FR2892515A1 - In vitro detection of presence and/or concentration of an antigen or particular antibody in a blood sample by immuno-chromatographic test, comprises applying blood sample and migration buffer to an absorbent element having marker - Google Patents

Abstract

The in vitro detection of presence and/or concentration of an antigen or particular antibody in a blood sample by immuno-chromatographic test, comprises applying blood sample and migration buffer to an absorbent element (100) having a marker. The absorbent element is placed on a distal part of a reactive band of an analytical test device by immuno-chromatographic test. The migration buffer is applied to the absorbent element that allows the migration of a specifically marked binding reactant towards a detection zone (103) of a porous support (102). The in vitro detection of presence and/or concentration of an antigen or particular antibody in a blood sample by immuno-chromatographic test, comprises applying blood sample and migration buffer to an absorbent element (100) having a marker. The absorbent element is placed on a distal part of a reactive band of an analytical test device by immuno-chromatographic test. The migration buffer is applied to the absorbent element that allows the migration of a specifically marked binding reactant towards a detection zone (103) of a porous support (102), which is directly/indirectly connected to the absorbent element. The detection zone is immobilized to allow specifically non-marked binding reactant. The antigen or particular antibody present in the blood sample is determined by observing a level of the specifically marked binding reactant, which is immobilized on the detection zone. The applying step of migration buffer is carried out by contacting the absorbent element with cotton/celluloid matrix soaked with the migration buffer while placing the absorbent element in a tube/receptor. The porous support comprises a nitrocellulose layer and a control zone (106), which allows the user to determine the test function. The specifically marked binding reactant is directly/indirectly coupled with a latex colored particle marker. The specifically marked binding reactant is maintained to a dry state in a macroporous body having a porous size, which is ten times higher than the size of the latex colored particle marker. The macroporous body of the blood sample is crossed before the migration of the porous support with the absorbent element. The latex colored particle marker is indirectly coupled with the specifically marked binding reactant. The indirect coupling is obtained by the specifically marked binders, which are coupled with the binding reagent and the latex colored particle marker. The latex colored particle marker is coupled with a first binder, which is maintained to a dry state in the macroporous body. The specifically marked binder reactant is coupled with a second binder that is present in the migration buffer. Absorbent layer (105) is directly placed in the distal zone of the analytical test device with the porous support in which the layer has an absorption capacity to absorb excess binding reactant in the detection zone. Independent claims are included for: (1) an analytical immuno-chromotographic test device; and (2) an analytical immuno-chromotographic test kit.

Description

METHOD AND DEVICE FOR IMMUNO-CHROMATOGRAPHIC DETECTION

  The invention relates to the general field of immunological tests for diagnosis. More specifically, the invention relates to a kit and a method using immunochromatographic assay techniques useful in immunoassays for diagnosis and using liquid samples as a preferentially tested sample. Many methods and analytical devices are commonly used in tests to determine the presence and / or concentration of substances that may be present in biological fluids or other materials. Such substances are usually referred to as analytes and include, for example, antibodies, antigens, drugs, hormones, etc. Immunological testing techniques are based on the mechanisms of the immune systems of higher organisms in which antibodies are produced in response to the presence of antigens, which antigens are pathogenic or foreign to the organisms. Antibodies and antigens (i.e., immunoreactors) are capable of binding to one another by a highly specific reaction mechanism that can be used in vitro to determine the presence or concentration of a particular antigen or antibody in a biological sample. Many diagnostic immunoassays generally use the specific binding characteristics that exist between an analyte and a specific antigen or antibody associated with a tracer element. Such tests include, for example, radioimmunoassays (RIAs) and enzyme immunoassays (EIAs), which include a tracer element during the detection phase on a solid support, immunological agglutination techniques in which The interaction between the antigen and the antibody occurs in solution and immunochromatographic techniques in which the interaction with the tracer elements in the form of antibodies or labeled antigens takes place in the porous membrane. Samples used in many immunological techniques, including immunochemical tests, may include environmental or organic materials or liquid extracts thereof and body fluids including serum, plasma, urine, cerebrospinal fluid ( CSF), saliva, sweat, milk, eye fluid and whole blood. Some tested samples, such as whole blood, are used preferentially for the specific detection of antibodies, such as anti-tetanus antibodies or other antibodies and antigens that are present at higher concentrations in whole blood or that are not present in other samples in viable or intact form. Samples tested, such as whole blood, urine or saliva, are especially preferred as suitable samples in diagnostic locations, such as hospital emergency departments, doctors' offices, veterinarians and farms. where sample processing equipment is not widely available or where time constraints make the use of such instrumentation impractical. The immuno-chromatographic test platforms used for the detection of analytes may be cassette-type in which the test components are contained in a solid plastic compartment or dipstick where the test components are not contained in the test. a solid housing but rather are layered on a plastic support material attached to the test components. The method of applying the sample to the immunochromatographic test cassette comprises adding the sample to a tube or a secondary container, followed by the addition of buffers or diffusion activators to said tube or container so that to improve the diffusion and the application of the sample to the membrane followed by the application of the dipstick in the tube or container.

  However, the methods and kits of the prior art do not always make it possible to obtain a satisfactory sensitivity and / or a result with great speed. The inventors have now demonstrated that a direct application of a blood sample on a test strip, and thus without prior dilution of said sample, makes it possible to obtain a diagnosis with greater sensitivity than the tests of the prior art. . Accordingly, a first object of the invention is a method of detecting an analyte comprising the steps of: 1) directly applying a liquid biological sample capable of containing said analyte to an absorbent member (100) corresponding to 30 the distal portion of the test strip of an immunochromatographic analytical test device; and applying a migration buffer on said absorbent member (100) to permit migration of a specific labeled binding reagent to the detection zone (103) of the porous support (102), in which detection zone (103) is permanently immobilized a specific unlabeled binding reagent and which porous carrier (102) is connected directly or indirectly to the absorbent member (100); the presence of said analyte in the liquid biological sample being determined by observing the level (if any) to which the specific labeled binding reagents are immobilized on the detection zone. By directly applying the liquid biological sample, it is intended to apply said liquid biological sample without prior dilution thereof. The present invention provides an improved method for applying samples to immunochromatographic test strips. The method thus provides increased sensitivity, lower detection limits, and improved development times compared to test results using the prior art application techniques. Advantageously, said liquid biological sample is selected from the group consisting of blood, urine, saliva and cerebrospinal fluid. Preferably, said liquid biological sample is a blood sample. Because of the coagulation characteristics of the blood, it was expected that its direct application on a test strip would cause its coagulation and counteract its diffusion, ultimately resulting in a lower sensitivity of a detection method using such a direct application step. On the contrary, the inventors have demonstrated that the method according to the invention makes it possible to obtain a greater sensitivity to the processes of the prior art. The immunochromatographic analytical test devices using test strips are well known to those skilled in the art. By way of example of such devices, mention may be made of the devices described in patents EP 0383619 B1, EP 0560411 B1, EP 0679893 B1 and in patent application EP 1248112 A2. Absorbent element (100) is understood to mean an element based on porous or fibrous material and capable of rapidly absorbing the biological biological sample. Examples of such materials include porous plastics such as those based on polypropylene, polyethylene, ethylene vinyl acetate, acrylonitrile and polytetrafluoroethylene, and also cellulose. Preferably, said absorbent element (100) is based on cellulose. Said absorbent element (100) can be saturated in liquid biological sample in a few seconds, said liquid biological sample is then free to migrate to the porous support (102) with which it is directly or indirectly in contact. Advantageously, the application step 1) is maintained for a time sufficient for a given volume of liquid biological sample to be absorbed by the absorbent element. This time is a function of the volume of the absorbent element, the viscosity characteristics of the liquid biological sample and the potential concentration of the analyte in said liquid biological sample. The skilled person will be able to simply determine the time required for his general knowledge. By way of example and for a blood sample, this time is between one second and one minute, preferably between 2 and 30 seconds and particularly preferably between 5 and 10 seconds. Advantageously, the absorption capacity of the absorbent element (100) is limited such that regardless of the quantity of liquid biological sample applied, in particular a large drop of blood, the quantity of liquid biological sample actually absorbed will be constant. According to a preferred embodiment of the process according to the invention, the absorbent element comprises a control mark (104). Stage 1) of application of the liquid biological sample is maintained until the liquid biological sample migrates into the absorbent element to the control mark (104).

  In the case of colored liquid biological samples such as blood, the migration of these can be simply followed. In the case of colorless liquid biological samples such as saliva, the absorbent member (100) may comprise color-changing compounds in the presence of the liquid biological sample. Such compounds may especially correspond to compounds that change color depending on the pH or on their state of hydration.

  The positioning of the control mark (104) makes it possible to indicate the minimum volume of liquid biological sample to be deposited. Said positioning is a function of the volume of the absorbent element and the volume of liquid biological sample necessary for a correct detection of the analyte. The positioning of this control mark (104) on the absorbent element can be carried out simply by those skilled in the art with regard to his general knowledge and simple routine experiments. By way of example, said control mark (104) corresponds to the boundary between the external part of the absorbent element which is discovered and the internal part which is covered by an adhesive film. Migration buffers for permitting migration of a labeled binding reagent specific for the absorbent member (100) to the porous support (102) are well known to those skilled in the art. By way of example of such migration buffers, mention may be made of phosphate buffer (PBS). Said migration buffer can also comprise at least one migration activator such as surfactants (for example TRITON X100).

  The method according to the invention can be used for the detection of a wide variety of analytes by selecting the appropriate specific binding reagents. The analytes may be, for example, proteins, haptens, immunoglobulins, polynucleotides, hormones, antigens specific for infectious agents. Step 2) of applying said pad is carried out by placing said absorbent member (100) in a tube or receptacle comprising said pad or by contacting said absorbent member (100) with a cotton or celluloid matrix soaked with said pad buffer. Advantageously, the porous support (102) comprises a chromatographic layer, preferably a nitrocellulose layer.

  Nitrocellulose is indeed a particularly advantageous carrier compared to conventional materials such as paper because of its natural ability to bind proteins without prior activation. Specific binding reagents of a protein-like analyte such as immunoglobulins can thus be directly applied and immobilized thereon. No chemical treatment likely to interfere with the binding activity is therefore required. The binding sites not used on nitrocellulose can be blocked later, in particular using proteins (bovine serum albumin (BSA) or milk proteins), or with polyvinyl alcohol (PVA) ethanolamine or a combination thereof. In addition, the nitrocellulose has the advantage of being available with a large range of pore size, which makes it possible to choose a porous support adapted to a particular use.

  Preferably, the nitrocellulose has a pore size of at least one m, preferably greater than 5 m, and particularly preferably between 8 and 12 m. Such nitrocelluloses are especially available from SCHLEICHER and SCHUELL GMBH or Advanced Microdevices Pvt. Ltd. More preferably, the nitrocellulose has a pore size of less than 20 m. Advantageously, the porous support (102) comprises at least one layer intended to increase its resistance and covering the nitrocellulose layer. For example, there may be mentioned a plastic layer such as polyester, which further has the advantage of being impermeable to moisture. According to a second preferred embodiment of the method according to the invention, the specific binding reagents (labeled or unlabeled) correspond to immunoglobulins specific for the analyte to be detected. The labeled binding reagent, the analyte and the unlabeled binding reagent then participate in a sandwich reaction corresponding to the immobilization of the labeled binding reagent at the detection zone and this in the case where the analyte is present in the liquid biological sample. The two specific binding reagents must then bind to different epitopes of the analyte to be detected. According to a third preferred embodiment of the method according to the invention, the specific labeled binding reagent corresponds to the analyte itself or an analogue thereof, which analyte or the like is coupled directly or indirectly with a marker. . In this embodiment, the labeled analyte (or analog) thereof will migrate into the porous support (102) to the detection zone and bind to the unlabeled binding reagent specific for said analyte, which may be an immunoglobulin. specific. Any analyte present in the liquid biological sample will compete with the labeled binding reagent specific for the binding reaction to the unlabeled binding reagent immobilized in the detection zone (103). Such competition will result in a reduction in the amount of specific labeled binding reagents immobilized at the detection zone and in a corresponding decrease in the signal observed at the detection zone in comparison with the signal obtained in the absence of analyte. According to a fourth preferred embodiment of the method according to the invention, the unlabeled binding reagent immobilized at the level of the detection zone corresponds to an analyte or an analogue thereof and the labeled binding reagent corresponds to a specific immunoglobulin. said analyte. When a liquid biological sample is applied to the device, competition between the immobilized analyte and the free analyte reduces the level at which the labeled binding reagent will be immobilized on the detection zone. The specific labeled binding reagent comprises a specific binding reagent directly or indirectly coupled to a particulate marker.

  According to a fifth preferred embodiment of the method according to the invention, the analyte corresponds to a specific immunoglobulin and the unlabeled binding reagent at the detection zone and the labeled binding reagent correspond to the specific antigen of the said immunoglobulin or one of its derivatives. Such a process is referred to as a double sandwich (see Example 1). Such particulate markers, also called direct markers are well known to those skilled in the art and include in particular the colored latex beads, colloidal gold and coal particles. The markers can be used to produce an instantaneous result without the need for reagents to provide a detectable signal when immobilized in the detection zone.

  Preferably, said particulate marker is a colored latex particle. Said particle has a spherical or quasi-spherical shape with a diameter of less than 0.5 m. Advantageously, said colored latex particle has a diameter of between 0.05 m and 0.5 m. According to a sixth preferred embodiment of the method according to the invention, the coupling of the particulate marker to the specific binding reagent corresponds to a direct coupling, which is carried out by covalent bridging or by hydrophobic interaction. Such techniques are well known to those skilled in the art. In the case of colored latex particles, hydrophobic bridging is preferred. Advantageously, the specific labeled binding reagent is kept dry in a separate macroporous body (101) having a pore size at least ten times greater than the size of the particulate label and in which macroporous body (101) is present. Liquid biological sample must pass through before migrating into the porous support (102), which porous carrier (102) is bonded via the macroporous body (101) to the absorbent member (100) to which the liquid biological sample is applied. Preferably, the macroporous body has a pore size greater than 10 m and particularly preferably greater than 100 m. The macroporous body (101) should comprise materials having no binding capacity for the proteins and should be capable of being simply neutralized with agents such as BSA and PVA so as to minimize nonspecific binding and migration of the specific labeled binding reagent after the liquid biological sample has been applied. Such a macroporous body (101) may comprise plastic or mineral materials, especially glass fiber. Such a macroporous body (101) is described in EP 0679893 B1. According to a seventh preferred embodiment of the method according to the invention, the coupling of the particulate marker to the specific binding reagent corresponds to an indirect coupling, which indirect coupling is obtained by the binding between two specific partners A and B, one of which is coupled to the binding reagent and the other is coupled to the particulate label. As before, said couplings can be carried out by covalent bonding or by hydrophobic interaction. By way of example of such specific partners, mention may be made of the biotin / streptavidin pair. Advantageously, the particulate marker coupled to the first partner (eg, strepatvidin) is kept in the dry state in a macroporous body (101) as described above and the specific binding reagent coupled to the second partner (eg, biotin) is present in the migration buffer used in step 2).

  According to an eighth embodiment of the method according to the invention, an absorbent layer (105) is positioned in the distal zone of the device in direct contact with the porous support (102), which absorbent layer has a sufficient absorption capacity to absorb the excess of specific labeled binding reagent unreacted at the detection zone (103). According to a ninth preferred embodiment of the method according to the invention, the porous support (102) comprises a control zone (106), which control zone must allow the user to determine that the test has worked well. For example, the control zone (106) may comprise an antibody that will bind to the specific labeled binding reagent (for example, an anti-mouse IgG antibody if such an antibody is used as a specific labeled binding reagent) or a nonspecific labeled control reagent present in the analytical test device at the same location as the specific labeled binding reagent (for example, an anti-rabbit IgG antibody with the addition of labeled rabbit immunoglobulins with latex colored in area (101)).

  Advantageously, said control zone (106) is located downstream of the detection zone (103). FIG. 1 shows a preferred embodiment of an immuno-chromatographic analytical test device used in the process according to the invention with an absorbent element (100), a macroporous body (101), a porous support (102), a detection zone (103), a control mark (104), an absorbing layer (105) and a control zone (106).

  A second subject of the invention relates to an analytical immuno-chromatographic test device comprising i) an absorbent element (100) on which a liquid biological sample may be applied, capable of containing an analyte to be detected, ii) a porous support ( 102), which porous support is connected directly or indirectly to the absorbent member (100); iii) a detection zone (103) on said porous support (102), on which detection zone (103) is permanently immobilized a specific unlabeled binding reagent; and iv) a free-mobility labeled specific binding reagent in the porous support (102) in the wet state, wherein the labeled and unlabeled specific binding reagents are capable of participating in either a sandwich reaction or a competitive reaction in a presence of the analyte; Which device is characterized in that said absorbent member (100) comprises a control mark (104) for determining that the directly applied liquid biological sample volume is sufficient to detect the analyte possibly contained in said sample. The characteristics of the device according to the invention are as previously described. Preferably, the marker of the labeled specific binding reagent is a particulate marker and particularly preferably a colored latex ball. Advantageously, the device according to the invention comprises a distinct macroporous body (101) having a pore size at least ten times greater than the size of the particulate marker and in which macroporous body (101) the liquid biological sample must pass before migrating. in the porous carrier (102), which porous carrier (102) is bonded via the macroporous body (101) to the absorbent member (100) to which the liquid biological sample is applied. Advantageously, the marker of the labeled specific binding reagent is kept dry in said macroporous body (101).

  According to a particular embodiment, the device according to the invention further comprises an absorbent layer (105) which is positioned in the distal zone of the device in direct contact with the porous support (102), which absorbent layer has a capacity of Absorption sufficient to absorb the excess of specific unreacted labeled binding reagent at the detection zone (103). According to a second particular embodiment, the device according to the invention further comprises a control zone (106) in the porous support (102), which control zone must allow the user to determine that the device has worked well. A third object of the invention is a kit comprising i) a device as previously described and ii) a migration buffer. According to a particular embodiment of the kit according to the invention, the marker and the specific binding reagent forming the labeled specific binding reagent are coupled indirectly. The present invention is illustrated by the following examples, which are provided without limitation. Example 1: Detection of immunity to tetanus toxin a) Introduction: Clostridium tetani toxin is a toxin that binds to peripheral nerve cell membranes and inhibits neurotransmitter delivery from synaptic neurons. cells of the central nervous system. Tetanus toxin affects the nerve endings of all peripheral neurons (motor, sensory and adrenergic neurons). The anti-tetanus toxoid antibodies can be produced by the immune system in humans and other mammals by vaccination (purified toxoid injection) in order to stimulate the immune system to produce these antibodies thereby making the subject immune to all the effects of tetanus toxin. In addition, direct injection of specific anti-tetanus gammaglobulin antibodies of human origin can also be performed to counteract the effects of acute tetanus toxin infection. In the context of emergency medicine, knowledge of the immune status in subjects at risk (deep wounds) can make it possible to choose the best prophylaxis (tetanus vaccination and possibly anti-tetanus gammaglobulin). b) Protocol: The finger of a patient is cleaned and the pulp of it is stitched with a disposable lancet.

  The following two protocols were used on the same strips: 1) Deposition of the blood directly on the strip with maintaining contact for 5 to 10 seconds, and finally immersion of the end of the strip in a tube containing 6 drops of migration buffer. 2) Mixing the same volume of blood with 6 drops of migration buffer in a tube and immersion of the end of the strip in the tube. The results are readable in the fifth minute. C) Results: The results showed that the intensity of staining in the zone (103) is systematically more intense with the first protocol.

  In addition to the first protocol, 7 samples are positive against only 4 with the second protocol which demonstrates a lowering of the detection threshold with the first protocol.

  Example 2) Detection of Plasmodium falciparum in a blood sample: a) Principle: A capture monoclonal antibody is immobilized on the nitrocellulose membrane. The red blood cells are lysed by the lysis buffer, releasing the Plasmodium falciparum-specific PfHRP II antigen (Histidin rich protein II), which will bind to the capture antibody during migration of the migration fluid along the nitrocellulose membrane. . The colored signal is generated by black latex particles on which is immobilized a specific monoclonal antibody which will bind to the antibody / antigen complex thereby causing the appearance of a black band. The presence of a purple internal control band at the top of the test validates the proper functioning of the test. b) Protocol: 15 l of a blood sample are placed on the lower end of a test strip and the contact is maintained for 5 to 10 seconds or the lower end of the strip is directly brought into contact with the test strip. a drop of capillary blood for about 5 seconds. The lower end of the test strip is then introduced into a tube comprising migration buffer.

  The result is readable in the fifth minute. This same protocol was performed on 49 other blood samples. c) Results: The results showed a sensitivity of 100% of the test and a specificity of 97.7% of the latter.

Claims (14)

  A method of detecting an analyte comprising the steps of: i) directly applying a liquid biological sample capable of containing said analyte to an absorbent member (100) corresponding to the distal portion of the test strip of a test device; analytical test by immuno-chromatography; and ii) applying a migration buffer on said absorbent member (100) to permit migration of a specific labeled binding reagent to the detection zone (103) of the porous support (102), on which detection zone ( 103) is permanently immobilized a specific unlabeled binding reagent and which porous carrier (102) is connected directly or indirectly to the absorbent member (100); the presence of said analyte in the liquid biological sample being determined by observing the level (if any) to which the specific labeled binding reagents are immobilized on the detection zone.   2) The method according to claim 1, characterized in that said liquid biological sample is a blood sample.   3) The method according to any one of claims 1 or 2, characterized in that said absorbent element (100) comprises a control mark (104).   4) The method according to any one of claims 1 to 3, characterized in that the step 2) of applying said migration buffer is carried out by placing said absorbent element (100) in a tube or a receptacle comprising said buffer migrating or contacting said absorbent member (100) with a cotton or celluloid matrix soaked with said migration buffer.   5) The method according to any one of claims 1 to 4, characterized in that said porous support (102) comprises a chromatographic layer, preferably a layer of nitrocellulose.   6. The method according to any one of claims 1 to 5, characterized in that the specific labeled binding reagent comprises a specific binding reagent coupled directly or indirectly to a particulate marker.   7) The method according to any one of claims 1 to 6, characterized in that said particulate marker is a colored latex particle. 25   The method according to any one of claims 1 to 7, characterized in that the specific labeled binding reagent is kept dry in a separate macroporous body (101) having a pore size at least ten times greater than the size of the particulate label and in which macroporous body (101) the liquid biological sample must pass before migrating into the porous support (102), which porous support (102) is bound via the macroporous body (101) to the absorbent member (100) to which the liquid biological sample is applied.   9) The method according to any one of claims 1 to 7, characterized in that the coupling of the particulate marker to the specific binding reagent corresponds to an indirect coupling, which indirect coupling is obtained by the connection between two specific partners A and B one of which is coupled to the binding reagent and the other is coupled to the particulate label and in that the particulate label coupled to the first partner is maintained in a dry state in a macroporous body (101) and the specific binding reagent coupled to the second partner is present in the migration buffer used in step 2).   10) The method according to any one of claims 1 to 9, characterized in that an absorbent layer (105) is positioned in the distal region of the device in direct contact with the porous support (102), which absorbent layer has a Absorption capacity sufficient to absorb the excess of specific unreacted labeled binding reagent at the detection zone (103).   11) The method according to any one of claims 1 to 10, characterized in that the porous support (102) comprises a control zone (106), which control zone must allow the user to determine that the test has worked well. .   12) An immuno-chromatographic analytical test device comprising: i) an absorbent element (100) on which a liquid biological sample may be applied which may contain an analyte to be detected; ii) a porous support (102), which porous support is connected directly or indirectly to the absorbent element (100); iii) a detection zone (103) on said porous support (102), on which detection zone (103) is permanently immobilized a specific unlabeled binding reagent; and iv) a free-mobility-labeled specific binding reagent in the porous support (102) in the wet state, wherein the labeled and unlabeled specific binding reagents are capable of participating in either a sandwich reaction or a competitive reaction. in the presence of the analyte; wherein said device is characterized in that said absorbent member (100) comprises a control mark (104) for determining that the directly applied liquid biological sample volume is sufficient to detect the analyte possibly contained in said sample. 25   13) An immuno-chromatographic analytical test kit comprising i) a device according to claim 12 and ii) a migration buffer.   14) The kit according to claim 13, characterized in that the marker and the specific binding reagent forming the labeled specific binding reagent are coupled indirectly by the binding between two specific partners A and B, one of which is coupled to the reagent and the other is coupled to the particulate label and in that the particulate label coupled to the first partner is kept dry in a macroporous body (101) of said device and the specific binding reagent coupled to the second partner is present. in said migration buffer.