WO1997036618A1 - Composition diagnostique pour maladies s'accompagnant de reactions auto-immunes declenchees par la decarboxylase d'acide 65k-glutamique - Google Patents
Composition diagnostique pour maladies s'accompagnant de reactions auto-immunes declenchees par la decarboxylase d'acide 65k-glutamique Download PDFInfo
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- WO1997036618A1 WO1997036618A1 PCT/JP1997/001104 JP9701104W WO9736618A1 WO 1997036618 A1 WO1997036618 A1 WO 1997036618A1 JP 9701104 W JP9701104 W JP 9701104W WO 9736618 A1 WO9736618 A1 WO 9736618A1
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- skin
- gad65
- partial peptide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0006—Skin tests, e.g. intradermal testing, test strips, delayed hypersensitivity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
Definitions
- the present invention relates to a method and a composition for diagnosing a disease associated with an autoimmune reaction caused by 65K glutamate decarboxylase.
- Diagnosis in daily practice of insulin-dependent diabetes is based on test results such as blood glucose, urine and glucose tolerance tests. These are mostly studies reflecting the condition of insulin deficiency, and it is difficult to diagnose using these tests early on in diseases where insulin secretion is not very impaired.
- Today, insulin-dependent diabetes mellitus is recognized as an endocrine disease in which auxin beta cells are destroyed by an autoimmune mechanism, resulting in an absolute lack of insulin. Therefore, it was hoped that the diagnosis could be made earlier by incorporating a test for detecting this autoimmune process.
- glutamate decarboxylase which is abundant in the brain, and attempts to detect an immune response to this molecule have begun (Nature 347: 151-156, 1990).
- Glutamate decarboxylase is an enzyme that produces the neurotransmitter 7-aminobutyric acid from glutamate. This enzyme has two isozymes with molecular weights of 65 K daltons and 67 K daltons, 65 K glutamate decarboxylase (GAD 65) and 67 K glutamate decarboxylase (GAD 67), respectively. is called. Subsequent searches have revealed that it is GAD 65 that elicits a stronger autoimmune response (J.C1 in. Invest. 1993: 91: 2084-2090). A humoral immune reaction, which is relatively easy to detect among the immune responses to GAD65, that is, an anti-GAD65 antibody was first searched.
- an ELISA (enzyme linked immunosorbent assay) method, an RIA (radioimmuno assay) method, an immunoprecipitation method, an immunoblot method, and the like are used as a method for measuring the anti-GAD65 antibody titer.
- an ELISA (enzyme linked immunosorbent assay) method, an RIA (radioimmuno assay) method, an immunoprecipitation method, an immunoblot method, and the like are used.
- insulin-dependent diabetes mellitus there is a form in which the disease develops in the early stage of the disease in non-insulin-dependent diabetes mellitus, and thereafter gradually decreases insulin secretion and shifts to insulin-dependent diabetes mellitus (slowly progressive disease).
- Insulin dependent diabetes mellitus This type of diabetes is prevalent in Japan and is estimated to account for about 5% of non-insulin-dependent diabetes (Diab TES CARE, 1993; 16: 780-788, Internal Medicine, 1995; 76: 31-35). .
- measuring the anti-GAD65 antibody titer early in the disease is thought to be useful for accurate diagnosis, but even in this case, the case with a higher antibody titer is earlier in insulin. It does not lead to lack. In such cases, it is important to understand the dynamics of not only humoral immunity but also cellular immunity in order to determine prognosis and select appropriate treatment.
- T cells can be calculated the material to become when the c capturing thymidine, tritium, thymidine amount labeled radioactive thymidine incorporated Ri by the keeping mixed into the medium at a beam of DNA synthesis when dividing.
- mononuclear cells are separated from about 10 ml of peripheral blood by density gradient centrifugation.
- thymidine After culturing for 3 to 7 days in an appropriate medium containing a partial peptide of GAD65, add radioactive thymidine and further culture for about 18 hours. After that, the amount of thymidine incorporated is calculated by collecting the cell fractions and measuring the radioactivity therein, and used as an index of cellular immunity.
- the current method of measuring cellular immunity by incorporating radioactive thymidine is difficult to use for actual diagnosis in the following points.
- radioactive thymidine is only permitted in certain controlled areas. Therefore, it cannot be performed in general clinical laboratories.
- a method using fluorescently labeled thymidine without using a radioactive substance has been devised, but is inferior to the radioactive thymidine method in terms of sensitivity and is not widely used.
- the required blood volume is at least 10 ml, and there is a problem in blood sampling in testing children who are the main target of insulin-dependent urinary disease.
- the culture of mononuclear cells from the patient's peripheral blood generally contains fetal calf serum.
- the reactivity of mononuclear cells to fetal bovine serum varies from one fetal serum to another, and it is not possible to simply compare the results of tests performed at different facilities. This is a consequence of the standardization of this inspection.
- the present invention provides a simple method for evaluating cell-mediated immunity against GAD65, thereby providing a disease associated with an autoimmune reaction induced by 65K glutamate decarboxylase. It is an object to provide a composition that allows the diagnosis of a disease.
- the present invention provides a method for evaluating cell-mediated immunity to GAD65 by a simple operation, and an autoimmune reaction induced by 65K glutamate decarboxylase by using the method. Disclosure of invention c also aims to provide a method for diagnosing a disease associated
- the present inventors injected GAD65 partial peptides intradermally into NOD mice, which are model animals that naturally develop insulin-dependent diabetes. Two days later, the skin at the injection site was observed to show changes such as swelling and redness. This indicates that it is possible to evaluate cell-mediated immunity by the skin reaction generated by administration of the GAD65 partial peptide, and the present invention has been completed based on such findings. .
- the present invention relates to a 65K-glutamic acid decarboxylase (65K-glutamic acid) capable of reacting with T cells in the skin of a mammal and at the surface of Z or skin to cause inflammation in the skin of the mammal.
- An acid decarboxylase: GAD65 or a partial peptide thereof is provided.
- a composition for diagnosing a disease associated with an autoimmune reaction induced by GAD65 by a percutaneous reaction is provided.
- the present invention also provides a method for producing an inflammation in the skin of a mammal by reacting with T cells in the skin of the mammal and / or on the surface of the skin, and in the skin of the mammal and / or on the surface of the skin.
- the obtained GAD65 or a partial peptide thereof is administered, and the size of a dermatitis caused by a percutaneous reaction between the GAD65 or the partial peptide and a T cell in the subject is measured.
- a method for diagnosing a disease associated with an autoimmune reaction caused by GAD65 is provided.
- the present invention provides a method for producing inflammation in the skin of a mammal by reacting with T cells in the skin of the subject, on the skin or on the surface of the skin, on the skin of the mammal, and on the surface of the skin or Z.
- Administering GAD65 or a partial peptide thereof, and measuring the magnitude of inflammation of the skin / ⁇ caused by a skin reaction between the GAD6 ⁇ 5 or a partial peptide thereof and T cells in the subject. Provides a method for evaluating cell-mediated immunity to GAD65.
- the present invention relates to a method for diagnosing a disease associated with an autoimmune reaction caused by GAD65 by a skin reaction, by reacting with T cells in the skin of a mammal and on the surface of the skin or the skin of the mammal. It also provides the use of GAD 65 or a partial peptide thereof, which can cause inflammation in the skin.
- the present invention also relates to a method for producing a composition for diagnosing a disease associated with an autoimmune reaction elicited by GAD65 by a skin-capture reaction, wherein the disease is intradermally and / or on the surface of a mammal. Also provided is the use of GAD65 or a partial peptide thereof, which can react with cells to cause inflammation in the mammalian seedlings.
- FIG. 1 is a diagram showing the time course of footpad swelling of NOD mice to which GAD65 partial peptide was administered.
- FIG. 2 is a diagram showing swelling of the footpad of a NOD mouse by administration of various peptide antigens (GAD65 partial peptide, cyclophilin and carboxyl carrier protein).
- Fig. 3 shows the footprint of the GAD65 partial peptide in mice of each strain (NOD, C3H / He, C57BL, Ba1b / c, BIO.A and DBA).
- Fig. 4 shows the T cell embryos induced by GAD65 partial peptide administration in mice of each strain (NOD, C3H / He, C57BL, Ba1b / c, BIO.A and DBA). It is a figure which shows a weakening reaction.
- FIG. 5 shows the foot-pat swelling response of the GAD65 partial peptide in mice of each strain (NOD, C3H / He, C57BL, BalbZc, BIOA and DBA).
- FIG. 4 is a diagram showing a correlation between and the T cell embryogenic reaction.
- GAD glutamic acid decarboxylase
- GAD67 and GAD65 there are two isoforms, GAD67 and GAD65, and in the present invention, GAD65 having a molecular weight of 65,000 and consisting of 585 amino acids is used.
- GAD65 of human, rat, mouse, mouse, etc. has been isolated and its amino acid sequence has been determined (see Fig. 2 of Proc. Natl. Acad.
- GAD65 is human, rat, Because of high homology between mice, any of these can be used. From the viewpoint of early diagnosis and prognosis of human insulin-dependent diabetes, the use of human GAD65, which is the original recognition antigen of human T cells, is preferred in terms of improving detection sensitivity. No. In the present invention, GAD 65 does not necessarily have to be a protein having the entire amino acid sequence.
- the peptide may be a partial peptide, for example, a peptide selected from a range of up to about 120 amino acids as counted from the terminal side of 0805 (: SEQ ID NO: 1 And a peptide consisting of the amino acid sequence of SEQ ID NO: 2.
- GAD65 is used in view of peptide length, which is easily synthesized by a solid phase method.
- a mixture of peptides consisting of 10 to 30 amino acids, preferably 20 to 26 amino acids, selected from the range of about 120 amino acid sequences counted from the C-terminal side is used.
- An example thereof is a peptide consisting of the amino acid sequence of SEQ ID NO: 3 to 6.
- peptides are amino acids 473 to 498 of the human GAD65 partial peptide of SEQ ID NO: 2, respectively.
- Acid sequence, amino acids 494-518 The amino acid sequence corresponds to the amino acid sequence at positions 514 to 538 and the amino acid sequence at positions 534 to 558.
- Amino acid sequence at positions 508 to 509 in the amino acid sequence of the human GAD65 partial peptide of SEQ ID NO: 2 (SEQ ID NO: 2) Is Tyr-Ile, but it is Phe-Val in the mouse amino acid sequence (the 9th to 10th sequence of the amino acid sequence of SEQ ID NO: 1) at the corresponding site. You may change it.
- a mixture of peptides of about 30 amino acids can be easily obtained by chemical synthesis of each peptide by a solid phase method.
- GAD65 or its partial peptide can be in any form as long as it can be administered to the skin of mammals such as humans and / or to the surface of the skin. Preferably, it is in a dosage form that can be administered. Specifically, for example, 25 to 75% by weight, preferably about 40 to 60% by weight of GAD 65, which is a diagnostically effective amount, is dissolved or suspended in a physiological saline solution or the like. Things can be mentioned.
- potassium alum can be added to prevent diffusion in the skin. For this purpose, a precipitate is formed with a 10% solution of carimyoban, a 10% solution of sodium carbonate and an antigen peptide, and the precipitate is further resuspended in a physiological saline solution.
- the final amount of potassium alum in the injection solution is 0.2 to 0.4%.
- the solubility of GAD 65 or its partial peptide in the preparation is low, and A suspension of GAD65 or a partial peptide thereof is preferred.
- the above-mentioned preparation by adjusting the pH so as to be in the range of 6.0 to 7.0.
- a composition containing GAD65 or a partial peptide thereof is administered intradermally and Z or to the surface of the skin of a test individual, and the GAD65 or a partial peptide thereof and T cells in the test-individual are administered.
- GAD65-induced diseases associated with autoimmune reactions such as insulin-dependent diabetes mellitus, SUffman syndrome, and certain types of autoimmunity, by measuring the magnitude of skin inflammation caused by the f-reaction.
- Early diagnosis of thyroid disease, etc. Can be prognostic.
- Examples of the subject include mammals such as humans, mice, and rats.
- the diagnosis of insulin-dependent diabetes mellitus can be made by the following method. That is, the above-mentioned GAD65 or a partial peptide thereof is intradermally administered to the subject by subcutaneous injection or the like. Then, usually two days later, the skin is measured for changes such as swelling or redness. Alternatively, the above-mentioned GAD65 or its partial peptide may be adhered to the skin by pulling the skin (scratch test). Then, usually 48 hours later, changes such as swelling or redness of the skin are measured. This change, such as swelling or redness, may be due to the following mechanism.
- IDDM insulin-dependent diabetes mellitus
- T cells After GAD65 or its partial peptide is taken up by antigen-presenting cells and undergoes the process, antigen stimulation is transmitted to T cells.
- the resulting activated T cells trigger an inflammatory response, such as the release of various cytokines and the recruitment of mononuclear and multinuclear cell populations.
- the degree of the change such as swelling or redness of the skin corresponds well to the current IDDM diagnosis by radioactive thymidine incorporation, so the degree of the change in advance, for example, redness If a correlation between the size and degree of induration and the symptoms of IDDM is determined, diagnosis can be made easily.
- RNA was isolated from mouse brain and cDNA was synthesized using oligo dT primer and reverse transcriptase (this procedure was described in J.G1 in. Invest. 90, 160-164, 1992). According to the method). Using this cDNA solution as type II, PCR was performed using the two chemically synthesized primers described below.
- SEQ ID NO: 8 A DNA fragment (about 300 bp) obtained by double digesting the obtained PCR product with the restriction enzymes BamHI and Ndel was separated by agarose gel electrophoresis, and then the DNA purification kit from Qiagen was purified. Was used to purify the DNA.
- the expression vector pET14b (manufactured by Novagen, USA) was double-digested with the restriction enzymes BamHI and Ndel, and ligated to the approximately 300 bp DNA fragment obtained in the previous section using DNA ligase.
- a vector having the circularized DNA fragment incorporated therein was introduced into Escherichia coli BL21, which was used for expression. Specifically, this Escherichia coli was cultured at 37 ° C in a broth medium supplemented with 50 / zg / ml ampicillin until the absorbance at 600 mn became 0.7, and then IPTG was added to a final concentration of 0.5 mM. After further culturing at 37 ° C for 3 hours, the cells were collected.
- the peptide solution eluted from the '(GSSHHHHHHSSGLVPRGSH V-) (SEQ ID NO: 9) column was dialyzed against phosphate buffered saline (PBS). At this time, the peptide is insolubilized. Resuspended in PBS. In this state, it can be stably stored at 180 ° C for a long time.
- Example 2 To determine whether the hind limb swelling seen in Example 2 was a specific response to the GAD65 partial peptide, an experiment was performed using two peptides other than the GAD65 partial peptide. Select the rat cyclophilin (molecular weight 12000) and the carboxyl carrier protein (CCP: molecular weight 22000) of Escherichia coli that are not homologous in primary structure to GAD65. In the same manner as in Example 1), the expression vector pET14b was used to express in escherichia coli a form containing six histidine residues at the N-terminus, followed by purification using a nickel chelate column.
- CCP carboxyl carrier protein
- Example 5 T cell blastogenesis during antigen stimulation by GAD65 partial peptide
- the incorporation of radioactive thymidine was measured using the same GAD65 partial peptide as the stimulating antigen used in the experiment of Example 2 ( J. Clim. Invest. 94, 2125-2129, 1994 J. Ex P. Med. 180, 1979-1984, 1994).
- the mice used were N0D mice, four types of strains that do not spontaneously develop insulin-dependent diabetes: C3H / He, Ba1b / c, BIO.A, C57BL, and insulin. A total of six DBAs were spontaneously developing non-dependent diabetes.
- the uptake of radioactive thymidine in NOD mice, which spontaneously develop insulin-dependent diabetes mellitus was significantly higher than in other strains of mice.
- the cell-mediated immune response was found to be higher than in other strains of mice.
- Example 6 Correlation between footpad swelling reaction and uptake of radioactive thymidine Incorporation of radioactive thymidine (the experiment in Example 5), which is a method for examining existing cellular immunity, and a method for evaluating cellular immunity by the method of the present invention
- the correlation with the result of the intradermal reaction was examined. As shown in FIG. 5, a significant correlation was found between the two with a correlation coefficient (R) power of .809 and a P value of 0.05 or less. Therefore, it was found that the GAD65-based intradermal reaction method can sufficiently evaluate the cellular immune response instead of the conventional radioactive thymidine incorporation method.
- a composition comprising GAD65 or a partial peptide thereof of the present invention is placed in the skin and / or on the surface of the skin of a subject, and after an appropriate period of time, the swelling and redness of the skin captive
- a very simple operation of observing the change allows the evaluation of cellular immunity against the antigen.
- pre-onset diagnosis and onset of diseases such as insulin-dependent diabetes, which were difficult only by measurement of humoral immunity, were made possible. Later prognosis can be determined.
- it has the potential to be used for screening purposes in school children and non-insulin-dependent diabetic patients because it requires less time and effort than conventional measurement of humoral immunity.
- Organism name human
- Organism name human
- Sequence type nucleic acid
- Sequence type other nucleic acid synthetic DNA
- Sequence type nucleic acid PT
- Sequence type other nucleic acid synthetic DNA
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU20441/97A AU2044197A (en) | 1996-04-01 | 1997-03-31 | Diagnostic composition for diseases accompanied by autoimmune reactions caused by 65k-glutamic acid decarboxylase |
EP97908553A EP0923944A4 (en) | 1996-04-01 | 1997-03-31 | DIAGNOSTIC COMPOSITION FOR DISEASES ACCOMPANIED BY AUTO-IMMUNE REACTIONS TRIGGERED BY 65K-GLUTAMIC ACID DECARBOXYLASE |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP7887896 | 1996-04-01 | ||
JP8/78878 | 1996-04-01 |
Publications (1)
Publication Number | Publication Date |
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WO1997036618A1 true WO1997036618A1 (fr) | 1997-10-09 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/JP1997/001104 WO1997036618A1 (fr) | 1996-04-01 | 1997-03-31 | Composition diagnostique pour maladies s'accompagnant de reactions auto-immunes declenchees par la decarboxylase d'acide 65k-glutamique |
Country Status (3)
Country | Link |
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EP (1) | EP0923944A4 (ja) |
AU (1) | AU2044197A (ja) |
WO (1) | WO1997036618A1 (ja) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06505385A (ja) * | 1991-02-22 | 1994-06-23 | アムラド・コーポレイション・リミテッド | グルタミン酸デカルボキシラーゼ自己抗原関連疾患の診断及び処置方法 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3626054A (en) * | 1967-10-25 | 1971-12-07 | Bard Hamilton Co Inc | Lupus erythematosus skin test |
US5674978A (en) * | 1990-09-21 | 1997-10-07 | The Regents Of The University Of California | Peptides derived from glutamic acid decarboxylase |
CN1112933A (zh) * | 1994-01-20 | 1995-12-06 | 伯伦格·曼海姆有限公司 | 抗原特异性活化t淋巴细胞,其检测和用途 |
DE19526561A1 (de) * | 1995-07-20 | 1997-01-23 | Boehringer Mannheim Gmbh | In-vivo-Diabetes-Test |
-
1997
- 1997-03-31 EP EP97908553A patent/EP0923944A4/en not_active Withdrawn
- 1997-03-31 WO PCT/JP1997/001104 patent/WO1997036618A1/ja not_active Application Discontinuation
- 1997-03-31 AU AU20441/97A patent/AU2044197A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06505385A (ja) * | 1991-02-22 | 1994-06-23 | アムラド・コーポレイション・リミテッド | グルタミン酸デカルボキシラーゼ自己抗原関連疾患の診断及び処置方法 |
Non-Patent Citations (3)
Title |
---|
JOURNAL OF AUTOIMMUNITY, 7, (1994), pages 643-653. * |
NATURE, 347, (13-9-1990), pages 151-156. * |
See also references of EP0923944A4 * |
Also Published As
Publication number | Publication date |
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AU2044197A (en) | 1997-10-22 |
EP0923944A4 (en) | 2000-05-03 |
EP0923944A1 (en) | 1999-06-23 |
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