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 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
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
Application number
EP78100134A
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German (de)
English (en)
French (fr)
Other versions
EP0000134A1 (de
Inventor
Hans Dr. Bohn
Wilhelm Winckler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens Healthcare Diagnostics GmbH Germany
Original Assignee
Behringwerke AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Behringwerke AG filed Critical Behringwerke AG
Publication of EP0000134A1 publication Critical patent/EP0000134A1/de
Application granted granted Critical
Publication of EP0000134B1 publication Critical patent/EP0000134B1/de
Expired legal-status Critical Current

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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)
EP78100134A 1977-06-15 1978-06-09 Glykoprotein, Verfahren zu seiner Herstellung, seine Verwendung zur Gewinnung von Antiserum sowie das Antiserum Expired EP0000134B1 (de)

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)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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

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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