1
Device and method for immunoassays
The present invention has as its object a device and a method for
5 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.
10 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
15 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
20 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
25 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
30 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
35 2004/003559, WO 2006/092103, WO 2007/081330 and US 2004/0161859 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.
5
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
10 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:
15 - a) a support,
- b) a matrix 1, fixed on the support, which allows the migration
of the liquid sample, said matrix comprising:
- (i) a liquid sample application zone 2,
- (ii) a marking zone 3 comprising at least a first ' marked binding 20 partner which is able to bind to said at least one analyte, if
it is present in the liquid sample, and which is able to bind to
at least one analogue of the analyte, and
- (iii) at least one reaction zone 4 comprising:
- a test results display zone 5 comprising at least a second
imrnobilised binding partner which is able to bind to said at
least one analyte, and
- a monitoring zone 6 downstream of the results display zone 5 or
parallel with the test results display zone 5 which allows
monitoring of the functioning of the device and which comprises
at least one analogue of the at least one analyte which is able
to bind to said at least first marked binding partner;
- said liquid sample application zone 2, marking zone 3 and
reaction zone 4 zone being in fluid communication.
35. 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.
5 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.
10
I 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
15 at least one analogue is a reagent which is identical to the second
binding partner of the test results display zone.
I , In another embodiment of the device of the invention, in the
monitoring zone 6 the analogue of the analyte is immobilised directly
20 or indirectly on the matrix.
I 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
25 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,
30 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
35 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:
5 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
10 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
15 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.
20 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.
25 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
30 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).
35 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
* 5
(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).
5 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
10 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
15 divided into two parts adjacent and parallel with each other. The
I 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
20 sample migration monitoring zone 7. Zone 4B 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
25 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 3Af
the test results display zone 5, and preferably the liquid sample
migration monitoring zone 7. Part 1B comprises an analogue application
30 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
35 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.
5 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
10 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
15 peritoneal fluid. However the biological sample also comprises semisolid
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
20 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.
25 "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
30 or to an antigen.
The binding partner antibodies are for example either polyclonal
antibodies, or monoclonal antibodies.
Polyclonal antibodies can be obtained by imrnunisation of an animal
with the appropriate immunogen, followed by the recovery of the
35 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
5 the appropriate imrnunogen, 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
1 10 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 imrnunotransfer (Western blot) in one or two dimensions, by
15 imrtiunofluorescence, or using a biocaptor. The monoclonal antibodies
thus selected are subsequently purified in particular by the affinity
I
I chromatography technique described above.
The monoclonal antibodies can also be recombinant antibodies
obtained by genetic engineering, by techniques well known to the man
20 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
25 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 NanofitinesTM, small proteins marketed by the company
30 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
35 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.
A non-limiting list of these marker reagents consists in:
- metallic or alloy particles, such as particles of colloidal
gold,
polymer particles, such as coloured latex particles,
magnetic particles,
fluorescent molecules,
chemiluminescent molecules.
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.
FIGURES
20 Figure 1 :
Figure 1A is a view from above of an embodiment of the device of the
invention, before application of the sample. The device of the
invention comprises a support (not shown), a matrix 1 comprising a
sample application zone 2, a marking zone 3, a reaction zone 4
25 comprising a test results display zone 5 comprising means for
displaying the test results and a positive monitoring zone 6,
downstream of the test results display zone 5, comprising means for
performing positive monitoring and means for displaying the correct
functioning of the device and of the test. Optionally, the reaction
30 zone 4 can in addition comprise a migration monitoring zone 7 and
means for displaying the sample migration. Optionally, the matrix 1
can also comprise a sample absorption zone 8.
Figure 1B shows the results obtained with the device of figure 1~
after application of a negative sample for an analyte to be
35 determined.
Figure 1C shows the results obtained with the device of figure 1A
after application of a positive sample for an analyte to be
determined.
Figure ID shows a particular case in which the user cannot produce
results.
Although shown respectively at 7 and 8 in figures 1B to ID, the
migration monitoring zone 7 and absorption monitoring zone 8 are
optional in this embodiment.
~igure 2:
Figure 2 is a side view of the device of figure 1A.
Figure 3:
Figure 3 is a view from above of a particular embodiment of the device
of the invention, in which the matrix 1 is in the form of a U on its
side. The matrix 1 comprises the necessary means to display the test
results in zone 5 and means for performing the positive monitoring and
for displaying the correct functioning of the device and of the test.
In figure 3, the reaction zone 4 is shown in grey. The absorption zone
8 is optional. Figure 3 shows the results obtained after application
of a positive sample for an analyte to be determined.
Figure 4:
Figure 4 shows an embodiment of the invention, seen from above, in
which the matrix 1 is divided into two adjacent and parallel parts 1A
and 1B which are not in fluid communication with each other. The
matrix 1 comprises a sample application zone 2 divided into two parts
2A and 2B, a marking zone divided into two parts 3A and 3B and a
reaction zone divided into two parts 4A and 4B. Reaction zone 4A
comprises the test results display zone 5, a sample migration
monitoring zone 7 and the means for displaying the test results and
for displaying the sample migration. Reaction zone 4B comprises the
positive monitoring zone 6, the means for performing the positive
monitoring and the means for displaying the correct functioning of the
device and of the test in the monitoring zone 6. The test results
display zone 5 and monitoring zone 6 are adjacent and parallel with
each other. Optionally, the matrix 1 comprises in addition an
absorption zone 8 divided into two parts shown respectively at 8A and
8B. The liquid sample application zone 2, marking zone 3 and reaction
zone 4 are in fluid communication. Figure 4 shows the results obtained
after application of a positive sample for an analyte to be
determined.
Figure 5 :
Figure 5 shows another embodiment of the device according to the
invention which includes two adjacent and parallel reaction zones 4A
5 and 4B which are not in fluid communication with each other so that
the liquid sample migration occurs simultaneously and independently in
said zones. The matrix 1 comprises a sample application zone 2, a
marking zone 3 and a reaction zone 4 divided into 2 parts 4A and 4B.
Reaction zone 4A comprises the test results display zone 5, a sample
10 migration monitoring zone 7 and the means for displaying the test
results and for displaying the sample migration. Reaction zone 4B
comprises the positive monitoring zone 6 and the means for performing
the positive monitoring and for displaying the correct functioning of
the device and of the test in the monitoring zone 6. The test results
15 display zone 5 and monitoring zone 6 are adjacent and parallel with
each other. Optionally the matrix 1 comprises in addition an
absorption zone 8 divided into two parts 8A and 8B. The liquid sample
application zone 2, marking zone 3 and reaction zone 4 are in fluid
communication. Figure 5 shows the results obtained after application
1 20 of a positive sample for an analyte to be determined.
Figure 6:
Figure 6 shows an embodiment of the invention in which the matrix 1 is
divided into two adjacent and parallel parts 1A and 1C which are not
in fluid communication with each other. The matrix 1 comprises a
25 sample application zone 2, a marking zone divided into two parts 3A
and 3C and a reaction zone 4 divided into two parts 4A and 4B.
Reaction zone 4A comprises the test results display zone 5, a sample
migration monitoring zone 7 and the means for displaying the test
results and for displaying the sample migration. Reaction zone 4B
30 comprises the monitoring zone 6 and the means necessary to perform the
positive monitoring and for displaying the correct functioning of the
device and of the test. The test results display zone 5 and monitoring
zone 6 are adjacent and parallel with each other. The matrix 1, in its
part lC, comprises an analogue deposition zone 9. Optionally the
35 matrix 1 comprises an absorption zone 8 divided into two parts 8A and
8B. The liquid sample application zone 2, marking zone 3 and reaction
zone 4 are in fluid communication. Figure 6 shows the results obtained
after application of a positive sample for an analyte to be
determined.
5 EMBODIMENTS
In one embodiment, with reference to figure 1, the matrix 1 is shown
in the form of a rectangular strip the longitudinal axis of which is
in the horizontal position. Zones 2, 3 and 4 are in fluid
communication. Zone 3 comprises the first marked binding partner, for
10 example an antibody carrying a visible marker, such as a particle of
coloured latex, a particle of gold etc ... This reagent can migrate
freely across the matrix in the presence of the liquid sample
deposited in zone 2 and react with the analyte (antigen) to be
determined if it is present. In zone 5 of the matrix 1, the second
15 binding partner, for example an antibody having a specificity for an
epitope of the antigen which is different from that recognised by the
first marked antibody, is immobilised. In zone 6 of the matrix 1, an
analogue of the antigen is either imrnobilised directly or indirectly
or can migrate freely in the presence of the fluid flow in zone 6
20 until it is immobilised by a capture reagent which is identical to the
I second antibody.
Figures 1B and 1C show the functioning of the test in the presence of
negative control and of positive samples.
As shown in figure lB, the sample being a negative control, there is
25 no emission of a detectable signal in the results display zone 5. On
the contrary, there is emission of a detectable signal in the positive
monitoring zone 6, revealed, for example, by a line perpendicular to
the direction of the flow of the liquid sample, which means on the one
hand that the negative control sample has indeed migrated to zone 6
30 and on the other hand the device is operational.
As shown in figure I C , the sample being positive, There is emission of
a detectable signal in the results display zone 5 which is revealed by
a line perpendicular to the direction of flow of the liquid sample.
There is also emission of a detectable signal in the positive
35 monitoring zone 6, revealed, for example, by a line perpendicular to
the direction of the flow of the liquid sample, which means on the one
hand that the sample has indeed migrated and on the other hand that
the device is operational. The intensity of the signal in zones 5 and
6 will be a function of the sample load, as explained in more detail
in the following examples.
As shown in figure ID, there is no emission of detectable signal
5 either in the test results display zone 5, or in the monitoring zone
6. The results are uninterpretable and the test must be performed
again. In the case in which migration monitoring is provided in zone
7, the display of a migration signal allows it to be established that
these results are not due to physical malfunction of the device.
10
In another embodiment shown in figure 4, the matrix 1 is shown in
the form of two strips 1A and lB, the longitudinal axes of which are
in the horizontal position. The two strips 1A and 1B are adjacent and
parallel and are not in fluid communication with each other. Zones 2A,
15 3A and 4A are in fluid communication. Zones 2B, 3B and 4B are in fluid
communication. Zones 3A and 3B respectively comprise the first marked
binding partner, for example an antibody carrying a visible marker,
such as a particle of coloured latex, a particle of gold etc ... This
reagent can migrate freely across the matrix 1A in the presence of the
20 liquid sample deposited in the application zone 2A and react with the
I
1 analyte (antigen) to be determined if it is present. In zone 5 of the
matrix lA, the second binding partner, for example an antibody having
a specificity for an epitope of the antigen which is different from
that recognised by the first marked antibody, is immobilised. If the
25 antigen is present, there is emission of a detectable signal in the
results display zone 5 which is revealed, for example, by a line
perpendicular to the direction of the flow of the liquid sample, as
shown in figure 4. In addition, whether the sample is negative or
positive, a sample migration signal must appear in the migration
30 monitoring zone 7. The matrix 1B comprises in addition an analogue of
the antigen which is either immobilised directly or indirectly or can
migrate freely in the presence of the flow of the sample in zone 6
until it is immobilised by a capture reagent which is identical to the
second antibody.
In another embodiment shown in figure 5, the liquid sample is
deposited in the sample application zone 2 by any appropriate means.
After application, the liquid sample starts to migrate across the
matrix, enters into contact with the first marked binding partner
(marked antibody) in the marking zone 3 so that a marked analyte/first
binding partner complex is formed if the analyte is present in the
5 sample. The marked complex migrates with the flow of the sample to
reaction zones 4A and 4B respectively. The marked complex is on the
one hand immobilised in the test display zone 5 by binding with the
second specific binding partner of the analyte (antigen), which is
immobilised in this zone, so that a signal is generated in the results
10 display zone, preferably in the form of a line perpendicular to the
direction of displacement of the sample. The remaining fluid,
comprising the first marked binding partner and the marked complex on
the other hand migrates to the monitoring zone 6 comprising an
analogue of the antigen which is either immobilised directly or
15 indirectly in the matrix, or which is free to migrate with the flow of
the sample until it is immobilised in zone 6 by a capture reagent
which is identical to the second antibody, so that a signal is
generated in monitoring zone 6, preferably in the form of a line
perpendicular to the direction of displacement of the fluid and
20 parallel with the test line.
In another embodiment and with reference to figure 6, the matrix
1 is composed of two adjacent and parallel strips 1A and 1B which are
not in fluid communication with each other. Zones 2A, 3A and 4A are in
25 fluid communication. Zones 2C, 3C and 4C are in fluid communication.
Zones 3A and 3B respectively comprise the first marked binding
partner, for example an antibody carrying a visible marker, such as a
particle of coloured latex, a particle of gold etc ... This reagent can
migrate freely across the matrix 1A in the presence of the liquid
30 sample deposited in the application zone 2A and react with the analyte
(antigen) to be determined if it is present. In zone 5 of the matrix
lA, the second binding partner, for example an antibody having a
specificity for an epitope of the antigen which is different from that
recognised by the first marked antibody, is immobilised. If the
35 antigen is present, there is emission of a detectable signal in the
results display zone 5 which is revealed, for example, by a line
perpendicular to the direction of the flow of the liquid sample. In
1 addition, whether the sample is negative or positive, a sample
migration signal must appear in the migration monitoring zone 7. The
matrix 1C comprises the analogue of the antigen which is either
deposited in liquid form in a deposition zone 9, or is present in dry
I 5 form in zone 9 and is reconstituted by any appropriate means. The
matrix 1C comprises in addition an analogue of the antigen which is
either immobilised directly or indirectly or can migrate freely in the
presence of the flow of liquid in zone 6 until it is immobilised by a
capture reagent which is identical to the second antibody.
10
EXAMPLES
I
Example 1 - T e s t i n g for the antigen HBs
15 Testing for the antigen HBs by the test illustrated in figure 1
consists in a sandwich type immunological reaction in one step based
on an immunochromatographic technique.
Red latex particles marketed by the company Magsphere (trade name) are
coated with a mixture of two anti-HBs monoclonal antibodies
20 (bioMerieux, 2G2G10A12 and 6H6B6), at a respective concentration of
500 pg/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
25 bioMerieux, which is coated on a nitrocellulose membrane CN 140
(Sartorius - 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 101011FFU04), developed by
30 biomerieux, 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 mrn 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
35 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 Fran~ais du Sang (EFS) of the Rh8ne-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.
i 20
i The results are presented in table 1 below:
Table 1
The results show that in the case of a negative sample, only the
25 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
Samples
Map60 (high positive)
Map59 (high positive)
Map60 (medium positive)
Map59 (medium positive)
Map64 (low positive)
Map60 (low positive)
Negative serum pool
HBs Ag batch 101011FFU04 (bioMerieux)
Test Line
15 minutes
L 7
L 7
L5
L5
L 4
L4
L1
Positive Control Line
15 minutes
L7
L7
L8
L8
L8
L8
L8
* 16
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
5 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
10 form a second complex at the control line 6.
Example 2 - Testing for the influenza A antigen
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
15 anti-influenza A monoclonal detection antibody (bioMerieux, 15C9H2) is
immobilised on red particles (Magsphere). 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, bioMerieux) immobilised directly on the
20 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 mrn 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
25 deposition of the sample.
A concentration range of the recombinant protein (Nucleoprotein INF A,
bioMerieux) was tested. The negative sample tested is a PBS buffer.
The results are presented in table 2 below:
Table 2
Samples
NP A INF (40 pg/ml)
NP A INF (1 pg/ml)
NP A INF (50 ng/ml)
Influenza A batch 101011FFU05 (bioMerieux)
Test Line
10 minutes
L10
L10
L7
Positive .Control Line
10 minutes
L6
L8
L10
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
5 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
10 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
pg/ml to 10 ng/ml), antibodies on the particle can remain available
15 and form a second complex at the control line 6.
Example 3 - Testing for anti-HIV-1 group M antibody
I The detection is based on the same principles as those described in
examples 1 and 2, i.e. one-step sandwich type immunochromatographic
20 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.
L10
L10
NP A INF (10 ng/ml)
bioMerieux buffer batch
100923FFU03
Blue latex particles marketed by the company VARIAN (trade name) are
25 coated with specific peptides of the HIV-1 group M virus. These
L4
L1
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
30 monoclonal antibody (bioMerieux, P12GllB10) 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 RhBne Alpe
region.
The results are presented in table 3 below:
Table 3
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.
HIV 1/2
MARHIV0023 (SCI13) HIVl M
MARHIV0024 (SCI14) HIVl M
MARHIV0025 (SCI15) HIV2
MARHIV0032 (SCI22) HIV group 0
Negative serum pool
HIV sZ batch 101011FFU03
Test Line
Reading time
30 minutes
L10
L10
L9
L 8
L1
Positive Control Line
Reading time 30 minutes
L1
L1
L6
L6
L6

CLAIMS
Device for performing a test to determine the presence or the
absence of at least one
analyte in a liquid sample comprising:
a) a support,
b)' a matrix (I), fixed on the support, which allows the
migration of the liquid sample, said matrix comprising:
(i) a liquid sample application zone (2),
10 - (ii) a marking zone (3) comprising at least a first marked
binding partner which is able to bind to said at least one
analyte, if it is present in the liquid sample, and which is
able to bind to at least one analogue of the analyte, and
- (iii) at least one reaction zone (4) comprising:
15 - a test results display zone (5) comprising at least a second
immobilised binding partner which is able to bind to said at
I least one analyte, and
I - a monitoring zone (6) downstream of the results display zone (5)
or parallel with the results display zone (5) which allows
20 monitoring of the correct functioning the device and which
comprises at least one analogue of said at least one analyte
which is able to bind to said at least first marked binding
partner;
- said liquid sample application zone ( 2 ) , marking zone (3) and
25 reaction zone (4) being in fluid communication.
2. Device according to claims 1, in which the first binding partner
and the second binding partner are selected from the group consisting
in antibody, antibody mixture, antibody fragment, mixture of antibody
30 fragments, antibody analogue, mixture- of antibody analogues, antigen,
mixture of antigens, protein, mixture of proteins, polypeptide,
mixture of polypeptides, peptide and mixture of peptides.
3. Device according to claim 1, in which the monitoring zone (6)
35 comprises in addition a capture reagent of the analogue, immobilised
on the matrix, and to which said one analogue is able to bind.
I 20
4. Device according to claim 1, in which in the zone (6) the analogue
is irnmobilised on the matrix directly or indirectly.
5. Device according to claim 3, in which the capture reagent of the
5 analogue is a reagent which is identical to the second binding partner
of the test results display zone.
I 6. Device according to claim 4, in which the analogue of the analyte
I is immobilised on the matrix by a reagent selected from the group 1 10 consisting in antibody, mixture(s) of antibodies, antibody fragment,
I mixture of antibody fragments, antibody analogue, mixture of antibody
analogues, antigen, m.. .i xture of antigens, protein, mixture of proteins,
polypeptide and mixture of polypeptides, peptide, mixture of peptides,
and biotin/steptavidin and biotin/avidin receptor.
I 7. Device according to claim 1, in which the analogue of the analyte
is 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,
i 20 protein, mixture of proteins, polypeptide, mixture of polypeptides,
peptide and mixture of peptides.
8. Device according to claim 1, in which
(i) the first binding partner is an antibody, an antibody fragment or
25 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, or
(ii) the first binding partner is a protein, a polypeptide or a
peptide, the second binding partner is a protein, a polypeptide or a
30 peptide, and the analogue of the analyte is an antibody, an antibody
fragment or an antibody analogue.
9. Device according to claim 8, in which the analogue of the analyte
can be immobilised on the matrix, by a capture reagent which is an
35 identical reagent to the second binding partner.
10. Device according to claim 1, in which the first binding partner is
marked by a detectable marker. u
. 11. Device according to claim 10, in which the detectable marker is a '
compound, a substance or a particle which can be detected by visual,
5 fluorescent or instrumental means and in particular the detectable
marker can be a coloured latex particle, a gold particle, or a
magnet-ic particle.
12. Device according to any one of the preceding claims in which the
10 correct functioning of the device and of the test is revealed in the
form of a detectable line which is substantially perpendicular to the
direction of the flow of the liquid sample.
13. Device according to any one of the preceding. claims, in which the
15 monitoring zone (6) is downstream of the results display zone (5).
14. Device according to claim 1, in which the reaction zone 4
comprises two adjacent and parallel reaction zones (4A and 4B) which
are not in fluid communication with each other.
20
15. Device according to claim 14, in which the monitoring zone (6) is
parallel with the results display zone (5).
16. Device according to claim 14, comprising in addition monitoring of
25 liquid sample migration monitoring ( 7 ) .
17. Method for perfo2ing a test to determine the presence or the
absence of an analyte in a liquid sample comprising the steps of:
placing the liquid sample in contact with a device such as defined in
30 any one of the preceding claims and determining whether the sample
does not comprise or comprises the analyte by reference to the absence
or to the presence of a detectable signal in the test results display
zone, and when there is no signal in the test results display zone,,
confirming the correct functioning of the test by reference to the
35 presence of a signal in the monitoring zone.
A
- - -___ . . _ _ _ - _ .. . . _ ._
Dated . this 15.05.2013 W>d&
[NEHA SRNASTAVA]
OF REMFRY &, SAGAR
ATTORNEY FOR THE APPLICANT[S]