EP2010907A1 - Procede de detection de molecules biologiques presentes dans des cellules - Google Patents

Procede de detection de molecules biologiques presentes dans des cellules

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Publication number
EP2010907A1
EP2010907A1 EP07728160A EP07728160A EP2010907A1 EP 2010907 A1 EP2010907 A1 EP 2010907A1 EP 07728160 A EP07728160 A EP 07728160A EP 07728160 A EP07728160 A EP 07728160A EP 2010907 A1 EP2010907 A1 EP 2010907A1
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EP
European Patent Office
Prior art keywords
cells
reagent
area
solid support
antigen
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
EP07728160A
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German (de)
English (en)
Inventor
Roberta Carbone
Andrea Zanardi
Pier-Giuseppe Pelicci
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Tethis SpA
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TETHIS Srl
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Filing date
Publication date
Application filed by TETHIS Srl filed Critical TETHIS Srl
Priority to EP07728160A priority Critical patent/EP2010907A1/fr
Publication of EP2010907A1 publication Critical patent/EP2010907A1/fr
Ceased 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells

Definitions

  • the present invention relates to a method for the detection of biological molecules in or on cells and a kit for carrying out the method.
  • the method comprises the steps of
  • the mis-regulation or hyperactivity of intracellular and extracellular signalling cascades may represent the molecular signature of human diseases: the capability of monitoring the complexity of all biochemical networks involved in specific pathologies is essential for the realisation of patient-tailored therapies and for the characterization of drug responses.
  • Kinase activity, post-translational modification, protein overexpression- downreguiation or derealization are only few of the putative mechanisms that can be relevant to human diseases (Megan et al. (2006) Nature Reviews Cancer 6,184- 192; Jonathan et al. (2006) Nature Reviews Cancer 6, 146-155; McLean et al. (2005) Nature Reviews Cancer 5, 505-515; Colombo et al. (2006) Cancer Res. 66 (6),3044- 50).
  • reverse microarrays (Nishizuka et al. (2003) Proc. Natl. Acad. Sci. USA 100 (24), 14229-14234) represent one approach to analyse tissue or cell lysates with a large number of antibodies or patient sera to allow the identification of new biomarkers, the analysis of protein expression profiles and drug candidates' efficacy and toxicity.
  • a multiplexed reverse phase protein microarray (RPP) has been developed by Chan and co-workers (2004, Nature Med., 10 (12), 1390-1396) and applied in the study of different molecular pathways.
  • the reverse microarray technology detects molecules in cell lysates, it does not allow the detection of multiple events in a "cellular" context of compartments and organels, where phenomena need to be analyzed for the comprehension of the biological networks, either in normal or pathological conditions.
  • US Patent Application No. 2003/0189850 A1 discloses a method for carrying out continuous or simultaneous detection of a subject for assay in plural cells by chemiluminescence, bioluminescence or fluorescence comprising a device which comprises a substrate, and an array of spatially-separated plural sections formed on the substrate, each of the sections having an area enough to immobilise plural independent cells.
  • the cells are immobilised in an array of small spots and are, without further proliferation, analysed, e.g. with respect to DNA ploidy, chromosomal aberration, or antibody expression.
  • Stephan et al. (2002, Am. J. Pathol. 161 , 787-797) describe the development of a frozen cell array for performing high-throughput cell-based analysis.
  • the construction of the frozen cell array is carried out by a relatively complicated method involving several steps performed under dry ice conditions.
  • the technical problem underlying the present invention is the provision of an improved system for carrying out multiple analyses of biologically relevant molecules in a cellular context.
  • the present invention provides a method for detecting biological molecules in/on cells comprising, in the following order, the steps of
  • any kind of culture cells can be used. Suitable cells can be obtained from American Type Culture Collection (ATCC), Manassas, VA, USA. Preferred cells are mammalian cells such as osteoblasts, melanocytes, keratinocytes, fibroblasts, haematopoietic cells, epithelial cells, endothelial, neuronal, macrophage, dendritic cells, embryonal/adult stem cells, hepatocytes etc. Especially preferred are cells capable of adherent proliferation.
  • the solid support according to step (a) of the present method may be any support suitable for receiving the cells and allowing proliferation thereof. Therefore, preferred materials of the solid support are biocompatible. If the support material itself is not biocompatible, it is desirable to cover the primary support or at least the area for receiving the cells with a biocompatible material. Preferred embodiments of the solid support are supports made of glass, ceramics, silicon, plastics etc. As mentioned before, the surface of the solid support but at least the at least one distinct area for receiving living adherent cells may be surface treated with suitable agents such as materials favouring the adherence of the cells.
  • the one or more area(s) on the solid support or the complete upper surface of the support is/are coated with a nanostructured TiO 2 film.
  • a nanostructured TiO 2 film Preferred embodiments of such a nanostructured TiO 2 film are disclosed in European Patent Application No. 05 015 869.0 the respective contents of which is herewith enclosed in the present description by reference.
  • a "nanostructured" TiO 2 film means an at least partial coating of the at least one area with nanoparticles of TiO 2 which are preferably deposited by supersonic cluster beam deposition (SCBD) as described in US 6,392,188 wherein the nanoparticles are produced by a pulsed microplasma cluster source (PMCS) and selected by aerodynamic separation effects.
  • SCBD supersonic cluster beam deposition
  • the deposited film is a complex mixture where amorphous material ("matrix”) typically coexists, at the nanoscale, with anatase and rutile crystal phases.
  • the nanocrystalline fraction of such a film is usually characterised by crystal sizes of between about 5 nm (or even less) to about 100 nm, preferably about 10 nm to about 20 nm.
  • the spreading of the crystals in the amorphous matrix leads to a nanoporous structure having a large surface area.
  • Such films show, at the nanoscale, a porosity and granularity comparable to typical extracellular matrix (ECM) structures.
  • ECM extracellular matrix
  • the solid support according to the present invention may have any suitable shape such as squares, triangles, circles and so on. The same holds true for the at least one area for receiving the living adherent cells.
  • the at least one area of the solid support is treated with a suitable reagent promoting the adhesion of the cells.
  • suitable reagents are for example gelatine or solutions thereof, polyornitine, polylysine, matrigel ® , aminosilane, fibronectin, vitronectin, laminin, collagen, vitronectin, biocompatible polymers that favour adhesion (see Anderson, D.G. (2004) Nature Biotechnology 22 (7), 863-866).
  • the at least one area containing the medium is inoculated with a suitable number of cells such as between about 4x10 5 to about 6x10 5 cells, preferably 5x10 5 to about 6x10 5 cells depending on the type of cells, later incubation conditions, growth rate, medium, and surface area to be finally covered.
  • a suitable number of cells such as between about 4x10 5 to about 6x10 5 cells, preferably 5x10 5 to about 6x10 5 cells depending on the type of cells, later incubation conditions, growth rate, medium, and surface area to be finally covered.
  • the solid support containing the inoculated cells is incubated under suitable conditions allowing the proliferation of the cells.
  • the incubation is typically carried out in a suitable growth medium (e.g. DMEM, optionally supplemented with fetal calf serum (FCS), antibiotics, growth factors etc.) and, in particular for mammalian cells, under atmospheric conditions of 5 % CO 2 in a humidified atmosphere.
  • a suitable growth medium e.g. DMEM, optionally supplemented with fetal calf serum (FCS), antibiotics, growth factors etc.
  • FCS fetal calf serum
  • the incubation time is chosen such that the cells on the at least one distinct area reach a suitable percentage of confluence (i.e.
  • percentage of coverage of the surface area in terms of a cell monolayer such as at least about 50%, more preferred at least 60%, even more preferred at least 70% and particularly preferred at least 80% or even 90 % depending on the cell type, conditions of the further method steps (reagents, application techniques for the reagents, detection means etc.). It is, however, as a rule, not desirable to proliferate the cells to such an extent that the at least one distinct area is more than covered with a monolayer of the cells. Suitable growth media, proliferation conditions etc. are described, e.g. in Bonifacino et al. (eds.) Current Protocols in Cell Biology, 1998-2005, John Wiley & Sons, New York.
  • the cells are treated with at least one biochemically active compound or means during step (d), i.e. during the proliferation step.
  • Suitable biochemically active compounds or means are any compounds or means that at least potentially exert an effect on the cells that is measurable by any biochemical, chemical or physical method.
  • the biochemically active compound may be selected from the group consisting of pharmaceutical drugs or candidates thereof, nucleic acids, peptides, proteins, antibodies, chemokins, antibiotics, growth factors, X rays, UV, nanoparticles, vital stainings, free radicals releasing compounds, dendrimers, quantum dot-conjugated molecules.
  • the cells can be treated with suitable factors which at least are potentially capable of changing the biochemical status of the cells before the cells are further processed during the next steps.
  • the cells may be treated in any way that is imaginable in a cell biological/biochemical context.
  • the cells can be treated with pharmaceutical drugs or at least candidates thereof in order to later detect the effect of such drugs or drug candidates on specific cell components or signalling pathways.
  • Other exemplary cell biological treatments are by influencing the expression profile of the cells or by starting or preventing the expression of endogenous or exogenous genes contained in the cells.
  • Exogenous genes are genes present on exogenous DNA or RNA vehicles such as plasmids, viruses and so on.
  • Preferred exogenous gene vehicles are recombinant constructs.
  • Transfection of cells to be analysed by the inventive method may be carried out by any method known by a person skilled in the art (see, e.g., Bonifacino et al., supra).
  • Preferred methods of gene delivery into cells according to the present invention are based on viral infection.
  • viral infection of the proliferated or proliferating cells is improved by the use of a nanostructured T ⁇ O2 film, preferably deposited on the at least one distinct area by cluster supersonic cluster beam deposition (SCBD), e.g. using the apparatus disclosed in US 6,392,188 (see also European Patent Application No. 05 015 869.0).
  • SCBD cluster supersonic cluster beam deposition
  • the method of the present invention preferably comprises the sub-step of contacting the cells with an infectious viral particle.
  • Particularly preferred viruses are retroviruses.
  • a so-called "reverse infection” process which comprises contacting the ns-Ti ⁇ 2 coated area(s) of the support with the virus (usually the virus is present in form of a virus supernatant) such that the virus adheres to the area (preferably, the ns-Ti ⁇ 2 coated area is coated by a suitable adhesion molecule such as streptavidin and the virus is derivatised by the corresponding counterpart of a binding pair such as biotin) whereafter the cells are administered to the area(s) and incubated under appropriate conditions.
  • the virus usually the virus is present in form of a virus supernatant
  • a suitable adhesion molecule such as streptavidin and the virus is derivatised by the corresponding counterpart of a binding pair such as biotin
  • the reverse infection mechanism especially in combination with ns-Ti ⁇ 2 films, provides for very high gene transduction efficiencies and avoids the use of toxic polycations leading to apoptosis and DNA damage.
  • a detailed description of "reverse infection" of cells on ns-Ti ⁇ 2 -coated substrates is disclosed in EP 05 015 869.0 the disclosure content of which is hereby incorporated into the present description by reference.
  • multiple predetermined spatially distinct sites of the at least one area comprising the proliferated cells are reacted with at least one reagent capable of interacting at least temporarily with one or more components of the cells.
  • the term "reacted with” means that the at least one reagent (typically a suitable solution of a molecule capable of interacting at least temporarily with one or more components of the cells) is spotted onto the multiple predetermined spatially distinct sites such that said at least one reagent is restricted to said multiple predetermined spatially distinct sites.
  • the spotting of the reagents according to the present invention is typically carried out by applying a small volume, generally not more than 200 nl, e.g.
  • the volume of the spotted solution correlates with the desired density of the spots.
  • the interaction between the at least one reagent and the component of the cell can be of any type but will usually be sufficient such that the interaction can be detected by suitable means.
  • the interaction may be by hydrogen bonding, an electrostatic interaction, a covalent bonding or through Van der Waals interactions.
  • reagents capable of interacting with a component of the cells are antigen-specific binding molecules such as immunoglobulins or antigen- binding fragments thereof.
  • antigen-specific binding molecules such as immunoglobulins or antigen- binding fragments thereof.
  • Particular preferred reagents are antibodies which may be polyclonal, monoclonal or recombinant.
  • Antibodies for use in the present invention may be directed against any cellular component (examples of typical antigens are given in Tables 1 and 2 herein below).
  • the term “antibody” comprises polyclonal as well as monoclonal antibodies, chimeric antibodies, humanised antibodies.
  • an "antibody” according to the present invention may be a fragment or derivative of the afore-mentioned species. Such antibodies or antibody fragments may also be present as recombinant molecules, e.g. as fusion proteins with other (proteinaceous) components.
  • Antibody fragments are typically produced through enzymatic digestion, protein synthesis or by recombinant technologies known to a person skilled in the art. Therefore, antibodies for use in the present invention may be polyclonal, monoclonal, human or humanised or recombinant antibodies or fragments thereof as well as single chain antibodies, e.g. scFv-constructs, or synthetic antibodies.
  • Polyclonal antibodies are heterogeneous mixtures of antibody molecules being produced from sera of animals which have been immunised with the antigen.
  • Subject of the present invention are also polyclonal monospecific antibodies which are obtained by purification of the antibody mixture (e.g. via chromatography over a column carrying peptides of the specific epitope.
  • a monoclonal antibody represents a homogenous population of antibodies specific for a single epitope of the antigen.
  • Monoclonal antibodies can be prepared according to methods described in the prior art (e.g.
  • RNA is prepared by lysing the cells using guanidinium thiocyanate, acidification with sodium acetate, extraction with phenol, chloroform/isoamyl alcohol, precipitations with mit isopropanol and washing with ethanol.
  • mRNA is typically isolated from the total RNA by chromatography over or batch absorption to oligo-dT-coupled resins (e.g. sepharose).
  • the cDNA is prepared from the mRNA by reverse transcription.
  • cDNA can be inserted into suitable vectors (derived from animals, fungi, bacteria or virus) directly or after genetic manipulation by "site directed mutagenesis" (leading to insertions, inversions, deletions or substitutions of one or more bases pairs) and expressed in a corresponding host organism.
  • suitable vectors and host organisms are well known to the person skilled in the art.
  • Vectors derived from bacteria or yeast such as pBR322, pUC18/19, pACYC184, Lambda oder yeast mu vectors may be mentioned as preferred examples.
  • Such vectors are successfully used for cloning the corresponding genes and their expression in bacteria such as E. coli or yeast such as Saccharomyces cerevisiae.
  • Antibodies for use in the present invention can belong to any one of the following classes of immunoglobulins: IgG, IgM, IgE, IgA, GILD and, where applicable, a subclass of the afore-mentioned classes, e.g. the sub-classes of the IgG class.
  • IgG and ist sub-classes such as IgGI , lgG2, lgG2a, lgG2b, lgG3 or IgGM, are preferred.
  • IgG subtypes lgG1/k or lgG2b/k are especially preferred.
  • a hybridoma clone which produces monoclonal antibodies for use in the present invention can be cultured in vitro, in situ oder in vivo. High titers of monoclonal antibodies are preferably produced in vivo or in situ.
  • Chimeric antibodies are species containing components of different origin (e.g. antibodies containing a variable region derived from a murine monoclonal antibody, and a constant region derived from a human immunoglobulin). Chimeric antibodies can be employed in order to improve the production yield. For example, in comparison to hybridoma cell lines, murine monoclonal antibodies give higher yields.. A further example is a monoclonal antibody in which the hypervariable complementarity defining regions (CDR) of a murine monoclonal antibody are combined with the further antibody regions of a human antibody. Such an antibody is called a humanised antibody. Chimeric antibodies and methods for their production are described in the prior art (see e.g. EP-A- 125023 and Harlow und Lane, Antibodies: A Laboratory Manual, supra). The disclosure content of the cited documents is incorporated in the present description by reference.
  • CDR hypervariable complementarity defining regions
  • the term ..antibody comprises complete antibody molecules as well as fragments thereof being capable of binding to a cellular component.
  • Antibody fragments comprise any deleted or derivatised antibody moieties having one or two binding site(s) for the antigen.
  • Specific examples of such antibody framents are Fv, Fab or F(ab') 2 fragments or single strand fragments such as scFv. Double stranded fragments such as Fv, Fab or F(ab')2 are preferred.
  • Fab und F(ab') 2 fragments have no Fc fragment contained in intact antibodies.
  • Such fragments may be produced from intact antibodies by proteolytic digestion using proteases such as papain (for the production of Fab fragments) or pepsin (for the production of F(ab')2 fragments), or chemical oxidation.
  • antibody fragments or antibody constructs are produced through genetic manipulation of the corresponding antibody genes.
  • Recombinant antibody constructs usually comprise single-chain Fv molecules (scFvs, -3OkDa in size), in which the V H and V L domains are tethered together via a polypeptide linker to improve expression and folding efficiency.
  • scFvs single-chain Fv molecules
  • the monomeric scFv fragments can be complexed into dimers, trimers or larger aggregates using adhesive protein domains or peptide linkers.
  • An example of such a construct of a bivalent scFv dimer is a 60 kDa diabody in which a short, e.g.
  • linker between V H - and VL-domains of each scFv prevents alignment of V-domains into a single Fv module and instead results in association of two scFv molecules.
  • Diabodies have two functional antigen-binding sites. The linkers can also be reduced to less than three residues, which prevents the formation of a diabody and instead directs three scFv molecules to associate into a trimer (90 kDa triabody) with three functional antigen-binding sites. Association of four scFvs into a tetravalent tetrabody is also possible.
  • Further antibody constructs for use in the present invention are dimers of scFv-CH3 fusion proteins (80 kDa; so-called "minibodies"
  • the method of the present invention is particularly useful for testing antibodies or antigen-binding fragments thereof, e.g. with respect to their antigen specificity in the cellular context.
  • reagents it is possible to apply more than one reagent to the multiple predetermined spatially distinct (separated) sites in order to analyse more than one specific component of the cells at the same time.
  • more than one antibody each specific for a particular cell component may be spotted onto the predetermined sites, whereafter the binding of these reagents can be detected, e.g. by indirect detection using differently labelled secondary antibodies (e.g. green, blue, red, far red such as appropriately chosen fluorescent markers).
  • reagents typically used for detecting cellular components such as organels, cytoskeletal molecules etc. are dyes, in particular fluorescent dyes, specifically binding or localising to a compartment or even single molecules. Suitable dyes for such purposes are commercially available and may be obtained from Invitrogen Moleculare Probes, Invitrogen Corp., Carlsbad, CA, USA. Furthermore, fluorescent ligands of cellular components (such as receptors, enzymes etc.) and quantum dot- conjugated reagents can be used as well.
  • the reagent capable of interacting with a component of the cell may be selected from nucleic acids for suitable hybridisation techniques. Therefore, the method according to the present invention can take the form of an in situ hybridisation protocol.
  • the method according to the present invention provides a highly flexible system in that not only the multiple spatially distinct sites can be reacted with different reagents capable of binding to cellular components. Also, it is possible to contact the cells on the multiple spatially distinct sites with different amounts of the reagents. Furthermore, it is of course possible to form sub-groups of the multiple spatially distinct sites which may be reacted with different reagents or different amount of the reagent(s).
  • the cells are fixed and/or permeabilised between steps (d) and (e) of the inventive method.
  • Reagents for fixation such as paraformaldehyde or glutaraldehyde solutions or acetone/methanol or methanol
  • permeabilisation such as Tween ® or Triton-X 100, saponin
  • DPBS Dulbecco's Phosphate Buffered Saline
  • step (f) of the protocol according to the present invention comprises a direct or indirect detection of whether or not the at least one reagent capable of binding to, i.e. interacting with, a component of the cell has factually reacted with this component.
  • Direct detection means that the reagent itself is typically labelled with a detectable marker which may be a radioactive label or dye, in particular a luminescent dye, more preferably a fluorophor.
  • a detectable marker which may be a radioactive label or dye, in particular a luminescent dye, more preferably a fluorophor.
  • the reagent is a proteinaceous molecule such as an antibody or at least an antigen-specific binding molecule or an antigen-binding fragment of such a molecule
  • the detection of whether or not the reagent has factually interacted or in other words reacted with a cellular component generally comprises the use of a (secondary) antibody directed against the reagent such as the antibody or fragment thereof.
  • step (f) comprises the sub-step of reacting one or more of the multiple spatially distinct sites with a molecule capable of interacting with the reagent(s).
  • the (second) molecule such as a secondary antibody is typically labelled with a detectable marker which may take the forms as described above.
  • a detectable marker which may take the forms as described above.
  • fluorescent dyes are preferred for such purposes. Suitable fluorescent dyes and other components of detectable markers may be obtained from Invitrogen Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA.
  • detection step (f) it is finally necessary to detect the signal arising from the interaction of the reagent with the cellular component and/or the interaction of the secondary detection molecule with the initial reagent.
  • Appropriate detecting means depend on the signal generated by the interaction or marker. In the case of fluorescent dyes fluorescence microscopes or spectrophotometers are suited for this purpose.
  • the method of the present invention typically comprises one or more washing steps between steps (d) and (e) and/or (e) and (f). Therefore, it is generally preferred to decrease the background of the final signal by washing away unbound (that is non-interacting) reagents (the first capable of interacting with the cellular component as well as optionally the secondary molecule in the case of indirect detection techniques) by using large volumes of suitable buffer solutions (e.g. PBS, DPBS, TBS, TBST etc.). Further washing steps are typically performed after fixation and/or permeabilisation steps.
  • suitable buffer solutions e.g. PBS, DPBS, TBS, TBST etc.
  • the surface of the at least one area is treated with a blocking solution before performing step (e) in order to block non-specific binding sites for the reagent(s) capable of interacting with a cellular component as well as of the secondary molecules in the case of indirect detection techniques.
  • Suitable blocking solutions are phosphate or Tris buffered solutions of BSA or milk powder.
  • the method of the present invention comprises a further step (g) of detecting the localisation of the reagent within/on the cells.
  • microscope equipment is a suitable means for detecting the localisation of cellular components interacting with the reagents.
  • automated fluorescence microscopes optionally equipped with a CCD camera, and other well known high resolution techniques (confocal microscopes).
  • Step (e) of the present invention means that the at least one reagent capable of interacting with a cellular component is applied to the multiple predetermined spatially distinct sites of the at least one area comprising the proliferated cells in the form of multiple spots.
  • state of the art arraying techniques are used for the application of the reagent on a multitude of spatially distinct sites, e.g. by contact and/or non-contact spotting.
  • Corresponding arrayers and accessory equipment are commercially available, e.g. from BioDot, Inc., Irvine, CA, USA.
  • spatially distinct means that the predetermined multiple sides are spatially separated from one another. Preferred are regular arrangements, i.e. arrays of e.g. 8 x 8, 8 x 16 etc. spots, more preferred high density arrays wherein the multiple spatially distinct sides are separated by not more than about 2 mm, preferably not more than about 1 mm.
  • Separatated means that the centres of the multiple spatially distinct sites have a certain distance in a cartesian (x-y) system (also called “pitch”) such that the spatially distinct sites do not overlap. Depending on the spotting technique it is possible to achieve much lower distances between the spots, e.g. about 500 ⁇ m or even less.
  • step (e) is carried out by spotting, preferably non-contact spotting using suitable and commercially available equipment. This technique is also preferably used in step (f) in the case of the application of suitable molecules for indirect detection (e.g. secondary antibodies) of the reagent capable of interacting with a cellular component.
  • suitable molecules for indirect detection e.g. secondary antibodies
  • the solid support for carrying out the method of the present invention may be composed of different materials.
  • glass slides typically have dimensions such that they are compatible with usual high-throughput equipment for microarray purposes (typical surface dimensions of about 25 mm x 76 mm).
  • microarray glass slides are commercially available, e.g. from TeleChem International, Inc., Arraylt ® Division, Sunnyvale, CA, USA.
  • a solid support can comprise more than one distinct area for receiving living adherent cells.
  • the upper limit of the number of distinct areas is not critical for performing the invention and depends on the surface area of the solid support.
  • a further parameter determining the upper limit of distinct areas for receiving living adherent cells are the type of the cells.
  • the method of the present invention can be performed according to a multiplexed microarray format.
  • the present invention provides a kit for performing the above-defined method, wherein the kit comprises a solid support having at least one distinct area for receiving living cells; a reagent providing a medium for the proliferation of cells; at least one strain of culture cells; - at least one reagent capable of binding to a biological molecule within and/or on the cells, which reagent is to be applied onto multiple predetermined spatially distinct sites of the at least one area; means for the detection of the at least one reagent; and an instruction manual containing information for carrying out the method as defined above.
  • Fig. 1 shows immunocell-arrays of 8x8 immunostained spots of 100-150 cells each.
  • Primary fibroblasts were fixed and permeabilized and stained in alternate spots with a monoclonal antibody against PML and BSA 1 as control for specific staining in localised spots.
  • Panel A image of a 2x2 array (enlarged magnification of the original image) where bright white staining indicates detection of PML, dark gray indicates detection of nuclei, not stained with the antibody.
  • Panel B image of an array of 8X8 immunostained spots of 100-150 cells each showing only PML staining in bright white;
  • panel C image enlargement of an area of panel B to exclude possible cross-contaminations among adjacent spots; inset: magnification of two cells of one spot. In panel B images were acquired at 2Ox magnification.
  • Panel A shows an image of an 8x17 immunocell-array of NIH3T3 cells stained with 17 different antibodies; in panel B magnification of small areas of different spots are shown to evaluate specific protein localisation in detail (E3B1 , clathrin, vinculin, p-tyrosine, phalloidin, profilin, Wip, Vasp, AP1 -alpha, Eps8,
  • Panel C shows an image of an 8X16 immunocell-array of adult primary melanocytes with 15 different antibodies (BSA was spotted as control in the last lane); in panel D magnification of small areas of different spots are shown to evaluate specific protein localisation in detail (clathrin, tubulin, NPM, phalloidin, Eps8, Eps15). In panels A and C images were acquired at 2Ox magnification.
  • FIG. 3 shows images of an 8x24 immunocell-array of U2OS cells treated with bleomycin taken at different time points (control, 8h and 24h).
  • Panel A example of the 8x24 array.
  • Panel B small areas of different spots corresponding to the immunostained proteins in control and 24 h bleomycin treated slides. Differences in staining intensity or specific localisation of proteins can be observed.
  • Fig. 4 shows diagrams of statistical analyses of bleomycin response after image acquisition.
  • Graph 1 shows an analysis of the mean fluorescence intensity of acetylases/deacetylases at 0-8-24h after treatment.
  • Graph 2 shows an analysis of the mean fluorescence intensity of the other series of antigens involved in DNA damage response.
  • the present invention is further illustrated by the following non-limiting example.
  • Arraylt ® SuperClean2 glass slides are put into Vivascience ® Vivadish plates, microbiological grade.
  • the glass slides are covered with a sterile solution of 0,2% Sigma ® type B gelatin from bovine skin in IxDPBS and incubated at room temperature for 30 min. After incubation the slides are washed once with IxDPBS. Then, growing cells (typically about 80% confluent) are collected from their growth dish and typically 4x10 5 to 6x10 5 cells are plated per slide in a volume of 4 ml of growth medium.
  • a Biodot ® BioJet Plus spotter with Axsys software aspirating antibodies from a 96 well plate and dispensing drops on a slide holder, was used. Usually, lines of 10 drops are spotted for each antibody with a pitch of 2 mm. For each drop 30 nl are generally dispensed. Usually, the humidifier is switched on 10' before spotting in order to reach constant and uniform 65% humidity in the spotting chamber. After drying, the slides are rapidly placed in the spotting chamber and left there for rehydration (in about 5 min slides lose their opacity which is indicative that the cells are not dry anymore).
  • Fig. 1 it was possible to specifically identify, by analysis using automated time-lapse microscopy, PML stained spots with no contamination of staining in BSA spots (see panel C at 10X and panel D at 2OX magnification).
  • the inset on panel D shows the PML localisation in nuclear bodies.
  • NIH3T3 cells represent a suitable model for cytoskeleton-associated signalling pathways. Therefore, an 8x17 immunocell-array with 12 different cytoskeleton related antibodies (see Table 1) was performed. As shown in Fig. 2 panel B specific staining is obtained.
  • Bleomycin treated U2OS cells show deacetylation of chromatin and modulation of acetylase-deacetylase enzymes.
  • U2OS cells were treated for 8 and 24 hours, processed for staining with antibodies against 21 different antigens (see Table 2) and analysed by time lapse microscopy. After image analysis a series of graphs representing the response in terms of Cy3 intensity/DAPI staining of each antibody were obtained (see Table 3 and Fig. 4).
  • the present invention provides a novel cell array-based method which can be conveniently used for immunofluorescence analysis of target proteins in different cellular models.
  • the present technology provides many advantages over existing immunofluorescence methods: it decreases the cost of the assay in terms of antibodies and cells, provides a flexible high content approach "on chip" for multiple targets and different kinetics, data can be analyzed through high resolution confocal microscopy, and finally the present method ensures a high degree of reproducibility and no well to well variability.
  • This technology can be used inter alia for the molecular dissection of signalling pathways (kinase response, post-translational modifications, protein localization and protein profiling) upon drug response in normal and disease states, for the characterization of "cancer" signatures (protein overexpression, mis-localization) on patient cells samples and, ultimately, for the screening of monoclonal and polyclonal antibodies for immunofluorescence applications.
  • signalling pathways kinase response, post-translational modifications, protein localization and protein profiling
  • cancer protein overexpression, mis-localization
  • a pathway of deacetylation in mammalian cells upon DNA damage could be identified, which probably involves specific chromatin modifying enzymes.
  • Detailed biochemical analysis at sites of DNA double strand breaks will help in elucidating specific molecular changes in chromatin state.
  • the present technology could accelerate the drug development process and offer new tools to patient monitoring during disease therapy.
  • the present invention there is provided a methodology based on the investigation of living cells "on chip" where, typically upon fixation, it is possible to detect simultaneously, e.g. by using specific antibodies, the localisation and state of a multitude such as hundreds or more of cell components, in particular proteins, involved in different biochemical (e.g. signalling, metabolic etc.) pathways. Furthermore, at the same time, reagents and cell consumption are minimised and high quality detection signals, e.g. high resolution images by using corresponding microscopic equipment, are routinely obtainable. As an example, by using the method of the present invention a previously unidentified signalling pathway of deacetylation of chromatin upon DNA damage in mammalian cells was identified. The present invention thus permits the detailed analysis of dynamical events such as protein profiling, protein post-translational modification, protein migration and transportation inside cells, even at the single molecule level.
  • biochemical e.g. signalling, metabolic etc.

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Abstract

La présente invention concerne un procédé de détection de molécules biologiques présentes dans ou sur des cellules et un kit permettant de mettre en œuvre le procédé. Ledit procédé comprend les étapes consistant à : (a) disposer d'un support solide pourvu au moins d'une zone distincte en vue de recevoir les cellules vivantes ; (b) traiter au moins la zone du support solide avec un réactif stimulant l'adhésion des cellules ; (c) inoculer la zone dotée de cellules vivantes ; (d) incuber le support solide dans des conditions permettant la prolifération des cellules ; (e) localiser au moins un réactif capable d'interagir avec un composant des cellules sur de multiples sites prédéterminés spatialement distincts de la zone comprenant les cellules ayant proliférées ; et (f) détecter si le réactif a réagi ou non avec un composant des cellules.
EP07728160A 2006-04-20 2007-04-16 Procede de detection de molecules biologiques presentes dans des cellules Ceased EP2010907A1 (fr)

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EP07728160A EP2010907A1 (fr) 2006-04-20 2007-04-16 Procede de detection de molecules biologiques presentes dans des cellules

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EP06112860 2006-04-20
PCT/EP2007/053695 WO2007122133A1 (fr) 2006-04-20 2007-04-16 Procede de detection de molecules biologiques presentes dans des cellules
EP07728160A EP2010907A1 (fr) 2006-04-20 2007-04-16 Procede de detection de molecules biologiques presentes dans des cellules

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US20070111201A1 (en) * 2001-04-30 2007-05-17 Benjamin Doranz Reverse transfection of cell arrays for structural and functional analyses of proteins
WO2002088165A1 (fr) * 2001-04-30 2002-11-07 Doranz Benjamin J Elucidation de la fonction de genes
US20030032203A1 (en) * 2001-07-10 2003-02-13 Sabatini David M. Small molecule microarrays

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