WO2004019039A2 - Procede et kit permettant de realiser des essais fonctionnels sur des cellules biologiques qui ont ete immobilisees sur un reseau par des molecules « eboueurs » - Google Patents

Procede et kit permettant de realiser des essais fonctionnels sur des cellules biologiques qui ont ete immobilisees sur un reseau par des molecules « eboueurs » Download PDF

Info

Publication number
WO2004019039A2
WO2004019039A2 PCT/EP2003/007440 EP0307440W WO2004019039A2 WO 2004019039 A2 WO2004019039 A2 WO 2004019039A2 EP 0307440 W EP0307440 W EP 0307440W WO 2004019039 A2 WO2004019039 A2 WO 2004019039A2
Authority
WO
WIPO (PCT)
Prior art keywords
test cells
array
cells
capture molecules
test
Prior art date
Application number
PCT/EP2003/007440
Other languages
German (de)
English (en)
Other versions
WO2004019039A3 (fr
Inventor
Hugo Hämmerle
Thomas Joos
Markus Templin
Hansjürgen VOLKMER
Kerstin Ragnitz
Susanne Stumpf
Cornelia Kuschel
Original Assignee
NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen
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 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen filed Critical NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen
Priority to CA002494558A priority Critical patent/CA2494558A1/fr
Priority to EP03792191A priority patent/EP1527344A2/fr
Priority to AU2003246668A priority patent/AU2003246668A1/en
Publication of WO2004019039A2 publication Critical patent/WO2004019039A2/fr
Publication of WO2004019039A3 publication Critical patent/WO2004019039A3/fr
Priority to US11/051,315 priority patent/US20050214832A1/en

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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or 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 and a kit for carrying out functional tests on biological cells, in which an array of measuring points is used, with at least one capture molecule or binding partner for the biological cells to be tested being immobilized at each measuring point.
  • functional test means, for example, but not exclusively or in a restrictive manner, such experiments, tests or measurements in which certain properties or features of the cells or the change in these properties or features as a function of a treatment the cells and / or the type of capture molecules and / or the addition of substances are recorded or evaluated.
  • the treatment of the cells is e.g. radiation with high-energy radiation, such as is used in radiotherapy.
  • the addition of substances includes e.g. in the context of pharmaceutical screening, the administration of pharmaceutical preparations whose effect on the cells is examined, for example, in the course of a dose-finding study, or the addition of antibodies which are screened against cell surface receptors.
  • the choice of the type of capture molecules affects e.g. Components of extracellular matrix molecules for simulating the natural microenvironment of the cells in order to test their reaction in vitro to radiation, stimulation by ligands and / or added pharmaceuticals under conditions of the natural microenvironment.
  • biological cells to be tested include, for example, but not exclusively or in a restrictive manner, primary animal, in particular human cells, plant or bacterial cells, cell lines, genetically modified cells, cells made of biopsy material, healthy un-degenerate cells, in particular tumor cells , Cells from peripheral blood etc. understood. These cells are referred to below as test cells.
  • the "properties and characteristics" of the test cells include, but are not limited to, their proliferation ability, their viability, the pattern of their cell surface molecules, their ability to exchange signals or to interact with other cells, a possible pathological condition, a genetic degeneration, their genetic profile, their expression profile, their ability to bind to certain substances.
  • Carrier plates with arrays which are suitable for carrying out such methods and examples of such functional tests can be found, for example, in WO 02/02226 by the applicant or WO 00/39580.
  • the carrier plates are usually Glass or plastic platelets which have a functionalized, for example aldehyde-activated surface, on which capture molecules or binding partners for the test cells are immobilized at mutually separated measuring points. The surface between the measuring points is blocked to prevent non-specific binding of test cells or other substances.
  • the measuring points have a diameter of 200 to 800 ⁇ m and a center distance of, for example, 500 ⁇ m, so that 100 measuring points can be accommodated on an area of 1 x 1 cm.
  • different capture molecules are immobilized on the measuring points.
  • a solution with test cells is then placed on the carrier plate, which is then incubated for a certain period of time before the test cells that are not immobilized on capture molecules are washed off again.
  • the bound test cells are then recorded optically in a spatially resolved manner in order to determine to which capture molecules the test cells have bound.
  • the optical detection can take place, for example, using bright field microscopy or fluorescence measurements, but other measurement principles can also be used, as described, for example, in WO 02/02226 or WO 00/39580.
  • This publication describes a method in which more than 50 CD antigens are detected on leukocytes.
  • an array of different, at different Measuring points immobilized antibodies against the respective CD antigens are used, to which a suspension of test cells is placed, the test cells only binding to those measuring points at which antibodies are immobilized, for which they express the corresponding CD antigens.
  • the bound test cells are recorded optically with spatial resolution.
  • the resulting pattern of measuring points occupied with test cells represents the patient's phenotype from which the test cells originate.
  • the antibody array covers a total of 60 measuring points on an area of 0.72 cm x 0.4 cm, to which 5nl antibody solution was added.
  • the diameter of a measuring point is approx. 400 ⁇ m.
  • 100 ⁇ l of a cell suspension with a concentration of 10 7 test cells / ml were added to the array. The authors report that around 600 test cells were bound at this concentration per measuring point, while around 100 test cells are bound at 10 ⁇ test cells / ml.
  • the present invention has for its object to provide a method of the type mentioned, in which the reliability and reproducibility between the measurement results of measurement points in an array and in different arrays is so great that a reliable comparison of the measurement results and the creation a reliable gradation of the measurement results is possible.
  • this object is achieved on the one hand by a method for carrying out functional tests on test cells, with the steps:
  • step b) contacting the mixture from step b) with the array so that test cells and reference particles can bind to each measuring point
  • the inventors of the present application have recognized that the reproducibility and reliability of the measurement results depend on the varying size of the measurement points and the lack of homogeneity of the immobilized capture molecules, and that the use of the reference particles makes it possible to influence the different sizes of the surfaces of the Calculate measuring points and other inhomogeneities, for example in the local concentration of the capture molecules, from the measurement results.
  • the inventors are therefore not taking the path that is actually available on the basis of the inventors' findings to minimize the variability by means of more complex production processes which lead to more uniform areas of the measuring points and a uniform local concentration of the capture molecules, but instead use reference particles.
  • the number of test cells bound per measuring point and thus the respective measuring signal in the known methods according to the knowledge of the inventors of the present application depends not only on the binding properties between the test cells and the capture molecules but also on the density of the capture molecules per measurement point the size of the area of the measuring points and the homogeneity of the concentration of the capture molecules.
  • the quotient of the measurement signal for the test cells and the measurement signal for the reference particles at a specific measurement point can be taken as a measure for the binding of the test cells to the capture molecules of this measurement point.
  • the measurement signal for the reference particles is, so to speak, a measure of the number of capture molecules in a measurement point.
  • bound test cells of interest are understood to mean test cells that have deposited from the suspension on measuring points and adhere there.
  • the ratio of bound test cells to bound reference particles then serves to compare the adhesion behavior of the test cells to different capture molecules to be able to.
  • test cells of interest are, for example, all bonded test cells in the context of adhesion tests, the apoptosis rate, viability, the ability to bind to antibodies, exchange signals, etc. in the context of other functional tests.
  • Artificial spheres for example latex spheres, can be used as reference particles, which carry surface molecules that enable a binding to the catcher molecules that is comparable to that of the surface cells of the test cells. These balls are inexpensive and easy to manufacture and can be stored for a long time; they are known from other applications in the prior art and have a size that can correspond to that of the test cells.
  • Another advantage of the Ku geln is that their binding behavior to the capture molecules is not influenced by subsequently added substances or, for example, radioactive or UV radiation, so that after mixing and, if necessary, immobilizing test cells and spheres, the influence of this measure on the test cells and, for example, theirs Binding or apoptosis rate can be tracked without affecting the binding of the balls, so that the reference is retained.
  • biological reference cells as reference particles which can be distinguished from the test cells in terms of measurement technology, preferably optically, but which bind to capture molecules like the test cells.
  • the reference cells can be untreated test cells, which can be distinguished by measurement technology from the test cells to be examined and treated before mixing.
  • test cells and the reference particles are preferably mixed with one another in a ratio of 1: 1, so that they are the same. Probability to hit the catcher molecules.
  • the test cells can be distinguished from the reference particles by measurement technology, preferably optically, in that the test cells are labeled with a marker other than the reference particles, preferably a fluorescent marker or a genetic marker. It is advantageous here that the array can be read out with two different excitation waves and / or emission filters, with successively or simultaneously spatially resolved optical signals being recorded, from which the ratio of the test cell of interest to the reference particle can then be calculated.
  • test cells can also be labeled instead of or different from the test cells.
  • a genetic marker such as a reporter gene such as GFP (green fluorescent protein).
  • GFP green fluorescent protein
  • test cells can be distinguished from the reference particles by measurement technology in that the test cells are provided with a different radioactive marker than the reference particles.
  • radioactive and optical markings together, that is to say test cells and reference cells, to radioactively and optically mark, or to use them in a mixed manner, for example to optically mark the test cells and the reference particles radioactively.
  • the reference cells are preferably genetically different from the test cells in such a way that they do not, or are known to react differently to substances and / or radiation whose effect on the test cells is to be investigated.
  • the advantage here is that after the test and reference cells have been mixed, this mixture can be treated in the manner indicated without impairing the binding to the capture molecules or other properties of the reference cells examined in the course of the functional tests.
  • radiation can, for example, lead to a certain percentage of the test cells being killed or its binding properties to the catcher molecules being changed such that it binds poorly or better to the catcher molecules, the binding of the reference cells to the catcher molecules remains unchanged and is therefore a measure of the number of capture molecules per measuring point.
  • test cells and reference cells are also possible to divide a mixture of test cells and reference cells into two arrays and to irradiate only one array after or during the incubation or to bring it into contact with a test substance.
  • the untreated array then serves as a reference for the effect on test cells and on reference cells.
  • Two or more types of reference particles can also be mixed with the test cells, the different types of reference particles being distinguishable from one another and from the test cells, for example by three different "colors". It is thus possible to use so-called “beads” as the first type of reference particles, which are not influenced by the treatment and serve as a reference between different arrays, and as second type of reference particles to use reference cells that are used to calculate the variation within an array.
  • a reference measuring point can be provided on each array, to which only reference particles bind. This can be used as a reference between different arrays, the reference measuring point being isolated from the other measuring points, so that only reference particles are applied to it.
  • the object on which the invention is based is achieved by a method for carrying out functional tests on test cells, with the steps:
  • the inventors of the present application have recognized that the uneven deposition of test cells on the measuring points, as can be seen from the publication by Belov et al., Loc. Cit., Is not solely due to the inhomogeneity of the measuring points, but also due to the fact that the Test cells in the suspension are not distributed homogeneously, but preferentially deposit at certain points on the array. However, this means that the local number of test cells on the array is not the same everywhere. In other words, for example, despite a strong bond between test cells and capture molecules, a weaker measurement signal can result for a specific measuring point than for a measuring point at which the binding is weaker because the local concentration of the test cells is lower than at the first measuring point the second measurement point.
  • the distribution of a (logical) measurement point over a number of measurement areas at different locations in the array averages the statistical probability of adhesion for different measurement points, that is to say averaged over the assigned measurement areas. If the individual measuring surfaces become so small that, in the statistical sense, it is no longer possible for a sufficient number of test cells to bind in order to generate a reliable measurement signal, the reference particles and measures discussed in detail above can also be used here, which leads to a synergistic effect.
  • a specific ratio between the area (F) of a measuring point and the number (N) of test cells in the suspension is required, which depends on the adhesive area (H) of the respective cells and is selected as follows:
  • either the area F of a measurement point is chosen larger or several measurement areas are combined to form a logical measurement point with a total area F. Under these conditions, almost all test cells can bind from the overhang without interfering with each other.
  • This choice of the ratio between the number and type of test cells and the size of the surface of a measuring point, which determines the adhesive surface is in itself new and inventive and, together with one or both of the above-mentioned measures, namely the reference particles and / or the distributed measuring surfaces, leads to one synergistic effect.
  • the above ratio should be applied to the sum of the test cells and reference particles present in the suspension as follows:
  • NT and HT denote the number and the adhesive surface of the test cells and NR and HR the number and adhesive surface of the reference particles and a is a factor of 0.5, preferably approximately 1.0.
  • the amount of test cells and possibly reference particles in the suspension / mixture to be applied to the array must be selected in such a way, according to the knowledge of the inventors of the present application, that the above ratio is maintained in order to To achieve a situation in which almost all test cells / reference particles can bind to a measuring point, so that there is a competition between the measuring points around the cells.
  • This ratio is advantageous, for example, if a few cells are available, as in tumor biopsies, or if the "homing" of stem cells is to be investigated.
  • a preference of the test cells for certain catcher molecules can only be determined quantitatively in the low numbers of test cells used according to the invention. This also applies if a subpopulation is to be examined separately in mixed cell populations.
  • the inventors of the present application were able to show that with larger amounts of test cells they also bind to substrates for which they have no proven preference.
  • the array is moved after contacting with the suspension / mixture, i.e. during the incubation with the test cells and possibly the reference particles, for example on a vibrator or a cradle or by means of a pump, for example via microfluidic flow to reduce the local concentration differences in the test cells and possibly reference particles.
  • new test cells or reference particles are also repeatedly brought to the measurement points, so that a larger number of them have the chance to bind to capture molecules.
  • shaking surprisingly increases the number of bound test cells and possibly reference particles, as the inventors of the present application were able to show.
  • this measure is also new and inventive in itself, but is preferably used together with one or more of the above-mentioned measures.
  • the array is either applied to a carrier plate, as is the case in WO 00/39580 and WO 02/02226 mentioned at the outset, or that it is a logical array of individual balls which are loaded with capture molecules and in the usual way, for example can be distinguished from one another by color markings.
  • a measuring point then corresponds to either one sphere or several spheres, each of which represents a measuring surface in the above sense.
  • balls are used as an array, in the simplest case they are added to the solution / mixture and incubated, for example on a shaker, with gentle agitation.
  • test cells are subjected to a treatment before or after contacting the array, which treatment is selected from the group: irradiation with high-energy radiation, for example UV light or radioactive radiation, contact with test substances such as pharmaceutical active ingredients, etc.
  • high-energy radiation for example UV light or radioactive radiation
  • test substances such as pharmaceutical active ingredients, etc.
  • Different capture molecules are immobilized at the measuring points, which are preferably selected from the group: protein such as, for example, components of extracellular matrix proteins, receptors.
  • Ligands poly-lysine, peptides from laminin sequences, Control peptides, peptidomimetics, antibodies, lectins, antigens, allergens.
  • the invention further relates to a kit with an array of mutually separate measuring points, at which capture molecules are immobilized, to which test cells can bind, and with reference particles, which bind to the capture molecules.
  • the reference particles are preferably the reference particles described in more detail above, with further capture molecules preferably being immobilized at the measuring points, which are preferably selected from the group: protein such as, for example, components of extracellular matrix proteins, receptors.
  • protein such as, for example, components of extracellular matrix proteins, receptors.
  • the array is either applied to a carrier plate or it is a logical array of individual balls that are loaded with catcher molecules, further preferably at least one measuring point with its associated catcher molecules being divided over several measuring surfaces in the array, which are located at different locations in the array are arranged.
  • FIG. 1 shows a schematic example of an array of measuring points arranged on a carrier plate, the measuring points being distributed over different measuring surfaces;
  • FIG. 2 shows a schematic side view of the carrier plate from FIG. 1;
  • Fig. 5 is a diagram showing the binding of test cells to different capture molecules depending on the shows the number of cells used in the applied suspension.
  • Fig. 6 is a diagram showing the settlement of a measurement area depending on the number of cells used in the applied suspension with and without movement.
  • Example I Carrier plate with an array of measuring points
  • 10 denotes a rectangular support plate made of glass or plastic, on which some measuring surfaces 11 are arranged in an array, to which biological cells, not shown in FIG. 1 - hereinafter: test cells - or reference particles can bind. Areas 12 of the carrier plate 10 are provided between the measuring surfaces 11, to which test cells and reference particles cannot bind.
  • the measuring surfaces have a diameter of 500 ⁇ m and an edge distance of 250 ⁇ m, so that there is a center distance of 750 ⁇ m. In this way, 96 measuring surfaces 11 can be accommodated on a carrier plate 10 with an edge length of 6 mm ⁇ 9 mm.
  • the carrier plate can be designed as the base plate of a cell culture vessel.
  • the carrier plate 10 bears on the section 17 a functionalized surface 18 on which capture molecules 19, 21, 22 are immobilized in the measuring points 11.
  • the catcher molecules 19, 21, 22 generally differ from measuring surface 11 to measuring surface 11, it being possible for different measuring surfaces 11 to be combined to form a logical measuring point.
  • the measuring surfaces 11 of a logical measuring point carry identical catcher molecules 19, 21 or 22 and are statistically distributed over the carrier plate 10.
  • Test cells 23 can bind to the catcher molecules 19, 21 and 22, and their binding behavior to the catcher molecules 19, 21, 22 or their reaction to co-stimulation by catcher molecules 19 and test substances or to a treatment such as radiation should be examined.
  • the functionalized surface 18 is blocked by molecules 24, so that the test cells 23 can only be bound in the area of the measurement areas 11.
  • a reference particle is indicated at 25 in FIG. 2, which binds to the capture molecules 22.
  • Biological cells or plastic spheres are suitable as reference particles 25, which carry molecules on their surface with which they bind to the capture molecules 19, 21, 22.
  • the reference particles 25 are provided as an internal reference in order to be able to calculate out fluctuations in the size of the measurement areas 11 or the number of capture molecules in the measurement area both within an array and between different arrays.
  • Example 1 of the aforementioned WO describes how such a carrier plate 10 with measuring surfaces 11 can be produced 02/02226, the disclosure of which is hereby expressly incorporated by reference.
  • Example II Incubation of a Mixture of Test Cells and Reference Cells on an Array with Measuring Points Distributed on Different Measuring Surfaces
  • test and reference cells are marked with different membrane dyes.
  • Hu AO SMC or GLZ are used as test cells, which are labeled with the Vibrant Dil Red Fluorescent Cell Linker Kit (V22885Y MoBiTec) according to the manufacturer's instructions.
  • PC12 serve as reference cells, which are marked with the Vibrant DiO Green Fluorescent Cell Linker Kit (V22886Y MoBiTec) according to the manufacturer's instructions.
  • both cell types were stained blue with the Vibrant Cell Labeling Solution DAPI.
  • FIG. 3 shows, by way of example, for a measuring point with laminin from the human placenta as a capture molecule, that both test cells (GLZ) and reference cells (PC 12) bind to the measuring point, but the reference cells in a significantly smaller number than the test cells. It can be seen that the measuring point is evenly populated with capture molecules.
  • FIG. 3A shows a bright field measurement for both cells
  • FIG. 3B shows a fluorescence image of the measuring point with a blue filter for test and reference cells after DAPI staining
  • FIG. 3C shows a fluorescence image with a red filter that is permeable to the emission of the test cells
  • FIG. 3D shows one Corresponding image with a green filter permeable to the emission of the reference cells.
  • the total number and percentage of the bound cells is given as an example for some measuring points, each with laminin (human placenta) as a capture molecule for a mixed suspension of test and reference cells, 100,000 cells of each type being found for PC12 with GLZ an array was given and for PC12 with hu AO SMC 35,000 cells of each type.
  • test cells glioma cell line GLZ
  • reference cells neurovascular cell line PC12
  • test cells smooth muscle cells huAO SMC
  • reference cells neurovascular cell line PC12
  • the ratio between the two respective cell types is approximately constant if the ratio F> N x H is maintained, regardless of the number of cells per measurement area and regardless of whether the two cell types compete for a capture molecule. This makes it possible to use reference cells to calculate the variation between measuring points.
  • the percentage or the ratio between bound test and reference cells is therefore a more reliable measure of the binding of the test cells to the individual measuring points than the absolute number of bound test cells.
  • Example III Incubation with and without shaking
  • Test cells were in a concentration of 0.5 to 50 x 10 s cells / ml on measuring surfaces of the area 280,000 ⁇ m 2 with different capture molecules, namely collagen I, collagen II and collagen III, incubated for 4 h each, the carrier plates being shaken during the incubation in one case and left to rest in the other case. For shaking, the carrier plate was moved manually at intervals of 10 min to mix the cell suspension over the arrays.
  • the upper line shows the binding to collagen I, the middle one the binding to collagen II and the lower one the binding to collagen III. Incubation without shaking is shown on the left for A, C and E and incubation with shaking on the right for ⁇ B, D and E.
  • FIG. 5 shows the number of cells per measuring surface as a function of the number of cells used and the binding of the test cells to the three capture molecules mentioned in FIG. 5.
  • FIG. 6 shows the number of cells per measuring surface as a function of the number of cells used and the binding to the laminin substrate and the influence of the substrate movement.
  • the shaking ensures that a measuring area is populated with cells at most.
  • a "saturation" of the measuring surfaces already occurs at a concentration of 20 x 10 5 cells / ml, which can be seen from the fact that the number of bound cells does not increase any further from this concentration.
  • concentrations that is to say with a lower number of cells per measurement point, there is virtually no binding to thrombospondin, but rather that almost all cells bind to laminin or collagen I. This is also to be expected, because PC12 binds to thrombospondin considerably less than to the other capture molecules. Only through a high number of cells in the supernatant, the effect of competition is weakened and the cells' also bind to thrombospondin.
  • Example IV Determination of the sensitivity of cells in their natural microenvironment
  • An example of how the method according to the invention can be used is the investigation of how tumor and normal tissues react to radiation or the addition of toxic substances, depending on their natural microenvironment. It is known that cells whose radiation sensitivity is tested in a cell culture without the addition of extracellular matrix components (ECM) are more radiation-sensitive than parallel cultures which have been cultivated on ECM. This means that the composition of the extracellular matrix is of crucial importance for the cell-specific reactivity of both tumor and normal tissue. Furthermore, the combination effect of radiation and chemotherapy can be further optimized in the natural microenvironment.
  • ECM extracellular matrix components
  • test cells are placed in a mixed suspension with reference cells on an array of different capture molecules and the number of bound test cells as well of the bound reference cells is determined and from this the normalized number of test cells per measuring point is calculated.
  • the test cells are then treated with staurospondin and incubated. After a certain incubation period, the number of dead test cells per measurement point is determined and the apoptosis rate is determined from this number and from the normalized number determined before the incubation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé permettant de réaliser des essais fonctionnels sur des cellules test. Ce procédé consiste à: a) préparer un réseau composé de points de mesure distants, au niveau desquels sont respectivement immobilisés des molécules « éboueurs » auxquelles les cellules d'essai peuvent se lier, b) à créer un mélange de cellules d'essai et de particules de référence capables de se lier aux molécules « éboueurs » et distinctes des cellules d'essai et, c) à mettre en contact le mélange obtenu en b) avec le réseau, de telle façon que des cellules d'essai et des particules de référence puissent se lier à chaque point de mesure, et d) à déterminer les rapports entre les cellules test liées pertinentes et les particules de référence liées au moins pour quelques points de mesure. Dans le cas d'un autre procédé, au moins un point de mesure et ses molécules « éboueurs » associées sont répartis sur plusieurs surfaces de mesure du réseau, lesquelles surfaces sont situées à différents emplacements du réseau. Le réseau est déplacé lors d'un autre procédé après le contact avec les cellules d'essai et éventuellement les particules de référence.
PCT/EP2003/007440 2002-08-05 2003-07-09 Procede et kit permettant de realiser des essais fonctionnels sur des cellules biologiques qui ont ete immobilisees sur un reseau par des molecules « eboueurs » WO2004019039A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002494558A CA2494558A1 (fr) 2002-08-05 2003-07-09 Procede et kit permettant de realiser des essais fonctionnels sur des cellules biologiques qui ont ete immobilisees sur un reseau par des molecules <= eboueurs >=
EP03792191A EP1527344A2 (fr) 2002-08-05 2003-07-09 Procede et kit permettant de realiser des essais fonctionnels sur des cellules biologiques qui ont ete immobilisees sur un reseau par des molecules eboueurs
AU2003246668A AU2003246668A1 (en) 2002-08-05 2003-07-09 Method and kit for performing functional tests on biological cells immobilized on an array of scavenger molecules
US11/051,315 US20050214832A1 (en) 2002-08-05 2005-02-04 Method and kit for performing functional tests on biological cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2002136101 DE10236101A1 (de) 2002-08-05 2002-08-05 Verfahren und Kit zur Durchführung funktioneller Tests an biologischen Zellen
DE10236101.0 2002-08-05

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/051,315 Continuation US20050214832A1 (en) 2002-08-05 2005-02-04 Method and kit for performing functional tests on biological cells

Publications (2)

Publication Number Publication Date
WO2004019039A2 true WO2004019039A2 (fr) 2004-03-04
WO2004019039A3 WO2004019039A3 (fr) 2004-04-08

Family

ID=30775054

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2003/007440 WO2004019039A2 (fr) 2002-08-05 2003-07-09 Procede et kit permettant de realiser des essais fonctionnels sur des cellules biologiques qui ont ete immobilisees sur un reseau par des molecules « eboueurs »

Country Status (5)

Country Link
EP (1) EP1527344A2 (fr)
AU (1) AU2003246668A1 (fr)
CA (1) CA2494558A1 (fr)
DE (1) DE10236101A1 (fr)
WO (1) WO2004019039A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3502274A1 (fr) * 2017-12-22 2019-06-26 Attomol GmbH Support d'échantillon et procédé d'obtention dudit support

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020019018A1 (en) * 1998-12-23 2002-02-14 Christopherson Richard Ian Assay to detect a binding partner

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020019018A1 (en) * 1998-12-23 2002-02-14 Christopherson Richard Ian Assay to detect a binding partner

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BELOV LARISSA ET AL: "Immunophenotyping of leukemias using a cluster of differentiation antibody microarray" CANCER RESEARCH, Bd. 61, Nr. 11, 1. Juni 2001 (2001-06-01), Seiten 4483-4489, XP002257440 ISSN: 0008-5472 *
CHANG T-W: "BINDING OF CELLS TO MATRICES OF DISTINCT ANTIBODIES COATED ON SOLID SURFACE" JOURNAL OF IMMUNOLOGICAL METHODS, Bd. 65, Nr. 1-2, 1983, Seiten 217-224, XP009018805 ISSN: 0022-1759 *
MATTIS ANITA E ET AL: "Analyzing cytotoxic T lymphocyte activity: A simple and reliable flow cytometry-based assay" JOURNAL OF IMMUNOLOGICAL METHODS, Bd. 204, Nr. 2, 1997, Seiten 135-142, XP002257441 ISSN: 0022-1759 *
SCHWEITZER BARRY ET AL: "Measuring proteins on microarrays" CURRENT OPINION IN BIOTECHNOLOGY, Bd. 13, Nr. 1, Februar 2002 (2002-02), Seiten 14-19, XP002257442 ISSN: 0958-1669 *
SCHWENK JOCHEN M ET AL: "Cell microarrays: An emerging technology for the characterization of antibodies." SUPPLEMENT TO BIOTECHNIQUES, Nr. December, 2002, Seiten S54-S61, XP009018784 *

Also Published As

Publication number Publication date
WO2004019039A3 (fr) 2004-04-08
CA2494558A1 (fr) 2004-03-04
DE10236101A1 (de) 2004-02-26
AU2003246668A1 (en) 2004-03-11
EP1527344A2 (fr) 2005-05-04

Similar Documents

Publication Publication Date Title
DE60307645T2 (de) Hoch-durchsatz assay zur messung von zellulären antworten mit hilfe von mikroarrays
DE3606124A1 (de) Kolorimetrische pruefvorrichtung zur bestimmung einer spezifischen substanz in einem fluessigen medium, und verfahren zur verwendung der vorrichtung
DE2005543A1 (de) Obj ektivträger
WO2003056335A2 (fr) Reseau de micropuits permettant de trier ou separer des cellules en suspension dans un fluide en ecoulement en fonction de leur taille et procede pour analyser l&#39;activite fonctionnelle de cellules individuelles
EP0957359A2 (fr) Quantification de l&#39;activité de cellules sanguines utilisant une méthode ELISPOT et une plaque de microtitrage
EP2356215A1 (fr) Substrats permettant de sélectionner et d&#39;influencer spécifiquement le fonctionnement de cellules
WO2004019039A2 (fr) Procede et kit permettant de realiser des essais fonctionnels sur des cellules biologiques qui ont ete immobilisees sur un reseau par des molecules « eboueurs »
DE102010060498B4 (de) Isolierung und funktionale Identifizierung ganzer Zellen
DE19751581C2 (de) Verfahren und Vorrichtung zur Prüfung von Materialien hinsichtlich ihrer potentiellen antimikrobiellen Wirksamkeit und der Adhäsion auf ihrer Oberfläche
WO2001054707A2 (fr) Utilisation d&#39;au moins un inhibiteur d&#39;aminopeptidase
EP1660886B1 (fr) Dispositifs pour detecter une activite biochimique au moyen de jonctions communicantes
DE60110598T2 (de) Quantitatives fluoreszenz-bestimmungsverfahren für transportproteinenaktivität
WO2002088734A1 (fr) Systeme pour mesurer la permeation membranaire
EP1599727B1 (fr) Procede de fabrication d&#39;un support comportant des virus immobilises et utilisation d&#39;un tel support
EP1159443A1 (fr) Procede pour tester des principes actifs in vitro, dispositif approprie et son utilisation
US20050214832A1 (en) Method and kit for performing functional tests on biological cells
WO2007028380A1 (fr) Dosage immunologique in vitro fonctionnel
DE19709019C2 (de) Verfahren zur Bestimmung der Verträglichkeit, insbesondere auch der Toxizität von gasförmigen, flüssigen und/oder viskosen Stoffen für den menschlichen oder tierischen Organismus
WO2001082265A1 (fr) Dispositif et procede de simulation
EP0971232B1 (fr) Détermination luminométrique d&#39;ATP
EP1402258A2 (fr) Procede pour determiner des interactions entre des keratinocytes et des neurones
WO2003054548A1 (fr) Procede de determination analytique quantitative d&#39;agglomerats
EP2406626B1 (fr) Procédé et moyens de détection de l&#39;activité d&#39;ostéoclastes
DE102015100171A1 (de) Verfahren zur markierung einer zelle
DE102022100146A1 (de) Verfahren zur analyse von personalisierten anti-krebs-wirkstoffen in zellkulturen

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2494558

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2003246668

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 11051315

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2003792191

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2003792191

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2003792191

Country of ref document: EP