IE83806B1 - Solid phase analytical device - Google Patents
Solid phase analytical device Download PDFInfo
- Publication number
- IE83806B1 IE83806B1 IE1998/0110A IE980110A IE83806B1 IE 83806 B1 IE83806 B1 IE 83806B1 IE 1998/0110 A IE1998/0110 A IE 1998/0110A IE 980110 A IE980110 A IE 980110A IE 83806 B1 IE83806 B1 IE 83806B1
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- IE
- Ireland
- Prior art keywords
- porous carrier
- test device
- sample
- specific binding
- analytical test
- Prior art date
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Abstract
ABSTRACT An analytical test device incorporating a dry porous carrier to which a liquid sample, eg. urine, suspected of containing an analyte such as HCG or LH can be applied indirectly, the device also incorporating a labelled specific binding reagent which is freely mobile in the porous carrier when in the moist state, and an unlabelled specific binding reagent which is permanently immobilised in a detection zone on the carrier material, the labelled and unlabelled specific binding reagents being capable of participating in either a sandwich reaction or a competition reaction in the presence of the analyte, in which prior to the application to the device of a liquid sample suspected of containing the analyte, the labelled specific binding reagent is retained in the dry state in a macroporous body, eg. of plastics material having a pore size of (10) microns or greater, through which the applied liquid sample must pass en route to the porous carrier material, the labelled specific binding reagent being freely soluble or dispersible in any liquid sample which enters the macroporous body.
Description
PATENTS ACT 1992
980110
SOLID PHASE ANALYTICAL DEVICE
UNILEVER PLC
This patent application is a divisional of Irish Patent
Application No. 592/90, corresponding to European patent
application No. 9030l697.0, now granted as EP 383619 B.
The present invention relates to assays involving specific
binding, especially immunoassays.
In particular, the invention relates to analytical devices
which are suitable for use in the home, clinic or doctor's
surgery and which are intended to give an analytical result
rapidly and which require the minimum degree of skill and
involvement from the user. The use of test devices in the
home to test for pregnancy and fertile period (ovulation) is
now commonplace.
In the specification of UK patent application GB-A—2204398
we describe test devices which are readily usable even by an
unskilled person and which typically merely require that
some portion of the device is contacted with a sample (e.g.
urine in the case of a pregnancy or ovulation test) and
thereafter no further actions are required by the user
before an analytical result can be observed. The analytical
result can be observable within a matter of minutes
following sample application, e.g. ten minutes or less.
The use of reagent—impregnated test strips in specific
binding assays, such as immunoassays, has previously been
proposed. In such procedures a sample is applied to one
portion of the test strip and is allowed to permeate through
usually with the aid of an eluting
the strip material,
solvent such as water. In so doing, the sample progresses
into or through a detection zone in the test strip wherein a
specific binding reagent is immobilised. Analyte present in
the sample can participate in a sandwich or a competition
reaction within the detection zone, with a labelled reagent
which can also be-incorporated in the test strip or applied
thereto. Examples of prior proposals utilising these
principles are given in Thyroid Diagnostics Inc GB 1589234,
(USA)
Boots—Celltech Diagnostics Limited EP 0225054, Syntex
Inc EP 0183442, and Behringwerke AG EP 0186799.
Other examples of analytical devices, comprising multiple
zones or layers of porous membranes or matrices arranged in
series, through which reagents can migrate, are described in
EP-A2—299359, W0 86/04683 and WO 89/06799.
are especially suited to the use of enzyme labels.
These devices
The use
of insoluble particulate labels is not contemplated, whereas
such particulate labels the
described in GB-A-2204398 mentioned above.
are preferred in -devices
(3) discloses
EPC,
containing a
EP-A-323605, citable under Article
assay devices having an application
The label can be particulate,
and the application pad can be made from
labelled reagent. for example
a selenium colloid,
porous polyethylene frit, i.e. a macroporous body comprising
plastics material.
In the patent application now granted as IE 81184
(corresponding to EP 383619 B), of which this present
application is a divisional, we claim an
analytical test device incorporating a dry porous carrier to
which device a liquid sample suspected of containing an
the device also incorporating a
analyte can be applied,
specific binding reagent attached to a particulate label
which labelled specific binding reagent is freely mobile in
carrier when in the moist state, and an
the porous
reagent which is permanently
unlabelled
immobilised in a detection zone on the carrier material,
labelled unlabelled
capable of participating in either a sandwich reaction or a
the the
specific binding
and specific binding reagents being
competition reaction in presence of analyte,
characterised in that prior to the application to the device
of the liquid sample, the particle-labelled specific binding
reagent is retained within the device in the dry state in a
separate macroporous body comprising plastics material
having a pore size not less than 10 times greater than the
maximum particle size of the particulate label, through
which macroporous body the applied liquid sample must pass
thus facilitating uptake of
by the
en route to the porous carrier,
the particle-labelled specific binding reagent
applied liquid sample, and in that the porous carrier is
linked via the macroporous body to a porous receiving member
to which the liquid sample can be applied and from which the
sample can permeate into the porous carrier.
In the present application we provide an analytical test
device incorporating a dry’ porous carrier strip to which
device an aqueous liquid sample suspected of containing an
analyte can be applied, the device also incorporating a
specific binding reagent attached to a particulate label
which labelled specific binding reagent is freely mobile in
the porous carrier strip when in the moist state, and an
unlabelled
immobilised in a detection zone on the porous carrier strip,
the labelled and unlabelled specific binding reagents being
specific binding reagent which is permanently
capable of participating in either a sandwich reaction or a
the of the
characterised in that prior to the application to the device
the particle-labelled specific binding
competition reaction in presence analyte,
of the liquid sample,
reagent is retained in the dry state in a separate
macroporous body comprising plastics material having a pore
size not less than 10 times greater than the maximum
particle size of the particulate label, through which
macroporous body the applied liquid sample must pass en
route to the proximal end of the porous carrier strip, and
in that there is an absorbent sink at the distal end of the
the absorbent sink not being merely a
length of which the
detection zone and extends beyond the detection zone.
porous carrier strip,
porous carrier material contains
The invention therefore encompasses the use of a macroporous
body to facilitate the uptake of a labelled specific binding
agent by a liquid sample before such a sample is analysed on
a test strip or the like.
Preferably, the dry porous carrier material comprises a
chromatographic strip, sudu as a strip of nitrocellulose.
If desired, the nitrocellulose can be backed with moisture
impermeable material, such as polyester sheet. Using
nitrocellulose as the porous carrier material has
considerable advantage over more conventional strip
materials, such as paper, because nitrocellulose has a
natural ability to bind proteins without requiring prior
sensitisation. Specific binding reagents, such as
immunoglobulins, can be applied directly to nitrocellulose
and immobilised thereon. No chemical treatment is required
which xnight interfere with the essential specific binding
activity of the reagent. Unused binding sites on the
nitrocellulose can thereafter be blocked using simple
materials, such as polyvinylalcohol. Moreover,
nitrocellulose is readily available in a range of pore sizes
and this facilitates the selection of a carrier material to
suit particularly requirements such as sample flow rate.
Preferably the nitrocellulose has a pore size of at least
one um. Preferably the nitrocellulose has a pore size not
greater than about 20pm.
The labelled specific binding reagent comprises a specific
attached to a label. Such
e.g.
non-metallic colloids,
binding particulate
“direct labels”,
reagent
coloured latex particles, gold sols,
and dye sols, are already known per
instant analytical
se. They can be used to produce an
result without the need to add further reagents in order to
develop a detectable signal. They are robust and stable and
can therefore be used readily in an analytical device which
is stored in the dry state. Their release on contact with
an aqueous sample can be modulated, for example by the use
of soluble glazes. Preferably, the particulate label isea
latex particle, such as a coloured latex particle which can
be readily visible to the eye if it becomes bound in the
If desired,
by colour reflectance.
detection zone. the assay result can be read
instrumentally, e.g. Alternatively,
the latex particle can incorporate a fluorescent compound
which can respond to applied electromagnetic energy such as
ultraviolet light or visible light, to provide an emitted
that
particularly preferred embodiment,
signal can be measured instrumentally. In a
the direct label is a
coloured latex particle of spherical or near-spherical shape
and having a maximum diameter of not greater than about
.5pm. An ideal size range for such particles is from about
.05 to about O.5um.
We have found that use of a macroporous body as the portion
of the device wherein the applied liquid sample encounters
the particulate label considerably facilitates the ease with
which the label is up by the
compared to the situation that usually prevails if
particulate taken liquid
sample,
the particulate label is incorporated as a pre—dosed reagent
on the dry porous carrier strip. To enable the particulate
label to migrate freely out of the macroporous body with the
liquid sample, the macroporous body has a pore size at least
times greater than the maximum particle size of the
particulate label. More preferably, the macroporous body
comprises plastics material having an average pore size of
because such
not less than 10pm, and ideally about 100nm,
larger pore sizes give better release of the labelled
reagent. The plastics material should not be protein-
binding, or should be easily blockable by means of reagents
such as BSA or PVA, to minimise non-specific binding to
facilitate free movement of the labelled redagent after the
macroporous body has become moistened with the liquid
The
surface active agent or solvent,
more hydrophilic and to promote rapid uptake of the liquid
sample. plastics material can be pre—treated with
if necessary, to render it
sample. Alternatively, if desired, a surface active agent
can be incorporated in the solution containing the labelled
reagent when this is applied to the macroporous material
during manufacture of the device.
The labelled incorporated in
macroporous material in bulk,
reagent is preferably
large sheet, form before
e.g.
it is subdivided into individual bodies for use in a testing
device of the invention.
After a solution containing the labelled reagent has been
saturate the material, the
allowed to macroporous
macroporous material should be dried, e.g. by vacuum or air-
drying, or preferably by freeze-drying. Optionally, the
solution can also contain a surface active agent, such as a
detergent, and/or a glazing material, such as a sugar, e.g.
The presence of the glazing material appears to
the labelled
specific binding reagents
SUCIOSE .
enhance release of reagent and promotes
stability of delicate such as
antibodies.
labelled
rather than pre-dosed onto the carrier
the the
By incorporating the reagent in a separate
macroporous body,
material that also incorporates detection zone,
following advantages can be obtained:
because a substantial
able to
sensitivity of the test,
of the
Enhanced
take
the
enhancing potential reaction
quantity liquid sample is up the
labelled reagent..before ,migrating through carrier
material to the detection zone,
time without significantly increasing overall test time.
Also, the liquid which permeates the carrier is of a more
uniform and consistent composition. Whereas the test
devices as described in our earlier patent application GB—A—
are primarily, although not exclusively, suited to
qualitative assays, those of the present invention are
especially suitable for quantitative assays as well as for
qualitative assays.
For example,
and the
Enhanced perceived performance of the test.
the
detection zone comprises a line of immobilised reagent,
when device incorporates a carrier strip
the label is a visible direct label, a positive result shows
up more clearly, with much reduced temporary background
caused by the visible labelled reagent being progressively
conveyed past the detection zone.
Ease of manufacture, because the incorporation of the
labelled reagent in the separate macroporous body avoids the
need to apply the labelled reagent in a special zone in the
as described
carrier, which may need careful pre—treatment,
in our GB-A—2204398.
If the assay device is intended to identify more than one
the body
incorporate several labelled specific binding reagents each
single macroporous can
analyte in a sample,
carrying a different label, eg. having different colours or
fluorescent properties. This will facilitate the
manufacture of a multiple analyte testing device.
Ideally, the macroporous body is in direct moisture-
conductive contact with the porous material, and~ the
detection zone on the porous carrier material is spaced away
from the region of contact between the porous carrier
In such an embodiment,
the
material and the macroporous body.
the
macroporous body is preferably not less than the quantity of
quantity of liquid sample required to saturate
liquid sample capable of being absorbed by the mass of
porous carrier material linking the macroporous body and the
detection zone. In other words, the liquid capacity of the
macroporous body is at least equal to the liquid capacity of
the working portion of the porous carrier.
The invention also provides an analytical method in which a
contacted with an aqueous
such that
device as set forth above is
liquid sample suspected of containing the analyte,
the sample permeates by capillary action via the macroporous
body through the porous solid carrier into the detection zne
and the labelled reagent migrates therewith to the detection
the presence of analyte in the sample being determined
to which the labelled
Zone,
by observing the extent (if any)
reagent becomes bound in the detection zone.
In one embodiment of the invention, the labelled reagent is
The labelled
immobilised
a specific binding partner for the analyte.
reagent, the analyte (if present) and the
unlabelled specific binding reagent cooperate together in a
“sandwich” reaction. This results in the labelled reagent
being bound in the detection zone if analyte is present in
the The
specificities for different epitopes on the analyte.
sample. two binding reagents must have
In another embodiment of the invention, the labelled reagent
is either the analyte itself which has been conjugated with
a label, or is an analyte_analogue, ie. a chemical entity
having the identical specific binding characteristics as the
analyte, and which similarly has been conjugated with a
label. In the latter case, it is preferable that the
properties of the analyte analogue which influence its
solubility or dispersibility in an aqueous liquid sample and
its ability to migrate through the moist porous solid phase
material should be identical to those of the analyte itself,
similar. In this second
or at least very closely
embodiment, the labelled analyte or analyte analogue will
migrate through the porous carrier into the detection zone
and bind with the immobilised reagent. Any analyte present
in the sample will compete with the labelled reagent in this
Such will
reduction in the amount of labelled reagent binding in the
binding reaction. competition result in a
detection zone, and a consequent decrease in the intensity
of the signal observed in the detection zone in comparison
with the signal that is observed in the absence of analyte
in the sample.
In a further alternative embodiment, an analyte or analyte
analogue is immobilised in the detection zone, and the
labelled reagent is specific for the ,analyte. If an
analyte—containing sample is applied to the device,
competition between the immobilised and free analyte reduced
the extent to which the labelled reagent may become bound in
the detection zone.
In a further embodiment of the present invention, the porous
carrier is linked via the macro—porous body to a porous
receiving member to which the liquid sample can be applied
into the porous
and from which the sample can permeate
carrier. Preferably, the porous carrier and the macroporous
body are contained within a nwisture—impermeable casing or
housing and the porous receiving member extends out of the
housing and can act as a means for permitting a liquid
sample to enter the housing and reach the porous carrier.
The should be with e.g.
appropriately placed apertures, which enable the detection
provided means,
housing
zone of the porous solid phase carrier material (carrying
the immobilised unlabelled specific binding reagent) to be
observable from outside the housing so that the result of
the assay can be observed. If desired,
be provided with further means which enable a further zone
the housing may also
of the porous solid phase carrier material to be observed
from outside the housing and which further zone incorporates
one or more control reagents which enable an indication to
be iven as to the
Preferably the
whether assay procedure has been
completed. housing is provided with a
removable cap or shroud which can protect the protruding
porous receiving member during storage before use. If
desired, the cap or shroud can be replaced over the
protruding porous receiving member, after sample
application, while the assay procedure is being performed.
An important embodiment of the invention is a pregnancy
testing device comprising a hollow elongated casing
containing a dry porous nitrocellulose carrier which
communicates indirectly with the exterior of the casing via
a bibulous urine receiving member which protrudes from the
casing, the porous nitrocellulose carrier and the sample
receiving member being linked via a macroporous body such
that any sample reaching the porous carrier must first pass
through the macroporous body, the sample receiving member
and the macroporous body together acting as a reservoir from
which the. carrier, the
highly-specific anti-hCG
label, the labelled
antibody being freely mobile within the macroporous body and
urine .is .released. into
body
antibody bearing a coloured
porous
macroporous containing a
“direct”
the porous carrier when in the moist state, and in a
detection zone on the carrier spatially distant from the
macroporous body an highly-specific unlabelled anti—hCG
antibody which is permanently immobilised on the carrier
material and is therefore not mobile in the moist state, the
labelled and unlabelled antibodies having specificities for
different hCG the
opaque or translucent material‘ incorporating =at- least one
epitopes, casing being constructed of
aperture through which the analytical result may be
observed, together with a removable and replaceable cover
for the protruding bibulous urine receiving member. A
fertile period prediction device, essentially as just
defined except that the analyte is LH, is an important
alternative.
Such devices can be provided as kits suitable for home use,
comprising’ a plurality (e.g. two) of devices individually
wrapped in moisture impervious wrapping and packaged
together with appropriate instructions to the user.
The porous sample receiving member can be made from any
bibulous, porous or fibrous material capable of absorbing
The of the
(ie. with pores or fibres running wholly or
liquid rapidly. porosity material can be
unidirectional
predominantly parallel to an axis of member) or
multidirectional (omnidirectional, so that the member has an
amorphous sponge-like structure). Porous plastics material,
such as polypropylene, polyethylene (preferably of very high
molecular weight), polyvinylidene fluoride, ethylene
vinylacetate, acrylonitrile and polytetrafluoroaethylene can
be used. It can be advantageous to pre—treat the member
with a surface—active agent during manufacture, as this can
reduce inherent hydrophobicity in the member and
therefore enhance its ability to take up and deliver a moist
any
sample rapidly and efficiently. Porous sample receiving
members can also be made from paper or other cellulosic
such as nitro-cellulose. Materials that are now
used in the
particularly sutable and such materials can be shaped or
materials,
nibs of so—called fibre tipped pens are
cross-sections
Preferably the
extruded in a lengths
variety of
appropriate in the context of the invention.
material comprising the porous receiving member should be
chosen such that the porous member can be saturated with
aqueous liquid within a matter of seconds. Preferably the
material remains robust when moist, and for this reason
paper
embodiment wherein the porous
and similar materials are less preferred in any
receiving member protrudes
_ 13 _
from a housing. The liquid must thereafter permeate freely
from the porous sample receiving member into the macroporous
body.
If present, the “control” zone can be designed merely to
convey an unrelated signal to the user that the device has
worked. For example, the control zone can be loaded with an
antibody that will bind to the labelled reagent, e.g. an
“anti—mouse” antibody if the labelled reagent is an antibody
that has been derived using a murine hybridoma, to confirm
that the sample has permeated the test strip.
Alternatively, the control zone can contain an anhydrous
when moistened, produces a colour change or
e.g.
turn blue when mostened by an aqueous sample.
reagent that,
colour formation, anhydrous copper sulphate which will
As a further
could immobilised
_zone_ contain
alternative,“_ai.control
analyte which will react with excess labelled reagent from
the first zone. As the purpose of the control zone is to
indicate to the user that the test has been completed, the
control zone should be located downstream from the detection
zone in which the desired test result is recorded. A
positive control indicator therefore tells the user that the
sample has permeated the required distance through the test
device.
The label is a direct label,
ie. an entity which, in its natural state, is readily
visible either to the naked eye, or with the aid of an
optical filter and/or applied stimulation, e.g. UV light to
promote fluorescence. For example, minute coloured
particles, such as dye sols, metallic sols (e.g. gold), and
coloured latex particles, are very suitable. Of these
options, coloured latex particles are most preferred.
Concentration of the label into a small zone or volume
should give rise to a readily detectable signal, e.g. a
strongly-coloured area. This can be evaluated by eye, or by
instruments if desired.
Coupling of the label to the specific binding reagent can be
by covalent bonding, if desired, or by hydrophobic bonding.
Such techniques are commonplace in the art, and form no part
of the present invention. Because the label is a direct
label such as a coloured latex particle, hydrophobic bonding
is preferred.
In all embodiments of the invention, it is essential that
the labelled reagent migrates with the liquid sample as this
The flow of
continues beyond the detection zone and sufficient sample is
sample
to the detection zone.
progresses
applied to the porous carrier material in order that this
may occur and that any excess labelled reagent which does
_15_
not participate in any binding reaction in the detection
flushed detection by this
away from the zone
zone is
continuing flow. An absorbent “sink” is provided at
distal end of the carrier material. The absorbent sink may
comprise, for example, Whatman 3MM chromatography paper, and
should provide sufficient absorptive capacity to allow any
unbound conjungate 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 materials which extends beyond
but this alternative is not within the
this
the detection zone,
scope of the invention claimed in divisional
application.
The presence or intensity of the signal from the label which
becomes bound in the detection zone can provide a
qualitative or iquantitative measurement of analyte in ‘the
A plurality of detection zones arranged in series
which the
can also be
sample.
on the porous solid phase material, through
aqueous liquid sample can pass progressively,
used to provide a quantitative measurement of the analyte,
loaded with different
binding agents to provide a multi—analyte test.
or can be individually specific
The immobilised reagent in the detection zone is preferably
a highly specific antibody, and more preferably a monoclonal
antibody. In the embodiment of the invention involving the
sandwich reaction, the labelled reagent is also preferably a
highly specific antibody, and more preferably a monoclonal
antibody.
Preferably the porous carrier material is in the form of a
strip or sheet to which during manufacture of the device,
one or more reagents can be applied in spacially distinct
_ 16 _
zones. During use, the liquid sample is allowed to permeate
through the sheet or strip from one side or end to another.
If desired, a device according to the invention can
incorporate two or more discrete bodies of porous solid
phase carrier material, e.g. separate strips or sheets, each
These discrete bodies can be
that a
carrying immobilised reagents.
arranged in parallel, for example, such single
application of liquid sample to the device initiates sample
flow ixx the discrete bodies simultaneously. The separate
analytical results that can be determined in this way can be
used as control results, or if different reagents are used
on the different carriers, the simultaneous determination of
a plurality of analytes in a single sample can be made.
Alternatively, multiple samples can be applied individually
to an array of carriers and analysed simultaneously.
The material comprising the porous solid phase is preferably
nitrocellulose. This has the advantage that proteinaceous
in the detection zone can be
If the
reagents, such as an antibody,
immobilised firmly without prior chemical treatment.
porous solid. phase materiall comprises paper, for example,
the immobilisation of an antibody in the second zone needs
to be performed by chemical coupling using, for example,
CNBr, carbonyldiimidazole, or tresyl chloride.
Following the application of the specific binding reagent to
the detection zone, the remainder of the porous solid phase
material should be treated to block any remaining binding
sites elsewhere. Blocking can be achieved by treatment with
protein (e.g. bovine serum albumin or milk protein), or with
polyvinylalcohol or ethanolamine, or any combination of
these agents, for example. Between these process steps the
porous solid phase carrier material should be dried.
Preferably the porous solid phase material is nitrocellulose
sheet having a pore size of at least about 1pm, even more
preferably of greater than about Sum, and yet more
preferably about 8—l2um. Very suitable nitrocellose sheet
having a nominal pore size of up to approximately 12pm, is
available commercially from Schleicher and Schuell GmbH.
the nitrocellulose sheet is “backed”, e.g. with
This can
Preferably,
plastics sheet, to increase its handling strength.
thin layer of
The actual
be manufactured easily by forming a
nitrocellulose on a sheet of backing material.
pore size of the nitrocellulose when backed in this manner
will than that of ‘the
unbacked material.
tend to be lower corresponding
Alternatively, a pre-formed sheet of nitrocellulose can be
tightly sandwiched between two supporting sheets of solid
material, e.g. plastics sheets.
It is preferable that the flow rate of an aqueous sample
through the porous solid phase material should be such that
in the untreated material, aqueous liquid migrates at a rate
of 1cm in not more than 2 minutes, but slower flow rates can
be used if desired.
The spatial separation between the macroporous body and the
and the flow rate characteristics of the
detection zone,
porous carrier material, can be selected to allow adequate
reaction times during which the necessary specific binding
-18....
can occur. Further control over these parameters can be
achieved by the incorporation of viscosity modifiers (e.g.
sugars and modified celluloses) in the sample to slow down
the reagent migration.
Preferably, the immobilised reagent in the detection zone is
impregnated throughout the thickness of the carrier in the
detection zone throughout the thickness of the sheet
(e.g.
or strip if the carrier is in this form). Such impregnation
can enhance the extent to which the immobilised reagent can
capture any analyte or labelled reagent, present in the
migrating sample.
Reagents can be applied to the porous carrier material in a
variety of ways. Various “printing” techniques have
previously been proposed for application of liquid reagents
to carriers, e.g. micro-syringes, pens using metered pumps,
and ink-jet printing, and any of these
direct printing
context. To
(e.g. sheet)
treated with the reagents and then subdivided into smaller
the
can be used in present
techniques
facilitate manufacture, the carrier can be
small narrow strips each embodying
(e.g.
required reagent-containing zones)
portions
to provide a plurality of
identical carrier units.
An assay based on the above principles can be used to
determine a wide variety of analytes by choice of
appropriate specific binding reagents. The analytes can be,
for example, proteins, haptens, immunoglobulins, hormones,
infectious disease agents
Streptoccus,
polynucleotides, steroids, drugs,
(e.g. of bacterial or viral origin) such as
Neisseria and Chlamydia.
be performed for analytes such as hCG, LH, and infectious
Sandwich assays, for example,
disease agents, whereas competition assays, for example, may
be carried out for analytes such as E—3—G and P—3—G.
The determination of the presence (if any) of more than one
analyte in sample can have significant clinical utility.
For example, the ratio of the levels of apolipoproteins A1
and B can be indicative of susceptibility to coronary heart
disease. Similarly, the ratio of the levels of glycated
haemoglobin (HbA) to unglycated (HbAo) or total (Hb)
haemoglobin can aid in the management of diabetes.
Additionally it is possible to configure tests to measure
two steroids simultaneously, e.g. E-3—G and P—3—G.
The determination of the presence of more than two (ie.
significant
may have
the"
multiple) analytes in any sample
clinical utility. For example, detection of
presence of various different sereotypes of one bacterium,
or the detection of the presence of soluble serological
markers in humans may be useful. By way of example, a
multiple analyte test for the detection of the presence of
different Streptococcus
A, B, C and D. A cocktail of monoclonal antibodies,
specific for various _pathologically
serotypes of can be prepared for
groups ,
each important group
serotypes, or a polyclonal antiserum raised against a
Streptococcal group,
porous carrier strip as a line extending the width of the
particular can be dispensed onto a
strip of approximately lmm zone length. Multiple lines can
be dispensed in spatially discrete zones, each zone
containing immunochemically reactive component(s) capable of
binding the analyte of interest. Following the application
of the multiple zones, via a suitable application procedure
(e.g. ink—jet printing, metered pump and pen, airbrush), the
remainder of the porous material should be treated with a
_ 20 _
reagent (e.g. bovine serum albumin, polyvinylalcohol,
ethanolamine) to block any remaining binding sites
elsewhere.
some features of the invention will
detail with the the
The described devices do not include
By way of example only,
now be described in reference to
accompanying drawings.
an absorbent sink.
Embodiment 1
Figure 1 of the accompanying drawings represents an
isometric view of an assay device, and Figure 2 represents a
cross-sectional side elevation of the device shown in Figure
Referring to Figure 1, the device comprises a housing or
casing 100 of elongate rectangular form having at one end
101 a portion 102 of reduced cross-sectional area. A cap
103 can be fitted onto portion 102 and can abut against the
shoulder 104 at end 101 of the housing. Cap 103 is shown
Extending beyond end 105 of
When cap 103
it covers porous
100
The housing is
separated front housing 100.
portion 102 is a porous sample collector 106.
is fitted onto portion 102 of the housing,
106. face 107
incorporates two apertures 108 and 109.
sample collector Upper of housing
constructed of an upper half 110 and a lower half 111.
Referring to Figure 2, it can be seen that housing 100 is of
hollow construction. Porous sample collector 106 extends
The inner end 112 of sample collector 106
body 113 of
applied to
into housing 100.
accommodate a macroporous
is recessed to
plastics material. Aqueous liquid sample
collector 106 can pass freely into macroporous body 113,
rapidly saturating it. In turn, macroporous body 113 is in
liquid permeable contact with a strip of porous carrier
material 114. The housing is constructed of an upper half
110 and a lower half 111 and strip 114 overlap to ensure
that there is adequate contact between these two components
and that a liquid sample applied to sample collector 106 can
permeate via macroporous body 113 and into strip 114. Strip
114 extends further into housing 100. To help ensure that
no liquid sample reaches Strip 114 without first passing
a gap 115 can be left in the
114 to
Strip 114 is
through macroporous body 113,
strip overlap
“backed”
by a supporting strip 116 formed of transparent mosture-
housing 100 by arranging for the
macroporous body 113 only partially.
impermeable plastics material. Strip 114 extends beyond
apertures.108 and 109.. Means are provided within housing
100 by webbs 117 and 118 to hold strip 114 firmly in place.
In this respect, the internal constructional details of the
housing are not a significant aspect of the invention as
the held firmly the
sample collector 106 is firmly retained in the
long as strip is in place within
housing,
housing, and adequate fluid permeable contact is maintained
between sample collector 106, macroporous body 113 and strip
114.
and apertures 108 and 109 and can act as a seal against
The transparent backing strip 116 lies between strip
ingress of mosture from outside the housing 100 via these
If desired, the residual space 119 within the
apertures.
housing can contain moisture-absorbant material, such as
silica gel, to help maintain the strip 114 in the dry state
The reagent-containing detection zone in
but the
during storage.
strip 114 is
containing the immobilised unlabelled reagent will lie in
not depicted in Figure 2, zone
the region exposed through aperture 108 in order that when
...22_
the device has been used in an assay, the result can be
observed through aperture 108. Aperture 109 provides means
through which a control zone containing further reagents
which may enable the adequate permeation of sample through
the strip to be observed.
In operation, the protective cap 103 is removed from the
holder and
sample e.g. by being placed in a urine stream in the case of
sample collector 106 is exposed to a liquid
a pregnancy test. After exposing sample collector 106 to
the liquid sample for a time sufficient to ensure that the
collector 106 is saturated with the sample, the cap 103 can
be replaced and the device placed aside by the user for an
while
two or three minutes)
114 to
After the appropriate time,
(e.g.
test
appropriate period time
the provide
strip
sample permeates
analytical result. the user can
observe the test strip through apertures 108 and 109 and can
ascertain whether the assay has been completed by observing
the control zone through aperture 109, and can ascertain the
result of the assay’ by observing the second zone through
aperture 108.
During manufacture, the device can be readily assembled
from, for example, plastics material with the housing 100
being moulded in two parts (e.g. upper and lower halves 110
and lll)
ultrasonic welding)
body and test
halves and then sandwiched between the two halves.
(e.g. by
macroporous
which can be securely fastened together
after the sample collector,
one of the
The act
strip have been placed within
of forming this sandwich construction can be used to “crimp”
body and test
the collector strip
together to ensure adequate contact between them. Cap 103
If desired,
sample macroporous
can be moulded as a separate complete item.
_23_
apertures 108 and 109 can be provided with transparent
inserts which may insure greater security against ingress of
extraneous moisture from outside the housing. By providing
a tight fit between the end 105 of housing 100 and the
protruding sample collector 106, the application of sample
to the protruding member will not result in sample entering
the device directly and by—passing collector 106. Collector
therefore provides the sole route of access for the
sample to the strip within the housing, and can deliver
sample to the strip in a controlled manner. The device as a
whole therefore combines the functions of sampler and
analyser.
By using the test strip materials and reagents as herein
described, a device in accordance with Figures 1 and 2 can
for use as a
be produced which His .eminently. suitable
pregnancy test kit or fertile period test kit for use in the
The user 1nerely' needs to apply a urine
then (after
home or‘ clinic.
the and
sample to exposed porous member
optionally replacing the cap) can observe the test result
through aperture 108 within a matter of a few minutes.
Although described .with particular reference to pregnancy
tests and fertile period tests, it will be appreciated that
the device, as just described, can be used to determine the
presence of a very wide variety of analytes if appropriate
reagents are incorporated in the test strip. It will be
further appreciated that aperture 109 is redundant and may
be omitted if the test strip does not contain any control
means. Further, the general shape of the housing and cap,
both in terms of their length, cross-section and other
physical features, can be the subject of considerable
variation.
-24..
Figure 3 of the accompanying drawings shows an enlarged view
of the sample collector, macroporous body and test strip in
the device illustrated in Figures 1 and 2.
is
strip 114,
linked to the
backed by the
collector
and test
bibulous sample
body 113
transparent plastics sheet 116,
The
macroporous
such that liquid can flow in
the direction shown by the arrows from the sample collector
through the macroporous body and into the porous strip.
Test zone 120 incorporates the immobilised specific binding
reagent, and control zone 121 contains a reagent to indicate
that the sample has permeated a sufficient distance along
the test strip.
An aqueous sample deposited in collector 106 can flow into
macroporous body 113 and take up labelled reagent therein.
The sample can permeate from macroporous body 113 along the
length of strip 114 and in doing so will carry the labelled
reagent along the strip and through zone 120.
If desired, e.g. for ease of manufacture, the collector 106
need not be recessed to accommodate the macroporous body
113.
overlapping arrangement,
and pressed together during assembly of the complete device.
This will
which the liquid path will be essentially as depicted in
Instead, these components can simply be placed in an
together with the porous strip 114,
in practice provide a physical arrangement in
Figure 3.
Embodiment 2
._25_
Figures 4 and 5 illustrate another test device, which is
seen in plan view in Figure 4 and in cross-section in Figure
, the cross-section being an elevation on the line A seen
Figure 4.
Referring to Figure 4, the test device comprises a flat
rectangular casing 400 incorporating a centrally disposed
rectangular aperture 401, adjacent the left hand end 402,
and two further apertures 403 and 404 near the mid point of
403 and 404
the device and arranged such that apertures 401,
the device
lie on central longitudinal axis of
corresponding to line A. Although all three apertures are
illustrated as being rectangular, their actual shape is not
critical.
Referring to the cross-section seen in Figure 5, the device
is hollow and incorporates within it a nacroporous sample
receiving member 405 adjacent end 402 of casing 400 and
lying directly beneath 401.
member 405 is in liquid—conductive contact with one end of a
aperture Sample receiving
test strip 406 back by a transparent plastics sheet 407 also
400, the
extreme other end of the casing.
sheet 407 is in firm Contact with the upper inner surface
408 of casing 400,
and 404 to prevent ingress of moisture or sample into the
Although not shown in the drawings, the porous test
contained within casing and which extends to
The transparent backing
and provides a seal against apertures 403
casing.
strip 406 incorporates a test zone and a control zone placed
in a
appropriately in relation to apertures 403 and 404,
manner analagous to that described in Embodiment 1.
macroporous sample receiving member incorporates a labelled
reagent which is readily soluble or dispensable in an
applied liquid sample.
_ 25 -
In operation, an sample can be applied through
aqueous
aperture 401, e.g. by means of a syringe, to saturate porous
receiving member 405 which contains labelled reagent which
can be taken up by the sample. Thereafter, the aqueous
sample can permeate the test strip and, after an appropriate
time, the test result can be observed through apertures 403
and 404.
Example
This example does not include a sink, but illustrates other
features of the invention.
detergent
(1.4 mm thick) of commercially—available,
macroporous polyethylene having a pore size of
A sheet
pre—treated,
about lO0um was saturated with an aqueous suspension of
blue—coloured latex particles (prepared as described in GB
A) or particle size about 0.4um. The latex particles
carried an anti—beta LH monoclonal antibody. The solution
also contained 3% BSA and 4% sugar. The sheet was then
freeze—dried and cut into portions each 6 x 12mm, having a
liquid capacity of about 50uL. These were incorporated in
test devices as described above under embodiment l, with the
test strip comprising backing nitrocellulose with an anti-
alph LH monoclonal antibody immobilised in the test zone.
of the test
The liquid capacity of the “working length”
strip between the macroporous body and the detection zone
was about 40uL.
When a LH-containing urine sample was applied to the device,
a positive result showed up as a very clear blue line, with
negligible background bluecolour being visible in the
detection window while the assay was being run.
Claims (15)
1. An analytical test device incorporating a dry porous carrier strip (114) to which device an aqueous liquid sample suspected of containing an analyte can be applied, the device also incorporating a specific binding reagent attached to a particulate label which labelled specific binding reagent is freely mobile in the porous carrier strip when in the moist state, and an unlabelled specific binding reagent which is permanently immobilised in a detection zone the labelled capable of (119) on the carrier strip, and unlabelled participating in either a sandwich reaction or a competition porous specific binding reagents being reaction in the presence of the analyte, characterised in that prior to the application to the device of the liquid the particle-labelled sample, specific binding reagent is retained in the dry state ix: a separate nacroporous body (113) less than 10 times greater than the maximum particle size of comprising plastics material having a pore size not the particulate label, through which nmcroporous body the applied liquid sample must pass en route to the proximal end of the porous carrier strip, and in that there is an absorbent sink at the distal end of the porous carrier strip, the absorbent sink not being merely a length of porous carrier" material which contains the detection zone and extends beyond the detection zone.
2. An test device according to claim 1, characterised in that the nacroporous body has an average analytical pore size of not less than 10 um. - 29 _
3. An analytical test device according to claim 1 or claim 2, characterised in that the macroporous body has an average pore size of about 100 pm.
4. An analytical test device according to any one of the preceding claims, characterised in that the plastics material has been pre—treated to render it more hydrophilic.
S. An analytical test device according to any one of the preceding claims, characterised in that the plastics material is polyethylene.
6. An analytical test device according to any one of. the preceding claims, characterised in that the particulate label is~ selected from the group consisting of metallic (e.g. gold) sols, non-metallic sols, dye sols, and latex particles having a maximum diameter of not greater than about 0.5 p.
7. An analytical test device according to claim 6, characterised in that the particulate label is a gold sol.
8. An analytical test device according to claim 6, characterised in that the particulate label is coloured latex.
9. An analytical test device according to any one of the preceding claims, characterised in that the porous carrier strip is nitrocellulose, having a pore size of at least lum.
10. An analytical test device according to any one of the preceding claims, characterised in that the macroporous body is in direct moisture-conductive contact with the proximal end of the porous carrier strip, and the detection zone on the porous carrier strip is spaced away from the region of Contact of the porous carrier material with the macroporous body.
11. An analytical test device according to any one of the preceding claims, characterised in that the macroporous body and porous carrier strip are contained within a casing or (400) and having a sample entry port constructed of xnoisture—impermeable material (401) the casing or housing also incorporating housing communicating with the macroporous body, (403) from outside the casing or housing. means to enable the detection zone to be qbservable
12. An analytical test device according to claim 11, characterised in that the casing or housing is provided with means (404, 109) which enable a further zone (120) of the porous carrier strip to be observed from outside the housing and which further zone incorporates one or more control indication to be given as to reagents which enable an whether the assay procedure has been completed.
13. An analytical test device according to any one of the preceding claims, characterised in that the liquid sample is urine. device according to claim 13,
14. An test characterised in that the analyte is hCG. analytical
15. An analytical test device according to claim 1, substantially as hereinbefore described with particular reference to and as illustrated in the accompanying drawings. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBUNITEDKINGDOM17/02/19898903627.1 | |||
GB898903627A GB8903627D0 (en) | 1989-02-17 | 1989-02-17 | Assays |
Publications (3)
Publication Number | Publication Date |
---|---|
IE19980110A1 IE19980110A1 (en) | 2000-02-23 |
IE980110A1 IE980110A1 (en) | 2000-02-23 |
IE83806B1 true IE83806B1 (en) | 2005-02-23 |
Family
ID=
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