EP0267918A1 - Procede de production de recepteurs et/ou ligands et/ou anticorps purs, materiaux ainsi produits et leur utilisation - Google Patents

Procede de production de recepteurs et/ou ligands et/ou anticorps purs, materiaux ainsi produits et leur utilisation

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Publication number
EP0267918A1
EP0267918A1 EP19860905726 EP86905726A EP0267918A1 EP 0267918 A1 EP0267918 A1 EP 0267918A1 EP 19860905726 EP19860905726 EP 19860905726 EP 86905726 A EP86905726 A EP 86905726A EP 0267918 A1 EP0267918 A1 EP 0267918A1
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EP
European Patent Office
Prior art keywords
ligand
receptor
receptors
known per
antibodies
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.)
Withdrawn
Application number
EP19860905726
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German (de)
English (en)
Inventor
Hermann J. Wolter
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Individual
Original Assignee
Individual
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Filing date
Publication date
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Publication of EP0267918A1 publication Critical patent/EP0267918A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Definitions

  • the invention relates to methods for the production of native receptors and / or ligands and ligand antibodies binding to them, the use of the pure ligands, receptors or ligand antibodies produced and ligands, receptors and antibodies.
  • ligands are, for example, peptide hormones or the like, but can also be foreign materials, such as viruses, chemotherapeutics or the like).
  • ligand antibodies that selectively recognize and bind native ligands are very important for the study of and intervention in ligand / receptor interactions.
  • receptors from biological material have been obtained by attaching a carrier material such as Sephadex or the like. bound ligand was brought into contact with the receptor-containing material and, after incubation with it, the specific receptors bound to the immobilized ligands were isolated from the mixture.
  • the immobilized ligands can be placed, for example, in columns through which the biological material runs.
  • the receptors were then separated from the ligands bound to the support material by incubation with a buffer which led to the dissociation of the receptors from the ligands (Fujicka, T. et al., Biochem. Biophys. Res. Comrnun., Vol. 131 (1985 ), Pp. 640-646).
  • the previous method was disadvantageous in that an already modified ligand was used, of which it was not exactly known whether it binds to its receptor with the same specificity and sensitivity as the native ligand. Due to its connection to the carrier material, the modified ligand is additionally structurally changed, so that it cannot be said with certainty whether it binds to the desired receptor in this modified form or whether it occupies another binding site.
  • the receptors produced according to the state of the art were therefore always subject to changes in the molecule.
  • the investigation and structure elucidation of peptide ligands has been carried out exclusively by using peptide ligands which have been isolated from biological materials by chromatographic methods and which, after their synthesis, have been tested for their receptor binding capacity under various conformation changes.
  • Carrier material is bound and that
  • Serum containing ligand antibodies is incubated with it. The remaining serum components are washed by washing the immobilized antibody-ligand-ligand-antibody
  • Ligand antibody detached from the ligand by elution. There with the binding of the ligand to the carrier material, its binding ability and specificity to its specific antibody can be reduced and also ligand antibodies can be bound which bind their native or synthetic ligand only to a reduced extent (e.g. the conformity parameters of the native peptide ligand are used for antibody recognition and retention may be of importance, cannot be taken into account sufficiently because they are not sufficiently known).
  • the receptors and ligands produced by the method according to the prior art were therefore always subject to changes in the molecule.
  • antibody II is understood to mean material which is capable of binding to the ligand antibody, which also includes substances such as protein A or protein G.
  • Ligand / receptor complexes are prepared which are incubated with a ligand-antibody-linked support material. This removes receptors that have no binding sites for the ligand from the mixture. The ligand-bound receptors are then labeled with a
  • Dissociation buffer detached from its ligands and separated from the rest of the material by dialysis. This makes it possible for the first time to obtain only bioactive receptors that were previously capable of binding with unmodified ligands.
  • a preferred variant of the method according to the invention for producing bindable ligands comprises the incubation of solubilized cell membranes to whose receptors the native ligand is bound, carrier material linked to a ligand antibody and the removal of non-binding receptors by washing. Incubation and washing are done with an antibody binding buffer. The receptor is detached from its native ligand by incubation with a dissociation buffer. The native ligand is now separated from its antibody by elution and separated from the rest of the material by dialysis or molecular exclusion chromatography.
  • the method according to the invention for the production of ligand antibodies includes the connection of receptors which are shown purely according to the invention and which may already be linked to their specific ligands, with a carrier material and subsequently with a specific ligand, which can be native or synthetic.
  • This complex is incubated with sera that contain ligand antibodies and were obtained in a conventional manner. This results in a stable carrier-receptor-ligand-ligand-antibody complex to be removed.
  • the specific ligand antibody is then separated in a manner known per se from its receptor-bound ligand by elution with a buffer and the antibody with coeluted ligands is separated, for example by molecular exclusion chromatography or dialysis or similar suitable methods, as is familiar to any person skilled in the art in this field.
  • the biological material Before carrying out the method steps described above, it may be advisable to enrich the receptors in the biological material by incubating the biological material beforehand with an elution or dissociation buffer in order to thereby detach the native, receptor-bound ligand.
  • the detached ligand can be separated from the receptors by dialysis or centrifugation. Removal of ligand binding sites with binding constants other than the receptors, such as materials that synthesize, transport and / or store the ligand, can also be achieved from the biological material.
  • the biological material is incubated with ligand antibody-linked carrier material.
  • the ligand-binding materials with a greater ligand binding capacity than the receptor are bound to the immobilized ligand antibodies and the ligand-free receptors whose native ligand has been detached by treatment with a dissociation or elution buffer remain in solution.
  • This ligand-free receptor material can then be subjected to one of the further methods according to the invention.
  • the receptor-ligand bonds can be stabilized in a known manner by cnemic cross-linking ("crosslinking", S. Brenner, VB et al., Cell Vol. 40 (1985), pp. 183-190).
  • crosslinking S. Brenner, VB et al., Cell Vol. 40 (1985), pp. 183-190.
  • the linkage can take place between primary and secondary aliphatic amines on the receptor and ligand side (Tesser, GI et al. Hoppe. Seyler's Z. Physiol. Chem. Vol. 355 (1975) pp. 1624-1629).
  • DSP dithiobis (succinimidyl) propionate
  • cross-linker which has an internally reducible disulfide bond can achieve a stable receptor-ligand bond which can be achieved under reduction conditions (e.g.
  • dithiothreitoi or Mercaptoethanol is cleavable again.
  • the incubation with the reducing agents also cleaves disulfide bonds in the receptor molecule and ligand molecule, which can impair the bioactivity of the receptor or ligand if they are essential for its function.
  • the invention further relates to the use of such pure ligands, receptors or ligands;
  • the receptors according to the invention for the production of poly- or monoclonal receptor antibodies for the isolation and identification of receptor cDNA clones from clone mixtures ("clone immunoprecipitated probes"), such as for the isolation and identification of human glucocorticoid and estrogen receptors (Weinberger , D. et al., Science, Vol. 228 (1985), pp. 740-742 and Walter, P. et al., Proc. Natl. Acad. Sci. USA, Vol. 82 (1985), p.
  • the receptor clones can also be isolated and characterized as such by formation of a clone-ligand-ligand antibody-Sepharose-4B complex according to the invention - it also being possible to use the ligands and ligand antibodies according to the invention. This complex formation for clone isolation is unknown up to now.
  • the ligand antibodies according to the invention can be used to isolate and characterize ligand cDNA clones by forming a clone-ligand antibody-protein-A-Sepharose-4B complex (Young TA et al., Science, Vol. 222 (1983), p.
  • the receptors, ligands and ligand antibodies according to the invention can also be used to distinguish normal and pathologically modified biological material by conventional methods (RIA) or immunohistochemical methods and the like. are used, with the pure bioactive native substances according to the invention for the first time having a very high specificity and sensitivity.
  • RIA conventional methods
  • immunohistochemical methods and the like are used, with the pure bioactive native substances according to the invention for the first time having a very high specificity and sensitivity.
  • Specific ligand binding sites on receptors and ligands can also be determined and poly- or monoclonal antibodies against these ligand or receptor binding sites can be produced.
  • Receptors, ligands and ligand antibodies can be used to simplify and more reliably elucidate the mechanisms of action of synthetic or natural active substances and their derivatives (for example tests for
  • Binding ability of different ligands through receptor blockade via specific receptor antibodies can be determined via fluorescence activated cell sorter (FACS) or immunoprecipitation (Brenner, MB et al, Nature Vol. 322 (1986), pp. 145-149) using specific receptor antibodies, which are produced by using receptors according to the invention, are isolated.
  • FACS and immunoprecipitation analyzes can be carried out with the aid of ligands and ligand antibodies according to the invention which are labeled.
  • medicaments which are receptors, ligands or ligand antibodies produced according to the invention, for example poly- or monoclonal antibodies against receptors according to the invention, receptor binding sites or ligand binding sites (for example for blocking receptors on cell surfaces).
  • peptide ligands can be elucidated by substances produced according to the invention. So far, peptide ligands have been isolated chromatographically from biological materials and post-synthesized, after synthesis they have been tested for various conformational changes and receptor binding behavior.
  • antibody II means any material that specifically binds to "antibody I", the ligand antibody, including proteins such as protein A or protein G.
  • the production of the pure receptors opens up a wealth of possibilities, for example to investigate synthetic ligands; to investigate the presence of receptor sites by producing native ligands.
  • FIG. 1 shows a schematic representation of a method according to the invention for producing native receptors
  • FIG. 2 shows a schematic representation of a variant of the method according to the invention for producing bindable ligands
  • Figure 4 shows a pre-cleaning step of the inventive method for the
  • the invention is illustrated below using the example of the receptor / ligand system of dynorphin-dynorphin receptor, in particular the region of the octapeptide fragment dynorphin-A-1-8
  • Dynorphin is an opioid peptide with 17 amino acids, the receptors of which are widely used in the central and peripheral nervous system. DA8 has no internal disulfide bonds. The dynorphin 1-8 receptor is a
  • the invention can, however, also be applied to other ligand / receptor systems familiar to the person skilled in the art, such as cDNA / mRNA; Virus / cell surfaces etc. and in no way limited to this application.
  • ligand / receptor systems familiar to the person skilled in the art, such as cDNA / mRNA; Virus / cell surfaces etc. and in no way limited to this application.
  • example 1
  • the coupling buffer was removed and the reaction was stopped with 15 ml of 0.2 M glycine (pH 8.0) at 20 ° C. for 2 h; then the gel thus provided with a coupled peptide was transferred to a 10 x 0.7 cm Econo column (BIORAD Laboratories, Ricnmonc, USA), at 20 degrees C three times with coupling buffer - alternating with 0.1 M sodium acetate pH 4.0 and 0.5 M etc.Cl been and cas gel at 4 degrees C in 50 mM TRIS-HCl pH 5, 7 with 0.05 wt. / vol. % Sodium azide saved.
  • Washed 50 ml of a Tris buffer (50 mM TRIS-HCl, pH 7.6, 1 vol.% Triton X -100), 2 mg peptide (DA8-linked DA8 receptors or Dynorphin-A (1-8) antibodies) in 5 ml of 0.1M sodium bicarbonate (pH 8.0) and 0.5 M NaCl were applied to the column and incubated with recirculation for 16 h at 4 ° C (flow rate: 6-7 ml / h); the eluate was collected and the column was washed with 40 ml of a buffer solution (50 mM TRIS-HCl, pH 7.6 with 0.5 M NaCl, 0.1% by volume Triton X-100 and 1 mM PMSF), the peptide with 40 ml of an elution buffer A (0.1 M glycine-HCl, pH 5.0, with 1.5 mol NaCl, 0.1 mmol PMSF, 0.2 vol.% Triton X-100,
  • Synthetic pig DA8 contains) incubated.
  • the DA8 receptors are then dissolved in an antibody binding buffer (0.1 M sodium bicarbonate, pH 8.0 with 0.5 NaCl and 0.2% by volume Triton-X-100) and Sepharose-4B linked with DA8 for 16 hours at 4 degrees Celsius incubated.
  • an antibody binding buffer 0.1 M sodium bicarbonate, pH 8.0 with 0.5 NaCl and 0.2% by volume Triton-X-100
  • the DA8 receptors are incubated (30 min at 20 degrees Celsius) with a dissociation buffer (50mM TRIS-HCl, pH 7.5, with 5 ⁇ mol synthetic pig DA8, 5 mM Mg 2+ , 5 mM EGTA and 5 ⁇ mol 5 ' -Guanylylimido diphosphate (Gpp (NH) p) separated from synthetic DA8, the now free DA8 receptor can now be dialyzed from the rest of the material.
  • a dissociation buffer 50mM TRIS-HCl, pH 7.5, with 5 ⁇ mol synthetic pig DA8, 5 mM Mg 2+ , 5 mM EGTA and 5 ⁇ mol 5 ' -Guanylylimido diphosphate (Gpp (NH) p
  • DA8-bound DA8 receptors can also be first used with free D A8 antibodies (diluted 1: 100 with an antibody binding buffer - 0.1M sodium bicarbonate, pH 8.0, with 0.5 M NaCl and 0.2 vol.% Triton X -100) for 15 hours Incubated 4 degrees C and the DA8-bound DAG antibodies are separated from the rest of the material by dialysis (see FIG. 1 of the drawing). This receptor-ligand-ligand antibody is subsequently described
  • Protein-A-Sepharose-4B (Stephenson, F.a. et al. J. Neurochem. 46 (1986), pp. 854-861) can also be used instead of porcine anti-rabbit immunoglobulin antibody Sepharose 4B.
  • the DA8 receptors are separated from the DA8, as described above, by the dissociation buffer.
  • the DA8 receptors can be separated with the elution buffer A in fractions of 500 ⁇ l from the DA8 and neutralized immediately with the same volume of the neutralization buffer (pH 7.6).
  • the DA8 receptors can be removed from the coeluted material either by dialysis (if DA8 antibody-Se ⁇ harose-4B was used) or by
  • Incubation with DA8 can also take place before the production of cell membranes or before the cell membranes are solubilized.
  • Solubilized cell membranes containing only the endogenous receptor-bound DA8 are treated with Sepharose 4B-linked DA8 antibodies in the presence of an antibody binding buffer (0.1M sodium bicarbonate, pH 8.0, with 0.5 M NaCl and 0.2 vol.% Triton X-100) for 16 hours. incubated at 4 degrees C. Thereafter, the DA8 receptors are incubated with the dissociation buffer (50mM TRIS-HCl, pH 7.6, with 5 ⁇ M welding ne-DA8, 5mM Mg 2+ , 5mM EGTA and 5 ⁇ Mol Gpp (NH) p) for 30 min. at 20 degrees C separated from the native DA8.
  • an antibody binding buffer 0.1M sodium bicarbonate, pH 8.0, with 0.5 M NaCl and 0.2 vol.% Triton X-100
  • the DA8 receptor detached from the endogenous DA8 can now be separated from the rest of the material by dialysis or molecular exclusion chromatography.
  • the endogenous DA8 is then detached from the Sepharose 48-linked DA8 antibodies by the elution buffer B, collected in fractions of 500 ⁇ l, immediately neutralized with the same volume of neutralization buffer and separated from the rest of the material by dialysis.
  • DA8-bound DA8 receptors can also be used beforehand
  • DA8 antibody-containing serum Incubate DA8 antibody-containing serum in the previously described manner in the antibody binding buffer.
  • the receptor-native DA8 ligand-antibody complex is now incubated with porcine anti-rabbit immunoglobulin antibodies, which are linked to activated Sepharose-4B as described in Example 1, for 16 hours at 4 ° C.
  • Porcine anti-rabbit immunoglobulin antibody Sepharose-4B can also be used Protein A-Sepharose-4B.
  • the unbound serum components eg nonimmune immunoglobulins
  • the DA receptor is separated from the endogenous DA8 by incubation with the dissociation buffer as before.
  • the DA8 receptor can be separated from the rest of the material by dialysis.
  • the native DA8, which is still linked to the DA8 antibody, can now, together with the DA8 antibodies from the swine anti-rabbit immunoglobulin antibody linked Sepharose-4B with elution buffer (0.1M glycine-HCl, pH 2.5, with 0.1 mmol PMSF and 0.2 vol.% Triton X-100) can be removed.
  • the eluate contains endogenous, formerly receptor-bound DA8 and specific DA8 antibodies.
  • Subsequent dialysis can be used to display the serum-isolated specific DA8 antibodies and, by molecular exclusion chromatography, the native formerly receptor-blown DA8 ⁇ .
  • the endogenous DA8 can be separated from its DA8 receptor and its DA8 antibody by incubation with the elution buffer B (0.1 M glycine-HCl, pH 2.5, with 0.1 mmol PMSF and 0.2% by volume Triton X-100) with the neutralization buffer neutralized and separated from the coeluted material by molecular exclusion chromatography. Dialysis gives the DA8 receptor which, when using the elution buffer A is bioactive. Formerly receptor-bound, endogenous DA8, DA8 receptors and DA8 antibodies, which are specific for formerly receptor-bound DA8, have not yet been shown. Both the endogenous DA8 and the DA8 antibodies and the DA8 receptors according to the invention have high sensitivities and specificities.
  • the elution buffer B 0.1 M glycine-HCl, pH 2.5, with 0.1 mmol PMSF and 0.2% by volume Triton X-100
  • Solubilized DA8 receptors which were connected to activated Se ⁇ harose-4B in the manner described above (Example 1), are incubated with the ligand binding buffer at 20 ° C. for 2 hours. Then the DA8-bound DA8 receptors are treated with a serum that is in the
  • Antibody binding buffer is dissolved, incubated (16 hours at 4 degrees C), which contains specific DA8 antibodies.
  • the specifically DA8-bound DA8 antibodies are detached from DA8 with the elution buffer B (0.1M glycine-HCl, pH 2.5 with 0.1 mol PMSF and 0.2% pre-Triton X - 100) and immediately neutralized with the appropriate neutralization buffer.
  • Coeluted DA8 can be separated from the DA8 antibodies by dialysis in the usual way. Incubation with synthetic or endogenous DA8 can also take place before the production of the cell membranes or before the solubilization of DA8 receptors. Furthermore, instead of solubilized DA8 receptors, DA8 can be bound to Sepharose 4B-linked, DA8 receptor-bearing, non-solubilized biological materials.
  • DA8 antibodies which were purified according to the method presented here, show a higher specificity and sensitivity to synthetic pig DA8 than DA8 antibodies, which were purified in the usual way.
  • DA8 antibodies and endogenous formerly receptor-bound DA8 may need to a) remove such biological material that
  • DA8 material synthesized transported or stored. This is done by pre-incubating the biological material with the dissociation buffer (30 min at 20 degrees C). The biological material is then dissolved in the antibody binding buffer, incubated with DA8-antibody-linked Sepharose-4B for 16 hours at 4 ° C. and the unbound biological material is removed by washing. In the washing solution there is that for the purification of DA8 antibodies,
  • DA8 receptor-bearing, non-solubilized cell membranes from its receptor by incubation with the dissociation buffer and then remove them by centrifugation (2x30 min, 20,000 g, 4 degrees C) and washing or by dialysis.
  • DA8 receptor-DA8 complexes are incubated (30 min. At 20 degrees C) with 50 ⁇ g / ml DSP (in 0.01 M phosphate buffer, pH 8.0, with 0.5 M NaCl, 5 mM Mg 2+ and 0.1 vol.% Triton-X-100 is cross-linked.
  • the cross-linking is separated after the Dynorphi nA (1-8) antibody has been bound by incubation with either 3 mM dithiotreitol or 5% mercaptoethanol (both dissolved in 0.01 M phosphate buffer with 0.5 M NaCl, 5 mM Mg 2+ and 0.1 vol.% Triton X-100).

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Abstract

Un procédé de production de récepteurs bioactifs purs comprend l'incubation de récepteurs solubilisés et liés à des ligands avec des anticorps des ligands (AK I) liés à un support, par exemple un tampon de liaison d'anticorps, suivie de l'incubation du complexe récepteur-ligand-anticorps de ligands-support immobilisé ainsi obtenu, selon un procédé connu, avec par exemple un tampon de dissociation des ligands, et la dissolution de la liaison entre les récepteurs et les ligands. Les récepteurs libérés et non-immobilisés sont ensuite séparés selon un procédé connu et le cas échéant, les récepteurs obtenus sont purifiés selon un procédé connu. Une variante du procédé comprend l'incubation du complexe récepteur-ligand-anticorps de ligands avec un support lié selon un procédé connu à des anticorps qui se lient spécifiquement aux anticorps des ligands (AK II). Les récepteurs sont séparés par incubation selon un procédé connu, avec un tampon de dissociation de ligands par exemple, les récepteurs du complexe immobilisé ligands-anticorps I-anticorps II-supports sont séparés selon un procédé connu et les récepteurs purs obtenus sont purifiés le cas échéant selon un procédé connu.
EP19860905726 1985-08-28 1986-08-28 Procede de production de recepteurs et/ou ligands et/ou anticorps purs, materiaux ainsi produits et leur utilisation Withdrawn EP0267918A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19853530870 DE3530870A1 (de) 1985-08-28 1985-08-28 Verfahren zur herstellung von spezifisch rezeptor-ligand-ligandantikoerper (rll)- markierten geweben, zellen, zellbestandteilen und zellstoffen (biologisches material) und dessen reindarstellung
DE3530870 1985-08-28

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EP0267918A1 true EP0267918A1 (fr) 1988-05-25

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EP19860905726 Withdrawn EP0267918A1 (fr) 1985-08-28 1986-08-28 Procede de production de recepteurs et/ou ligands et/ou anticorps purs, materiaux ainsi produits et leur utilisation

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EP (1) EP0267918A1 (fr)
DE (1) DE3530870A1 (fr)
WO (1) WO1987001459A1 (fr)

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Publication number Priority date Publication date Assignee Title
US5236830A (en) * 1988-11-10 1993-08-17 Eiji Ishikawa Method of assay for antigen

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Publication number Priority date Publication date Assignee Title
EP0169729A3 (fr) * 1984-07-23 1988-08-03 Becton Dickinson and Company Récupération de récepteurs cellulaires

Non-Patent Citations (1)

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Title
See references of WO8701459A1 *

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WO1987001459A1 (fr) 1987-03-12
DE3530870C2 (fr) 1987-12-17
DE3530870A1 (de) 1987-03-12

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