U.S. patent application number 12/987503 was filed with the patent office on 2012-01-19 for assay device with shared zones.
Invention is credited to Stephen Paul Sharrock.
Application Number | 20120015448 12/987503 |
Document ID | / |
Family ID | 39812288 |
Filed Date | 2012-01-19 |
United States Patent
Application |
20120015448 |
Kind Code |
A1 |
Sharrock; Stephen Paul |
January 19, 2012 |
ASSAY DEVICE WITH SHARED ZONES
Abstract
Disclosed is an assay device for determining the presence and/or
extent of one or more analytes in liquid sample containing a) first
and second assays each comprising a flow-path having a detection
zone for immobilising a labelled binding reagent, wherein detection
of a labelled binding reagent at one or both detection zones is
indicative of the presence and/or extent of one or more analytes;
b) a shared reference zone; c) one or more light sources to
illuminate the detection zones and the reference zone; d) one or
more photodetectors to detect light from the detection zones and
the reference zone, which photodetector/s generate a signal, the
magnitude of which signal is related to the amount of light
detected; and e) signal processing means for processing signals
from the photodetector/s.
Inventors: |
Sharrock; Stephen Paul;
(Bedford, GB) |
Family ID: |
39812288 |
Appl. No.: |
12/987503 |
Filed: |
January 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12199284 |
Aug 27, 2008 |
7879624 |
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12987503 |
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60991543 |
Nov 30, 2007 |
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Current U.S.
Class: |
436/501 ;
422/401 |
Current CPC
Class: |
G01N 21/6428 20130101;
Y10S 436/805 20130101; G01N 33/54366 20130101; G01N 2201/062
20130101; G01N 2021/6439 20130101; G01N 33/545 20130101; G01N
2201/12 20130101; Y10S 435/808 20130101; G01N 21/76 20130101; G01N
2458/00 20130101; G01N 33/558 20130101; G01N 21/8483 20130101; G01N
33/76 20130101 |
Class at
Publication: |
436/501 ;
422/401 |
International
Class: |
G01N 21/75 20060101
G01N021/75 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 1, 2007 |
GB |
0717045.9 |
May 31, 2008 |
GB |
0809994.7 |
Claims
1. An assay device for determining the presence and/or extent of
one or more analytes in a liquid sample comprising: a) first and
second assays each comprising a flow-path having a detection zone
for immobilising a labelled binding reagent, wherein detection of a
labelled binding reagent at one or both detection zones is
indicative of the presence and/or extent of one or more analytes;
b) a shared reference zone; c) one or more light sources to
illuminate the detection zones and the reference zone; d) one or
more photodetectors to detect light from the detection zones and
the reference zone, which photodetector generates a signal, the
magnitude of which signal is related to the amount of light
detected; and e) signal processing means for processing signals
from the photodetector.
2. The assay device according to claim 1, wherein the first and/or
second assay comprises a labelled binding reagent for an analyte or
analyte analogue provided in a mobilisable form upstream from the
detection zone in a dry state prior to use of the device.
3. The assay device according to claim 1, wherein the first and/or
second assay comprises a binding reagent for an analyte or a
labelled binding reagent provided in an immobilised form at the
detection zone.
4. The assay device according to claim 1, further comprising a
shared control zone.
5. The assay device according to claim 1, wherein the shared
reference zone is comprised as part of either the first or second
assay.
6. The assay device according to claim 4, wherein the control zone
is comprised as part of either the first or second assay.
7. The assay device according to claim 4, wherein the control zone
is comprised as part of one assay and the shared reference zone is
comprised as part of the other assay.
8. The assay device according to claim 1, wherein the first assay
and second assay are capable of detecting the presence of an
analyte in different concentration ranges.
9. The assay device according to claim 1, wherein the flow-paths of
the first and second assays each comprise a porous carrier.
10. The assay device according to claim 9, wherein the porous
carrier comprises nitrocellulose.
11. The assay device according to claim 1, comprising a single
porous sample receiver provided upstream from the first and second
assays.
12. The assay device according to claim 1, further comprising a
sink provided at the distal end of the assay flow-paths.
13. The assay device according to claim 1, wherein the first and
second assays each comprise different amounts of labelled binding
reagent.
14. The assay device according to claim 1, comprising a first assay
for the detection of an analyte in a lower range and a second assay
for the detection of analyte in a higher range.
15. The assay device according to claim 14 wherein the second assay
has a greater amount of labelled binding reagent than the first
assay.
16. The assay device according to claim 1, wherein the first assay
comprises a labelled binding reagent for the analyte provided
upstream from a detection zone and the second assay comprises a
labelled binding reagent for the analyte and a second binding
reagent for the analyte provided upstream from the detection
zone.
17. The assay device according to claim 16, wherein the first assay
comprises a shared reference zone and the second assay comprises a
shared control zone.
18. The assay device according to claim 17, wherein the reference
zone is provided downstream from the detection zone.
19. The assay device according to claim 17, wherein the control
zone is provided downstream from the detection zone.
20. The assay device according to claim 1, wherein a photodetector
detects light from a plurality of zones.
21. The assay device according to claim 20, comprising a single
photodetector to detect light from the two detection zones, the
reference zone and the control zone.
22. The assay device according to claim 21, comprising four light
sources to illuminate the two detection zones, the reference zone
and the control zone.
23. The assay device according to claim 20, wherein the amount of
light detected by the photodetector from the detection zones and
the reference zone is measured prior to addition of sample to the
assay device and again after addition of sample to the assay
device, and a ratio of the two measurements calculated for each
zone.
24. The assay device according to claim 20, wherein a normalised
percentage relative attenuation (% A) is calculated for the
detection and/or control zones wherein: HS t ( % A ) = Ref ratio t
- HS test ratio t Ref ratio t .times. 100 % ##EQU00003## LS t ( % A
) = Ref ratio t - LS test ratio t Ref ratio t .times. 100 %
##EQU00003.2## Ctrl t ( % A ) = Ref ratio t - Ctrl ratio t Ref
ratio t .times. 100 % ##EQU00003.3##
25. The assay device according to claim 1, wherein the light source
comprises one or more LEDs.
26. The assay device according to claim 1, wherein the analyte to
be determined is hCG.
27. The assay device according to claim 1, wherein the liquid
sample is urine.
28. A method of performing an assay to determine the presence
and/or extent of an analyte in a liquid sample, the method
comprising the step of contacting the sample with the assay device
of claim 1.
29. An assay device for determining the presence and/or extent of
one or more analytes in a liquid sample comprising: a) first and
second assays each comprising a flow-path having a detection zone
for immobilising a labelled binding reagent, wherein detection of a
labelled binding reagent at one or both detection zones is
indicative of the presence and/or extent of one or more analytes;
b) a shared control zone; c) one or more light sources to
illuminate the detection zones and the control zone; d) one or more
photodetectors to detect light from the detection zones and the
control zone, which photodetector generates a signal, the magnitude
of which signal is related to the amount of light detected; and e)
signal processing means for processing signals from the
photodetector.
Description
RELATED APPLICATIONS
[0001] The present application claims benefit of priority to U.S.
Provisional Patent Application No. 60/991,543, filed on Nov. 30,
2007; Great Britain Application 0809994.7, filed May 31, 2008; and
Great Britain Application 0717045.9, filed Sep. 1, 2007, the
contents of which are incorporated by reference herein in their
entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to an assay device, kit and
method for determining the presence or extent of an analyte. In
particular it relates to the determination of an analyte over an
extended concentration range.
BACKGROUND OF THE INVENTION
[0003] Simple lateral flow immunoassay devices have been developed
and commercialised for detection of analytes in fluid samples, see
for example EP291194. Such devices typically comprise a porous
carrier comprising a dried mobilisable labelled binding reagent
capable of binding to the analyte in question, and an immobilised
binding reagent also capable of binding to the analyte provided at
a detection zone downstream from the labelled binding reagent.
Detection of the immobilised labelled binding reagent at the
detection zone provides an indication of the presence of analyte in
the sample.
[0004] Alternatively, when the analyte of interest is a hapten, the
immunoassay device may employ a competition reaction wherein a
labelled analyte or analyte analogue competes with analyte present
in the sample for an immobilised binding reagent at a detection
zone. Alternatively the assay device may employ an inhibition
reaction whereby an immobilised analyte or analyte analogue is
provided a detection zone, the assay device comprising a
mobilisable labelled binding reagent for the analyte.
[0005] An assay device may determine more than one analyte. For
example in the case of assays for the determining the presence of
drugs of abuse, the device may be capable of determining a whole
panel of drugs. Such lateral flow immunoassay devices are provided
with multiple detection zones, such zones being provided on a
single or multiple lateral flow carriers.
[0006] Determination of the result of the assay has been
traditionally carried out by eye. However such devices require the
result to be interpreted by the user which introduces an
undesirable degree of subjectivity.
[0007] As such, digital devices have been developed comprising an
optical detection means arranged to determine the result of the
assay as well as a display means to display the result of the
assay. Digital assay readers for use in combination with assay
test-strips for determining the concentration and/or amount of
analyte in a fluid sample are known as are assay devices comprising
an integral digital assay reader.
[0008] Light from a light source, such as a light emitting diode
(LED), is shone onto a portion of the porous carrier and either
reflected or transmitted light is detected by a photodetector.
Typically, the reader will have more than one LED to illuminate
various zones of the carrier, and a corresponding photodetector is
provided for each of the plurality of LEDs. EP1484601 discloses an
optical arrangement for a lateral flow test strip digital reading
device comprising a baffle arrangement allowing for the possibility
of reducing the number of photodetectors in the device.
[0009] Such devices are often designed to be single use and
therefore it is desirable to keep the costs of such devices as low
as possible, especially where expensive optical and electronic
components are involved.
SUMMARY OF THE INVENTION
[0010] It is an object according to an aspect of the invention to
provide an assay device having one or more shared zones enabling a
reduction in the number of optical components that are required for
an assay device comprising two or more assay flow-paths.
[0011] According to a first aspect, the invention provides an assay
device for determining the presence and/or extent of one or more
analytes in a liquid sample comprising:
a) first and second assays each comprising a flow-path having a
detection zone for immobilising a labelled binding reagent, wherein
detection of a labelled binding reagent at one or both detection
zones is indicative of the presence and/or extent of one or more
analytes; b) a shared reference zone; c) one or more light sources
to illuminate the detection zones and the reference zone; d) one or
more photodetectors to detect light from the detection zones and
the reference zone, which photodetector/s generate a signal, the
magnitude of which signal is related to the amount of light
detected; and e) signal processing means for processing signals
from the photodetector/s.
[0012] It is a further object of the invention to provide an assay
reader for use with one or more assay test-strips comprising two or
more assay flow-paths, the assay reader having a reduction in the
number of optical components that are typically required.
[0013] According to a second aspect, the invention provides an
assay reader for reading the result of first and second assays each
comprising a flow-path, each flow-path comprising a detection zone
for immobilising labelled binding reagent, wherein detection of
labelled binding reagent at one or both detection zones is
indicative of the presence and/or extent of one or more analytes,
and a shared reference zone; said assay reader comprising: [0014]
a) one or more light sources to illuminate the detection zones, and
the shared reference zone; [0015] b) one or more photodetectors to
detect light from the detection zones and the reference zone, which
photodetector/s generate a signal, the magnitude of which signal is
related to the amount of light detected; and signal processing
means for processing signals from the photodetector/s, wherein the
signal obtained from the shared reference zone is used to
compensate the values of the signals obtained from the detection
zones.
[0016] The first and/or second assay may comprise a labelled
binding reagent provided in a mobilisable form upstream from the
detection zone in a dry state prior to use of the device.
[0017] The shared reference zone may be comprised as part of either
the first or second assay. Alternatively the reference zone may be
provided on a subsidiary flow-path to the first and second assay.
The reference zone may be chosen from a portion of the flow-path
not corresponding to a detection zone, or, where a dried labelled
reagent is present upstream from the detection zone, a portion not
corresponding to where the dried labelled reagent is present. The
reference zone may be provided downstream or upstream from the
detection zone. Measurement of the reference zone enables
measurement of the background levels of reflected or transmitted
light from the flow-path. The background level may be affected by,
for example, the optical reflectance of the porous carrier, the
presence of liquid sample, or of components of the assay such as a
labelled binding reagent. The levels of light measured at the
detection zone may therefore be corrected with respect to the
levels of background light to provide a compensated signal more
accurately indicative of the amount of labelled binding reagent
present at the detection zone. Measurement at the reference zone
also compensates for any variation between fluid samples applied to
assay devices, for example urine samples may vary widely in colour.
The value of the signal obtained at the reference zone for one
assay is used to compensate the value of the signal obtained at the
detection zone for the other assay. As such the reference zone is
"shared" between both assays. The provision of a shared reference
zone can reduce the number of components required for the assay
device, since each reference zone would typically require a light
source.
[0018] The concept of a shared reference zone is rather
counter-intuitive. The purpose of a reference measurement is to
allow for variations in the background readings of signals which
can arise, inter alia, as a result of variations in reagent or
assay strip composition. Accordingly, the normal practice is to use
a separate reference zone on each assay, so that a "dedicated"
reference measurement can be made for each assay.
[0019] The present inventors have found however that separate
reference zones can be dispensed with and instead a single shared
reference zone will suffice.
[0020] The light source is conveniently an LED. A plurality of LEDs
may be employed. In an embodiment each zone in the assay
(detection, reference or control zone) is illuminated by a
respective LED. The one or more photodetectors may conveniently
comprise a photodiode. In a preferred embodiment a single
photodiode or other photodetector is employed. In one embodiment
there are four LEDs and a single photodiode.
[0021] The assay device may further comprise a control zone which
may be a single control zone provided as part of either the first
or second assay. Alternatively the control zone may be provided on
a subsidiary flow-path to the first and second assay. Provision of
a single control zone reduces further the number of light sources
that are needed. The purpose of the control zone is to indicate
that the assay has been carried out correctly, namely that fluid
sample has been applied to the device and that labelled binding
reagent has moved along the flow-path to some extent. The control
zone may be provided downstream from the detection zone. A suitable
control zone is disclosed in EP291194 and may comprise an
immobilised binding reagent for a labelled binding reagent. A
separate population of labelled binding reagent may be provided
upstream from the detection and control zones wherein said separate
population of labelled binding reagent is capable of being
immobilised at the control zone but does not become immobilised at
the detection zone in the presence or absence of analyte. The
control zone is typically provided downstream from the detection
zone. The signal obtained at the control zone may also be
referenced with respect to the signal obtained at the reference
zone.
[0022] Thus measurement of the signal at the control zone provides
a value or indication that the test has been carried out correctly
(or incorrectly) for that assay. If for example, the control zone
indicates that the test has been carried out correctly for one
assay, an assumption is made that the test has been carried out
correctly at the other assay. Hence the control zone may be thought
of as being "shared" between the assays of the assay device. As in
the case of a shared reference zone, provision of a single or
"shared" control zone enables a reduced number of optical
components to be used in the device. The rationale for making this
assumption is that it is highly likely that if liquid sample has
been applied to one assay flow-path, that liquid sample has been
applied to the other flow-path, especially so if the two assay
flow-paths are connected, for example by a common sample receiving
means such as a porous sample receiver. Furthermore, if the assay
device has for example been subjected to conditions, such as
ingress of moisture which may for example result in poor
resuspension of the mobilisable reagents, or high temperature which
may denature the binding reagents, it is likely that both
flow-paths will be affected. As in the case of a shared reference
zone, the concept of a shared control is also counter-intuitive.
The signal at the control zone is calculated with respect to the
signal at the reference zone.
[0023] The reference and control zones may be provided as part of
the same assay or provided as part of different assays. In an
exemplary embodiment, the shared reference and control zones are
each provided in separate assays, e.g. one assay comprises a
detection zone and a reference zone, and the other assay comprises
a detection zone and a control zone.
[0024] According to a third aspect the invention provides an assay
device for determining the presence and/or extent of one or more
analytes in a liquid sample comprising:
a) first and second assays each comprising a flow-path having a
detection zone for immobilising a labelled binding reagent, wherein
detection of a labelled binding reagent at one or both detection
zones is indicative of the presence and/or extent of one or more
analytes; b) a shared control zone; c) one or more light sources to
illuminate the detection zones and the control zone; d) one or more
photodetectors to detect light from the detection zones and the
control zone, which photodetector/s generate a signal, the
magnitude of which signal is related to the amount of light
detected; and e) signal processing means for processing signals
from the photodetector/s.
[0025] According to a fourth aspect, the invention provides an
assay reader for reading the result of first and second assays each
comprising:
a flow-path, each flow-path comprising a detection zone for
immobilising labelled binding reagent, wherein detection of
labelled binding reagent at one or both detection zones is
indicative of the presence and/or extent of one or more analytes;
and a shared control zone; said assay reader comprising: [0026] a)
one or more light sources to illuminate the detection zones, and
the shared control zone; [0027] b) a stored control signal
threshold [0028] c) one or more photodetector/s to detect light
from the detection zones and the control zone, which
photodetector/s generate a control signal and detection signals,
the magnitude of which signals are related to the amount of light
detected; and [0029] d) signal processing means to process signals
from the photodetector/s and to compare the signal obtained from
the control zone to the control signal threshold, and to determine
that both assays have been carried out correctly if the control
signal is equal to or greater than the control signal
threshold.
[0030] The assay device and reader according to the first, second
and third aspects of the invention may comprise a control signal
threshold.
[0031] The control signal threshold may be stored in the device or
reader. The signal measured from the control zone may be compared
to the control signal threshold to determine whether sufficient
labelled binding reagent has become immobilised at said zone. If
the value of the control signal is equal to or exceeds the control
signal threshold, the device or reader may determine that the assay
has been carried out satisfactorily. If the control signal is less
than the control signal threshold, the device or reader may
determine that the assay has been not been carried out
satisfactorily and will provide an error message.
[0032] The signal detected from the control zone may be referenced
to a signal obtained from a reference zone.
[0033] The assay device according to the third aspect may also
comprise a shared reference zone.
[0034] The first and/or second assay may conveniently comprise a
binding reagent for an analyte or a labelled binding reagent
provided in an immobilised form at the detection zone.
[0035] By employing a shared reference and/or a shared control
zone, the assay device of the invention provides for reduced number
of zones that need to be interrogated and consequently the number
of optical components that need to be employed. The use of shared
zones is most effective when the assay architectures of the first
and second assays are very similar or, if the reference and/or
control zone is provided on one or more subsidiary flow-paths, when
the assay architecture of the one or more subsidiary flow-paths is
similar to the first and second assays. Thus, for example, the
assays will typically both comprise porous carriers of similar
material (e.g. both comprise nitrocellulose carriers). It is also
advantageous to use the same liquid sample for each assay. This may
be conveniently achieved by providing a common sample application
region that is in fluid communication with both assays. Thus a
single liquid sample applied to the device via the common sample
application region may flow through both the first and second
assays. In cases where the first and second assays are
non-identical, it may be acceptable to provide a shared reference
zone as long as background levels of light that would be detected
at each assay are sufficiently similar to each other.
[0036] The signal processing means may comprise a central processor
unit which is able to process the signals obtained from the
photodetectors from the respective zones and calculate values
obtained at the test zone with respect to the reference zone. The
measurement data may be taken at various times during the assay and
may be taken after the device has been switched on but before fluid
sample has been applied to the device, in order to obtain optical
values of light transmission or reflectance in the dry state.
[0037] An absorbent "sink" can be provided at the distal end of the
assay flow-paths. A common sink may be provided or a sink may be
provided at the distal end of each assay. The absorbent sink may
preferably comprise a highly absorbent material such as, for
example, CF7 Whatman paper, and should provide sufficient
absorptive capacity to remove any unbound conjugate from the
vicinity of the detection zones, the reference zone and the control
zone. As an alternative to such a sink it can be sufficient to have
a length of porous solid phase material which extends beyond the
detection zone. An advantage of providing a highly absorbent sink
is that it removes or substantially removes excess labelled binding
reagent from the flow-paths of the respective assays. This has the
effect of minimising the extent of unbound labelled binding reagent
in the vicinity of respective zones and therefore enables assay
flow paths to be employed in the device that may have differing
amounts of labelled binding reagent.
[0038] As an alternative to providing an immobilised binding
reagent at the detection zone, the binding reagent may be provided
in a mobilisable form which is capable of binding to an
analyte-labelled binding reagent complex. The binding reagent may
for example be conjugated to a large particle such as agarose and
the detection zone may comprise a filter whose pore-size has
dimensions smaller than the large particle, but larger than the
size of the labelled binding reagent, such that the filter is able
to trap any labelled binding reagent/analyte/binding reagent
complex present, any labelled binding reagent that is not complexed
to the capture reagent being able to pass through the filter. Yet
alternatively a reagent may be provided in an immobilised form at
the detection zone that is capable of binding a mobilisable
labelled binding reagent/analyte/binding reagent complex. For
example the binding reagent may be provided in a mobilisable form
and conjugated to a binding species such as biotin, the reagent
immobilised at the detection zone being a complementary binding
partner such as streptavidin.
[0039] The assay device may employ a sandwich immunoassay and/or a
competitive/inhibition assay for the determination of an analyte.
An example of a sandwich immunoassay is where a labelled binding
reagent/analyte/binding reagent complex is formed. The device will
typically comprise a labelled binding reagent for the analyte in a
mobilisable form provided upstream from a detection zone comprising
an immobilised binding reagent for the analyte. Alternatively, in
particular when the analyte of interest is a hapten, the
immunoassay device may employ a competition reaction wherein a
labelled analyte or labelled analyte analogue competes with analyte
present in the sample for an immobilised binding reagent at a
detection zone. The labelled analyte or labelled analyte analogue
may be provided in a mobilisable form upstream from the detection
zone. Yet alternatively the assay device may employ an inhibition
reaction wherein an immobilised analyte or analyte analogue is
provided at detection zone, the assay device comprising a
mobilisable labelled binding reagent for the analyte.
[0040] The term "flow-path" for the purposes of this invention
refers to a substrate that is able to convey a liquid from a first
position to a second position and may be for example a capillary
channel, a microfluidic pathway, or a porous carrier such as a
lateral flow porous carrier. The porous carrier may comprise one or
a plurality of porous carrier materials which may overlap in a
linear or stacked arrangement or which are fluidically connected.
The porous carrier materials may be the same or different. The
first and second assays may be provided on separate substrates or
they may be provided on a common substrate such that liquid being
conveyed along a flow-path of the first assay is not able to cross
over to the flow-path of the second assay. For example, the first
and second assays may be provided on the same porous carrier such
that the first and second flow-paths are isolated from each other.
This may be achieved for example by laser cutting parts of the
porous carrier to make it non-porous, thus separating the first and
second assays. Alternatively, a non-porous blocking material may be
applied along a strip to provide two (typically essentially
parallel) flow paths on the same porous carrier.
[0041] In particular the flow-path may be a lateral flow porous
carrier. Suitable materials that may be employed as a porous
carrier include nitrocellulose, acetate fibre, cellulose or
cellulose derivatives, polyester, polyolefin or glass fibre. The
porous carrier may comprise nitrocellulose. This has the advantage
that a binding reagent can be immobilised firmly without prior
chemical treatment. If the porous solid phase material comprises
paper, for example, the immobilisation of the antibody in the
second zone needs to be performed by chemical coupling using, for
example, CNBr, carbonyldiimidazole, or tresyl chloride.
[0042] The term "binding reagent" refers to a member of a binding
pair, i.e., two different molecules wherein one of the molecules
binds with the second molecule through chemical and/or physical
means. The two molecules are related in the sense that their
binding with each other is such that they are capable of
distinguishing their binding partner from other assay constituents
having similar characteristics. The members of the binding pair are
referred to as ligand and receptor (antiligand), a binding pair
member and binding pair partner, and the like. A molecule may also
be a binding pair member for an aggregation of molecules; for
example an antibody raised against an immune complex of a second
antibody and its corresponding antigen may be considered to be an
binding pair member for the immune complex.
[0043] In addition to antigen and antibody binding pair members,
other binding pairs include, as examples without limitation, biotin
and avidin, carbohydrates and lectins, complementary nucleotide
sequences, complementary peptide sequences, effector and receptor
molecules, enzyme cofactors and enzymes, enzyme inhibitors and
enzymes, a peptide sequence and an antibody specific for the
sequence or the entire protein, polymeric acids and bases, dyes and
protein binders, peptides and specific protein binders (e.g.,
ribonuclease, S-peptide and ribonuclease S-protein), and the like.
Furthermore, specific binding pairs can include members that are
analogues of the original specific binding member.
[0044] "Label" when used in the context of a labelled binding
reagent, refers to any substance which is capable of producing a
signal that is detectable by visual or instrumental means. Various
labels suitable for use in the present invention include labels
which produce signals through either chemical or physical means,
such as being optically detectable. Such labels include enzymes and
substrates, chromogens, catalysts, fluorescent compounds,
chemiluminescent compounds, electroactive species, dye molecules,
radioactive labels and particle labels. The analyte itself may be
inherently capable of producing a detectable signal. The label may
be covalently attached to the binding reagent. In particular the
label may be chosen from one that is optically detectable.
[0045] The label may comprise a particle such as gold, silver,
colloidal non-metallic particles such as selenium or tellurium,
dyed or coloured particles such as a polymer particle incorporating
a dye, or a dye sol. The dye may be of any suitable colour, for
example blue. The dye may be fluorescent. Dye sols may be prepared
from commercially-available hydrophobic dyestuffs such as Foron
Blue SRP (Sandoz) and Resolin Blue BBLS (Bayer). Suitable polymer
labels may be chosen from a range of synthetic polymers, such as
polystyrene, polyvinyltoluene, polystyrene-acrylic acid and
polyacrolein. The monomers used are normally water-insoluble, and
are emulsified in aqueous surfactant so that monomer micelles are
formed, which are then induced to polymerise by the addition of
initiator to the emulsion. Substantially spherical polymer
particles are produced. An ideal size range for such polymer
particles is from about 0.05 .mu.m to about 0.5 .mu.m. According to
an exemplary embodiment the label is a blue polymeric particle.
[0046] The dried binding reagents may be provided on a porous
carrier material provided upstream from a porous carrier material
comprising the detection zone. The upstream porous carrier material
may be macroporous. The macroporous carrier material should be low
or non-protein-binding, or should be easily blockable by means of
reagents such as BSA or PVA, to minimise non-specific binding and
to facilitate free movement of the labelled reagent after the
macroporous body has become moistened with the liquid sample. The
macroporous carrier material can be pre-treated with a surface
active agent or solvent, if necessary, to render it more
hydrophilic and to promote rapid uptake of the liquid sample.
Suitable materials for a macroporous carrier include plastics
materials such as polyethylene and polypropylene, or other
materials such as paper or glass-fibre. In the case that the
labelled binding reagent is labelled with a detectable particle,
the macroporous body may have a pore size at least ten times
greater than the maximum particle size of the particle label.
Larger pore sizes give better release of the labelled reagent. As
an alternative to a macroporous carrier, the labelled binding
reagent may be provided on a non-porous substrate provided upstream
from the detection zone, said non-porous substrate forming part of
the flow-path.
[0047] The porous carrier may comprise a glass-fibre macroporous
carrier provided upstream from and overlapping at its distal end a
nitrocellulose porous carrier.
[0048] The liquid sample can be derived from any source, such as an
industrial, environmental, agricultural, or biological source. The
sample may be derived from or consist of a physiological source
including blood, serum, plasma, interstitial fluid, saliva, sputum,
ocular lens liquid, sweat, urine, milk, mucous, synovial liquid,
peritoneal liquid, transdermal exudates, pharyngeal exudates,
bronchoalveolar lavage, tracheal aspirations, cerebrospinal liquid,
semen, cervical mucus, vaginal or urethral secretions and amniotic
liquid. In particular the source may be human and in particular the
sample may be urine.
[0049] "Light" as used herein is intended to encompass any suitable
electromagnetic radiation, regardless of wavelength.
Notwithstanding this, the invention is primarily intended to
utilise light in the visible part of the spectrum, and "light
source" and "photodetector" should be construed accordingly as
encompassing respectively any source of, and means for detecting,
electromagnetic radiation, but especially relating to radiation of
visible wavelengths (i.e. in the range of about 390-800 nm).
[0050] The photodetector/s will detect light from one or more zones
of the assay device. The light may actually originate from those
zones, for example, if the label is fluorescent or the like. More
normally however, the photodetector/s will detect light which
appears to emanate from those zones i.e., light which originates
from the light source and is reflected and/or transmitted by the
zone onto the photodetector.
[0051] Analytes include, but are not limited to, toxins, organic
compounds, proteins, peptides, micro-organisms, bacteria, viruses,
amino acids, nucleic acids, carbohydrates, hormones, steroids,
vitamins, drugs (including those administered for therapeutic
purposes as well as those administered for illicit purposes),
pollutants, pesticides, and metabolites of or antibodies to any of
the above substances. The term analyte also includes any antigenic
substances, haptens, antibodies, macromolecules, and combinations
thereof.
[0052] The assay device may determine one or more analytes.
[0053] The assay device may be capable of determining the amount or
presence of an analyte over an extended analyte range, wherein the
first assay is capable of determining the level of analyte at a
lower concentration range and the second assay is capable of
determining the level of analyte in a liquid sample at a higher
concentration range.
[0054] There are several ways in which an assay may be prepared in
order to measure analyte at a higher analyte range.
[0055] For example, the assay device may comprise a scavenger assay
comprising a labelled binding reagent for the analyte and a
scavenger binding reagent for the analyte, provided upstream from
the detection zone. The scavenger binding reagent serves to remove
excess analyte and lower the sensitivity of the assay. This has the
effect of increasing the dynamic range of the assay enabling
measurement at higher analyte levels. The scavenger binding reagent
may be may be immobilised, mobilisable or both. The scavenger
binding reagent may be provided at either the same region of the
porous carrier as the mobilisable labelled binding reagent,
upstream from it or downstream from it. The scavenger binding
reagent may bind to the same binding region of the analyte as the
mobilisable labelled binding reagent or to a different region of
the analyte than the labelled binding reagent. The scavenger
reagent may have a different affinity for the analyte than the
mobilisable labelled binding reagent of the second assay. In an
exemplary embodiment, the scavenger binding reagent has a higher
affinity for the analyte than the mobilisable binding reagent of
the second assay. The amount of scavenger binding reagent may be
varied to change the sensitivity of the assay to analyte
concentration. Increasing the amount of scavenger binding reagent
present lowers the sensitivity of the assay due to the fact that
the scavenger binding reagent is able to bind more analyte,
effectively lowering the proportion of labelled binding reagent
that is able to bind to the detection zone.
[0056] In order to increase the dynamic range of the assay, the
assay device may for example comprise multiple detection zones,
wherein each detection zone is capable of binding analyte at
different analyte concentration levels. For example the respective
zones may comprise binding reagent for the analyte having a
differing affinities for the analyte.
[0057] Other ways to increase the dynamic range of the assay are to
provide an assay device comprising a sandwich binding assay and a
competition or inhibition assay. For example, the sandwich assay
may be the high sensitivity assay, namely it is capable of
measuring analyte at a lower concentration range and the inhibition
or competition assay may be a low sensitivity assay, namely it is
capable of measuring analyte at a higher concentration range. A
further way is to alter the affinity or amount of the labelled
binding reagent or the immobilised reagent at the detection zone. A
high affinity binding reagent will have a higher analyte
sensitivity than a lower affinity binding reagent. Similarly a low
concentration of binding reagent will have a lower analyte
sensitivity than a high concentration of binding reagent. The assay
sensitivity can be changed by altering the ratio of binding reagent
to the label of the labelled binding reagent. If a particle is used
as the label, then the quantity of the binding reagent applied to
the label can be altered to alter assay sensitivity. A further way
to manipulate the sensitivity of an assay is to vary the quantity
of the label used in the assay. For example the sensitivity of an
assay may be lowered by reducing the ratio of binding reagent to
labelled species for the labelled binding reagent.
[0058] A further means of manipulating the sensitivity of an assay
is to alter the optical density of a label. The assay sensitivity
can be lowered by use of a label with a low optical density. This
may be achieved for example by provision of a polymer particle
label having a low concentration of dye or by use a coloured label
which is less sensitive to an optical detector.
[0059] Yet a further way to measure high analyte levels is to
employ a non-particulate labelled binding reagent. High levels of
analyte when measured by way of a sandwich binding assay require
high levels of binding reagent. In the case wherein the label is a
particle label, provision of high levels of analyte within or on
the porous carrier can give rise to steric hindrance resulting in
poor assay sensitivity. Conversely, at lower analyte levels, the
use of a non-particle labelled binding reagent can give rise to a
low signal due to the low optical density. However, at high analyte
levels, non-particle labels may be present at sufficiently high
levels to be readily detected. An example of a optically detectable
non-particulate label may be a dye. The dye may be fluorescent.
[0060] Assay sensitivity may be influenced by the flow rate of the
porous carrier. A way to lower the sensitivity of the assay is to
employ a porous carrier (such as nitrocellulose) having a higher
flow rate.
[0061] The sensitivity of an assay may be further manipulated by
modifying the rate at which the labelled binding reagent is
released from its origin. A further way to lower analyte
sensitivity is to provide for a rapid release of the labelled
binding reagent from the porous carrier during contact with the
liquid sample. The release of the labelled binding reagent can be
modified by the provision of sugars, proteins or other polymeric
substances such as methylcellulose within the device.
[0062] According to a particular embodiment, the assay device
comprises a scavenger assay comprising a mobilisable second
(scavenger) binding reagent for the analyte and a mobilisable
binding reagent for the analyte provided upstream from the
detection zone.
[0063] According to a particular embodiment the first assay is
capable of measuring analyte in a lower analyte concentration range
and the second assay is capable of measuring analyte in a higher
analyte concentration range. The first assay may comprise a shared
reference zone and the second assay may comprise a shared control
zone.
[0064] The first assay may comprise a labelled binding reagent
provided upstream from a detection zone and the second assay may
comprise a labelled binding reagent and a mobilisable scavenger
binding reagent provided upstream from a detection zone. The
scavenger binding reagent may be provided at the same position or
in the region of the labelled binding reagent upstream from the
detection zone.
[0065] In particular the analyte to be determined is hCG and the
liquid sample is urine.
[0066] In order to measure an analyte concentration over a certain
range it is important to ensure that there is sufficient labelled
binding reagent present such that the assay signal does not become
saturated. Measurement of large amounts of analyte often requires a
corresponding increase in the amount of labelled binding reagent to
avoid the so-called "hook effect" or saturation of the assay signal
with increasing analyte concentration. Variation in the control
signal has been shown to occur particularly in the case where there
is an increased amount of binding reagent present.
[0067] Where first and second assays are provided having differing
amounts of labelled binding reagent, it has been shown to be
advantageous to provide the reference zone as part of the assay
having a lower level of labelled binding reagent.
[0068] According to an embodiment, the assay device is capable of
measuring analyte at a higher analyte range. There are several ways
of providing such a device.
[0069] For example, the assay device may comprise a labelled
binding reagent for the analyte and a second binding reagent for
the analyte, provided upstream from the detection zone. The second
binding reagent serves to remove excess analyte and lower the
sensitivity of the assay. This has the effect of increasing the
dynamic range of the assay enabling measurement at higher analyte
levels. The second binding reagent may be may be immobilised,
mobilisable or both. The second binding reagent may be provided at
either the same region of the porous carrier as the mobilisable
labelled binding reagent, upstream from it or downstream from it.
The second binding reagent may bind to the same binding region of
the analyte as the mobilisable labelled binding reagent or to a
different region of the analyte than the labelled binding reagent.
The second reagent may have a different affinity for the analyte
than the mobilisable labelled binding reagent of the second assay.
In an exemplary embodiment, the second binding reagent has a higher
affinity for the analyte than the mobilisable binding reagent of
the second assay. The amount of second binding reagent may be
varied to change the sensitivity of the assay to analyte
concentration. Increasing the amount of second binding reagent
present lowers the sensitivity of the assay due to the fact that
the second binding reagent is able to bind more analyte,
effectively lowering the proportion of labelled binding reagent
that is able to bind to the detection zone.
[0070] In order to increase the dynamic range of the assay, the
assay device may for example comprise multiple detection zones,
wherein each detection zone is capable of binding analyte at
different analyte concentration levels. For example the respective
zones may comprise binding reagent for the analyte having a
differing affinities for the analyte.
[0071] Other ways to increase the dynamic range of the assay are to
provide an assay device comprising a sandwich binding assay and a
competition or inhibition assay. For example, the sandwich assay
may be the high sensitivity assay, namely it is capable of
measuring analyte at a lower concentration range and the inhibition
or competition assay may be a low sensitivity assay, namely it is
capable of measuring analyte at a higher concentration range. A
further way is to alter the affinity or amount of the labelled
binding reagent or the immobilised reagent at the detection zone. A
high affinity binding reagent will have a higher analyte
sensitivity than a lower affinity binding reagent. Similarly a low
concentration of binding reagent will have a lower analyte
sensitivity than a high concentration of binding reagent. The assay
sensitivity can be changed by altering the ratio of binding reagent
to the label of the labelled binding reagent. If a particle is used
as the label, then the quantity of the binding reagent applied to
the label can be altered to alter assay sensitivity. A further way
to manipulate the sensitivity of an assay is to vary the quantity
of the label used in the assay. For example the sensitivity of an
assay may be lowered by reducing the ratio of binding reagent to
labelled species for the labelled binding reagent.
[0072] A further means of manipulating the sensitivity of an assay
is to alter the optical density of a label. The assay sensitivity
can be lowered by use of a label with a low optical density. This
may be achieved for example by provision of a polymer particle
label having a low concentration of dye or by use a coloured label
which is less sensitive to an optical detector.
[0073] Yet a further way to measure high analyte levels is to
employ a non-particulate labelled binding reagent. High levels of
analyte when measured by way of a sandwich binding assay require
high levels of binding reagent. In the case wherein the label is a
particle label, provision of high levels of analyte within or on
the porous carrier can give rise to steric hindrance resulting in
poor assay sensitivity. Conversely, at lower analyte levels, the
use of a non-particle labelled binding reagent can give rise to a
low signal due to the low optical density. However, at high analyte
levels, non-particle labels may be present at sufficiently high
levels to be readily detected. An example of a optically detectable
non-particulate label may be a dye. The dye may be fluorescent.
[0074] Assay sensitivity may be influenced by the flow rate of the
porous carrier. A way to lower the sensitivity of the assay is to
employ a porous carrier (such as nitrocellulose) having a higher
flow rate.
[0075] The sensitivity of an assay may be further manipulated by
modifying the rate at which the labelled binding reagent is
released from its origin. A further way to lower analyte
sensitivity is to provide for a rapid release of the labelled
binding reagent from the porous carrier during contact with the
liquid sample. The release of the labelled binding reagent can be
modified by the provision of sugars, proteins or other polymeric
substances such as methylcellulose within the device.
[0076] According to a particular embodiment, the assay device
comprises a mobilisable second binding reagent for the analyte and
a mobilisable binding reagent for the analyte provided upstream
from the detection zone. The second binding reagent may be provided
at the same or similar position upstream from the detection zone as
the labelled binding reagent.
[0077] According to a particular embodiment, the assay device
comprises two assays each comprising an flow-path, wherein the
first assay is capable of measuring analyte in a lower analyte
concentration range and the second assay is capable of measuring
analyte in a higher analyte concentration range. The first assay
may comprise a shared reference zone and the second assay may
comprise a shared control zone.
[0078] The assay device of the invention may be used to measure the
extent or presence of hCG over an extended concentration range. The
range may vary from between about 10 mIU to about 250,000 mIU.
[0079] The second assay may comprise a labelled binding reagent for
the analyte and a second binding reagent for the analyte. The first
assay may comprise labelled binding reagent for the analyte
provided upstream from the detection zone.
[0080] The assay device may comprise one or more further
measurement threshold values to indicate the level of analyte in a
certain analyte range. In an embodiment, the assay device comprises
a first and second measurement thresholds, wherein an analyte
measurement signal of less than the first measurement threshold is
indicative of the absence of analyte or the absence of analyte
above a certain level and wherein an analyte measurement signal
greater than the second threshold is indicative of the level of
analyte in a second concentration range and a measurement signal of
less than the second threshold is indicative of the level of
analyte in a first concentration range. According to a particular
embodiment, the assay device additionally comprises a third
measurement threshold, wherein an analyte measurement signal
greater than the third threshold is indicative of the level of
analyte in a third concentration range.
[0081] In particular the assay device may be capable of measuring
the presence and extent of the analyte hCG analyte in a liquid
sample, in particular urine, of a female mammalian subject. The
assay device may comprise a first measurement threshold, wherein
hCG analyte signal levels of below the threshold are indicative or
being not pregnant and wherein hCG analyte signal levels greater
than or equal to the first measurement threshold are indicative of
being pregnant, wherein the device comprises at least a further
measurement threshold. In addition the assay device may provide an
indication of the extent of pregnancy. The assay device may provide
a time-based indication to the user, such as the extent of
pregnancy in units of days or weeks.
[0082] A typical full assay development time for an assay test for
the determination of hCG in urine is 3 minutes.
[0083] It is an desirable object of the invention to reduce the
number of optical components, this may be conveniently achieved,
where the reference zone is provided as part of one assay and the
control zone is provided as part of the other assay. According to
an embodiment, the reference zone is provided as part of a first
assay having a lower level of labelled binding reagent and the
control zone is provided as part of a second assay having a higher
level of binding reagent.
[0084] According to an embodiment, the assay device comprises four
light sources, wherein the light sources are arranged to illuminate
the detection zones of the first and second assay and the shared
control and reference zones, each zone being illuminated by a
respective light source. One or more photodetectors may be
positioned to detect reflected and/or transmitted light from the
respective zones. According to an embodiment, a single
photodetector may be employed to detect light from all of the
zones. This may be achieved by, for example, illuminating the
respective zones sequentially such that the device is able to
recognise from which zone light detected at the photodetector is
emanating. The sequential illumination process may be repeated with
a fixed or varied frequency during the duration of the assay such
that the levels of signal over time at each zone may be monitored.
In addition the change in levels of light detected from one or more
zones may be used to determine whether and when a fluid sample has
been applied to the device and to determine the flow-rate of liquid
sample along the device. Determination of the flow-rate may be used
as a further quality control check, for example the assay may be
rejected if the flow-rate is either greater than or less than set
levels. A suitable flow-rate detection method and means is
disclosed by EP1484641.
[0085] The labelled binding reagent typically accumulates at the
detection zone over a period of time for a sandwich immunoassay and
thus the rate of increase of signal over time may be monitored. The
device may determine the result after the signal has reached
equilibrium or more typically before the reaction has reached
equilibrium. The device may provide a quantitative result such as a
individual value, a semi-quantitative result or range such as 1-10,
11-20 and so on, or a qualitative result such as YES/NO. The device
may determine the result with respect to one or more signal
thresholds. The device may have a fixed measurement time or provide
an early result before the fixed measurement time has elapsed. An
early result may for example be given in the case where the device
determines that the signal level will never exceed a particular
threshold or exceeds a particular threshold at an early stage. In
these particular cases the device may call an early NO or YES
measurement, indicating the absence or presence of analyte with
respect to a particular base level (which may be zero). An assay
device employing an early result determination method is disclosed
by EP1464613.
[0086] The assay device may be used to determine whether a subject
is pregnant or not (namely whether the liquid sample contains hCG
above a certain level) and may also employ further thresholds
indicating to the user the extent of pregnancy. The extent of
pregnancy may be displayed in terms of a time-based or
concentration-based measurement.
[0087] The assay device will typically comprise a housing. The
housing may be fluid impermeable and constructed from a suitable
plastics material, such as ABS. The assay device may further
comprise a sample receiving member for receiving the fluid sample.
The sample receiving member may extend from the housing.
[0088] The housing may be constructed of a fluid impermeable
material. The housing will also desirably exclude ambient light.
The housing or casing will be considered to substantially exclude
ambient light if less than 10%, preferably less than 5%, and most
preferably less than 1%, of the visible light incident upon the
exterior of the device penetrates to the interior of the device. A
light-impermeable synthetic plastics material such as
polycarbonate, ABS, polystyrene, polystyrol, high density
polyethylene, or polypropylene containing an appropriate
light-blocking pigment is a suitable choice for use in fabrication
of the housing. An aperture may be provided on the exterior of the
housing which communicates with the assay provided within the
interior space within the housing. Alternatively the aperture may
serve to allow a porous sample receiver to extend from the housing
to a position external from the housing.
[0089] Also provided within the housing will typically be a power
source. The device will typically comprise a display means to
display the result of the assay as well as a memory means to store
data. Conveniently the display means comprises an LCD.
[0090] The display means may further display further information
such as an error message, personal details, time, date, and a timer
to inform the user how long the assay has been measured for. The
information displayed by the assay may be indicated in words,
numbers or symbols, in any font, alphabet or language, for example,
"positive", "negative", "+", "-", "pregnant", "not pregnant", "see
your doctor", "repeat the test".
[0091] The assay device may comprise a porous sample receiver in
fluid connection with and upstream from the flow-path. The assay
device may comprise more than one assay flow-path each comprising a
detection zone, in which case a single porous sample receiver may
be provided which is common to the multiple assay flow paths. Thus
a fluid sample applied to the porous sample receiver of the device
is able to travel along the flow-paths of the respective assays to
the respective detection zones. The porous sample receiver may be
provided within a housing or may at least partially extend out of
said housing and may serve for example to collect a urine stream.
The porous sample receiver may act as a fluid reservoir. The porous
sample receiving member can be made from any bibulous, porous or
fibrous material capable of absorbing liquid rapidly. The porosity
of the material can be unidirectional (i.e. with pores or fibres
running wholly or predominantly parallel to an axis of the member)
or multidirectional (omnidirectional, so that the member has an
amorphous sponge-like structure). Porous plastics material, such as
polypropylene, polyethylene (preferably of very high molecular
weight), polyvinylidene fluoride, ethylene vinylacetate,
acrylonitrile and polytetrafluoro-ethylene can be used. Other
suitable materials include glass-fibre. Provision of a common
porous sample receiver enables a single sample to be provided
simultaneously to the flow-paths of the first and second assays and
further increases the effectiveness of providing a shared reference
and/or shared control zone.
[0092] In a fifth aspect, the invention provides a method of
performing an assay to determine the presence and/or extent of one
or more analytes, the method comprising the step of contacting a
liquid sample with an assay device in accordance with the first and
third aspects of the invention.
OVERVIEW OF THE FIGURES
[0093] FIG. 1 is a view of an assay device in accordance with the
invention;
[0094] FIG. 2 is a schematic view of the assay flow-paths according
to an exemplary embodiment in accordance with the invention;
[0095] FIG. 3 is a view of the arrangement of light sources and
photodetector of the embodiment shown in FIG. 2;
[0096] FIG. 4 is a schematic cross-sectional view of part of one
embodiment of the assay device illustrating the relative positions
of some of the assay components;
[0097] FIGS. 5a and 5b are views of the underside of a baffle
arrangement also showing some of the optical components of the
embodiment shown in FIG. 3; and
[0098] FIG. 6 is a top view of the part of the assay device
embodiment depicted in preceding figures, and illustrating a
lateral flow test-strip in situ in the assay device.
DETAILED DESCRIPTION
[0099] An external top view of an assay device is shown in FIG. 1.
The device (10) is elongate having a length of about 14 cm and a
width of about 25 mm. The casing (11) may be formed of a suitable
liquid impermeable casing such as polycarbonate, ABS, polystyrene,
high density polyethylene, or polypropylene. The external porous
sample receiver (12) may be formed of any bibulous, porous or
fibrous material capable of absorbing liquid rapidly. Also shown is
an LCD display (15) for displaying the results of the assay. Also
provided within the assay device and not shown, are the assay
flow-paths, light sources, photodetector, a power source and
associated electronic components.
[0100] FIG. 2 shows the layout of the photodetector and the
individual assay porous carriers of an assay device according to an
exemplary embodiment. Assay device (20) has a common sample
application region (21) which fluidically connects first and second
assays (22) and (23). A single photodetector (4) is provided
between the two assays to detect light from the respective zones.
Zones (24) and (25) correspond respectively to a detection and
control zone on first assay (22). Zones (26) and (27) correspond
respectively to a detection and reference zone on second assay
(23). Not shown are the corresponding four LEDs which each
illuminate a respective zone through appropriately positioned
windows.
[0101] FIG. 3 shows a view of the arrangement according to an
exemplary embodiment comprising a single photodetector (32) and
four LEDs (31). The active area of the photodetector is 1.5
mm.times.1.5 mm.
[0102] FIG. 4 shows a cross-sectional schematic view of the assay
device (40) showing the relative positions of some of the
components. Light from LED (41) illuminates a zone of strip (42)
and light reflected from the zone is detected by the photodetector
(44). Similarly, light from LED (45) illuminates a zone of strip
(46) and reflected light is detected by the photodetector. Provided
are dividers (47) which prevent light from the LED being directly
incident on the photodetector. Also provided is a sloping member
(48) which serves to prevent illumination of strip (46) by LED (41)
and correspondingly the illumination of strip (42) by LED (45)
whilst allowing light reflected from the respective test strips to
be detected by the photodetector. The sloping member also serves to
guide reflected light from the test-strips onto the photodetector.
The LEDs are mounted on a surface (49) made from printed circuit
board.
[0103] FIG. 5a illustrates an underside view of the baffle
arrangement of the exemplified embodiment. Light from the LEDs, of
which one (denoted by reference numeral 51) is shown, illuminates a
zone of an assay strip (not shown) through an aperture. Each LED is
associated with a respective aperture. In the Figure, an exemplary
aperture is denoted by reference numeral 55. Light is reflected
from the strip onto photodetector (52). Also shown is the sloping
member (53) and divider (56). Adjacent LEDs are screened from one
another by baffles (54).
[0104] FIG. 5b shows an underside view of the baffle arrangement of
the exemplified embodiment from a different perspective. The
sloping member (53) is symmetrical about axis (57) and serves to
guide reflected light from all four LEDs (not shown) onto the
photodetector (not shown).
[0105] FIG. 6 shows top view of the assay device looking down onto
a test-strip (61) located over the apertures (62) and held in
position by locating pins (63). The LEDs and photodetector can be
partially seen through the apertures.
Example 1
[0106] The value of the signal determined from the respective zones
for an assay device comprising a low sensitivity test-zone (for
measuring high analyte concentration) and a high sensitivity
test-zone (for measuring low analyte concentration) is determined
by the signal computation means as follows:
[0107] The use of the strips and windows are defined in the table
below (see FIG. 2)
TABLE-US-00001 Strip Window 1 Window 2 A Low Sensitivity test line
(LS) Control line (Ctrl) B High Sensitivity test line (HS)
Reference window (Ref)
[0108] Measurements of the light reflected from each window are
taken approximately twice a second and a low pass digital filter is
used to reject noise and smooth the data. Filtered values are used
for detecting flow and determining the result and are expressed in
terms of normalised percentage relative attenuation (% A). This
takes into account and minimises any variations in the optical
components both within the device and between devices.
[0109] The measured value is inversely proportional to the quantity
of light reflected.
[0110] For each window, the window ratio at the reference, control,
and test windows is equal to the measured value when the porous
carrier is dry, t=0 (prior to addition of sample), divided by the
measured value at time t after addition of sample:
Calculation of Filtered Window Ratios
[0111] For each time point t the window ratios for each window are
evaluated as follows:
Ref ratio t = filtered reference window value time = 0 filtered
reference window value time = t ##EQU00001## HS ratio t = filtered
HS test window value time = 0 filtered HS test window value time =
t ##EQU00001.2## LS ratio t = filtered LS test window value time =
0 filtered LS test window value time = t ##EQU00001.3## Ctrl ratio
t = filtered Ctrl window value time = 0 filtered Ctrl window value
time = t ##EQU00001.4##
Calculation of Filtered % A Values
[0112] The normalised percentage relative attenuation (% A) is
given by the difference of the reference (ref.) window ratio and
the window ratio being considered (control or test windows) divided
by the reference window ratio and multiplied by 100%.
[0113] For each time point t, % A values are calculated for the HS
test line, LS test line and control line, wherein:
HS t ( % A ) = Ref ratio t - HS test ratio t Ref ratio t .times.
100 % ##EQU00002## LS t ( % A ) = Ref ratio t - LS test ratio t Ref
ratio t .times. 100 % ##EQU00002.2## Ctrl t ( % A ) = Ref ratio t -
Ctrl ratio t Ref ratio t .times. 100 % ##EQU00002.3##
Construction of Assay Devices
[0114] An assay device according to the first aspect of the
invention was constructed comprising a first assay test-strip
comprising a labelled binding reagent provided upstream from a
detection zone and a second assay test-strip comprising a labelled
binding reagent and a second (scavenger) binding reagent for the
analyte as well as labelled binding reagent for a control zone
provided upstream from a detection zone and a control zone.
Preparation of the First Assay Test-Strip
[0115] The detection zone was prepared by dispensing a line of
anti-.beta.-hCG antibody (in-house clone 3468) at a concentration
of 3 mg/ml in PBSA buffer, at a rate of 1 .mu.l/cm on onto bands of
nitrocellulose of dimensions 350 mm length.times.40 mm width
(Whatman) having a pore-size of 8 microns and a thickness between
90-100 microns which had been laminated to a 175 micron backing
layer. The anti-.beta.-hCG antibody was applied using the Biodot
xyz3050 dispensing platform as a line .about.1.2 mm in width and
.about.300 mm in length at a position of 10 mm along the length of
the nitrocellulose.
[0116] The bands of nitrocellulose were dried using Hedinair drying
oven serial #17494 set at 55.degree. C. and speed 5 (single
pass).
[0117] The nitrocellulose was subsequently blocked using a blocking
buffer comprising a mixture of 5% ethanol (BDH Analar 104766P) plus
150 mM Sodium Chloride (BDH Analar 10241AP) plus 50 mM trizma base
from (Sigma T1503) plus Tween 20 (Sigma P1379) and 1% (w/v)
polyvinyl alcohol (PVA, Sigma 360627).
[0118] The blocking buffer was applied at a rate of 1.75 .mu.l/mm
to the proximal end of the band. Once the blocking solution had
soaked into the membrane a solution of 2% (w/v) sucrose (Sigma
S8501 in deionised water) was applied using the same apparatus at a
rate of 1.6 .mu.l/mm and allowed to soak into the nitrocellulose
membrane for .about.5 minutes).
[0119] The bands of NC were then dried using a Hedinair drying oven
serial #17494 set at 75.degree. C. and speed 5 (single pass).
Preparation of the Mobilisable Labelled Binding Reagent on the
First Porous Carrier Material.
[0120] Labelled binding reagent was prepared according to the
following protocol:
[0121] Coating Latex Particles with Anti-.alpha.hCG [0122] 1.
Dilute blue latex particles from Duke Scientific (400 nm in
diameter, DB1040CB at 10% solids (w/v)) to 2% solids (w/v) with 100
mM di-sodium tetra borate buffer pH 8.5 (BDH AnalaR 102676G) (DTB).
[0123] 2. Wash the diluted latex by centrifuging a volume of (2
mls) of diluted latex in two Eppendorf centrifuge tubes at 17000
rpm (25,848 rcf) for 10 minutes on an Heraeus Biofuge 17RS
centrifuge. Remove and discard the supernatant and re-suspend the
pellets in 100 mM DTB to give 4% solids (w/v) in a total volume of
1 ml. [0124] 3. Prepare a mixture of ethanol and sodium acetate
(95% Ethanol BDH AnalaR 104766P with 5% w/v Sodium Acetate Sigma
S-2889). [0125] 4. Add 100 .mu.ls ethanol-sodium acetate solution
to the washed latex in step 2 (this is 10% of the volume of latex).
[0126] 5. Dilute the stock antibody (in-house clone 3299) to give
.about.1200 .mu.g/ml antibody in DTB. [0127] 6. Heat a volume of 1
ml of the diluted antibody from step 5 in a water bath set at
41.5.degree. C. for .about.2 minutes. Also heat the washed latex
plus ethanol-sodium acetate from step 4 in the same water bath for
2 minutes. [0128] 7. Add the diluted antibody to the latex plus
ethanol-acetate, mix well and incubate for 1 hour in the water bath
set at 41.5.degree. C. whilst mixing using a magnetic stirrer and a
magnetic flea placed in the mixture. [0129] 8. Prepare 40 mg/ml
Bovine Serum Albumin (BSA) Solution (Intergen W22903 in de-ionised
water). Block the latex by adding an equal volume of 40 mg/ml BSA
to the mixture of latex/antibody/ethanol-acetate and incubate in
the water bath at 41.5.degree. C. for 30 minutes with continued
stirring. [0130] 9. Centrifuge the mixture at 17000 rpm for 10
minutes as in step 2, (split the volume into 1 ml lots between
Eppendorf tubes). Remove and discard the supernatant and re-suspend
the pellet in 100 mM DTB. Repeat the centrifugation as in step 2,
remove and discard the supernatant and re-suspend in pellet in Air
Brushing Buffer (20% (w/v) Sucrose Sigma 58501, 10% BSA (w/v) in
100 mM Trizma Base Sigma T1503 pH to 9). Add Air Brushing Buffer to
give 4% solids (w/v) latex.
[0131] The conjugated latex was and sprayed in a mixture of BSA and
sucrose onto a glass-fibre porous carrier (F529-09, Whatman) at a
rate of 50 g/hr and 110 mm/s and dried using a Hedinar Conveyor
Oven Serial number 17494 set at 65.degree. C. and speed 5 (single
pass).
[0132] The zone chosen as the reference zone was at a distance of
13 mm along the nitrocellulose, namely downstream of the detection
zone.
[0133] The glass fibre material comprising the labelled binding
reagent was attached to the nitrocellulose membrane using a clear
adhesive coated laminate film (Ferrisgate, 38 mm wide) arranged
such that the labelled reagent was uppermost and the glass fibre
overlapped the surface of the nitrocellulose by .about.2 mm along
the length (350 mm) of the band of nitrocellulose membrane. The
glass fibre was attached to the end of the nitrocellulose such that
it was upstream of the detection zone.
[0134] The laminated sheet was subsequently cut into test-strips
comprising a glass fibre porous carrier material having a width of
6 mm and a length 25 mm, with the labelled reagents having been
applied 20 mm along the length of the glass fibre, provided
upstream from and overlapping by 2 mm, a nitrocellulose membrane
having a width of 6 mm and a length of 40 mm.
Preparation of the Second Assay Test-Strip.
[0135] The detection zone was prepared as follows:
[0136] MAb mouse anti-human .beta.-hCG antibody (clone 3468) 3
mg/ml in PBSA buffer was plotted at 1 .mu.l/cm onto nitrocellulose
(of type and dimensions as that according to the first assay) at
the 10 mm position using a Biodot XYZ3050 dispensing platform to
provide a sole detection zone for the first assay.
[0137] The control zone was prepared as follows:
[0138] Goat-anti-Rabbit antibody (Lampire) at 2 mg/ml in PBSA
buffer was plotted at 1 .mu.l/cm onto the same nitrocellulose as
used for the second assay, at the 13 mm position using a Biodot
XYZ3050 dispensing platform to provide a sole control zone for the
assay device.
[0139] Mouse-anti-human .alpha.-hCG mAb (clone 3299) conjugated to
400 nm blue polystyrene latex (Duke Scientific) was mixed with
scavenger antibody mAb mouse anti-human .beta.-hCG (in-house clone
3468) at 3 mg/ml to give a final % blue latex of 3%, a final 3468
concentration of 0.075 mg/ml and 0.06 mg/ml concentration of the
free anti-.beta. hCG antibody. The resulting mixture was airbrushed
onto Whatman glass fibre (F529 25 mm wide reels) using the BIODOT
XYZS (serial number 1673) at 90 g/hr sprayed at 2.02 .mu.g/cm onto
F529-09 glass fibre at approximately the 20 mm position. The
sprayed solution spread out to form a band that was approximately 7
mm in length.
[0140] Labelled binding reagent for the control zone was also
deposited onto the same region of the porous carrier as the
labelled binding reagent for the analyte as follows: Rabbit IgG
(Dako) was conjugated to 400 nm blue latex polystyrene latex (Duke
Scientific) in BSA/sucrose to give a final % blue latex of 0.7%
solids and sprayed at 65 g/hr onto glass fibre.
[0141] The glass fibre was dried using a Hedinar Conveyor Oven
Serial number 17494 set at 65.degree. C. and speed 5 (single pass).
A second pass of latex was deposited onto the glass fibre by
repeating the above however at an offset of .about.0.8 mm from the
original position of spray (further downstream of the glass fibre).
The glass fibre as dried as described above.
[0142] The glass fibre material with sprayed latex was attached to
the nitrocellulose membrane using a clear adhesive coated laminate
film (Ferrisgate, 38 mm wide) arranged such that the sprayed latex
was uppermost and the glass fibre overlapped the surface of the
nitrocellulose by approximately 2 mm along the length (350 mm) of
the band of nitrocellulose membrane. The glass fibre was provided
upstream from the nitrocellulose membrane and the binding reagents
were provided towards the distal end of the glass fibre.
[0143] The laminated sheet was subsequently cut into test-strips
comprising a glass fibre porous carrier material having a width of
6 mm and a length 25 mm, with the labelled reagents having been
applied 20 mm along the length of the glass fibre, provided
upstream from and overlapping by 2 mm, a nitrocellulose membrane
having a width of 6 mm and a length of 40 mm. A porous sample
receiver (Filtrona) of 45 mm length, 12 mm width and a thickness of
approximately 2.5 mm was provided upstream from and overlapping the
first porous carrier material by approximately 3 mm.
[0144] Labelled binding reagent for the control zone was also
deposited onto the same region of the porous carrier as the
labelled binding reagent for the analyte as follows:
[0145] Rabbit IgG (Dako) was conjugated to 400 nm blue latex
polystyrene latex (Duke Scientific) in BSA/sucrose to give a final
% blue latex of 0.7% solids and sprayed at 65 g/hr onto glass fibre
(F529-09).
[0146] The first and second assay test-strips were positioned
parallel to one another and a common polyester sample application
pad (505521, Filtrona) was overlaid at the upstream ends of both
assays. A common cotton absorbent sink pad (CF7, Whatman) was
overlaid downstream of the reference and control zones.
[0147] The assay device was prepared by mounting the assay strips
in a parallel configuration into a plastic housing comprising the
optical components. The LEDs were arranged such that the four LEDs
were positioned in close proximity to the respective four zones (2
detection zones and the reference and control zones) in an offset
position and above the plane of the assays. A single photodetector
was positioned between and above the plane of the two assays and
positioned in the middle of the assay strips (see FIG. 2).
EQUIVALENTS
[0148] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
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