EP1611443A1 - Procede et trousse pour bioanalyse d'analytes cellulaires - Google Patents

Procede et trousse pour bioanalyse d'analytes cellulaires

Info

Publication number
EP1611443A1
EP1611443A1 EP04725223A EP04725223A EP1611443A1 EP 1611443 A1 EP1611443 A1 EP 1611443A1 EP 04725223 A EP04725223 A EP 04725223A EP 04725223 A EP04725223 A EP 04725223A EP 1611443 A1 EP1611443 A1 EP 1611443A1
Authority
EP
European Patent Office
Prior art keywords
cell
binding
solid support
bound
ligand
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
EP04725223A
Other languages
German (de)
English (en)
Inventor
Robert Karlsson
Pascale Richalet-Secordel
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.)
Cytiva Sweden AB
Original Assignee
Biacore AB
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
Priority claimed from SE0301058A external-priority patent/SE0301058D0/xx
Application filed by Biacore AB filed Critical Biacore AB
Publication of EP1611443A1 publication Critical patent/EP1611443A1/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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • the present invention relates to a method of analysing a cell sample for cell surface-bound and intracellularly bound analytes as well as to a kit for cell characterisation.
  • the cell walls of living organisms expose a number of cell surface elements of different functions such as, for example, receptors that interact with specific signal species in the cell environment, and antigens having specific immune reactions. Information on the presence and concentration of such surface elements in a cell sample is sought for various reasons. For instance, many disease states in particular animals, such as humans, have a genetic basis and can be characterised by changes in the patterns or levels of expression of various genes. Some of the genetic changes are expressed by alterations in cell surface antigens. For example, leukemia may be diagnosed on the basis of expression of specific CD, lymphoid and myeloid antigens.
  • the assay device comprises an array of different molecules having a binding partner in a biological sample, wherein the pattern of binding of the binding partners, which may be cells or components released by the cells, to the respective molecules on the array provides an indication of a normal condition or disease condition or a propensity therefor to develop in an animal, avian or plant species, or the presence of a particular cell type or microbial, viral, parasitic or other pathogenic agent.
  • WO 96/38729 discloses the use of an inhibition type immunoassay to detect pathogens in a sample suspension.
  • the suspension is first reacted with a predetermined excess of antibodies against the pathogen, and all pathogens are then removed from the suspension, e.g. by filtration or centrifugation.
  • the amount of antibody in the resulting pathogen-free solution is then determined by contacting the solution with a sensor surface having immobilised receptor to the antibody.
  • WO 90/05305 discloses the use of bifunctional molecules for functionalising sensing surfaces.
  • Each bifunctional molecule has a function for immobilizing the molecule to the sensing surface, and a bioselective function for interaction with a target biomolecule.
  • the above and other objects and advantages are obtained by a novel method for analysing a cell sample for cell surface-bound analytes as well as intracellularly bound analytes.
  • the method is based on contacting the cell sample with multiple ligands specific to different receptors on or within the cell, wherein each ligand in addition to the cell-specific part comprises a portion specifically binding to a particular member of an array.
  • the ligands may be bound to the array after (inhibition assay) or before (direct assay) the contacting with the cell sample.
  • the present invention therefore provides a method for analysing a cell sample fluid for cell surface-bound and/or intracellularly bound analytes.
  • this method comprises the steps of:
  • each binding agent comprises one member of a specific binding pair
  • each ligand comprising a first part capable of specifically binding to a specific analyte selected from cell surface-bound analytes and intracellularly bound analytes of a defined cell type, and a second part which comprises the other member of each specific binding pair, such that each ligand binds through its specific binding pair part to a specific position on the solid support surface;
  • step (iii) determining the amount of binding of each ligand to the solid support surface; (iv) incubating a cell sample-containing fluid with a set of ligands identical to that in step (ii) to permit the ligands to bind to cell surface-bound or intracellularly bound analytes of cells present in the cell sample fluid; (v) contacting the cell sample fluid with a solid support surface according t ⁇ > step (i) to permit ligands that have not bound to cell surface-bound or intracellularly boxind analytes to bind to the solid support surface; and
  • step (vi) determining the amount of binding of each ligand obtained in step (v) and comparing that binding amount with the amount of binding of the same ligand obtained in step (iii), reduced binding in step (v) indicating the presence of ligand-specific cell surface-bound analytes or intracellularly bound analytes in the cell sample.
  • the method comprises the steps of:
  • each binding agent comprises one member of a specific binding pair
  • step (iii) contacting a cell sample-containing fluid with the solid support surface resulting from step (ii) having the ligands bound thereto to permit cells or cell fragments in the sample to bind to ligands on the solid support surface through cell surface-bound or intracellularly bound analytes;
  • the present invention provides an assay kit for cell characterisation, which kit comprises:
  • each binding agent comprises one member of a specific binding pair
  • each ligand comprising a first part capable of specifically binding to a specific analyte selected from cell surface-bound analytes and intracellularly bound analytes of a defined cell type, and a second part which comprises the other member of a respective one of the specific binding pairs, such that each ligand may bind through its specific binding pair part to a specific position on the solid support surface.
  • the present invention provides an assay kit for cell characterisation, which kit comprises: (i) a solid support having on a surface thereof an array of different binding agents immobilized at defined positions on the surface, wherein each binding agent comprises one member of a specific binding pair; and
  • a set of binding elements each of which comprises the other member of a respective one of the specific binding pairs and a reactive group that permits chemical coupling of the binding element to a ligand which is capable of specifically binding to a specific analyte selected from specific cell surface-bound analytes and intracellularly bound analytes of a defined cell type.
  • Figure 1 is a schematic illustration of a solid support surface having an array of ligand binding agents immobilized thereto.
  • Figure 2 is the array surface in Fig. 1 with ligands immobilized thereto.
  • Figure 3 is a schematic illustration of a cell having the same ligands as in Fig. 2 bound to different receptors on the cell surface.
  • Figure 4 is a diagram which shows detector response versus cell concentration at a sensor surface with immobilized anti-mouse IgG antibody for a fluid containing anti- EGF-antibody (at 5 nM concentration) after contact thereof with cells that do not express EGF-receptor (Jurkat cells) and cells that express EGF-receptor (A431 cells).
  • array and “array surface” as used herein are to be interpreted broadly and generally relate to a linear or two-dimensional array of discrete defined regions (here at least two), each having a finite area, formed on a solid support, usually on a continuous surface thereof, and supporting one or more binding agents. Ordered arrays of nucleic acids, proteins, small molecules, cells or other substances on a solid support enable parallel analysis of complex biochemical samples.
  • the density of discrete regions, or spots is typically at least lOO/cm ⁇ , and the discrete regions typically have a diameter in the range of about 10-500 ⁇ m and are separated from other regions in the array by about the same distance.
  • Defined region as used herein relates to a localized area on the solid support surface.
  • the defined region may have any desired shape, such as circular, rectangular, elliptical, etc, and is often referred to as a "spot”.
  • Solid support as used herein is meant to comprise any solid (flexible or rigid) substrate onto which it is desired to apply an array of one or more binding agents.
  • the substrate may be biological, non-biological, organic, inorganic or a combination thereof, and may be in the form of particles, strands, precipitates, gels, sheets, tubings, spheres, containers, capillaries, pads, slices, films, plates, slides, etc, having any convenient shape, including disc, sphere, circle, etc.
  • the substrate surface supporting the array may have any two-dimensional configuration and may include, for example steps, ridges, kinks, terraces and the like and may be the surface of a layer of material different from that of the rest of the substrate.
  • Specific binding pair (abbreviated “sbp”) as used herein describes a pair of molecules (each being a member of a specific binding pair) which are naturally derived or synthetically produced.
  • One of the pair of molecules has a structure (such as an area or cavity) on its surface that specifically binds to (and is therefore defined as complementary with) a particular structure (such as a spatial and polar organisation) of the other molecule, so that the molecules of the pair have the property of binding specifically to each other.
  • types of specific binding pairs are antigen-antibody, antibody-hapten, biotin-avidin, ligand-receptor (e.g.
  • hormone receptor peptide-receptor, enzyme-receptor
  • carbohydrate-protein carbohydrate-lipid
  • lectin-carbohydrate nucleic acid-nucleic acid (such as oligonucleotide-oligonucleotide).
  • Nucleic acid refers to a deoxyribonucleotide polymer (DNA) or ribonucleotide polymer (RNA) in either single- or double-stranded form, and also encompasses synthetically produced analogs that can function in a similar manner as naturally occurring nucleic acids. While natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, nucleotides or bases. These include, for instance, peptide nucleic acids (PNAs) as described in e.g. US-A- 5,948,902 and the references cited therein; pyranosyl nucleic acids (p-NAs) as described in e.g.
  • PNAs peptide nucleic acids
  • p-NAs pyranosyl nucleic acids
  • WO 99/15540 p-RNAs
  • WO 99/15539 p-RNAs
  • WO 00/11011 p- DNAs
  • LNAs locked nucleic acids
  • Oligonucleotide refers to single stranded nucleotide multimers of from about 5 to about 100 nucleotides.
  • Ligand refers to a heterobifunctional molecule that, on one hand, has affinity for a given cell surface-bound or intracellularly bound analyte and, on the other hand, is capable of specifically binding to a moiety immobilized on a solid support.
  • the ligand may be a naturally occurring molecule or one that has been synthesized.
  • Antibody as used herein means an immunoglobulin which may be natural or partly or wholly synthetically produced and also includes active fragments, including Fab antigen-binding fragments, univalent fragments and bivalent fragments. The term also covers any protein having a binding domain which is homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources, or partly or wholly synthetically produced. Exemplary antibodies are the immunoglobulin isotypes and the Fab, Fab', F(ab')2, scFv, Fv, dAb,.and Fd fragments. "Hapten” as used herein means a low molecular species that may give rise to an immune response only when coupled to a larger molecule or cell or by aggregation. After immunisation, however, free haptens may react with antibodies.
  • Cell surface analyte refers to a molecule situated on the external surface of a cell.
  • the cell surface analyte may be an antigen having a specific immune reaction.
  • Cell surface antigens may, for example, consist of carbohydrates, lipids or proteins.
  • Intracellularly bound analyte refers to a molecule situated inside a cell and bound to an intracellular structure in such a way that it can not freely diffuse out of a cell wall that is permeable to specific binding partners.
  • the intracellularly bound analyte may be an antigen having a specific immune reaction.
  • Intracellularly bound analytes may, for example, consist of carbohydrates, lipids or proteins.
  • Cell sample as used herein means any sample of cells either from cell culture or isolated from an organism, an organ, a body liquid or a tissue.
  • a cell sample preparation may be in the form of a suspension or adherent to a surface.
  • the cell sample preparation may contain intact cells, or cells where the cell membrane has been rendered permeable so that specific binding partners to intracellular components can enter and leave the cell interior. This situation can for instance be achieved by fixation and permeabilization methods as described by Hedley, D.W., et al. in Cytometry, (Communications in clinical cytometry) 46:72-78 (2001).
  • the present invention relates to a method of analysing a cell sample for a set of cell surface-bound analytes or intracellularly bound analytes as well as to an array surface that can be used therefor.
  • the invention resides in (i) providing an array of immobilised specific binding agents for a set of different ligands, where each ligand is specific to a respective cell surface-bound or intracellularly bound analyte, and (ii) using the array and the set of ligands in either an inhibition type or a direct type assay format to determine cell surface-bound or intracellularly bound analytes in the cell sample. While the following description is primarily written with respect to cell surface-bound analytes, it is understood that the description is equally applicable to intracellularly bound analytes.
  • An array surface for use in the invention may be constructed by immobilising to defined areas of a solid support surface a number of different ligand recognition elements, which for the sake of illustration are named A, B, C, D..., and each of which is one member of a specific binding pair.
  • a schematic illustration of an array surface with immobilized recognition elements A, B, C, D... is shown in Fig. 1.
  • the corresponding other members of these specific binding pairs here named a, b, c, d..., are each attached (such as conjugated) to a respective one of a set of different ligands LI, L2, L3, L4..., which are directed against specific surface structures or elements (such as e.g.
  • modified ligands that may be present on the surface of a particular cell to form modified ligands aLl, bL2, cL3, dL4...
  • the specific structures or elements on the cell surface are below often referred to as analytes.
  • aLl combines with A on the surface
  • bL2 combines with B, etc, as is schematically illustrated in Fig. 2.
  • the specific binding of a modified ligand to its recognition element on the array surface may be detected by a variety of different methods well known in the art as will be described in more detail below.
  • the array surface is first contacted with the set of modified ligands aLl, bL2, cL3, dL4..., each ligand preferably being at a certain known concentration, and the responses at the different recognition elements A, B, C, D... are measured.
  • the array surface is then regenerated, i.e. the bound ligands are dissociated to expose the recognition elements A, B, C, D...
  • the binding between the modified ligands aLl, bL2, cL3, dL4...and the respective recognition elements A, B, C, D ... should be reversible.
  • ligands are incubated with a desired cell sample, which, for example, may be intact cells, expressing surface-bound analytes for the ligands aLl, bL2 etc. During the incubation, reactions will occur in solution and the ligands will bind to their respective cell surface analytes, as is schematically illustrated in Fig. 3.
  • the sample solution is then brought in contact with the regenerated array surface which, as described above, permits self-orientation of free ligands in the solution. It is, however, not necessary to regenerate the array surface used for the initial binding of the ligands, but a separate identical array surface with recognition elements A, B, C, D... may optionally be used instead.
  • suspended or emulsified matter including cells, membranes etc are removed from the sample solution by, for instance, filtration or centrifugation.
  • the sample solution may be passed over the array surface in a laminar flow that concentrates the suspended or emulsified matter in a central flow core spaced from the surface as described in WO 95/27208 (the disclosure of which is incorporated by reference herein).
  • the different ligands will, depending on the presence and amount of corresponding cell surface analytes, be bound to the respective cell surface analytes, and optionally be removed along with the cells in the separation step.
  • the binding of the ligands aLl, bL2 etc to the corresponding recognition elements A, B etc on the array surface will differ in rate and extent in comparison with the first-mentioned situation where the ligands were contacted with the array surface without preceding exposure to their respective cells surface analytes.
  • repeating the above procedure with different concentrations of the cells or membranes in the cell sample may provide a quantitative measure of cell surface analytes in a cell sample, such as e.g. the number of a particular receptor per cell.
  • the cell sample may be contacted with different concentrations of the ligands.
  • the procedure described above may thus be used to simultaneously determine or monitor a large number of cell surface-bound as well as intracellularly bound analytes and their concentrations.
  • an array may be distributed between two or more flow cells.
  • an array of eight ligands may be provided in, say, four flow cells, i.e. two ligands in each flow cell.
  • a direct type assay format may be used.
  • the array surface shown in Fig. 2 as obtained after contacting the surface with the set of modified ligands aLl, bL2, cL3, dL4..., is brought in contact with the sample, which in this case also may be a cell lysate (in addition to intact or permeable cells) to permit cells or cell fragments to bind to ligands on the array surface through cell surface-bound analytes.
  • the binding of cell surface-bound or intracellularly bound analytes to each ligand on the solid support surface is then determined.
  • Specific binding pairs Aa, Bb, Cc, Dd... that may be used are well known to the skilled person and may, for example, be hybridising strands of nucleic acids, usually oligonucleotides, one of the strands being linked (such as conjugated) to the ligands LI,
  • A, B, C, D... directed against haptens a, b, c, d... that are linked (such as conjugated) to the ligands LI, L2, L3, L4....Such heterobifunctional ligands and the preparation thereof are per se well known to the skilled person and need not be described any further herein.
  • the members of the specific binding pairs preferably bind reversibly to each other, so that the array surface may regenerated in the sense that the surface-bound member is prepared for renewed binding of the other member of the binding pair.
  • Conditions for cleaving or disrupting the binding between the members of each particular specific binding pair are well known to or may readily be established by the skilled person.
  • the strands of an oligonucleotide duplex may be separated at low or high pH conditions.
  • a kit for carrying out the above described assay procedure may comprise a sensor surface, such as a sensor chip, with immobilized recognition elements A, B, C,
  • the kit may comprise a sensor surface, such as a sensor chip, with immobilized recognition elements A, B, C, D..., and a set of corresponding binding elements a,b,c,d...that have been modified with a reactive group (e.g. succinimide ester, maleimide, dipyridyl-disulfide) so that they can readily be incorporated into ligands LI,
  • a reactive group e.g. succinimide ester, maleimide, dipyridyl-disulfide
  • Exemplary cell types that may be of interest for use in the assay include: liver cells, gastrointestinal cells, epithelial cells, endothelial cells, kidney cells, cancer cells, blood cells, stem cells, bone cells, smooth muscle cells, striated muscle cells, cardiac muscle cells, nerve cells.
  • Blood cells include e.g. leukocytes, such as neutrophils, lymphocytes, monocytes, eosinophils, basophils, macrophages.
  • a biosensor is broadly defined as a device that uses a component for molecular recognition (for example a layer with immobilised antibodies) in conjunction with a solid state physicochemical transducer.
  • Biosensors may be based on a variety of detection methods. Typically such methods include, but are not limited to, mass detection methods, such as piezoelectric, optical, thermo-optical and surface acoustic wave (SAW) device methods, and electrochemical methods, such as potentiometric, conductometric, amperometric and capacitance methods.
  • mass detection methods such as piezoelectric, optical, thermo-optical and surface acoustic wave (SAW) device methods
  • electrochemical methods such as potentiometric, conductometric, amperometric and capacitance methods.
  • representative methods include those that detect mass surface concentration, such as reflection-optical methods, including both internal and external reflection methods, angle, wavelength or phase resolved, for example ellipsometry and evanescent wave spectroscopy (EWS), the latter including surface plasmon resonance (SPR) spectroscopy, Brewster angle refractometry, critical angle refractometry, frustrated total reflection (FTR), evanescent wave ellipsometry, scattered total internal reflection (STIR), optical wave guide sensors, evanescent wave-based imaging such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, and the like.
  • reflection-optical methods including both internal and external reflection methods, angle, wavelength or phase resolved
  • EWS evanescent wave spectroscopy
  • SPR surface plasmon resonance
  • Brewster angle refractometry critical angle refractometry
  • FTR frustrated total reflection
  • evanescent wave ellipsometry scattered total internal reflection (STIR)
  • optical wave guide sensors evanescent wave
  • photometric methods based on, for example, evanescent fluorescence (TIRF) and phosphorescence may also be employed, as well as waveguide interferometers.
  • TIRF evanescent fluorescence
  • phosphorescence evanescent fluorescence
  • waveguide interferometers evanescent fluorescence (TIRF) and phosphorescence
  • TIRF evanescent fluorescence
  • phosphorescence phosphorescence
  • waveguide interferometers evanescent fluorescence
  • SPR-based biosensors is sold by Biacore AB (Uppsala,
  • biosensors utilize a SPR based mass-sensing technique to provide a "real-time" binding interaction analysis between a surface bound ligand and an analyte of interest.
  • Example Instrumentation A BIACORE® 3000 instrument (Biacore AB, Uppsala, Sweden) was used. BIACORE® instruments are based on surface plasmon resonance (SPR) detection at gold surfaces, and a micro-fluidic system is used for passing samples and running buffer through four individually detected flow cells (one by one or in series), with very high precision and with small sample volumes needed. As sensor chip was used Series S CM5 (Biacore AB, Uppsala, Sweden) which has a gold-coated surface with a covalently linked carboxymethyl-modified dextran polymer hydrogel. The output from the instrument is a "sensorgram” which is a plot of detector response (measured in
  • resonance units As a function of time.
  • An increase of 1000 RU corresponds to an increase of mass on the sensor surface of approximately 1 ng/mm ⁇ .
  • A431 human epidermoid carcinoma cell line (ATCC #CRL-2592) which overexpresses epidermal growth factor (EGF), and a human T-cell leukaemia Jurkat cell line (ATCC#CRL -10915) which does not express the EGF receptor were each grown in culture media.
  • A431 cells were grown in DMEM/FCS and Jurkat cells in RPMI/Lglu FCS. Jurkat cells were harvested after three days, and A431 cells when they were 90-100% confluent.
  • A431 cells were detached using Versene (EDTA solution) at a 1:5000 dilution.
  • BR-1000-55 immobilized (18400 RU) to detect free antibody in the supernatants, i.e. antibody that had not bound to cell surface EGF.
  • Cell samples were injected for two minutes. Between cell sample injections the sensor surface was regenerated by an injection of 10 mM glycine-HCl at pH 1.2 for 10 seconds.
  • Fig. 4 The results are shown in Fig. 4. As can be seen from the graph, the level of captured antibody when Jurkat cells were used was constant and independent of cell concentration. In contrast, the level of captured antibody was reduced when A431 cells were used and more so at higher cell concentration, reflecting that the antibody was partially bound to EGF-receptor on cell surfaces.

Abstract

La présente invention a trait à un procédé permettant l'analyse d'un échantillon cellulaire pour des analytes liés à la surface ou de liaison intracellulaire par la mise à disposition d'un jeu ordonné d'échantillons d'agents de liaison spécifiques immobilisés pour un ensemble de ligands différents, dans lequel chaque ligand est spécifique à un analyte respectif lié à la surface ou de liaison intracellulaire, et par l'utilisation du jeu ordonné d'échantillons et de l'ensemble de ligands dans un format de bioanalyse de type inhibition ou de type direct en vue de déterminer les analytes liés en surface ou de liaison intracellulaire dans l'échantillon cellulaire. L'invention a également trait à des trousses de bioanalyse pour la caractérisation cellulaire.
EP04725223A 2003-04-04 2004-04-01 Procede et trousse pour bioanalyse d'analytes cellulaires Ceased EP1611443A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US46053903P 2003-04-04 2003-04-04
SE0301058A SE0301058D0 (sv) 2003-04-10 2003-04-10 Method and kit for cell analyte assay
PCT/SE2004/000501 WO2004088318A1 (fr) 2003-04-04 2004-04-01 Procede et trousse pour bioanalyse d'analytes cellulaires

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EP1611443A1 true EP1611443A1 (fr) 2006-01-04

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EP3548891A1 (fr) * 2016-11-29 2019-10-09 The Charles Stark Draper Laboratory, Inc. Biocapteur régénérable et méthodes d'utilisation de ce dernier

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US4659678A (en) * 1982-09-29 1987-04-21 Serono Diagnostics Limited Immunoassay of antigens
FR2634891B1 (fr) * 1988-08-01 1994-05-06 Biotrol Laboratoires Procede et dispositif pour la determination qualitative et/ou quantitative d'un ligand dans un fluide
SE9502024D0 (sv) * 1995-06-02 1995-06-02 Pharmacia Biosensor Ab Pathogen assay method
PT1151300E (pt) * 1998-12-23 2010-06-21 Medsaic Pty Ltd Ensaio para detectar um parceiro de ligação
US20020146745A1 (en) * 2001-04-03 2002-10-10 Surromed, Inc. Methods and reagents for multiplexed analyte capture, surface array self-assembly, and analysis of complex biological samples

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