Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-01
2003-05-27
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S389100
Reexamination Certificate
active
06569634
ABSTRACT:
TECHNICAL FIELD
This invention relates to an immunological method for measuring human thymidylate synthase and also to a kit for the same.
BACKGROUND ART
Thymidylate synthase (EC2.1.1.45, hereinafter called “TS”) is an enzyme that catalyzes a reaction in which thymidylic acid is formed from deoxyuridylic acid, plays a role to supply thymine which is a base specific to DNAs, and is one of principal rate-limiting enzymes for a DNA precursor supply pathway. Accordingly, its activity is known to become higher in normal or tumor tissues where cell growth is active.
On the other hand, fluoropyrimidine antitumor drugs such as 5-fluorourasil and 5-fluorodeoxyuridine act against TS as a target enzyme, and for example, 5-fluorodeoxyuridine changes into fluorodeoxyuridylic acid in vivo and inhibits TS. In particular, fluoropyrimidine antitumor drugs are known to show high administration effect and marked life prolongation effect for patients with a small amount of TS in tumor cells but to exhibit low administration effect for patients with TS in a large amount [“Gan to Kagaku Ryoho (Cancers and Chemotherapy)”, 24(6), 705-712 (1997)]. The importance of TS is therefore high, for example, an advance measurement of the amount of TS in an excised tumor upon treatment of a tumor patient gives indications for the determination of a treatment method and for the selection of an antitumor drug.
As conventional TS measuring methods, methods which involve biochemical measurement of its enzyme activity are practiced primarily, including, for example, a method in which formation of dihydrofolic acid as a reaction product is measured by an absorbance at a specific wavelength and a method in which the amount of radiolabeled fluorodeoxyuridylic acid (FdUMP) (for example, [3H]FdUMP) which binds to TS is measured.
On the other hand, immunological measuring methods making use of anti-TS antibodies have also been reported. Known methods include, for example, a method in which TS is measured by using anti-TS monoclonal antibodies M-TS-4 and M-TS-9 [Malgorzata M. Jastreboff et al., Biochemistry, 1985(24), 587-592]; and a method in which TS is detected using anti-TS monoclonal antibodies TA 102, TS 105, TS 106, TS 109, TS 110, TS 11A and TS 11B [Japanese Language Laid-Open Publication (PCT) No. HEI 6-507314]. Also known are a blotting method in which various homogenized tumor cells are subjected to electrophoresis and subsequent to transferring, an anti-TS-IgG antibody and a labeled anti-IgG are used as a primary antibody and a secondary antibody, respectively [“Gan to Kagaku Ryoho (Cancers and Chemotherapy”, 24(6), 705-712 (1997)]; a method in which wells are coated with a standard TS antigen and a TS-containing test sample and an anti-TS antibody are then added to cause competition; and a method in which wells are coated with a TS-containing test sample, an anti-TS antibody is added and bound, and labeled anti-mouse IgG is then reacted.
However, the above-described methods which biochemically measure the enzyme activity of TS are accompanied by many problems in that inter alia the sensitivity is insufficient to measure enzyme activity of low level, use of fresh samples is needed, test samples must be carefully handled to avoid enzymatic degradation, test samples are required in large amounts, and special techniques and facilities are needed if a radioactive substance is handled.
Further, the conventional immunological measurement methods are not considered to be sufficient in the irksomeness of procedures and the accuracy of measurements. Described specifically, for example, the method in which a detection is performed by blotting subsequent to electrophoresis is accompanied by a problem in that it is poor in quantitativeness although its procedures are very complex, the method in which a test sample and an antibody are competitively reacted on antigen-coated wells involves a problem in that it has poor measurement sensitivity, and even the method in which a diluted solution of a test sample is coated on wells as it is involves a problem in that TS cannot necessarily be coated in its entirety, resulting in poor reliability. It is therefore the current situation that there is an outstanding demand for a simple and high-accuracy method for the measurement of TS.
DISCLOSURE OF THE INVENTION
With the foregoing circumstances in view, the present inventors have proceeded with various investigations. As a result, it has been found that use of an anti-human TS polyclonal antibody immobilized on an insoluble carrier is very effective for specifically and efficiently capturing a trace amount of human TS from a test sample which contains a great number of components (impurities), leading to the completion of the present invention.
The present invention therefore provides a method for immunologically measuring human thymidylate synthase with an anti-human TS antibody, wherein as the antibody, at least an anti-human TS polyclonal antibody immobilized on an insoluble carrier is used.
The present invention also provides a method for evaluating sensitivity of cancer cells to a fluoropyrimidine antitumor drug, which comprises measuring an amount of human TS contained in a sample of the cancer cells by the above-described method and determining the sensitivity from results of the measurement.
The present invention also provides a human TS measuring kit comprising an insoluble carrier with at least one anti-human TS polyclonal antibody immobilized thereon.
REFERENCES:
patent: 5998151 (1999-12-01), Johnston et al.
patent: 31 15 115 (1982-02-01), None
patent: 0126173 (1984-11-01), None
Tolleson et al. Bioconjugate Chem. 2:327-332, 1991.*
Johnston et al. Biochemical Pharmacology, 45/12:2483-2486, Jun. 1993.*
Okabe et al. Oncology Reports 4:685-690, 1997.*
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Okabe et al., Jpn J Cancer & Chemotherapy, vol. 24, No. 6, (Apr., 1997) pp 705-712.
Yamachika et al., Cancer, “A New Prognostic Factor for Colorectal Carcinoma, Thymidylate Synthase, and Its Therapeutic Significance”, 82,1 (Jan., 1998) pp. 70-77.
Men'eki Kagakuteki Duteihou, J. Clausen, 3rdEdition (1993), p. 182, including a copy of the cover page and imprint of the book, and an English translation of the relevant portion.
XP-002156191, Patrick G. Johnston, James C. Drake, Jane Trepel, and Carmen J. Allegra “Immunological Quantitation of Thymidylate Synthase Using the Monoclonal Antibody TS 106 in 50fluorouracil-sensitive and -resistant Human Cancer cells”, Cancer Research, vol. 52, pp. 4306-4312, Aug. 15, 1992.
XP002926146, Patrick G. Johnston, Chi-Ming Liang, Sally Henry, Bruce A. Chabner, and Carmen J. Allegra “Production and Characterization of Monoclonal Antibodies That Localize Human Thymidylate Synthase in the Cytoplasm of Human Cells and Tissue”, Cancer Research, vol. 51, 6668-6676, Dec. 15, 1991.
XP002926147, Malgorzata J. Jastreboff, Mary B. Todd, Harry L. Malech and Joseph R. Bertino “Isolation and Functional Effects of Monoclonal Antibodies Binding to Thymidylate Syntase”, Biochemistry 1985, vol. 24, pp. 587-592, 1985.
Fukushima Masakazu
Hoshino Nobuhiro
Matsuya Takeshi
Okabe Hiroyuki
Huff Sheela
Sughrue & Mion, PLLC
Taiho Pharmaceutical Co. Ltd.
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