Patent Application
20130224884
The present invention has as its object a device and a method for performing a test known as a lateral flow test to determine the presence or the absence of an analyte in a sample. In particular, the invention relates to novel improved monitoring of the correct functioning of the device.
Lateral flow tests, also called rapid tests, are currently used in the fields of clinical, food, pharmaceutical and chemical analyses. Thus, rapid test devices are used to determine the presence of a large number of analytes, such as antibodies, antigens, hormones, proteins and chemical molecules in liquid samples. These devices generally comprise a support and a matrix which permits the migration of the liquid sample. Conventionally, a plurality of zones is distinguished in the matrix, namely an application zone of the liquid sample, a marking zone and a reaction zone, the latter comprising a capture zone and a monitoring zone. These different zones are in fluid, communication. Thus, the analyte to be detected, if it is present in the sample deposited in the application zone, binds to a first marked binding partner in the marking zone, the complex thus formed then migrates to the reaction zone where it is immobilised in the capture zone by reaction with a second binding partner and the user can determine whether the analyte is indeed present from the appearance of a detectable signal which is determined by the type of marker associated with the first binding partner. Generally, the presence of the analyte in the sample is revealed in the form of a detectable line, usually called a test line. The reaction zone also comprises a sample migration monitoring zone which will indicate to the user that at least a part of the sample has indeed passed across the matrix, upstream of the monitoring zone and in particular in the capture zone. This can be for example by the disclosure of a control line of a predetermined colour. By way of example, patent applications WO 2004/003559, WO 2006/092103, WO 2007/081330 and US 2004/0161855 can be cited. The limits of the monitoring means currently used in rapid tests on a strip, which may or may not be integrated in a cassette, are that they can only verify that the migration of the fluid has indeed taken place by capillary action from the application zone to the reaction zone and cannot monitor the correct functioning of the device and of the test.
The present invention now provides a device which integrates true positive monitoring. The positive monitoring of the invention permits verification on the one hand of the integrity and the functioning of the physical elements of the device and on the other hand verification of the functionality of the biological elements of the device, in order to monitor the correct functioning of the device and of the test.
The device of the invention comprises:
The first binding partner and the second binding partner are selected from the group consisting in antibody, mixture of antibodies, antibody fragment, mixture of antibody fragments, antibody analogue, mixture of antibody analogues, antigen, mixture of antigens, protein, mixture of proteins, polypeptide, mixture of polypeptides, peptide and mixture of peptides.
The analogue of the analyte is immobilised either directly or indirectly in the monitoring zone, or is able, in the monitoring zone, to be carried by the flow of the liquid sample to a determined region of the monitoring zone in which it is immobilised to reveal the control line.
Consequently, in one embodiment of the device of the invention, the monitoring zone 6 comprises in addition a capture reagent immobilised on the matrix of said at least one analogue to which said at least one analogue is able to bind. In particular, the capture reagent of said at least one analogue is a reagent which is identical to the second binding partner of the test results display zone.
In another embodiment of the device of the invention, in the monitoring zone 6 the analogue of the analyte is immobilised directly or indirectly on the matrix.
The analogue of the analyte can thus be immobilised on the matrix by a reagent selected from the group consisting in antibody, antibody mixture, antibody fragment, mixture of antibody fragments, antibody analogue, mixture of antibody analogues, antigen, mixture of antigens, protein, mixture of proteins, polypeptide, mixture of polypeptides, peptide, mixture of peptides, and biotin/steptavidin and biotin/avidin receptor.
The analogue of the analyte can also be immobilised on the matrix, by a capture reagent which is an identical reagent to the second binding partner.
Preferably, the analogue of the analyte is an antibody, a mixture of antibodies, an antibody fragment, a mixture of antibody fragments, an antibody analogue, a mixture of antibody analogues, an antigen, a mixture of antigens, a protein, a mixture of proteins, a polypeptide, a mixture of polypeptides, a peptide or a mixture of peptides or their associations.
Two preferred embodiments of the device according to the invention are described below:
in one embodiment the first binding partner is an antibody, an antibody fragment or an antibody analogue, the second binding partner is an antibody, an antibody fragment, or an antibody analogue and the analogue of the analyte is a protein, a polypeptide or a peptide, and
in another embodiment the first binding partner is a protein, a polypeptide or a peptide, the second binding partner is a protein, a polypeptide or a peptide, and the analogue of the analyte is an antibody, an antibody fragment or an antibody analogue.
The first binding partner is marked by a detectable marker, i.e. a compound, a substance or a particle which can be detected by visual, fluorescent, or instrumental means and in particular the detectable marker can be a coloured latex particle, a gold particle, or a magnetic particle.
The correct functioning of the device can be displayed, by the formation of a detectable positive control line which is substantially perpendicular and preferably perpendicular to the direction of the flow of the liquid sample.
In another embodiment of the device according to the invention, the latter device comprises two adjacent and parallel reaction zones 4A and 4B which are not in fluid communication with each other so that the migration of the liquid sample takes place simultaneously and independently in said zones. It is desirable, in this embodiment, for the device to comprise in addition liquid sample migration monitoring (7).
In the above-mentioned devices the monitoring zone (6) is preferably downstream of the results display zone (5).
In another embodiment of the invention, the reaction zone 4 comprises two adjacent and parallel reaction zones (4A and 4B) which are not in fluid communication with each other and the monitoring zone (6) is parallel with the results display zone (5). It is desirable in this embodiment for the device to comprise in addition monitoring of migration of the liquid sample (7).
In another particular embodiment of the device of the invention, the matrix 1 is divided into at least two adjacent and parallel parts 1A and 1B which are not in fluid communication with each other. Part 1A comprises the liquid sample application zone 2A, the marking zone 3A, the test results display zone 5, and preferably the sample migration monitoring zone 7. Part 1B comprises the liquid sample application zone 2B, the marking zone 3B and the monitoring zone 6 which permits monitoring of the device.
In a modified embodiment of the device, the matrix 1 is partially divided into two parts adjacent and parallel with each other. The matrix 1 comprises the liquid sample application zone 2 and the marking zone 3. The reaction zone 4 is divided into two adjacent and parallel parts which are not in fluid communication with each other. Zone 4A comprises the test results display zone 5, and preferably the sample migration monitoring zone 7. Zone 4E comprises the monitoring zone 6 which permits monitoring of the correct functioning of the device.
In another modified embodiment of the device of the invention, the matrix 1 is divided into at least two adjacent and parallel parts 1A and 1C which are not in fluid communication with each other. Part 1A comprises the liquid sample application zone 2, the marking zone 3A, the test results display zone 5, and preferably the liquid sample migration monitoring zone 7. Part 1B comprises an analogue application zone 9, the marking zone 3C and zone 6 which allows monitoring of the correct functioning of the device. In this embodiment, the analogue of the analyte can be present in dehydrated form in which case it is taken up by any appropriate means, for example by a buffer or by the sample. The analogue of the analyte can also be deposited in the application zone 9 in liquid form, in particular after being taken up by an appropriate liquid medium before it is deposited in the application zone 9.
The term “matrix” refers to any type of material which is capable of ensuring the flow and the transfer of a fluid. The transfer of the fluid can be effected by capillary force. The matrix may be, for example made of at least one bibulous material. Bibulous materials are materials which easily absorb a liquid and across which the liquid is transported by capillary action. Non-limiting examples of bibulous materials include nitrocellulose, polyester, glass fibres, etc.
“Liquid sample” means any sample taken from a patient or individual, and able to contain an analyte such as defined below. This sample can in particular be a liquid biological sample such as one of blood, serum, plasma, saliva, urine, cerebrospinal fluid, pleural fluid, or peritoneal fluid. However the biological sample also comprises semi-solid or solid samples insofar as that they can be converted into a liquid sample by any appropriate method, for example a food sample, a stool sample, a tissue sample, cell cultures, or a mucous sample. This biological sample is prepared by any type of sampling known to the man skilled in the art. The sample can also be a sample of environmental origin, i.e. a liquid, solid or semi-solid sample from the environment, such as effluents, muds, soils, plants etc . . . . Of course, when the sample is solid or semi-solid, it must be pre-treated to be converted into a liquid sample.
“Analyte” means principally an antigen, an antibody, a hormone, a protein or a chemical molecule.
When the analyte is a protein or an antigen it can be detected by binding partners, for example receptors, antibody, antibody fragments, antibody analogue and any other ligand capable of binding to a protein or to an antigen.
The binding partner antibodies are for example either polyclonal antibodies, or monoclonal antibodies.
Polyclonal antibodies can be obtained by immunisation of an animal with the appropriate immunogen, followed by the recovery of the antibodies sought in purified form, by taking serum from said animal, and separation of said antibodies from the other constituents of the serum, in particular by affinity chromatography on a column on which is fixed an antigen specifically recognised by the antibodies.
Monoclonal antibodies can be obtained by the hybridoma technique the general principle of which is below.
In a first stage, an animal, generally a mouse, is immunised with the appropriate immunogen, the B lymphocytes of which are then capable of producing antibodies against this antigen. These antibody producing lymphocytes are then fused with “immortal” myeloma cells (mouse cells in the example) to produce hybridomas. From the heterogeneous mixture of the cells thus obtained, a selection is then performed of the cells capable of producing a particular antibody and of reproducing indefinitely. Each hybridoma is reproduced in clone form, each leading to the production of a monoclonal antibody the recognition properties of which with regard to the protein will be testable for example by ELISA, by immunotransfer (Western blot) in one or two dimensions, by immunofluorescence, or using a biocaptor. The monoclonal antibodies thus selected are subsequently purified in particular by the affinity chromatography technique described above.
The monoclonal antibodies can also be recombinant antibodies obtained by genetic engineering, by techniques well known to the man skilled in the art.
“Antibody analogues” means biological and/or chemical compounds which have the same binding abilities as the antibodies or antibody fragments or similar binding capacities. In particular antibody analogues include small proteins which like antibodies are capable of binding to a biological target thus permitting its detection, its capture or quite simply its targeting in an organism or a biological sample. The fields of applications of these antibody analogues are practically as vast as those of the antibodies. By way of example can be cited the Nanofitines™, small proteins marketed by the company AFFILOGIC.
The specific binding partners of the protein or of the antigen sought in the method of the invention can be used as a capture reagent, as a detection reagent or as capture and detection reagents.
The display of the immunological reactions, i.e. of the protein/binding partner or antigen/binding partner binding can be performed by any detection means employing marking of the binding partner.
Marking means the fixing of a marker reagent capable of generating a detectable signal, i.e. a compound, a substance or a particle which can be detected by visual, fluorescent or instrumental means.
In the embodiments of the invention, the signal generated in the results display zone and the signal generated in the positive monitoring zone will, preferably, be of the same nature and will exhibit the same colours.
By way of an example of immunological tests such as defined above, can be cited the “sandwich” and “competitive” methods.
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In one embodiment, with reference to
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Testing for the antigen HBs by the test illustrated in
Red latex particles marketed by the company Magsphère (trade name) are coated with a mixture of two anti-BBs monoclonal antibodies (bioMérieux, 2G2G10A12 and 6H6B6), at a respective concentration of 500 μg/ml. The particles are then distributed by means of a BIODOT (trade name) apparatus on a polyester membrane (Ahlstrom—trade name). The membrane is dried for one night at 37° C.
The capture antibody is a polyclonal goat anti-HBs, produced by biomérieux, which is coated on a nitrocellulose membrane CN 140 (Sartorins—trade name) at a concentration of 1 mg/ml. The distribution is performed with the BIODOT apparatus.
The positive control of the HBs Ag test or analogue of the native HBs antigen is a recombinant HBs antigen (batch 101011FF004), developed by biomérieux, which is immobilised directly, in the monitoring zone, at a concentration of 1 mg/ml by distribution with a BIODOT apparatus on the nitrocellulose membrane, at a distance of 5 mm from the polyclonal anti-HBs capture antibody. After distribution of the capture antibody and of the positive control of the test, the membrane is dried for one night at 37° C.
The two polyester and nitrocellulose membranes are then assembled on a rapid test support (backing, of the company G&L (trade name)). They are mounted in the cassettes after cutting into strip form.
The samples tested are well-characterised positive samples for the HBs Ag antigen.
Dilutions in negative serum (Scantibodies—trade name) are performed to obtain high, medium and low levels of positivity. The negative sample tested corresponds to a negative serum pool from the Etablissement Francais du Sang (EFS) of the Rhône-Alpes region.
The reading time after deposition of the sample in the sample deposition well of the cassette is 15 minutes.
Reading is performed visually by means of a reading card which is used to attribute signal intensities depending on the intensity of the red colour observed.
This card is graduated from L1 to L10. A sample is considered positive if a red colour appears with an intensity corresponding to at least L3 on the reading scale.
The results are presented in table 1 below:
The results show that in the case of a negative sample, only the positive control is detected with a high colour intensity (L8). This result provides confirmation that the absence of signal at the test line 5, which corresponds to the capture polyclonal, is due to the negativity of the sample and not to a functional defect of the cassette used for the testing. Indeed, in case of a negative serum, the monoclonal antibodies coupled to the red particles are available and a complex is formed with the recombinant HBs antigen upon migration of the particles at the control line 6.
Conversely, in case of a positive sample, the particles fixing the antigen present in the sample form a complex with the capture polyclonal at the test line 5, depending on the level of positivity of the sample, and antibodies at the particle can remain available and form a second complex at the control line 6.
Testing for the influenza A antigen is based on the same principle as that described in example 1 for testing for the HBs Ag antigen. An anti-influenza A monoclonal detection antibody (bioMérieux, 15C9H2) is immobilised on red particles (Magsphère). The same antibody is used for capture on the nitrocellulose membrane. The positive control (analogue of the antigen) is a recombinant protein (Nucleoprotein Influenza A batch 101011FFU05, bioMérieux) immobilised directly on the nitrocellulose membrane in the monitoring zone, at a concentration of 1 mg/ml. The analogue is distributed by the BIODOT apparatus on the nitrocellulose membrane at a distance of 5 mm from the anti-influenza A capture monoclonal. After assembly and mounting in the cassettes, the tests are performed with a reading time at 10 minutes after deposition of the sample.
A concentration range of the recombinant protein (Nucleoprotein INF A, bioMérieux) was tested. The negative sample tested is a PBS buffer.
The results are presented in table 2 below:
The results show that in the case of a negative sample, only the positive control is detected with a high colour intensity (L10). This result provides confirmation that the absence of signal at the test line 5 (corresponding to the capture monoclonal), is due to the negativity of the sample and not to a functional defect of the cassette used for the testing. Indeed, in case of a negative serum, the monoclonal antibodies coupled to the red particles are available and a complex is formed with the recombinant Influenza A protein upon migration of the particles at the control line 6. Conversely, in case of a positive sample, the particles fixing the antigen present in the sample form a complex with the capture monoclonal at the test line 5. Depending on the level of positivity of the sample tested, (from 40 μg/ml to 10 ng/ml) , antibodies on the particle can remain available and form a second complex at the control line 6.
The detection is based on the same principles as those described in examples 1 and 2, i.e. one-step sandwich type immunochromatographic testing. The only difference resides in the fact that in this test the presence of an antibody is tested for, i.e. an anti-HIV-1 group M antibody.
Blue latex particles marketed by the company VARIAN (trade name) are coated with specific peptides of the HIV-1 group M virus. These particles are then distributed on a polyester membrane (Ahlstrom). The capture peptides are coated on the nitrocellulose membrane (Millipore, 135UF).
The positive control of the HIV test is an anti-HIV-1 group M monoclonal antibody (bioMérieux, P12G11B10) coated on the nitrocellulose membrane at a concentration of 1 mg/ml. The positive control antibody is distributed by the apparatus at a distance of 5 mm from the capture peptides.
After distribution of the capture peptides and of the positive control of the test, the membrane is dried for one night at 37° C.
After assembly and mounting in the cassettes, the tests are performed. The reading of the signal is taken 30 minutes after the deposition of the sample.
The samples tested are well-characterised HIV positive samples. The negative sample is a negative serum pool from EFS of the Rhône Alpe region.
The results are presented in table 3 below:
The results show that in the case of a negative sample, only the positive control is detected. This result provides confirmation that the absence of signal at the test line 5 (corresponding to the capture peptides), is due to the negativity of the sample and not to a functional defect of the cassette used for the testing. Indeed, in case of a negative serum, the HIV peptides coupled to the blue particles are available and a complex is formed with the control anti-HIV monoclonal antibody upon migration of the particles at the control line 6.
Conversely, in case of a positive sample, the particles fixing the anti-HIV antibodies present in the sample form a complex, with the capture peptides at the test line 5, depending on the level of positivity of the sample, the peptides on the particle are saturated or remain partially available and form a second complex at the control line 6.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1059431 | Nov 2010 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR2011/052663 | 11/16/2011 | WO | 00 | 4/26/2013 |
