EP0000134B1 - Glycoprotein, its preparation, its use for the manufacture of antiserum and the antiserum - Google Patents

Glycoprotein, its preparation, its use for the manufacture of antiserum and the antiserum Download PDF

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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
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Prior art keywords
glycoprotein
solution
protein
antiserum
molecular weight
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EP78100134A
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German (de)
French (fr)
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EP0000134A1 (en
Inventor
Hans Dr. Bohn
Wilhelm Winckler
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Siemens Healthcare Diagnostics GmbH Germany
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Behringwerke AG
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    • 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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Description

Die Erfindung betrifft ein neues Glykoprotein, das im Blutserum, im Urin und im Extrakt menschlicher Plazenten nachgewiesen und daraus isoliert werden kann, sowie ein Verfahren zu dessen Herstellung.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.

Die durch die wässrige Extraktion menschlicher Plazenten erhältliche Proteinlösung enthält bekanntlich eine Vielzahl von Komponenten, die teilweise den Serumproteinen zuzuordnen und zum anderen Teil Gewebeproteine sind.As is known, 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.

Die Erfindung hat sich die Aufgabe gestellt, ein bisher noch nicht bekanntes Serum-Glykoprotein aus dem Extrakt menschlicher Plazenten zu isolieren, damit spezifisch gegen das neue Glykoprotein gerichtete Antiseren herzustellen, womit das Glykoprotein im Serum und im Urin qualitativ nachgewiesen oder quantitativ bestimmt werden kann.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.

Gegenstand der Erfindung ist ein neues Glykoprotein, welches aus Blutserum, aus Urin und dem Extrakt menschlicher Plazenten erhältlich ist. Es ist gekennzeichnet durch einen Proteinanteil, im wesentlichen bestehend aus a-Aminosäuren von 75± 6%, einen Kohlenhydratanteil von 24,6 ± 5,2%, davon Hexosen 8,9 ± 2%, N-acetyliertes Hexosamin 7,1 ± 1,5%, Fucose 0,2± 0,2%, N-acetylierte Neuraminsäure 8,4 ± 1,5%, einen Sedimentationskoeffizienten s20w von 2,5 ± 0,3 S, ein Molekulargewicht von 35000 ±5 000 aufgrund der Bestimmung in der Ultrazentrifuge bzw. ein Molekulargewicht von 65 000 ±10 000 aufgrund der Bestimmung im Natriumdodezylsulfat-haltigen Polyacrylamidgel, einen isoelektrischen Punkt von pH 3,4 ± 0,4, einen Extinktionskoeffizienten

Figure imgb0001
(280 nm) von 1,9% ± 0,3, eine elektrophore- . tische Beweglichkeit im Bereich zwischen den a\-und den a2-Globulinen und eine spezifische immunologische Reaktion mit einem spezifisch gegen das Glykoprotein gerichteten Antikörper. Zur Erläuterung der kennzeichnenden Merkmale des Glykoproteins sei folgendes ausgeführt: Die Bestimmung der Sedimentationskoeffizienten wurde in einer analytischen Ultrazentrifuge der Firma Beckman (Spinco-Apparatur, Modell E) bei 6000 UpM in Doppelsektorzellen mit Hilfe der UV-Scanner Technik bei 280 nm durchgeführt. Als Lösungsmittel diente ein 0,05 M Phosphatpuffer (pH 6,8) der 0,2 Mol/l NaCI enthielt. Die Proteinkonzentration betrug 2%. Die Sedimentationskoeffizienten sind auf die Basis von Wasser bei 21) °C umgerechnet worden.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
Figure imgb0001
 (280 nm) of 1.9% ± 0.3, an electrophoretic. table mobility in the area between the a \ and a 2 globulins and a specific immunological reaction with an antibody specifically directed against the glycoprotein. To explain the characteristic features of the glycoprotein, the following should be carried out: The determination of the sedimentation coefficients was carried out in an analytical ultracentrifuge from Beckman (Spinco apparatus, model E) at 6000 rpm in double-sector cells using the UV scanner technology at 280 nm. A 0.05 M phosphate buffer (pH 6.8) which contained 0.2 mol / l NaCl was used as solvent. The protein concentration was 2%. The sedimentation coefficients have been converted on the basis of water at 2 1 ) ° C.

Zur Ermittlung der Molekulargewichte wurde die Sedimentationsgleichgewichtsmethode und die Polyacrylamidgel-Elektrophorese herangezogen. Die Bestimmung in der Ultrazentrifuge wurde bei 9000 UpM vorgenommen. Die Auswertung erfolgte unter Zugrundelegung eines partiellen spezifischen Volumens (partial specific volume) von 0,74 mg/g. In der Ultrazentrifuge ergab sich ein Molekulargewicht von 35 000 ± 5000.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.

Für die Polyacrylamidgel-Elektrophorese wurden zwei Verfahren angewandt. Die Auftrennung im normalen Polyacrylamid (PAA)-Gel erfolgte nach der Methode von Zwisler und Biel, Z.klin. Chem. 4, Seite 58, (1966). Zur Untersuchung im Natriumdodecylsulfat-haltigen Gel wurde ein Gel mit 7,5% PAA, das 0,1% Natriumdodecylsulfat (SDS) enthielt, verwendet. Zur Reduktion sind die Proteine in 1 % SDS mit 1% Merkaptoäthanol inkubiert worden. Das Anfärben der Proteine geschah mit Amidoschwarz. Aus der Wanderung im SDShaltigen PAA-Gel wurde für das Glykoprotein ein Molekulargewicht von 65 000 ±10 000 abgeleitet.Two methods were used for polyacrylamide gel electrophoresis. The separation in normal polyacrylamide (PAA) gel was carried out according to the method of Zwisler and Biel, Z.klin. Chem. 4, page 58, (1966). A gel with 7.5% PAA, which contained 0.1% sodium dodecyl sulfate (SDS), was used for the investigation in the gel containing sodium dodecyl sulfate. For reduction, the proteins were incubated in 1% SDS with 1% mercaptoethanol. The proteins were stained with amido black. A molecular weight of 65,000 ± 10,000 was derived for the glycoprotein from the migration in the PAA gel containing SDS.

Die Bestimmung des isoelektrischen Punktes wurde mit einer Säule (440 ml) der Firma LKB Stockholm, durchgeführt. Das sogenannte Ampholin-Gemisch hatte bei der Untersuchung des Glykoproteins einen pH-Bereich von 2,5 bis 4,0.The isoelectric point was determined using a column (440 ml) from LKB Stockholm. The so-called ampholine mixture had a pH range of 2.5 to 4.0 when examining the glycoprotein.

Die Untersuchung der elektrophoretischen Beweglichkeit erfolgte in der Mikromodifikation von Beckmann Instruments auf Zelluloseazetatfolien mit Natriumdiäthylbarbiturat-Puffer pH 8,6.The electrophoretic mobility was examined in the micromodification by Beckmann Instruments on cellulose acetate films with sodium diethyl barbiturate buffer pH 8.6.

Die Bestimmung der Kohlehydrate erfolgte nach der von H. E. Schultze, R. Schmidtberger, H. Haupt, Biochem. Z. 329, Seite 490, (1958), beschriebenen Methode.The carbohydrates were determined according to that of H.E. Schultze, R. Schmidtberger, H. Haupt, Biochem. Z. 329, page 490, (1958).

Die Aminosäurenanalyse wurde nach S. Moore, D. H. Spackmann, W. H. Stein, Anal. Chem. 30, Seite 1185, (1958), unter Verwendung des Flüssigkeitschromatographen Multichrom B der Firma Beckmann durchgeführt. 1/2 Cystin wurde nach Oxydation der Proteine mit Perameisensäure (S. Moore et al., Anal. Chem. 30, Seite 1185, (1958) ) und nachfolgender Chromatographie (S. Moore, J. Biol. Chem. 238, Seite 235, (1963) ) als Cysteinsäure bestimmt. Der Tryptophangehalt ist mit der direkten photometrischen Bestimmung nach H. Edelhoch, Biochemistry 6, Seite 1948, (1967) ermittelt worden.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).

Die immunologische Charakterisierung der Substanz erfolgt am einfachsten durch ein bekanntes Diffusionsverfahren, bei welchen Antigen, d.h. das Glykoprotein und ein gegen das Glykoprotein gerichteter Antikörper bzw. das hinsichtlich der Antikörper nicht angereicherte Antiserum gegeneinander in einem Trägermedium, wie Agar, diffundieren. Treffen die beiden Reaktionskomponenten in einem günstigen Verhältnis aufeinander, bildet sich ein sichtbares Präzipitat aus. Nach dieser Kenntnis ist es für den Fachmann einleuchtend, dass alle immunologischen Techniken zum Nachweis und zur Bestimmung sowohl des neuen Glykoproteins, als auch der gegen das Glykoprotein gerichtete Antikörper möglich sind.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.

Eine einfache und in der Regel ausreichend genaue Methode zur quantitativen Bestimmung des Glykoproteins in Körperflüssigkeiten oder in Gewebeextrakten stellt die sogenannte Laurell-Technik dar. Sie ist beschrieben in Analyt. Biochem. (New York), 15, Seite 45 (1966).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).

Gegenstand der Erfindung ist ferner ein Verfahren zur Herstellung des oben charakterisierten Glykoproteins, dadurch gekennzeichnet, dass Körperflüssigkeiten oder Extrakte von Organen, welche das Glykoprotein enthalten, unter Zugrundelegung folgender erfindungsgemäss gefundener Kriterien fraktioniert werden.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.

Das Glykoprotein ist mit Neutralsalzen fällbar. Mit dem üblicherweise für derartige Fällungen verwendeten Ammoniumsulfat wird es bei einer Sättigungskonzentration des Salzes von 30 bis 60% in einem pH-Bereich in der Nähe des Neutralpunktes gefällt.The glycoprotein can be precipitated with neutral salts. With the 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.

Entsprechend seinem Molekulargewicht kann das Glykoprotein durch Massnahmen, die zur Abtrennung von Substanzen mit Molekulargewichten zwischen 25000 und 75000 geeignet sind, gewonnen werden. Vorteilhaft werden hierfür die Methoden der Gel-Filtration oder Ultrafiltration eingesetzt.Depending on its molecular weight, 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.

Das Glykoprotein wird bei neutralem oder schwach alkalischem pH-Wert an schwach basische Ionenaustauscher adsorbiert. Vorteilhaft wird dabei eine vergleichsweise wenig konzentrierte Pufferlösung verwendet, denn durch Erhöhung der Salzkonzentration oder auch durch Erniedrigung des pH-Wertes kann die Adsorption verhindert werden. Andererseits bietet sich bei Kenntnis dieses Verhaltens die Möglichkeit an, das Glykoprotein zu adsorbieren und unter Verwendung von höherkonzentrierten Salzlösungen bzw. von Pufferlösungen mit erniedrigtem pH-Wert zu eluieren.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. On the other hand, if 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.

Es hat sich gezeigt, dass das neue Glykoprotein mit den wasserlöslichen organischen Basen der Acridin- und Chinolinreihe, die für Protein-Fällungsverfahren üblicherweise Verwendung finden, nicht präzipitiert wird. Es bleibt in den bei diesem Verfahren üblichen Konzentrationen im wässrigen Überstand. Danach kann eine Acridinbase, wie 2-Äthoxy-6,9-Diaminoacridinlactat oder eine Chinolinbase, wie Bis-(2-methyl-4-aminochi- nolyl-6)-carbamid-hydrochlorid, zur Fällung von begleitenden Proteinen verwendet werden, wobei das erfindungsgemässe Glykoprotein im Oberstand verbleibt.It has been shown that 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.

Ähnliche Überlegungen können gelten bei Verwendung von Hydroxylapatit als Adsorbens für Proteine. Das neue Glykoprotein zeigt keine Affinität zum Hydroxylapatit, wohingegen eine Reihe von Begleitproteinen von Hydroxylapatit festgehalten wird. Das Glykoprotein gehört somit zu den Hydroxylapatit-passierenden Globulinen. Der Erfinder schlägt vor, es als Hydroxylapatit passierendes Globulin (HPG-2) zu bezeichnen.Similar considerations may apply when using 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.

Aufgrund der Kenntnis der elektrophoretischen Beweglichkeit kann für die Anreicherung bzw. Isolierung des Glykoproteins die präparative Zonenelektrophorese eingesetzt werden.Based on the knowledge of the electrophoretic mobility, the preparative zone electrophoresis can be used for the enrichment or isolation of the glycoprotein.

Die Affinität des Glykoproteins aufgrund seines immunologischen Verhaltens kann dafür eingesetzt werden, das Glykoprotein mit Hilfe von sog. Immun-Adsorptionsverfahren anzureichern. Hierfür kann in an sich bekannter Weise ein Immunadsorbens d.h. ein trägergebundener Antikörper, gegen das neue Glykoprotein hergestellt werden, welches das Glykoprotein spezifisch zu binden vermag. Das Glykoprotein kann danach durch Änderung der Milieubedingungen wieder eluiert werden, wie dies mehrfach beschrieben ist.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. For this purpose, 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.

Durch eine ausgewählte Kombination der genannten Methoden, die einerseits zur Anreicherung des Glykoproteins, andererseits zu einer Abtrennung von übrigen Begleitproteinen führen, kann die Isolierung der erfindungsgemässen Substanzen erfolgen. Demzufolge ist der Gegenstand der vorliegenden Erfindung in den einzelnen Anreicherungsschritten für das neue Glykoprotein und in den durch Kombination der Massnahmen zur Anreicherung sich ergebenden Verfahren zu dessen Reinigung zu sehen.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.

Die Leitlinie für das Verfahren zur Herstellung des Glykoproteins besteht darin, dass jeweils derjenige Anteil gewonnen wird, welcher eine positive immunologische Reaktion mit einem gegen das neue Glykoprotein gerichteten Antiserum ergibt.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.

Nach Durchführung der genannten Verfahrensschritte zeigt es sich zuweilen, dass das Glykoprotein noch von anderen immunologisch nachweisbaren Begleitproteinen verunreinigt ist. In diesem Falle werden die Verunreinigungen durch deren spezifische Adsorption entfernt. Man bedient sich dabei gängiger Techniken der Immunadsorption, bei welchen nach beschriebenen Verfahren an einen. Träger gebundene Antikörper gegen das zu entfernende Protein als Adsorbenzien eingesetzt werden. Häufig ist das weitgehend reine neue Glykoprotein noch mit Spuren des schwangerschafts-spezifischen ßi-Glykoproteins und/oder des ai-B-Glykoproteins, auch als leicht fällbares ai-Glykoprotein bezeichnet, vorgesellschaftet. Zu deren Abtrennung können gegen die Proteine gerichteten Immunoglobuline, welche kovalent an vernetzte Agarpräparation, z.B. Sepharose, gebunden sind, verwendet werden.After performing the above-mentioned process steps, it is sometimes shown that the glycoprotein is still contaminated by other immunologically detectable accompanying proteins. In this case, 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 β 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.

Die auf eine Säule, welche mit dem spezifischen Immunadsorbens gefüllt wurde, aufgetragene Proteinlösung läuft insoweit unbehelligt durch die Säule, als nur diejenigen Komponenten gebunden werden, gegen die der Träger einen immunologisch aktiven Partner enthält. Das neue Glykoprotein kann auf diese Weise von den Verunreinigungen befreit werden.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.

Zur Herstellung des neuen Glykoproteins werden mehrere der angeführten Massnahmen miteinander kombiniert und dabei jeweils diejenige Fraktion weiterverarbeitet, in der immunologisch das neue Glykoprotein nachgewiesen werden kann, während die übrigen Fraktionen verworfen werden.To produce the new glycoprotein, several of the measures listed are combined with one another and the fraction in which the new glycoprotein can be detected immunologically while the other fractions are discarded is further processed.

Als Ausgangsmaterial für die Herstellung des neuen Glykoproteins kann jede Körperflüssigkeit oder jeder Organextrakt verwendet werden, in welchem es gelingt, das Glykoprotein immunologisch nachzuweisen. Bevorzugt werden Extrakte menschlicher Plazenten verwendet, die durch Zerkleinerung und Extraktion mit Wasser oder einer verdünnten, zweckmässig einer weniger als 10%igen Salzlösung, vorteilhaft mit einer 0,5%igen Neutralsalzlösung, wie Natriumchlorid, gewonnen werden können. Zweckmässigverwendet man auf 1 kg Plazenten etwa 1-5 Liter der Extraktionslösung. Die nicht gelösten Anteile werden durch Zentrifugation oder Filtration von dem Extrakt abgetrennt.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.

Das Verfahren zur Anreicherung ist gekennzeichnet durch die Anwendung mindestens einer der folgenden Massnahmen auf Körperflüssigkeiten, welche das neue Glykoprotein enthalten und die anschliessende Gewinnung der bezüglich des Glykoproteins angereicherten Fraktion:

  • a) Zugabe von wasserlöslichen Derivaten einer Acridin- oder Chinolinbase, vorzugsweise des 2-Äthoxy-6,9-Diaminoacridin-lactat, im pH-Bereich von 5-10, vorzugsweise bei etwa pH 8, bis zu einer Endkonzentration von etwa 0,8% (Gewicht zu Volumen), wobei das Glykoprotein im wesentlichen im Überstand verbleibt.
  • b) Zugabe von Neutralsalzen bis zur Ausfällung des Glykoproteins, vorzugsweise von Ammoniumsulfat, bei etwa neutralem pH-Wert von 5-8 bis zu 30-60% der Sättigungskonzentration des Ammoniumsulfats.
  • c) Adsorption des Glykoproteins an einem schwach-basischen lonenaustauscher, wie Di- äthylaminoäthyl-cellulose, bei einer Leitfähigkeit der Lösung von 0-2 mS und neutralem oder schwachalkalischem pH-Wert (6-9). Beispielsweise unter Verwendung eines etwa 0,01 M Puffers vom pH-Wert von etwa 8. Ein bevorzugt zu verwendender Puffer ist Tris-hydroxymethylaminomethan-HCI. Die Elution des Glykoproteins kann durch Senkung des pH-Wertes unter pH 7,0 oder durch Erhöhung der Leitfähigkeit über 5 mS erreicht werden.
  • d) Trennung aufgrund der Molekülgrösse (Molekularsiebfraktionierung). Besonders geeignet ist die Gelfiltration in einer Säule gefüllt mit einem Polymeren entsprechender Porengrösse, beispielsweise mit Epichlorhydrin-vernetztem Dextran, als Sephadex@ hergestellt von der Firma Pharmacia, Uppsala, mit dem Ziel der Anreicherung von Proteinen mit einem Molekulargewicht von etwa 50 000. Aber auch Produkte wie Ultro- ge18 von LKB, Bromma oder Bio-Gef" von Bio-Rad Laboratories, Richmond, Calif. können eingesetzt werden.
  • e) Durchführung einer Adsorption mit Hydroxylapatit. Da das Glykoprotein in verdünnter Phosphatpufferlösung von Hydroxylapatit nicht gebunden wird, ist Hydroxylapatit ein geeignetes Agens, um Begleitproteine des Glykoproteins aus der Lösung zu entfernen. Die Proteinlösung wird hierfür zweckmässig auf einen pH-Wert um den Neutralpunkt eingestellt und die Leitfähigkeit der Lösung auf etwa 1 mS gehalten.
  • f) Präparative Zonenelektrophorese.
The enrichment process is characterized by the application of at least one of the following measures to body fluids containing the new glycoprotein and the subsequent recovery of the fraction enriched with respect to the glycoprotein:
  • a) addition of water-soluble derivatives of an acridine or quinoline base, preferably of 2-ethoxy-6,9-diaminoacridine lactate, in the pH range from 5-10, preferably at about pH 8, to a final concentration of about 0.8 % (Weight to volume), the glycoprotein remaining essentially in the supernatant.
  • b) adding neutral salts until the precipitation of the glycoprotein, preferably ammonium sulfate, at an approximately neutral pH of 5-8 up to 30-60% of the saturation concentration of the ammonium sulfate.
  • c) adsorption of the glycoprotein on a weakly basic ion exchanger, such as diethylaminoethyl cellulose, with a solution conductivity of 0-2 mS and neutral or weakly alkaline pH (6-9). For example, using an approximately 0.01 M buffer with a pH of approximately 8. A preferred buffer to use is tris-hydroxymethylaminomethane-HCl. Elution of the glycoprotein can be achieved by lowering the pH below pH 7.0 or by increasing the conductivity above 5 mS.
  • d) separation based on molecular size (molecular sieve fractionation). Gel filtration in a column filled with a polymer of appropriate pore size, for example with epichlorohydrin-crosslinked dextran, as Sephadex®, manufactured by Pharmacia, Uppsala, with the aim of enriching proteins with a molecular weight of approximately 50,000, is particularly suitable, but also products like Ultro- ge1 8 of LKB, Bromma or organic Found "by Bio-Rad Laboratories, Richmond, Calif can. be used.
  • e) performing an adsorption with hydroxyapatite. Since the glycoprotein is not bound by hydroxyapatite in dilute phosphate buffer solution, hydroxyapatite is a suitable agent for removing accompanying proteins of the glycoprotein from the solution. For this purpose, the protein solution is expediently adjusted to a pH around the neutral point and the conductivity of the solution is kept at approximately 1 mS.
  • f) Preparative zone electrophoresis.

Geeignet für die Durchführung einer Elektrophorese ist eine das Glykoprotein enthaltende Lösung, vorzugsweise eine alkalische Pufferlösung, beispielsweise in einem Natriumdiäthylbarbiturat-puffer von pH 8,6, und einer lonenstärke=0,1. Die Lösung wird in eine Apparatur zur präparativen Elektrophorese eingetragen, wie sie beispielsweise von N. Heimburger und R. Schmidtberger in Behringwerke-Mitteilungen, Heft 43, Seite 83 ff., insbesondere auf Seite 119-120, beschrieben wird. Bei dem Gerät handelt es sich um die horizontale Anordnung einer Trägerelektrophorese in einem offenen Trog, in welchem das Trägermaterial zur Abführung der bei der Elektrophorese auftretenden Joul'schen Wärme auf unter 10 °C gekühlt wird. Als Trägermaterial dienen gegenüber Proteinen indifferente Substanzen, vorteilhaft Polyvinylchlorid, oder dessen Copolymerisate in Form eines Granulats.Suitable for performing electrophoresis is a solution containing the glycoprotein, preferably an alkaline buffer solution, for example in a sodium diethyl barbiturate buffer of pH 8.6, and an ionic strength = 0.1. 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. Substances which are indifferent to proteins, advantageously polyvinyl chloride, or its copolymers in the form of granules, serve as carrier material.

Es ist empfehlenswert, die Elektrophorese im alkalischen pH-Bereich, vorteilhaft bei etwa pH 8,6, bei einer Ionenstärke von 0,08-0,12 und bei einer Feldstärke von 4-6 Volt/cm vorzunehmen. Bei Verwendung von 0,1 M Natriumdiäthylbarbituratpuffer vom pH-Wert 8,6 wandert das Glykoprotein im elektrischen Feld in den zwischen ai-und a2-Globulinen liegenden Bereich der Plasmaproteine.It is recommended to carry out electrophoresis in the alkaline pH range, advantageously at about pH 8.6, with an ionic strength of 0.08-0.12 and with a field strength of 4-6 volts / cm. When 0.1 M sodium diethyl barbiturate buffer with a pH of 8.6 is used, the glycoprotein migrates in the electric field into the region of the plasma proteins lying between a i and a 2 globulins.

Für die Gewinnung des neuen Glykoproteins wird die betreffende Zone herausgeschnitten und vom inerten Trägermaterial mit Hilfe von Wasser oder wässriger Salzlösungen, beispielsweise einer 0,5 bis 1%igen Kochsalzlösung, eluiert.To obtain the new glycoprotein, 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.

Das erfindungsgemäss hergestellte Glykoprotein hat antigene Eigenschaften. Eine damit durchgeführte Immunisierung von Tieren nach bekannten Methoden führte zur Bildung von spezifischen Antikörpern im Blut der immunisierten Tiere. Deren Seren können nach üblichen Verfahren gewonnen und die darin enthaltenen Antikörper angereichert werden. Die Antiseren können in bekannten immunologischen Verfahren zum Nachweis und zur Bestimmung des neuen Proteins in Körperflüssigkeiten, insbesondere in dem Blutserum, Verwendung finden.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.

Die Erfindung wird in dem nachstehenden Beispiel näher erläutert.The invention is explained in more detail in the example below.

Beispielexample

150 kg tiefgefrorene Plazenten werden zerkleinert und mit 150 I einer 0,5%igen wässrigen Natriumchloridlösung extrahiert. Der Extrakt wird mit 2n-Natriumhydroxyd auf pH 8 eingestellt und mit 50 I einer 3%igen wässrigen Lösung von Diaminoäthoxyacridin-lactat versetzt. Nach einer Wartezeit von 1 Stunde wird der Überstand der das erfindungsgemässe Glykoprotein (HPG-2) enthält, abgehebert, mit 5% festem Natriumchlorid (11 kg) zur Abscheidung des noch in Lösung verbliebenen Diaminoäthoxyacridin-lactats versetzt, filtriert und mit 30% - bezogen auf das Gewicht der Flüssigkeit - festem Ammoniumsulfat versetzt und gut durchgerührt. Nach 1 Stunde wird der Niederschlag abfiltriert.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. After a waiting time of 1 hour, the supernatant, which contains the glycoprotein according to the invention (HPG-2), is siphoned off, mixed with 5% solid sodium chloride (11 kg) to separate the diaminoethoxyacridine lactate still in solution, filtered and obtained with 30% to the weight of the liquid - solid ammonium sulfate added and stirred well. After 1 hour the precipitate is filtered off.

500 g des auf dem Filter gesammelten Niederschlages werden in 500 ml destilliertem Wasser gelöst und gegen eine 0,01 molare Tris-(hydroxymethyl)-aminomethan-Salzsäure-Pufferlösung vom pH-Wert 7,0, die 0,05% Natriumazid enthält, dialysiert. Die dialysierte Lösung wird zentrifugiert und der Überstand wird mit der gleichen PufferLösung auf 2000 ml aufgefüllt, mit 0,1 n Natriumhydroxydlösung auf pH 8,0 eingestellt und mit 500 g feuchter Diäthylaminoäthylcellulose (Firma Serva, Heidelberg) 1 Stunde verrührt.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.

Dann wird die Diäthylaminoäthylcellulose durch Filtrieren von der Lösung abgetrennt, zweimal mit je 1 Liter 0,01 molarem Tris-(hydroxymethyl)-aminomethan-Salzsäure-Puffer vom pH-Wert 8,0 gewaschen und danach dreimal mit je 500 ml 0,02 molarem Tris-(hydroxymethyl)-ami- nomethan-Salzsäure-Puffer, pH 6,5, der 0,85% Natriumchlorid und 0,05% Natriumazid enthält, eluiert.Then the 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.

Den vereinigten Eluaten werden 30% Ammonsulfat, bezogen auf das Flüssigkeitsgewicht, zugesetzt und das ganze wird verrührt. Der Niederschlag, der das Protein (HPG-2) enthält, wird in 300 ml destilliertem Wasser gelöst. Die Proteinlösung wird gegen Tris-hydroxymethyl-Aminomethan-Salzsäure-Puffer von pH 8,0, der 1,0 Mol Natriumchlorid/1 enthält, dialysiert und auf eine mit Sephadex G-150 gefüllte Säule (100X20 cm) aufgetragen und mit dem genannten Puffer eluiert. Während der Elution findet eine Fraktionierung der Proteine nach deren Molekülgrösse statt.30% ammonium sulfate, based on the liquid weight, are added to the combined eluates and the whole is stirred. The precipitate containing the protein (HPG-2) is dissolved in 300 ml of distilled water. The protein solution is dialyzed against tris-hydroxymethyl-aminomethane-hydrochloric acid buffer of pH 8.0, which contains 1.0 mol of sodium chloride / 1, and applied to a column (100 × 20 cm) filled with Sephadex G-150 and with the buffer mentioned eluted. During the elution, the proteins are fractionated according to their molecular size.

Anschliessend werden die Eluate mit spezifischem Antiserum getestet, die das Glykoprotein (HPG-2) enthaltenden Fraktionen werden gesammelt und daraus die Proteine nochmals, wie oben beschrieben, mit 30% festem Ammonsulfat ausgefällt.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.

Zur Weiterreinigung wird die Fällung in 50 ml Wasser gelöst, gegen einen 0,005 m Phosphatpuffer, pH 6,8, dialysiert und auf eine mit Hydroxylapatit gefüllte Säule, 3x23 cm, gegeben. Die Entwicklung der Säule erfolgt mit dem 0,005 m Phosphatpuffer, pH 6,8.For further purification, 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.

Das Glykoprotein (HPG-2) erscheint im Durchlauf; dieser wird auf einem Ultrafilter eingeengt. Das Konzentrat wird anschliessend gegen einen 0,01 M Tris-HCI-Puffer, pH 7,0, dialysiert und an Deae-Sephadex (Säule 3 X 23 cm) adsorbiert. Zur Elution und Auftrennung der adsorbierten Proteine dient ein NaCI-Gradient von 0-2%. Die das Glykoprotein (HPG-2) enthaltenden Eluatfraktionen werden gesammelt eingeengt.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.

Zur Weiterreinigung wird das eingeengte Eluat in einer 0,075 m Ammoniumbikarbonatlösung aufgenommen und einer präparativen Zonenelektrophorese unterworfen. Die das HPG-2 enthaltende Zone wird nach der Auftrennung herausgeschnitten und mit physiologischer Kochsalzlösung eluiert; die Eluate engt man anschliessend auf dem Ultrafilter ein.For further purification, 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.

Das als Verunreinigung noch vorhandene a,B-Glykoprotein wird mit Hilfe eines entsprechenden Immunadsorbens entfernt. Zur Herstellung des Immunadsorbens werden Antikörper gegen das α1B-Glykoprotein kovalent an Sepharose gebunden und das so erhaltene Adsorbens mit dem Eluat im Batch-Verfahren oder in einer Säule in Berührung gebracht. Dabei wird das a,B-Glykoprotein an die trägergebundenen Antikörper adsorbiert, während das Glykoprotein HPG-2 in Lösung bleibt. Die Lösung, die dann nur noch HPG-2 enthält, wird gegen Wasser dialysiert und lyophilisiert. Man erhält etwa 10 bis 30 mg des neuen Glykoproteins HPG-2.The a, B-glycoprotein still present as an impurity is removed with the aid of an appropriate immunoadsorbent. To prepare the immunoadsorbent, antibodies against the α 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.

Es zeigt folgende Aminosäure-Zusammensetzung (Häufigkeit mit Variationskoeffizienten (VK in %):

Figure imgb0002
Das Protein kann in ähnlicher Weise aus Serum, auch aus Männerserum, gewonnen werden. Dazu werden als Ausgangsmaterial 100 Liter Serum eingesetzt, das mit 100 I destilliertem Wasser verdünnt wird. Das Protein wird analog Beispiel 1 mit Rivanol gefällt und weitergereinigt.It shows the following amino acid composition (frequency with coefficient of variation (CV in%):
Figure imgb0002
 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.

Claims (5)

1. Glycoprotein, characterized by
a) a protein proportion of 75 ± 6%;
b) a carbohydrate proportion of 24,6 ± 5,2%; of which 8,9 ± 2% of hexoses, 7,1 ± 1,5% N-acetylated hexosamine, 0,2 ± 0,2% of fucose and 8,4 ± 1,5% of N-acetylated neuraminic acid;
c) a sedimentation coefficient S20w of 2,5 ± 0,3 S;
d) a molecular weight of 35 000 ± 5000 determined in the ultracentrifuge;
e) an iso-electric point of pH 3,4 ± 0,4;
f) an extinction coefficient
Figure imgb0005
 (280 mm) of 1,9 ± 0,3;
g) an electrophoretic mobility in the range between ai- and a2-globulines;
h) a specific immunologic reaction with an antibody directed specifically against the glykoprotein.
2. Process for enriching the glycoprotein defined in claim 1, which comprises subjecting a protein solution containing that glycoprotein to at least one of the following measures and isolating the fraction enriched with that glycoprotein:
a) addition of neutral salts until the glycoprotein is precipitated;
b) molecular sieve fractionation and isolation of the fraction having a molecular weight of between 25 000 and 75 000;
c) adsorption of the glycoprotein on a weakly basic ion exchanger and elution therefrom;
d) addition of water-soluble derivatives of an acridine or quinoline base in a pH-range of from 5 to 10 until a final concentration of about 0,8% is reached;
e) treatment of the glycoprotein solution with hydroxyl-apatite;
f) preparative zone electrophoresis and isolation of the zone between a,- and a2-globulines;
g) treatment of the glycoprotein solution with an immuno adsorbant.
3. A process as claimed in claim 2, wherein said protein solution is an extract of human placentas.
4. The use of the protein defined in claim 1 for the preparation of antisera.
5. An antiserum obtained by immunization of vertebrates with the glycoprotein defined in claim 1.
EP78100134A 1977-06-15 1978-06-09 Glycoprotein, its preparation, its use for the manufacture of antiserum and the antiserum Expired EP0000134B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2726886 1977-06-15
DE19772726886 DE2726886A1 (en) 1977-06-15 1977-06-15 NEW GLYCOPROTEIN AND METHOD FOR THE PRODUCTION THEREOF

Publications (2)

Publication Number Publication Date
EP0000134A1 EP0000134A1 (en) 1979-01-10
EP0000134B1 true EP0000134B1 (en) 1981-10-28

Family

ID=6011534

Family Applications (1)

Application Number Title Priority Date Filing Date
EP78100134A Expired EP0000134B1 (en) 1977-06-15 1978-06-09 Glycoprotein, its preparation, its use for the manufacture of antiserum and the antiserum

Country Status (14)

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US (1) US4269825A (en)
EP (1) EP0000134B1 (en)
JP (1) JPS548717A (en)
AT (1) AT366063B (en)
AU (1) AU523785B2 (en)
CA (1) CA1110617A (en)
DE (2) DE2726886A1 (en)
DK (1) DK267278A (en)
ES (1) ES470653A1 (en)
IE (1) IE47092B1 (en)
IT (1) IT1113082B (en)
MX (1) MX5459E (en)
NZ (1) NZ187548A (en)
ZA (1) ZA783431B (en)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2842467A1 (en) * 1978-09-29 1980-04-10 Behringwerke Ag NEW UBIQUITAER GEWEBSPROTEIN PP TIEF 8
DE2946458A1 (en) * 1979-11-17 1981-06-11 Behringwerke Ag, 3550 Marburg NEW PROTEIN PP (DOWN ARROW) 11 (DOWN ARROW)
DE2952792A1 (en) * 1979-12-31 1981-07-02 Behringwerke Ag, 3550 Marburg NEW PROTEIN (PP (DOWN ARROW) 15 (DOWN ARROW)) WITH IMMUNE SUPPRESSIVE EFFECT
DE3013724A1 (en) * 1980-04-10 1981-10-15 Behringwerke Ag, 3550 Marburg NEW PROTEIN PP (DOWN ARROW) 9 (DOWN ARROW), METHOD FOR ITS ENRICHMENT AND PRODUCTION AND ITS USE
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 (en) * 1981-03-13 1982-09-23 Behringwerke Ag, 3550 Marburg "NEW PROTEIN (PP (DOWN ARROW) 1 (DOWN ARROW) (DOWN ARROW) 6 (DOWN ARROW)), METHOD FOR ITS PREPARATION AND PRODUCTION AND ITS USE"
US4481137A (en) * 1982-02-26 1984-11-06 Mochida Pharmaceutical Co., Ltd. Glycoproteins and processes for their production
DE3315000A1 (en) * 1983-04-26 1984-10-31 Behringwerke Ag, 3550 Marburg TISSUE PROTEIN PP (DOWN ARROW) 4 (DOWN ARROW), METHOD FOR ITS RECOVERY AND USE
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 (en) * 1983-09-23 1985-04-04 Behringwerke Ag, 3550 Marburg MEMBRANE ASSOCIATED PROTEINS (MP (DOWN ARROW) 2 (DOWN ARROW)), METHOD FOR THEIR EXTRACTION AND USE
JPS60199819A (en) * 1984-03-23 1985-10-09 Kowa Co Thrombin binding substance and preparation thereof
JPS61294366A (en) * 1985-06-24 1986-12-25 Toubishi Yakuhin Kogyo Kk Reagent for detecting antibody specific to sugar chain and its use
DE3602688A1 (en) * 1986-01-30 1987-08-06 Behringwerke Ag METHOD FOR OBTAINING AND PRODUCING A PASTEURIZED PREPARATE OF (ALPHA) (ARROW DOWN) 2 (ARROW DOWN) -ANTIPLASMINE
JPH0266456A (en) * 1988-08-31 1990-03-06 Saikin Kagaku Kenkyusho:Kk Primary diagnostic method for disease animal and diagnostic reagent used therein and its production
US5497003A (en) * 1995-02-15 1996-03-05 Servo Corporation Of America Pyroelectric detector array with optical filter elements
BG65084B1 (en) * 2002-03-13 2007-02-28 Закрьiтое Акционерное Общество, Производственное Предприятие"Эндо-Фарм-А" New class of bioactive glycoproteins

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2221261A1 (en) * 1972-04-29 1973-11-15 Behringwerke Ag AP GLYCOPROTEINS AND PROCEDURES FOR THEIR ISOLATION
DE2256168A1 (en) * 1972-11-16 1974-06-27 Behringwerke Ag Histidine-rich 3,8 s-alpha 2-glucoprotein separation - from human serum by adsorption, extn, concn., zone electrophoresis, separation of alpha 2-globulin zone and re-concn.

Also Published As

Publication number Publication date
CA1110617A (en) 1981-10-13
JPS6361319B2 (en) 1988-11-28
IE47092B1 (en) 1983-12-14
JPS548717A (en) 1979-01-23
ATA433878A (en) 1981-07-15
AU3708678A (en) 1979-12-20
IE781198L (en) 1978-12-15
IT1113082B (en) 1986-01-20
ES470653A1 (en) 1979-09-01
DK267278A (en) 1978-12-16
MX5459E (en) 1983-08-11
AT366063B (en) 1982-03-10
DE2861256D1 (en) 1982-01-07
IT7824531A0 (en) 1978-06-13
AU523785B2 (en) 1982-08-12
US4269825A (en) 1981-05-26
DE2726886A1 (en) 1979-01-18
NZ187548A (en) 1984-05-31
EP0000134A1 (en) 1979-01-10
ZA783431B (en) 1979-06-27

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