvinyl group, 2-phenyl-2-methylvinyl group,
|
|
2-(dimethylamino-phenyl)-2-phenylvinyl group,
|
|
2-(diethylaminophenyl)-2-phenylvinyl group,
|
|
2-(dibenzylaminophenyl)-2-phenylvinyl group, 2,2-di(diethylaminophenyl
|
|
)vinyl group, 2,2-di(methoxyphenyl)vinyl group, 2,2-di(ethoxylphenyl)vinyl
|
|
group, 2-(dimethylaminophenyl)-2-methylvinyl group,
|
|
2-(diethylaminophenyl)-2-ethylvinyl group, and the like).
|
|
PAR r.sub.1 ' and r.sub.2 ' individually represent hydrogen atom or alkyl group
|
|
(for example, methyl group, ethyl group, n-propyl group, iso-propyl group,
|
|
n-butyl group, sec-butyl group, iso-butyl group, t-butyl group, n-amyl
|
|
group, t-amyl group, n-hexyl group, n-octyl group, t-octyl group and the
|
|
like); and additionally, other alkyl groups such as for example a
|
|
substituted alkyl group (for example, 2-hydroxyethyl group,
|
|
3-hydroxypropyl group, 4-hydroxybutyl group, 2-acetoxyethyl group,
|
|
carboxymethyl group, 2-carboxyethyl group, 3-carboxypropyl group,
|
|
2-sulfoethyl group, 3-sulfopropyl group, 4-sulfobutyl group, 3-sulfate
|
|
propyl group, 4-sulfate butyl group, N-(methylsulfonyl)-carbamylmethyl
|
|
group, 3-(acetylsulfamyl)propyl group, 4-(acetylsulfamyl)butyl group and
|
|
the like); cyclic alkyl group (for example, cyclohexyl group), allyl group
|
|
(CH.sub.2 .dbd.CH--CH.sub.2 --), alkenyl group (vinyl group, propenyl
|
|
group, butenyl group, pentenyl group, hexenyl group, heptenyl group,
|
|
octenyl group, dodecyl group, prenyl group and the like), aralkyl group
|
|
(for example, benzyl group, phenethyl group, .alpha.-naphthylmethyl group,
|
|
.beta.-naphthylmethyl group and the like), and a substituted aralkyl group
|
|
(for example, carboxybenzyl group, sulfobenzyl group, hydroxybenzyl group
|
|
and the like).
|
|
PAR A, B, D, r.sub.1 ' and r.sub.2 ' preferably contain one or more well-known
|
|
polar groups in order to impart water solubility to the labeling agent
|
|
(dye) of the general formula (IV). The reactive group includes for example
|
|
hydroxyl group, alkylhydroxyl group, sulfone group, alkyl sulfone group,
|
|
carboxyl group, alkylcarboxyl group, tetra-ammonium base and the like. A,
|
|
B, D, r.sub.1 ' and r.sub.2 ' preferably contain one or more well-known
|
|
reactive groups in order that the labeling agent of the general formula
|
|
(IV) can form a covalent bond with a substance from a living organism.
|
|
PAR The reactive group includes the reactive sites of isocyanate,
|
|
isothiocyanate, succinimide ester, sulfosuccinimide ester, imide ester,
|
|
hydrazine, nitroaryl halide, piperidine disulfide, maleimide, thiophthal
|
|
imide, acid halide, sulfonyl halide, aziridine, azide nitrophenyl, azide
|
|
amino, 3-(2-pyridyldithio) propionamide and the like. In these reactive
|
|
sites, the following spacer groups
|
|
##STR35##
|
|
(n=0, 1 to 16) may be interposed in order to prevent the steric hindrance
|
|
on the bonding of a labeling agent and a substance from a living organism.
|
|
PAR Preferable such reactive groups include isothiocyanate, sulfosuccinimide
|
|
ester, succinimide ester, maleimide and the like.
|
|
PAR The k in the general formula (IV) is 0 or 1 and 1 is 1 or 2.
|
|
PAR X.sub.2.sup..crclbar. represents an anion including chlorine ion, bromine
|
|
ion, iodine ion, perchlorate ion, benzenesulfonate ion, p-toluene
|
|
sulfonate ion, methylsulfate ion, ethylsulfate ion, propylsulfate ion,
|
|
tetrafluoroborate ion, tetraphenylborate ion, hexafluorophosphate ion,
|
|
benzenesulfinic acid ion, acetate ion, trifluoroacetate ion, propionate
|
|
ion, benzoate ion, oxalate ion, succinate ion, malonate ion, oleate ion,
|
|
stearate ion, citrate ion, monohydrogen diphosphate ion, dihydrogen
|
|
monophosphate ion, pentachlorostannate a ion, chlorosulfonate ion,
|
|
fluorosulfonate ion, trifluoromethane sulfonate ion, hexafluoroantimonate
|
|
ion, molybdate ion, tungstate ion, titanate ion, zirconate ion and the
|
|
like.
|
|
PAR Specific examples of these labeling agents are illustrated in Table 5, but
|
|
are not limited thereto.
|
|
TBL3 TABLE 5
|
|
- No A B D E r.sub.1 ' r.sub.2
|
|
' X.sub.2
|
|
28 ph .rarw. .rarw.
|
|
##STR36##
|
|
-- -- BF.sub.4 k = 0,l =
|
|
1
|
|
29
|
|
##STR37##
|
|
.rarw. .rarw.
|
|
##STR38##
|
|
-- -- BF.sub.4 k = 0,l =
|
|
1
|
|
30
|
|
##STR39##
|
|
##STR40##
|
|
(CH.sub.3).sub.2N
|
|
##STR41##
|
|
-- -- I k = 0,l =
|
|
1gleaming
|
|
31
|
|
##STR42##
|
|
##STR43##
|
|
(C.sub.2 H.sub.5).sub.2
|
|
N
|
|
##STR44##
|
|
H CH.sub.3 AsF.sub.6 k = 1,l =
|
|
0
|
|
32
|
|
##STR45##
|
|
.rarw. .rarw.
|
|
##STR46##
|
|
-- -- FSO.sub.3 k = 0,l =
|
|
1
|
|
33
|
|
##STR47##
|
|
##STR48##
|
|
##STR49##
|
|
##STR50##
|
|
-- --
|
|
##STR51##
|
|
k = 0,l =
|
|
1
|
|
34
|
|
##STR52##
|
|
Ph
|
|
##STR53##
|
|
##STR54##
|
|
-- --
|
|
##STR55##
|
|
k = 0,l =
|
|
1
|
|
35
|
|
##STR56##
|
|
.rarw. .rarw. .rarw. H
|
|
##STR57##
|
|
BF.sub.4 k = 1,l =
|
|
0
|
|
36
|
|
##STR58##
|
|
H
|
|
##STR59##
|
|
.rarw. -- -- AsF.sub.6 k = 0l =
|
|
0
|
|
37
|
|
##STR60##
|
|
.rarw. .rarw. .rarw. H
|
|
##STR61##
|
|
BF.sub.4 k = 1,l =
|
|
0
|
|
38
|
|
##STR62##
|
|
.rarw.
|
|
##STR63##
|
|
.rarw. -- -- FSO.sub.3 k = 0,l =
|
|
1
|
|
39
|
|
##STR64##
|
|
.rarw. .rarw. .rarw. H C.sub.2 H.sub.5 BF.sub.4 k = 1,l =
|
|
0
|
|
40
|
|
##STR65##
|
|
.rarw. .rarw.
|
|
##STR66##
|
|
-- -- BF.sub.4 k = 0,l =
|
|
1
|
|
41
|
|
##STR67##
|
|
.rarw. .rarw. .rarw. H
|
|
##STR68##
|
|
BF.sub.4 k = 1,l =
|
|
0
|
|
42
|
|
##STR69##
|
|
CH.sub.3
|
|
##STR70##
|
|
CH.sub.3 H CH.sub.3 FSO.sub.3 k = 1,l =
|
|
0
|
|
43
|
|
##STR71##
|
|
C.sub.3
|
|
H.sub.7
|
|
##STR72##
|
|
C.sub.3
|
|
H.sub.7 H
|
|
##STR73##
|
|
BF.sub.4 k = 1,l =
|
|
0
|
|
44
|
|
##STR74##
|
|
##STR75##
|
|
##STR76##
|
|
##STR77##
|
|
-- --
|
|
##STR78##
|
|
k = 0,l =
|
|
1
|
|
45
|
|
##STR79##
|
|
##STR80##
|
|
##STR81##
|
|
##STR82##
|
|
-- -- AsF.sub.6 k = 0,l =
|
|
1
|
|
46
|
|
##STR83##
|
|
##STR84##
|
|
##STR85##
|
|
##STR86##
|
|
--
|
|
##STR87##
|
|
I k = 0,l =
|
|
1
|
|
47
|
|
##STR88##
|
|
##STR89##
|
|
##STR90##
|
|
##STR91##
|
|
--
|
|
##STR92##
|
|
I k = 1,l =
|
|
0
|
|
48
|
|
##STR93##
|
|
##STR94##
|
|
##STR95##
|
|
##STR96##
|
|
-- -- BF.sub.4 k = 0,l =
|
|
1
|
|
PAR These illustrated labeling agents absorb light in a near-infrared
|
|
wavelength region of 670 to 900 nm, and the molar absorption coefficient e
|
|
is in the region of 50,000 to 300,000 1/mol.cm. Some of the illustrated
|
|
labeling agents generate intense fluorescence.
|
|
PAR The dye No. 30 illustrated in Table 5 exhibits the maximum absorption at a
|
|
wavelength of 819 nm in a near-infrared region and emits fluorescence. The
|
|
maximum fluorescence wavelength (.lambda.em) is 864 nm (medium;
|
|
dichloromethane).
|
|
PAR In accordance with the present invention, the labeling agents described
|
|
above are immobilized onto a substance from a living organism, but the
|
|
substance from a living organism to be immobilized is selectively
|
|
determined based on a substance to be analyzed or a subject sample. That
|
|
is, if a substance is selected from a living organism having a biological
|
|
specificity to a subject sample, the substance to be analyzed can be
|
|
detected with specificity. By the term "substance from a living organism"
|
|
is meant naturally occurring or synthetic peptides, proteins, enzymes,
|
|
sugars, rectins, viruses, bacteria, nucleic acids, DNA, RNA, antigens
|
|
(including for example recombinant antigens), antibodies and the like. The
|
|
substances specifically useful in terms of clinical pathology include the
|
|
following; immunoglobulin such as IgG, IgM, IgE and the like; plasma
|
|
proteins and antibodies thereof, such as compliments, CRP, ferritin,
|
|
.alpha.1-microglobulin, .beta.2-microglobulin, and the like; tumor markers
|
|
and antibodies thereof, such as .alpha.-fetoprotein, carcinoembryonic
|
|
antigen (CEA), prostate acid phosphatase (PAP), CA19-9, CA-125 and the
|
|
like; hormones and antibodies thereof such as luteinizing hormone (LH),
|
|
follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG),
|
|
estrogen, insulin and the like; substances in relation with virus
|
|
infection and antibodies thereof, such as HBV-related antigens (HBs, HBe,
|
|
HBc), HIV, ATL and the like; bacteria and antibodies thereof, such as
|
|
Corynebacterium diphteriae, Clostridium botulinum, mycoplasma, Treponema
|
|
pallidum and the like; protozoae and antibodies thereof such as Toxoplasma
|
|
gondii, Trichomonas, Leishmania, Tripanozoma, malaria protozoa and the
|
|
like; pharmaceutical agents and antibodies thereof, such as antileptic
|
|
agents including phenytoin, phenobarbital and the like, cardiovascular
|
|
agents including quinidine and digoxin, antasthmatic agents including
|
|
theophylline, antibiotics including chloramphenicol and gentamycin; as
|
|
well as, enzymes, enterotoxin (streptolysin O) and the antibodies thereof.
|
|
Depending on the type of sample, a substance which can incur the
|
|
antigen-antibody reaction with a substance to be measured in a sample is
|
|
appropriately selected for use.
|
|
PAR In accordance with the present invention, the following known method can be
|
|
utilized in order to immobilize a labeling agent onto a substance from a
|
|
living organism such as a physiological active substance.
|
|
PAR There are illustrated for example i) ion bonding method, ii) physical
|
|
absorption method, iii) covalent bonding method and the like.
|
|
PAR The ion bonding method comprises electrostatically bonding a labelling
|
|
agent having principally a positive charge to a substance from a living
|
|
organism such as proteins, DNA, RNA and the like.
|
|
PAR The physical absorption method comprises utilizing the hydrophobic bond
|
|
between the lipophilic part of a labeling agent and the lipophilic part of
|
|
a protein.
|
|
PAR The reaction process of bonding is simple in accordance with the ion
|
|
bonding method and physical absorption method, but the bonding strength of
|
|
a labeling agent and a substance from a living organism is weak.
|
|
PAR On contrast, the covalent bonding method comprises bonding a highly
|
|
reactive functional group to at least one of a labeling agent and a
|
|
substance from a living organism, and covalently bonding the two through
|
|
the functional group whereby a highly intense bonding strength can be
|
|
generated. In bonding a labeling agent with a substance from a living
|
|
organism such as physiological active substances via covalent bonds, the
|
|
functional groups being present in the substance from a living organism
|
|
and which can be involved in the bonding, include free amino group,
|
|
hydroxyl group, phosphate group, carboxyl group, the sulfhydryl group of
|
|
cysteine, the imidazole group of histidine, phenol group of tyrosine, the
|
|
hydroxyl group of serine and threonine, and the like.
|
|
PAR These functional groups react with a variety of diazonium salts, acid
|
|
amides, isocyanate, active-type halogenated alkyl groups, active-type
|
|
ester groups and the like. Therefore, by a variety of methods, dyes can be
|
|
immobilized onto a substance from a living organism by introducing these
|
|
functional groups into a labeling agent. Alternatively, the conformation
|
|
of a substance from a living organism, specifically that of proteins, is
|
|
readily damaged because it is retained through relatively weak bonds such
|
|
as hydrogen bond, hydrophobic bond, ion bond and the like. Thus, the
|
|
immobilization with a labeling agent preferably should be carried out
|
|
under mild conditions, without processing by means of high temperatures,
|
|
strong acids and strong alkalis.
|
|
PAR One method of carrying out the immobilization under mild conditions
|
|
includes the use of bifunctional cross-linking agents which react with a
|
|
labeling agent and with the functional groups of a substance from a living
|
|
organism. The bifunctional cross-linking agents include, for example,
|
|
carbodiimide represented by the general formula R--N.dbd.C.dbd.N--R',
|
|
dialdehyde represented by the general formula CHO--R--CHO, diisocyanate
|
|
represented by O.dbd.C.dbd.N--R--N.dbd.C.dbd.O (wherein R and R' represent
|
|
individually the same or a different substituted or unsubstituted alkyl
|
|
group, aryl group alkylaryl group or aryl alkyl group), and the like.
|
|
PAR The analysis of a certain particular objective substance is conducted by
|
|
using the resulting labeled complex in which a labeling agent is
|
|
immobilized onto a substance from a living organism.
|
|
PAR If a target (analytical subject) is one species of cell, the labeled
|
|
complex is bonded to a specific substance on the cell complimentary to the
|
|
substance from a living organism bonded to the labeled complex via a
|
|
specific bonding such as an antigen-antibody reaction or the hydrogen
|
|
bonding between nucleic acids. Then, the amount of such antigen, antibody
|
|
or nucleic acids can be measured based on the fluorescence or absorbance
|
|
of the system.
|
|
PAR If the analysis is effected of a target in relation with an antigen and an
|
|
antibody, a complex bonded through the labeling agent to an antigen (or an
|
|
antibody) and an antibody (an antigen if a labeling agent is immobilized
|
|
onto the antibody) to be measured are subjected to antigen-antibody
|
|
reaction. The complex (B; bonded type) bonded to the antibody (antigen) is
|
|
then separated from the complex (F; free type) which is not bonded to the
|
|
antibody (antigen) (B/F separation). Thereafter, the amount of the complex
|
|
(B) is determined based on the fluorescence or absorbance. The technique
|
|
utilizing the antigen-antibody reaction described above is described in
|
|
details in "Examination and Technology", Vol. 16, No. 7 (1988).
|
|
PAR In terms of detection sensitivity, furthermore, it is preferable that two
|
|
or more, preferably 10 or more labeling agents are bonded to one molecule
|
|
of a substance from a living organism. In terms of synthesis and
|
|
sensitivity, preferably 10 to 100, more preferably 20 to 50 such agents
|
|
may be bonded to one molecule thereof.
|
|
PAR The present invention will now be explained with reference to examples.
|
|
PAC EXAMPLE 1
|
|
PAR Anti-human CRP sheep serum (IgG fraction; manufactured by Cooper Biomedical
|
|
Inc.) was diluted with phosphate buffer, pH 8.0, to a concentration of 0.5
|
|
mg/ml, to prepare an antibody solution. To 8 ml of the antibody solution
|
|
were added 0.2 mg of a labeling agent No. 3 of Table 1 (.lambda.max=833
|
|
nm) and 0.09 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
|
|
hydrochloride (referred to as WSC hereinafter) (manufactured by Dojin
|
|
Chemicals, Co. Ltd.) for reaction at room temperature for three hours, to
|
|
generate a labeling agent-antibody complex. The labeling agent-antibody
|
|
complex was separated and purified from unreacted substances by gel
|
|
filtration chromatography on a column packed with Sepharose 6B. The
|
|
bonding molar ratio of the labeling agent and the antibody in the complex
|
|
thus obtained was 2.1:1. By using a spectrophotometer Shimadzu UV-3100S,
|
|
the absorbance of the complex was measured at wavelengths .lambda.=833 nm
|
|
and .lambda.=280 nm, separately, to calculate the molar ratio of the
|
|
labeling agent and the antibody.
|
|
PAC EXAMPLE 2
|
|
PAR Rectin. Concanavalin A (manufactured by E. Y. Laboratories Co. Ltd.) was
|
|
diluted with phosphate buffer, pH 8.2, to a concentration of 0.2 mg/ml, to
|
|
prepare a rectin solution.
|
|
PAR With 10 ml of the rectin solution was reacted 0.2 mg of a labeling agent
|
|
No. 6 of Table 1 (.lambda.max=825 nm) at room temperature for three hours.
|
|
The labeling agent-rectin complex was separated and purified on a gel
|
|
filtration chromatocolumn packed with Sepharose 6B. The molar ratio of the
|
|
labeling agent and the rectin in the complex obtained was 3.7:1. The
|
|
absorbances at wavelengths .lambda.=825 and .lambda.=280 nm were measured
|
|
by a spectrophotometer Shimadzu UV-3100S, to calculate the molar ratio of
|
|
the labeling agent and the rectin.
|
|
PAC EXAMPLE 3
|
|
PAR Anti-human HCG monoclonal antibody (manufactured by ZyMED Lab. Inc.) was
|
|
diluted with phosphate buffered physiological saline (PBS), pH 7.2, to a
|
|
concentration of 0.2 mg/ml, to prepare a monoclonal antibody solution.
|
|
PAR To 8 ml of the antibody solution was added 0.3 mg of a labeling agent No.
|
|
12 of Table 1 (.lambda.max=705 nm) for agitation at room temperature for
|
|
three hours. The labeling agent-antibody complex was separated and
|
|
purified by gel filtration chromatography on a column packed with
|
|
Sepharose 6B.
|
|
PAR The molar ratio of the labeling agent and the antibody in the labeling
|
|
agent-antibody complex thus obtained was 1.7:1. By using a
|
|
spectrophotometer 1 Shimadzu UV-3100S, the absorbance of the complex was
|
|
measured at wavelengths .lambda.=705 nm and .lambda.=280 nm, separately,
|
|
to calculate the molar ratio of the labeling agent and the antibody.
|
|
PAC EXAMPLE 4
|
|
PAR M13mp18 single-strand DNA (7249 bases) (manufactured by TAKARA Liquor KK.)
|
|
(0.1 mg) was diluted with 5 mmol phosphate buffer, pH 6, to prepare a DNA
|
|
solution. A labeling agent No. 5 (0.1 mg) shown in Table 1
|
|
(.lambda.max=796 nm) was dissolved in 5 ml of distilled water, and
|
|
subsequently, 5 ml of the DNA solution was gradually added dropwise to the
|
|
resulting dye solution. Agitation was further effected at room temperature
|
|
for 2 hours, to produce a DNA-labeling agent complex.
|
|
PAR To the solution of the DNA-labeling agent complex described above was added
|
|
further 40 ml of ethanol, to precipitate the DNA-labeling agent complex.
|
|
The DNA-labeling agent complex was separated on a filter, followed by
|
|
washing with ethanol. The DNA-labeling agent complex after the washing was
|
|
again dissolved in 2 ml of the phosphate buffer, pH 6. The amount of the
|
|
labeling agent bonded to that of the DNA was 0.5 .mu.g per .mu.g.DNA. The
|
|
absorbance of the complex was measured at wavelengths .lambda.=705 nm and
|
|
.lambda.=280 nm, separately, to calculate the concentrations of the
|
|
labeling agent and the DNA.
|
|
PAC EXAMPLE 5
|
|
PAR A 20-mer oligonucleotide having a base sequence partially complimentary to
|
|
the base sequence of a model target nucleic acid M13mp18 ss DNA was
|
|
synthesized by a DNA synthesizer 381 A, manufactured by ABI Co. Ltd. Then,
|
|
a primary amine was introduced into the 5' terminus of the oligonucleotide
|
|
by using a N-MMT-hexanol amine linker manufactured by Milligen Co. Ltd.,
|
|
instead of general amidide reagents. A predetermined protocol was followed
|
|
to perform cutting out from the CPG-support, deprotection (including the
|
|
deprotection of monomethoxytrityl group as a protective group of the
|
|
primary amine), and the purification by high-performance liquid
|
|
chromatography.
|
|
PAR After mixing together 200 .mu.g of the oligonucleotide, 100 .mu.l of 1M
|
|
sodium carbonate buffer, pH 9.0, and 700 .mu.l of water, 2 mg of a
|
|
labeling agent No. 27 (.lambda.max=826 nm) shown in Table 1, which had
|
|
preliminarily been dissolved in 200 .mu.l of dimethyl formamide, was
|
|
gradually added under agitation. After the reaction at room temperature
|
|
for 24 hours, the peak of the nucleic acid was decreased on a
|
|
high-performance liquid chromatogram, whereas a new peak having the
|
|
absorbances of the nucleic acid and the labeling agent developed. Thus,
|
|
the reaction solution was nearly purified on a gel filtration column,
|
|
NAP-50, manufactured by Pharmacia, which was then purified by HPLC to
|
|
obtain 175 .mu.g of the nucleic acid-labeling agent complex.
|
|
PAC COMPARATIVE EXAMPLE 1
|
|
PAR The chemical structure of a well-known cyanine-type near-infrared
|
|
absorption dye NK-1967 (manufactured by Nippon Photosensitive Dye Research
|
|
Institute) is depicted hereinbelow.
|
|
##STR97##
|
|
PAR To 5 ml of the antibody solution prepared in Example 1 was added 0.3 mg of
|
|
the cyanine dye, and agitated at room temperature for 3 hours, to generate
|
|
a labeling agent-antibody complex.
|
|
PAR The labeling agent-antibody complex was separated and purified by gel
|
|
filtration chromatography on a column packed with Sepharose 6B.
|
|
PAR The molar ratio of the labeling agent and the antibody was 1.7:1. The
|
|
absorbances at wavelength p=747 nm and p=280 nm were measured by a
|
|
spectrophotometer Shimadzu UV-3100S to calculate the molar ratio of the
|
|
labeling agent and the antibody. Complex stability under storage
|
|
PAR In order to examine the complex stability under storage, the following
|
|
experiments were carried out.
|
|
PAR The labeled complexes prepared in Examples 1 to 5 and Comparative Example 1
|
|
were prepared to predetermined concentrations with 10 mmol phosphate
|
|
buffer, pH 7.2. The solutions of the labeled complexes were kept in dark
|
|
at 7.degree. C. for three days. At the initiation and termination of the
|
|
test of complex stability under storage, the absorbance was measured at
|
|
predetermined wavelengths to calculate the ratio of the absorbance at the
|
|
termination, provided that the absorbance at the initiation was designated
|
|
as 100.
|
|
PAR For the complexes exhibiting fluorescence, the ratio of the fluorescence
|
|
intensity at the termination was calculated, provided that the
|
|
fluorescence intensity at the initiation was designated as 100.
|
|
PAR The results are shown in Table 6.
|
|
TBL TABLE 6
|
|
______________________________________
|
|
Stability under storage of labeled complexes
|
|
Change in
|
|
Change in fluorescence
|
|
absorbance*
|
|
intensity**
|
|
(wavelength
|
|
(wavelength
|
|
Concentration
|
|
in nm) in nm)
|
|
______________________________________
|
|
Example
|
|
1 0.4 g/ml 93.4 (833)
|
|
94 (875)
|
|
2 0.4 g/ml 91.9 (825)
|
|
90 (870)
|
|
3 0.5 g/ml 94.2 (705)
|
|
--
|
|
4 0.4 g/ml 95.1 (796)
|
|
--
|
|
5 0.5 g/ml 96.1 (826)
|
|
93 (870)
|
|
Comparative
|
|
Example
|
|
1 0.5 g/ml 71.2 (747)
|
|
63 (820)
|
|
______________________________________
|
|
*The initial absorbance was designated as 100.
|
|
**The initial fluorescence was designated as 100.
|
|
PAR As is shown in Table 6, the labeled complexes of the present invention
|
|
showed lower change of the absorbance or fluorescence intensity in water
|
|
than those of Comparative Example.
|
|
PAC EXAMPLE 6
|
|
PAR Anti-human CRP sheep serum (IgG fraction; manufactured by Cooper Biomedical
|
|
Inc.) was diluted with PBS, pH 7.2, to a concentration of 0.5 mg/ml, to
|
|
prepare an antibody solution. To 8 ml of the antibody solution were added
|
|
0.2 mg of a labeling agent No. 29 of Table 5 (.lambda.max=819 nm) and 0.09
|
|
g of WSC for reaction at room temperature for three hours, to generate a
|
|
dye-antibody complex. The dye-antibody complex was separated and purified
|
|
from unreacted substances by gel filtration chromatography on a column
|
|
packed with Sepharose 6B. The molar ratio of the dye and the antibody in
|
|
the complex thus obtained was 2.5:1. By using a spectrophotometer,
|
|
Shimadzu UV-3100S, the absorbance of the complex was measured at
|
|
wavelength .lambda.=819 nm and .lambda.=280 nm, separately, to calculate
|
|
the molar ratio of the dye and the antibody.
|
|
PAC EXAMPLE 7
|
|
PAR Anti-human HCG monoclonal antibody (manufactured by ZyMED Lab, Inc.) was
|
|
diluted with PBS to a concentration of 0.4 mg/ml, to prepare a monoclonal
|
|
antibody solution. To 2 ml of the monoclonal antibody solution were added
|
|
0.3 mg of a dye No. 32 of Table 5 (.lambda.max=825 nm) and 0.10 g of
|
|
Woodward reagent (manufactured by Tokyo Chemicals, Co. Ltd.) for reaction
|
|
at room temperature for three hours. The dye-antibody complex was
|
|
separated and purified by gel filtration chromatography on a column packed
|
|
with Sepharose 6B. The molar ratio of the dye and the antibody in the
|
|
dye-antibody complex thus obtained was 3.1:1. By using a spectrophotometer
|
|
Shimadzu UV-3100S, the absorbance of the complex was measured at
|
|
wavelengths .lambda.=825 nm and .lambda.=280 nm, separately, to calculate
|
|
the molar ratio of the dye and the antibody.
|
|
PAC EXAMPLE 8
|
|
PAR Rectin.Concanavalin A (manufactured by E. Y. Laboratories Co. Ltd.) was
|
|
diluted with PBS to a concentration of 0.2 mg/ml, to prepare a rectin
|
|
solution. With 10 ml of the rectin solution were added 0.2 mg of a dye No.
|
|
40 of Table 5 (.lambda.max=805 nm) and 10 ml of 0.05 M sodium borate
|
|
buffer, pH 8.0 containing 1% glutaraldehyde at room temperature for one
|
|
hour. The dye-rectin complex was separated and purified on a gel
|
|
filtration chromatocolumn packed with Sepharose 6B. The molar ratio of the
|
|
dye and the rectin in the complex obtained was 1.7:1. The absorbances at
|
|
wavelengths .lambda.=805 and .lambda.=280 nm were measured by a
|
|
spectrophotometer Shimadzu UV-3100S, to calculate the molar ratio of the
|
|
dye and the rectin.
|
|
PAC EXAMPLE 9
|
|
PAR M13mp18 single-strand DNA (7249 bases) (manufactured by TAKARA Liquor KK.)
|
|
(0.1 mg) was diluted with 5 mmol phosphate buffer, pH 6, to prepare a DNA
|
|
solution. A dye No. 35 (0.1 mg) shown in Table 5 (.lambda.max=780 nm) was
|
|
dissolved in 2 ml of ethanol, followed by gradual dropwise addition of 5
|
|
ml of the DNA solution to the resulting dye solution under stirring.
|
|
Agitation was further effected at room temperature for 2 hours, to produce
|
|
a DNA-dye complex.
|
|
PAR To the solution of the DNA-dye complex described above was added further 40
|
|
ml of ethanol, to precipitate the DNA-dye complex. The DNA-dye complex was
|
|
separated on a filter, followed by washing several times with ethanol. The
|
|
DNA-dye complex after the washing was again dissolved in 2 ml of the
|
|
phosphate buffer, pH 6. The amount of the dye bonded to that of the DNA
|
|
was 0.5 .mu.g per .mu.g.DNA. The absorbance of the complex was measured at
|
|
wavelengths .lambda.=780 nm and .lambda.=260 nm, separately, to calculate
|
|
the concentrations of the dye and the DNA.
|
|
PAC EXAMPLE 10
|
|
PAR A 20-mer oligonucleotide having a base sequence partially complimentary to
|
|
the base sequence of a model target nucleic acid M13mp18 ss DNA was
|
|
synthesized by a DNA synthesizer 381 A, manufactured by ABI Co. Ltd. Then,
|
|
a primary amine was introduced into the 5' terminus of the oligonucleotide
|
|
by using a N-MMT-hexanol amine linker manufactured by Milligen Co. Ltd.,
|
|
instead of general amidide reagents. A predetermined protocol was followed
|
|
to perform cutting out from the CPG-support, deprotection (including the
|
|
deprotection of monomethoxytrityl group as a protective group of the
|
|
primary amine), and the purification by high-performance liquid
|
|
chromatography.
|
|
PAR After mixing together 200 .mu.g of the oligonucleotide, 100 .mu.l of 1M
|
|
sodium carbonate buffer, pH 9.0, and 700 .mu.l of water, 2 mg of a dye No.
|
|
46 (.lambda.max=810 nm) shown in Table 5, which had preliminarily been
|
|
dissolved in 200 N1 of dimethyl formamide, was gradually added under
|
|
agitation. After the reaction at room temperature for 24 hours, the peak
|
|
of the nucelic acid was decreased on a high-performance liquid
|
|
chromatogram, whereas a new peak having the absorbances of the nucleic
|
|
acid and the dye developed. Thus, the reaction solution was nearly
|
|
purified on a gel filtration column, NAP-50, manufactured by Pharmacia,
|
|
which was then purified by HPLC to obtain 175 .mu.g of the nucleic
|
|
acid-dye complex. Complex stability under storage
|
|
PAR In order to examine the stability under storage of dye complexes, the
|
|
following experiments were carried out.
|
|
PAR The labeled dye complexes prepared in Examples 6 to 10 were prepared to
|
|
predetermined concentrations with 10 mmol phosphate buffer, pH 7.2. The
|
|
solutions of the labeled complexes were kept in dark at 7.degree. C. for
|
|
three days. At the initiation and termination of the test of complex
|
|
stability under storage, the absorbance was measured at predetermined
|
|
wavelengths. Then, the ratio of the absorbance at the termination was
|
|
calculated, provided that the absorbance at the initiation was designated
|
|
as 100.
|
|
PAR The results are shown in Table 7.
|
|
TBL TABLE 7
|
|
______________________________________
|
|
Stability under storage of labeled dye complexes
|
|
Change in absorbance
|
|
Wavelength
|
|
(initial absorbance
|
|
for was designated
|
|
Example
|
|
Concentration
|
|
measurement
|
|
as 100)
|
|
______________________________________
|
|
6 0.5 g/ml 819 95.1
|
|
7 0.5 g/ml 825 94.5
|
|
8 0.5 g/ml 805 91.3
|
|
9 0.4 g/ml 780 95.7
|
|
10 0.4 g/ml 810 93.9
|
|
______________________________________
|
|
PAR As is shown in Table 7, the labeled dye complexes of the present invention
|
|
showed lower change of the absorbance in water than those of Comparative
|
|
Example.
|
|
PAC EXAMPLE 11
|
|
PAR A 20-mer oligonucleotide having a base sequence partially complimentary to
|
|
the base sequence of a model target nucleic acid M13mp18 ss DNA was
|
|
synthesized by a DNA synthesizer 381 A, manufactured by ABI Co. Ltd. Then,
|
|
by using a deoxyuridylic acid derivative monomer:
|
|
##STR98##
|
|
with an amino group introduced, instead of general amidide reagents, 20
|
|
such deoxyuridylic acid derivatives each having a primary amine group were
|
|
added to the 5' terminus of the oligonucleotide. Routine method was
|
|
followed to perform cutting out from the CPG-support, deprotection
|
|
(including the deprotection of trifluoroacetyl group as a protective group
|
|
of the primary amine), and the purification by high-performance liquid
|
|
chromatography.
|
|
PAR After mixing together 200 .mu.g of the oligonucleotide bonding the primary
|
|
amines, 100 .mu.l of 1M sodium carbonate buffer, pH 9.0, and 700 .mu.l of
|
|
water, 5 mg of a dye No. 27 (.lambda.max=826 nm) shown in Table 1, which
|
|
had preliminarily been dissolved in 200 .mu.l of dimethyl formamide, was
|
|
gradually added under agitation. After the reaction at 40.degree. C. for
|
|
24 hours, the peak of the nucleic acid was decreased on a high-performance
|
|
liquid chromatogram, whereas a new peak having the absorbances of the
|
|
nucleic acid and the labeling agent developed. Thus, the reaction solution
|
|
was nearly purified on a gel filtration column, NAP-50, manufactured by
|
|
Pharmacia, which was then purified by HPLC to obtain 350 .mu.g of the
|
|
nucleic acid-labeling agent complex. The absorbance of the nucleic
|
|
acid-labeling agent complex at 826 nm had the intensity about 20-fold that
|
|
of the nucleic acid-labeling agent shown in Example 5.
|
|
PAR In accordance with the present invention, a stable complex can be formed
|
|
with less decomposition of dyes, and hence with less change in absorbance
|
|
or with less change in fluorescence, by bonding a labeling agent of a
|
|
particular structure to a substance from a living organism.
|
|
PAR Therefore, the complex of the present invention can provide a reagent with
|
|
excellent stability under storage for the application to microanalysis.
|
|
CLMS
|
|
STM What is claimed is:
|
|
NUM 1.
|
|
PAR 1. A labeled complex for detecting a subject compound to be analyzed by
|
|
means of optical means using near-infrared radiation which complex
|
|
comprises a substance from a living organism and a labeling agent fixed
|
|
onto the substance, the substance capable of specifically binding to the
|
|
subject compound, wherein the labeling agent comprises a compound
|
|
represented by the general formula (IV):
|
|
##STR99##
|
|
wherein A, B, D and E are independently selected from the group consisting
|
|
of hydrogen atom, a substituted or an unsubstituted alkyl group having two
|
|
or more carbon atoms, alkenyl group, aralkyl group, aryl group, styryl
|
|
group and heterocyclic group, and at least one of A and B is a substituted
|
|
or unsubstituted aryl group, and at least one of D and E is a substituted
|
|
or unsubstituted aryl group;
|
|
PA1 r.sub.1 ' and r.sub.2 ' are individually selected from the group consisting
|
|
of hydrogen atom, a substituted or an unsubstituted alkyl group, cyclic
|
|
alkyl group, alkenyl group, aralkyl group and aryl group; k is 0 or 1; is
|
|
0, 1 or 2; and X.sub.2.sup..crclbar. represents an anion.
|
|
NUM 2.
|
|
PAR 2. The labeled complex according to claim 1, wherein the substance from a
|
|
living organism is an antibody or an antigen.
|
|
NUM 3.
|
|
PAR 3. The labeled complex according to claim 1, wherein the substance from a
|
|
living organism is a nucleic acid.
|
|
NUM 4.
|
|
PAR 4. The labeled complex according to claim 1, wherein the substituted aryl
|
|
group constituting at least one of A and B is phenyl group substituted by
|
|
dialkylamino group.
|
|
NUM 5.
|
|
PAR 5. The labeled complex according to claim 1, wherein the substituted aryl
|
|
group constituting at least one of D and E is phenyl group substituted by
|
|
dialkylamino group.
|
|
NUM 6.
|
|
PAR 6. The labeled complex according to claim 4 or 5, wherein the dialkylamino
|
|
group is a diethylamino group.
|
|
NUM 7.
|
|
PAR 7. The labeled complex according to claim 1, wherein each of A, B and D is
|
|
dimethylaminophenyl group, E is aminophenyl group, k is 0 and l is 1.
|
|
NUM 8.
|
|
PAR 8. The labeled complex according to claim 1, wherein each of A, B and D is
|
|
diethylaminophenyl group, E is phenyl group substituted by carboxyl group,
|
|
k is 0 and l is 1.
|
|
NUM 9.
|
|
PAR 9. The labeled complex according to claim 1, wherein each of A, B, D and E
|
|
is diethylaminophenyl group, k is 1 and l is 0.
|
|
NUM 10.
|
|
PAR 10. The labeled complex according to claim 1, wherein each of A, B, and D
|
|
is diethylaminophenyl group, E is aminophenyl group, K is 0 and l is 1.
|
|
NUM 11.
|
|
PAR 11. The labeled complex according to claim 1, wherein A is
|
|
dimethylaminophenyl group, each of B and E is ethoxyphenyl group, k is 0,
|
|
1 is l and D is represented by the following formula:
|
|
##STR100##
|
|
NUM 12.
|
|
PAR 12. A method of detecting a subject compound to be analyzed in a sample
|
|
comprising the steps of:
|
|
PA1 providing a labeled complex comprising a substance from a living organisms
|
|
and a labeling agent fixed onto the substance, the substance being capable
|
|
of specifically binding to the subject compound;
|
|
PA1 binding the labeled complex to the subject compound; and
|
|
PA1 detecting the labeled complex to which the subject compound is bonded by
|
|
means of optical means, wherein the labeling agent comprises a compound
|
|
represented by the general formula (IV):
|
|
##STR101##
|
|
wherein A, B, D and E are independently selected from the group consisting
|
|
of hydrogen atom, a substituted or an unsubstituted alkyl group having two
|
|
or more carbon atoms, alkenyl group, aralkyl group, aryl group, styryl
|
|
group and heterocyclic group, and at least one of A and B is a substituted
|
|
or unsubstituted aryl group, and at least one of D and E is a substituted
|
|
or unsubstituted aryl group;
|
|
PA1 r.sub.1 ' and r.sub.2 ' are individually selected from the group consisting
|
|
of hydrogen atom, a substituted or an unsubstituted alkyl group, cyclic
|
|
alkyl group, alkenyl group, aralkyl group and aryl group; k is 0 or 1; is
|
|
0, 1 or 2; and X.sub.2.sup..crclbar. represents an anion.
|
|
NUM 13.
|
|
PAR 13. The method according to claim 12, wherein the substance from a living
|
|
organism is an antibody or an antigen.
|
|
NUM 14.
|
|
PAR 14. The method according to claim 12, wherein the substance from a living
|
|
organism is a nucleic acid.
|
|
NUM 15.
|
|
PAR 15. The analyzing method according to any one of claims 12, 13 and 14,
|
|
wherein the optical means is an optical means using near-infrared ray.
|
|
NUM 16.
|
|
PAR 16. The method according to claim 12, wherein each of A, B and D is
|
|
dimethylaminophenyl group, E is aminophenyl group, k is 0 and l is 1.
|
|
NUM 17.
|
|
PAR 17. The method according to claim 12, wherein each of A, B and D is
|
|
diethylaminophenyl group, E is phenyl group substituted by carboxyl group,
|
|
k is 0 and l is 1.
|
|
NUM 18.
|
|
PAR 18. The method according to claim 12, wherein each of A, B, D and E is
|
|
diethylaminophenyl group, k is 1 and l is 0.
|
|
NUM 19.
|
|
PAR 19. The method according to claim 12, wherein each of A, B and D is
|
|
diethylaminophenyl group, E is aminophenyl group, k is 0 and l is 1.
|
|
NUM 20.
|
|
PAR 20. The method according to claim 12, wherein A is dimethylaminophenyl
|
|
group, each of B and E is ethoxyphenyl group, k is 0, l is 1 and D is
|
|
represented by the following formula:
|
|
##STR102##
|