Note: Descriptions are shown in the official language in which they were submitted.
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Assay Method
The present invention relates to an improved assay
method and to devices for use in such methods. In
particular the invention relates to assay methods,
especially immunoassays, in which soluble releasable
reagents are used.
The method and devices are, in certain embodiments,
intended for use in specific binding assay procedures,
in particular immunoassay procedures. Examples of such
procedures in which soluble releasable reagents may be
employed are cited in EP-A-0171148, W092/09892,
W093/25892 and W093/25908.
In the assay procedures disclosed in EP-A-0171148
certain ancillary reagent(s) are employed and can be in
the form of a releasable reagent coating e.g. a coating
of releasable antigen or antibody, or derivative
thereof. In W092/09892, in which a device is described
possessing one or more calibration regions for the
purposes of internal referencing of an assay method, the
use of a polyvinyl alcohol (PVA) capping layer is
disclosed, in order to delay the dissolution of the
soluble reagent for a few seconds after the addition of
the sample to the device. This delayed-release is to
prevent the reagents washing from one zone to another
thereby precluding an accurate assay. However, although
limited effectiveness has been achieved by use of such a
capping layer, problems of poor reproducibility and low
sensitivity have still been encountered.
In the assay procedures disclosed in W092/09892 the
success of the method of assay depends on the spatial
separation (i.e. non-mixing) C' the various soluble
reagents released into the sample solution. However, in
other assay techniques involving only one soluble
reagent it is advantageous to ensure a maximum amount of
the released reagent remains in a certain defined area
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to ensure high assay precision and sensitivity.
Additionally, WO-A-93/025908 (ARS Holdings NV)
refers generally to the delayed release properties of a
coated patch and suggests PVA as a suitable material for
such a patch. However, there is no suggestion that
cross-linked PVA may be used as a delayed release agent.
We have now found that by employing alternative
reagents for delaying the release of soluble reagents in
assays unexpected improvements in the assay precision
and sensitivity can be achieved as compared with the
existing methods used.
Thus according to a first aspect of the present
invention we provide in assays utilising one or more
soluble releasable reagents the use of cross-linked PVA
or of copolymers of methacrylic acid or methacrylate
esters in order to achieve the delayed-release of said
soluble releasable reagents.
According to a further aspect of the present
invention we provide a method of improving assay
precision in assays utilising one or more soluble
releasable reagents in which the release of said
reagents is delayed by means of cross-linked PVA or
copolymers of methacrylic acid or methacrylate esters.
The present technique may be used for a wide
variety of chemical or biochemical test procedures but
is especially useful in connection with clinical test
procedures, most especially immunoassays.
According to a further aspect of the present
invention there is provided a sensor device for an assay
as defined above which carries on a surface thereof one
or more soluble releasable reagents coated with or
incorporated in cross-linked PVA or copolymers of
methacrylic acid or methacrylate esters.
The present method is applicable to a wide variety
of devices including, for example, dip-stick or test-
strip sensors, devices using a "sample flow-through"
configuration or devices employing sample containment.
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Sample containment.devices are preferred for carrying
out the method of the invention, with a more preferred
device being a capillary fill device, especially a
fluorescence capillary device, for example the type of
device described in EP-A-171148 or in WO-90/14590. Such
capillary fill devices may be used singly or in a
suitable holder such as described in WO-90/11830.
As described in EP-A-171148, a capillary fill
device (hereinafter CFD) typically consists of two
plates of transparent material, e.g. glass, separated by
a narrow gap or cavity. One plate acts as an optical
waveguide and carries an immobilised reagent appropriate
to the test to be carried out in the device. As
described in WO-90/14590, the other transparent plate
can carry on its surface remote from the cavity a layer
of light-absorbing or opaque material. For use in a
competition assay, the immobilised reagent may for
example be a specific binding partner to the ligand
desired to be detected and one of the plates may carry a
dissoluble reagent comprising ligand analogue, labelled
with a fluorescent dye (the ancillary reagent) When a
sample is presented to one end of the CFD it is drawn
into the gap by capillary action and dissolves the
ancillary reagent. In a competition assay for an
antigen, the fluorescently labelled antigen analogue
will compete with sample antigen for the: limited number
of antibody binding sites immobilised onto the
waveguide. Because the capillary gap is narrow
(typically about 100 microns) the reaction will
generally go to completion in a short time, possibly
less than 5 minutes depending upon the sample matrix,
assay type (e.g. sandwich or competitive immunoassay)
and antibody affinity. Thus for a competition assay,
the amount of fluorescently labelled antigen which
becomes indirectly bound to the waveguide by virtue of
complex formation will be inverselv proportional to the
concentration of antigen in the sample. In a sandwich
2 ~ ~ PCT/GB95/02236
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assay, the waveguide will carry a specific binding
partner for the ligand desired to be detected and one of
the plates will carry a dissoluble reagent comprising a
further specific binding partner labelled with a
fluorescent dye (the ancillary reagent). In a sandwich
immunoassay for an antigen, a sample antigen will form a
sandwich complex with a fluorescently labelled antibody
and an antibody immobilised on the waveguide. Thus, for
a sandwich immunoassay, the amount of fluorescently
labelled antibody which becomes indirectly bound to the
waveguide by virtue of complex formation will be
directly proportional to the concentration of antigen in
the sample.
In the above assay techniques, it is important that
the soluble releasable fluorescently labelled reagent
does not dissolve instantaneously and get washed down to
one end of the device away from the region of the
capture antibody during filling of the CFD. If this
does happen then poor assay signals are obtained with
very high imprecision producing a meaningless result.
The method of the present invention ensures that the
wash-down of the soluble reagent is minimised.
Thus, according to a further aspect of the present
invention we provide a specifically-reactive sample-
collecting and testing device possessing a cavity or
cavities each having a dimension small enough to enable
sample liquid to be drawn into the cavity by capillary
action wherein a surface of the cavity carries an
immobilised reagent appropriate to the assay to be
carried out in the device, and wherein said surface is a
surface of a transparent solid plate which in use acts
as a light transmissive waveguide and which forms a wall
of the cavity, and wherein the cavity surface(s) have
one or more zones comprising, in releasable form,
ancillary reagent(s) suitable for the desired assay,
said ancillary reagent(s) being coated with or
incorporated in cross-linked PVA or copolymers of
i
r
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WO 96/09549
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methacrylic acid or methacrylate esters.
To provide a suitable delayed release of the
soluble reagent by means of cross-linked PVA two methods
can be employed. In a first method the soluble reagent
is microdosed on the device. The reagent is dissolved
in a buffer solution containing PVA. A further layer of
PVA is then coated, suitably spray-coated, over the
printed conjugate, this PVA layer subsequently being
cross-linked, suitably by spray coating with a cross-
linking reagent. In a second method the soluble reagent
is microdosed on the device. The reagent is dissolved
in a buffer solution containing PVA. A cross-linking
reagent is then applied, suitably by spray-coating,
cross-linking the PVA present in the initial solution.
Both methods result in the production of a cross-
linked film of PVA on the surface of the device (coating
the soluble reagent or incorporating it). In use in
assay techniques a further coating of humectant can be
applied if desired, e.g. by spray-coating a
sucrose/lactose solution. This aids wetting of the
device by the sample, facilitates filling of a device of
the sample-containment type, and improves the stability
of the reagents on storage.
The preferred reagent for cross-linking the PVA is
a tetraborate solution e.g. sodium tetraborate, although
other cross-linking reagents can be employed. It has
been found that using about an 0.5-2o solution of
tetraborate provides good results, with the best results
being obtained by using about a 1% solution.
To provide a suitable delayed release of the
soluble reagent by means of a copolymer of methacrylic
acid or methacrylate esters the soluble reagent is
microdosed on the device. The reagent is dissolved in a
buffer solution (the solution possibly also containing
PVA), a film of polymer solution is then applied,
preferably by spray-coating. Again a suitable humectant
coating can be applied if desired.
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Suitable polymers have the required properties of
swellability/porosity and preferred copolymers of
methacrylic acid or methacrylate esters are those that
have a time constant of swelling within the time taken
for the assay to be performed and swell to give a pore
size suitable to allow the soluble reagent to diffuse
out. Especially useful are the reagents marketed as
EUDRAGITSTm- by R6hm Pharma. Eudragit NE 30 D provides
especially good results. Also of use is Eudragit RL.
The polymer reagents should desirably be pH
independent. Alternatively, particularly for blood or
serum based assays the polymer reagents desirably swell
between pH 7 and 8. For urine samples there is often a
wide variation in the pH of the samples., Use of
polymers which are pH-dependent i.e. which swell at
various pH levels enables one to achieve a pH cut off
for assays i.e. to select only those samples which have
the desired pH range.
The method of the invention is particularly
applicable to assays of antigens or antibodies, i.e. tc
immunoassays, and in a preferred embodiment of the
invention the ligand under assay is an antigen and the
specific binding partner comprises an antibody to the
said antigen. However, the invention is not to be taken
as limited to assays of antibodies or antigens.
Examples of ligands which may be assayed by the improvec.
assay method of the invention are given in Table A
below, together with an indication of a suitable
specific binding partner in each instance.
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Table A
Ligand Specific Binding Partner
antigen specific antibody
antibody antigen
hormone hormone receptor
hormone receptor hormone
polynucleotide strand complementary
polynucleotide strand
avidin biotin
biotin avidin
protein A immunoglobulin
immunoglobulin protein A
enzyme enzyme cofactor
(substrate) or inhibitor
enzyme cofactor enzyme
(substrate) or inhibitor
lectins specific carbohydrate
specific carbor.vdrate lectins
of lectins
The method of the invention has very broad
applicability but in particular may be used in assays
for: hormones, including peptide hormones (e.g. thyroid
stimulating hormone (TSH), luteinizing hormone (LH),
human chorionic gonadotrophin (hCG), follicle
stimulating hormone (FSH), insulin and prolactin) or
non-peptide hormones (e.g. steroid hormones such as
cortisol, estradiol, progesterone and testosterone, or
thyroid hormones such as thyroxine (T4) and
triiodothyronine), proteins (e.g. carcinoembryonic
antigen (CEA) and antibodies, aiphafetoproteir. (AFP) and
prostate specific antigen (PSA)), drugs (e.g. diaoxin;
drugs of abuse), sugars, toxins, vitamins, viruses such
as influenza, para-influenza, adeno-, hepatitis,
respiratory and AIDS viruses, virus-like particles or
microorgaaisms.
It will be understood that the term "antibodlr" usee
WO 96/09549 2200690 PCT/GB95/02236
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herein includes within its scope:
(a) any of the various classes or sub-classes of
immunoglobulin, e.g. IgG, IgA, IgM, or IgE derived
from any of the animals conventionally used, e.g.
sheep, rabbits, goats or mice,
(b) monoclonal antibodies,
(c) intact molecules or "fragments" of antibodies,
monoclonal or polyclonal, the fragments being those
which contain the binding region of the antibody,
i.e. fragments devoid of the Fc portion (e.g. Fab,
Fab', F(ab')2), the so-called "half-molecule"
fragments obtained by reductive cleavage of the
disulphide bonds connecting the heavy chain
components in the intact antibody or fragments
obtained by synthetic methods,
(d) antibodies produced or modified by recombinant DNA
techniques, including "humanised antibodies".
The method of preparation of fragments of
antibodies is well known in the art and will not be
described herein.
The term "antigen" as used herein will be
understood to include both permanently antigenic species
(for example, proteins, peptides, bacteria, bacterial
fragments, cells, cell fragments and viruses) and
haptens which may be rendered antigenic under suitable
conditions.
The method of the present invention is applicable
to the normal range of sample types e.g. urine, serum-
based and whole-blood samples. However, particularly
striking improvements over the prior art techniques are
found when performing assays on whole-blood samples.
For a better understanding of the present
invention, reference is made to the accompanying
drawings wherein:-
Figure 1 shows a diagrammatic section through a
fluorescence capillary device (hereinafter FCFD)
according to one embodiment of the present invention.
WO 96/09549 Z2 0~ 0 90 PCT/GB95/02236
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Figure 2 shows a diagrammatic section through an
FCFD used to illustrate the method of the present
invention.
Figure 3 illustrates schematically an example of
the regions of an FCFD possessing a calibration region
according to one embodiment of the present invention.
Figure 4 shows a diagrammatic section through a
dip-stick type device additionally illustrating
schematically an example of the regions of such a device
possessing a calibration region according to one
embodiment of the present invention.
In Figures 3 and 4, the symbols illustrated denote
the following entities:
p Antigen under assay
--~ fluorescent label
p-~ fluorescently labelled antigen
analogue
-{ or 0--~ specific antibody to antigen
under assay
specific antibody to specific
antibody to antigen under assay.
Referring to Fig. 1, the device depicted comprises
an upper plate 2 fashioned of transparent material (e.g.
of plastic material, quartz, silica or glass) carrying
on its external face an opaque coating 8, and a lower
plate 4 fashioned of transparent material, both plates
being around 1 mm thick and fixed together in
substantially parallel relationship, less than 1 mm
apart by means of bonding tracks of suitable adhesive
containing spacer means (not shown). In the embodiment
shown, the cell cavity 6 so formed is open to the
surroundings at both ends, so that when liquid sample is
drawn into one opening of the cavity by means of
capillarity, air may escape through the other opening.
In the embodiment shown, the two plates are offset.
PCT/GB95/02236
WO 96/09549 L2 O ~ '
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Carried on the inner surface of the upper plate 2
is a patch of reagent 12 appropriate to the test
being carried out. The reagent is contained within the
device in a soluble releasable form but such release is
delayed according to the method of the present
invention.
Carried on the inner surface of the lower plate 4
is a patch of reagent 10 appropriate to the test being
carried out, said patch 10 being directly below patch 12
on the plate 2. In the case of an immunoassay, the
patch 10 will carry, for example, an amount of relevant
immobilised antibody or antigen or hapten.
The operation in use of an embodiment of the device
shown in Fig. 1 will now be described. Although the
following description relates to the use of a device in
a'labelled-antigen format competition-type immunoassay,
it should be understood that devices according to the
invention are also suitable for use in labelled-antibody
format immunoassays (both competition-type and sandwich-
type) and in other types of assay (sandwich-type or
competition-type) or in other types of chemical or
biochemical tests.
The sample liquid passes into the device in the
direction of the arrow shown in Fig. 1. A short time
after the cavity 6 fills with sample liquid, the patch
12 of material dissolves, releasing the reagents
contained therein into the liquid.
As mentioned hereinbefore, the patch 12 is carried
on the upper plate 2 by means of suitable soluble
material(s). Suitable soluble materials include
humectant coatings, e.g. sucrose- or sorbitol-based.
The reagent in patch 12 is coated with or is
incorporated in cross-linked PVA or copolymers of
methacrylic acid or methacrylate esters to provide
delayed release of the reagent within the patch. A
suitable coating according to the present invention
would take typically 2-10 seconds to dissolve after
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WO 96/09549 PCT/GB95/02236
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initial contact with a sample liquid.
In one embodiment of the device of the type shown
in Fig. 1 which is set up for a competition-type
immunoassay for an antigen, patch 12 may contain a
fluorescently labelled antigen analogue. Patch 10 would
then comprise an amount of immobilised specific binding
partner being a specific antibody to the antigen under
assay. Thus, after introduction of the sample liquid,
the patch 12 dissolves, releasing antigen analogue into
the sample liquid. Antigen introduced in the sample
liquid competes with antigen analogue for epitopic
binding sites on the specific antibody to the antigen
contained in patch 10. The amount of fluorescent
material which becomes bound to the immobilised specific
antibody in patch 10 will t:_=efore be a function of the
concentration of antigen in :.he sample liquid.
Conventional competition-type optical immunoassays
involve this type of competitive equilibrium.
The delayed-release of the reagent in patch 12
ensures that a maximum amount of fluorescent material
remains in the region bounded by patches 10 and 12 after
the device has filled and this minimising of the
washdown of the reagent provides an increase in assay
precision and sensitivity.
Referring to Figure 2, the device depicted is not
one which would be used in an assay method but is
designed to demonstrate the effectiveness of the present
invention. The device is essentially the same as that
illustrated in Figure 1, except patch 12 is offset as
compared to patch 10. Thus, in use in an assay
procedure, on filling the device with sample and
performing the assay the amount of fluorescently
labelled reagent which becomes bound in patch 10 will be
a measure of the washdown of reagent from patch 12. By
comparing existing assays, including those utilising
known delayed-release techniques, with those according
to the present invention the improvement provided by the
WO 96/09549 L. 200690 12 - PCT/GB95/02236
-
present invention can be demonstrated.
In Figure 3 the device depicted comprises an upper
plate 2, and a lower plate 4 as in the device of
Figure 1. Carried on the inner surface of plate 2 is a
patch 12 and carried on the inner surface of plate 4 is
a patch 10, these patches and the reagents contained
therein being as described above in respect of Figure 1.
Patches 9 and 13 carried on plates 2 and 4 as shown
comprise a calibration region. The release of the
reagents in patches 12 and 13 in use is delayed
according to the method of the present invention. In
use, in the region bounded by the pair of patches 9 and
13 an initial high signal will arise from the region 9
due to binding of the complex from patch 13 to the
immobilised reagent in patch 9. This signal will
decrease over time as ligand competes with the labelled
ligand analogue in the complex in patch 9. The delayed
release of the reagents from patches 12 and 13 ensures
that a maximum amount of the respective reagents is
released into the regions bounded by the pair of patches
10 and 12 and 9 and 13. A minimum of washdown of the
reagent from patch 13 to the adjacent region occurs.
These factors maximise the precision and sensitivity of
the assay.
In Figure 4, the device depicted comprises only a
lower plate 4, as in the device in Figure 1. Carried on
the surface of plate 4 is a zone 10 containing a patch
of reagents appropriate to the test being carried out.
In the case of an immunoassay, the zone will carry, for
example an amount of unlabelled relevant immobilised
antibody directed to a first epitope of the ligand under
assay and an amount of a labelled antibody directed to a
second epitope of the ligand under assay, the labelled
antibody being present in soluble releasable form, but
the release of the reagents in use being delayed
according to the method of the present invention. Also
carried on the surface of plate 4 is a calibration zone
9 containing a patch of reagents appropriate to the test
being carried out. In the case of an immunoassay, the
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WO 96/09549 - 13 PCT/GB95/02236
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zone will carry, for example, an amc..-..t of unlabelled
relevant immobilised antibody direct~d to a first
epitope of the ligand under assay, an amount of a
labelled antibody directed to a second epitope of the
ligand under assay and an amount of the antigen under
assay such that a 1:1:1 complex between the antigen and
the two antibodies forms under the operation of the
assay, the labelled antibody and the antigen under assay
being present in soluble releasable form, but the
release of the reagents in use being delayed according
to the method of the present invention.
The operation in use of an embodiment of the device
shown in Figure 4 will now be described. Although the
examples of reagents and the following description
relates to the use of a device in a labelled antibody
format sandwich-type immunoassay, it should be
understood that the devices are also suitable in
labelled antigen format immunoassays and in other types
of assay (competition-type) or in other types of
chemical or biochemical tests.
The device is dipped into the sample liquid and a
short time thereafter the soluble reagents in patches 9
and 10 dissolve. The delayed release of these reagents
according to the present invention ensures that the
reagents remain substantially within the regions shown.
Thus region 10 provides the assay measurement and region
9 provides a calibration region with an initial high
signal. The limited transfer of soluble reagents
between the regions maximises the precision and
sensitivity of the assay
The following Examples serve to illustrate the
applicability of the method of the present invention
without, however, limiting it.
Comparative Examgle 1
Preparation of starting materials:
CA 02200690 2005-04-18
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1.1 Fabrication of antibody-coated optical waveguides:
Anti-PSA monoclonal antibodies were supplied by Serono
Diagnostics S A, Coinsins, Switzerland. A sheet of
PermablocTM glass (Pilkington Glass Ltd., St. Helens, UK)
having a thickness of about 1 mm was cleaned with
detergent (e.g. TweenTM 20) in ultra-pure water with
ultrasonic agitation. The surface of the glass was
activated by incubating it in a 2% solution of
aminopropyltrimethoxysilane in water (pH 3-4) for two
hours at 75 C. After rinsing in water the glass sheet
was dried at 115 C for at least four hours. The glass
was then incubated for 60 minutes in a 2.5a solution of
glutaraldehyde in a 0.05 M phosphate buffer (pH 7) and
then washed thoroughly with distilled water. Anti-PSA
antibody was patterned onto the glass by discretely
dosing a 1o solution of the antibody in phosphate buffer
(pH 7) onto the glass and incubating it for 2 to 4 hours
(to form patch 10) after which the glass sheet was
washed with buffer solution. Unwanted adsorbed protein
was removed by soaking with 6 M urea sol'ution in a known
manner. Finally a layer of sucrose/lactose was formed
over the surface of the glass sheet by spin coating.
This formed plate 4 of the FCFD test- device as
illustrated in Figure 1.
1.2. Preparation of anti-PSA antibody'conjugated to
allophycocyanin (APC):
A second anti-PSA monoclonal antibody, which recognises
a different epitope on the PSA molecule to the antibody
used in 1.1 above, was conjugated to allophycocyanin
(kex = 650 nm, Xem; = 660 nm) by Molecular Probes Inc.,
Eugene, Oregon, USA and was used as supplied.
1.3. Microdosing of the specific reagents over a
discrete zone cf anti-PSA antibody:
An opapue coatinc was screen printed onto a c7.ean sheet
CA 02200690 2005-04-18
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of Permabloc glass as described in WO-90/14590. The
measurement zone of the device was fabricated by
microdosing a layer of allophycocyanin/anti-PSA antibody
conjugate in buffer containing polyvinyl alcohol in an
area 3 x 7 mm onto the glass over the zone. After the
conjugate was air dried a layer of polyvinyl alcohol (40
in buffer) was microdosed over the conjugate (to form
patch 12). Finally the whole sheet of glass was coated
in a layer of sucrose/lactose by spray coating. This
formed plate 2 of the FCFD test device as illustrated in
Figure 1.
1.4. Fabrication of FCFD test devices:
FCFD test devices such as have been described in
EP-A-0171148 were fabricated by screen printing onto the
waveguide resulting from 1.1 above bonding tracks of an
ultraviolet curing glue (LNS 91TM , Norland Inc., USA)
containing glass microspheres of 100 um diameter
(Jencons Ltd., UK) in a pattern defining the long edges
of the capillary cell devices. A sheet of glass as
defined in 1.3 above was then placed overthe waveguide
and a vacuum applied to the laminate. As a result.of
the vacuum the upper sheet of glass was caused to press
down onto the glue, the glass microspheres defining a
gap of 100 pm between the glass sheets. The laminate
was then exposed to an ultraviolet light source to cure,
the glue. Finally, the laminate sheet was broken into.
individual test devices as described in EP-A-0171148.
1.5. Apparatus used in the measurement of the PSA assay:
A simple fluorimetry apparatus as described in
W092/09892 was used to make suitable assay measurements
as described in WO-90/14590.
Assav Procedure for PSA:
Samples of heparinised whole blood containing knowr_
()
WO 96/09549 2~~ J ~~ 16 PCT/GB95/02236
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amounts of PSA were added to the FCFD device and
incubated at room temperature for 20 minutes. Six FCFDs
were used to produce a"reduced" standard curve (i.e. 0,
and 100 ng/mL PSA) with pairs of devices being filled
5 with the same PSA concentration. The data presented in
Table 1 shows that a poor standard curve results, there
being a low top signal and poor replication between
pairs of FCFD devices. This is caused by the whole
blood sample entering the device and "instantly"
10 dissolving the allophycocyanin/anti-PSA antibody
conjugate and transporting down the length of the FCFD
away from the region in which the capture antibody is
located.
Comparative Example 2
FCFD whole blood assay for PSA without using any delayed
release chemistry and having no microdosed assay
reagents
2.1. Fabrication of antibody-coated optical waveguides:
As for Example 1.
2.2. Preparation of anti-PSA antibody conjugated to
allophycocyanin (APC):
As for Example 1.
2.3. Fabrication of FCFD test devices:
As for Example 1.
2.4. Apparatus used in the measurement of the PSA assay:
As for Example 1.
Assay Procedure for PSA:
22 0 0 69 0
WO 96/09549 - 17 _ PCT/GB95/02236
Equal volumes of the allophycocyanin/anti-PSA antibody
conjugate were mixed with whole blood samples containing
PSA and added to the FCFD. The assays were read in the
same way as in Example 1. The data show (Table 2) that
there is a good dose/response curve for the FCFD whole
blood assay.
ExamBle 3
3.1. Fabrication of antibody-coated optical waveguides:
As for Example 1.
3.2. Preparation of anti-PSA conjugated to
allophycocyanin (APC):
As for Example 1.
3.3. Microdosing of the specific reagents over a
discrete zone of anti-PSA antibody:
As for Example 1 except that, after the antibody was
microdosed, eudragit NE 30D (Rohm Pharma, Germany) was
spray-coated on top of the conjugate.
3.4. Fabrication of FCFD test devices:
As for Example 1.
3.5. Apparatus used in the measurement of PSA assay:
As for Example 1.
Assay Procedure for PSA:
As for Example 1 except that, due to the presence of the
eudragit, the dissolution of the antibody/fluorophore
conjugate was delayed until the sample had filled the
FCFD. This resulted in the PSA assay showing better
~JJ~
WO 96/09549 _ 18 - PCT/GB95/02236
precision between replicates and giving a higher top
signal (Table 3 ) .
Example 4
4.1. Fabrication of antibody-coated optical waveguides:
As for Example 1.
4.2. Preparation of anti-PSA conjugated to
allophycocyanin (APC):
As for Example 1.
4.3. Microdosing of the specific reagents over a
discrete zone of anti-PSA antibody:
As for Example 1 except that, after the antibody was
microdosed, a 0.2401 solution of tetraborate in water was
spray-coated on top of the conjugate to cross link the
polyvinyl alcohol present in the microdosing solution.
4.4. Fabrication of FCFD test devices:
As for Example 1.
4.5. Apparatus used in the measurement of the PSA assay:
As for Example 1.
Assay Procedure for PSA:
As for Example 1 except that, due to the presence of the
cross-linked polyvinyl alcohol, the dissolution of the
antibody/fluorophore conjugate was delayed until the
sample had filled the FCFD. This resulted in the PSA
assay showing better precision between replicates and
giving a higher top signal (Table 4).
l . ,
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WO 96/09549 - 19 - PCT/GB95/02236
Exam8le 5
Optimisation of tetraborate concentration:
5.1. Fabrication of antibody-coated optical waveguides:
As for Example 1.
5.2. Preparation of anti-PSA antibody conjugated to
allophycocyanin (APC):
As for Example 1.
5.3. Microdosing of the specific reagents over a
discrete zone of anti-PSA antibody:
As for Example 1 except that, after the antibody was
mi::rodosed, solutions of a range of tetraborate
concentrations in water were spray-coated on top of the
conjugate to cross-link the polyvinyl alcohol present in
the microdosing solution.
5.4. Fabrication of FCFD test devices:
As for Example 1.
5.5. Apparatus used in the measurement of the PSA assay:
As for Example 1.
Assay Procedure for PSA:
As for Example 1 except that, due to the presence of the
cross-linked polyvinyl alcohol, the dissolution of the
antibody/fluorophore conjugate was delayed until the
sample had filled the FCFD. The optimum delayed release
was given by lo tetraborate (Table 5).
Although the figures in Table 5 suggest that using 0.50
CA 02200690 2005-04-18
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tetraborate gives a higher top signal/background signal
ratio, repeating the assay did not give such good
reproducability as was found when using 1% tetraborate.
Hence using 1o tetraborate gave better assay precision.
Examule 6
FCFD whole blood assay for PSA using kriown and present
delayed release methods
6.1. Fabrication of antibody coated optical waveguides:
As for Example 1 except that the capture antibody was
immobilised over a region to form patch 10 of a device
as illustrated in Figure 2.
6.2. Preparation of anti-PSA antibody conjugated to
allophycocyanin (APC):
As for Example 1.
6.3. Microdosing of the specific reagents over a
discrete zone of anti-PSA antibody:
As for Example 1 except that the specific reagents were
microdosed in a region to form patch 12 of a device as
illustrated in Figure 2. After microdosing of the
reagents some devices were treated with tetraborate
whilst others were not.
6.4. Fabrication of FCFD test devices:
As for Example 1.
6.5. Apparatus used in the measurement of the PSA assay:
As for Example 1.
Assav Procedure fcr PSA:
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WO 96/09549 - 21 _ PCT/GB95/02236
Whole blood samples containing PSA were added to the
FCFD and the assay signal from region 10 was read.
Where only a known PVA capping layer was present there
was in effect no delayed release within the FCFD and the
soluble reagents washed down the device giving a
dose/response curve for the PSA assay (Table 6). When
the delayed release method of the present invention was
employed in the FCFD the amount of reagent washed down
the device was less giving a much reduced dose/response
curve (Table 6).
The figure of 8.580 at a PSA concentration of 50 ng/ml
in Table 6 shows the imprecision of the existing
technique of merely including a PVA capping layer.
Similarly the assay curve in such a method tends to be
skewed i.e. showing a large background signal.
It should be noted that in the Examples given above
different conjugates were used in various of the
Examples, the conjugates being of varying quality i.e.
giving a different maximum signal. Some Examples used a
conjugate with a lower colour intensity, others used a
conjugate with a higher colour intensity. Thus a
straight comparison of the top signals obtained in the
assays should not in general be made between the
Examples since this factor of varying intensity will not
be taken into account in such a comparison.
WO 96/09549 ~Z2 GO 0 6' - 22 - PCT/GB95/02236
PSA Concentration Signal (Arbitrary Mean Signal
(ng/mL) Units) (Arbitrary Units)
0.465
0 0.384 0.412
0.386
1.259
1.458 1.174
10 0.805
1.430
100 1.432 1.823
2.607
Table 1. Known PVA capping layer present in the FCFD
PSA concentration Signal (Arbitrary Mean signal
(ng/mL) units) (Arbitrary units)
0 0.581 0.545
0.508
10 1.531 1.506
1.480
100 4.666 4.688
4.710
Table 2. Dose/response characteristics for an FCFD assay
for PSA in whole blood using pre-mixed reagents
PSA Concentration Signal (Arbitrary Mean Signal
(ng/mL) Units) (Arbitrary Units)
0.665
0 0.561 0.646
0.713
1.262
10 1.226 1.287
1.373
2.069
100 2.010 2.012
1.956
Table 3. Eudragit delayed release reagent present in the
FCFD.
22ru) 0"
WO 96/09549 - 23 _ PCT/GB95/02236
PSA Concentration Signal (Arbitrary Mean Signal
(ng/tnL) Units) (Arbitrary Units)
0.665
0 0.561 0.646
0.713
1.262
1.226 1.287
10 1.373
2.069
100 2.010 2.012
1.956
Table 4. Cross-linked polyvinyl alcohol delayed-release
reagent present in the FCFD
Tetraborate PSA concentration Signal (Arbitrary
concentration (o) (ng/mL) units)
0 0.525
0.5 10 2.554
100 6.872
0 0.479
1.0 10 2.138
100 6.089
0 0.481
1.5 10 1.961
100 4.913
0 0.459
2.0 10 1.857
100 5.398
Table 5. Effect of variations in the tetraborate
concentration.
r)., ,-
WO 96/09549 L~00" C~ u - 24 - PCT/GB95/02236
Treatment PSA Concentration Signal (Arbitrary
(ng/mL) units)
0 1.546
known PVA capping 5 2.539
layer 10 4.086
(essentially no 50 8.580
delayed release 100 6.195
chemistry)
0 0.514
PVA plus 5 0.850
tetraborate 10 1.855
(present delayed 50 1.980
release method) 100 2.390
Table 6. Effect of PVA/tetraborate delayed release
method.
