WO2000037940A1 - Diagnostic method for autoimmune arthritis - Google Patents
Diagnostic method for autoimmune arthritis Download PDFInfo
- Publication number
- WO2000037940A1 WO2000037940A1 PCT/US1999/001499 US9901499W WO0037940A1 WO 2000037940 A1 WO2000037940 A1 WO 2000037940A1 US 9901499 W US9901499 W US 9901499W WO 0037940 A1 WO0037940 A1 WO 0037940A1
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- WO
- WIPO (PCT)
- Prior art keywords
- collagen
- type
- antibody
- elisa
- reactivity
- Prior art date
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Definitions
- the present invention relates to a method of assaying with high sensitivity an antibody to a type II collagen for diagnosis of the probability of the onset of autoimmune arthritis, for diagnosis of autoimmune clinical rheumatoid arthritis, and also for diagnosis of the severity of autoimmune clinical rheumatoid arthritis.
- Another object of the present invention is to provide an agent for conducting the above- mentioned assay method.
- Fig. 1 is a schematic illustration of examples of ELISA plates for assaying antibodies to chick (CII), bovine(BII), and porcine(PII) and human type II collagen (HII) in human sera.
- Fig. 2 is a graph showing a typical standard curve obtained by the ELISA of the present invention.
- Fig. 3A and Fig. 3B are graphs showing the assay results with respect to patients with RA, and negative control sera, obtained by a conventional ELISA using BSA-Tween as a blocking agent and an improved ELISA using heterologous animal serum as a blocking agent, which is used as a blocking agent in the present invention.
- Fig. 4 is a graph showing standard curves for the assay of human type II collagen antibodies obtained by conventional ELISA using Peroxidase-conjugated rabbit anti-human IgG antibodies as a secondary antibody, further improved ELISA using an Avidin-Biotin system (A-B), and the ELISA of the present invention.
- Fig. 5 shows graphs indicating a correlation between serum IgG autoantibody levels to type II collagen and arthritis markers in patients with RA (SJ: the number of swollen joints, ESR: erythrocyte sedimentation rate, CRP: concentration of C-reactive protein, RF: rheumatoid factor).
- Fig. 6 shows graphs indicating a correlation between serum IgG autoantibody level to type II collagen in patients with RA and serum immunoglobulin levels thereof.
- Fig. 7 shows graphs indicating the changes in the antibody levels in sera from patients with relapsing polychondritis who were treated with prednisolone.
- Fig. 8 shows the results of tests analyzing the antibody reactivity to CB-peptide fragments of human type II collagen in individual patients with RA. DESCRIPTION OF THE PREFERRED EMBODIMENTS
- the inventor of the present invention has discovered a method of assaying type II collagen with high assay sensitivity in the course of this study of removing various shortcomings of the conventional method. More specifically, the present invention provides a method of assaying cross-reactivity between an antibody to a heterologous type II collagen and an antibody to an autologous type II collagen, which can be performed by ELISA.
- the present invention provides an ELISA in which such a type II collagen is assayed under conditions where the presence of bacteria is suppressed, based on the discovery that non-specific reactions are induced in the course of the assay of the type II collagen.
- the ELISA which is performed under conditions where the presence of bacteria is suppressed makes it possible to assay with high assay sensitivity the antibody to the type II collagen, so that the diagnosis of the probability of the onset of autoimmune arthritis, the diagnosis of autoimmune clinical rheumatoid arthritis, and also the diagnosis of the severity of autoimmune clinical rheumatoid arthritis, which cannot be performed by the conventional method, can be performed by the method of the present invention.
- (1) assaying cross-reactivity between an antibody to a heterologous type II collagen and an antibody to an autologous type II collagen, and (2) determining the ratio of the reactivity of an antibody to a heterologous type II collagen to the reactivity of an antibody to an autologous type II collagen for diagnosing early or autoimmune clinical rheumatoid arthritis, are carried out by an immunological method.
- immunological methods Varieties of immunological methods can be employed in the present invention.
- ELISA is preferable for use in the present invention because non-specific reactions can be easily controlled.
- type II collagen for use in the present invention for instance, human, chick, bovine, and porcine type II collagens, can be employed. These collagens are easily available.
- the reaction is carried out in a buffer, preferably in a phosphate buffer. It is preferable to add an antibacterial agent such as sodium azide to such a buffer to prevent the buffer from being contaminated with bacteria or to prevent the growth of bacteria in the buffer, in an amount sufficient for exhibiting a sufficient antibacterial effect, but which does not have adverse effects on immunological reactions and assays, usually in a range of about 0.005% to 0.5%.
- an antibacterial agent such as sodium azide
- a mixed solution is incubated in the well at a temperature in a range of 0 to 35 C , preferably at about 4 C , for 2 to 24 hours. Furthermore, the surface of the type II collagen bound well is then treated with animal serum. It is preferable that the animal serum used here be serum from the same animal as that derived from a second antibody used for the detection of human immunoglobulin.
- a sample such as serum is diluted with buffered animal serum containing an antibacterial agent such as azide and placed in the wells of an ELISA plate, and incubated at a temperature in a range of 0 to 35 C , preferably at about 4 C, for 2 to 24 hours. After incubation, any unreacted antibody is removed by washing the well.
- an antibacterial agent such as azide
- conjugated antibody (the second antibody) is then added to the well and incubated. After incubation, any unreacted conjugated antibody is removed by washing the well. This incubation is carried out at about room temperature for about 30 minutes to 12 hours.
- conjugated antibodies for use in the assay include enzyme-, biotin-, and hap ten-conjugated antibodies.
- conjugated antibodies are animal derived antibodies such as anti-human IgG antibody, anti-human IgA antibody, and anti-human IgM antibody. Such antibodies may be antibody fragments such as Fab' and F(ab')2. It is preferable that the antibodies be antibodies specifically bound to an Fc portion of human immunoglobulin.
- the activity of the enzyme can be measured by adding a substrate to the enzyme.
- enzymes are peroxydase (POD), alkali phosphatase, and /A-galactosidase.
- substrates are varieties of coloring substrates, fluorescent substrates, and luminescent substrates.
- biotin or hapten is used as the labeling material, the assay can be performed using enzyme-conjugated avidin, enzyme-conjugated streptoavidin, and enzyme-conjugated anti-hapten antibody.
- the enzyme the above-mentioned enzymes can be used, and as the substrate, the above-mentioned corresponding substrate can be used.
- the result of the measured cross-reactivity between the antibody to the heterologous type II collagen and an antibody to the autologous type II collagen be used for diagnosing the probability of the onset of autoimmune arthritis in comparison with the type of a major histocompability complex (MHC).
- MHC major histocompability complex
- the result of the measured ratio of the reactivity of the antibody to a heterologous type II collagen to the reactivity of the antibody to the autologous type II collagen can be used for diagnosing early or autoimmune clinical rheumatoid arthritis in comparison with the type of a major histocompability complex (MHC).
- MHC major histocompability complex
- the ratio of the reactivity can be calculated by dividing the antibody level for the autologous type II collagen by the antibody level for the heterologous type II collagen.
- ELISA plates were coated overnight at 4 C with chick (CII) or human (H ⁇ I)type II collagen (5 g/ml) dissolved in 0.15M phosphate buffer containing 0.05% of sodium azide, with the respective solution placed in the wells thereof. The wells of the plates were washed 3 times with wash buffer. With the placement of
- ELISA plates for reference blank wells were prepared by repeating the above- mentioned procedure except that neither the chick (CII) nor human (H ⁇ I)type II collagen (5 g/ml) was dissolved in the 0.15M phosphate buffer containing the 0.05% of sodium azide therein, and dissolved in the 0.15M phosphate buffer without containing the 0.05% of sodium azide therein.
- Each serum sample was diluted 100 fold and 100 1 of each diluted serum sample was placed in each well of the ELISA plates and incubated at 4 C overnight. Each well of the ELISA plates was washed with wash buffer.
- Biotin-conjugated goat anti-human IgG (5 1, Fc specific) antibody was dissolved in 10 ml of 25%> NGS, and added to each of the wells (100 1/well).
- the ELISA plates were incubated at room temperature for 2 hours, and were then washed 6 times with wash buffer.
- one Urea-H 2 O 2 plus buffer tablet and one o-phenylenediamine (OPD) tablet (Sigma, St. Louis, MO) were dissolved in 20 ml of glass- distilled water, and 100 1 of the solution was added to each of the wells.
- the ELISA plates were incubated at room temperature for 30 minutes so as to develop a color in each of the wells. The incubation was stopped by adding 50 1 of 2.5N sulfuric acid to each well.
- the ELISA plates were coated overnight at 4 C with chick (CII), bovine (BII), porcine (PII) or human (HII) type II collagen (5 g/ml) dissolved in 0.15M phosphate buffer containing 0.05% of sodium azide.
- CII chick
- BII bovine
- PII porcine
- HII human type II collagen
- well for this purpose were not coated with collagen but were treated with buffer only containing sodium azide, with the placement of the respective solution in the wells of each ELISA plate.
- the wells of the plates were washed 3 times with wash buffer. After the addition of 100 1 of full strength buffered NGS to the wells, the plates were incubated at room temperature for 1 hour and blocked. The wells were then washed 3 times with wash buffer.
- Reference standards were prepared from the serum of a single monkey which was immunized with CII/CFA.
- the serum was diluted 1 : 100,000 with buffered NGS and defined as 16 units/ml and kept at -20 C as a stock solution. This stock solution was diluted serially with buffered NGS to make 8, 4, 2, 1, 0.5 and 0.25 units/ml just before use.
- the diluted samples (100 1) were placed in the above-mentioned type II collagen- coated wells and also in uncoated wells.
- the reference standards (0.25-16 units/ml, 100 1) were added to the standard strips as shown in Fig.1.
- Biotin-conjugated goat anti-human IgG (5 1, Fc specific) antibody was dissolved in 10 ml of 25% NGS, and placed in all wells (100 1/well).
- the plates were incubated at room temperature for 2 hours and washed 6 times with wash buffer. Furthermore, 2.5 1 of Strept-avidin-Peroxidase was dissolved in 10 ml of 1% OVA-0.05% Tween 20 solution, pH 7.5, and added to all wells (100 1/well). The plates were incubated at room temperature for 1 hour and washed with wash buffer.
- one Urea-H 2 O 2 plus buffer tablet and one OPD tablet were dissolved in 20 ml of distilled water, and 100 1 of the solution was added to each of the wells. The plates were incubated at room temperature for 30 minutes so as to develop a color in each of the wells.
- the reaction was stopped by adding 50 1 of 2.5N sulfuric acid to each well.
- OD values were determined at 490 nm (a 650 nm filter was used as a reference). When the OD values were more than the OD value of the highest reference standard (16 units/ml), all the samples were re-assayed at a higher dilution (1 :200 - 1 : 10,000).
- OD values of reference standards calculated in the above Step 1 were plotted as the ordinate against the units/ml of antibody standard as the abscissa.
- Fig. 2 shows the results of a representative experiment where the standard range was from 0.25 to 16 units/ml.
- anti-human type II collagen antibodies in cynomolgus monkeys were assayed by the ELISA of the present invention and also by a conventional ELISA, whereby the standard curve of the ELISA of the present invention and the standard curve of a conventional ELISA as shown in Fig. 4 were obtained.
- the ELISA of the present invention exhibits an assay sensitivity as high as 100 times the assay sensitivity of the improved ELISA, and an assay sensitivity as high 10 times the assay sensitivity of the further improved ELISA (A-B).
- the anti-collagen antibodies in sera from patients with RA were assayed by the ELISA of the present invention and the prevalence of the anti-collagen antibodies determined by the ELISA of the present invention and that determined by the prior ELISA (A-B) were compared.
- ELISA plates were coated overnight at 4 C with chick (CII), bovine (BII), porcine (PII) or human (HII) type II collagen (5 g/ml) dissolved in 0.15M phosphate buffer, with the placement of each of the respective solutions in the wells of the ELISA plates, and the coated ELISA plates were blocked, using a buffered NGS at room temperature for 1 hour.
- serum samples were diluted 1 :100, whereas the conventional ELISA (A-B), serum samples were diluted 1 :50.
- the prevalence of the autoantibodies to IgA and IgG anti-type II collagens were respectively determined to be only 23.6% and 13.1% of patients with RA by the further improved ELISA (A-B).
- A-B further improved ELISA
- the ELISA of the present invention with respect to IgG antibodies (10 units/ml or more), the prevalence was determined to be as high as 83% of Japanese patients with RA and 90% of American patients with RA.
- IgG antibodies 26 units/ml or more
- the prevalence was determined to be as high as 77% of Japanese patients with RA and 63% of American patients with RA.
- IgG antibodies to CII and BII were detected in 90% or more of patient sera by the ELISA of the present invention. This indicates that antibody production to dietary collagen is a very common biological phenomenon in humans. Antibodies to heterologous type II collagen cross-reacted with autologous human type II collagen to some degree in most patients with RA.
- IgG anti-HII antibody levels in patients with DR-0405/0405 (Gl: HLA +/+) and DR0405/1405, and those in patients with 0405/1501 (G2: HLA +/-) were respectively as low as 98 ⁇ 75 and 37 ⁇ 9 units/ml, since the cross-reactivity of anti-BII or CII antibodies to HII was less than 20% ("+” denotes alleles that are believed to be RA-susceptible, "-" denotes alleles that are believed not to be RA- susceptible).
- IgG antibody levels in patients with RA-susceptible types such as 0401, 0404, 0405 or 10101 were not always high, while the levels in patients with non-RA types were not always low.
- High IgG anti-HII antibody levels were observed in patients with a particular combination of HLA-DR types, such as 0401/0701 and 0404/03 (HLA +/-), and 1301/0701 in HLA (-/-). The results are shown in TABLE 5.
- HLA DR-type influences the epitope specificity of type II collagen antibodies.
- the reactivity of type II collagen antibodies to HII are regulated by epitope specificity to type II collagen.
- no differences were observed in species- specificity or antibody levels to heterologous CII or BII among individual groups; some patients had high levels of antibodies to CII, while others had high levels to BII even though their DR alleles were identical.
- humanized HLA-DRwl2 transgenic mice are resistant to CIA, since DRwl2, an RA-resistant type, down-regulates the immune-responses to type II collagen.
- IgG anti-HII antibody levels in individual patients were compared to clinical markers in these patients by regression analysis. As shown in Fig.
- the combined information of DR-types and serum IgG anti-HII antibody levels may provide important information in order to predict the risk of the onset of RA, distinguish the type of RA, and to predict disease severity. Such information is useful for preventing the onset of arthritis in patients. The results are shown in TABLE 6.
- Serum antibody levels to chick (CII) and human type II (HII) collagen in these patients were determined and in both the patients, a significant decrease in antibody levels was observed over the course of treatment. Notably, the decrease of antibody level in patient Ju4 was remarkable and was almost undetectable after 14 months. This patient was released from the hospital due to a complete remission. In contrast to this, Tj7, who had a remaining high level of anti-collagen antibodies died at 10 months. The data implicate the pathological role of anti-type II collagen antibodies in RP and their measurement can be used as a diagnostic marker for immunomodulating therapies.
- ELISA plates were coated with the renatured CB-peptides (10 g/ml) dissolved in phosphate buffer, pH 7.5 at 4 C overnight.
- Antibodies purified from RA sera by affinity chromatography and extracted from RA cartilage were diluted with full-strength buffered NRS and were allowed to react with each CB-peptide at 4 C overnight.
- Antibodies bound to CB-peptides were detected by peroxidase- conjugated anti-human IgG antibodies diluted 1:2,000 with 25% NRS. The results of this assay are shown in Fig. 8, which indicate that the antibodies in each RA serum recognized various antigenic determinants in different regions of HII.
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000589946A JP2002533677A (en) | 1998-12-22 | 1999-01-22 | How to diagnose autoimmune arthritis |
EP99905464A EP1141715A1 (en) | 1998-12-22 | 1999-01-22 | Diagnostic method for autoimmune arthritis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US21955198A | 1998-12-22 | 1998-12-22 | |
US09/219,551 | 1998-12-22 |
Publications (1)
Publication Number | Publication Date |
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WO2000037940A1 true WO2000037940A1 (en) | 2000-06-29 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US1999/001499 WO2000037940A1 (en) | 1998-12-22 | 1999-01-22 | Diagnostic method for autoimmune arthritis |
Country Status (3)
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EP (1) | EP1141715A1 (en) |
JP (1) | JP2002533677A (en) |
WO (1) | WO2000037940A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CA2651185C (en) * | 2006-05-09 | 2022-08-30 | The University Of British Columbia | Dissolved protein arthritis markers |
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1999
- 1999-01-22 EP EP99905464A patent/EP1141715A1/en not_active Withdrawn
- 1999-01-22 JP JP2000589946A patent/JP2002533677A/en not_active Ceased
- 1999-01-22 WO PCT/US1999/001499 patent/WO2000037940A1/en not_active Application Discontinuation
Non-Patent Citations (4)
Title |
---|
BIOLOGICAL ABSTRACTS, vol. 86, Philadelphia, PA, US; abstract no. 125313, XP002114367 * |
DATABASE MEDLINE 1 January 1900 (1900-01-01), XP002114366, Database accession no. 96197047 * |
K. TERATO ET AL.: "The mechanism of autoantibody formation to cartilage in rheumatoid arthritis: possible cross-reaction of antibodies to dietary collagens with autologous type II collagen.", CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY, vol. 79, no. 2, 1996, New York NY USA, pages 142 - 154 * |
R. HOLMDAHL ET AL.: "Immunogenetics of type II collagen autoimmunity and susceptibility to collagen arthritis.", IMMUNOLOGY, vol. 65, no. 2, 1988, London UK, pages 305 - 310 * |
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JP2002533677A (en) | 2002-10-08 |
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