EP0784792A2 - Procede de recherche de l'interaction entre des biomolecules par resonance de plasmon superficiel - Google Patents

Procede de recherche de l'interaction entre des biomolecules par resonance de plasmon superficiel

Info

Publication number
EP0784792A2
EP0784792A2 EP95933414A EP95933414A EP0784792A2 EP 0784792 A2 EP0784792 A2 EP 0784792A2 EP 95933414 A EP95933414 A EP 95933414A EP 95933414 A EP95933414 A EP 95933414A EP 0784792 A2 EP0784792 A2 EP 0784792A2
Authority
EP
European Patent Office
Prior art keywords
peptide
group
chelating agent
poly
biosensor unit
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.)
Ceased
Application number
EP95933414A
Other languages
German (de)
English (en)
Inventor
Peter Steinlein
Wolfgang Zauner
Bianca Habermann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boehringer Ingelheim International GmbH
Original Assignee
Boehringer Ingelheim International GmbH
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 Boehringer Ingelheim International GmbH filed Critical Boehringer Ingelheim International GmbH
Publication of EP0784792A2 publication Critical patent/EP0784792A2/fr
Ceased legal-status Critical Current

Links

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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/26—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one amino group bound to the carbon skeleton, e.g. lysine

Definitions

  • the invention relates to the study of
  • Macromolecules e.g. (Poly) peptide (poly) peptide or (poly) peptide-DNA interactions have been reported in the past
  • a recently developed method is based on the optical phenomenon of surface plasmon resonance ("Surface Plasmon Resonance", SPR).
  • SPR Surface Plasmon Resonance
  • Glass support which has a gold layer on one side, to which a hydrogel matrix made of carboxymethyl-dextran is covalently attached via a barrier layer provided with linker groups
  • the hydrogel matrix serves on the one hand
  • hydrogel matrix is generally a (poly) peptide, the following methods have hitherto been used on the hydrogel matrix:
  • Process 1) is associated with the risk that, due to the non-directional chemical reaction between the hydrogel-forming substance and the
  • (Poly) peptide takes place, the biological and / or biophysical properties of which are influenced in a way that is difficult or impossible to define. That can have the consequence that the immobilized (poly) peptide is not or not completely in its native, biologically active form because, for example, the
  • Section of the molecule that is to interact with the reactant has been blocked by a chemical grouping or has become inaccessible due to a change in the conformation of the molecule.
  • Sandwich immunoassays e.g. in the ELISA (Enzyme linked Immunosorbent Assay) technique.
  • ELISA Enzyme linked Immunosorbent Assay
  • the present invention was based on the object of a method for investigating the interaction of (poly) peptides with reactants using SPR provide the disadvantages of the known
  • affinity peptides Use of genetic engineering methods as fusion proteins with sequence segments that have high affinity for a ligand (so-called affinity peptides)
  • Immobilized metal chelate affinity chromatography is a wide one
  • nitrilotriacetic acid derivatives have particularly advantageous properties, which include the extremely high affinity for certain metal ions, for example Cu 2+ , Ni 2+ or Zn 2+ . So far, this method has been widely used using nickel as the metal ion and
  • Nitrilotriacetic acid as a complexing agent in the
  • the present invention thus relates to a method for investigating the interaction of a (poly) peptide with a reaction partner by means of a
  • Biosensor unit in which the through the
  • Interaction triggered surface plasmon resonance is determined in a metallic layer at the interface of two media which are permeable to electromagnetic radiation and have a different refractive index, the medium with a lower refractive index being an aqueous medium in which the (poly) peptide is present in an immobilized form and with the
  • Reaction partner is brought into contact.
  • the invention relates to a biosensor unit for examining the
  • Reaction partner by determining the surface plasmon resonance in a metallic layer at the interface of two media with different permeability for electromagnetic radiation
  • Refractive index is an aqueous medium.
  • Biosensor unit is characterized in that a chelating agent, possibly in a form complexed with a metal ion, is bound to its surface facing the aqueous medium.
  • the aqueous phase is a biocompatible porous matrix, in particular a hydrogel.
  • hydrogel formers there is basically no restriction with regard to the hydrogel formers, provided that they are suitable for the SPR process, especially with regard to the required diffusion of the biomolecules in the
  • Hydrogel matrix is basically given.
  • suitable hydrogel formers are polysaccharides, such as agarose, dextran, carragen, alginates, starch, cellulose or derivatives of these polysaccharides, such as e.g.
  • Carboxymethyl derivatives or water-swellable organic polymers, such as polyvinyl alcohol, polyacrylic acid,
  • Polyacrylamide or polyethylene glycol are examples of Polyacrylamide or polyethylene glycol.
  • a particularly suitable hydrogel consists of dextran, which with regard to the covalent binding of the
  • Hydrogel layer and its binding to the metal layer, which is optionally carried out via an organic barrier layer, was among others. in PCT application WO 90/05303 and by Löfas and Johnsson, 1990.
  • the chelating agent is bound to the reactive groups of the hydrogel.
  • the hydrogel former is a dextran which has carboxymethyl groups as reactive groups.
  • the (poly) peptide is a fusion (poly) peptide which, in addition to its biologically active section, has an affinity peptide which contains at least two adjacent histidine residues.
  • the chelating agent is on
  • NTA Nitrilotriacetic acid
  • R can be an alkylene bridge of the type (CH 2 ) n - which can be substituted or unsubstituted, with the proviso that the substituent does not adversely affect the function of the chelating agent, and n is an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 means, the alkylene group being of sufficient size
  • R can mean an aromatic bridge member which consists of one or more one or more
  • polynuclear aromatics which can optionally also be an aromatic heterocycle, is built up, or in which R can mean an aralkyl bridge member in which the aromatic part either directly or via an alkyl group of the type (CH 2 ) n -, in which n is a whole Number 1, 2, 3, 4 or 5 may mean to which Y or to which the ⁇ -C atom adjacent to the carboxyl group may be bound, and in which Y is a reactive group, in particular an NH 2 or an SH group.
  • R can mean an aralkyl bridge member in which the aromatic part either directly or via an alkyl group of the type (CH 2 ) n -, in which n is a whole Number 1, 2, 3, 4 or 5 may mean to which Y or to which the ⁇ -C atom adjacent to the carboxyl group may be bound, and in which Y is a reactive group, in particular an NH 2 or an SH group.
  • R examples are methylene, ethylene, n-propylene or iso-propylene or o-, m-, p-phenylene or ⁇ , ⁇ '-naphthylene.
  • the NTA derivative can also have the general formula Y-R1-CH (COOH) -N (CHR2COOH) 2 , in which R1 is a group with that indicated for R.
  • R2 can be an alkyl group of the type CH 3 (CH 2 ) n -, which is substituted, for example with OH or Cl, or may be unsubstituted, and n may be an integer 1, 2, 3, 4 or 5, or where R2 may be a branched alkyl group such as iso-propyl, t-butyl or iso-butyl, or where R2 is a aromatic bridge member of the meaning given for R, and wherein Y is a reactive group, in particular an NH 2 ⁇ or an SH group.
  • R and R1 are selected in such a way that on the one hand the ability to complex in comparison to
  • unbound NTA is influenced as little as possible and on the other hand the distance to the surface of the
  • Biosensor unit is small enough not to irritate the SPR phenomena.
  • R2 is selected so that the
  • the ability of the chelating agent to complex with the metal ion to be chelated and with the substance to be investigated is not adversely affected.
  • Ligands usually a His (6) modified
  • Y is a reactive group with which the chelating agent contacts the surface of the biosensor unit, in particular the reactive groups contained in the hydrogel matrix, is bound.
  • the reactive group of the chelating agent is thus placed on the reactive group surface of the biosensor unit, in particular the hydrogel matrix;
  • a particularly preferred reactive group Y is an NH 2 group attached to the modified ones
  • Carboxymethyl groups of dextran binds.
  • Other reactive groups Y which are suitable for covalent bonding, are SH groups, which are stable
  • reducing agents e.g. Mercaptoethanol, which is often used in the purification or synthesis of
  • the chelating agent can be reacted via its reactive groups using methods known for the coupling of (poly) peptides, e.g. by means of N-hydroxysuccinimide (NHS) and N-ethyl-N '- (dimethylaminopropyl) carbodiimide (EDC) (see e.g. Cuatrecasas and Parikh, 1972).
  • NHS N-hydroxysuccinimide
  • EDC dimethylaminopropyl carbodiimide
  • Transition metal ions are primarily suitable as metal ions, preferably
  • Transition metal ions of the fourth period are particularly preferred, of which Ni 2+ is particularly preferred.
  • NTA derivatives are the N- (5-amino-1-carboxypentyl) described by Hochuli et al., 1987
  • His-Tag proteins are used (so-called “His-Tag proteins”), reference is made to the European patent application EP-A-282 042; Examples include (His) 6 proteins in which the affinity peptide has six histidine residues side by side.
  • Biosensor chips possibly via a reactive one
  • hydrogel matrix is generally preferred, above all because its structure makes it a structure that is quite similar to the physiological conditions inside the cell and therefore for the
  • the present invention has the decisive advantage that the biosensor surface can be completely regenerated. This makes it possible to carry out series tests under comparable conditions.
  • the biosensor unit according to the invention fulfills the following conditions imposed on the regeneration: 1) The (poly) peptide bound to the metal chelate can be completely removed. 2) When reloaded, the surface of the biosensor unit does not lose any binding capacity for a (poly) peptide to be immobilized.
  • a metal ion-saturated chelate surface which contains a ligand, e.g. a His (6) protein has bound
  • several options are available. On the one hand, the ligand can pass through
  • Acid treatment e.g. 10 mM acetic acid
  • the metal ion load remains largely stable, depending on the metal ion used.
  • the bound protein can be combined with the metal ion by adding another strong chelating agent, e.g. 100 mM EDTA, from the
  • the stability of the metal ion bond to the chelating agent must be determined in individual cases and is, among other things. depends on the stability of the ligand metal ion complex.
  • the method of regenerating the chelate surface with other chelating agents is preferable to the first method, considering the reproducibility of the method.
  • Macromolecule with affinity for ligand 1, but not for the metal chelate surface can be achieved under conditions that remove ligand 2
  • Affinity chromatography can be applied.
  • the present invention can advantageously be used for biochemical cleaning
  • the invention relates to a biosensor kit for investigating the interaction of a (poly) peptide with a reaction partner using SPR.
  • the kit contains an SPR biosensor unit in a first one, a chelating agent in another container, and a salt for complexing with the chelating agent in another container
  • Container a reagent for the regeneration of the surface and optionally one or more reference proteins in one or more further containers.
  • a surface of the biosensor unit is preferably a hydrogel layer. The is convenient
  • Chelating agent in the form of a deep-frozen solution the concentration and buffer solution being switched off with regard to the coupling to the surface of the biosensor unit.
  • Preferred chelating agents are
  • Nitrilotriacetic acid derivatives especially N- (5-amino- 1-carboxypentyl) iminodiacetic acid and
  • the metal salt is preferably nickel sulfate, preferably in the form of a stock solution which can be diluted with a view to the desired degree of loading of the surface.
  • a surface of the biosensor unit consists of a hydrogel with reactive groups, in particular of a carboxymethylated dextran, these are
  • N-ethyl-N '- (dimethylaminopropyl) carbodiimide which are preferably in the form of frozen solutions which are suitable for activation in terms of concentration and buffer solution.
  • the reagent for deactivating the N-hydroxysuccinimide groups remaining after coupling of the (poly) peptide to the biosensor surface is ethanolamine in a suitable concentration and
  • Buffer solution preferably also in frozen form.
  • the reagent for regenerating the biosensor surface is preferably a chelating agent, in particular EDTA.
  • test proteins which may be present in further containers preferably have an affinity peptide with several histidine residues and are in the form of
  • Hydrogel layer from the bisosensor unit appears to be difficult to carry out in a reproducible manner.
  • Biosensor unit can be used.
  • the chelating agent was immobilized according to the method recommended by the manufacturer of the biosensor chips carried out. Because of the low relative
  • BSA bovine serum albumin
  • Chelating agent modified biosensor chip surface is very small, no binding to the modified surface was observed.
  • the binding capacity of the surface for the test protein is, as shown in Example 4, directly correlated with that
  • Nickel ion concentration used for loading The setting of the nickel ion concentration enables simple adjustment of the
  • the present invention relates to the nitrilotriacetic acid derivative of the formula N ⁇ , N ⁇ -Di (1-carboxyethyl) -2,6-diaminohexanoic acid.
  • This chelation can be like the well known
  • Nitrilotriacetic acid derivatives also for the
  • immobilized metal chelate affinity chromatography can be used to purify proteins and peptides.
  • Fig. 1 Immobilization of N- (5-amino-1-carboxypentyl) iminodiacetic acid on the dextran surface of a biosensor unit
  • Fig. 2 Indirect determination of the loading of the
  • Fig. 3 Regeneration with bovine serum albumin
  • Fig. 4 Determination of the non-specific binding of a
  • Trifluoromethanesulfonic acid (Merck) was slowly added dropwise and the mixture was stirred at room temperature for 1 hour. The precipitate formed was separated off, the solution was mixed with 30 ml of water and almost to dryness
  • the solution was added dropwise to 10 volumes of diethyl ether for 1.5 h at room temperature.
  • the precipitation was washed 3 times with ether and dried.
  • the precipitate formed was separated off, the solution was mixed with
  • HBS buffer 10 mM HEPES (2- [4- (2-hydroxyethyl) -1-piperazino] ethanesulfonic acid), 150 mM NaCl and 5 mM MgCl 2 , pH 7.4) at 25 ° C
  • a CM5 biosensor chip was used for immobilization
  • This biosensor unit has a hydrogel surface made of carboxymethylated dextran.
  • the activation of the hydrogel surface was carried out by injecting 35 ⁇ l of a 0.05 M N-hydroxysuccininimide (NHS) /0.2 M N-ethyl-N '- (dimethylaminopropyl) carbodiimide (EDC) solution (Flow rate 5 ⁇ l / min).
  • NHS N-hydroxysuccininimide
  • EDC dimethylaminopropyl
  • the surface was 35 ul of a solution of 15 mg / ml
  • N- (5-Amino-1-carboxypentyl) iminodiacetic acid in 1 M NaOH was injected at a flow rate of 3 ul / min.
  • Unsaturated surface binding sites were found by injecting 35 ⁇ l of a 1 M solution of
  • Immobilization is indicated in the figure.
  • Bovine serum albumin (BSA, Sigma) was injected into 100 ml running buffer.
  • the sensorgram (Fig. 2) shows
  • Running buffer injected and regenerated with EDTA (ethylenediamine-N, N, N ', N'-tetraacetic acid). For calculating 3 (baseline,
  • Injection maximum and bound protein t 1) the resonance was 30 s before the injection of bovine serum albumin (baseline), 30 s before the end of the injection of the
  • Bovine serum albumin injection maximum
  • Example 1 To determine the non-specific binding of the protein to the surface, the surface described in Example 1 was regenerated with EDTA, as described in Example 3, and 20 ⁇ l of a test protein solution were injected at a flow rate of 5 ⁇ l / min. After renewed regeneration of the surface with EDTA and loading with
  • Binding behavior of different proteins were 40 ⁇ l of solutions (10 ⁇ g / ml in running buffer) of a chicken protein in the sequence listing
  • the protein was further purified via a
  • Bovine serum albumin in contrast to the His (6) - modified chicken protein, saturates the
  • the surface loaded with the His (6) -modified chicken protein for saturation can be used to search for interacting with this protein
  • Proteins e.g. Cell culture supernatants from
  • Cell culture supernatants from different cell lines are injected.
  • the binding of an interacting partner can be demonstrated by increasing the resonance after the injection.
  • the sensorgram is shown in FIG. 7.
  • the surface shows a binding behavior comparable to that described in Example 2 and modified with N- (5-amino-1-carboxypentyl) iminodiacetic acid.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Food Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé et une unité de biodétection pouvant être régénérée, ainsi que des matériels appropriés, pour la recherche de l'interaction entre des biomolécules par résonance de plasmon superficiel (SPR). Un des réactifs, un (poly)peptide, est couplé au moyen d'un chélate métallique à la surface de l'unité de biodétection. Les agents de formation de chélate utilisés sont, de préférence, des dérivés d'acide nitrilonitriacétique auxquels peuvent être liées des protéines présentant un peptide d'affinité contenant des radicaux histidine.
EP95933414A 1994-09-23 1995-09-21 Procede de recherche de l'interaction entre des biomolecules par resonance de plasmon superficiel Ceased EP0784792A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE4433980A DE4433980C2 (de) 1994-09-23 1994-09-23 Verfahren und Biosensorhit zur Untersuchung der Wechselwirkung von Biomolekülen mittels Oberflächen-Plasma-Resonanz
DE4433980 1994-09-23
PCT/EP1995/003731 WO1996009547A2 (fr) 1994-09-23 1995-09-21 Procede de recherche de l'interaction entre des biomolecules par resonance de plasmon superficiel

Publications (1)

Publication Number Publication Date
EP0784792A2 true EP0784792A2 (fr) 1997-07-23

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ID=6528988

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95933414A Ceased EP0784792A2 (fr) 1994-09-23 1995-09-21 Procede de recherche de l'interaction entre des biomolecules par resonance de plasmon superficiel

Country Status (12)

Country Link
EP (1) EP0784792A2 (fr)
JP (1) JPH10505910A (fr)
KR (1) KR970706494A (fr)
CN (1) CN1158658A (fr)
AU (1) AU3608995A (fr)
BR (1) BR9509077A (fr)
CA (1) CA2198615A1 (fr)
CO (1) CO4410395A1 (fr)
DE (1) DE4433980C2 (fr)
PL (1) PL319354A1 (fr)
WO (1) WO1996009547A2 (fr)
ZA (1) ZA958024B (fr)

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US6107080A (en) * 1996-04-25 2000-08-22 Mcgill University Biosensor device and method
US5955379A (en) * 1996-04-25 1999-09-21 Mcgill University Biosensor device and method
US6130037A (en) * 1996-04-25 2000-10-10 Pence And Mcgill University Biosensor device and method
US6165335A (en) 1996-04-25 2000-12-26 Pence And Mcgill University Biosensor device and method
AU6883198A (en) * 1997-04-24 1998-11-13 American Home Products Corporation Method for the identification and characterization of nuclear receptor ligands
IL141759A0 (en) * 1998-09-03 2002-03-10 Trellis Bioinformatics Inc Multihued labels
AUPP856399A0 (en) * 1999-02-08 1999-03-04 Australian Membrane And Biotechnology Research Institute Improved compounds for protein binding
US6787368B1 (en) 1999-03-02 2004-09-07 Helix Biopharma Corporation Biosensor method for detecting analytes in a liquid
WO2005038442A1 (fr) * 2003-10-16 2005-04-28 Kabushiki Kaisha Nard Kenkyusho Procede de mesure de la resonance du plasmon de surface et compose de metal noble a utiliser dans ce procede
US7804592B2 (en) 2003-10-16 2010-09-28 Nard Institute, Ltd. Method for measuring a surface plasmon resonance and noble metal compound used for the same
CN101261226B (zh) * 2007-03-08 2010-12-08 北京宏荣博曼生物科技有限责任公司 一种基于聚乙二醇的表面等离子共振仪芯片
JP2010197046A (ja) * 2007-05-28 2010-09-09 Tanaka Holdings Kk バイオセンサー
EP2015071A1 (fr) 2007-07-13 2009-01-14 FUJIFILM Corporation Support, son procédé de production, bioréacteur et puce pour analyse par résonance à plasmons de surface
KR100953558B1 (ko) 2007-12-12 2010-04-21 한국전자통신연구원 표면 플라즈몬 공명을 이용한 ｐｓ-ｓｐｃｌ 탐색 장치 및방법
CN114797804B (zh) * 2022-03-29 2023-08-04 翌圣生物科技(上海)股份有限公司 一种具有长连接臂的nta色谱介质及其制备方法

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IL85137A (en) * 1987-01-21 1992-02-16 Ares Serono Res & Dev Ltd Method of assaying for a ligand using surface plasmon resonance effect
CA1340522C (fr) * 1987-03-10 1999-05-04 Heinz Dobeli Proteins hybrides renfermant des histidines voisines pour une purification amelioree
DE58905660D1 (de) * 1988-04-25 1993-10-28 Hoffmann La Roche Diagnose-Hilfsmittel.
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Also Published As

Publication number Publication date
CO4410395A1 (es) 1997-01-09
JPH10505910A (ja) 1998-06-09
KR970706494A (ko) 1997-11-03
BR9509077A (pt) 1997-11-25
WO1996009547A2 (fr) 1996-03-28
ZA958024B (en) 1996-03-25
CN1158658A (zh) 1997-09-03
WO1996009547A3 (fr) 1996-05-30
PL319354A1 (en) 1997-08-04
CA2198615A1 (fr) 1996-03-28
DE4433980A1 (de) 1996-03-28
DE4433980C2 (de) 1996-08-22
AU3608995A (en) 1996-04-09

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