Classifications

• • 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.)
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/04—Endocrine or metabolic disorders
  • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

• This invention relates to methods for the identification of persons who have diabetes or a prediabetic status, and in particular it is directed to a diagnostic assay which will provide a simple and effective means of screening large populations of individuals to detect both diabetes and prediabetes (the early stages of diabetes prior to the onset of the clinical disease).
• Diabetes mellitus is a heterogeneous disorder.
• Insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) are subtyped physiologically according to patients' dependence for survival on treatment with insulin 1 .
• IDDM is attributed to autoimmunity 2 by reason of disease associations, autoantibodies to pancreatic islet cell antigens, similarities with animal models of IDDM and HLA linkages.
• the disease-relevant autoantigen or antigens in IDDM include a 64,000- M r protein demonstrable by immunoprecipitation from pancreatic islets 3 but hitherto unidentified because of low abundance and assay difficulties.
• Immunoassay for antibody to the 64,000-M r antigen indicated that it was a specific marker for IDDM and preceded symptomatic onset of disease 4 .
• the recognition in cases of the Stiff Man syndrome of coexisting IDDM and a neural antigen of 64,000-65,000-/Vf r identified as glutamic acid decarboxylase (GAD) led Baekkeskov et al. 5 to establish the coidentity of their 64,000-M r antigen and GAD.
• Rowley et al. 6 described a high frequency of positive results by radioimmunopreciprtation assay with IDDM sera and a GAD preparation from pig brain and proposed detection of antibodies to GAD as a useful diagnostic test as an autoimmune marker associated with IDDM.
• Insulin-dependent diabetes mellitus is a frequently occurring disease that predominantly affects young individuals, less than age 15 years.
• the disease can, however, have an onset in early or even mid-adult life, hence there is a need for a test that has diagnostic and predictive capacity for IDDM in both childhood and in adult life.
• IDDM occurs at highest frequencies in the developed and economically advanced communities in the world. There is a high incentive to diagnose IDDM at the earliest stage, and even in the preclinical stage before the disease can be recognised by conventional laboratory tests.
• the recognition of antibodies to GAD by a routinely applicable, sensitive and specific immunoassay kit would fulfil this need.
• NIDDM non-insulin dependent form
• LADA latent autoimmune diabetes in adults
• the antibody could be readily and specifically detected by an immunoprecip tation assay.
• the 64,000-/W r antigen is an enzyme molecule as are various other disease-relevant autoantigens, most notably thyroid peroxidase in autoimmune thyroiditis 14 , H + K* ATPase in autoimmune gastritis 11 , and pyruvate dehydrogenase in primary biliary cirrhosis 12 .
• GAD is not species specific and, in fact, cDNAs for brain GAD from humans 13 , rat 14 , and cat 15 have such close homology that antibody reactivity could be expected with any mammalian GAD preparation.
• the autoantigen is clearly not organ specific because the source used by Rowley et al. 6 was brain.
• GAD exists as at least two isoforms 18 , and the Rowley et al. 6 preparation contained both representatives, as indicated by data by polyacrylamide gel electrophoresis (PAGE).
• GAD ⁇ -aminobutyric acid
• the enzyme has a restricted tissue distribution and is highly expressed in the cytoplasm of GABA-secreting neurons and pancreatic ⁇ -cells.
• the two isoforms of GAD are referred to as GAD 65 and GAD 67 according to their relative molecular masses. These isoforms are the products of two different genes and differ substantially only in their amino-terminal region.
• GAD appears to exist as an integral membrane protein with hydrophobic properties, and is present as a non-covalently linked dimer of MW 9G,60 °.
• This purified membrane form of GAD has been found to be reactive with IDDM sera, however no sequence data are yet available for this integral membrane protein 20 .
• the GAD autoantibody is a valuable predictive marker of the disease, and in a study in Finland 23 based on serum samples obtained from apparently healthy pregnant women who subsequently developed IDDM, the predictive capacity for IDDM of a positive test for anti-GAD was remarkably high a relative risk versus a negative test for anti-GAD being 800- fold.
• a study on stored sera from women with gestational diabetes likewise showed that a positive test for anti- GAD was predictive of the occurrence later in life of IDDM in such women 24 .
• WO 92/04632 discloses assays for the detection of diabetes or a prediabetic status relying on exposing patient serum samples to purified ligand capable of binding autoantibodies specific for the 64 kD autoantigen present on pancreatic ⁇ -cells.
• a method for detecting autoantibodies to glutamic acid decarboxylase (GAD) in the serum of a patient comprising contacting a serum sample from the patient with a GAD antigen and detecting binding of autoantibodies to GAD in said sample by said GAD antigen, characterised in that said GAD antigen comprises a GAD preparation containing an enhanced amount of dimer(s) or oligomer(s) of the 65 kD and /or 67 kD isoforms of GAD.
• GAD glutamic acid decarboxylase
• serum is used as a non-limiting term, and that the present invention also extends to the detection of autoantibodies to GAD in "blood” samples in general, including blood serum and plasma.
• the method of the invention is carried out under conditions under which the dimeric or oligomeric form of the GAD is retained and is not broken down into monomeric GAD.
• the method of the invention is carried out under non- denaturing conditions, and particularly under non-reducing conditions.
• the detection of binding between the autoantibodies and the GAD antigen is indicative of the presence of the autoantibodies in the serum sample, and hence is diagnostic of a diabetic or prediabetic condition in the patient.
• the present invention provides a non-naturally occurring preparation of GAD antigen which contains an enhanced amount of dimer(s) or oligomer(s) of the 65 kD and/or 67 kD isoforms of GAD.
• the present invention further extends to a diagnostic kit for the detection of autoantibodies to GAD in the serum of a patient, comprising a GAD antigen and means for detecting binding of autoantibodies to GAD in a serum sample from the patient by said GAD antigen, characterised in that said GAD antigen comprises a GAD preparation containing an enhanced amount of dimer(s) or oligomer(s) of the 65 kD and/or 67 kD isoforms of GAD.
• an "enhanced amount" of dimer(s) or oligomer(s) in the GAD preparation indicate that the amount of dimer(s) or oligomer(s) in the GAD preparation is greater than the amount of dimer(s) or oligomer(s) in preparations of naturally occurring GAD.
• Such greater amounts may be achieved by separation of naturally occurring GAD into fractions having an "enhanced amount” of dimer(s) or oligomer(s), for example by electrophoretic separation on SDS-PAGE gels or by size exclusion chromatography.
• such an "enhanced amount" of dimer(s) or oligomer(s) may be achieved by augmenting the content of dimeric or oligomeric GAD in naturally occurring GAD.
• references herein to GAD preparations having an enhanced amount of dimer(s) or oligomer(s) also include preparations of "substantially pure" GAD having greater than 50% by weight, more particularly greater than 70% by weight or even greater than 90% by weight, of the dimeric or oligomeric form of GAD, with the balance monomeric GAD.
• Such "substantially pure" dimeric or oligomeric GAD may be produced by removal of appropriate amounts of monomeric GAD from naturally occurring GAD using known separation techniques. Alternatively, however, the dimeric or oligomeric GAD may be synthesised by appropriate cross-linking, for example by forming disulphide bonds or by other means, of naturally occurring or recombinantly produced monomeric GAD.
• the present invention provides a GAD preparation which comprises substantially pure dimer(s) or oligomer(s) of the 65 kD and/or 67 kD isoforms of GAD, and in particular a GAD preparation which comprises substantially pure heterodimer or heterooligomer of the 65 kD and 67 kD isoforms of GAD.
• the dimer(s) or oligomer(s) in the GAD preparation used as antigen in this invention may be an aggregation of one or other of the two isoforms of GAD, or it may be a heterodimer or heterooligomer of both isoforms (65 kD or 67 kD) of GAD.
• the GAD may be either human or non-human mammalian, native or recombinant, and it may be either pancreatic or brain (or other CNS) GAD.
• the GAD preparation used in the work leading to the present invention was obtained from porcine brain and was purified as described in Rowley et al. 6 on an affinity column that contained a monoclonal antibody GAD1 reactive with mammalian GAD 19 .
• This preparation contained predominantly monomeric GAD (65 kD and 67 kD) but also contained some dimeric and oligomeric GAD.
• this preparation may be further processed to provide a preparation containing an enhanced amount of dimeric or oligomeric GAD, for example, by electrophoretic separation.
• the essential characteristics of the diagnostic assay for autoantibodies to GAD in accordance with the present invention are the use of an antigen preparation containing an enhanced amount of dimeric or oligomeric GAD instead of monomeric GAD, together with appropriate reaction conditions which avoid the destruction of the dimers or oligomers.
• any procedure that enhances the amount of dimeric or oligomeric GAD in a GAD preparation would generate an antigenic preparation that would bind even more strongly and more specifically with anti-GAD present in IDDM sera compared with preparations used hitherto. This in turn provides to a more sensitive assay with greatly improved diagnostic utility.
• Enrichment of the dimeric/oligomeric fraction of GAD can be achieved by a number of methods obvious to one skilled in the art of protein biology. These procedures include the purification of the dimeric/oligomeric forms of GAD which can be accomplished by a number of methods including gel filtration, HPLC, affinity chromatography, ion exchange chromatography, etc. The enrichment may also be achieved by the classical cross-linking of monomeric GAD using hetero-bi functional or homo-bi functional reagents known to form co-valent intra-molecular and inter-molecular protein-protein associations.
• the assays of the present invention can be performed using conventional protocols for immunoassays, such as competitive immunoassays, radio-immunoassays, enzyme-linked immunosorbent assays, and the like. Further details of suitable assay protocols in accordance wrth this invention are described, by way of example, in International Patent Specification No. PCT/US91 /06438 (WO 92/04632), the disclosure of which is incorporated herein by reference.
• IDDM sera react preferentially with dimeric or oligomeric forms of molecular GAD.
• any method or procedure that provides a preparation in which the amounts or proportions of dimeric/oligomeric GAD are enhanced would create a more effective reactant for the serological diagnosis of IDDM.
• Such methods for the isolation of GAD include both chemical methods and immunochemicai separations such as affinity chromatographic procedures that may be modified to give higher yields of dimers.
• GAD in its monomeric form may be susceptible to the folding influences of enzymes such as PDI, so creating an enhanced reactant with IDDM serum. It is further to be noted that both homodimers and heterodimers of GAD 65 and GAD 67 may be prepared, and that the preparation of heterodimers of GAD 65-GAD 67 may provide a reactant of particular utilrty for serological testing.
• Figure 1 shows the results by autoradiography of 125 I-GAD that was precipitated wrth three representative IDDM sera and analysed by SDS-PAGE under reducing or non-reducing condrtions (see text).
• Lanes 1 and 5 contain non- immunoprecipitated 125 I-GAD and lanes 2, 3, 4 and 6, 7 8 contain 125 I-GAD precipitated wrth three representative sera from patients wrth IDDM. It is clearly seen that under reducing condrtions there is a strong signal at - 64 kD, and weak signals at 110-130 kD, whereas under non-reducing conditions there is a weak signal at 64 kD and a strong signal at 110-130 kD.
• Figure 2 shows end-point titrations for 68 IDDM sera, including one wrth Stiff Man Syndrome (SMS), plotted against the value in reference units for the same sera at a 1 :2 dilution. Note that a plot of mean values reaches a plateau corresponding to 5-10% of the total GAD added, indicating that not all of the GAD in the preparation can be precipitated, even wrth high potency sera, as judged by high titre.
• Figure 3 shows the results by autoradiography of 125 I-GAD that was precipitated wrth three anomalous sera and analysed by SDS-PAGE under reducing and non-reducing condrtions.
• the precipitated 125 I-GAD migrates only as a - 64 kD component.
• Lanes 1-3 and 5-7 are the anomalous sera.
• Lanes 4 and 8 are a typical IDDM serum.
• the typical IDDM serum (lane 8) has reactivity, as expected, only with the 110-130 kD component under non-reducing conditions, with a weak signal at the exposure time used for this autoradiograph (arrow).
• Figure 4 shows the result by Western blotting after SDS-PAGE electrophoresis of GAD using antibodies to GAD raised experimentally (A) and in humans (B), with serum dilutions of 1 :200.
• A Monoclonal mouse antibody GAD-6 raised against GAD 65, and polyclonal rabbit antibody K2 raised against GAD 67;
• B lane 1, healthy person; lane 2, patient wrth SDS and IDDM; lanes 3-10, patients wrth IDDM.
• Figure 5 shows the results by dot blotting whereby GAD is spotted onto a nitrocellulose filter and then exposed to the test sera.
• the upper row shows the reaction of IDDM sera with GAD under non-denaturing condrtions (see text), wherein all sera show strong reactivrty, and in contrast, the lower row (spots 2-10) shows the reaction of the same IDDM sera with GAD treated with ME and boiling wherein only serum 2, and serum 3 (weak) show reactivity.
• Spot 1 represents normal serum.
• the sera depicted here are the same as those depicted in Figure 4.
• Figure 6 shows the results by autoradiography of biosynthetically labelled recombinant GAD ⁇ S-GAD) prepared so as to retain the normal structure of native GAD by expression using rabbit reticulocyte lysate (RRL); precipitation was wrth a representative positive IDDM serum and a negative serum, and analysed by SDS-PAGE under reducing and non-reducing condrtions.
• lane 1 contains the RRL recombinant GAD (19 hour exposure)
• lane 2 contains RRL-expressed GAD precipitated with IDDM serum (7 day exposure)
• lane 3 contains RRL-expressed GAD preciprtated with a normal serum (7 day exposure).
• the major signal corresponds to a M.W. of 110-130 kD, indicative of a dimeric form of GAD.
• This Example demonstrates that autoantibodies in sera from patients with IDDM react wrth dimeric/oligomeric forms of porcine brain GAD as shown by autoradiography of immunopreciprtates in SDS-polyacrylamide gels.
• the nature of the GAD antigen reacting in the radioimmunopreciprtation assay described by Rowley et al. 6 was investigated as follows. The pellet consisting of antigen and antibody that was deposited after immunopreciprtation with Sepharose-protein A was exposed to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), and the gel was examined by autoradiography. The 25 I-GAD antigen in the precipitate was compared with the GAD labelled wrth 125 l that had been initially added to the assay.
• SDS-PAGE sodium dodecyl sulphate
• Electrophoresis samples were dissolved in a buffer that contained 64 mM Tris-HCI, pH 6.8, 2% SDS, and 10% glycerol with or without 5% 2-mercapto- ethanol. Electrophoresis was performed on 10% separating gels. The gels were dried and examined by autoradiography for periods of up to 2 weeks.
• GAD was immunopreciprtated wrth the IDDM serum and the precipitate was analysed by gel electrophoresis with or without the reducing agent, 2- mercaptoethanol, i.e. reducing or non-reducing condrtions. The gel was exposed for autoradiography for 2 weeks.
• lanes 1 and 5 contain non- immunopreciprtated 125 I-GAD and lanes 2, 3, 4 and 6, 7, 8 contain 125 I-GAD precipitated wrth three representative sera from patients with IDDM.
• the non-precipitated GAD and the immunoprecipitated GAD have a similar migration corresponding to a MW of - 64 kD.
• the non-precipitated GAD appears as two components that have a migration corresponding to MW of -64 kD and - 110-130 kD, whereas the preciprtated GAD has a migration exclusively as a component corresponding to a MW of - 110-130 kD.
• EXAMPLE 2 This Example demonstrates that diabetic sera immuno-precipitate only a small fraction of the total amount of iodinated GAD in a test preparation of affinity- purified porcine brain GAD, irrespective of the amount or potency of serum used for the immunoprecipitation.
• EXAMPLE 3 This Example shows that the removal of the dimeric and oligomeric forms of GAD from the GAD preparations abrogates reactivity of IDDM sera wrth GAD.
• the residual supernatant after immunoprecipitation of 125 I-GAD by IDDM serum regularly contains most of the inrtial radioactivrty added (see above).
• Autoradiography of the residual 125 l-GAD after electrophoresis shows a signal corresponding to a ⁇ 64 kD component, this being precisely comparable with the - 64 kD signal in the original GAD preparation.
• no other signals corresponding to components of higher molecular weights these components having been completely removed by the first immunoprecipitation.
• the radioimmunoprecipitation assay wrth 125 l-GAD is carried out under conditions of antibody excess, such that these sera remain reactive at dilutions of 1:100 or 1 :1000 or even higher, yet most IDDM sera react with only -5-10% of the total iodinated GAD in the preparation.
• IDDM serum was significantly reduced after the inrtial preciprtation.
• EXAMPLE 5 This Example shows that the dimeric/oligomeric forms of GAD that contain both GAD 65 and GAD 67 react with sera from patients with IDDM.
• GAD preparations derived from porcine brain contain both GAD 65 and
• GAD 67 This indicates that there would be a substantial amount of the heterodimeric form of GAD, GAD 65-GAD 67 in a porcine brain GAD preparation, given that the monoclonal antibody GAD 1 that is used in the affinity purification is reported to react wrth only GAD 65. Thus GAD 67 could become bound to the affinity column only through its linkage to GAD 65.
• an inrtial preciprtation of a GAD preparation was made wrth various reactants, the monoclonal antibody GAD 6 against GAD 65, the polyclonal rabbit antibody K2 against GAD 67, IDDM serum or normal serum, and followed by a second preciprtation with an IDDM serum or normal serum.
• the results show that the capacrty of GAD to react with IDDM serum was moderately influenced by serum K2 and markedly by GAD 6 and the IDDM serum. From the finding that GAD 6 and K2 are totally depleting, the inference is that homodimers and heterodimers of GAD 65/GAD 67 are present in the GAD preparation and both homo- and heterodimeres are reactive.
• “Western” transfer immunoblotting is a standard procedure for examining the reactivrty of autoimmune sera with protein components, including intracellular proteins. This procedure is usually performed under condrtions in which the antigenic protein is denatured by exposure to heat (boiling), a reducing agent (2- mercaptoethanol) and the detergent sodium dodecyl sulphate (SDS).
• the polypeptide epitopes so defined are therefore regarded as “linear” rather than “conformational”, although some degree of renaturation may occur after transfer of the proteins from the gel to the nrtrocellulose filter.
• This Example shows that recombinant GAD produced in a bacterial expression system is inactive in the immuno-precipitation assay, although the recombinant protein has strong enzymatic activity, and hence must retain most of the native configuration of the molecule.
• the action of an enzyme is highly dependent on the conformational structure of the molecule since the activity of most enzymes is readily destroyed when the conformational structure is lost, and GAD is completely inactivated by heating for 2 hours at 45° C 20 . Accordingly, an enzymatically active molecule is usually considered to exist folded in its native conformation 30 .
• Recombinant GAD 65 was prepared in the bacterial expression system pMAL (New England Biolabs 1 .
• the recombinant protein was shown to have strong enzymatic activity as measured by the production of GABA from glutamate labelled with 3 H, according to the procedure described by Rowley et al. 6 , and the specific activity of the recombinant protein was much greater than the specific activity of the purified porcine brain GAD. Nonetheless, when the recombinant GAD antigen was labelled wrth 125 i and used as antigen in the standard immuno ⁇ preciprtation assay, it proved to be poorly reactive.
• Recombinant GAD has been used previously with some claimed success in ELISA systems, but the performance of such ELISAs using recombinant GAD is quite inferior to the performance of radioimmunoprecipitation assays using native porcine brain GAD, as judged by published results 32 and results from the First International GAD Antibody Workshop 33 .
• the conformational requirement of the GAD molecule for reactivrty with sera from patients wrth IDDM differs from the conformational requirement for enzymatic activity, indicating that the additional conformational requirement for immunopreciprtation is crrtically dependent on a dimeric/oligomeric configuration of the GAD molecule.
• This purified, eukaryotically expressed ⁇ S-GAD was used as an antigen in the immunopreciprtation assay wrth IDDM and normal sera as described previously.
• the result was that, as for affinity purified brain GAD, only a small proportion of the total radioactivrty could be immunopreciprtated.
• the immunopreciprtated pellet (GAD-anti-GAD) was examined by autoradiography after separation of proteins by electrophoresis under non-reducing condrtions and, as previously, only the dimeric form of GAD was immunopreciprtated. It is important to note that most of the ⁇ S-GAD in the preparation was in the monomeric form, yet this major proportion proved to be non-preciprtable.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Hematology (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Biochemistry (AREA)
• Organic Chemistry (AREA)
• Analytical Chemistry (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Cell Biology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Pathology (AREA)
• General Physics & Mathematics (AREA)
• General Engineering & Computer Science (AREA)
• Rehabilitation Therapy (AREA)
• Rheumatology (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Priority Applications (6)

Applications Claiming Priority (2)

Publications (1)

Family

ID=3776693

Family Applications (1)

Country Status (12)

Cited By (1)

Families Citing this family (3)

Family Cites Families (3)

• 1994
  • 1994-02-08 IL IL108587A patent/IL108587A/en not_active IP Right Cessation
  • 1994-02-08 ZA ZA94845A patent/ZA94845B/xx unknown
  • 1994-02-08 TW TW083101065A patent/TW305936B/zh active
  • 1994-02-09 AT AT94906796T patent/ATE229650T1/de not_active IP Right Cessation
  • 1994-02-09 EP EP94906796A patent/EP0686262B1/en not_active Expired - Lifetime
  • 1994-02-09 NZ NZ261541A patent/NZ261541A/en unknown
  • 1994-02-09 US US08/495,584 patent/US5770381A/en not_active Expired - Fee Related
  • 1994-02-09 CA CA002155677A patent/CA2155677A1/en not_active Abandoned
  • 1994-02-09 DE DE69431874T patent/DE69431874D1/de not_active Expired - Lifetime
  • 1994-02-09 WO PCT/AU1994/000056 patent/WO1994018568A1/en not_active Ceased
  • 1994-02-09 SG SG1996000807A patent/SG45161A1/en unknown
  • 1994-02-09 JP JP51744894A patent/JP3510889B2/ja not_active Expired - Fee Related

Non-Patent Citations (7)

Also Published As

Similar Documents

Legal Events