U.S. patent application number 11/595820 was filed with the patent office on 2007-11-08 for assay reagents and devices.
This patent application is currently assigned to Inverness Medical Switzerland GmbH.Unipath. Invention is credited to Yvonne E. Penfold, David A. Percival.
Application Number | 20070259446 11/595820 |
Document ID | / |
Family ID | 8225101 |
Filed Date | 2007-11-08 |
United States Patent
Application |
20070259446 |
Kind Code |
A1 |
Penfold; Yvonne E. ; et
al. |
November 8, 2007 |
Assay reagents and devices
Abstract
A reagent useful in immunoassays, comprising a direct
particulate level co-sensitised with a specific binding agent
having specificity for an analyte or analyte analogue and with a
non-specific protein which can participate in a control reaction
with another specific binding agent which does not bind to the
first specific binding agent nor participate in the formation of a
complex by means of which detection of the analyte or analyte
analogue is accomplished. Preferably the first specific binding
agent is an antibody raised in a first species and the non-specific
protein is an immunoglobulin from another species. Optionally, the
reagent additionally comprises a second population of the direct
particulate label sensitised solely with the non-specific
protein.
Inventors: |
Penfold; Yvonne E.;
(Bedford, GB) ; Percival; David A.; (Harwarden,
GB) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP (w/ISA)
155 SEAPORT BLVD.
BOSTON
MA
02210-2600
US
|
Assignee: |
Inverness Medical Switzerland
GmbH.Unipath
Bedford
GB
|
Family ID: |
8225101 |
Appl. No.: |
11/595820 |
Filed: |
November 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09557955 |
Apr 25, 2000 |
7153681 |
|
|
11595820 |
Nov 9, 2006 |
|
|
|
08935537 |
Sep 23, 1997 |
6133048 |
|
|
09557955 |
Apr 25, 2000 |
|
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Current U.S.
Class: |
436/501 ;
422/68.1 |
Current CPC
Class: |
G01N 33/558 20130101;
G01N 33/54393 20130101; G01N 33/585 20130101; Y10S 435/805
20130101; Y10S 435/97 20130101; Y10S 436/81 20130101 |
Class at
Publication: |
436/501 ;
422/068.1 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 27, 1996 |
EP |
EP 96307078.4 |
Claims
1. A reagent useful in immunoassays, comprising a direct
particulate label co-sensitised with (i) a specific binding agent
having specificity for an analyte or analyte analogue, and (ii) a
non-specific protein which can participate in a control reaction
with another specific binding agent which does not bind to said
first specific binding agent nor participate in the formation of a
complex by means of which detection of said analyte or analyte
analogue is accomplished.
2. A reagent according to claim 1, wherein said first specific
binding agent is an antibody raised in a first species and said
non-specific protein is an immunoglobulin from another species.
3. A reagent according to claim 2, wherein said first specific
binding agent is a murine antibody.
4. A reagent according to claim 2, wherein said non-specific
protein is a rabbit immunoglobulin.
5. A reagent according to claim 1, additionally comprising a second
population of said direct particulate label sensitised solely with
said non-specific protein.
6. A reagent according to claim 2, comprising coloured latex
particles of diameter less than 0.5 micron, co-sensitised with an
anti-hCG murine monoclonal antibody and with rabbit IgG.
7. A reagent according to claim 6, additionally comprising
same-coloured latex particles of diameter less than 0.5 micron
sensitised solely with rabbit IgG, the ratio of said co-sensitised
particles to said second particles being at least 2:1.
8. An assay device according to claim 7, wherein said ratio is
about 3:1.
9. An assay device of the type wherein a sample liquid
reconstitutes a labelled reagent and carries it into a detection
zone and a control zone, binding of said labelled reagent in these
zones revealing the assay result, wherein said labelled reagent is
as claimed in claim 1.
10. An assay device according to claim 9, wherein said detection
zone contains an immobilised specific binding agent which acts as a
direct or indirect capture means for said analyte or analyte
analogue but which does not bind to said non-specific protein, and
said control zone contains a specific binding agent which binds
said non-specific protein but does not bind said specific binding
agent co-sensitised on said first particle.
11. An assay device according to claim 9, wherein said detection
zone contains an immobilised specific binding agent which acts as a
direct or indirect capture means for hCG, said control zone
contains an immobilised anti-rabbit IgG antibody, and said labelled
reagent is coloured latex particles of diameter less than 0.5
micron, co-sensitised with an anti-hCG murine monoclonal antibody
and with rabbit IgG.
Description
FIELD OF THE INVENTION
[0001] This invention relates to reagents useful in immunoassays
and to assay devices using such reagents.
BACKGROUND TO THE INVENTION
[0002] Many assays are now available which utilise the technology
described in EP-A-291194, wherein a particulate direct label such
as a gold sol or coloured latex particle is used to reveal the
result of an assay conducted in a porous carrier such as a porous
strip. Concentration of the particulate label in a comparatively
small detection zone in the strip reveals the assay result. It is
common practice for the strip to include a control zone, normally
located downstream from the detection zone, in which a coloured
signal is also generated to reassure the user that the test has
been correctly performed. In most commercial products based on this
concept, the test and control signals are generated using the same
particulate label.
[0003] This technology copes very well with most assay situations,
but there can be extreme situations in which the clarity of the
signals could be improved For example, the control zone signal may
appear rather feint if the concentration of analyte is very high
and is causing most of the particle label to become bound in the
detection zone, leaving insufficient label to carry through and
provide a strong control signal.
[0004] An objective of the present invention is to provide reagents
and assay devices in which improved clarity of assay signals are
obtained irrespective of the amount of analyte that may be present
in a sample being tested.
[0005] A further objective is to provide good clear assay signals
without the use of excessive amounts of labelled reagent.
GENERAL DESCRIPTION OF THE INVENTION
[0006] By the invention we provide a reagent useful in
immunoassays, comprising a direct particulate label co-sensitised
with a specific binding agent having specificity for an analyte or
analyte analogue and with a non-specific protein which can
participate in a control reaction with another specific binding
agent which does not bind to the first specific binding agent nor
participate in the formation of a complex by means of which
detection of the analyte or analyte analogue is accomplished.
[0007] Preferably the quantity of specific binding agent on the
co-sensitised particle exceeds the quantity of non-specific protein
thereon. Preferably there is at least a 2:1 ratio by weight between
these two materials on the particle. More preferably at least about
5:1, ideally about 10:1. The primary specific activity, in terms of
analyte-binding and control signal formation, is therefore heavily
biased in favour of analyte-binding.
[0008] Optionally, the reagent additionally comprises a second
population of the direct particulate label sensitised solely with
the non-specific protein.
[0009] Preferably the first specific binding agent is an antibody
raised in a first species and the non-specific protein is an
immunoglobulin from another species. For example, the first
specific binding agent can be a murine antibody.
[0010] The non-specific protein can be a rabbit immunoglobulin, for
example, but any immunoglobulin from any species can be used
provided that it does not bind either to the analyte or to any
reagent that participates in detection of the analyte.
[0011] A preferred reagent according to the invention comprises
coloured latex particles of diameter less than 0.5 micron,
co-sensitised with an anti-hCG murine monoclonal antibody and with
rabbit IgG. Preferably this reagent additionally comprises
same-coloured latex particles of diameter less than 0.5 micron
sensitised solely with rabbit IgG, the weight ratio of the
co-sensitised particles to the second particles being at least 2:1,
more preferably about 3:1.
[0012] Another embodiment of the invention is an assay device of
the type wherein a sample liquid reconstitutes a labelled reagent
and carries it into a detection zone and a control zone, binding of
the labelled reagent in these zones revealing the assay result,
characterised in that the labelled reagent is as described in any
one of the foregoing paragraphs.
[0013] Preferably the detection zone contains an immuobilised
specific binding agent which acts as a direct or indirect capture
means for the analyte or analyte analogue but which does not bind
to the non-specific protein, and the control zone contains a
specific binding agent which binds the non-specific protein but
does not bind the specific binding agent co-sensitised on the first
particle.
[0014] The particles can be any micro-particles that can be used as
mobile labels in strip-format assays. Such assays are described in
many publications, including EP-A-291194, EP-A-383619, WO 96/09553
and WO 96/09546. Appropriate particles include latex (polystyrene)
particles, usually of diameter less than about 0.5 micron, metal
sols, such as gold sols, non-metallic elemental sols, such as
selenium or carbon, and dye sols.
[0015] The invention will be described with particular reference to
test kits useful in monitoring of body fluid analytes, and
especially to home monitoring of urinary analytes of relevance to
the determination of pregnancy (hCG) or of the fertility status of
the human ovulation cycle (by measuring LH and/or E3G and/or P3G,
for example). This is by way of example only, and it will be
appreciated that the invention is useful in many other contexts
where other sample liquids and analytes are involved, such as
assays for cancer markers, cardiac markers, blood glucose, drugs of
abuse, hormones, infectious disease markers, tests in therapeutic
drug monitoring, manufacturing and raw material quality control,
and tests for effluent and pollution levels.
[0016] Preferably the detection zone contains an immobilised
specific binding agent which acts as a direct or indirect capture
means for hCG, the control zone contains an immobilised anti-rabbit
IgG antibody, and the mobile labelled reagent comprises coloured
latex particles of diameter less than 0.5 micron, co-sensitised
with an anti-hCG murine monoclonal antibody and with rabbit
IgG.
[0017] Preparation of the novel reagents of the invention, and the
manufacture of assay devices using these novel reagents, can both
be accomplished using conventional procedures. The co-sensitised
particulate label can be prepared by contacting
commercially-available particulate labels, such as latex
(polystyrene) particles of appropriate dimension, in aqueous
suspension with a mixture of the two materials with which the
particles must be sensitised. For example, these materials can be a
mixture of a murine monoclonal antibody directed against the
alpha-chain of hCG, together with a non-specific polyclonal
antibody such as rabbit IgG. These materials need to be present in
an appropriate weight ratio, as described elsewhere herein. The
co-sensitisation needs to be conducted under buffered conditions,
as is standard practice. Following a sufficient time interval to
allow the materials to deposit onto the particles, unbound
materials can be separated by conventional procedures such as
centrifugation, filtration and/or ultra-filtration and the
co-sensitised particles resuspended in a conventional storage
buffer solution ready for use in the preparation of an assay. A
specific example of a co-sensitisation procedure is given
below.
[0018] In order to describe the benefits of the invention in more
detail, we can consider as an example its applicability in the
context of a pregnancy test based on the immunochromatographic
format using coloured latex particles as a mobile direct label in
an assay strip. The assay strip, which is, for example, made from
nitrocellulose of pore size about 8 microns backed with "Mylar"
polyester, includes two transverse lines of deposited immobilised
specific binding reagents, namely a test line containing an
anti-.beta. hCG murine monoclonal and a control line downstream
from the test line containing an immobilised murine monoclonal
raised against rabbit IgG. At a location upstream from the test
line is a mobile reagent comprising coloured latex particles of
diameter approximately 0.3 microns. This location can be on the
nitrocellulose or in a separate pad or wick of porous material
which is upstream in the flow path by which sample liquid (urine)
can reach the nitrocellulose. The mobile reagent comprises two
populations of latex particles, namely: [0019] a) particles
co-sensitised with an anti-.alpha. hCG murine monoclonal and with
the same rabbit IgG against which the control line antibody was
raised; and [0020] b) a second population of the same latex
particles simply bearing the rabbit IgG.
[0021] Application of a urine sample will mobilise the latex
particles. If the urine contains hCG, a sandwich complex can form
between the anti-hCG monoclonal on the co-sensitised particle and
the anti-hCG antibody in the test line. In consequence, at least
some of the co-sensitised particles will become bound in the test
line to provide a coloured signal indicative of the presence of
hCG. Remaining mobilised co-sensitised particles which do not
become bound in the test line, eg. because they are in excess
relative to the amount of hCG present in the sample, can become
bound downstream in the control line by interaction between the
control line antibody and the rabbit IgG on the co-sensitised
particles. In addition, the second population of latex particles
bearing only the rabbit IgG will be mobilised and carried past the
test line to reinforce the control signal.
[0022] We have found in practice that if the hCG concentration in
the urine sample is comparatively high (although not so high as to
induce the well-known "hook effect" which causes a drop in the
apparent hCG signal) most of the anti-hCG particles are bound in
the test line. Typically this will occur if the hCG concentration
lies between about 5000 and about 15000 mIU/ml. Under these
circumstances there would be a strong test line signal but a rather
feint or completely absent control line signal. However, the a
provision of the second population of latex particles, which cannot
possibly bind in the test line, ensures that a strong control line
signal is still obtained even under these circumstances. An
appropriate blend of the co-sensitised particles and the
mono-sensitised particles is about 3:1. In a sandwich-format assay
this combination of particles provides a good clear test signal and
control signal under most assay conditions, with the exception of
extremely high analyte concentrations, while requiring the minimum
total number of particles. Substantially more particles would be
required if the test signal and control signal were generated by
completely separate populations.
EXAMPLE
[0023] This example describes a sensitisation procedure which can
be used to prepare reagents in accordance with the invention.
[0024] A latex particle reagent co-sensitised with a murine
anti-.alpha. hCG monoclonal antibody and with rabbit immunoglobulin
can be prepared as follows.
[0025] 10 ml of a commercially available suspension (10% solids) of
blue coloured latex particles of diameter about 0.3 microns is
added to 40 ml of 100 mM borate buffer pH 8.5 and stirred
vigorously. This mixture is centrifuged for 10 minutes at 13500 rpm
and the supernatant liquid removed. The latex pellet is
re-suspended in 20 ml of the same buffer.
[0026] To a separate 20 ml of the same buffer are added 400g/ml of
a murine anti-.beta. hCG monoclonal antibody and 50 .mu.g/ml rabbit
IgG. The latex-containing buffer and the antibody-containing buffer
are both heated in a water bath to 40.degree. C. and, on reaching
this temperature, 5 ml of ethanol is added to each. The antibody
solution is then immediately added to the latex suspension, mixed
using a magnetic stirrer, and incubated in the water bath for 60
minutes. At this point 50 ml of a solution of bovine serum albumin
(BSA) in the same buffer pre-warmed to 40.degree. C. is added and
the incubation continued at 40.degree. C. for a further 30 minutes.
Thereafter the solution is centrifuged for 25 minutes at 13500 rpm
and the supernatant removed. The pellet is resuspended in 50 ml of
100 mM Tris buffer pH 9.0 to provide a suspension containing 2%
solids. Optionally preservatives such as sucrose at 20% (w/v) and
BSA at 10% (w/v) can be added. This latex suspension is ready for
use in the preparation of an assay device.
[0027] An identical procedure can be used to prepare latex
particles sensitised solely with the rabbit immunoglobulin.
[0028] In this instance the 20 ml suspension of latex particles is
combined with 20 ml buffer containing 150 .mu.g/ml rabbit IgG.
[0029] In the subsequent preparation of an assay device using a
combination of the co-sensitised and mono-sensitised particles, the
two suspensions of sensitised particles can be combined in
appropriate proportions (to provide an appropriate blend, e.g. 3 to
1 of the two populations of particles) and applied as a single
combined reagent on a test strip or in a separate pad or wick
forming part of an assay device.
* * * * *