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WO1992007254A2 - Use of disulphide bidged substances - Google Patents

Use of disulphide bidged substances Download PDF

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Publication number
WO1992007254A2
WO1992007254A2 PCT/EP1991/001877 EP9101877W WO9207254A2 WO 1992007254 A2 WO1992007254 A2 WO 1992007254A2 EP 9101877 W EP9101877 W EP 9101877W WO 9207254 A2 WO9207254 A2 WO 9207254A2
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Prior art keywords
substances
antibodies
oxidation
antigen
pbs
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Application number
PCT/EP1991/001877
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German (de)
French (fr)
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WO1992007254A3 (en
Inventor
Kurt Lang
Thomas Subkowski
Juergen Schweden
Original Assignee
Basf Aktiengesellschaft
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Publication of WO1992007254A2 publication Critical patent/WO1992007254A2/en
Publication of WO1992007254A3 publication Critical patent/WO1992007254A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • C07K14/575—Hormones
    • C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • 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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to new applications of disulfide-bridged substances.
  • S-S bridges Most proteins and many peptides contain cysteine residues that can be linked by S-S bridges. These S-S bridges can be both intra- and intermolecular. If they are intermolecular, two or more protein molecules can be connected to one another.
  • Antibodies and suitable as calibration substances for the determination of molecular weights of higher molecular substances are provided.
  • the present invention is particularly suitable for those proteins or peptides which, in the native state, where the S-S bridges are generally intermolecular, are not immunogenic or are only extremely weak.
  • hirudin intermolecularly linked via S-S bridges is preferred for the production of antibodies against hirudin.
  • the starting point is the SH proteins or SH peptides, which e.g. in an aqueous buffer at pH values of 5 to 10, preferably 7.5 to 9, in a known manner using oxidizing agents such as oxygen or hydrogen peroxide to give S-S-di- or polymers.
  • the reaction is generally carried out at a temperature of 4 ° C to 45 ° C.
  • the optimal reaction temperature should be determined for each protein in a test range. Denatured and reduced proteins and peptides are particularly suitable for the production of intermolecular disulfide bridges.
  • the degree of polymerization can be determined by varying the concentration of SH protein or oxidizing agent in the oxi set dations approach. The degree of polymerization increases with increasing concentration of protein or peptide or oxidizing agent.
  • the reaction can e.g. by dilution, separation of the reaction components (e.g. gel filtration, precipitation, extraction, dialysis) or by acidification to pH values less than 5.0.
  • separation of the reaction components e.g. gel filtration, precipitation, extraction, dialysis
  • acidification to pH values less than 5.0.
  • non-proteinogenic substances with at least 2 SH groups can also be oligomerized in the same way and used according to the invention.
  • SH groups can subsequently be introduced by derivatizing other functional groups.
  • Compounds containing amino groups can e.g. by 2-iminothiolane, N-succinimidyl-S-acetylthioacetate (SATA) or N-succinimidyl-3'-2'-pyridyldithio-propionate (SPDP) to form SH-containing compounds.
  • SATA N-succinimidyl-S-acetylthioacetate
  • SPDP N-succinimidyl-3'-2'-pyridyldithio-propionate
  • the degree of polymerization should be such that the molecular weight range of the polymers includes the molecular weight of the substance to be examined.
  • the highest possible degree of polymerization should be achieved for the induction of antibodies.
  • polymerized proteins especially those with a
  • Aprotinin was dissolved to a concentration of 100 mg / ml in 6 M guanidinium chloride (GdmCl), 0.2 M Tris / HCl, 0.4 M dithiothreitol (DTT), pH 8.5 and incubated at 37 ° C. for 2 h.
  • the oxidation was initiated by adding 20% H 2 O 2 to a concentration of 1% in the aprotinin solution with constant stirring. After 30 min, the oxidation solution was diluted 10-fold by the addition of 20 mM Tris / HCl, pH 8.6 and then dialyzed against the same buffer.
  • the oxidation products are cross-linked oligomers of denatured aprotinin through intermolecular disulfide bridges.
  • Ribonuclease A was dissolved in 6 M GdmCl, 0.2 M Tris / HCl, 0.4 M DTT, pH 8.7 to a concentration of 100 mg / ml and incubated for 2 hours.
  • the oxidation was carried out by adding 20% H 2 O 2 to a concentration of 1% to the ribonuclease solution with stirring. After 30 min, the oxidation solution was diluted 1:10 with 20 mM Tris / HCl and dialyzed against the same buffer.
  • the oxidation products are predominantly cross-linked ribonuclease oligomers via intermolecular disulfide bridges.
  • game 3
  • Desulfatohirudin produced by technical means was dissolved at 60 or 90 mg / ml in 6 M GdmCl, 0.2 M Tris / HCl, 0.4 M DTT, pH 8.7 and incubated at 37 ° C. for 2 h.
  • the oxidation was then carried out by adding 20% H2O2 to a concentration of 1% in the hirudin solution, with stirring. After 30 minutes, the oxidation batch was 1:10 diluted with water or with 20 mM Tris / HCl, pH 8.6 and dialyzed against water or the latter buffer.
  • the oxidation product consists predominantly of oligomers of denatured hirudin which are crosslinked via intermolecular disulfide bridges.
  • the oxidation products of the denatured and reduced proteins aprotinin, ribonuclease A and desulfatohirudin prepared in Examples 1, 2 and 3 were after dilution to 10 mg protein / ml in 20 mM Tris / HCl, pH 8.6 and dialysis against the same buffer SDS gel electrophoresis separated.
  • the oxidation products are primarily oligomers crosslinked via disulfide bridges, which differ in their molecular weight by two or more times the molecular weight of the respective monomeric protein (ribonuclease A: 13700 D; hirudin: 6960 D; aprotinin: 6150 D).
  • the semi-logarithmic plot of the molecular weights of the various oligomers as a function of the distance traveled in the gel shows that there is a linear dependence between these parameters over a wide range of molecular weights.
  • hirudin or desulfatohirudin is very suitable for therapeutic use as an anticoagulant.
  • antibodies are required for the sensitive detection of hirudin in the serum of treated patients by means of an ELISA specific for hirudin.
  • the induction of antibodies against hirudin has been problematic due to the low immunogenicity of hirudin.
  • Spinner et al. Thrombosis Research, 51, 617 (1988) antibodies to hirudin can only be produced in 2 of 11 sheep.
  • microtiter plates from Falcon were first coated with the antigen (oxidized desulfatohirudin or native desulfatohirudin) (2 h, 37 ° C., 5 ⁇ g / l 50 mM sodium carbonate buffer, pH 9.0). Unspecific binding sites on the microtiter plate were saturated by subsequent incubation with 3% BSA / PBS. The mixture was then incubated with various dilutions of the antiserum for 2 h and then the bound
  • antigen oxidized desulfatohirudin or native desulfatohirudin
  • Big-ET prepared by solid phase peptide synthesis was dissolved in 6 M GdmCl; 0.2 M Tris / HCL; 0.4 DTT; pH 8.7 dissolved to a concentration of 50 mg / ml and incubated for 2 hours at 37 ° C.
  • the oxidation was carried out by adding 20% H2O2 to a concentration of 1% with stirring. After 30 minutes the
  • Oxidation solution diluted 1:10 with 20 mM Tris / HCl and dialyzed against the same buffer.
  • the dialysis tube had an exclusion volume of 5000 Da, so that only cross-linked oligomers were retained intermolecularly by disulfide bridges and the by-products (intramolecular disulfide bridges) are separated.
  • the spleen was removed on day 45. Fusion and selection of the anti-big-ET secreting hybridomas is carried out according to the standard rules described (monoclonal antibodies: principles and practice; James W. Goding; Academic Press 1983).
  • Amiopterin was at a concentration of 20 mg / ml 50 mM triethanolamine pH 8.0; 10 mM DTT dissolved and with 2-iminothiolane (Final concentration 11.5 mg / ml) reacted for 4 hours at room temperature.
  • the SH groups of the aminopterin modified on the amino groups were oxidized by adding 10% H2O2 to a final concentration of 0.5%. After 30 minutes, the solution was diluted 1:10 with 50 mM triethanolamine pH 8.0 and dialyzed against the same buffer (exclusion volume of the dialysis membrane: 1000 Da).

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Abstract

The use is disclosed of substances that are linked at the intermolecular level by S-S bridges for producing antibodies, as well as the use of these substances as calibrating substances in molecular weight determination.

Description

Verwendung von disulfidverbrückten SubstanzenUse of disulfide-bridged substances

Beschreibungdescription

Die vorliegende Erfindung betrifft neue Anwendungen von disulfid¬ verbrückten Substanzen.The present invention relates to new applications of disulfide-bridged substances.

Die meisten Proteine und viele Peptide enthalten Cystein-Reste, die über S-S-Brücken miteinander verbunden sein können. Diese S-S-Brücken können sowohl intra- als auch intermolekular vor¬ liegen. Liegen sie intermolekular vor, so können zwei oder mehr Proteinmoleküle miteinander verbunden sein.Most proteins and many peptides contain cysteine residues that can be linked by S-S bridges. These S-S bridges can be both intra- and intermolecular. If they are intermolecular, two or more protein molecules can be connected to one another.

Es wurde nun gefunden, daß sich intermolekular über S-S-Brücken verknüpfte Proteine und Peptide sehr gut zur Herstellung vonIt has now been found that proteins and peptides intermolecularly linked via S-S bridges are very good for the production of

Antikörpern und als Eichsubstanzen zur Bestimmung von Molekular¬ gewichten von höher molekularen Substanzen eignen.Antibodies and suitable as calibration substances for the determination of molecular weights of higher molecular substances.

Die vorliegende Erfindung ist besonders für solche Proteine oder Peptide geeignet, die im nativen Zustand, wo die S-S-Brücken in der Regel intermolekular vorliegen, nicht oder nur äußerst schwach immunogen sind.The present invention is particularly suitable for those proteins or peptides which, in the native state, where the S-S bridges are generally intermolecular, are not immunogenic or are only extremely weak.

Bevorzugt ist die Verwendung von intermolekular über S-S-Brücken verknüpftem Hirudin zur Herstellung von Antikörpern gegen Hirudin.The use of hirudin intermolecularly linked via S-S bridges is preferred for the production of antibodies against hirudin.

Zur Herstellung von intermolekular über S-S-Brücken verknüpften Proteinen und Peptiden geht man von den SH-Proteinen bzw. SH-Peptiden aus, die z.B. in einem wäßrigen Puffer bei pH-Werten von 5 bis 10, bevorzugt von 7,5 bis 9, in bekannter Weise mit Oxidationsmitteln wie Sauerstoff oder Wasserstoffperoxid zu S-S-Di- oder Polymeren oxidiert werden. Die Reaktion wird im allgemeinen bei einer Temperatur von 4°C bis 45°C durchgeführt. Die optimale Reaktionstemperatur sollte für jedes Protein in einer Versuchsreiche ermittelt werden. Zur Herstellung von inter¬ molekularen Disulfidbrücken sind insbesondere denaturierte und reduzierte Proteine und Peptide geeignet.For the production of proteins and peptides linked intermolecularly via S-S bridges, the starting point is the SH proteins or SH peptides, which e.g. in an aqueous buffer at pH values of 5 to 10, preferably 7.5 to 9, in a known manner using oxidizing agents such as oxygen or hydrogen peroxide to give S-S-di- or polymers. The reaction is generally carried out at a temperature of 4 ° C to 45 ° C. The optimal reaction temperature should be determined for each protein in a test range. Denatured and reduced proteins and peptides are particularly suitable for the production of intermolecular disulfide bridges.

Der Grad der Polymerisation läßt sich durch Variation der Konzentration an SH-Protein oder Oxidationsmittel im Oxi- dationsansatz einstellen. Mit steigender Konzentration an Protein bzw. Peptid oder Oxidations ittel steigt der Polymerisationsgrad.The degree of polymerization can be determined by varying the concentration of SH protein or oxidizing agent in the oxi set dations approach. The degree of polymerization increases with increasing concentration of protein or peptide or oxidizing agent.

Nach Erreichen des gewünschten Oxidationsgrades kann die Reaktion z.B. durch Verdünnen, Abtrennung der Reaktionskomponenten (z.B. Gelfiltration, Fällung, Extraktion, Dialyse) oder durch Ansäuern auf pH-Werte kleiner 5,0 abgestoppt werden.After reaching the desired degree of oxidation, the reaction can e.g. by dilution, separation of the reaction components (e.g. gel filtration, precipitation, extraction, dialysis) or by acidification to pH values less than 5.0.

Auch andere, nicht proteinogene Substanzen mit mindestens 2 SH-Gruppen, können in gleicher Weise oligomerisiert und erfindungsgemäß verwendet werden.Other non-proteinogenic substances with at least 2 SH groups can also be oligomerized in the same way and used according to the invention.

Falls die zu oligomerisierende Substanz weniger als 2 SH-Gruppen enthält, können durch Derivatisierung anderer funktioneller Gruppen nachträglich SH-Gruppen eingeführt werden. Aminogruppen enthaltende Verbindungen können z.B. durch 2-Iminothiolan, N-Succinimidyl-S-acetylthioacetat (SATA) oder N-Succin- imidyl-3'-2'-pyridyldithio-propionat (SPDP) zu SH-haltigen Ver¬ bindungen umgesetzt werden. Die Reaktionsbedingungen werden zweckmäßigerweise so gewählt, wie sie vom Hersteller empfohlen werden (z.B. Pierce).If the substance to be oligomerized contains fewer than 2 SH groups, SH groups can subsequently be introduced by derivatizing other functional groups. Compounds containing amino groups can e.g. by 2-iminothiolane, N-succinimidyl-S-acetylthioacetate (SATA) or N-succinimidyl-3'-2'-pyridyldithio-propionate (SPDP) to form SH-containing compounds. The reaction conditions are expediently chosen as recommended by the manufacturer (e.g. Pierce).

Für die Verwendung als Eichsubstanz zur Bestimmung von Molekular¬ gewichten sollte der Polymerisationsgrad so liegen, daß der Molekulargewichtsbereich der Polymerisate das Molekulargewicht der zu untersuchenden Substanz einschließt. Für die Induktion von Antikörpern sollte ein möglichst hoher Polymerisationsgrad er¬ zielt werden.For use as a calibration substance for determining molecular weights, the degree of polymerization should be such that the molecular weight range of the polymers includes the molecular weight of the substance to be examined. The highest possible degree of polymerization should be achieved for the induction of antibodies.

Die polymerisierten Proteine, insbesondere solche mit einemThe polymerized proteins, especially those with a

Molekulargewicht von über 10 kD, sind wesentlich besser als das nicht polymerisierte Protein oder Peptid dazu geeignet, Anti¬ körper auch gegen das nicht polymerisierte Protein oder Peptid zu induzieren. Die sonst üblichen Methoden zur Induktion von Antikörpern durch Kopplung an Trägerproteine, z.B. BSA (bovines Serumalbumin) oder KLH (keyhole limpet hemocyanine) haben den Nachteil, daß ein großer Anteil der gebildeten Antikörper gegen das Trägerprotein gerichtet ist. Dies wird durch die Verwendung von disulfidverbrückten Proteinen vermieden. Gegen viele nichtproteinogene Substanzen oder auch niedermolekulare proteinogene Substanzen, z.B. Neuropeptide, Hormone, ist es schwierig bzw. unmöglich, auch bei Verwendung von KLH- oder BSA-Konjugaten, spezifische Antikörper zu erhalten. Mit Hilfe der intermolekular disulfidverbrückten Substanzen lassen sich sowohl poly- als auch monoklonale Antikörper herstellen. Die erhaltenen Antikörper sind wichtig z.B. zum Nachweis der Substanzen in Körperflüssigkeiten bzw. zu therapeutischen und diagnostischen Zwecken.Molecular weights of more than 10 kD are much better than the unpolymerized protein or peptide suitable for inducing antibodies against the unpolymerized protein or peptide. The otherwise usual methods for inducing antibodies by coupling to carrier proteins, for example BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanine) have the disadvantage that a large proportion of the antibodies formed are directed against the carrier protein. This is avoided by using disulfide-bridged proteins. It is against many non-proteinogenic substances or even low-molecular proteinogenic substances, eg neuropeptides, hormones difficult or impossible to obtain specific antibodies even when using KLH or BSA conjugates. With the help of the intermolecularly disulfide-bridged substances, both poly and monoclonal antibodies can be produced. The antibodies obtained are important, for example for the detection of the substances in body fluids or for therapeutic and diagnostic purposes.

Beispiel 1example 1

Oxidation von reduziertem und denaturiertem AprotininOxidation of reduced and denatured aprotinin

Aprotinin wurde zu einer Konzentration von 100 mg/ml in 6 M Guanidiniumchlorid (GdmCl), 0,2 M Tris/HCl, 0,4 M Dithiothreitol (DTT), pH 8,5 gelöst und 2 h bei 37°C inkubiert. Die Oxidation wurde durch die Zugabe von 20%igem H202 zu einer Konzentration von 1% in der Aprotininlösung unter stetem Rühren eingeleitet. Nach 30 min wurde die Oxidationslösung durch die Zugabe von 20 mM Tris/HCl, pH 8,6 10-fach verdünnt und anschließend gegen den- selben Puffer dialysiert.Aprotinin was dissolved to a concentration of 100 mg / ml in 6 M guanidinium chloride (GdmCl), 0.2 M Tris / HCl, 0.4 M dithiothreitol (DTT), pH 8.5 and incubated at 37 ° C. for 2 h. The oxidation was initiated by adding 20% H 2 O 2 to a concentration of 1% in the aprotinin solution with constant stirring. After 30 min, the oxidation solution was diluted 10-fold by the addition of 20 mM Tris / HCl, pH 8.6 and then dialyzed against the same buffer.

Die Oxidationsprodukte sind durch intermolekulare Disulfidbrücken quervernetzte Oligo ere von denaturiertem Aprotinin.The oxidation products are cross-linked oligomers of denatured aprotinin through intermolecular disulfide bridges.

Beispiel 2Example 2

Oxidation von denaturierter und reduzierter Ribonuklease A aus Rinderpankreas:Oxidation of denatured and reduced ribonuclease A from bovine pancreas:

Ribonuklease A wurde in 6 M GdmCl, 0, 2 M Tris/HCl, 0,4 M DTT, pH 8,7 zu einer Konzentration von 100 mg/ml gelöst und 2 h inku¬ biert. Die Oxidation erfolgte durch Zugabe von 20%igem H2θ2 bis zu einer Konzentration von 1% zur Ribonuklease-Lösung unter Rühren. Nach 30 min wurde die Oxidationslösung 1:10 mit 20 mM Tris/HCl verdünnt und gegen denselben Puffer dialysiert.Ribonuclease A was dissolved in 6 M GdmCl, 0.2 M Tris / HCl, 0.4 M DTT, pH 8.7 to a concentration of 100 mg / ml and incubated for 2 hours. The oxidation was carried out by adding 20% H 2 O 2 to a concentration of 1% to the ribonuclease solution with stirring. After 30 min, the oxidation solution was diluted 1:10 with 20 mM Tris / HCl and dialyzed against the same buffer.

Die Oxidationsprodukte sind überwiegend über intermolekulare Disulfidbrücken quervernetzte Ribonukleaseoligomere. Bei spi el 3The oxidation products are predominantly cross-linked ribonuclease oligomers via intermolecular disulfide bridges. In game 3

Oxidation von denaturiertem und reduziertem Desulfatohirudin:Oxidation of denatured and reduced desulfatohirudin:

Gεntechnisch hergestelltes Desulfatohirudin wurde zu 60 bzw. 90 mg/ml in 6 M GdmCl, 0,2 M Tris/HCl, 0, 4 M DTT, pH 8,7 gelöst und 2 h bei 37°C iπkubiert. Anschließend erfolgte die Oxidatior durch die Zugabe von 20 igem H2O2 bis zu einer Konzentration von 1% in der Hirudinlösung, wobei gerührt wurde. Nach 30 min wurde der Oxidationsansatz 1 : 10 mit Wasser oder mit 20 mM Tris/HCl, pH 8,6 verdünnt und gegen Wasser oder letzteren Puffer dia¬ lysiert.Desulfatohirudin produced by technical means was dissolved at 60 or 90 mg / ml in 6 M GdmCl, 0.2 M Tris / HCl, 0.4 M DTT, pH 8.7 and incubated at 37 ° C. for 2 h. The oxidation was then carried out by adding 20% H2O2 to a concentration of 1% in the hirudin solution, with stirring. After 30 minutes, the oxidation batch was 1:10 diluted with water or with 20 mM Tris / HCl, pH 8.6 and dialyzed against water or the latter buffer.

Das Oxidationsprodukt besteht aus überwiegend über intermole- kulare Disulfidbrücken quervernetzten Oligomeren des denaturier¬ ten Hirudins.The oxidation product consists predominantly of oligomers of denatured hirudin which are crosslinked via intermolecular disulfide bridges.

Beispiel 4Example 4

Laufverhalten der Oxidationsprodukte von verschiedenen im dena¬ turierten und reduzierten Zustand oxidierten Proteinen in der SDS-Gelelektrophorese:Running behavior of the oxidation products of various proteins oxidized in the denatured and reduced state in SDS gel electrophoresis:

Die in den Beispielen 1, 2 und 3 hergestellten Oxidationsprodukte der denaturierten und reduzierten Proteine Aprotinin, Ribo¬ nuklease A und Desulfatohirudin wurden nach Verdünnung auf 10 mg Protein/ml in 20 mM Tris/HCl, pH 8,6 und Dialyse gegen denselben Puffer durch SDS-Gelelektrophorese aufgetrennt.The oxidation products of the denatured and reduced proteins aprotinin, ribonuclease A and desulfatohirudin prepared in Examples 1, 2 and 3 were after dilution to 10 mg protein / ml in 20 mM Tris / HCl, pH 8.6 and dialysis against the same buffer SDS gel electrophoresis separated.

Die Oxidationsprodukte sind vor allem über Disulfidbrücken quer¬ vernetzte Oligomere, die sich in ihrem Molekulargewicht um das zwei- oder vielfache des Molekulargewichts des jeweiligen mono- meren Proteins (Ribonuklease A: 13700 D; Hirudin: 6960 D; Aprotinin: 6150 D) unterscheiden. Die halblogarithmische Auftra- gung der Molekulargewichte der verschiedenen Oligomeren in Ab¬ hängigkeit von der jeweils zurückgelegten Laufstrecke im Gel zeigt, daß über einen weiten Molekulargewichtsbereich eine lineare Abhängigkeit zwischen diesen Parametern besteht. Bei spiel 5The oxidation products are primarily oligomers crosslinked via disulfide bridges, which differ in their molecular weight by two or more times the molecular weight of the respective monomeric protein (ribonuclease A: 13700 D; hirudin: 6960 D; aprotinin: 6150 D). The semi-logarithmic plot of the molecular weights of the various oligomers as a function of the distance traveled in the gel shows that there is a linear dependence between these parameters over a wide range of molecular weights. In game 5

Induktion .von Antikörpern durch im denaturierten und reduzierten Zustand oxidiertes DesulfatohirudinInduction of antibodies by desulfated hirudin oxidized in the denatured and reduced state

Hirudin bzw. Desulfatohirudin ist aufgrund seiner geringen Immunogenität für eine therapeutische Verwendung als Blut¬ gerinnungshemmstoff sehr geeignet. Für den empfindlichen Nachweis von Hirudin im Serum von behandelten Patienten mittels eines für Hirudin spezifischen ELISA sind jedoch Antikörper erforderlich. Die Induktion von Antikörpern gegen Hirudin war bisher aufgrund der geringen Immunogenität von Hirudin problematisch. So konnten von Spinner et al. (Thrombosis Research, 51, 617 (1988)) nur in 2 von 11 Schafen Antikörper gegen Hirudin erzeugt werden.Because of its low immunogenicity, hirudin or desulfatohirudin is very suitable for therapeutic use as an anticoagulant. However, antibodies are required for the sensitive detection of hirudin in the serum of treated patients by means of an ELISA specific for hirudin. The induction of antibodies against hirudin has been problematic due to the low immunogenicity of hirudin. For example, Spinner et al. (Thrombosis Research, 51, 617 (1988)) antibodies to hirudin can only be produced in 2 of 11 sheep.

Zur Induktion von Antikörpern gegen Desulfatohirudin wurde das gemäß Beispiel 3 hergestellte oxidierte Desulfatohirudin als Antigen verwendet. Es wurden Kaninchen wie folgt intramuskulär immunisiert:To induce antibodies against desulfatohirudin, the oxidized desulfatohirudin prepared according to Example 3 was used as the antigen. Rabbits were immunized intramuscularly as follows:

Tag 0: 500 μg Antigen + 500 μl PBS + 500 μl CFADay 0: 500 μg antigen + 500 μl PBS + 500 μl CFA

Tag 14 500 μg Antigen + 500 μl PBS + 500 μl IFA Tag 31 250 μg Antigen + 500 μl PBS + 500 μl IFA Tag 45 250 μg Antigen + 500 μl PBSDay 14 500 μg antigen + 500 μl PBS + 500 μl IFA Day 31 250 μg antigen + 500 μl PBS + 500 μl IFA Day 45 250 μg antigen + 500 μl PBS

Am 53. Tag wurden den Kaninchen je 2 ml Blut entnommen und der Antikörpertiter im Serum wie üblich bestimmt.On the 53rd day, 2 ml of blood were taken from each rabbit and the antibody titer in the serum was determined as usual.

Dazu wurden Mikrotiterplatten (Fa. Falcon) zunächst mit dem Antigen (oxidiertes Desulfatohirudin oder natives Desulfato¬ hirudin) beschichtet (2 h, 37°C, 5 μg/ l 50 mM Natriumcarbonat- puffer, pH 9,0). Unspezifische Bindungsstellen der Mikrotiter- platte wurden durch anschließende Inkubation mit 3% BSA/PBS abgesättigt. Daraufhin wurde mit verschiedenen Verdünnungen des Antiserums für 2 h inkubiert und anschließend die gebundenenFor this purpose, microtiter plates (from Falcon) were first coated with the antigen (oxidized desulfatohirudin or native desulfatohirudin) (2 h, 37 ° C., 5 μg / l 50 mM sodium carbonate buffer, pH 9.0). Unspecific binding sites on the microtiter plate were saturated by subsequent incubation with 3% BSA / PBS. The mixture was then incubated with various dilutions of the antiserum for 2 h and then the bound

Antikörper mit einem "goat-anti-rabbit-Peroxidase"-Konjugat (Fa. Bio Rad) nachgewiesen.Antibodies with a "goat-anti-rabbit-peroxidase" conjugate (Bio Rad) detected.

Die induzierten Antikörper reagieren mit dem oxidierten und dem nativen Desulfatohirudin mit annähernd derselben Empfindlichkeit. Dies bedeutet, daß Antikörper gegen das oxidierte Desulfato¬ hirudin ebenfalls das native Protein erkennen. Beispiel 6The induced antibodies react with the oxidized and the native desulfatohirudin with approximately the same sensitivity. This means that antibodies against the oxidized desulfate hirudin also recognize the native protein. Example 6

Oxidation .von denaturiertem und reduziertem big-Endothelin (big-ET)Oxidation of denatured and reduced big endothelin (big ET)

Durch Festphasenpeptidsynthese hergestelltes big-ET wurde in 6 M GdmCl; 0,2 M Tris/HCL; 0,4 DTT; pH 8,7 zu einer Konzentration von 50 mg/ml gelöst und 2 Stunden bei 37°C inkubiert. Die Oxidation erfolgte durch Zugabe von 20 %igem H2O2 bis zu einer Konzentration von 1 % unter Rühren. Nach 30 Minuten wurde dieBig-ET prepared by solid phase peptide synthesis was dissolved in 6 M GdmCl; 0.2 M Tris / HCL; 0.4 DTT; pH 8.7 dissolved to a concentration of 50 mg / ml and incubated for 2 hours at 37 ° C. The oxidation was carried out by adding 20% H2O2 to a concentration of 1% with stirring. After 30 minutes the

Oxidationslösung 1 : 10 mit 20 mM Tris/HCl verdünnt und gegen den gleichen Puffer dialysiert. Der Dialyseschlauch hatte ein Aus¬ schlußvolumen von 5000 Da, so daß nur intermolekular durch Di¬ sulfidbrücken quervernetzte Oligomere zurückgehalten wurden und die Nebenprodukte (intramolekulare Disulfidbrücken) abgetrennt werden.Oxidation solution diluted 1:10 with 20 mM Tris / HCl and dialyzed against the same buffer. The dialysis tube had an exclusion volume of 5000 Da, so that only cross-linked oligomers were retained intermolecularly by disulfide bridges and the by-products (intramolecular disulfide bridges) are separated.

Beispiel 7Example 7

Induktion von monoklonalen Antikörpern, gerichtet gegen inter¬ molekular über Disulfidbrücken quervernetztes big-ETInduction of monoclonal antibodies directed against inter-molecular big-ET cross-linked via disulfide bridges

Mit den big-ET-Oligomeren wurden Balb c Mäuse nach folgendem Schema immunisiert:Balb c mice were immunized with the big ET oligomers according to the following scheme:

Tag 0 50 μg Antigen + 250 μl PBS + 250 μl CFADay 0 50 μg antigen + 250 μl PBS + 250 μl CFA

14 50 μg Antigen + 500 μl PBS14 50 µg antigen + 500 µl PBS

28 50 μg Antigen + 500 μl PBS28 50 μg antigen + 500 μl PBS

42 25 μg Antigen + 500 μl PBS 43 25 μg Antigen + 500 μl PBS42 25 μg antigen + 500 μl PBS 43 25 μg antigen + 500 μl PBS

44 25 μg Antigen + 500 μl PBS44 25 µg antigen + 500 µl PBS

Am Tag 45 wurde die Milz entnommen. Fusion und Selektion der anti-big-ET sezernierenden Hybridome erfolge nach beschriebenen Standardvorschriften (Monoclonal antibodies: principles and practice; James W. Goding; Academic Press 1983).The spleen was removed on day 45. Fusion and selection of the anti-big-ET secreting hybridomas is carried out according to the standard rules described (monoclonal antibodies: principles and practice; James W. Goding; Academic Press 1983).

Beispiel 8Example 8

Oxidation von modifiziertem AminopterinOxidation of modified aminopterin

Amiπopterin wurde zu einer Konzentration von 20 mg/ml 50 mM Triethanolamin pH 8,0; 10 mM DTT gelöst und mit 2-Iminothiolan (Endkonzentration 11,5 mg/ml) 4 Stunden bei Raumtemperatur umgesetzt. Die Oxidation der SH-Gruppen des an den Aminogruppen modifizierten Aminopterins erfolgte durch Zugabe von 10 %igem H2O2 bis zu einer Endkonzentration von 0,5 %. Nach 30 Minuten wurde die Lösung 1 : 10 mit 50 mM Triethanolamin pH 8,0 verdünnt und gegen den gleichen Puffer dialysiert (Ausschlußvolumen der Dialysemembran: 1000 Da).Amiopterin was at a concentration of 20 mg / ml 50 mM triethanolamine pH 8.0; 10 mM DTT dissolved and with 2-iminothiolane (Final concentration 11.5 mg / ml) reacted for 4 hours at room temperature. The SH groups of the aminopterin modified on the amino groups were oxidized by adding 10% H2O2 to a final concentration of 0.5%. After 30 minutes, the solution was diluted 1:10 with 50 mM triethanolamine pH 8.0 and dialyzed against the same buffer (exclusion volume of the dialysis membrane: 1000 Da).

Beispiel 9Example 9

Induktion von polyklonalen Antikörpern, gegen A inopterin. Mit den gemäß Beispiel 8 hergestellten Oligomeren wurden Kaninchen nach folgendem Schema immunisiert.Induction of polyclonal antibodies against A inopterin. Rabbits were immunized with the oligomers prepared according to Example 8 according to the following scheme.

Tag 0 100 μg Antigen + 500 μl PBS + 500 μl CFADay 0 100 μg antigen + 500 μl PBS + 500 μl CFA

14 100 μg Antigen + 500 μl PBS + 500 μl IFA14 100 μg antigen + 500 μl PBS + 500 μl IFA

28 100 μg Antigen + 500 μl PBS + 500 μl IFA28 100 μg antigen + 500 μl PBS + 500 μl IFA

42 100 μg Antigen + 1000 μl PBS42 100 µg antigen + 1000 µl PBS

Dann in 14-tägigem Rhythmus wie an Tag 42. Die resultierenden Antikörper reagieren sowohl mit Aminopterin, als auch mit Folsäure und dienen zur sensitiven immunologischen Detektion der Substanzen z.B. in Körperflüssigkeiten. Then every 14 days as on day 42. The resulting antibodies react both with aminopterin and with folic acid and are used for sensitive immunological detection of the substances e.g. in body fluids.

Claims

Patentansprüche Claims 1. Verwendung von intermolekular über S-S-Brücken verknüpften Substanzen als Eichsubstanzen zur Bestimmung von Molekular- gewichten.1. Use of substances intermolecularly linked via S-S bridges as calibration substances for determining molecular weights. 2. Verwendung von intermolekular über S-S-Brücken verknüpften Substanzen zur Herstellung von Antikörpern.2. Use of substances intermolecularly linked via S-S bridges for the production of antibodies. 3. Verwendung nach Anspruch 2, dadurch gekennzeichnet, daß als3. Use according to claim 2, characterized in that as Substanzen Polypeptide verwendet werden, die im nativen Zustand nicht oder schwach immunogen sind. Substances polypeptides are used which are not or are weakly immunogenic in the native state.
PCT/EP1991/001877 1990-10-10 1991-10-01 Use of disulphide bidged substances WO1992007254A2 (en)

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US4507233A (en) * 1981-04-22 1985-03-26 Oriental Yeast Co., Ltd. Colored molecular weight marker
US4713366A (en) * 1985-12-04 1987-12-15 The Ohio State University Research Foundation Antigenic modification of polypeptides
US5128319A (en) * 1987-08-28 1992-07-07 Board Of Regents, The University Of Texas System Prophylaxis and therapy of acquired immunodeficiency syndrome
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DK0380443T4 (en) * 1989-01-25 1999-11-29 Novartis Ag Monoclonal antibodies specific for hirudin
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