EP1342090A1 - Dispositif et procede d'analyse immunologique - Google Patents
Dispositif et procede d'analyse immunologiqueInfo
- Publication number
- EP1342090A1 EP1342090A1 EP01999821A EP01999821A EP1342090A1 EP 1342090 A1 EP1342090 A1 EP 1342090A1 EP 01999821 A EP01999821 A EP 01999821A EP 01999821 A EP01999821 A EP 01999821A EP 1342090 A1 EP1342090 A1 EP 1342090A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- zone
- reaction
- state
- reagent
- analyte
- 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.)
- Withdrawn
Links
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- 238000003018 immunoassay Methods 0.000 title description 3
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- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
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- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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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
- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Definitions
- the invention relates to a biological analysis device as well as to its implementation method.
- It relates in particular to immunological analyzes based on the reaction between an analyte present in a fluid, in particular a biological fluid, and a reagent capable of forming a complex with said analyte, in which the analyte and / or the reagent is in the form of figured elements.
- reaction zone and the immobilization zone are separated from each other by a specific medium.
- the medium then has the function on the one hand of isolating the immobilization zone from the reaction medium and on the other hand of allowing, under the effect of external forces, the selective passage of the figured elements complexed or not from the reaction zone towards the immobilization zone.
- This type of method is widely used because it has the advantage of avoiding washing and therefore of being able to be carried out in a single step, of being very sensitive and of being easy to use by requiring a reduced number of manipulations.
- the medium must completely cover the substance capable of specifically binding with the complexes possibly formed so that the latter is not inactivated by direct contact with the reaction medium.
- the media proposed in the prior art are not perfectly impermeable to the reaction medium, especially during the pouring of the latter into the reaction zone.
- this embodiment requires providing a particular shape of membrane depending on the shape and / or the size of the container.
- the invention therefore aims to remedy all of these drawbacks by proposing an immunological analysis device of the solid phase type which is simple to manufacture while being reliable and in which the separation medium between the reaction zone and the immobilization zone is impermeable to the reaction medium so as in particular to allow automated introduction of the reaction medium into the reaction zone.
- the separation medium can be made, in a simple and reliable manner, capable of letting the figured elements pass or not complexed from the reaction zone to the immobilization zone.
- the invention provides a biological analysis device of the type using a reaction between an analyte present in a fluid and a reagent capable of forming a complex with said analyte, in which the analyte and / or the reagent is in the form of figurative elements, said device comprising at least one reaction vessel provided with a reaction zone into which the fluid and the reagent are introduced, and an immobilization zone on which is fixed a substance capable of specifically binding with the complexes possibly formed, in which the reaction zone and the immobilization zone are separated from each other by a layer of a material, said material being capable of passing from a first state in which the layer is substantially impermeable to a second state in which the layer is capable of letting through the figured elements complexed or not.
- the invention provides a method of immunological analysis using such a device, providing the steps of: - introducing the fluid containing the analyte into the reaction zone;
- FIG. 1 represents, in section and schematically, a container of an immunological analysis device according to the invention.
- An immunological analysis device comprises at least one reaction container 1, for example of rigid plastic material, such as that shown in FIG. 1.
- the device comprises a plurality of reaction vessels 1 so as to allow the performance of several identical or different analyzes with the same device.
- the device comprises, for example, eight micro-wells 1 of a 96-well micro-titration plate with a unit capacity of between 300 and 350 ⁇ l, with a diameter of approximately 6 mm and a height of approximately 8 mm.
- the container 1 can be hermetically sealed with a peelable sheet, for example of special aluminum, so as to avoid possible pollution of its content.
- a peelable sheet for example of special aluminum, so as to avoid possible pollution of its content.
- Each container 1 is intended to allow a possible reaction between an analyte present in a fluid, in particular a biological fluid, and a reagent capable of forming a complex with said analyte.
- the terms “figured element” and “complex” refer respectively to the cellular elements of the biological fluid or of the reagent and to the complexes formed by specific bonds with these elements.
- the container 1 receives the reaction medium formed from the biological fluid and the reagent in a first zone 2 called the reaction zone.
- the biological fluid is blood or a component of the blood such as a plasma or a serum.
- Each container 1 also has the function of allowing the in situ revelation of the positive or negative nature of the test, that is to say of allowing the visualization of the presence or absence of complexes.
- the container 1 is provided with a so-called immobilization zone 3 to which a substance 4 is fixed capable of specifically binding with the complexes possibly formed.
- the container 1 has a U-shape, the opening part forming reaction zone 2 and the base part forming immobilization zone 3.
- the substance 4 capable of specifically binding with the complexes possibly formed is fixed on substantially the entire curved internal wall 5 of the base part.
- the immobilization zone 3 may include a collection zone 6 for the non-complexed elements which, in the embodiment shown in FIG. 1, is formed by the lowest central zone of the U.
- a collection zone 6 for the non-complexed elements which, in the embodiment shown in FIG. 1, is formed by the lowest central zone of the U.
- container 1 for example in a V shape, or alternatively containers 1 whose immobilization zone 3 is of convex shape.
- the biological fluid and the reagent include protein elements and / or figurative elements.
- the purpose of the analysis is to reveal the presence of a particular figurative element in the biological fluid.
- the reagent which is capable of specifically binding with the desired figured element is then in protein form.
- a particular example of such an analysis is when the analyte is a red cell carrying a blood group antigen and the reagent comprises a known antibody which is capable of binding to this antigen.
- This analysis makes it possible in particular to determine the group or the phenotype of the red blood cell.
- the purpose of the analysis is to reveal the presence of a particular protein element in the biological fluid.
- the reagent which is capable of specifically binding with the desired protein element is then in figurative form.
- a particular example of such an analysis is when the reagent comprises red cells carrying a known blood group antigen and the analyte is an antibody to a serum which is capable of binding to this antigen.
- This analysis makes it possible in particular to determine the presence and the nature of an antibody of the immune type prior to a transfusion.
- the analyte and the reagent are presented in figurative form, the analysis also having the aim of revealing the presence of the analyte in the biological fluid.
- a particular example of such an analysis is when the reagent comprises lymphocytes expressing a structure capable of recognizing surface molecules of another cell and the analyte is said other cell.
- the chemical and physicochemical nature of the plastic of the container 1 makes it possible to cover the latter with a layer 7 of active molecules of a substance 4 capable of specifically binding with the complexes possibly formed.
- Substance 4 is for example formed of antibodies of monoclonal and / or polyclonal origin, in particular anti-human immunoglobulin (AGH).
- AGH anti-human immunoglobulin
- substance 4 may include antibodies to determinants of serum complement proteins.
- the spaces of the internal wall 5 of the bottom of the container 1 which do not comprise any substance 4 can be saturated with the aid of the saturants conventionally used in techniques of the solid phase or ELISA (Enzyme Linked Immunosorbent Assay) type.
- This layer 7 which has been deposited, for example in the form of a monolayer, in the immobilization zone 3 is capable of recognizing any type of human antibody without specific isotypic specificity and, in the case of anti- complement, the C3 fraction thereof and more particularly the C3d and C3g fractions carried by the molecule C3.
- the fixing of the substance 4 on the internal wall 5 of the bottom of the container 1 can be carried out by non-specific means such as passive adsorption, in particular antibodies, or by techniques using bonds covalent and allowing the attachment of structures to plastic or other materials.
- This mono-layer 7 by interacting with the corresponding antigens, allows the fixing of the complexes possibly formed on the reactive surface.
- a solution of AGH and of anti-human complement antibodies at a concentration of between 1 and 10 ⁇ g / ml is prepared in 0.2M carbonate buffer pH 9.6.
- This solution is distributed in a volume of 75 ⁇ l in each well 1 of a round bottom micro-plate of the Maxisorp U8 NUNC type, then the plates are incubated overnight at 4 ° C.
- micro-wells 1 are then washed using a phosphate buffer solution (PBS 2.5 mM pH 7.4) in order to remove all the proteins not adsorbed directly to the plastic.
- a phosphate buffer solution PBS 2.5 mM pH 7.4
- micro-wells 1 are then treated using a albumin solution at 30 g / l in PBS buffer at the rate of 100 ⁇ l per micro-well.
- microwells 1 are again washed using phosphate buffer.
- the invention proposes that the reaction zone 2 and the immobilization zone 3 are separated from each other by a layer 8 of a material, in particular biological, which is, in a first state, substantially impermeable to any fluid.
- the figured elements complexed or not must be able to pass through the layer 8 so that the complexes possibly formed can bind to substance 4.
- the invention proposes that the material forming the layer 8 can pass into a second state in which it lets through the figured elements complexed or not.
- the layer 8 then has the function, in its first state, of acting as a physical barrier with respect to the reaction medium and, in its second state, of allowing the transfer, under the action of external forces, of the elements figured complexed or not.
- the biological material is, in its first state, in the form of a solid gel or of dense texture and, in its second state, in the form of a liquid.
- a biological material formed from a mixture of sodium alginates, bovine albumin, sodium pyrophosphate and calcium chloride.
- the biological material is introduced into the container 1 in liquid form and then the gelation is obtained after an incubation time greater than one hour.
- This gelation time allows the fluid to distribute well above the substance 4 with a good surface condition.
- the gel thus obtained allows the distribution of the reactants in the reaction zone 2 at speeds of the order of 400 ⁇ l / sec without causing any leakage of reaction medium in the immobilization zone 3.
- the immobilization zone 3 is filled with biological material so that it also serves to protect the substance 4 capable of specifically binding with the complexes possibly formed.
- the biological material does not directly cover the substance 4, for example by providing that another gel is disposed in the immobilization zone 3 prior to the introduction of the biological material.
- the transition between the first state and the second is achieved by a phase change of the biological material which is caused by the addition of a specific chemical substance in the reaction zone 2.
- the biological material also comprises the specific chemical substance capable of passing it from its first to its second state.
- radiation can initiate, at the desired moment, the action of the chemical substance on the material so as to cause it to pass from its first to its second state.
- the passage is caused only by the action of electromagnetic radiation, for example of the ultra sound or microwave type, and / or by a change in temperature of the biological material.
- the specific chemical substance capable of depolymerizing the gel described above is an agent complexing divalent ions, for example EDTA or sodium citrate, which causes its liquefaction.
- the sodium alginates used have the property of forming, in the presence of divalent ions, a dense network (first state of the biological material). This network is however reversible because in the presence of agents complexing divalent ions, there is a rearrangement and / or dissociation of the alginate chains causing a liquefaction of the gel (second state of the biological material).
- the density of the biological material in the second state can be between the density of the protein elements of the reaction medium and that of the figured elements so that on the one hand the protein elements remain in the reaction zone 2 and on the other hand the figured elements complexed or not pass through the immobilization zone 3.
- the density of the biological material in its second state may have a gradient in a longitudinal direction.
- Separation can also be obtained by a difference in physico-chemical affinity, for example by a difference in miscibility, between the biological material on the one hand and the figured elements or the protein elements on the other hand.
- the layer 8 in its second state has the function, due to its density and / or its composition, to allow, under the effect of external forces, the passage of the figured elements complexed or not while preventing the passage protein elements.
- this function may also be desirable, in order to avoid that the transfer of the figured elements complexed or not complexed, in particular by drainage effect, the protein elements in the immobilization zone 3.
- a 1.2% (weight / volume) solution of partially hydrolyzed sodium alginates (manuronic acid / guluronic acid ratio between 0.8 and 1) and 15 mM tetra-sodium pyrophosphate is prepared by dissolving an extract dry commercial alginates and sodium pyrophosphate in LISS buffer (Low lonic Strengh Solution). To ensure perfect dissolution of the product, vigorous stirring is carried out. Any bubbles formed are removed using softer stirring. This solution is stored at 4 ° C. Before use, the solution will be brought to room temperature.
- An albumin solution at 150 g / l is prepared by diluting the LISS buffer in half with a commercial albumin preparation at 300 g / l. This solution is stored at 4 ° C. Before use, the solution will be brought to room temperature.
- a LISS buffer solution of ethylene diamine tetra-acetic tetrasodium salt is prepared at a concentration of 100 mM. The pH of this solution is adjusted to 7. This solution is stored at 4 ° C. Before use, the solution will be brought to room temperature.
- a volume-to-volume mixture of the alginate and pyrophosphate solution and the albumin solution is carried out. After gentle stirring for a few minutes, to one volume of this solution is added one volume of the calcium chloride solution. This mixture is quickly homogenized and then distributed in micro-wells 1 at the rate of 100 ⁇ l per well before the start of gelation so as to completely cover the layer of AGH 7 (the time of preparation and handling of such a product must not exceed thirty minutes). A minimum one hour incubation provides a translucent and resistant gel.
- the gelling step takes place slowly enough to allow easy industrialization of the device and the absence of washing allows its implementation easily and possibly fully automated.
- micro-well 1 is then ready for use and must be stored at 4 ° C.
- the depolymerization of the gel is obtained by dispensing approximately 50 ⁇ l per well 1 of a solution containing 100 mM of EDTA (other products complexing calcium ions can also be used). Complete liquefaction of the gel is obtained after an incubation of approximately 15 minutes at a temperature of 20 ° C.
- the preparation and distribution of the layer 8 of biological material is thus carried out in one step and, the fact of mixing in a precise order and at given concentrations the different constituents of this layer 8 makes it possible to obtain a homogeneous gel throughout its mass and whose depolymerization is perfectly controllable and takes place simply in the axis of the microwells 1.
- reaction zone 2 - introduce the fluid into reaction zone 2;
- the reagent and the specific chemical can be introduced simultaneously so that the reactions on the one hand between the analyte and the reagent and on the other hand between the biological material and the chemical take place simultaneously.
- reaction mixture present in the reaction zone 2 is subjected to conditions favoring the reaction between the analyte and the reagent, for example an incubation so as to increase the speed of reactions to occur.
- the external force capable of allowing the passage of the figured elements complexed or not from the reaction zone 2 to the immobilization zone 3 through the layer 8 of biological material in its second state comprises a force centrifugal.
- the intensity, the direction and the duration of the centrifugal force are adjusted so as to allow the transfer of the figured elements, complexed or not, from the reaction zone 2 to the immobilization zone 3, as well as possibly to allow the collection of the non-complexed figured elements in the collection zone 6 and, on the other hand, to leave the protein elements in the reaction zone 2.
- the external force capable of allowing the passage of the figured elements complexed or not from the reaction zone 2 to the immobilization zone 3 through the layer 8 of biological material in its second state comprises, possibly in addition to a centrifugal force, a magnetic force.
- the magnetic force is created, for example by a permanent magnet or by an electromagnet, in a substantially longitudinal direction relative to the container 1.
- the figured elements must be or must be made sufficiently paramagnetic to migrate from the reaction zone 2 to the immobilization zone 3 under the effect of the magnetic force.
- the elements represented are red cells, as a reagent or as an analyte, a method is described below for increasing their magnetic susceptibility prior to their introduction into the reaction zone 2 without damaging the antigens that they wear.
- Paramagnetic particles are used which have as essential characteristics to have a very high homogeneity in size (approximately 200 nm), a high load of ferromagnetic material (approximately 75% by mass) and a rather hydrophobic surface state.
- These particles are fixed to the surface of the red blood cell by means, for example, of bovine serum albumin so as to create multiple bonds of low intensity between the surface of the red blood cell and the particles.
- the fixation takes place in two stages, the first consists in the activation of the particles using a sticky product and the second is the bringing into contact of these activated particles with a suspension of treated red blood cells or not by proteolytic enzymes.
- the red cells thus obtained are attracted by a magnetic field and can thus be used directly or, in a variant, treated with solutions of enzymes generally encountered in immuno-hematological tests.
- Particles of type P201 from the company Ademtech are placed in the presence of a solution of bovine albumin at 0.1% (weight / volume) in PBS buffer pH 7.2. After an incubation of thirty minutes at room temperature and with shaking (proscribe any magnetic shaking), the particles in suspension are attracted to a magnet and the particle-free supernatant is removed.
- the “glued” particle pellet can be used directly during the red blood cell awareness phase.
- Second step raising red blood cells
- the globular suspension buffered LISS at the appropriate concentration (possibility of working with cell suspensions between 0.6 to 10% and previously washed three times or not with physiological water for example). After having perfectly homogenized the suspension (check that there are no more particle aggregates), it is incubated for 30 minutes at room temperature with gentle but homogeneous stirring (the entire reaction volume must be set in motion) . The red cells are then washed with a PBS buffer pH 7.4 (two washings by centrifugation, three minutes at 500 g). The pellet of sensitized red cells can then be taken up at the concentration for carrying out the analysis using a LISS buffer.
- the ratio between the quantity of particles used and the quantity of red blood cells is between 600 and 1000 so as to obtain sufficient magnetization without risking degrading the antigens present on the surface of the red blood cell.
- red cells sensitized by paramagnetic particles then have the double property of being attracted by a magnetic field and also of possessing on their surface blood antigens (group and phenotype). They can then be used as a reaction support and vector for transporting the antigen-antibody couple and thus pass through the biological material in its second state.
- the red cells thus obtained can either be used directly as a reagent or as an analyte, or undergo treatment with proteolytic enzymes such as papain to perform a so-called enzymatic analysis.
- the antibodies are, prior to their introduction into reaction zone 2, treated so as to be made paramagnetic, for example by means of a method analogous to that presented above.
- red cells of which the group and the phenotype are known it is desired, using red cells of which the group and the phenotype are known, to detect and determine the nature of an antibody of the immune type, that is to say developed following immunization during a blood transfusion or pregnancy.
- the presence of such antibodies can seriously compromise any new incompatible blood transfusion in the system considered and in the event of pregnancy have serious consequences for the survival of the fetus.
- This regularly performed analysis is called Irregular Agglutinin Research (RAI).
- the reagent comprises red cells carrying a known blood group antigen and the analyte is an antibody capable of binding to this antigen.
- the operator then deposits in the reaction zone 2 a volume of serum or of biological medium to be studied (approximately 25 ⁇ l) and two volumes of indicator red cell solution, for example previously treated with paramagnetic particles.
- this solution may include the gel depolymerizing agent.
- the operator then deposits the microplate or the strip on a suitable form allowing, if necessary, an incubation at 37 ° C., then the assembly is subjected to a magnetic field and / or to a centrifugal force.
- the antibodies specifically linked to the red cell surface antigens will interact with the antibodies adsorbed on the internal wall 5 of the container 1.
- red blood cells will be formed, the uniformity and dimensions of which will depend on the quantity of antibodies sought. If the reaction is negative, a central point of red blood cells will appear in collection zone 6.
- the analyte is a red cell carrying a blood group antigen and the reagent can comprise a known antibody which is capable of binding to this antigen.
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0016024A FR2817969B1 (fr) | 2000-12-08 | 2000-12-08 | Dispositif et procede d'analyse immunologique |
FR0016024 | 2000-12-08 | ||
PCT/FR2001/003888 WO2002046773A1 (fr) | 2000-12-08 | 2001-12-07 | Dispositif et procede d'analyse immunologique |
Publications (1)
Publication Number | Publication Date |
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EP1342090A1 true EP1342090A1 (fr) | 2003-09-10 |
Family
ID=8857444
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP01999821A Withdrawn EP1342090A1 (fr) | 2000-12-08 | 2001-12-07 | Dispositif et procede d'analyse immunologique |
Country Status (5)
Country | Link |
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US (1) | US20040063218A1 (fr) |
EP (1) | EP1342090A1 (fr) |
AU (1) | AU2002217217A1 (fr) |
FR (1) | FR2817969B1 (fr) |
WO (1) | WO2002046773A1 (fr) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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FR2869996B1 (fr) * | 2004-05-05 | 2006-12-15 | Diagast Soc Par Actions Simpli | Utilisation de ferrofluides pour le phenotypage sanguin et applications derivees |
US20070059782A1 (en) * | 2005-09-13 | 2007-03-15 | Graham Henry A | Magnetic particle tagged blood bank reagents and techniques |
US9488665B2 (en) * | 2005-09-13 | 2016-11-08 | Chrome Red Technologies, Llc | Magnetic particle tagged reagents and techniques |
US20150140578A1 (en) * | 2012-05-29 | 2015-05-21 | Arryx, Inc. | Methods and devices for sample testing and evaluation |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
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US4522923A (en) * | 1983-10-03 | 1985-06-11 | Genetic Diagnostics Corporation | Self-contained assay method and kit |
US5013669A (en) * | 1988-06-01 | 1991-05-07 | Smithkline Diagnostics, Inc. | Mass producible biologically active solid phase devices |
US5279936A (en) * | 1989-12-22 | 1994-01-18 | Syntex (U.S.A.) Inc. | Method of separation employing magnetic particles and second medium |
FR2702050B1 (fr) * | 1993-02-26 | 1995-05-24 | Boy Inst Jacques | Procédé de groupage sanguin faisant appel à des réactions immunoenzymatiques. |
NL9400777A (nl) * | 1994-05-10 | 1995-12-01 | Stichting Centraal Lab | Vaste-fase filtratiemethode voor antigeen- en antistofbepalingen in de bloedgroepserologie, en testkit. |
US5536514A (en) * | 1995-05-11 | 1996-07-16 | The Nutrasweet Company | Carbohydrate/protein cream substitutes |
US5756304A (en) * | 1995-07-14 | 1998-05-26 | Molecular Solutions | Screening of microorganisms for bioremediation |
NL1003570C2 (nl) * | 1996-07-11 | 1998-01-15 | Stichting Centraal Lab | Methode voor antigeen- en antistofbepaling in de bloedgroepserologie. |
JP4397558B2 (ja) * | 1999-08-18 | 2010-01-13 | マイクロチップス・インコーポレーテッド | 熱駆動マイクロチップ化学送達デバイス |
-
2000
- 2000-12-08 FR FR0016024A patent/FR2817969B1/fr not_active Expired - Fee Related
-
2001
- 2001-12-07 AU AU2002217217A patent/AU2002217217A1/en not_active Abandoned
- 2001-12-07 WO PCT/FR2001/003888 patent/WO2002046773A1/fr not_active Application Discontinuation
- 2001-12-07 EP EP01999821A patent/EP1342090A1/fr not_active Withdrawn
- 2001-12-07 US US10/433,975 patent/US20040063218A1/en not_active Abandoned
Non-Patent Citations (2)
Title |
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None * |
See also references of WO0246773A1 * |
Also Published As
Publication number | Publication date |
---|---|
US20040063218A1 (en) | 2004-04-01 |
FR2817969B1 (fr) | 2003-02-28 |
AU2002217217A1 (en) | 2002-06-18 |
FR2817969A1 (fr) | 2002-06-14 |
WO2002046773A1 (fr) | 2002-06-13 |
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