[go: up one dir, main page]

WO1995004137A1 - Composition stable - Google Patents

Composition stable Download PDF

Info

Publication number
WO1995004137A1
WO1995004137A1 PCT/DK1994/000289 DK9400289W WO9504137A1 WO 1995004137 A1 WO1995004137 A1 WO 1995004137A1 DK 9400289 W DK9400289 W DK 9400289W WO 9504137 A1 WO9504137 A1 WO 9504137A1
Authority
WO
WIPO (PCT)
Prior art keywords
volume
composition according
gad
range
concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/DK1994/000289
Other languages
English (en)
Inventor
Kim Hejnaes
Alister J. Moody
Michael Owen Marshall
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Priority to AU72268/94A priority Critical patent/AU7226894A/en
Priority to EP94921606A priority patent/EP0717776A1/fr
Publication of WO1995004137A1 publication Critical patent/WO1995004137A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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.)
    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

  • the present invention relates to a stabilization of purified recombinant human islet glutamic acid decarboxylase 65 (GAD 65 , EC 4J J J5) and to a process for preparing stable enzymatically and antigenic active GADg5-
  • IDM Insulin-dependent diabetes mellitus
  • Circulating autoantibodies towards ⁇ -cell antigens have been detected in a number of newly onset diabetics (Bottazzo et aL, Lancet 1 (1980), 668 - 672; Palmer et aL, Science 222 (1983), 1137 - 1139; and Baskkeskov et aL; J.Clin.ln- yest. 79 (1987), 926 - 934).
  • the most reliable indicator of ongoing ⁇ -cell destruction is circulating antibodies towards the 64K antigen. These antibodies have been detected in 70 - 80 % of newly diagnosed patients and up to 8 years before the clinical manifestation of the disease (Baskkeskov et aL; J.CIin.lnvest.
  • the 64K islet cell antigen was recently identified as the enzyme glutamic acid decarboxylase, EC 4.1 J .15 (Baakkeskov et aL, Nature 347, 151 - 156) which converts L-glutamic acid into the neurotransmitter gamma-aminobutyric acid. With the recent cloning (Karlsen et aL, Proc.Natl.
  • GADg 5 is a complex molecule, containing 15 cysteine residues and several palmitoylated sites. In solution, GADg 5 aggregates rapidly to form both covalent and non- covalently bound multimers having reduced enzymatic activity and reduced ability to react with antibodies. These characteristics make the isolation of recombinant GAD ⁇ 5 difficult.
  • the material is often purified at high concentration (for example, on an immuneaffinity column) under which conditions its inherent tendency to irreversibly aggregate leads to the formation of unusable precipitates.
  • the known, purified or highly purified compositions of recombinant GADg are extremely unstable in aqueous solutions resulting in loss of enzymatic activity and loss of antigenic activity.
  • One object of this invention is to prepare stable, purified or highly purified compositions of recombinant GAD 65 SUMMARY OF THIS INVENTION
  • the invention described herein concerns a novel stable and biologically active recombinant human islet GADg 5 composition.
  • Purified recombinant GADg 5 is unstable in aqueous buffers and specific measures have to be taken to purify and stabilize recombinant human GADg 5 .
  • GAD 65 can be stabilized, resulting in preservation of the biological activity, by purifying GAD 65 under reducing conditions in the presence of detergents and that purified GADg can be kept biologically active under reducing conditions, preferably in the presence of a detergent, an alcohol or a divalent metal ion.
  • this invention relates to a glutamic acid decarboxylasegg composition containing a reducing agent and/or a detergent and/or an alcohol and/or a divalent metal ion.
  • the present invention deals with stabilization of GADg5 using specific immunological and biological activity as criteria for maintenance of stability.
  • the stabilized GADg 5 may be obtained by adding certain components during extraction and down stream processing.
  • GADg 5 - also as a highly purified composition - can be stabilized in the presence of reducing agents and, if preferred, in the presence of a detergent, an alcohol, or a divalent metal ion or mixtures thereof.
  • GAD is a firmly membrane anchored protein (Christgau et aL; J.Cell.Biol. 118(2)
  • GADg 5 is stable under reducing conditions in the presence of ionic detergents like SDS.
  • the compositions of this invention contain between 0.01 mg/ml and 10 mg/ml, more preferred between 0J mg/ml and 3 mg/ml, and most preferably between 1 and 3 mg/ml, of GADg 5 .
  • the compositions of this invention contain GAD in a very pure form, preferably GAD having a purity of more than 95 % (weight/weight) relative to the total amount of protein.
  • the preferred reducing agent to be added to the compositions of this invention is dithiothreitol (DTT), 2-mercaptoethanol, cysteine or cystamine, for example, at concentrations from 1 to 100 mM, preferably at concentrations below 20 mM.
  • DTT dithiothreitol
  • 2-mercaptoethanol 2-mercaptoethanol
  • cysteine or cystamine for example, at concentrations from 1 to 100 mM, preferably at concentrations below 20 mM.
  • the detergent to be added to the compositions of this invention may be non-ionic or ionic.
  • Preferred non-ionic detergents are triton X-114 (TX-114), triton X-100 (TX-114), triton X-100 (TX-114), triton X-114 (TX-114), triton X-100 (TX-114), triton X-114 (TX-114), triton X-100 (TX-114), triton X-114 (TX-114), triton X-100 (TX-
  • n-octyl glucoside for example, in a concentration in the range from 0.01 to 10 % (volume/volume), preferably at a concentration in the range from 0.5 to 5
  • the preferred ionic detergent to be added is sodiumdodecylsulphate
  • the preferred alcohol to be added to the compositions of this invention is glycerol or a polyethylene glycol, for example PEG 600.
  • the alcohol may be present in a concentration in the range from 20 to 70 % (volume/volume), preferably in a concentration less than 50 % (volume/volume).
  • the preferred divalent metal ion to be added to the compositions of this invention is Zn + + or Ni + + , for example, at concentrations from 0J to 1 mM.
  • This invention also relates to a process for isolating or purifying GAD 65 obtained from a crude material containing GADg 5 , for example, in the range from 0J to 20 % (weight/ weight).
  • GADgg is performed in a manner known p_er se including the presence of the co-factor, PLP, for example, at 0.2 to 1.0 mM concentration.
  • generel isolation or purification processes are extraction, phase separation, precipitation, filtration, ion exchange chromatography, gel permeation chromatography, affinity chromatography and hydrophobic interaction chromatography, which can be combined into different down stream processes.
  • the reducing agent is present during the whole isolation or purification procedure. If the reducing agent is not present during the whole isolation or purification process but is present only during the major part of the isolation or purification process, a fairly stable composition may be obtained.
  • An embodiment of this invention is purification of GAD 65 by affinity chromatography using elution at a pH value of about 9.5 or by anion exchange chromatography in the presence of a reducing agent and/or detergents.
  • compositions of this invention is to add, to a glutamic acid decarboxylase 6 5 composition, such an amount of a reducing agent and/or a detergent and/or an alcohol and/or a divalent metal ion as is stated herein to give the desired effect.
  • a glutamic acid decarboxylase 6 5 composition such an amount of a reducing agent and/or a detergent and/or an alcohol and/or a divalent metal ion as is stated herein to give the desired effect.
  • the preparation of such compositions is known to any skilled art worker.
  • the composition of this invention Being a major autoantigen in type 1 diabetes mellitus, the composition of this invention is of vital importance for the screening of specific autoantibodies in human serum and thereby for prediction of the disease.
  • a major advantage of this invention is that it provides the means to produce large quantities of highly purified stable enzymatically and antigenic active GADg5 which easily can be formulated and hereafter used in screening assays, tolerance experiments, T-cell experiments, immune invention, etc.
  • the novel GAD 65 compositions of this invention are used similar with the use of known GAD compositions, bearing the improved stability in mind.
  • BHK baby hamster kidney cells
  • GAD glutamic acid decarboxylase
  • DTT dithiotreitol
  • PLP pyridoxal-5'- phosphate
  • SDS sodium dodecyl sulphate
  • IDDM insulin dependent diabetes mellitus
  • NOD non-obese diabetic
  • BB Bio Breeding
  • EGTA is ethylene glycol- bis-( ⁇ -aminoethyl ether)
  • AET 2-aminoethylisothiouronium bromide hydrobromide
  • EDTA ethylenediaminetetraacetic acid
  • PEG polyethylene glycol
  • IgG immunoglobilin
  • ELISA enzyme-linked immunosorbent assay
  • PAGE polyacrylamide gelelectrophoresis
  • s.d. is standard deviation.
  • 100 ml BHK cell carrier pellets were homogenized with a glass homogenizer in 15 ml of 10 mM Hepes, 1 mM EGTA, 1 mM MgCI 2. 0.2 mM PLP, 1 mM AET, and 100 mg/1000 ml aprotinin (pH 7.4). The extract was centrifuged at 25000 rpm (SW25) at 4°C for 30 minutes.
  • the enzymatic activity of the control at a pH value of 7.4 was set to 100 %. All other activities were calculated relative to this sample. The experiment was repeated under reducing conditions, 10 mM DTT (see Table 1 , below).
  • 10 rat brains were homogenized with a polytrone in 100 ml of 10 mM Hepes, 1 mM EGTA, 1 mM MgCI 2 , 0.2 mM PLP, 1 mM AET, and 100 mg/1000 ml aprotinin (pH 7.4) and filtered on a 0.45 ⁇ Nalgene filter.
  • This extract was used as a reference material and analyzed as described above except that 180 ⁇ l of extract was mixed with 20 ⁇ l of 10 % solutions of TX-114, SDS, n-octyl glucoside (NOG), 10 mM solutions of NiCI 2 and ZnCI 2 and that 200 ⁇ l of ethanol, PEG 600 and glycerol were mixed with 200 ⁇ l of extract (see Table 2, below).
  • Rat brain GADgg stability in presence of various additives Rat brain GADgg stability in presence of various additives.
  • GAD1 -Sepharose-4B (coupled according to manufactures manual, Pharmacia, Sweden; Dr. A. Tobin, GAD1 :ATCC HB184) were washed twice in 10 mM Hepes, 10 mM cysteine, 10 mM benzamidine, 1 mM EDTA, 1 mM PLP, 1 mM AET, 1 mM glutamate, and 2 % (volume/volume) TX-114 (pH 7.4) and mixed with 1.0 ml of extract from BHK cell culture.
  • the buffers in Table 3 represent: 50 mM glycine buffer at pH 9.5 (1); glycine buffer with: 10 mM DTT (2); 10 mM DDT, 1 mM PLP (3); 10 mM DTT, 1 mM PLP, 1 % n-octyl glucoside (4); 10 mM DTT, 1 mM PLP, 1 % n-octyl glucoside, 1 mM NiCI 2 (5); 10 mM DTT, 1 mM PLP, 50 % glycerol (6); 10 mM DTT, 1 mM PLP, 1 mM NiCI 2 , 50 % glycerol (7).
  • 100 ml of BHK-carrier pellet were mixed with 100 ml of 40 mM Hepes, 20 mM cysteine, 20 mM benzamidine, 2 mM EDTA, 2 mM PLP, 2 mM AET, 2 mM glutamate, and 2 % (volume/volume) TX-114 (pH 7.4) for 1 hour at 4°C.
  • the extract was centrifuged for 10 minutes at 3300 x g at 4°C.
  • the supernatant was prefiltered on (AP2504200, Millipore) followed by filtration on a 1.2 ⁇ filter (RAWP 04700, Millipore).
  • the temperature was raised to 37°C in a water bath and the filtrate was centrifuged at 3300 x g for 5 minutes at 37°C.
  • the aqueous phase was extracted by adding TX-114 to 1 % (volume/volume), raising the temperature to 37°C and centrifugation as described above.
  • the combined detergent was cooled to 4°C, diluted 4-fold with 20 mM Hepes, 10 mM cysteine, 10 mM benzamidine, 1 mM EDTA, 1 mM PLP, 1 mM AET, and 1 mM glutamate (pH 7.4) and applied (0J5 ml/min) to a 1.6 x 1.5 cm GAD1-Sepharose-4B affinity column (5 mg GAD1/ml gel) with a 1.5 x 1.0 cm Sephadex G25 (Pharmacia, Sweden) precolumn.
  • the column system was preequilibrated in 20 mM Hepes, 10 mM cysteine, 10 mM benzamidine, 1 mM EDTA, 1 mM PLP, 1 mM AET, 1 mM glutamate, and 1 % (volume/volume) TX-114 (pH 7.4) and then washed in 20 mM Hepes, 10 mM DTT, and 1 mM PLP (pH 7.4).
  • GAD 65 was eluted with 50 mM glycine, and 10 mM DTT, (pH 9.5) into 200 ⁇ l of 0.5 M Hepes (pH 7.0). Fractions of 0.8 ml GAD 65 positive fractions were pooled and stored at -20°C in aliquots of 500 ⁇ l.
  • Data are based on extraction and purification of one batch of 100 ml BHK-carrier pellet.
  • the yield of GAD 65 was calculated relative to the bioactivity in the initial extract. 1 mU is defined as 1 nmol substrate converted per minute at 37°C.
  • AQ is aqueous phase and DP is detergent phase.
  • Cells were extracted (cells from 200 ml culture in 20 ml extraction buffer) by Dounce homogenization at 4°C in 40 mM HEPES, 5 mM DTT, 5 mM EDTA, and 50 ⁇ M pyridoxal phosphate, (pH 7J), containing 20 % (volume/volume) precondensed Triton X-114, and the protease inhibitors used in immunoprecipita- tion. The extract was centrifuged at 100,000 x g for 30 minutes at 4°C. The pellet was reextracted and the extract centrifuged as above.
  • the pooled supernatants were heated to 30°C for one to 2 minutes to induce phase separation of the Triton X-114 and centrifuged at 4000 x g at 30°C for 10 minutes.
  • the detergent- rich lower phase was collected.
  • Ten per cent (volume/volume) precondensed Triton X-114 was added to the upper aqueous phase that was then cooled to 4°C and then phase separated as above.
  • the two detergent-rich phases were pooled, diluted twice in extraction buffer and stored at -80°C.
  • GAD 65 was isolated from the detergent extract by ion-exchange chromatography at 4°C on a 2 cm 2 x 6 cm column of DEAE Superose High Flow. Yields and specific enzymatic activities are shown in Table 5, below.
  • Example 5 Measurement of enzymatic activity.
  • the enzymatic activity of GAD was assayed according to Wu J-Y. et aL; Methods in Enzvmoloqy 113 (1985), 3 - 10.
  • the incubation tube (Sarstedt 1.5 ml) contained 4 ⁇ l of L-(1- 14 C) glutamic acid (Amersham, U.K.) in 50 mM potassium phosphate, 1 mM EDTA, 1 % TX-114, 0.2 mM PLP, 1.0 mM AET (pH 7.2) and 21 ⁇ l of 5 mM L-glutamate (105 nmol).
  • the reaction was initiated by adding 125 ⁇ l of sample or enzyme standard.
  • Hyamine base (Packard 6006005) were pipetted into a filter disk (Whatman) placed in the lid. Immediately thereafter, the tubes were vortexed, closed with the filter and incubated first for two hours at 37°C and then for 60 minutes at 4°C. The filters were transferred to 2 ml of Ultima Gold scintillation liquid (Packard) and counted in a Liquid scintillation ⁇ -counter (Tri-carb 4530, Packard). One enzyme unit is defined as 1 ⁇ mol of product formed per minute at 37°C.
  • the assay was carried out at room temperature.
  • 96-plates Nunc Immuno-plates, Denmark
  • 96-plates Nunc Immuno-plates, Denmark
  • the plates were coated overnight with 100 ⁇ l/well of 5 ⁇ g/ml of purified GAD1 in 10 mM sodium phosphate, and 0J M NaCI (pH 6.5).
  • the plates were then blocked in 10 mM sodium phosphate, 0J M NaCI, 0.25 % Tween 20, and 1 % (weight/volume) bovine serum albumin (pH 6.5).
  • Reference and unknown samples were diluted in BSA buffer containing 2 % (volume/volume) Triton X-100 and 100 ⁇ l aliquots added to the wells, incubated for one hour and washed three times.
  • Biotinylated GAD6 (12.5 ng/ml in blocking buffer; Dr. D.I. Gottlieb and the Development Studies Hybridoma Bank, N01-HD-6-2915) was added to the plates, incubated for 1 hour, and the plates were washed three times before peroxidase labelled streptavidin (Amersham, U.K.) was added to the wells. Finally, after one hour of incubation, the wells were washed four times and peroxidase substrate 1 ,2-phenyldiamine dihydrochloride (DAKO, Denmark) dissolved in 72.9 mM sodium citrate, and 4.2 mM sodium borate (pH 5.0), was added.
  • DAKO peroxidase substrate 1 ,2-phenyldiamine dihydrochloride
  • the incubation was stopped after 10 minutes by the addition of 2N sulphuric acid and the optical density was measured at 490 and 650 nm.
  • the concentration of GAD 65 was determined by ELISA analysis using recombinant human islet GADg 5 isolated from SF9 cells. 1 ⁇ g of protein equals 2 units as calculated from amino acid analysis of the said material.
  • Example 7 1 D-SDS Electrophoresis and Western Blotting
  • One-dimensional polyacrylamide electrophoresis was carried out on a Mini- Protean ® II Dual Slab Cell (Bio-Rad) using 10 % SDS-PAG Plate 10 gels (Daiichi Pure Chemicals Co., LTD, Japan) according to Laemmli (Nature 227 (1970), 680 - 685).
  • Samples were prepared by adding 1 part of sample buffer (0.1 M Tris, 10 % SDS, and 50 % glycerol (pH 8.0)) to 4 parts of sample. Aliquots from 10 to 30 ⁇ l hereof were applied directly (non-reducing conditions) or boiled for 1 minute in the presence of 0.11 M DTT (reducing conditions).
  • proteins were transferred to PVDF membranes (Immobilon, Millipore) by electroblotting.
  • the membranes were incubated in 50 mM Tris, 0.15 M NaCI, 0.1 % (volume/volume) Tween 20, and 3 % (weight/volume) milk powder (pH 7.4) for 30 minutes, further incubated for 1 hour in rabbit antiserum (R1267, Petersen et aL; Diabetes 42 (1993), 484 -495) in the incubation buffer (dilution 1 :200), washed three times in 50 mM Tris, and 0.15 M NaCI (pH 7.4) and finally incubated with alkaline phosphatase labelled goat anti- rabbit IgG (Zymed) for 1.5 hours in the incubation buffer (dilution 1 :1000).
  • the cDNA coding for human islet GAD ⁇ 5 was cloned into the KH1117 transcription vector between a T7 RNA polymerase promotor and a poly A tail and two T7 transcription stop codons in tandem.
  • the cDNA was transcribed in vitro according to the manufactures instructions (Promega, Madison) and translated in vitro in the presence of ⁇ 5 S-
  • GAD 65 25 methionine labelled GAD 65 (see Table 6, below).
  • the immuneprecipitates were either counted directly or submitted to SDS-page and autoradiography. Values are mean of 3 experiments ⁇ s.d. Serum + 10 ⁇ l buffer: 3123 cpm. Table 6:
  • the Protein-A Sepharose with bound immunecomplexes was washed 5 times by centrifugation in 50 mM Tris, 150 mM NaCI, and 0J % (volume/volume) Tween 20 (pH 7.4), and boiled for 3 minutes in 1 M Tris, 10 mM DTT, 60 % (weight/volume) sucrose, 15 %
  • Table 8 Trapping of enzymatic activity with GAD 65 .

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Décarboxylase65 d'acide glutamique (GAD65) des îlots chez l'homme, pouvant être stabilisée par adjonction d'un agent réducteur, d'un détergent, d'un alcool ou d'un ion métallique bivalent.
PCT/DK1994/000289 1993-07-28 1994-07-19 Composition stable Ceased WO1995004137A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU72268/94A AU7226894A (en) 1993-07-28 1994-07-19 A stable composition
EP94921606A EP0717776A1 (fr) 1993-07-28 1994-07-19 Composition stable

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK0880/93 1993-07-28
DK88093A DK88093D0 (da) 1993-07-28 1993-07-28 Stabilt praeparat

Publications (1)

Publication Number Publication Date
WO1995004137A1 true WO1995004137A1 (fr) 1995-02-09

Family

ID=8098572

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1994/000289 Ceased WO1995004137A1 (fr) 1993-07-28 1994-07-19 Composition stable

Country Status (4)

Country Link
EP (1) EP0717776A1 (fr)
AU (1) AU7226894A (fr)
DK (1) DK88093D0 (fr)
WO (1) WO1995004137A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998049279A1 (fr) * 1997-04-25 1998-11-05 Diamyd Therapeutics Ab Utilisation de detergent/tensioactiof zwitterionique pour purifier l'acide glutamique decarboxylase (gad) et composition contenant cette enzyme gad
US5965389A (en) * 1995-11-09 1999-10-12 Zymogenetics, Inc. Production of GAD65 in methylotrophic yeast

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004632A1 (fr) * 1990-09-07 1992-03-19 The Regents Of The University Of California Procedes de diagnostic et de traitement de diabete
EP0519469A1 (fr) * 1991-06-18 1992-12-23 The Regents Of The University Of California Décarboxylase de l'acide glutamique cloné
WO1994010297A1 (fr) * 1992-11-04 1994-05-11 Elias Entwicklungslabor Für Immunoassays Gmbh & Co. Kg Procede de preparation de proteines gad-1 et gad-2 humaines de grande purete

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004632A1 (fr) * 1990-09-07 1992-03-19 The Regents Of The University Of California Procedes de diagnostic et de traitement de diabete
EP0519469A1 (fr) * 1991-06-18 1992-12-23 The Regents Of The University Of California Décarboxylase de l'acide glutamique cloné
WO1994010297A1 (fr) * 1992-11-04 1994-05-11 Elias Entwicklungslabor Für Immunoassays Gmbh & Co. Kg Procede de preparation de proteines gad-1 et gad-2 humaines de grande purete

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN, Vol. 13, No. 302, C-616; & JP,A,1 091 781 (RES ASSOC UTIL OF LIGHT OIL), 11 April 1989 (11.04.89). *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5965389A (en) * 1995-11-09 1999-10-12 Zymogenetics, Inc. Production of GAD65 in methylotrophic yeast
WO1998049279A1 (fr) * 1997-04-25 1998-11-05 Diamyd Therapeutics Ab Utilisation de detergent/tensioactiof zwitterionique pour purifier l'acide glutamique decarboxylase (gad) et composition contenant cette enzyme gad

Also Published As

Publication number Publication date
EP0717776A1 (fr) 1996-06-26
DK88093D0 (da) 1993-07-28
AU7226894A (en) 1995-02-28

Similar Documents

Publication Publication Date Title
Petersen et al. Neonatal tolerization with glutamic acid decarboxylase but not with bovine serum albumin delays the onset of diabetes in NOD mice
US6011139A (en) Hybridomas and monoclonal antibodies that specifically bind to glutamic acid decarboxylase peptides
Ikawa et al. Isolation and characterization of aldehyde dehydrogenase isozymes from usual and atypical human livers.
Rowley et al. Antibodies to glutamic acid decarboxylase discriminate major types of diabetes mellitus
Björk et al. Glucose regulation of the autoantigen GAD65 in human pancreatic islets
Yan et al. Multiple forms of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase from wheat seedlings. Regulation by fructose 2, 6-bisphosphate.
Allen et al. Increased membrane binding of erythrocyte catalase in hereditary spherocytosis and in metabolically stressed normal cells
Moody et al. Isolation by anion-exchange of immunologically and enzymatically active human islet glutamic acid decarboxylase 65 overexpressed in Sf9 insect cells
Noël et al. Human placental steroid sulfatase: purification and properties
Isono et al. Subparticles of Anabaena phycobilisomes: II. Molecular assembly of allophycocyanin cores in reference to “anchor” protein
Jefferson et al. Studies on ornithine decarboxylase activity in the isolated perfused rat liver
EP0717776A1 (fr) Composition stable
Vora et al. Characterization of the enzymatic defect in late-onset muscle phosphofructokinase deficiency. New subtype of glycogen storage disease type VII.
CA2070004C (fr) Glutamate-decarboxylase clone
Yeltman et al. Purification and characterization of aldolase from human erythrocytes
Shapiro et al. Immunoblot analysis of glutaminase peptides in intact and solubilized mitochondria isolated from various rat tissues
US5989551A (en) Materials and methods for detection and treatment of insulin-dependent diabetes
US6165738A (en) Expression in yeast of antigenically active, recombinant hybrid glutamic acid decarboxylase
Muraganandam et al. ELISA for in vivo assessment of nonenzymatically glycated platelet glutathione peroxidase
US5965372A (en) Genetic sequences encoding glucocorticoid dehydrogenases and uses thereof
EP0686262B1 (fr) Procedes de diagnostic du diabete et d'etats prediabetiques
US5883240A (en) Genetic sequences encoding glucocorticoid dehydrogenases and uses therefor
Bresolin et al. Phosphoglycerate kinase deficiency myopathy: biochemical and immunological studies of the mutant enzyme
Matsuda et al. Biochemical heterogeneity of ornithine carbamoyl transferase (OCT) in patients with OCT deficiency
Lopina et al. A Comparative Study of Na+/K+-ATPases of Duck Salt Gland and Canine Kidney: Implications for the Enzyme′ s Reaction Mechanism

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AU BB BG BR BY CA CN CZ FI GE HU JP KE KG KP KR KZ LK LT LV MD MG MN MW NO NZ PL RO RU SD SI SK TJ TT UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1994921606

Country of ref document: EP

ENP Entry into the national phase

Ref country code: US

Ref document number: 1996 586663

Date of ref document: 19960126

Kind code of ref document: A

Format of ref document f/p: F

WWP Wipo information: published in national office

Ref document number: 1994921606

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1994921606

Country of ref document: EP