EP0000134A1 - Glycoprotein, its preparation, its use for the manufacture of antiserum and the antiserum - Google Patents

Abstract

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, consisting essentially of α-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 o, 2 ± 0.2%, N-acetylated neuraminic acid 8.4 ± 1.5%, a sedimentation coefficient S20w of 2.5 ± 0.3 S, a molecular weight of 35,000 ± 5,000 based on the determination in the ultracentrifuge or a molecular weight of 65,000 ± 10,000 on the basis of the determination in polyacrylamide gel containing sodium dodezyl sulfate, an isoelectric point of pH 3.4 ± 0.4, an extinction coefficient E ??? (280 nm) of 1.9 ± 0.3, an electrophoretic mobility in the range between the α1 and the α2 globulins and a specific immunological reaction with an antibody specifically directed against the glycoprotein. The glycoprotein is obtained by fractionating body fluids or extracts from organs that contain the glycoprotein. To obtain antisera, vertebrates are immunized with the glycoprotein.

Description

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.

As is known, the protein solution obtainable by the 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 serum glycoprotein, which is not yet known, from the extract of human placentas, so that antisera directed specifically against the new glycoprotein can be produced, with which the glycoprotein in the serum and in the urine can be qualitatively detected or quantified .

(280 nm) von 1,9 ± 0,3, eine elektrophoretische Beweglichkeit im Bereich zwischen den α₁- und den α-₂-Globulinen und eine spezifische immunologische Reaktion mit einem spezifisch gegen das Glykoprotein gerichteten Antikörper.The invention relates to a new glycopronein; which is available from blood serum, urine and the extract of human placentas. It is characterized by a protein content, essentially consisting of α-amino acids of 75 ± 6%,
a carbohydrate content of 24.6 ± 5.2 of which hexoses 8.9 ± 2%, N-acetylated hexosamine 7.1 t 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 35,000 ± 5,000 based on the determination in the ultracentrifuge or a molecular weight of 65,000 ± 10,000 based on the determination in the sodium dodezyl sulfate-containing Polyacrylamide gel, an isoelectric point of pH 3.4 ^± 0.4, an extinction coefficient E

(280 nm) of 1.9 ± 0.3, an electrophoretic mobility in the range between the α ₁ - and the α- ₂ globulins and a specific immunological reaction with an antibody specifically directed against the glycoprotein.

• Die Bestimmung der Sedimentationskoeffizienten wurde in einer analytischen Ultrazentrifuge der Firma Beckman (Spinco-Apparatur, Modell E) bei 6.000 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 NaCl enthielt. Die Proteinkonzentration betrug 2 %. Die Sedimentationskoeffizienten sind auf die Basis von Wasser bei 20°C umgerechnet worden.

To explain the characteristic features of the glycoprotein, the following should be carried out:

• The sedimentation coefficients were determined in an analytical ultracentrifuge from Beckman (Spinco apparatus, model E) at 6,000 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 20 ° C.

The sediment equilibrium method and the polyacrylamide gel electrophoresis were used to determine the molecular weights. The determination in the ultracentrifuge was carried out at 9,000 rpm. The evaluation was carried out on the basis of a partial specific volume of 0.74 mg / g. In the Ultracentrifuge resulted in a molecular weight of 35,000 ^± 5,000.

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 containing 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 SDS-containing PAA gel.

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.

The electrophoretic mobility was examined in the micromodification by Beckmann Instruments on cellulose acetate films with sodium diethyl barbiturate buffer pH 8.6.

The carbohydrates were determined according to the method of H.E. Schultze, R. Schmidtberger, H. Haupt, Biochem. Z. 329, page 490, (1958).

The amino acid analysis was carried out according to S. Moore, DH Spackmann, WH 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 is with direct photometric determination according to H. Edelhoch, Biochemistry 6, page 1948, (1967).

The simplest way of immunologically characterizing the substance is with 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 against one another in a carrier medium, such as e.g. Agar, diffuse. 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 furthermore relates to a process for the preparation of the glycoprotein characterized above, 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. With the ammonium sulfate usually used for such precipitations, it is precipitated at a saturation concentration of 30 to 60% of the salt in a pH range near the neutral point.

von Substanzen mit Molekulargewichten zwischen 25.000 und 75.000 geeignet sind, gewonnen werden. Vorteilhaft werden hierfür die Methoden der Gel-Filtration oder Ultrafiltration eingesetzt.According to its molecular weight, the glycoprotein can

of substances with Molecular weights between 25,000 and 75,000 are suitable. 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, because 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.

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 to precipitate accompanying proteins, the glycoprotein according to the invention excess remains.

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 suggests to call it hydroxylapatite passing globulin (HPG-2).

Based on the knowledge of the electrophoretic mobility, 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. 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 in the specialist literature.

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 subject of the present invention is in the individual enrichment steps. to be seen for the new glycoprotein and in the processes for its purification resulting 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.

After carrying out 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. You use yourself common techniques of immunoadsorption, in which, according to the methods described, antibodies bound to a carrier against the protein to be removed are used as adsorbents. Often, the largely pure new glycoprotein is still associated with traces of the pregnancy-specific β ₁ glycoprotein and / or the α ₁ B glycoprotein, also known as easily precipitated a ₁ glycoprotein. For their separation, immunoglobulins directed against the proteins and which are covalently bound to crosslinked agar preparations, such as, for example, SEPHAROSE, can be used.

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.

To produce the new glycoprotein, several of the measures listed are combined with one another and in each case the fraction is further processed in which the new glycoprotein can be detected immunologically, while the other fractions are discarded.

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. It is advisable to use about 1 - 5 liters of the extraction solution on 1 kg of placenta. The undissolved portions are separated from the extract by centrifugation or filtration.

• a) Zugabe von wasserlöslichen Derivaten einer A^pridin-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 vom Ammoniumsulfat bei etwa neutralem pH-Wert von 5 - 8 bis zu 30 - 60 % der Sättigungskonzentration des Ammoniumsulfats.
• c) Adsorption des Glykoproteins an einem schwach-basischem Ionenaustauscher, wie Diäthylaminoäthyl-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 beispielsweise Tris-Hydroxy- ·methylaminomethan-HCl. 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^(R) hergestellt von der Firma Pharmacia, Uppsala, mit dem Ziel der Anreicherung von Proteinen mit einem Molekulargewicht von etwa 50.000. Aber auch Produkte wie ULTROGEL^(R) von _LKB, Bromma oder BIO-GEL^(R) 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. Geeignet für die Durchführung einer Elektrophorese ist eine das Glykoprotein enthaltende Lösung, vorzugsweise eine alkalische Pufferlösung, beispielsweise in einem Natriumdiäthylbarbituratpuffer von pH 8,6, und einer Ionenstärke = 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 Polyvinyl- chlorid; oder dessen Copolymerisate in Form eines Granulats.

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 in the glycoprotein:

• a) adding water-soluble derivatives of an ^A-p ridin or quinoline base, preferably, of 2-ethoxy-6,9-diaminoacridine lactate, in the pH range 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 glycoprotein is precipitated, preferably ammonium sulfate at an approximately neutral pH of 5-8 up to 30-60% of the saturation concentration of ammonium sulfate.
• c) Adsorption of the glycoprotein on a weakly basic ion exchanger, such as diethylaminoethyl cellulose, with a conductivity of the solution of 0-2 mS and a 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 be used is, for example, tris-hydroxy- · methylaminomethane-HCl. The 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 ^(R) manufactured by the Pharmacia, Uppsala, with the aim of enriching proteins with a molecular weight of about 50,000. But also products like ULTROGEL ^(R) from _LKB , Bromma or BIO-GEL ^(R) from 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. The protein solution is expediently adjusted to a pH around the neutral point and the conductivity of the solution is kept at about 1 mS.
• f) Preparative zone electrophoresis. 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, is suitable for carrying out electrophoresis. 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, serve as the carrier material; or its copolymers in the form of granules.

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 4 - 6 volts / cm. When using 0.1 M sodium diethyl barbiturate buffer with a pH of 8.6, the glycoprotein migrates in an electric field into the area of the plasma proteins between a ₁ and a ₂ globulins.

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.

The glycoprotein produced according to the invention has antigenic properties. An immunization of animals carried out by 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.

The invention is explained in more detail in the example below.

example

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 mixed with 50 liters of a 3% aqueous solution of diaminoethoxyacridine lactate. After a waiting time of 1 hour, the supernatant which contains the glycoprotein (HPG-2) according to the invention is siphoned off, mixed with 5% solid sodium chloride (11 kg) to separate the diaminoethoxyacridine lactate still in solution, filtered and obtained at 30% to the weight of the liquid - solid ammonium sulfate added and stirred well. After 1 hour the precipitate is filtered off.

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 (oxymethyl) 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 2,000 ml with the same buffer solution, adjusted to pH 8.0 with 0.1N sodium hydroxide solution and stirred with 500 g of moist diethylaminoethyl cellulose (Serva, Heidelberg) for 1 hour.

Then the diethylaminoethyl cellulose is separated from the solution by filtration, washed twice with 1 liter of 0.01 molar tris (oxymethyl) aminomethane-hydrochloric acid buffer with a pH of 8.0 and then three times with 500 ml of 0.02 molar Tris (oxymethyl) aminomethane hydrochloric acid buffer, pH 6.5, which contains: 0.85% sodium chloride and 0.05% sodium azide; eluted.

. Der Niederschlag, der das Protein (HPG-2) entwird in 300 ml destilliertem Wasser gelöst. Die Proteinlösung wird gegen Tris-Hydroxymethyl-Aminomethan-Salzsäure-Puffer von pH 8,0, der 1,0 Mol Natriumchlorid/l enthält, dialysiert und auf eine mit Sephadex G-150 gefüllte Säule (100 x 20 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

. The precipitate, which is 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 / l, and applied to a column (100 x 20 cm) filled with Sephadex G-150 and with the called buffer eluted. During the elution, the proteins are fractionated according to their molecular size.

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.

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.

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 NaCl gradient of 0-2% is used to elute and separate the adsorbed proteins. The eluate fractions containing the glycoprotein (HPG-2) are concentrated together.

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.

The α ₁ B glycoprotein still present as an impurity is removed with the aid of an appropriate immunoadsorbent. To prepare the immunoadsorbent, antibodies against the a 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 ₁ 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.

It shows the following amino acid composition (frequency with coefficients of variation (CV in%):

Claims (5)

1. New glycoprotein characterized by a) a protein content of 75 ± 6%; b) 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%. _c) a _sed i _men t _a t i o _{e onsk} ffizienten _{S20 w} of 2.5 ± 0.3 S; d) a molecular weight determined in the ultracentrifuge of 35,000 ± 5,000; e) an isoelectric point of pH 3.4 ± 0.4; f) an extinction coefficient E

(280 mm) of 1.9 ± 0.3; g) an electrophoretic mobility in the range between a ₁ - and the α ₂ , -globulins; h) a specific immunological reaction with an antibody specifically directed against the glycoprotein. 2. A method for enriching the glycoprotein according to claim 1, characterized in that a protein solution in which the glycoprotein can be detected immunologically is subjected to at least one of the following measures and the fraction enriched with respect to the glycoprotein is obtained: a) adding neutral salts until the glycoprotein is precipitated; b) molecular sieve fractionation and recovery of the fraction with a molecular weight of 25,000 to 75,000; c) adsorption of the glycoprotein on a weakly basic ion exchanger and elution thereof; d) addition of water-soluble derivatives of an acridine or quinoline base in the range of pH 5-10 to a final concentration of about 0.8%; e) treatment of the glycoprotein solution with hydroxyapatite; f) preparative zone electrophoresis and extraction of the zone between α ₁ and α ₂ globulins; g) treatment of the protein solution with an immunoadsorbent. 3. The method according to claim 2, characterized in that an extract from human placentas is used as the protein solution. 4. Use of the protein according to claim 1 for the production of antisera. 5. Antiserum obtainable by immunizing vertebrates with a glycoprotein according to claim 1.