U.S. patent application number 12/202639 was filed with the patent office on 2009-08-06 for multiple assay device.
This patent application is currently assigned to Inverness Medical Switzerland GmbH. Invention is credited to Saji Eapen, Ezra Linley, Balbir Raj.
Application Number | 20090196792 12/202639 |
Document ID | / |
Family ID | 39430746 |
Filed Date | 2009-08-06 |
United States Patent
Application |
20090196792 |
Kind Code |
A1 |
Raj; Balbir ; et
al. |
August 6, 2009 |
Multiple Assay Device
Abstract
Disclosed is an assay device for the determination of analyte in
a liquid sample over an extended concentration range comprising a
first assay and a second assay, wherein the first assay for an
analyte comprises a first flow-path having a sole detection zone
capable of immobilising a labelled binding reagent and the second
assay for said analyte comprises a second flow-path having a sole
detection zone capable of immobilising a labelled binding reagent,
wherein the presence of labelled binding reagent at the detection
zones provides an indication of the presence and/or extent of
analyte in said liquid sample.
Inventors: |
Raj; Balbir; (Bedford,
GB) ; Linley; Ezra; (Cardiff, GB) ; Eapen;
Saji; (Cambridge, GB) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP (w/ISA)
155 SEAPORT BLVD.
BOSTON
MA
02210-2600
US
|
Assignee: |
Inverness Medical Switzerland
GmbH
Zug
CH
|
Family ID: |
39430746 |
Appl. No.: |
12/202639 |
Filed: |
September 2, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60991531 |
Nov 30, 2007 |
|
|
|
Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01L 2300/0825 20130101;
B01L 2400/0406 20130101; G01N 33/76 20130101; Y10T 29/494 20150115;
B01L 2300/087 20130101; C12Q 2537/125 20130101; G01N 33/558
20130101; G01N 33/689 20130101 |
Class at
Publication: |
422/56 |
International
Class: |
G01N 31/22 20060101
G01N031/22 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 10, 2007 |
GB |
0706906.5 |
Sep 1, 2007 |
GB |
0717043.4 |
Claims
1. An assay device for determining the presence or concentration of
an analyte in a liquid sample over an extended concentration range
comprising: a) a first flow-path having a sole detection zone
capable of immobilising a labelled binding reagent, the first
flow-path capable of providing an indication of the level of
analyte in a first concentration range; and b) a second flow-path
having a sole detection zone capable of immobilising a labelled
binding reagent, the second flow-path capable of providing an
indication of the level of analyte in a second concentration range;
wherein the presence of labelled binding reagent at the detection
zones provides an indication of the presence or concentration of
analyte in said liquid sample.
2. (canceled)
3. The device according to claim 1 wherein the first and second
concentration ranges overlap.
4. The assay device according to claim 1 which is capable of
providing an indication of the level of analyte with respect to one
or more thresholds.
5. The device according to claim 1 wherein the first and/or second
flow-path comprises a porous carrier.
6. (canceled)
7. The device according to claim 1 wherein the first and/or second
flow-path comprises a mobilisable labelled binding reagent for the
analyte provided upstream from a detection zone.
8. The device according to claim 1 wherein the detection zone of
the first and/or second flow-path comprises an immobilised binding
reagent for the analyte.
9. The device according to claim 1 wherein the first flow-path
defines a sandwich assay and the second flow-path defines a
competition or inhibition assay.
10. The device according to claim 9 wherein the first flow-path
comprises a mobilisable labelled binding reagent for the analyte
provided upstream from a detection zone, said detection zone
comprising an immobilised binding reagent for the analyte and
wherein the second flow-path comprises a mobilisable binding
reagent for the analyte provided upstream from provided upstream
from a detection zone, said detection zone comprising an
immobilised analyte or analyte analogue for the mobilisable binding
reagent.
11. The device according to claim 9 wherein the first flow-path
comprises a mobilisable labelled binding reagent for the analyte
provided upstream from a detection zone, said detection zone
comprising an immobilised binding reagent for the analyte and
wherein the second flow-path comprises a mobilisable labelled
analyte or analyte analogue provided upstream from an immobilised
binding reagent for the analyte.
12. (canceled)
13. The device according to claim 1 wherein the first flow-path
comprises a mobilisable labelled binding reagent for an analyte
provided upstream from a detection zone, wherein the detection zone
comprises an immobilised non-labelled binding reagent for the
analyte; and wherein the second assay comprises a mobilisable
labelled binding reagent for a first binding region of the analyte
and a scavenger binding reagent for the analyte provided upstream
from a detection zone, wherein said detection zone is capable of
binding the labelled binding reagent.
14. (canceled)
15. (canceled)
16. The device according to claim 13 wherein the mobilisable
labelled binding reagent and the scavenger binding reagent of the
second flow-path are for respectively a first and second binding
region of the analyte, the detection zone comprising an immobilised
binding reagent for a second binding region of the analyte.
17. (canceled)
18. (canceled)
19. The device according to claim 13 wherein the mobilisable
binding reagents and the scavenger reagent are provided on or in a
first porous carrier material and the immobilised binding reagent
provided at the detection zone is provided on and/or in a second
porous carrier material.
20. The device according to claim 19 wherein the first porous
carrier material is glass-fibre and the second porous carrier
material is nitrocellulose.
21. (canceled)
22. (canceled)
23. (canceled)
24. The device according to claim 1 wherein the fluid sample is
urine.
25. The device according to claim 1 wherein the binding reagent is
labelled with an optically detectable particle.
26. The device according to claim 25 wherein the optically
detectable particle is a coloured polymer label.
27. The device according to claim 1, comprising a common sample
application region which serves to supply liquid sample to both
flow-paths.
28. (canceled)
29. An assay device for determining the presence or concentration
of an analyte in a liquid sample comprising a first flow-path and a
second flow-path wherein said first flow-path comprises a porous
carrier having a detection zone comprising an immobilised binding
reagent for an analyte and provided upstream from the detection
zone is a mobilisable labelled binding reagent for said analyte;
and wherein said second flow-path comprises a detection zone
comprising an immobilised binding reagent for the analyte and a
mobilisable labelled binding reagent upstream from the detection
zone for the analyte and a scavenger reagent for said analyte,
wherein detection of the presence of labelled binding reagent at
the detection zone provides an indication of the presence or
concentration of analyte in said liquid sample.
30. (canceled)
31. (canceled)
32. The device according to claim 1, wherein: a) the first
flow-path is capable of providing an indication of the level of hCG
in the sample in a first range of less than or equal to a first
threshold; and b) the second flow-path is capable of providing an
indication of the level of hCG in the sample in a second range of
above or equal to a second threshold; and c) the first and second
flow-paths together are capable of providing an indication of the
level of hCG in a third range of greater than the first threshold
but less than the second threshold.
33. (canceled)
34. (canceled)
35. An assay kit for determining the presence or concentration of
an analyte in a liquid sample comprising an assay device comprising
a first flow-path and a second flow-path wherein: a) said first
flow-path comprises: i) a porous carrier having a detection zone
comprising an immobilised binding reagent for an analyte; and ii) a
mobilisable labelled binding reagent for said analyte upstream from
the detection zone; and b) said second flow-path comprises: i) a
detection zone comprising an immobilised binding reagent for the
analyte; and ii) a mobilisable labelled binding reagent for the
analyte and a scavenger reagent for said analyte upstream from the
detection zone, wherein detection of the presence of labelled
binding reagent at the detection zones provides an indication of
the presence and/or extent of the analyte in the sample.
36-38. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application claims benefit of priority to U.S.
Provisional Patent Application No. 60/991,531, filed on Nov. 30,
2007; Great Britain Application 0706906.5, filed Apr. 10, 2007; and
Great Britain Application 0717043.4, 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 concentration 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 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] A sandwich immunoassay is often the assay of choice when
detecting analytes. However, a sandwich assay is not always
possible, for example in the case of small molecules such as
haptens which may not be large enough to allow the simultaneous
binding thereto of two different binding partners. A dose-response
curve prepared using a typical lateral flow device employing a
sandwich immunoassay shows increasing levels of signal with
increasing analyte up to the point where at higher analyte levels
the curve tends to plateau. At yet higher analyte levels, the
signal begins to decrease due to preferential capture at the
detection zone of analyte which has not yet bound to labelled
reagent. This phenomenon is known as the hook effect. Thus sandwich
immunoassays exhibit a limited assay range due to the fact that the
signal amount or intensity observed at higher analyte levels may be
the same, or even less, than that observed at lower analyte
levels.
[0006] A competition or inhibition assay typically provides a high
signal at zero or low levels of analyte. At increasing levels of
analyte the signal level may still be high depending upon the
amount of labelled binding species present compared to the amount
of analyte. At still increasing levels of analyte, the signal
starts to decrease as unbound analyte either competes with labelled
analyte or analyte analogue for the immobilised binding reagent or
binds to labelled binding reagent, lowering binding of the
unlabelled binding reagent at the detection zone.
[0007] Thus the above assay methods are not suitable for measuring
levels of analyte over an extended analyte range.
[0008] US2005/0112780 discloses an assay device and method for
extending the dynamic detection range of assay devices comprising a
flow through porous carrier comprising a detection zone and a
compensation zone provided downstream from the detection zone. The
detection involves a first binding reagent which binds a detection
probe to generate a detection signal having an intensity
proportional to the amount of analyte, and the compensation zone
comprises a second capture reagent which binds a detection probe to
generate a signal which is inversely proportional to the intensity
of the detection signal. The assay may further comprise a third
calibration zone which generates a signal. The first binding
reagent may be selected from a group including an antigen, hapten
or streptavidin. The first and second binding reagents may be
chosen from a number of species including an antigen, hapten or
streptavidin.
[0009] US2004/0197820 discloses a flow through porous carrier assay
device for reducing the hook effect comprising a detection zone
wherein the device may include a downstream calibration zone.
[0010] US2006/0019404 discloses an assay device with an extended
dynamic range comprising a lateral flow test-strip comprising a
plurality of detection zones with a progressively decreased
sensitivity to analyte concentration. The assay device may comprise
two carriers each having a plurality of detection zones. The amount
of label/signal present at the plurality of detection zones is
detected to determine the analyte concentration.
[0011] EP462376 discloses an assay device comprising a capture site
and a conjugate recovery site wherein the conjugate recovery site
receives and binds said conjugate or conjugate complexes which
migrate through said capture site and wherein immobilised conjugate
at both the conjugate recovery site and capture site is detected to
determine the amount of the analyte of interest.
[0012] The present inventors have shown that for assay devices
wherein multiple detection zones for the detection of an analyte
are provided on the same porous carrier, binding at an upstream
detection zone may change the binding characteristics at a
downstream detection zone and that any variation in binding at an
upstream detection zone may cause a compounded variation of binding
at a downstream detection zone. This is especially so at higher
analyte concentration levels and can give rise to poor assay
precision. Furthermore, it has been found that cross-binding may
occur between the respective binding reagents present in the
detection zones during running of the test and cross-binding has
also been observed during manufacture of the devices and whilst
they are stored in the dry state. This was shown to have an impact
on the levels of assay precision and sensitivity. These problems do
not appear to have been recognised previously in the prior art.
[0013] It is an object to provide an improved assay device, kit and
method for extending the analyte range of an assay.
SUMMARY OF THE INVENTION
[0014] An assay device for determining the presence or
concentration of an analyte such as hCG in a liquid sample over an
extended concentration range is provided. In one embodiment, the
device comprises a first flow-path having a sole detection zone
capable of immobilising a labelled binding reagent and a second
flow-path having a sole detection zone capable of immobilising a
labelled binding reagent, wherein the presence of labelled binding
reagent at the detection zones provides an indication of the
presence or concentration of analyte in said liquid sample.
[0015] In another embodiment, an assay device may comprise a first
flow-path and a second flow-path wherein said first flow-path
comprises a porous carrier having a detection zone comprising an
immobilised binding reagent for an analyte and provided upstream
from the detection zone is a mobilisable labelled binding reagent
for said analyte; and wherein said second flow-path comprises a
detection zone comprising an immobilised binding reagent for the
analyte and a mobilisable labelled binding reagent upstream from
the detection zone for the analyte and a scavenger reagent for said
analyte, wherein detection of the presence of labelled binding
reagent at the detection zone provides an indication of the
presence or concentration of analyte in said liquid sample.
[0016] In another aspect, the first flow-path is capable of
providing an indication of the level of analyte in a first
concentration range and the second flow-path is capable of
providing an indication of the level of analyte in a second
concentration range. The assay device according is capable of
providing an indication of the level of analyte with respect to one
or more thresholds. The first flow-path of the device may define a
sandwich assay, and the second flow-path may define a competition
or inhibition assay.
[0017] The first flow-path may comprise a mobilisable labelled
binding reagent for the analyte provided upstream from a detection
zone, wherein the detection zone comprises an immobilised binding
reagent for the analyte and wherein the second flow-path comprises
a mobilisable binding reagent for the analyte provided upstream
from provided upstream from a detection zone, said detection zone
comprising an immobilised analyte or analyte analogue for the
mobilisable binding reagent. The first flow-path may define a high
analyte sensitivity assay, and the second flow-path defines a low
analyte sensitivity assay.
[0018] Assay kits and methods for determining the presence or
concentration of an analyte in a liquid sample comprising an assay
device comprising the devices are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Aspects of the invention are further illustrated by
reference to the following figures:
[0020] FIG. 1 illustrates typical signal responses that are
observed for a typical assay compared with that of a typical
competition assay.
[0021] FIG. 2 illustrates plots of signal intensity vs hCG
concentration for Example 1 and Comparative Example 1.
[0022] FIG. 3 illustrates a plot of signal intensity vs hCG
concentration for the assay device according to Example 2.
[0023] FIG. 4 illustrates the effect of varying the amounts of
scavenger antibody for the assay device according to Example 2.
[0024] FIG. 5 illustrates the effect of varying latex spray
positions to reduce a hook effect using a device as described
herein.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Unless defined otherwise above, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
Where a term is provided in the singular, the inventor also
contemplates the plural of that term. The nomenclature used herein
and the procedures described below are those well known and
commonly employed in the art.
[0026] The singular forms "a", "an", and "the" include plural
references unless the context clearly dictates otherwise.
[0027] As used herein, the term "analyte" generally refers to a
substance to be detected. The term "analyte" includes, but is not
limited to, toxins, organic compounds, proteins, peptides,
microorganisms, 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. Specific examples of
analytes include but are not limited to ferritin; creatinine kinase
MB (CK-MB); digoxin; phenytoin; phenobarbitol; carbamazepine;
vancomycin; gentamycin; theophylline; valproic acid; quinidine;
luteinizing hormone (LH); follicle stimulating hormone (FSH);
estradiol, progesterone; C-reactive protein; lipocalins; IgE
antibodies; cytokines; vitamin B2 micro-globulin; glycated
hemoglobin; cortisol; digitoxin; N-acetylprocainamide (NAPA);
procainamide; antibodies to rubella, such as rubella-IgG and
rubella IgM; antibodies to toxoplasmosis, such as toxoplasmosis IgG
(Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone;
salicylates; acetaminophen; hepatitis B virus surface antigen
(HBsAg); antibodies to hepatitis B core antigen, such as
anti-hepatitis B core antigen IgG and IgM (Anti-HBC); human immune
deficiency virus 1 and 2 (HIV 1 and 2); human T-cell leukemia virus
1 and 2 (HTLV); hepatitis Be antigen (HBeAg); antibodies to
hepatitis Be antigen (Anti-HBe); influenza virus; thyroid
stimulating hormone (TSH); thyroxine (T4); total triiodothyronine
(Total T3); free triiodothyronine (Free T3); carcinoembryoic
antigen (CEA); lipoproteins, cholesterol, and triglycerides; and
alpha fetoprotein (AFP). Drugs of abuse and controlled substances
include, but are not intended to be limited to, amphetamine;
methamphetamine; barbiturates, such as amobarbital, secobarbital,
pentobarbital, phenobarbital, and barbital; benzodiazepines, such
as librium and valium; cannabinoids, such as hashish and marijuana;
cocaine; fentanyl; LSD; methaqualone; opiates, such as heroin,
morphine, codeine, hydromorphone, hydrocodone, methadone,
oxycodone, oxymorphone and opium; phencyclidine; and
propoxyhene.
[0028] "Antigen" shall mean any compound capable of binding to an
antibody, or against which antibodies can be raised.
[0029] "Antibody" shall mean an immunoglobulin having an area on
its surface or in a cavity that specifically binds to and is
thereby defined as complementary with a particular spatial and
polar organization of another molecule. The antibody can be
polyclonal or monoclonal. Antibodies may include a complete
immunoglobulin or fragments thereof, which immunoglobulins include
the various classes and isotypes, such as IgA (IgA1 and IgA2), IgD,
IgE, IgM, and IgG (IgG1, IgG2, IgG3, and IgG4) etc. Fragments
thereof may include Fab, Fv and F(ab').sub.2, Fab', and the like.
Antibodies may also include chimeric antibodies made by recombinant
methods.
[0030] A "binding reagent" refers to a member of a binding pair,
i.e., two different molecules wherein one of the molecules
specifically binds with the second molecule through chemical 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 specific binding
pair ("sbp") are referred to as ligand and receptor (antiligand),
sbp member and sbp partner, and the like. In addition to antigen
and antibody specific binding pair members, other specific binding
pairs include 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, for example an analyte-analogue or a specific binding
member made by recombinant techniques or molecular engineering. 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 a binding pair member for the immune complex. 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.
[0031] The terms "comprise" and "comprising" is used in the
inclusive, open sense, meaning that additional elements may be
included.
[0032] 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. A first
flow path of a device provides for a first assay, and a second flow
path of the device provides for a second assay.
[0033] "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.
[0034] "Labelled binding reagent" refers to any substance
comprising a detectable label attached to a binding reagent. The
attachment may be covalent or non-covalent. The label provides a
detectable signal that is related to the presence or amount of
analyte in the fluid sample. Various labels suitable for use
include labels which produce signals through either chemical,
biochemical or physical means. Such labels can include enzymes and
substrates, chromogens, catalysts, fluorescent compounds,
chemiluminescent compounds, and radioactive labels. Other suitable
labels include colloidal metallic particles such as gold or silver,
colloidal non-metallic particles such as selenium or tellurium,
dyed or colored particles such as a coloured polymer such as
polystyrene, a stained microorganism or dyesols, organic polymer
latex particles and liposomes, colored beads or electrochemically
detectable species. Of the above, colored polymer particles and
colloidal gold are preferred.
[0035] The term "non-labelled binding reagent" for the purposes of
this invention refers to a binding reagent that is either not
labelled with a detectable label or is labelled with a label which
is not detected in order to determine the analyte.
[0036] "Porous carrier" refers to a porous body capable of
transporting fluid sample.
[0037] The term "sample" refers to any sample potentially
containing an analyte. For example, a sample may be a bodily fluid
such as blood, urine, mucous or saliva, or a respiratory sample,
such as a nasopharyngeal wash or aspirate, nasal swab,
nasopharyngeal swab, nasal wash, throat swab, transtracheal
aspirate, bronchoalveolar lavage, elution buffer used to wash a
respiratory sample, etc.
[0038] For the purpose of this application the term "scavenger
binding reagent" denotes an additional binding reagent capable of
binding analyte and the term "scavenger" is used merely to
distinguish the binding reagents from the other binding reagents
present in the device.
[0039] According to a first aspect, the invention provides an assay
device for the determination of analyte in a liquid sample over an
extended concentration range comprising a first assay and a second
assay, wherein the first assay for an analyte comprises a first
flow-path having a sole detection zone capable of immobilising a
labelled binding reagent and the second assay for said analyte
comprises a second flow-path having a sole detection zone capable
of immobilising a labelled binding reagent, wherein the presence of
the labelled binding reagent at the detection zones provides an
indication of the presence and/or concentration of analyte in said
liquid sample.
[0040] The first assay may provide an indication of the level of
analyte in a first concentration range and the second assay may
provide an indication of the level of analyte in a second
concentration range.
[0041] The first and second concentration ranges differ from each
other. The first and second concentration ranges may overlap so as
to provide a continuous concentration range.
[0042] The assay device may be capable of providing an indication
of the level of analyte with respect to one or more thresholds. The
assay device may provide an indication of the level of analyte
below or above a plurality of thresholds. For example the number of
thresholds may be two three, four, five or greater.
[0043] The first and second assays may either independently or
together provide an indication of the level of analyte within a
certain range.
[0044] According to an embodiment, the first assay provides an
indication of the level of analyte of less than or equal to a first
threshold and the second assay provides an indication of the level
of analyte of above or equal to a second threshold. The first and
second assays together provide an indication of the level of
analyte of greater than the first threshold but less than the
second threshold.
[0045] A non-labelled binding reagent for the analyte or an analyte
analogue may be provided in an immobilised form at a detection
zone. The non-labelled binding reagent may be chosen from a binding
reagent for the analyte of interest, an analyte or analyte
analogue, depending upon whether the assay is a sandwich type assay
or a competition type assay. Similarly the labelled binding reagent
may comprise a labelled binding reagent for the analyte of
interest, a labelled analyte or labelled analyte analogue.
[0046] Alternatively a reagent may be provided in an immobilised
form at the detection zone that is capable of binding a labelled
binding reagent-analyte-non-labelled binding reagent complex. For
example the binding reagent may be conjugated or otherwise joined
to binding species such as biotin, the reagent immobilised at the
detection zone being a complementary binding partner such as
streptavidin.
[0047] The non-labelled binding reagent may be provided in a
mobilisable form which is capable of immobilising a labelled
binding reagent-analyte complex at a detection zone. For example
the non-labelled binding reagent may be attached to particle such
as agarose and the detection zone may comprise a filter of
dimensions smaller than the particle, but larger than the size of
the labelled binding reagent, such that the filter is able to trap
the any labelled binding reagent-analyte-non-labelled binding
reagent complex present, any labelled binding reagent that is not
complexed to the capture reagent being able to pass through the
filter.
[0048] The first and/or second assay may comprise a labelled
binding reagent provided in a mobilisable form upstream from the
detection zone reagent in the dry state prior to use of the
device.
[0049] The first and second assays may each comprise a mobilisable
labelled binding reagent provided upstream from an immobilised
non-labelled binding provided at each detection zone.
[0050] The assay device may comprise more than two assays, each
capable of detecting the analyte at a particular concentration
range or above or below one or more thresholds.
[0051] The first and second assays may individually or together
provide an indication of the particular level of analyte, or
whether the analyte is above or below a certain threshold.
[0052] The assay device may have a common sample application region
that fluidically connects the plurality of flow-paths. Thus a fluid
sample applied to sample application region of the device is able
to travel along the flow-paths of the respective assays to the
respective detection zones. The sample application region may
comprise a porous sample receiver. In the case where the
mobilisable labelled binding reagent is the same for both assays it
may be provided at the sample application region.
[0053] As an alternative to providing the first and second assays
within a single assay device, the assays may be provided as
separate assay devices, the results from the respective devices
when taken together being capable of providing an indication or
measurement of the level of analyte.
[0054] Thus according to a second aspect, the invention provides an
assay kit for the determination of an analyte over an extended
concentration range comprising a first assay device and a second
assay device, wherein the first and second assay devices comprise
respectively first and second assays according to the first aspect
of the invention.
[0055] According to a third aspect, the invention provides a method
for the determination of an analyte over an extended analyte range
comprising the steps of: [0056] a) adding a liquid sample to a
first assay comprising a mobilisable labelled binding reagent
provided upstream from a sole detection zone and to a second assay
comprising a mobilisable labelled binding reagent provided upstream
from a sole detection zone, said detection zones being capable of
immobilising labelled binding reagent, and wherein detection of
labelled reagent at the detection zones provides an indication of
the concentration and/or presence of an analyte in the liquid
sample. [0057] b) reading the result of the assay.
[0058] In the case where the level of analyte is known to vary as a
function of time, for example the pregnancy hormone hCG, the assay
device may provide a time-based indication to the user, such as the
duration of pregnancy in units of days or weeks.
[0059] The first and second flow paths 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 flow-paths. As yet a further
alternative, the first and second detection zones may be provided
on the same flow-path in substantially a side by side arrangement,
such that neither is provided downstream from the other.
[0060] 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.
[0061] 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-insuluble, and
are emulsified in aqueous surfactant so that monomer mycelles 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 to about 0.5 .mu.m. According to an
exemplary embodiment the label is a blue polymeric particle.
[0062] 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, ascots liquid, mucous,
synovial liquid, peritoneal liquid, transdermalexudates, pharyngeal
exudates, bronchoalveolar lavage, tracheal aspirations,
cerebrospinal liquid, semen, cervical mucus, vaginal or urethral
secretions and amniotic liquid. In particular the source is human
and in particular the sample is urine.
[0063] A particular analyte is human chorionic gonadotropin (hCG).
The analyte may have a sole binding region or epitope or may have
more than one binding region. For example the analyte hCG comprises
an alpha sub-unit identical to that of luteinising hormone (LH),
follicle stimulating hormone (FSH) and thyroid stimulating hormone
(TSH) and a beta sub-unit unique to hCG. Antibodies to the alpha
and beta sub-units may be used to bind to hCG in a sandwich
immunoassay format.
[0064] The assay device of the invention may be used to measure the
presence of hCG or concentration over an extended range. The range
may vary from between, for example, about 10 mIU to about 250,000
mIU, from between about 30 mIU to about 200,000 mIU, from between
about 50 mIU to about 175,000 mIU, from between about 100 mIU to
about 100,000 mIU, from between about 250 mIU to about 75,000 mIU,
from between about 300 mlU to about 50,000 mIU, or from between
about 500 mIU to about 25,000 mIU.
[0065] According to an embodiment, the device is able to measure
the amount of hCG in the fluid sample and to indicate to the user,
based upon stored reference values the duration of pregnancy in
time based units. The device may also indicate whether the subject
is pregnant or not, determined by whether the level of hCG is above
or below a base threshold. The reference and threshold values are
typically stored within the device as part of an algorithm. The
base threshold may typically range from 10-25 mIU/ml. The base
threshold may be, for example, 10, 15, 20 or 25 mIU/ml.
[0066] According to an embodiment, the first assay may provide
either an indication of whether the subject is pregnant or not,
based upon whether the level of hCG detected is respectively above
or below a base threshold, and/or if pregnant, the level of hCG in
a first range of less than or equal to a first threshold, the
second assay provides an indication of the level of hCG in a second
range of above or equal to a second threshold and wherein the first
and second assays together provide an indication of the level of
hCG in a third range of greater than the first threshold but less
than the second threshold.
[0067] The first and/or second assay may further comprise a control
zone to indicate that the reagents have mobilised and have been
transported along the flow path and that the assay test has been
carried out satisfactorily. The control zone is typically
positioned downstream from the detection zone and may for example
comprise an immobilised binding reagent for a labelled binding
reagent. The labelled binding reagent may be present in a
mobilisable form upstream from the control zone and detection zone.
The labelled binding reagent may the same or different to the
labelled binding reagent for the analyte.
[0068] The assay device may comprise a porous sample receiver in
fluid connection with and upstream from the first and second
flow-paths. The porous sample receiver may be provided within the
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 (ie 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 flouride, ethylene vinylacetate,
acrylonitrile and polytetrafluoro-ethylene can be used. Other
suitable materials include glass-fibre.
[0069] If desired, an absorbant "sink" can be provided at the
distal end of the carrier material. The absorbent sink may comprise
of, for example, Whatman 3MM chromatography paper, and should
provide sufficient absorptive capacity to allow any unbound
labelled binding reagent to wash out of the detection 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.
[0070] Following the application of a binding reagent to a
detection zone, the remainder of the porous solid phase material
may be treated to block any remaining binding sites. Blocking can
be achieved by treatment for example with protein (e.g. bovine
serum albumin or milk protein), or with polyvinylalcohol or
ethanolamine, or combinations thereof. To assist the free mobility
of the labelled binding reagent when the porous carrier is
moistened with the sample, the porous carrier may further comprise
a material such as sucrose or lactose. Such material may be
depositing for example as an aqueous solution in the region to
which the labelled binding reagent is to be applied. Alternatively
such material may be deposited upstream from or at the labelled
binding reagent.
[0071] The nitrocellulose porous carrier may have having a pore
size of at least about 1 micron, for example greater than about 5
microns, and for example about 8-12 microns.
[0072] The nitrocellulose porous carrier may be backed e.g. with a
plastics sheet, to increase its handling strength. This can be
manufactured easily by forming a thin layer of nitrocellulose on a
sheet of backing material such as Mylar.TM..
[0073] The labelled binding reagent may be provided on a separate
macroporous carrier material to that of the detection zone. 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 such as polyethylene 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.
[0074] The first and/or second assays may comprise a glass-fibre
macroporous carrier provided upstream from and overlapping at its
distal end a nitrocellulose porous carrier.
[0075] The assay device or kit may further comprise one or more
optical detection means such as a photodetector and one or more
light sources such as an LED positioned so as to optically
illuminate the detection zones and determine the presence and/or
amount of labelled species present. The assay device may further
comprise one or more of a power source, a computation means, a
signal transduction means, an algorithm, a display means, a memory
means and data in/out port. The assay device may comprise a housing
which serves to house the first and second assays as well as other
components of the device. The device may comprise stored threshold
values.
[0076] The first and second assays may be provided for example in a
side by side arrangement or in a face to face arrangement wherein
one assay is provided above the other. A sole optical means may be
arranged to detect both detection zones.
[0077] In addition to measuring the detection zones of the
respective assays as well as the control zones where present, the
optical means may also measure a reference zone, namely a portion
of the flow-path which is free from binding reagent in the dry
state.
[0078] The purpose of the reference zone is to provide a signal
value against which the signal value obtained at the detection zone
may be referenced. This takes into account any change in optical
signal due to for example, the wetting of the porous carrier any
differences in the optical absorption of the liquid sample.
[0079] An assay device for measuring the levels of analyte in a
liquid sample comprising an optical detection means arranged to
measure the intensity of light reflected from a detection zone,
control zone and reference zone of an assay device is described in
EP1484601.
[0080] The illumination process may be carried out sequentially
such that device is able to know which from which zone light is
being reflected from onto the photodetector. The assay strips may
be positioned in a side by side arrangement and the photodetector
and light sources positioned above the plane of the strips such
that the detection control and reference zones are positioned
towards the light sources and optical detectors.
[0081] The first assay differs from the second assay such that the
respective assays are capable of measuring analyte at different
levels.
[0082] For example the first and second assays may employ differing
assay architectures, such as the first assay employing a sandwich
binding reaction and the second assay employing a competition or
inhibition reaction. The first assay may comprise a mobilisable
labelled binding reagent for the analyte provided upstream from a
detection zone, said detection zone comprising a non-labelled
immobilised binding reagent for the analyte and the second assay
may comprise a mobilisable binding reagent for the analyte provided
upstream from an immobilised non-labelled binding reagent for the
mobilisable binding reagent. Alternatively the second assay may
comprise a mobilisable labelled analyte or analyte analogue reagent
provided upstream from an immobilised non-labelled binding reagent
for the analyte. The sandwich assay is the high sensitivity assay,
namely it is capable of measuring analyte at a lower concentration
range and the inhibition or competition assay is a low sensitivity
assay, namely it is capable of measuring analyte at a higher
concentration range.
[0083] The assay device may for example comprise first and second
assays wherein the non-labelled binding reagent of the first assay
differ from the non-labelled binding reagent of the second assay,
and/or the labelled binding reagent of the first assay differs from
the labelled binding reagent of the second assay. For example this
may be a difference in concentration, or a difference in affinity
for an analyte, analyte analogue or binding reagent. 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 first
and second assays may be varied in this way such that they are
capable of determining an analyte at different concentration
ranges.
[0084] Thus the assay device may comprise a high analyte
sensitivity first assay comprising a mobilisable labelled binding
reagent of a certain concentration or affinity provided upstream
from a detection zone and a low analyte sensitivity second assay
comprising a mobilisable labelled binding reagent having a lower
concentration or affinity provided upstream from a detection zone.
Alternatively or additionally, the first assay comprising an
immobilised binding reagent at a detection zone of a certain
concentration or affinity and a second assay may comprise an
immobilised binding reagent at a detection zone having a lower
concentration or affinity.
[0085] Another way to lower the sensitivity of an assay is to
reduce the binding reagent to particle loading. Thus the assay
device may comprise a first high analyte sensitivity assay and a
second low analyte sensitivity assay wherein the first assay
comprises a mobilisable particle labelled binding reagent provided
upstream from a detection zone having a ratio of binding reagent to
particle label and wherein the second assay comprises a mobilisable
particle labelled binding reagent provided upstream from a
detection zone having a lower ratio of binding reagent to particle
label than that of the first assay.
[0086] A further way to lower the sensitivity of an assay is to
provide a labelled binding reagent having a label of a low optical
density. This may be achieved for example by provision of a polymer
particle label having a low concentration of dye. Thus the assay
device may comprise a first high analyte sensitivity assay and a
second low analyte sensitivity assay wherein the first assay
comprises a mobilisable particle labelled binding reagent provided
upstream from a detection zone, said label having an optical
density and wherein the second assay comprises a mobilisable
particle labelled binding reagent provided upstream from a
detection zone wherein the label has a lower optical density than
that of the first assay.
[0087] 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 readily detected. Therefore the assay may comprise a
first high analyte sensitivity assay comprising an optically
detectable particle labelled binding reagent provided upstream from
a detection zone and a second low analyte sensitivity assay
comprising an optically detectable non-particle labelled binding
reagent provided upstream from a detection zone. An example of a
optically detectable non-particle label may be a dye. The dye may
be fluorescent.
[0088] A yet further way to lower the sensitivity of the assay is
to employ a porous carrier such as nitrocellulose having a higher
flow rate. Thus the assay device may comprise a first high analyte
sensitivity assay having a porous carrier having a flow-rate and a
second low analyte sensitivity assay having a porous carrier having
a higher flow rate than that of the first assay.
[0089] A further way to lower analyte sensitivity is to provide for
a fast release of the labelled binding reagent from the porous
carrier during contact with the liquid sample. Ways to increase the
release of labelled binding reagent are for example to increase the
levels of sugars or methylcellulose in the device.
[0090] A further way to lower analyte sensitivity is to employ a
labelled binding reagent having a lower optical sensitivity. One
way of achieving this to use a colour which is less sensitive to an
optical detector.
Use of a Scavenger Reagent
[0091] A further way to lower the analyte sensitivity is to provide
a scavenger binding reagent to bind to analyte. The scavenger
binding reagent may be provided upstream from a detection zone and
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 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. Either or both of the assays may employ a
scavenger binding reagent and the scavenger binding reagents may
differ from one another in terms of their concentration, affinity
or both.
[0092] According to an embodiment, the assay device comprises a
first assay comprising a first porous carrier comprising a
mobilisable labelled binding reagent provided upstream from a
detection zone and a second assay comprising a mobilisable labelled
binding reagent provided upstream from a detection zone and a
scavenger binding reagent also provided upstream from the detection
zone of the second assay. The first assay may be the high analyte
sensitivity assay and the second assay may be the low analyte
sensitivity assay.
[0093] The scavenger reagent may be provided in a mobilisable
form.
[0094] 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 scavenger binding reagent
may be varied to change the sensitivity of the second 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. The amount of labelled
binding reagent in the first and second assays may be varied.
Increasing the amount of labelled binding reagent has the tendency
to reduce the hook effect and the amount of labelled binding
reagent present, especially in the lower sensitivity assay, may be
varied depending upon the analyte range.
[0095] The scavenger binding reagent may be capable of binding to
the same or a different analyte binding region. In an exemplary
embodiment, the scavenger binding reagent is capable of binding to
a different binding region of the analyte. In particular where the
analyte to be determined is hCG, the scavenger binding reagent is
capable of binding to the beta-subunit, and the mobilisable
labelled binding reagent is capable of binding to the
alpha-subunit.
[0096] According to an exemplary embodiment, the assay device
comprises a first assay comprising a glass-fibre porous carrier
material comprising a mobilisable particle-labelled binding reagent
for an analyte and a nitrocellulose porous carrier material
provided downstream from the glass-fibre porous carrier material
having a detection zone comprising a immobilised non-labelled
binding reagent for the analyte and a second assay comprising a
glass-fibre porous carrier material comprising a mobilisable
particle-labelled binding reagent for a first binding region of the
analyte and a mobilisable scavenger binding reagent for a second
binding region of the analyte and a nitrocellulose porous carrier
material provided downstream from the glass-fibre porous carrier
material having a detection zone comprising an immobilised
non-labelled binding reagent for the second binding region of the
analyte.
[0097] It will be appreciated that the above ways to alter the
assay sensitivity of an assay are not be exhaustive. The assay
device may comprise one of more the above features to affect assay
sensitivity. The particular assay architecture chosen would depend
upon the analyte and its concentration range.
Other Embodiments
[0098] In addition to the embodiments, aspects and objects of the
present invention disclosed herein, including the claims appended
hereto, the following paragraphs set forth additional, non-limiting
embodiments and other aspects of the present invention.
[0099] In one aspect the invention provides a assay device for the
determination of analyte in a liquid sample over an extended
concentration range comprising a first assay and a second assay,
wherein the first assay for an analyte comprises a first flow-path
having a sole detection zone capable of immobilising a labelled
binding reagent and the second assay for said analyte comprises a
second flow-path having a sole detection zone capable of
immobilising a labelled binding reagent, wherein the presence of
labelled binding reagent at the detection zones provides an
indication of the presence and/or extent of analyte in said liquid
sample. The first assay is capable of providing an indication of
the level of analyte in a first concentration range and the second
assay is capable of providing an indication of the level of analyte
in a second concentration range. In certain embodiments the first
and second concentration ranges overlap. The device is capable of
providing an indication of the level of analyte with respect to one
or more thresholds. In some embodiments, the first and/or second
flow-path comprises a porous carrier, such as a lateral flow porous
carrier.
[0100] In certain embodiments, the first and/or second assay
comprises a mobilisable labelled binding reagent for the analyte
provided upstream from a detection zone. In other embodiments, the
detection zone of the first and/or second assay comprises an
immobilised binding reagent for the analyte. Optionally, the first
assay defines a sandwich assay and the second assay defines a
competition or inhibition assay. In another embodiment, the first
assay comprises a mobilisable labelled binding reagent for the
analyte provided upstream from a detection zone, said detection
zone comprising an immobilised binding reagent for the analyte and
wherein the second assay comprises a mobilisable binding reagent
for the analyte provided upstream from provided upstream from a
detection zone, said detection zone comprising an immobilised
analyte or analyte analogue for the mobilisable binding reagent. In
other embodiments, the first assay comprises a mobilisable labelled
binding reagent for the analyte provided upstream from a detection
zone, said detection zone comprising an immobilised binding reagent
for the analyte and wherein the second assay comprises a
mobilisable labelled analyte or analyte analogue provided upstream
from an immobilised binding reagent for the analyte.
[0101] In certain embodiments, the first assay is a high analyte
sensitivity assay and the second assay is a low analyte sensitivity
assay. In other embodiments, the first assay comprises a
mobilisable labelled binding reagent for an analyte provided
upstream from a detection zone, wherein the detection zone
comprises an immobilised non-labelled binding reagent for the
analyte; and wherein the second assay comprises a mobilisable
labelled binding reagent for a first binding region of the analyte
and a scavenger binding reagent for the analyte provided upstream
from a detection zone, wherein said detection zone is capable of
binding the labelled binding reagent. For example, the detection
zone of the first and/or second assays comprises an immobilised
binding reagent for the analyte. In another embodiment, for the
second assay, the scavenger binding reagent has a higher affinity
for the analyte than the mobilisable labelled binding reagent.
Similarly, the mobilisable labelled binding reagent and the
scavenger binding reagent of the second assay are for respectively
a first and second binding region of the analyte, the detection
zone comprising an immobilised binding reagent for a second binding
region of the analyte. In a related embodiment, the mobilisable
labelled binding reagent and the scavenger binding reagent of the
second assay are provided in the same region. In another related
embodiment, the scavenger binding reagent is provided in a
mobilisable form. In certain embodiments, the mobilisable binding
reagents and the scavenger reagent are provided on and/or in a
first porous carrier material and the immobilised binding reagent
provided at the detection zone is provided on and/or in a second
porous carrier material. The first porous carrier material is,
e.g., glass-fibre and the second porous carrier material is
nitrocellulose.
[0102] In some embodiments, the analyte is hCG. For example, the
first binding region of hCG is the alpha sub-unit and the second
binding region is the beta sub-unit. Optionally, the binding
reagents are antibodies, and/or the binding reagent is labelled
with an optically detectable particle such as a coloured polymer
label. In certain embodiments, the fluid sample is urine.
[0103] In some embodiments, the devices of the invention include a
common sample application region which serves to supply liquid
sample to both assays. In other embodiments, the device includes a
housing wherein the first and second assays are provided within
said housing.
[0104] In another aspect, the invention provides an assay device
for determining the presence and/or extent of an analyte in a
liquid sample comprising a first assay and a second assay wherein
said first assay comprises a porous carrier having a detection zone
comprising an immobilised binding reagent for an analyte and
provided upstream from the detection zone is a mobilisable labelled
binding reagent for said analyte; and wherein said second assay
comprises a detection zone comprising an immobilised binding
reagent for the analyte and provided upstream from the detection
zone is a mobilisable labelled binding reagent for the analyte and
a scavenger reagent for said analyte, wherein detection of the
presence of labelled binding reagent at the detection zone provides
an indication of the presence and/or extent of analyte in said
liquid sample. In some embodiments, the first assay is a high
sensitivity assay and the second assay is a low analyte sensitivity
assay. In other embodiments, the analyte is hCG.
[0105] The invention provides devices in which the first assay
either provides an indication of whether the subject is pregnant or
not, based upon whether the level of hCG detected is respectively
above or below a base threshold, and/or if pregnant, the level of
hCG in a first range of less than or equal to a first threshold,
the second assay provides an indication of the level of hCG in a
second range of above or equal to a second threshold and the first
and second assays together provide an indication of the level of
hCG in a third range of greater than the first threshold but less
than the second threshold. The invention also provides devices in
which for the second assay, the scavenger binding reagent has a
higher affinity for the analyte than the mobilisable labelled
binding reagent.
[0106] In a further aspect, the invention provides an assay kit for
determining the presence an/or extent of an analyte in a liquid
sample comprising a first assay and a second assay wherein said
first assay comprises a porous carrier having a detection zone
comprising an immobilised binding reagent for an analyte and
provided upstream from the detection zone is a mobilisable labelled
binding reagent for said analyte; and wherein said second assay
comprises a detection zone comprising an immobilised binding
reagent for the analyte and provided upstream from the detection
zone is a mobilisable labelled binding reagent for the analyte and
a scavenger reagent for said analyte, wherein detection of the
presence of labelled binding reagent at the detection zones
provides an indication of the presence and/or extent of the analyte
in the sample.
[0107] In another aspect, the invention provides a method of
fabricating an assay device to reduce a hook effect, wherein the
assay device comprises a first flow-path and a second flow-path,
comprising the steps of providing a first flow-path comprising a
porous carrier and a second flow-path comprising a porous carrier;
contacting the first flow-path with a first material so as to form
a first zone; and contacting the second flow-path with a first
material so as to form a second zone, wherein the first zone and
the second zone are separated by a distance of at least about 5 mm,
and whereby the hook effect is thereby reduced. In certain
embodiments, the first material contains latex.
Comparative Example 1
Preparation of an Assay Device Comprising a Single Porous Carrier
Comprising a First Upstream Detection Zone for a Sandwich Assay and
a Second Downstream Detection Zone for an Inhibition Assay
[0108] An assay test-strip comprising a first upstream detection
zone for a sandwich assay and the second downstream detection zone
for an inhibition assay and a mobilisable labelled binding reagent
provided upstream from said zones was prepared as follows:
[0109] A solution of 1.5 mg/ml mAb mouse anti-human .beta.-hCG
(clone 3468, supplied in house) in PBSA buffer and 7.2 KIU/ml hCG
(Scipac) in PBSA/ovalbumin was mixed for 1-hour to provide an
anti-.beta. hCG-hCG conjugate. The resulting solution was plotted
in the form of a line onto nitrocellulose strips (Whatman) of
dimensions 40 mm length.times.6 mm width, 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 conjugate was
dispensed at a concentration of 1.5 mg/ml and a rate of 1 .mu.l/cm
at the 16 mm position (distance along the nitrocellulose) using a
Biodot xyz3050 dispensing platform to form a second downstream
detection zone.
[0110] The first detection zone was prepared by plotting a line of
anti-human .beta.-hCG antibody (clone 3468) at a concentration of 3
mg/ml in PBSA buffer and a rate of 1 .mu.l/cm on the nitrocellulose
at the 10 mm position using the Biodot xyz3050 dispensing platform.
The nitrocellulose was heated briefly to 55.degree. C. and blocked
with a solution of 1% w/w PVA in 0.05% w/w Tween-20 in
pH9Tris-buffered saline/5% ethanol, followed by treatment with 2%
w/w sucrose in de-ionised water. The nitrocellulose was thereafter
heated briefly at 75.degree. C.
[0111] Mouse-anti-human .alpha.-hCG mAb (clone 3299, supplied
in-house) was conjugated to 400 nm blue latex polystyrene latex
(Duke Scientific) in BSA/sucrose and sprayed onto a glass-fibre pad
(F529-09, Whatman) at a rate of 50 g/hr and 110 mm/s. The glass
fibre pad was partially overlaid onto and upstream from the
nitrocellulose porous carrier.
Example 1
[0112] Assay devices were prepared in a similar manner to that of
Comparative Example 1 except that the first and second detection
zones were provided on respectively first and second test-strips,
wherein the first and second detection zones were provided on
nitrocellulose, each test-strip comprising glass-fibre sprayed with
mobilisable latex labelled .alpha.-hCG antibody provided upstream
from the nitrocellulose.
Preparation of the First Test-Strip
[0113] The detection zone was prepared by plotting a line of
anti-human .beta.-hCG antibody (clone 3468) at a concentration of 3
mg/ml in PBSA buffer and a rate of 1 .mu.l/cm on the nitrocellulose
at the 10 mm position using the Biodot xyz3050 dispensing platform.
The nitrocellulose was heated briefly to 55.degree. C. and blocked
with a solution of 1% w/w PVA in 0.05% w/w Tween-20 in
pH9Tris-buffered saline/5% ethanol, followed by treatment with 2%
w/w sucrose in de-ionised water. The nitrocellulose was thereafter
heated briefly at 75.degree. C.
[0114] Mouse-anti-human .alpha.-hCG mAb (clone 3299, supplied
in-house) was conjugated to 400 nm blue latex polystyrene latex
(Duke Scientific) in BSA/sucrose and sprayed onto a glass-fibre pad
(F529-09, Whatman) at a rate of 50 g/hr and 110 mm/s. The glass
fibre pad was partially overlaid onto and upstream from the
nitrocellulose porous carrier.
Preparation of Second Test-Strip
[0115] A solution of 1.5 mg/ml mAb mouse anti-human .beta.-hCG
(clone 3468, supplied in house) in PBSA buffer and 7.2 KIU/ml hCG
(Scipac) in PBSA/ovalbumin was mixed for 1-hour to provide an
anti-.beta. hCG-hCG conjugate. The resulting solution was plotted
in the form of a line onto nitrocellulose strips (Whatman) of
dimensions 40 mm length.times.6 mm width, 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 conjugate was
dispensed at a concentration of 1.5 mg/ml and a rate of 1 .mu.l/cm
at the 16 mm position (distance along the nitrocellulose) using a
Biodot xyz3050 dispensing platform to form the detection zone.
[0116] Mouse-anti-human a-hCG mAb (clone 3299, supplied in-house)
was conjugated to 400 nm blue latex polystyrene latex (Duke
Scientific) in BSA/sucrose and sprayed onto a glass-fibre pad
(F529-09, Whatman) at a rate of 50 g/hr and 110 mm/s. The glass
fibre pad was partially overlaid onto and upstream from the
nitrocellulose porous carrier.
[0117] The test-strips according to Example 1 and Comparative
Example 1 were tested using in-house readers with calibrated hCG
buffer standards at concentrations 0, 25 50, 100, 250, 500, 1000,
2500, 5000, 10000, 15000, 20000, 25000, 50000, 150000, 200000 and
250000 mIU/ml hCG.
[0118] The signal intensity measured at the inhibition detection
zones as a function of hCG concentration of the assays of example 1
(denoted by --.diamond-solid.--) and comparative example 1 (denoted
by --.box-solid.--) is shown in FIG. 2 as signal in arbitrary units
vs. mIU/ml hCG.
[0119] As can be seen from this Figure, the inhibition detection
zone of Comparative Example 1 shows an initial plateau at levels of
hCG ranging from 0-100 mIU/ml, followed by a decrease in the
intensity at higher levels of hCG as expected. However, at higher
levels still, the signal intensity was observed to increase. By
comparison, the signal intensity of Example 1 decreases at higher
hCG levels without the subsequent increase in signal intensity at
higher hCG levels. As can be seen, the inhibition zone of assay
device constructed according to Comparative Example 1 has a more
limited range over which hCG may be measured.
Example 2
Preparation of Assay Devices Comprising a First Test-Strip
Comprising a First Sandwich Assay and a Second Test-Strip
Comprising a Scavenger Assay in Addition to a Sandwich Assay
Preparation of the Second Test-Strip.
[0120] MAb mouse anti-human .beta.-hCG antibody (clone 3468) at a
concentration of 3 mg/ml in PBSA buffer was plotted onto
nitrocellulose (of type and dimensions as that according to
Comparative Example 1) at the 10 mm position at a rate of 1
.mu.l/cm using a Biodot XYZ3050 dispensing platform to provide a
sole detection zone for the first assay.
[0121] 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 (clone 3468) at
3 mg/ml to give a final % blue latex of 3% and a final 3468
concentration of 0.075 mg/ml. The resulting mixture was sprayed at
2.02 .mu.g/cm onto F529-09 glass fibre.
[0122] The glass fibre was partially overlaid and provided upstream
from the nitrocellulose to provide the first assay test-strip.
Preparation of the First Assay Test-Strip
[0123] The first assay test strip was prepared according to the
first assay test-strip according to that of Example 1.
Comparative Example 2
[0124] Assay devices constructed wherein both detection zones were
provided on the same porous carrier were not able to result in the
measurement of an analyte concentration over an extended analyte
range.
[0125] Assay devices according to Example 2 were tested using
in-house detection zone optical readers with calibrated hCG buffer
standards at 12 concentrations ranging from 0-250000 mIU/ml hCG. 10
replicates per concentration level were measured giving a total
number of assay devices that were tested of 120.
[0126] The signal intensity vs hCG concentration for the second
assay constructed according to Example 2 is shown in FIG. 3.
[0127] The first assay test-strip according to Example 2 was able
to determine the amount of hCG present up to about 400 mIU/ml
before the assay curve flattened off. The second assay test-strip
according to Example 2 was able to detect hCG levels of greater
than about 1000 mIU/ml. Measurement of the signals at both the
first and second assay test-strips enabled determination of the
level of hCG between about 400 mIU/ml and 1000 mIU/ml.
The Effect of Varying the Amounts of Scavenger Antibody
[0128] Second assay test-strips according to Example 2 were
prepared except the amount of scavenger antibody present was varied
during preparation of the strip to give a final 3468 concentration
of 0.12, 0.16, 0.2 and 0.24 mg/ml.
[0129] As can be seen from FIG. 4 increasing the amount of
scavenger antibody lowers the amount of analyte captured at the
detection zone.
Example 3
[0130] The aim of this Example is to assess the effect of changing
secondary latex spray position on assay curve performance, with the
intent of reducing the hook effect. Materials used are as
follows:
TABLE-US-00001 Latex 2 Reagent Composition Latex 1 (Secondary
latex) Test latex 3% solids, anti .beta. hCG 42.25 mls 42.25 mls
Control latex 0.7% solids, Goat anti none 16.83 mls rabbit IgG Free
antibody anti .beta. hCG 1.16 mls 1.16 mls
[0131] Two latex sprays were generated. For Latex spray 1, the
distance from base of glass fibre was held constant at 5 mm, while
for Latex spray 2, the distance from base of glass fibre was set at
7, 9, 10, 11, 13, 14, or 16 mm.
[0132] As shown below, the signal was measured at 3 minutes run
time as a percentage attenuation (% A) of the test line.
TABLE-US-00002 Distance from Base [hCG] mIU/ml of Glass Fibre (mm)
400 1000 2000 10000 50000 250000 7 17.74 29.66 39.44 49.80 48.15
30.10 9 14.49 22.54 31.07 54.90 50.98 34.23 10 15.63 23.11 32.76
59.34 61.04 42.57 11 16.68 24.62 33.11 58.65 61.47 46.54 13 13.88
20.71 29.33 57.66 62.56 47.99 14 17.35 22.82 31.87 58.40 64.42
48.82 16 14.69 19.86 28.75 56.55 63.81 49.20
[0133] Results shown above and in FIG. 5 demonstrate that if
secondary latex spray position is below 9 mm, the % A signal at the
higher end of the curve falls greatly (for example, signal at
250,000 mIU/ml hCG drops below signal 2000 mIU/ml). It can be
concluded that increasing latex spray position to 10 mm or greater,
e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
30 or greater than 30, significantly reduces the hook effect.
* * * * *