EP1342089A2

EP1342089A2 - Temporary separation barrier, container comprising same and method for carrying out a test in said container

Info

Publication number: EP1342089A2
Application number: EP01999433A
Authority: EP
Inventors: Frédéric BUFFIERE; Christine Betremieux; Jean Alain Chevaleyre; Sandie Menard; Gérard OVLAQUE; Christophe Vinzia
Original assignee: Diagast SAS
Current assignee: Diagast SAS
Priority date: 2000-12-08
Filing date: 2001-12-07
Publication date: 2003-09-10
Also published as: AU2002217218A1; WO2002045858A2; FR2817961A1; WO2002045858A3; US20040067168A1; FR2817961B1

Abstract

The invention concerns a barrier (2) temporarily separating a reaction container (1) into at least a first compartment (3) wherein a reaction medium comprising an analyte and optionally a reagent is introduced and a second compartment (4) for receiving at least one of the elements among the reagent, the analyte and the reaction product, said barrier (2) being made of a material adapted to shift, by the action of a specific chemical substance, from a first state wherein said material is substantially impermeable to the reaction medium to a second state wherein said material is capable of allowing through at least one of said elements among the reagent, the analyte and the reaction product. The invention also concerns a container comprising such a barrier and a method for carrying out an assay in said container.

Description

Temporary separation barrier, container comprising it and method for setting up a test in this container

The invention relates to a barrier for the temporary separation of a reaction vessel, to said reaction vessel as well as to a method for carrying out a test in the reaction vessel.

It particularly relates to barriers for the temporary separation of containers into two compartments intended to receive the reaction medium and certain elements of the reaction medium, respectively, from the analyte, the reagent and the product of their reaction.

In a particular example, the reaction takes place in the first compartment and the second compartment is arranged to allow the revelation of the possible reaction, in particular by specific bonding of the reaction product with a substance placed in the second compartment.

This type of barrier has the function on the one hand of isolating the second compartment from the reaction medium and on the other hand of allowing, under the effect of external forces and at the desired time, the selective passage from the first to the second compartment certain elements of the reaction medium among the analyte, the reagent and the product of their reaction.

Such containers are for example intended to allow carrying out biological tests based on the reaction between an analyte present in a biological fluid and a reagent capable of forming a complex with said analyte.

This type of test typically applies to research, in blood or in a blood component, of antigens present on the surface of red blood cells using known anti-erythrocyte antibodies (erythrocyte typing) or research anti-erythrocyte antibodies using red blood cells on which specific antigens are present and / or attached. Separation barriers of a physical type are already known, for example formed from a membrane or a sieve of plastic material, which are arranged between a first and a second compartment of a container.

This type of barrier has the disadvantage of not being modifiable after the introduction of the reaction medium into the first compartment.

They therefore need to be produced very precisely so as not to allow the diffusion of the undesirable elements in the second compartment while allowing the diffusion of the desired elements at the desired time.

The compromise between these two opposing constraints generally leads to a mediocre reliability barrier which either lets undesirable elements pass, in particular during the pouring of the reaction medium into the first compartment, or requires the application of a severe external force to allow passage desired items.

In addition, this type of embodiment requires providing a particular barrier shape depending on the shape and / or the size of the container.

Separation barriers are also known, for example formed from agars, which are temporary.

However, this type of barrier has the drawbacks of poor reliability and of a phase change linked to a change in temperature which may not be desirable for the reaction which has to take place in the container.

The invention therefore aims to remedy all of these drawbacks by proposing in particular a separation barrier which can pass, in a simple and reliable manner, from a state impermeable to the reaction medium to a state where it is capable of allowing passage from the first to the second compartment certain elements among the analyte, the reagent or the product of their reaction, and this at the chosen time and without any particular constraint for the reaction which must take place in the container.

To this end, and according to a first aspect, the invention provides a barrier for the temporary separation of a reaction vessel into at least a first compartment into which a reaction medium comprising an analyte and optionally a reagent is introduced and a second compartment intended receiving at least one of the elements from the reagent, the analyte and the reaction product, said barrier being formed of a material capable of passing, by the action of a specific chemical substance, from a first state in wherein said material is substantially impermeable to the reaction medium in a second state in which said material is capable of allowing at least one of said elements to pass through the reagent, the analyte and the product of the reaction.

According to a second aspect, the invention provides a reaction container comprising a barrier described above, said container being in the form of a micro-well of a micro-titration plate, said micro-well having a shape in U or V

According to a third aspect, the invention provides a method for carrying out a test in a reaction vessel described above, said method comprising the steps providing for:

- introduce the reaction medium into the first compartment;

- Passing the material forming the barrier from its first to its second state;

- Apply an external force capable of allowing the passage of one of the elements among the reagent, the analyte and the reaction product from the first compartment to the second compartment through the material barrier in its second state; - reveal the positive or negative nature of the test.

Other objects and advantages of the invention will appear during the description which follows with reference to the appended FIG. 1 which represents, in section and schematically, a container comprising a temporary separation barrier according to the invention.

In connection with FIG. 1, a reaction vessel is described, for example made of rigid plastic material, which is in the form of a micro-well of a micro-titration plate, said micro-well having a shape U.

In a particular example, the container is part of a device comprising eight micro-wells 1 of a micro-titration plate, and has a unit capacity of between 300 and 350 μl, a diameter of approximately 6 mm and a height about 8 mm.

In addition, and prior to its use, 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.

The container 1 is divided by a temporary separation barrier 2 into a first compartment 3 into which a reaction medium comprising an analyte and optionally a reagent is introduced and a second compartment 4 intended to receive at least one of the elements from the reagent, the analyte and the product of their reaction.

In a variant not shown, and depending on the nature of the test to be carried out, the container 1 can be divided into more than two compartments 3, 4.

For example, the container 1 can be divided into three compartments by two separate barriers 2, the first receiving the reaction medium, the second receiving the reaction product and containing a second reagent and the third receiving the reaction product to occur in the second compartment. In the embodiment shown in FIG. 1, the container 1 has a U-shape, the opening part forming the first compartment 3 and the base part forming the second compartment 4.

The container 1 has in particular the function of allowing a possible reaction between an analyte and a reagent.

To this end, the barrier 2 is formed from a material, in particular a biological material which, in a first state, is substantially impermeable to the reaction medium so as to isolate the first compartment 3 from the second compartment 4.

Thus, the biological material in its first state, by acting as a physical barrier vis-à-vis the reaction medium, allows the reaction to be carried out in the first compartment 3 without risk of diffusion of the reaction medium in the second compartment 4.

In the case where the reaction medium does not contain a reagent, the barrier 2 has the function of isolating the second compartment 4, for example to subject the reaction medium to a particular treatment.

The container 1 also has the function of allowing the passage of certain elements of the reaction medium into the second compartment 4 in order, for example, to allow them to be brought into contact with a substance 5 such as a reagent or a substance allowing the character to be revealed in situ. positive or negative of the test.

To this end, the biological material forming barrier 2 is capable of passing, by the action of a specific chemical substance, from the first state to a second state in which said material is capable of allowing at least one of the elements to pass through. reagent, the analyte and the product of their reaction. In a first particular example, the biological material in its second state allows the revelation of the test in the first or in the second compartment 3, 4 depending on the elements which it lets through.

In the case where it allows the reagent and the analyte to pass, the revelation will take place in the first compartment 3 by visualization of the possible presence of the product of the reaction.

In the case where it allows at least the reaction product to pass, the revelation will take place in the second compartment 4 by visualization of the possible presence of the reaction product.

In the latter case, a substance 5 capable of specifically binding to the reaction product can be placed in the second compartment 4, for example by being fixed on substantially the entire curved internal wall 6 of the base part 7 of the container 1.

This embodiment is particularly desirable when the biological material does not allow only the reaction product, but the reaction product and the reagent or the analyte, to allow visualization of the presence of the reaction product by specific fixation. of it on substance 5.

Alternatively, the second compartment 4 can be filled with the biological material forming the barrier 2 so that it also serves to protect the substance 5 capable of specifically binding to the reaction product.

In a second particular example, the container 1 allows the chain reaction to be carried out by providing that a second reagent to react with the product of the reaction is placed in the second compartment 4. As a variant of this example, provision may be made not to introduce a reagent in the first compartment 3 but for the latter to be disposed in the barrier 2 and / or in the second compartment 4.

Thus, only the biological fluid is introduced into the first compartment 3 and, after a possible treatment, at least the analyte passes through the barrier 2 in its second state in order to be brought into contact with the reagent.

This embodiment makes it possible to bring the reagent and the analyte into contact at a given time and optionally with a determined kinetics in the case where the reagent is placed in the barrier 3.

In a third particular example, the second compartment does not comprise any reagent or specific substance and the biological material in its second state only serves as a filter between the elements of the reaction medium.

According to one embodiment, the barrier also comprises the specific chemical substance capable of passing the material from its first to its second state.

In this embodiment, 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.

In other embodiments not shown, it is possible to envisage other forms of container 1, for example in V, containers 1 whose surface 6 on which the substance 5 is fixed is of convex shape, or even that the two compartments 3, 4 are not arranged vertically but for example transversely.

Described below is an embodiment of a container 1 intended to allow the realization and the development of a reaction between an analyte present in a biological fluid, in particular blood or a blood component such as a plasma or a serum, and a reagent.

In this type of reaction, the biological fluid and the reagent include protein elements and / or figurative elements.

Following a first use, the purpose of the analysis is to reveal the presence of a particular figurative element in the biological fluid. The reagent which is able to bind specifically with the desired figured element to form a complex with it 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.

In the description, 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.

According to a second use, the purpose of the analysis is to reveal the presence of a particular protein element in the biological fluid. The reagent which is able to bind specifically with the desired protein element to form a complex with it 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.

In the context of these two particular examples, an embodiment of the substance 5 capable of specifically binding this with the complexes possibly formed is for example formed of human anti-immunoglobulin (AGH) antibodies of monoclonal and / or polyclonal origin, optionally supplemented with antibodies directed against determinants of serum proteins of the complement type.

Thus, in the case of a positive reaction, we will observe a carpet of complexes covering the reactive surface 8 and, if not, we will observe a point of negativity in the bottom 7 of the second compartment 4.

An embodiment of the biological material forming the temporary separation barrier 2 is described below.

In this example, 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.

Biological material using a mixture of sodium alginates, bovine albumin, sodium pyrophosphate and calcium chloride is used.

The interactions between these different compounds are not completely known, but it can however be argued that the alginate chains interact through their hydrophobic zones with the albumin molecules and through their guluronic acid zones. with calcium ions. As for sodium pyrophosphate, it is possible to control the polymerization reaction between calcium and alginate which, without it, would be too rapid to produce a gel with homogeneous crosslinking.

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 5 with a good surface condition. The gel thus obtained allows the distribution of the reaction medium in the first compartment 3 at speeds of the order of 400 μl / sec without causing any leakage of reaction medium in the second compartment 4.

The transition between the first state and the second is achieved by a phase change of the biological material which is caused by a specific chemical substance.

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.

Indeed, 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 kinetics of this liquefaction is linked to the concentration of sequestering agent, to the temperature and to possible agitation.

From the moment when the gel liquefies completely, nothing prevents the transfer of at least the reaction product from the first compartment 3 to the second compartment 4.

This transfer can take place under the action of three forces, used in combination or separately:

- gravity, by natural settling of the reaction product;

- adapted centrifugal forces;

- the magnetic forces in the case where at least the product of the reaction has paramagnetic properties. In the case where the transfer takes place under the action of gravity and / or a centrifugal force, the density of the biological material in the second state may be different from the elements having to pass through the barrier 2. For example , the density of the biological material can be between that of the reaction product and that of the reagent so that on the one hand the reagent remains in the first compartment 3 and on the other hand at least the product of the reaction passes into the second compartment 4.

Alternatively, 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 product of the reaction or the reagent on the other hand.

In all these embodiments, the biological material in its second state has the function, by its density and / or its composition, of allowing, under the effect of external forces, the passage of at least the product of the reaction in order to allow the test to be carried out in the second compartment 4.

In the case of a transfer using a magnetic force, this function may also be desirable, in order to avoid that the transfer of the reaction product does not entail, in particular by drainage effect, the reagent in the second compartment 4.

As a variant, barrier 2 may also comprise an active product in the production and / or the development of the reaction between the reagent and the analyte, for example in the form of an enzymatic solution which will be released into the reaction medium when the biological material will pass into its second state. The preparation of the biological barrier 8 presented above is described below.

Preparation of different solutions

- Solution of alginates and tetra-sodium pyrophosphate

A 1.2% (weight / volume) solution of partially hydrolyzed sodium alginates (manuronic acid / guluronic acid ratio between 0.8 and 1) and of 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.

- Albumin solution

A 150 g / l albumin solution is prepared by diluting with a LISS half-buffer 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.

- Calcium chloride solution

A solution of LISS buffer of calcium chloride at a concentration of 10 mM is used. This solution is stored at 4 ° C. Before use, the solution will be brought to room temperature.

Tetra-sodium EDTA solution 4

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.

Gel preparation

The extemporaneous mixing before use is carried out according to a precise order.

First, 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 μl per well before the start of gelation so as to completely cover the layer of AGH 5 (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.

In addition, 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.

The micro-well 1 is then ready for use and must be stored at 4 ° C.

Depolymerization of the gel

The depolymerization of the gel is obtained by dispensing approximately 50 μl 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 barrier 2 of biological material are thus carried out in one step and, the fact of mixing in a precise order and at given concentrations the different constituents of this barrier 2 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.

The steps of the method for carrying out a test in the reaction vessel 1 described above which describes the steps providing for:

- Introduce the reaction medium into the first compartment 3;

- passing the material from its first state to its second state;

- apply an external force capable of allowing the passage of one of the elements among the reagent, the analyte and the reaction product from the first compartment 3 to the second compartment 4 through the barrier 2 of material in its second state;

- reveal the positive or negative nature of the test.

According to a first embodiment, the passage of the material from its first state to its second state is carried out by introduction of the specific chemical substance into the first compartment 3.

According to a second embodiment, the passage of the material from its first state to its second state is carried out by activation of the specific chemical substance, for example by means of radiation.

This embodiment is understood as well in the case where the specific chemical substance has been introduced into the first compartment as in the case where said substance is present in the barrier.

As a variant, the method comprises a prior step of placing the biological barrier 2 inside the container 1. In an exemplary embodiment, the reagent and the specific chemical substance 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 substance take place simultaneously.

Prior to the application of the external force, the reaction mixture present in the first compartment 3 can be subjected to conditions favoring the reaction between the analyte and the reagent, for example an incubation so as to increase the speed of the reactions before happen.

In a first embodiment, the external force comprises a centrifugal force whose intensity, direction and duration are adjusted to allow the transfer of the desired elements.

In a second embodiment, the external force 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.

For this purpose, the elements having to pass through the barrier must be or must be made sufficiently paramagnetic to migrate from the first compartment 3 to the second compartment 4 under the effect of the magnetic force.

A first and a second example of analysis carried out with this method are described below.

In the first example, 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 transfusion of incompatible blood 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).

For the implementation of this type of analysis, 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 first compartment 3 a volume of serum or of biological medium to be studied (approximately 25 μl) and two volumes of indicator red cell solution, for example previously treated with paramagnetic particles. Alternatively, 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.

When they come into contact with the AGH layer 5, the antibodies specifically linked to the red cell surface antigens will interact with the antibodies adsorbed on the internal wall 6 of the container 1.

Thus, in the case of a positive reaction, an image of red blood cell carpet 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 the bottom 7 of the container 1.

In the second example, we want to determine the group and phenotype of red blood cells, and in this case we use specific probes (monoclonal or polyclonal antibodies, plant lectins) of certain determinants of blood groups. For this purpose, 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.

The implementation of the analysis is then identical to that presented previously with the difference that, in the case of the use of a magnetic force, it is the red cells to be analyzed which can be treated so as to increase their magnetic susceptibility. .

The barrier 2 makes it possible, during an analysis, to carry out a temporary isolation of the reaction medium in the first compartment 3 in order to allow the reaction to be carried out or a treatment of the reaction medium and then, at the desired moment, to selectively pass a part of the elements present in the reaction medium to a second compartment 4 in order to allow the revelation of the positive or negative nature of the analysis or to allow the desired elements to be brought into contact with a reagent placed in the barrier 2 and / or in the second compartment 4.

In addition, barrier 2 allows

- A sequential analysis or a distribution sequence of the reaction medium which precedes bringing the reaction product into contact with a substance;

the protection of a reactive zone of the second compartment 4 which contains a fragile or excessively reactive substance and which must be protected at the start of the analysis;

- Avoid possible contamination of a reactive area of the second compartment 4 by the reaction medium.

Due to its fluidity during its implementation in the container 1, the barrier 2 can be used in containers 1 of any shape and in which the current solutions are not satisfactory.

Claims

1. Barrier (2) for temporary separation of a reaction vessel (1) into at least a first compartment (3) into which a reaction medium comprising an analyte and optionally a reagent is introduced and a second compartment (4) intended for receiving at least one of the elements from the reagent, the analyte and the reaction product, said barrier (2) being characterized in that it is formed of a material capable of passing, by the action of a specific chemical substance, from a first state in which said material is substantially impermeable to the reaction medium to a second state in which said material is capable of allowing some of said elements to pass through the reagent, the analyte and the product of the reaction.

2. Barrier according to claim 1, characterized in that the material is, in its first state, in the form of a solid gel and, in its second state, in the form of a liquid.

3. Barrier according to claim 2, characterized in that the material is of a biological nature formed from a mixture of sodium alginates, bovine albumin, sodium pyrophosphate and calcium chloride so as to form a gel with homogeneous crosslinking, the specific chemical being an agent complexing divalent ions, for example EDTA, capable of liquefying said gel.

4. Barrier according to any one of claims 1 to 3, characterized in that the density of the material in the second state is different from that of the elements having to pass through the barrier (2).

5. Barrier according to any one of claims 1 to 4, characterized in that it further comprises an active product in the production and / or the development of the reaction between the reagent and the analyte.

6. Barrier according to any one of claims 1 to 5, characterized in that it further comprises the specific chemical substance.

7. Reaction container (1) comprising a barrier (2) according to any one of claims 1 to 6, characterized in that it is in the form of a micro-well of a micro-titration plate , said micro-well having a U or V shape.

8. Container according to claim 7, characterized in that a substance (5) capable of specifically binding to the product of the reaction is arranged in the second compartment (4).

9. Container according to claim 8, characterized in that the second compartment (4) is filled with the material forming the barrier (2) so that it also serves to protect the substance (5) capable of specifically binding to the reaction product.

10. Container according to any one of claims 7 to 9, characterized in that the second compartment (4) comprises a reagent.

11. Method for carrying out a test in a reaction container (1) according to any one of claims 7 to 10, characterized in that it comprises the steps providing for:

- introduce the reaction medium into the first compartment (3); - passing the material forming the barrier (2) from its first to its second state;

- apply an external force capable of allowing the passage of one of the elements among the reagent, the analyte and the product of the reaction from the first compartment (3) to the second compartment (4) through the barrier (2 ) material in its second state;

- reveal the positive or negative nature of the test.

12. Method according to claim 11, characterized in that the passage of the material from its first state to its second state is carried out by introduction of the specific chemical substance in the first compartment (3).

13. Method according to claim 11 or 12, characterized in that the passage of the material from its first state to its second state is achieved by activation of the specific chemical substance, for example by means of radiation.

14. Method according to any one of claims 11 to 13, characterized in that the possible reaction between the reagent and the analyte occurs in the first compartment (3) and the revelation of the possible reaction is carried out in the second compartment (4), the material in its second state then being able to allow at least the reaction product to pass.

15. Method according to any one of claims 11 to 14, characterized in that it comprises a prior step of arrangement of the biological barrier (2) inside the container (1).

16. Method according to any one of claims 11 to 15, characterized in that the reagent comprises red cells carrying an antigen of cpnnu blood group and the analyte is an antibody capable of binding to this antigen, the method making it possible in particular to determine the presence and nature of an immune type antibody prior to a transfusion.

17. Method according to any one of claims 11 to 15, characterized in that 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, the method allowing including determining the group or phenotype of red blood cells.