WO1996032642A1 - Affinity chromatographic method for screening combinatorial libraries - Google Patents

Affinity chromatographic method for screening combinatorial libraries Download PDF

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Publication number
WO1996032642A1
WO1996032642A1 PCT/GB1996/000790 GB9600790W WO9632642A1 WO 1996032642 A1 WO1996032642 A1 WO 1996032642A1 GB 9600790 W GB9600790 W GB 9600790W WO 9632642 A1 WO9632642 A1 WO 9632642A1
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WO
WIPO (PCT)
Prior art keywords
biological
ligands
interest
affinity
column
Prior art date
Application number
PCT/GB1996/000790
Other languages
French (fr)
Inventor
Neil James Hales
Edward Clayton
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Zeneca Limited
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 Zeneca Limited filed Critical Zeneca Limited
Priority to JP8530797A priority Critical patent/JPH11503526A/en
Priority to EP96908266A priority patent/EP0820591A1/en
Publication of WO1996032642A1 publication Critical patent/WO1996032642A1/en

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials

Definitions

  • the invention relates to methods for determining the affinity of chemical compounds for a biological of interest under physiologically relevant conditions.
  • the invention relates to the deconvolution of library mixtures of chemical compounds.
  • a fragment of the DNA gyrase enzyme may be immobilised on the stationary phase of a high performance liquid chromatography column and successfully used to assay the affinity of novobiocin analogues for the DNA gyrase enzyme. Therefore in a first aspect of the present invention we provide an assay method for determining the affinity of low molecular weight ligands for a biological of interest, which method comprises immobilising the biological of interest on a solid phase matrix, contacting this with a mixture comprising the ligands, removing any unbound ligands from the matrix, eluting the ligands from the matrix and establishing their affinity for the biological of interest by reference to their order of retention on the column.
  • the biological of interest may be a peptide macromolecule or a synthetic or otherwise modified variant thereof.
  • a particular advantage of the method of the invention is that the identity, structure and/or function of the chosen macromolecule is not critical to the performance of the invention. It need not be structurally or functionally intact. It will be understood that the biological of interest may be attached to the solid phase matrix in a number of different ways to identify different ligands for different epitopes.
  • the biological of interest may have itself been isolated by its affinity, for example by affinity chromatography, for a known ligand whose properties, for example binding affinity, it is desired to imitate. It may be a fragment of a larger macromolecule; such fragments may lack the biochemical function of larger macromolecule(s) from which they are derived. In a particular aspect of the invention the biological of interest is an enzyme fragment.
  • the ligands are low molecular weight species to which antibodies cannot be readily raised. As such they may have a molecular weight of less than about 2000 daltons. In particular they have a molecular wieght of less than about 1200 daltons. More particularly the ligands have a molecular weight of less than about 1000 daltons, such as less than 800, 600, 400 or 200 daltons. They may be extracts of natural products, optionally chemically modified; alternatively they may be extracts of a synthetic product or products optionally metabolically modified. They may also be mixtures of peptides, such as peptide libraries, to which antibodies cannot readily be raised.
  • the low molecular weight ligands are potential drug candidates, ie. synthetic small molecule compounds prepared as mixtures, for example as combinatorial libraries, or mixtures prepared from separate optionally purified single compounds. Any convenient number of ligands may be present in the mixture, such as up to 5,10, 20, 30, 40, 50, 100, 200, 400, 1000 or up to 10,000 ligands..
  • the low molecular weight ligand is not a specific ligand or partner for the biological of interest as would be the case for example with antibodies and corresponding antigens.
  • the solid phase matrix is conveniently a column, such as an epoxide based column, to which the biological of interest has been bound.
  • the ligand containing mixture is passed through the column any convenient number of times, such as once, twice, or thrice.
  • Ligand(s) which show affinity for the biological of interest are retained on the column whereas those with negligible affinity are not retained and are washed off the column.
  • the biological of interest is covalently bound to the solid phase, for instance using reactive side chains on the accessible surface of the biological of interest and the solid phase.
  • Suitable materials and methods are set out, for example in "Affinity Chromatography, principles and methods", published by Pharmacia Fine Chemicals.
  • the solid phase may be activated to provide points of attachment such as epoxides or it may have intrinsically reactive surface functional groups such as carboxyl. It will be understood that the biological of interest will not normally be bound at a specific point of attachment but rather at one of a number of possible alternatives. If necessary a ligand binding site may be preserved by a known ligand during attachment of the biological of interest to the solid phase. Alternatively specific binding interactions may be used, for example biotin/avidin are used as the interface between the solid phase matrix and the biological of interest.
  • the retained ligands are then eluted and retained, preferably as ordered fractions for further analysis.
  • a particular advantage of the methods of the present invention is that ligands are contacted with the biological of interest under physiologically relevant conditions (pH, temperature, salt concentrations and the like, all as found in the human or animal body). There is no application, for example of electric current or field, which could affect the interaction between ligands and the biological of interest, as would be the case in affinity gel electrophoresis.
  • the retained ligands are trapped on one or more high performance liquid chromatography (hplc) columns. This facilitates their further analysis. Suitable trapping columns will be apparent to the scientist of ordinary skill, and include C18 reverse phase HPLC cartridge columns.
  • the retained ligands are eluted from the one or more hplc columns (after optional backflushing) into a mass spectrometer such as an electrospray mass spectrometer.
  • a mass spectrometer such as an electrospray mass spectrometer.
  • the solid phase matrix retains the biological of interest and it is not generally necessary to add further material to the column.
  • the method of the invention may be used to rank the relative affinity either singly or in admixture of a series of compounds (ligands) for the biological of interest; alternatively the method may be calibrated to give a measure of the absolute affinity of each of a series of ligands for the biological of interest.
  • ligands are of use pei s_e_ and also as starting points for the design of related ligands.
  • the biological of interest may have itself been isolated by its affinity, for example by affinity chromatography, for a known ligand whose properties, for example binding affinity, it is desired to imitate. Therefore, in a further aspect of the invention the biological of interest is firstly isolated by its affinity for a known ligand, such as a small molecule drug.
  • a known ligand such as a small molecule drug.
  • the ligands identified by the methods of the invention are functional analogues of the known ligand.
  • This is used as a template to "print" functional analogues of the known ligand.
  • Such analogues may be of diverse chemical structure.
  • ligands have -uses as biochemical tools, as diagnostic and analytical agents, and in particular as pharmaceutical and agrochemical agents.
  • Such ligands may for example be potential therapeutic agents or metabolites of such agents.
  • they are preferably chemical compounds of low molecular weight and potential therapeutic agents. They are for example of less than about 1000 daltons, such as less than 800, 600 or 400 daltons.
  • An affinity column was made by filling an epoxide based column (Hydropore EP 12 micron - Rainin, catalogue no. 83-A03-GTI) with a dialysed sample of the 24kD N-terminal 2-220 amino acid residue fragment of the gyrase B subunit, which had itself been prepared by affinity chromatography on novobiocin-sepharose by the method of Gilbert and Maxwell (E.J. Gilbert and A. Maxwell, Molecular Microbiology, 1994, 12, 365-374). After standing for 12 hours the column was washed with buffer (0.1M mixed potassium phosphate pH7) and shown to act as an affinity column for compounds binding to this protein by testing with novobiocin (E. J. Gilbert and A.
  • Maxwell, op cit Maxwell, op cit analogues which in a gyrase assay had IC50 values in the range from lug/mL to greater than 400ug/mL.
  • the gyrase assay procedure used was analogous to that disclosed by Gellert et al (Proc.Natl.Acad.Sci.USA, 1976, 21, 3872-3876).
  • the order of retention on the column was found to be equal to the order of enzyme inhibition.
  • One novobiocin analogue which has an enzyme inhibitory potency of approximately one tenth that of novobiocin was retained by between 4-10 column volumes beyond the void volume.
  • the affinity of this compound for the corresponding gyrase 24 kD fragment in solution was measured as -10- and the gyrase inhibitory potency was 1.6 ug/mL.
  • the column Having thus shown that the column behaved reproducibly as an affinity column and that this behaviour was consistent even in the presence of a 50 fold excess of a non-binding novobiocin structural analogue with negligible gyrase inhibitory activity, the column was used to examine three library mixtures each comprising nine differently substituted triaminotriazines. Using liquid chromatography (Hewlett-Packard 1050, uv detection at 305 nanometres) it was possible to identify which of the three libraries contained the active components by the tailing of the HPLC trace.
  • Hewlett-Packard 1050 Hewlett-Packard 1050, uv detection at 305 nanometres
  • This library was passed through the column a second time and the fraction whose elution time corresponded to the most strongly bound components passed directly through a short Cl 8 column (Hewlett-Packard, cartridge ODS Hypersil 5 micron 20x4mm) to trap these components.
  • This trap was then connected directly to the probe of an electrospray spectrometer (BioQ spectrometer Vacuum Generators Biotechnology - Fisons Instruments) and backflushed with acetonitrile/water (50:50) 0.5% formic acid.
  • the eluent was continuously monitored by mass spectrometry and after about 5 minutes a broad peak was detected on the total ion trace which clearly showed the presence of two major components.

Abstract

An assay method for determining the affinity of low molecular weight ligands for a biological of interest under physiologically relevant conditions, which method comprises immobilising the biological of interest on a solid phase matrix, contacting this with a mixture comprising the ligands, removing any unbound ligands from the matrix, eluting the ligands from the matrix and establishing their affinity for the biological of interest by reference to their order of retention on the column.

Description

AFFINITY CHROMATXXaiAPHIC METHOD FOR SCREE_NING COMBINATORIAL
LIBRARIES
The invention relates to methods for determining the affinity of chemical compounds for a biological of interest under physiologically relevant conditions. In particular the invention relates to the deconvolution of library mixtures of chemical compounds.
Zuckerman et al (Proc. Natl. Acad. Sci. USA, 1992, 89, 4505-4509) describe the use of affinity selection from a defined peptide library of peptidic ligands for a proteinaceous antibody by gel-filtration to give a mixture of complexes of the antibody and its selected ligand. The ligands are then dissociated from the antibody in a separate step and then subsequently identified by separate hplc analysis followed by mass spectrometry and amino-acid analysis. In a related method, Songyang et al Cell, 1993, 72, 767-778) describe the use of affinity chromatography to select ligands for the SH2 protein using GST-SH2 fusion protein immobilised on agarose-glutathione as the stationary phase in the affinity chromatography. The selected ligands are then subsequently concentrated and analysed by amino acid analysis.
However these methods have a number of disadvantages. For example, they are limited by the requirement for separate selection and identification steps. An identified and well characterised biological macromolecule is required as target. The methods do not rank the affinity of the most useful and strongly bound ligands. Also amino acid analysis has limitations as a method for identifying ligands having affinity for the target biological macromolecule.
We now disclose a universal method for selecting and identifying those components of a mixture of chemical compounds that have affinity for a target biological of interest.
This is based on our discovery that a fragment of the DNA gyrase enzyme may be immobilised on the stationary phase of a high performance liquid chromatography column and successfully used to assay the affinity of novobiocin analogues for the DNA gyrase enzyme. Therefore in a first aspect of the present invention we provide an assay method for determining the affinity of low molecular weight ligands for a biological of interest, which method comprises immobilising the biological of interest on a solid phase matrix, contacting this with a mixture comprising the ligands, removing any unbound ligands from the matrix, eluting the ligands from the matrix and establishing their affinity for the biological of interest by reference to their order of retention on the column.
The biological of interest may be a peptide macromolecule or a synthetic or otherwise modified variant thereof. A particular advantage of the method of the invention is that the identity, structure and/or function of the chosen macromolecule is not critical to the performance of the invention. It need not be structurally or functionally intact. It will be understood that the biological of interest may be attached to the solid phase matrix in a number of different ways to identify different ligands for different epitopes.
The biological of interest may have itself been isolated by its affinity, for example by affinity chromatography, for a known ligand whose properties, for example binding affinity, it is desired to imitate. It may be a fragment of a larger macromolecule; such fragments may lack the biochemical function of larger macromolecule(s) from which they are derived. In a particular aspect of the invention the biological of interest is an enzyme fragment.
The ligands are low molecular weight species to which antibodies cannot be readily raised. As such they may have a molecular weight of less than about 2000 daltons. In particular they have a molecular wieght of less than about 1200 daltons. More particularly the ligands have a molecular weight of less than about 1000 daltons, such as less than 800, 600, 400 or 200 daltons. They may be extracts of natural products, optionally chemically modified; alternatively they may be extracts of a synthetic product or products optionally metabolically modified. They may also be mixtures of peptides, such as peptide libraries, to which antibodies cannot readily be raised. In general antibodies cannot be raised to polypeptides of less than 20 residues, such as less than 12 or less than 7 residues. In particular the low molecular weight ligands are potential drug candidates, ie. synthetic small molecule compounds prepared as mixtures, for example as combinatorial libraries, or mixtures prepared from separate optionally purified single compounds. Any convenient number of ligands may be present in the mixture, such as up to 5,10, 20, 30, 40, 50, 100, 200, 400, 1000 or up to 10,000 ligands..
From the above it will be understood that the low molecular weight ligand is not a specific ligand or partner for the biological of interest as would be the case for example with antibodies and corresponding antigens.
We have found that the quantities of ligand required are appreciably less than those required for conventional methods of ligand determination and identification. Furthermore less time and effort are required to prepare and analyse ligand containing mixtures in comparison to conventional preparative and analytical methods. The solid phase matrix is conveniently a column, such as an epoxide based column, to which the biological of interest has been bound. The ligand containing mixture is passed through the column any convenient number of times, such as once, twice, or thrice. Ligand(s) which show affinity for the biological of interest are retained on the column whereas those with negligible affinity are not retained and are washed off the column. In particular the biological of interest is covalently bound to the solid phase, for instance using reactive side chains on the accessible surface of the biological of interest and the solid phase. Suitable materials and methods are set out, for example in "Affinity Chromatography, principles and methods", published by Pharmacia Fine Chemicals. The solid phase may be activated to provide points of attachment such as epoxides or it may have intrinsically reactive surface functional groups such as carboxyl. It will be understood that the biological of interest will not normally be bound at a specific point of attachment but rather at one of a number of possible alternatives. If necessary a ligand binding site may be preserved by a known ligand during attachment of the biological of interest to the solid phase. Alternatively specific binding interactions may be used, for example biotin/avidin are used as the interface between the solid phase matrix and the biological of interest.
The retained ligands are then eluted and retained, preferably as ordered fractions for further analysis. A particular advantage of the methods of the present invention is that ligands are contacted with the biological of interest under physiologically relevant conditions (pH, temperature, salt concentrations and the like, all as found in the human or animal body). There is no application, for example of electric current or field, which could affect the interaction between ligands and the biological of interest, as would be the case in affinity gel electrophoresis.
In a preferred aspect of the invention the retained ligands are trapped on one or more high performance liquid chromatography (hplc) columns. This facilitates their further analysis. Suitable trapping columns will be apparent to the scientist of ordinary skill, and include C18 reverse phase HPLC cartridge columns.
In a more preferred aspect of the invention the retained ligands are eluted from the one or more hplc columns (after optional backflushing) into a mass spectrometer such as an electrospray mass spectrometer. In a further aspect of the present invention we provide an hplc column on which a biological of interest is immobilised. In a still further aspect of the invention we claim the use of such a reactive column in affinity selection procedures and also in the deconvolution of chemical libraries. Advantageously we have found that, despite repeat procedures, the solid phase matrix retains the biological of interest and it is not generally necessary to add further material to the column.
The method of the invention may be used to rank the relative affinity either singly or in admixture of a series of compounds (ligands) for the biological of interest; alternatively the method may be calibrated to give a measure of the absolute affinity of each of a series of ligands for the biological of interest. Such ligands are of use pei s_e_ and also as starting points for the design of related ligands.
As previously mentioned, the biological of interest may have itself been isolated by its affinity, for example by affinity chromatography, for a known ligand whose properties, for example binding affinity, it is desired to imitate. Therefore, in a further aspect of the invention the biological of interest is firstly isolated by its affinity for a known ligand, such as a small molecule drug. This means that the ligands identified by the methods of the invention are functional analogues of the known ligand. For this aspect of the invention it is not necessary to know the identity of the biological of interest. This is used as a template to "print" functional analogues of the known ligand. Such analogues may be of diverse chemical structure. All the ligands have -uses as biochemical tools, as diagnostic and analytical agents, and in particular as pharmaceutical and agrochemical agents. Such ligands may for example be potential therapeutic agents or metabolites of such agents. In this regard they are preferably chemical compounds of low molecular weight and potential therapeutic agents. They are for example of less than about 1000 daltons, such as less than 800, 600 or 400 daltons.
The invention will now be illustrated but not limited by reference to the following non-limiting example:
Example 1
An affinity column was made by filling an epoxide based column (Hydropore EP 12 micron - Rainin, catalogue no. 83-A03-GTI) with a dialysed sample of the 24kD N-terminal 2-220 amino acid residue fragment of the gyrase B subunit, which had itself been prepared by affinity chromatography on novobiocin-sepharose by the method of Gilbert and Maxwell (E.J. Gilbert and A. Maxwell, Molecular Microbiology, 1994, 12, 365-374). After standing for 12 hours the column was washed with buffer (0.1M mixed potassium phosphate pH7) and shown to act as an affinity column for compounds binding to this protein by testing with novobiocin (E. J. Gilbert and A. Maxwell, op cit) analogues which in a gyrase assay had IC50 values in the range from lug/mL to greater than 400ug/mL. The gyrase assay procedure used was analogous to that disclosed by Gellert et al (Proc.Natl.Acad.Sci.USA, 1976, 21, 3872-3876).
The order of retention on the column was found to be equal to the order of enzyme inhibition. One novobiocin analogue which has an enzyme inhibitory potency of approximately one tenth that of novobiocin was retained by between 4-10 column volumes beyond the void volume. The affinity of this compound for the corresponding gyrase 24 kD fragment in solution was measured as -10- and the gyrase inhibitory potency was 1.6 ug/mL.
Having thus shown that the column behaved reproducibly as an affinity column and that this behaviour was consistent even in the presence of a 50 fold excess of a non-binding novobiocin structural analogue with negligible gyrase inhibitory activity, the column was used to examine three library mixtures each comprising nine differently substituted triaminotriazines. Using liquid chromatography (Hewlett-Packard 1050, uv detection at 305 nanometres) it was possible to identify which of the three libraries contained the active components by the tailing of the HPLC trace. This library was passed through the column a second time and the fraction whose elution time corresponded to the most strongly bound components passed directly through a short Cl 8 column (Hewlett-Packard, cartridge ODS Hypersil 5 micron 20x4mm) to trap these components. This trap was then connected directly to the probe of an electrospray spectrometer (BioQ spectrometer Vacuum Generators Biotechnology - Fisons Instruments) and backflushed with acetonitrile/water (50:50) 0.5% formic acid. The eluent was continuously monitored by mass spectrometry and after about 5 minutes a broad peak was detected on the total ion trace which clearly showed the presence of two major components.
Comparison of their masses with the molecular ions of the compounds in the library showed that these were in fact two components of the library (the analysis had been carried out blind). In a separate experiment the fractions correponding to the wash off from the affinity column before these two compounds were analysed by direct electrospray mass spectrometry or by trapping. Four major species were detected.

Claims

1. An assay method for determining the affinity of low molecular weight ligands for a biological of interest, which method comprises immobilising the biological of interest on a solid phase matrix, contacting this with a mixture comprising the ligands, removing any unbound ligands from the matrix, eluting the ligands from the matrix and establishing their affinity for the biological of interest by reference to their order of retention on the column.
2. An assay as claimed in claim 1 wherein the biological of interest is firstly identified by its affinity for a known ligand.
3. An assay as claimed in claim 1 wherein the solid phase matrix is an HPLC colum.
4. An assay as claimed in claim 1 wherein the ligands are eluted for identification by mass spectrometry.
5. The use of an HPLC column having a biological of interest covalently attached in the method of any one of claims 1-4.
6. A method as claimed in any one of the previous claims wherein the biological of interest is an enzyme or receptor fragment.
PCT/GB1996/000790 1995-04-08 1996-04-01 Affinity chromatographic method for screening combinatorial libraries WO1996032642A1 (en)

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JP8530797A JPH11503526A (en) 1995-04-08 1996-04-01 Affinity chromatography methods for screening combinatorial libraries
EP96908266A EP0820591A1 (en) 1995-04-08 1996-04-01 Affinity chromatographic method for screening combinatorial libraries

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GBGB9507366.4A GB9507366D0 (en) 1995-04-08 1995-04-08 Assay

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Cited By (5)

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US6054047A (en) * 1998-03-27 2000-04-25 Synsorb Biotech, Inc. Apparatus for screening compound libraries
JP2002501176A (en) * 1998-01-08 2002-01-15 ザ・ユニヴァーシティ・オヴ・リヴァプール Using mass fingerprinting to identify protein affinity ligands
US6607921B1 (en) 1998-03-27 2003-08-19 Ole Hindsgaul Methods for screening compound libraries
US6613575B1 (en) 1998-03-27 2003-09-02 Ole Hindsgaul Methods for screening compound libraries
US6627453B1 (en) 1998-03-27 2003-09-30 Ole Hindsgaul Methods for screening compound libraries

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WO1997043301A2 (en) * 1996-05-10 1997-11-20 Novartis Ag Identification of members of combinatorial libraries by mass spectrometry
ATE376183T1 (en) * 1999-04-15 2007-11-15 Univ Virginia PROTEOME DEGRADATION

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002501176A (en) * 1998-01-08 2002-01-15 ザ・ユニヴァーシティ・オヴ・リヴァプール Using mass fingerprinting to identify protein affinity ligands
US6054047A (en) * 1998-03-27 2000-04-25 Synsorb Biotech, Inc. Apparatus for screening compound libraries
US6355163B2 (en) 1998-03-27 2002-03-12 Ole Hindsgaul Apparatus for screening compound libraries
US6387257B1 (en) 1998-03-27 2002-05-14 Ole Hindsgaul Apparatus for screening compound libraries
US6395169B1 (en) 1998-03-27 2002-05-28 Ole Hindgual Apparatus for screening compound libraries
US6607921B1 (en) 1998-03-27 2003-08-19 Ole Hindsgaul Methods for screening compound libraries
US6613575B1 (en) 1998-03-27 2003-09-02 Ole Hindsgaul Methods for screening compound libraries
US6627453B1 (en) 1998-03-27 2003-09-30 Ole Hindsgaul Methods for screening compound libraries
US6649415B1 (en) 1998-03-27 2003-11-18 Ole Hindsgaul Methods for screening compound libraries
US6656739B2 (en) 1998-03-27 2003-12-02 Ole Hindsgaul Methods for screening compound libraries
US6720190B1 (en) 1998-03-27 2004-04-13 Ole Hindsgaul Methods for screening compound libraries
US6723235B2 (en) 1998-03-27 2004-04-20 Ole Hindsgaul Apparatus for screening compound libraries

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GB2300260A (en) 1996-10-30
EP0820591A1 (en) 1998-01-28
JPH11503526A (en) 1999-03-26
GB9507366D0 (en) 1995-05-31
GB2300260B (en) 1998-03-11
GB9606887D0 (en) 1996-06-05

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