EP0000134B1 - Glykoprotein, Verfahren zu seiner Herstellung, seine Verwendung zur Gewinnung von Antiserum sowie das Antiserum - Google Patents
Glykoprotein, Verfahren zu seiner Herstellung, seine Verwendung zur Gewinnung von Antiserum sowie das Antiserum Download PDFInfo
- Publication number
- EP0000134B1 EP0000134B1 EP78100134A EP78100134A EP0000134B1 EP 0000134 B1 EP0000134 B1 EP 0000134B1 EP 78100134 A EP78100134 A EP 78100134A EP 78100134 A EP78100134 A EP 78100134A EP 0000134 B1 EP0000134 B1 EP 0000134B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- glycoprotein
- solution
- protein
- antiserum
- molecular weight
- 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.)
- Expired
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- 102000003886 Glycoproteins Human genes 0.000 title claims description 74
- 108090000288 Glycoproteins Proteins 0.000 title claims description 74
- 238000002360 preparation method Methods 0.000 title claims description 3
- 238000004519 manufacturing process Methods 0.000 title description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 19
- 239000000284 extract Substances 0.000 claims description 10
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 9
- 230000007935 neutral effect Effects 0.000 claims description 9
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 9
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims description 8
- 210000002826 placenta Anatomy 0.000 claims description 8
- 238000001179 sorption measurement Methods 0.000 claims description 8
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 claims description 6
- 239000012460 protein solution Substances 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 5
- 238000004062 sedimentation Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 4
- 230000001900 immune effect Effects 0.000 claims description 4
- 238000007693 zone electrophoresis Methods 0.000 claims description 4
- 150000001720 carbohydrates Chemical class 0.000 claims description 3
- 150000002500 ions Chemical class 0.000 claims description 3
- 238000002955 isolation Methods 0.000 claims description 3
- MSWZFWKMSRAUBD-UHFFFAOYSA-N 2-Amino-2-Deoxy-Hexose Chemical compound NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 2
- CERZMXAJYMMUDR-QBTAGHCHSA-N 5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid Chemical compound N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO CERZMXAJYMMUDR-QBTAGHCHSA-N 0.000 claims description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 2
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims description 2
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 2
- 230000008033 biological extinction Effects 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 238000005194 fractionation Methods 0.000 claims description 2
- 150000002402 hexoses Chemical class 0.000 claims description 2
- 230000003053 immunization Effects 0.000 claims description 2
- 238000002649 immunization Methods 0.000 claims description 2
- 239000002808 molecular sieve Substances 0.000 claims description 2
- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 claims description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims 1
- 230000036647 reaction Effects 0.000 claims 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 13
- 239000000872 buffer Substances 0.000 description 12
- 210000002966 serum Anatomy 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 6
- 235000011130 ammonium sulphate Nutrition 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 5
- 210000001124 body fluid Anatomy 0.000 description 5
- 239000010839 body fluid Substances 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 229920002401 polyacrylamide Polymers 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 102000006395 Globulins Human genes 0.000 description 4
- 108010044091 Globulins Proteins 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 239000012266 salt solution Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- JVIPLYCGEZUBIO-UHFFFAOYSA-N 2-(4-fluorophenyl)-1,3-dioxoisoindole-5-carboxylic acid Chemical compound O=C1C2=CC(C(=O)O)=CC=C2C(=O)N1C1=CC=C(F)C=C1 JVIPLYCGEZUBIO-UHFFFAOYSA-N 0.000 description 3
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 3
- IYLLULUTZPKQBW-UHFFFAOYSA-N Acrinol Chemical compound CC(O)C(O)=O.C1=C(N)C=CC2=C(N)C3=CC(OCC)=CC=C3N=C21 IYLLULUTZPKQBW-UHFFFAOYSA-N 0.000 description 3
- 229920001425 Diethylaminoethyl cellulose Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 239000003463 adsorbent Substances 0.000 description 3
- 229960002319 barbital Drugs 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- VRQURTHVUQBIMS-UHFFFAOYSA-N 2-acridin-1-yloxyethane-1,1-diamine;2-hydroxypropanoic acid Chemical compound CC(O)C(O)=O.C1=CC=C2C=C3C(OCC(N)N)=CC=CC3=NC2=C1 VRQURTHVUQBIMS-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 239000012876 carrier material Substances 0.000 description 2
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical compound OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- LJGHYPLBDBRCRZ-UHFFFAOYSA-N 3-(3-aminophenyl)sulfonylaniline Chemical compound NC1=CC=CC(S(=O)(=O)C=2C=C(N)C=CC=2)=C1 LJGHYPLBDBRCRZ-UHFFFAOYSA-N 0.000 description 1
- HKJKONMZMPUGHJ-UHFFFAOYSA-N 4-amino-5-hydroxy-3-[(4-nitrophenyl)diazenyl]-6-phenyldiazenylnaphthalene-2,7-disulfonic acid Chemical compound OS(=O)(=O)C1=CC2=CC(S(O)(=O)=O)=C(N=NC=3C=CC=CC=3)C(O)=C2C(N)=C1N=NC1=CC=C([N+]([O-])=O)C=C1 HKJKONMZMPUGHJ-UHFFFAOYSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 241001441724 Tetraodontidae Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 235000013877 carbamide Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 150000003248 quinolines Chemical class 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/827—Proteins from mammals or birds
- Y10S530/829—Blood
- Y10S530/83—Plasma; serum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/827—Proteins from mammals or birds
- Y10S530/85—Reproductive organs or embryos
- Y10S530/851—Placenta; amniotic fluid
Definitions
- the invention relates to a new glycoprotein that can be detected in blood serum, urine and in the extract of human placentas and isolated therefrom, and a method for its production.
- the protein solution obtainable by aqueous extraction of human placentas contains a large number of components, some of which can be assigned to the serum proteins and some of which are tissue proteins.
- the object of the invention is to isolate a hitherto unknown serum glycoprotein from the extract of human placentas so that it can specifically produce antisera directed against the new glycoprotein, with which the glycoprotein in the serum and in the urine can be qualitatively detected or quantified.
- the invention relates to a new glycoprotein which can be obtained from blood serum, from urine and from the extract of human placentas. It is characterized by a protein content, essentially consisting of a-amino acids of 75 ⁇ 6%, a carbohydrate content of 24.6 ⁇ 5.2%, of which hexoses 8.9 ⁇ 2%, N-acetylated hexosamine 7.1 ⁇ 1 , 5%, fucose 0.2 ⁇ 0.2%, N-acetylated neuraminic acid 8.4 ⁇ 1.5%, a sedimentation coefficient s 20w of 2.5 ⁇ 0.3 S, a molecular weight of 35000 ⁇ 5,000 due to the Determination in the ultracentrifuge or a molecular weight of 65,000 ⁇ 10,000 on the basis of the determination in the polyacrylamide gel containing sodium dodezyl sulfate, an isoelectric point of pH 3.4 ⁇ 0.4, an extinction coefficient (280 nm) of 1.9% ⁇ 0.3
- the sedimentation equilibrium method and the polyacrylamide gel electrophoresis were used to determine the molecular weights.
- the determination in the ultracentrifuge was carried out at 9000 rpm.
- the evaluation was carried out on the basis of a partial specific volume of 0.74 mg / g.
- the ultracentrifuge had a molecular weight of 35,000 ⁇ 5000.
- PAA polyacrylamide gel electrophoresis
- the isoelectric point was determined using a column (440 ml) from LKB Sweden.
- the so-called ampholine mixture had a pH range of 2.5 to 4.0 when examining the glycoprotein.
- the electrophoretic mobility was examined in the micromodification by Beckmann Instruments on cellulose acetate films with sodium diethyl barbiturate buffer pH 8.6.
- the amino acid analysis was carried out according to S. Moore, D.H. Spackmann, W.H. Stein, Anal. Chem. 30, page 1185, (1958), using the Multichrom B liquid chromatograph from Beckmann. 1/2 cystine was obtained after oxidation of the proteins with performic acid (S. Moore et al., Anal. Chem. 30, page 1185, (1958)) and subsequent chromatography (S. Moore, J. Biol. Chem. 238, page 235 , (1963)) as cysteic acid.
- the tryptophan content was determined using the direct photometric determination according to H. Edelhoch, Biochemistry 6, page 1948, (1967).
- the simplest way of immunologically characterizing the substance is by a known diffusion method in which antigen, i.e. the glycoprotein and an antibody directed against the glycoprotein or the antiserum not enriched with respect to the antibodies diffuse against one another in a carrier medium, such as agar. If the two reaction components meet in a favorable ratio, a visible precipitate forms. According to this knowledge, it is obvious to the person skilled in the art that all immunological techniques for the detection and determination of both the new glycoprotein and the antibodies directed against the glycoprotein are possible.
- the Laurell technique is a simple and generally sufficiently precise method for the quantitative determination of glycoprotein in body fluids or in tissue extracts. It is described in analyte. Biochem. (New York), 15, page 45 (1966).
- the invention further relates to a method for producing the one characterized above Glycoprotein, characterized in that body fluids or extracts from organs which contain the glycoprotein are fractionated on the basis of the following criteria found according to the invention.
- the glycoprotein can be precipitated with neutral salts.
- ammonium sulfate usually used for such precipitations, it is precipitated at a saturation concentration of the salt of 30 to 60% in a pH range near the neutral point.
- the glycoprotein can be obtained by means of measures which are suitable for the separation of substances with molecular weights between 25,000 and 75,000.
- the methods of gel filtration or ultrafiltration are advantageously used for this.
- the glycoprotein is adsorbed onto weakly basic ion exchangers at neutral or weakly alkaline pH.
- a comparatively less concentrated buffer solution is advantageously used, since the adsorption can be prevented by increasing the salt concentration or also by lowering the pH.
- this behavior is known, there is the possibility of adsorbing the glycoprotein and eluting it using more concentrated salt solutions or buffer solutions with a reduced pH.
- the new glycoprotein is not precipitated with the water-soluble organic bases of the acridine and quinoline series, which are usually used for protein precipitation processes. It remains in the aqueous supernatant in the concentrations customary in this process. Thereafter, an acridine base such as 2-ethoxy-6,9-diaminoacridine lactate or a quinoline base such as bis- (2-methyl-4-aminoquinolyl-6) carbamide hydrochloride can be used for the precipitation of accompanying proteins, the Glycoprotein according to the invention remains in the supernatant.
- an acridine base such as 2-ethoxy-6,9-diaminoacridine lactate or a quinoline base such as bis- (2-methyl-4-aminoquinolyl-6) carbamide hydrochloride
- hydroxyapatite As an adsorbent for proteins.
- the new glycoprotein shows no affinity for hydroxyapatite, whereas a number of accompanying proteins are retained by hydroxyapatite.
- the glycoprotein is therefore one of the hydroxylapatite-passing globulins.
- the inventor proposes to call it globulin (HPG-2) passing through hydroxyapatite.
- the preparative zone electrophoresis can be used for the enrichment or isolation of the glycoprotein.
- the affinity of the glycoprotein due to its immunological behavior can be used to enrich the glycoprotein with the help of so-called immune adsorption processes.
- an immunoadsorbent i.e. a carrier-bound antibody against which new glycoprotein are produced, which is able to bind the glycoprotein specifically.
- the glycoprotein can then be eluted again by changing the milieu conditions, as described several times.
- the substances according to the invention can be isolated by means of a selected combination of the methods mentioned, which on the one hand lead to the enrichment of the glycoprotein and on the other hand to a separation of other accompanying proteins. Accordingly, the object of the present invention is to be seen in the individual enrichment steps for the new glycoprotein and in the processes for the purification thereof which result from the combination of the enrichment measures.
- the guideline for the process for the production of the glycoprotein is that the portion is obtained which gives a positive immunological reaction with an antiserum directed against the new glycoprotein.
- the glycoprotein is still contaminated by other immunologically detectable accompanying proteins.
- the impurities are removed by their specific adsorption.
- Common techniques of immunoadsorption are used, in which the described method is applied to one.
- Carrier-bound antibodies against the protein to be removed can be used as adsorbents.
- the largely pure new glycoprotein is often pre-associated with traces of the pregnancy-specific ⁇ i glycoprotein and / or the a i -B glycoprotein, also known as easily precipitable a i glycoprotein.
- Immunoglobulins which are directed against the proteins and are covalently bound to crosslinked agar preparation, for example Sepharose, can be used to separate them.
- the protein solution applied to a column which has been filled with the specific immunoadsorbent passes through the column undisturbed insofar as only those components against which the carrier contains an immunologically active partner are bound. In this way, the new glycoprotein can be freed from the impurities.
- Any body fluid or organ extract can be used as the starting material for the production of the new glycoprotein, in which it is possible to detect the glycoprotein immunologically.
- Extracts of human placentas are preferably used, which can be obtained by comminution and extraction with water or a dilute, advantageously a less than 10% salt solution, advantageously with a 0.5% neutral salt solution, such as sodium chloride. Use appropriately about 1-5 liters of the extraction solution are found on 1 kg of placenta. The undissolved portions are separated from the extract by centrifugation or filtration.
- the solution is introduced into an apparatus for preparative electrophoresis, as described, for example, by N. Heimburger and R. Schmidtberger in Behringwerke-Mitteilungen, number 43, page 83 ff., In particular on page 119-120.
- the device is a horizontal arrangement of a carrier electrophoresis in an open trough, in which the carrier material is cooled to below 10 ° C. in order to dissipate the Joule heat that occurs during electrophoresis.
- the zone in question is cut out and eluted from the inert support material with the aid of water or aqueous salt solutions, for example a 0.5 to 1% saline solution.
- the glycoprotein produced according to the invention has antigenic properties.
- An immunization of animals carried out in this way according to known methods led to the formation of specific antibodies in the blood of the immunized animals. Their sera can be obtained by conventional methods and the antibodies contained therein can be enriched.
- the antisera can be used in known immunological methods for the detection and determination of the new protein in body fluids, in particular in the blood serum.
- 150 kg of frozen placenta are crushed and extracted with 150 l of a 0.5% aqueous sodium chloride solution.
- the extract is adjusted to pH 8 with 2N sodium hydroxide and 50 I of a 3% aqueous solution of diaminoethoxyacridine lactate are added.
- the supernatant which contains the glycoprotein according to the invention (HPG-2)
- HPG-2 glycoprotein according to the invention
- 500 g of the precipitate collected on the filter are dissolved in 500 ml of distilled water and dialyzed against a 0.01 molar tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution of pH 7.0, which contains 0.05% sodium azide .
- the dialyzed solution is centrifuged and the supernatant is made up to 2000 ml with the same buffer solution, adjusted to pH 8.0 with 0.1N sodium hydroxide solution and with 500 g of moist diethylaminoethyl cellulose (from Serva, Heidelberg) are stirred for 1 hour.
- diethylaminoethyl cellulose is separated from the solution by filtration, washed twice with 1 liter of 0.01 molar tris (hydroxymethyl) aminomethane-hydrochloric acid buffer with a pH of 8.0 and then three times with 500 ml of 0.02 molar Tris (hydroxymethyl) aminomethane hydrochloric acid buffer, pH 6.5, which contains 0.85% sodium chloride and 0.05% sodium azide, eluted.
- the eluates are then tested with specific antiserum, the fractions containing the glycoprotein (HPG-2) are collected and the proteins are precipitated again with 30% solid ammonium sulfate, as described above.
- the precipitate is dissolved in 50 ml of water, dialyzed against a 0.005 m phosphate buffer, pH 6.8, and placed on a 3 ⁇ 23 cm column filled with hydroxyapatite.
- the column is developed using the 0.005 m phosphate buffer, pH 6.8.
- the glycoprotein (HPG-2) appears in the run; this is concentrated on an ultrafilter.
- the concentrate is then dialyzed against a 0.01 M Tris-HCl buffer, pH 7.0 and adsorbed on Deae-Sephadex (column 3 ⁇ 23 cm).
- a NaCI gradient of 0-2% is used to elute and separate the adsorbed proteins.
- the eluate fractions containing the glycoprotein (HPG-2) are concentrated in a concentrated manner.
- the concentrated eluate is taken up in a 0.075 m ammonium bicarbonate solution and subjected to preparative zone electrophoresis.
- the zone containing the HPG-2 is cut out after separation and eluted with physiological saline; the eluates are then concentrated on the ultrafilter.
- the a, B-glycoprotein still present as an impurity is removed with the aid of an appropriate immunoadsorbent.
- an appropriate immunoadsorbent antibodies against the ⁇ 1 B glycoprotein are covalently bound to Sepharose and the adsorbent thus obtained is brought into contact with the eluate in a batch process or in a column.
- the a, B glycoprotein is adsorbed onto the carrier-bound antibodies, while the glycoprotein HPG-2 remains in solution.
- the solution, which then only contains HPG-2, is dialyzed against water and lyophilized. About 10 to 30 mg of the new glycoprotein HPG-2 are obtained.
- the protein can be obtained in a similar manner from serum, including male serum. For this purpose, 100 liters of serum are used as the starting material, which is diluted with 100 l of distilled water. The protein is precipitated with Rivanol analogously to Example 1 and further purified.
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Pregnancy & Childbirth (AREA)
- Gynecology & Obstetrics (AREA)
- Immunology (AREA)
- Reproductive Health (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19772726886 DE2726886A1 (de) | 1977-06-15 | 1977-06-15 | Neues glykoprotein und verfahren zu dessen herstellung |
DE2726886 | 1977-06-15 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0000134A1 EP0000134A1 (de) | 1979-01-10 |
EP0000134B1 true EP0000134B1 (de) | 1981-10-28 |
Family
ID=6011534
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP78100134A Expired EP0000134B1 (de) | 1977-06-15 | 1978-06-09 | Glykoprotein, Verfahren zu seiner Herstellung, seine Verwendung zur Gewinnung von Antiserum sowie das Antiserum |
Country Status (14)
Country | Link |
---|---|
US (1) | US4269825A (ja) |
EP (1) | EP0000134B1 (ja) |
JP (1) | JPS548717A (ja) |
AT (1) | AT366063B (ja) |
AU (1) | AU523785B2 (ja) |
CA (1) | CA1110617A (ja) |
DE (2) | DE2726886A1 (ja) |
DK (1) | DK267278A (ja) |
ES (1) | ES470653A1 (ja) |
IE (1) | IE47092B1 (ja) |
IT (1) | IT1113082B (ja) |
MX (1) | MX5459E (ja) |
NZ (1) | NZ187548A (ja) |
ZA (1) | ZA783431B (ja) |
Families Citing this family (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2842467A1 (de) * | 1978-09-29 | 1980-04-10 | Behringwerke Ag | Neues ubiquitaeres gewebsprotein pp tief 8 |
DE2946458A1 (de) * | 1979-11-17 | 1981-06-11 | Behringwerke Ag, 3550 Marburg | Neues protein pp(pfeil abwaerts)11(pfeil abwaerts) |
DE2952792A1 (de) * | 1979-12-31 | 1981-07-02 | Behringwerke Ag, 3550 Marburg | Neues protein (pp(pfeil abwaerts)15(pfeil abwaerts)) mit immunsuppressiver wirkung |
DE3013724A1 (de) * | 1980-04-10 | 1981-10-15 | Behringwerke Ag, 3550 Marburg | Neues protein pp (pfeil abwaerts)9(pfeil abwaerts), verfahren zu seiner anreicherung und gewinnung sowieseine verwendung |
US4350687A (en) * | 1980-02-10 | 1982-09-21 | Research Corporation | Platelet derived cell growth factor |
JPS56145297A (en) * | 1980-04-11 | 1981-11-11 | Kureha Chem Ind Co Ltd | Preparative method of glycoprotein having immunosupressing activity |
DE3109629A1 (de) * | 1981-03-13 | 1982-09-23 | Behringwerke Ag, 3550 Marburg | "neues protein (pp(pfeil abwaerts)1(pfeil abwaerts)(pfeil abwaerts)6(pfeil abwaerts)), verfahren zu seiner anreicherung und gewinnung sowie seine verwendung" |
US4481137A (en) * | 1982-02-26 | 1984-11-06 | Mochida Pharmaceutical Co., Ltd. | Glycoproteins and processes for their production |
DE3315000A1 (de) * | 1983-04-26 | 1984-10-31 | Behringwerke Ag, 3550 Marburg | Gewebeprotein pp(pfeil abwaerts)4(pfeil abwaerts), verfahren zu seiner gewinnung sowie seine verwendung |
US4554256A (en) * | 1983-07-21 | 1985-11-19 | The Idaho Research Foundation, Inc. | Antigen associated with early detection of mammalian pregnancy |
US4524026A (en) * | 1983-08-29 | 1985-06-18 | Yoshio Sakagami | Novel proteinous cancer-cell proliferation inhibitory factors |
DE3334405A1 (de) * | 1983-09-23 | 1985-04-04 | Behringwerke Ag, 3550 Marburg | Membranassoziierte proteine (mp(pfeil abwaerts)2(pfeil abwaerts)), verfahren zu ihrer gewinnung sowie ihre verwendung |
JPS60199819A (ja) * | 1984-03-23 | 1985-10-09 | Kowa Co | トロンビン結合性物質およびその製法 |
JPS61294366A (ja) * | 1985-06-24 | 1986-12-25 | Toubishi Yakuhin Kogyo Kk | 糖鎖に特異的な抗体の検出用試薬及びその使用 |
DE3602688A1 (de) * | 1986-01-30 | 1987-08-06 | Behringwerke Ag | Verfahren zur gewinnung und herstellung eines pasteurisierten praeparates von (alpha)(pfeil abwaerts)2(pfeil abwaerts)-antiplasmin |
JPH0266456A (ja) * | 1988-08-31 | 1990-03-06 | Saikin Kagaku Kenkyusho:Kk | 疾病動物の一次診断方法、それに使用する診断試薬及びその製造方法 |
US5497003A (en) * | 1995-02-15 | 1996-03-05 | Servo Corporation Of America | Pyroelectric detector array with optical filter elements |
BG65084B1 (bg) * | 2002-03-13 | 2007-02-28 | Закрьiтое Акционерное Общество, Производственное Предприятие"Эндо-Фарм-А" | Нов клас биоактивни гликопротеини |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2221261A1 (de) * | 1972-04-29 | 1973-11-15 | Behringwerke Ag | Ap-glykoproteine und verfahren zu ihrer isolierung |
DE2256168A1 (de) * | 1972-11-16 | 1974-06-27 | Behringwerke Ag | Histidinreiches 3,8 s-alpha tief 2glykoprotein und verfahren zu seiner isolierung aus humanserum |
-
1977
- 1977-06-15 DE DE19772726886 patent/DE2726886A1/de not_active Withdrawn
-
1978
- 1978-06-09 EP EP78100134A patent/EP0000134B1/de not_active Expired
- 1978-06-09 DE DE7878100134T patent/DE2861256D1/de not_active Expired
- 1978-06-09 ES ES470653A patent/ES470653A1/es not_active Expired
- 1978-06-13 IT IT24531/78A patent/IT1113082B/it active
- 1978-06-13 NZ NZ187548A patent/NZ187548A/en unknown
- 1978-06-14 IE IE1198/78A patent/IE47092B1/en unknown
- 1978-06-14 CA CA305,433A patent/CA1110617A/en not_active Expired
- 1978-06-14 AT AT0433878A patent/AT366063B/de not_active IP Right Cessation
- 1978-06-14 AU AU37086/78A patent/AU523785B2/en not_active Expired
- 1978-06-14 ZA ZA00783431A patent/ZA783431B/xx unknown
- 1978-06-14 DK DK267278A patent/DK267278A/da not_active Application Discontinuation
- 1978-06-14 MX MX787147U patent/MX5459E/es unknown
- 1978-06-15 JP JP7157678A patent/JPS548717A/ja active Granted
-
1979
- 1979-12-19 US US06/105,416 patent/US4269825A/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
JPS548717A (en) | 1979-01-23 |
ATA433878A (de) | 1981-07-15 |
EP0000134A1 (de) | 1979-01-10 |
IE781198L (en) | 1978-12-15 |
IT7824531A0 (it) | 1978-06-13 |
AU523785B2 (en) | 1982-08-12 |
US4269825A (en) | 1981-05-26 |
DK267278A (da) | 1978-12-16 |
CA1110617A (en) | 1981-10-13 |
ZA783431B (en) | 1979-06-27 |
IT1113082B (it) | 1986-01-20 |
AU3708678A (en) | 1979-12-20 |
NZ187548A (en) | 1984-05-31 |
AT366063B (de) | 1982-03-10 |
MX5459E (es) | 1983-08-11 |
JPS6361319B2 (ja) | 1988-11-28 |
IE47092B1 (en) | 1983-12-14 |
DE2861256D1 (en) | 1982-01-07 |
ES470653A1 (es) | 1979-09-01 |
DE2726886A1 (de) | 1979-01-18 |
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