U.S. patent application number 12/624191 was filed with the patent office on 2010-03-18 for hybrid phase lateral flow assay.
This patent application is currently assigned to Quidel Corporation. Invention is credited to Cem Gokhan.
Application Number | 20100068826 12/624191 |
Document ID | / |
Family ID | 34963546 |
Filed Date | 2010-03-18 |
United States Patent
Application |
20100068826 |
Kind Code |
A1 |
Gokhan; Cem |
March 18, 2010 |
HYBRID PHASE LATERAL FLOW ASSAY
Abstract
The invention relates to devices for performing single step
assays for the determination of the presence or absence of an
analyte in a liquid sample, and methods of determining the presence
or absence of such analytes using such devices. Devices disclosed
comprise a labeled analyte-binding reagent reversibly-immobilized
on a non-porous solid material, which solid material is in physical
contact with a dry porous carrier bearing an immobilized
analyte-binding reagent. Also provided are quantitative assay
devices.
Inventors: |
Gokhan; Cem; (Frankfurt,
DE) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Quidel Corporation
San Diego
CA
|
Family ID: |
34963546 |
Appl. No.: |
12/624191 |
Filed: |
November 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11088579 |
Mar 23, 2005 |
7632687 |
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12624191 |
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10854876 |
May 27, 2004 |
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11088579 |
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60555612 |
Mar 23, 2004 |
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Current U.S.
Class: |
436/518 ;
422/400 |
Current CPC
Class: |
G01N 33/558 20130101;
G01N 33/54386 20130101; Y10S 436/807 20130101; Y10S 435/823
20130101; Y10T 436/25125 20150115; Y10S 435/805 20130101; G01N
2500/10 20130101; Y10S 436/823 20130101 |
Class at
Publication: |
436/518 ;
422/56 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 21/00 20060101 G01N021/00 |
Claims
1. An analytical device for the single-step determination of the
presence of an analyte in a liquid sample comprising a test strip
comprising: a) a first piece of non-porous solid surface
comprising, in a reversible immobilization zone, a reversibly
immobilized labeled reagent that binds said analyte to form a
complex; b) a porous carrier comprising, in a detection zone, an
immobilized reagent that binds said complex, wherein said porous
carrier is in contact with said non-porous solid surface; and c) a
second piece of non-porous solid surface placed over (a) and
(b).
2. The device of claim 1 wherein, rather than being reversibly
immobilized upon the first piece of non-porous solid material, said
labeled reagent is reversibly immobilized in a reversible
immobilization zone on the surface of said second piece of
non-porous solid material that faces said first piece of non-porous
material, so that the first piece of non-porous solid material is
on the bottom and the labeled reagent is reversibly immobilized on
the underside of said second piece.
3. A method of detecting an analyte, the method comprising a)
contacting a liquid sample to be tested for said analyte with the
analytical device of claim 1; and b) permitting said liquid sample
to traverse said test strip such that said reversibly immobilized
labeled reagent is mobilized by the sample and flows to said
detection zone, wherein, if analyte is present in said liquid
sample, a detectable signal is generated indicative of the presence
of said analyte.
4. A device for the single step quantitative measurement of an
analyte in a liquid sample, the device comprising: a) a piece of
non-porous solid surface comprising, in an immobilization zone, a
reversibly immobilized labeled reagent that binds said analyte to
form a complex; and b) a porous carrier comprising a binding
reagent and being in contact with said non-porous solid surface,
wherein said binding reagent is applied to and immobilized in a
detection zone having a longitudinal axis parallel to a flow of a
liquid sample, wherein when said liquid sample comprises the
analyte, the binding results in a signal, and wherein the distance
from the point at which said liquid sample entered said detection
zone to the furthest point in said detection zone at which said
signal is detected above background is indicative of the amount of
analyte present in said sample.
5. The device of claim 4 wherein said detection area is at least
1.5 times as long as it is wide.
6. The device of claim 4 wherein said detection area extends over
at least 50% of the length of said porous carrier.
7. The device of claim 4 wherein said detection area extends over
the full length of said porous carrier.
8. A method of detecting an analyte, the method comprising a)
contacting a liquid sample to be tested for said analyte with the
analytical device of claim 4; and b) permitting said liquid sample
to traverse said test strip such that said reversibly immobilized
labeled reagent is mobilized by the sample and flows to said
detection zone, wherein, if analyte is present in said liquid
sample, a detectable signal is generated indicative of the presence
or quantity of said analyte.
9. An analytical device for the single-step determination of the
presence or absence of an analyte in a liquid sample comprising a
test strip comprising: a non-porous solid surface comprising, in a
reversible immobilization zone, a reversibly immobilized labeled
reagent, that competes with said analyte for binding sites present
in the detection zone, and a porous carrier comprising, in a
detection zone, an immobilized reagent that binds said labeled
reagent and analyte in a liquid sample, wherein the absence of
analyte generates a detectable signal, wherein said porous carrier
is in contact with said non-porous surface.
10. A method of detecting an analyte, the method comprising a)
contacting a liquid sample to be tested for said analyte with the
analytical device of claim 9; and b) permitting said liquid sample
to traverse said test strip such that said reversibly immobilized
labeled reagent is mobilized by the sample and flows to said
detection zone, wherein, analyte present in said liquid sample will
compete for binding sites in the detection zone with said labeled
reagent, and a detectable signal is generated indicative of the
absence of said analyte.
Description
[0001] This application is a divisional of U.S. Non-Provisional
application Ser. No. 11/088,579, filed Mar. 23, 2005, now allowed,
which is a continuation-in-part of U.S. Non-Provisional application
Ser. No. 10/854,876, filed May 27, 2004, which claims the priority
of U.S. Provisional Application No. 60/555,612, filed Mar. 23,
2004, the entirety of these applications is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Lateral-flow immunoassays, with their ease of use, speed and
reliability, are widely used for self-testing and in the clinical
setting. Lateral-flow immunoassays are probably the most common
non-electrical method used in rapid medical diagnostics to detect
the presence of a specific analyte in a liquid sample.
[0003] In the general method, a liquid sample suspected of
containing the analyte is applied to a porous carrier. Different
porous materials are commonly used for the porous carrier, and can
differ in pore size, flow rate, protein-binding specifications and
pre-treatment, etc. Essentially, all of the physical activities
(e.g., liquid migration) and chemical reactions take place in the
porous carrier, in the following order.
[0004] First a liquid sample to be tested is introduced to a
designated area in the sampling-end (also referred as the "proximal
end" or "wet end") of the porous carrier, for a measured time e.g.
S seconds or in a measured volume e.g. 2 drops. From this point
forward, the liquid sample migrates within the porous carrier to
the direction of the dry end (also referred as the "distal end").
At the outset of the migration, the liquid sample is frequently
optimized for reaction by means of chemicals e.g. pH agents or
buffers, surfactants, and/or blockers impregnated into the porous
carrier.
[0005] Second, while migrating in the porous carrier, the sample
mobilizes a labeled reagent that has been reversibly (temporarily)
immobilized in the porous carrier. The zone where the mobilizable
labeled reagent is located is often referred to as the "labeling
zone", but can be referred to as the "reversible immobilization
zone" or "mobilization zone"--the terms are equivalent.
[0006] Third, while analyte is reacting with the mobilized labeled
reagent, the liquid sample and mobilized labeled reagent migrates
further within the porous carrier to the detection zone, (which may
also be referred to the "irreversible immobilization zone" or
merely the "immobilization zone") where reagent that binds the same
analyte is fixed or immobilized, usually in the form of a line.
When analyte is present in the liquid sample, a "sandwich" in the
form of the mobilized labeled reagent:analyte:immobilized reagent
is formed, and the resulting concentration of the labeled reagent
leads to a visible line appearing in the detection zone, which is
indicative of a positive result.
[0007] Lastly, remaining sample liquid, together with the rest of
the labeled reagent further migrates to a control zone, where a
second line appears indicating that sample has progressed through
the detection and control zones and that the assay has provided a
valid test result. The rest of the sample and the remaining labeled
reagent then migrate to a porous sink. Labeled reagent remaining in
the porous carrier (other than in the detection zone, control zone
or sink) makes up any background signal. In some instances where
the migration direction reverses, so called "back flow," occurs.
Furthermore, the porous carrier can be pre-treated with chemicals
e.g. surfactants.
[0008] Lateral-flow immunoassays can also function on the basis of
competitive binding of the analyte. In these devices, lack of the
test line generally indicates a positive result.
[0009] The most common example of a lateral-flow immunoassay device
is a pregnancy test. These devices are commonly provided for home
use, in a plastic housing with a fibrous or a porous extension,
which can be held to a urine stream to collect urine sample into
the housing. The urine sample collected this way then migrates to
the porous carrier, which contains the labeled reagent and the
series of events mentioned above starts. The analyte detected in a
pregnancy test is Human Chorionic Gonadotropin (hCG) and the
reagents commonly used are anti-hCG monoclonal or polyclonal
antibodies. The most common labels are gold or latex particles.
[0010] Another known example of a lateral-flow immunoassay device
commonly provided for home use is an ovulation test, the analyte
being Luteinizing Hormone (LH) and reagents being anti-LH, and the
rest of the device being similar to a pregnancy test.
[0011] A professional format of lateral-flow immunoassay devices
commonly referred to as cassette tests, have smaller housings and a
sample application orifice instead of the fibrous extension. The
sample orifice exposes part of the porous carrier where a liquid
sample can be dispensed with a pipette, directly to the porous
carrier.
[0012] A low cost format of lateral-flow immunoassay devices is a
dipstick test in the shape of a strip.
[0013] A diversity of test designs, against an ever increasing
number of analytes, especially in the cassette and dipstick format,
exists in the market indicating the wide acceptance of the
method.
SUMMARY OF THE INVENTION
[0014] The present invention provides a new assay, applicable to
all known analytes, through a reversible reagent immobilization on
a non-porous solid surface and an irreversible reagent
immobilization on/in a porous media, wherein non-capillary liquid
transport may be achieved on the non-porous solid surface by means
of momentum of sampling and/or gravity. The use of a nonporous
surface reduces the opportunities for non-specific binding of
labeled reagent, thereby facilitating its ready mobilization by
applied liquid sample. The increased binding surface area of the
porous media enables a higher irreversible immobilization and
therefore signal intensity. The use of a minimal number of device
components decreases production related complications and the cost,
and the use of a non-capillary liquid transport means reduces the
residual liquid volume not contributing to the assay chemical
reaction and reduces the back-flow.
[0015] The present invention also provides improvements in
quantitative, dipstick and mid-stream test device designs.
[0016] The general reactions between the different analytes,
labeled reagents, capture reagents and the control reagents are
known to those skilled in art and are not the focus of this
invention.
[0017] In one aspect, the invention encompasses an analytical test
device, which, in the presence of a specific analyte in a liquid
sample, is capable of producing a detectable signal, the device is
characterized in that it comprises a non-porous solid surface and a
porous carrier. The non-porous solid surface comprises a reversibly
immobilized labeled reagent against the analyte in a designated
reversible immobilization zone, also referred to as mobilization
zone or labeling zone, and the porous carrier comprises an
immobilized reagent against the analyte in a designated detection
zone also referred to as an irreversible immobilization zone, or
merely as an immobilization zone. The liquid sample taken into the
device can initiate the mobilization of the labeled reagent from
the non-porous solid surface, and the labeled reagent-sample mix
can move to the detection zone via the porous carrier to produce
the signal.
[0018] The reversibly immobilized reagent in the reversible
immobilization or labeling zone is loosely attached to the
non-porous surface such that when sample is applied the reagent
releases from the surface and mixes within the sample.
[0019] In contrast, the immobilized reagent in the detection,
immobilization, or irreversible immobilization zone is designed to
remain substantially in place throughout the entire duration of the
assay.
[0020] In another aspect, the liquid sample, taken into the device,
moves along the non-porous surface through the mobilization zone,
by non-capillary liquid transport which is achieved by means of
momentum sampling and/or gravity means until sample reaches the
porous carrier, which then draws the fluid into and over the porous
material by capillary or wicking means.
[0021] It is a further embodiment of the present invention that
either the housing or the bottom surface of the non-porous solid
surface include an attached elevation mechanism or foot which
causes the device to rest in such a manner that after the liquid
sample is applied the liquid sample may flow downhill with the
assistance of gravity to thaw the liquid sample through the
device.
[0022] In another embodiment, the device is capable of producing a
separate control signal, either in the detection zone or in an
additional designated control zone, to confirm the passage of
adequate amount of the labeled reagent through the immobilization
zone to indicate the completion of the assay and/or the ability of
the device to produce the signal in the presence of the
analyte.
[0023] In other embodiments, the device can have, in addition to
the non-porous carrier bearing a reversibly immobilized labeled
reagent and a porous carrier bearing an immobilized reagent, any of
the numerous aspects of lateral flow assay devices known to those
of skill in the art. These include, among others, adaptations that
permit the measurement of multiple analytes in the same sample.
[0024] In another embodiment, the device comprises two non-porous
solid surfaces, positioned parallel to each other, at least one of
which comprises the labeled reagent in dry state in the
mobilization zone. In a preferred embodiment, the second non-porous
surface is sized wider and longer than the first non-porous
surface, such that when the second non-porous surface is placed
over the first non-porous surface, no edge of the first non-porous
surface extends beyond an edge of the second non-porous surface,
thus providing improved handling and protection from undesired
wetting in midstream sample application. In such a device design,
the sample is taken onto the sample reception zone through an
orifice in the enlarged non-porous surface.
[0025] In an alternately preferred embodiment, the second nonporous
surface-attached to a device is not sized wider and longer than the
first non-porous surface but is the same width and is at least as
long as the length of the porous surface and may extend in length
from the porous surface either partially or entirely covering the
non-porous surface.
[0026] In another embodiment, the device comprises a housing with
an orifice to intake liquid sample and a window to observe the
signal. In a preferred embodiment, at least one element of the
housing comprises the labeled reagent in a dry state in the
mobilization zone.
[0027] It is further preferred that the device in the housing is
adapted into various handling accessories, designed for different
test applications, such as home use, professional laboratory use
and patient side use, wherein the device with a minimum housing is
contained in different accessory devices.
[0028] The invention further encompasses an analytical test device
in the strip format, characterized in that the device comprises two
parallel non-porous solid surfaces attached to each other or the
continuation of each other or overlap with each other, in the
sample receiving end of the device, forming a sample reservoir.
[0029] In one embodiment, the reservoir comprises an orifice on
either of the non-porous solid surfaces, which may provide means
for sample dispensing and ventilation to release the liquid sample
from the reservoir.
[0030] In one embodiment, the device further comprises a sink,
distal to and in fluid contact with the porous carrier, the sink
capable of absorbing excess liquid sample after the sample
traverses the test strip.
[0031] The invention further encompasses a test device,
characterized in that the device comprises a housing comprising an
evaporation opening for the sink.
[0032] The invention further encompasses a test device,
characterized in that the device comprises a housing assembled into
a second housing or into an accessory device.
[0033] The invention further encompasses an analytical test device
in the strip format, characterized in that the device comprises two
parallel non-porous solid surfaces, one of the said surfaces being
absent from part of the sink area thus enabling efficient
evaporation.
[0034] The invention further encompasses a test strip in a
midstream test format, comprising an enlarged solid surface
comprising an orifice, the enlarged surface providing protection
from undesired wetting during the necessary sampling and handling
steps.
[0035] The invention further encompasses a test device according to
preceding aspects, characterized in that either or both sides of
the detection zones are transparent or comprise means to enable the
observation of the test result e.g. a window and all above
mentioned solid parts, non-porous or porous, can be attached to
each other by known techniques such as ultra sonic welding, hot
laminating, adhesives or mechanical means.
[0036] In another aspect, the invention encompasses an analytical
device for the single step determination of the presence of a
specific analyte in a liquid sample comprising a test strip
comprising: a first piece of non-porous solid surface comprising,
in a reversible immobilization zone, a reversibly immobilized
labeled reagent that binds the analyte to form a complex; and a
porous carrier comprising, in a detection zone, an immobilized
reagent that binds the analyte, wherein the porous carrier is in
contact with the non-porous solid surface; wherein liquid sample
applied to the non-porous solid surface mobilizes the reversibly
immobilized labeled reagent, whereupon analyte in the liquid sample
is permitted to bind the labeled reagent to form a complex which,
together with sample liquid, flows into or onto the porous carrier,
wherein the immobilized reagent binds the complex, thereby
generating a detectable signal indicative of the presence of the
analyte.
[0037] In another embodiment, the porous carrier further comprises,
at a site separate from and distal to the site of immobilization of
the immobilized reagent, a second immobilized binding reagent that
binds an agent other than the analyte, and wherein binding of the
second immobilized binding reagent to the agent generates a
detectable signal that confirms passage of liquid sample to the
site separate from and distal to the site of immobilization of the
immobilized reagent. In other embodiments, the device is capable of
producing a separate control signal, either in the immobilization
zone or in an additional designated control zone, to confirm the
passage of adequate amount of the labeled reagent through the
immobilization zone and/or the ability of the device to produce the
signal in the presence of the analyte.
[0038] In another embodiment, the device further comprises a porous
sample application member. The porous sample application member
either overlaps and is in physical contact with the reversibly
immobilized reagent on the non-porous solid material, or overlaps
and is in physical contact with at least a portion of the porous
carrier, which portion overlaps and is in contact with the
reversibly immobilized reagent on the non-porous solid
material.
[0039] In other embodiments, the device can have, in addition to
the non-porous carrier bearing a reversibly immobilized labeled
reagent and a porous carrier bearing an immobilized reagent, any of
the numerous aspects of lateral flow assay devices known to those
of skill in the art. These include, without limitation, adaptations
that permit the measurement of multiple analytes in the same
sample.
[0040] In another embodiment, the reversible immobilization or
mobilization zone comprises a plurality of labeled reagents. In
another embodiment, each of the plurality of labeled reagents binds
a different analyte.
[0041] In another embodiment, the porous carrier comprises, in the
detection zone, a plurality of immobilized binding reagents. In
another embodiment, each of the plurality of immobilized binding
reagents is located in a spatially distinct site and each binds a
different analyte to generate a signal.
[0042] In another embodiment, the device further comprises a
plurality of the test strip assemblies, each member of the
plurality bearing reagents that identify the presence of one or
more different analytes.
[0043] In another embodiment, the device comprises a second piece
of non-porous solid material, sized wider and longer than the first
non-porous surface, wherein the second piece of non-porous solid
material is positioned parallel to the first piece, the second
piece having an aperture through which sample is introduced, and
wherein the second piece is positioned such that the aperture is
located upstream of the reversible immobilization or mobilization
zone and such that the second piece provides protection to the
first non-porous surface from undesired wetting during mid-stream
sample application.
[0044] In another embodiment, the device further comprises a sample
reservoir, proximal to the reversible immobilization or
mobilization zone and formed from a continuation of the first piece
of non-porous solid material or from the juxtaposition of a second
piece of non-porous solid material with the first piece of
non-porous solid material.
[0045] In another embodiment, the device further comprises a
housing comprising a non-porous solid material.
[0046] In another embodiment, at least one element of the housing
constitutes the reversible immobilization or mobilization zone, the
zone comprising the reversibly immobilized labeled reagent in a dry
state.
[0047] In another embodiment, the flow of liquid sample applied to
the non-porous solid surface, from the site of application to the
reversible immobilization or mobilization zone and subsequently to
the porous carrier, is provided by the kinetic force of sample
application, such as by mid stream sample application, pipetting,
or by a swab.
[0048] The invention further encompasses an analytical device for
the single step determination of the presence of a specific analyte
in a liquid sample comprising a test strip comprising: a) a first
piece of non-porous solid material comprising on its surface, in a
reversible immobilization or mobilization zone, a reversibly
immobilized labeled reagent that binds the analyte; b) a porous
carrier comprising, in a detection zone, an immobilized reagent
that binds the analyte, wherein the porous carrier is in contact
with the non-porous solid surface; and c) a second piece of
non-porous solid material, placed over (a) and (b), wherein liquid
sample applied to the first non-porous solid material mobilizes the
reversibly immobilized labeled reagent, whereupon analyte in the
liquid sample is permitted to bind the labeled reagent to form a
complex which, together with sample liquid, flows into the porous
carrier, wherein the immobilized reagent binds the complex, thereby
generating a detectable signal indicative of the presence of the
analyte.
[0049] In another embodiment, rather than being reversibly
immobilized upon the first piece of non-porous solid surface, the
labeled reagent is reversibly immobilized in a reversible
immobilization or mobilization zone on the surface of the second
piece of non-porous solid material that faces the first piece of
non-porous material, such that when the test strip assembly is
oriented the first piece of non-porous solid material is on the
bottom, and the labeled reagent is reversibly immobilized on the
underside of the second piece.
[0050] It is a further embodiment of the present invention that the
detection zone be placed at the proximal portion of the porous
carrier, thus being closer to the non-porous mobilization zone.
[0051] In another aspect, the invention encompasses a device for
the single step quantitative measurement of an analyte in a liquid
sample comprising: a) a piece of non-porous solid surface,
comprising in a reversible immobilization or mobilization zone, a
reversibly immobilized labeled reagent that binds the analyte to
form a complex; and b) a strip of porous material, the strip
comprising a binding reagent and being in contact with the
non-porous solid surface, wherein the binding reagent is applied to
and immobilized on the strip of porous material in a detection area
having a longitudinal axis parallel to a flow of a liquid sample,
wherein when the liquid sample comprises an analyte-label conjugate
that is bound by the binding reagent, the binding results in a
signal, and wherein the distance from the point at which the liquid
sample entered the detection zone to the furthest point in the
detection area at which the signal is detected above background is
indicative of the amount of analyte present in the sample.
[0052] In one embodiment, the detection area is at least 1.5 times
as long as it is wide. In another embodiment, the detection area
extends over at least 50% of the length of the porous
chromatographic material. In another embodiment, the detection area
extends over at least 60%, 70%, 80%, 90% or over the full length of
the porous material.
[0053] In another aspect, the invention encompasses a method of
detecting an analyte, the method comprising a) contacting a liquid
sample to be tested for said analyte with an analytical device as
described herein, and b) permitting the liquid sample to traverse
the test strip assembly of the device such that reversibly
immobilized, labeled reagent is mobilized and thereafter conveyed
to the detection zone of the device, whereat, if analyte is present
in the liquid sample, a detectable signal is generated, the signal
indicative of the presence of the analyte. Analytical test devices
incorporating any or all of the embodiments described above or
elsewhere herein can be employed in the analytical methods
disclosed herein.
[0054] As used herein, the term "test strip" refers to a non-porous
solid surface upon which a reversibly immobilized dried reagent is
present, and a porous carrier strip with a detection zone which is
in contact with the non-porous solid material. Test strips can
additionally have a sink element, and/or a second piece of
non-porous solid material overlaid upon the assembly of the first
non-porous solid material and the porous carrier. A "test strip"
can also include a porous sample receiving element in contact with
the non-porous solid material upon which the reversibly immobilized
reagent is present. Furthermore, a "test strip" may comprise of one
piece of material which through processing or chemical treatment
comprises both non-porous and porous physical properties, or
multiple pieces including different materials.
[0055] As used herein, the term "non-porous" refers to a surface
which does not permit liquid sample to enter into or to pass
through it under normal assay conditions. A non-porous surface does
not absorb liquid sample under normal assay conditions and is
non-chromatographic. A non-porous surface is preferably inert,
impermeable to humidity and low in protein binding. For a
"non-porous" surface as the term is used herein, any pores present
are smaller than the smallest reagent used and/or are smaller than
any component of the sample. By "smaller than" is meant that the
smallest reagent or component of the sample will not pass through
any such pore, in any appreciable amount, under liquid flow
conditions applied in the routine or optimal operating conditions
for the device. "Non-porous" materials can be made of any low
protein binding, non-water absorbing, solid material such as
thermoform or thermoplastic polymers (e.g. polystyrene,
polyethylene, polycarbonate, polypropylene, fluoropolymer, or
polyester, or a combination) or glass or metals or ceramics or
composite materials well known to those skilled in art. The
non-porous surface can also be made of any material which is
surface coated with low protein binding, non-water absorbing
material e.g. Teflon.RTM. fluoropolymer resin or Mylar.RTM.
polyester film coated cellulose. A porous material, such as certain
porous plastics or nitrocellulose, may be made non-porous through
pressing or chemical treatment such that the surface is rendered
smooth and inert, and reagents and liquids are incapable of
penetrating the surface of the "non-porous" material.
[0056] As used herein, the term "porous" when used in relation to a
porous carrier strip means that the material has pores through
which the largest reagents or sample components for a given
immunoassay device will pass under liquid flow conditions applied
in the routine or optimal operating conditions for the device. The
term "porous" refers also to a material with pores which permit or
conduct the flow of liquid through the body of the material. It is
to be understood that "porous" materials can also permit the flow
of liquid across the outer surface of the material, as long as they
also permit the passage of the liquid through the body of the
material Porous materials with varying ranges and tolerances of
pore size are well known in the art, and include, for example,
nitrocellulose, glass fiber, porous plastics, such as polyethylene,
and various chromatographic papers, among others.
[0057] As used herein, the term "open," when used in reference to a
side of a test device assembly, means that an edge is not sealed or
adhered to another, such that air or liquid can pass. Thus, an
assembly which is "open on at least three sides" has an unsealed
opening at an inlet, an outlet and at least one side.
[0058] As used herein, the term "overlap" means that when an
assembly is viewed from the side, one material, e.g., a sheet of
material, extends over top of another. Overlap can but need not
necessarily include complete overlap, i.e., the top material covers
the full length of the bottom material.
[0059] As used herein, the term "at least a portion of said porous
carrier," when used in reference to overlap by, e.g., a sample
receiving member or element, means that the sample receiving member
or element overlaps the porous carrier but does not obscure the
detection zone.
[0060] As used herein, the term "into," when used in reference to
the passage of a liquid sample and materials dispersed within a
liquid sample "into" a porous material, means that the sample
penetrates a porous material such that it is carried within the
pores of the porous material as the liquid sample flows. The term
"into" when used in this manner does not exclude the simultaneous
passage of liquid sample over the surface of a porous carrier
material "into" which the sample is flowing. In preferred aspects,
however, the passage of liquid over the surface of the porous
carrier material is reduced by the presence of a non-porous backing
layer and/or the presence of an additional non-porous material
overlaid on the porous carrier material.
[0061] As used herein, the term "reversible immobilization" refers
to the deposition of a reagent on a non-porous solid surface in a
manner such that it is readily solubilized or dispersed by liquid
sample as applied to an assay device as described herein. By
readily solubilized or dispersed is meant that at least 80%, and
preferably at least 90% or even 100% of the reversibly immobilized
material is solubilized or dispersed into the liquid by the
application of liquid sample under the liquid flow conditions
applied in the routine or optimal operating conditions for the
device.
[0062] As used herein, the term "reversible immobilization zone"
refer to the discrete situs on a non-porous solid material at which
the labeled reagent is reversibly immobilized. The reversible
immobilization zone is also referred to herein as the "mobilization
zone," or the "labeling" zone.
[0063] By "mobilized" is meant at least the partial dispersal or
solubilization of a labeled reagent from the dry, reversibly
immobilized state to the mobile state capable of flowing to and
through a porous carrier in an assay device as described herein. By
"mobile" is meant that the labeled reagent is moving in a lateral
direction relative to a surface, for example, the reagent is moving
in or with a flowing liquid.
[0064] As used herein, the relative terms "proximal" and "distal"
are based upon the direction of flow--liquid sample flows from the
proximal end of a test strip assembly to the distal end.
[0065] As used herein, the term "detection zone" refers to the
region or regions on a porous carrier as described herein that
comprise immobilized analyte binding reagent, located in one or
more discrete locations. The detection zone is proximal to any
control zone or zones and proximal to the sink. In the quantitative
assay embodiments described herein, the detection zone can extend
up to the full length of the porous carrier material.
[0066] As used herein, the term "in contact" refers to direct
contact between two entities, but also includes the use of
intervening structures between entities described herein, so long
as there is touching or contact between the elements and/or fluid
flow amongst the elements.
[0067] As used herein, the term "immobilized," when used in
reference to an immobilized reagent (as opposed to a reversibly
immobilized reagent) means that the reagent is not appreciably
solubilized or displaced by the passage of liquid sample under the
liquid flow conditions applied in the routine or optimal operating
conditions for the device. By "not appreciably solubilized or
displaced" is meant that most of the immobilized reagent remains
attached to the porous carrier during and after passage of the
liquid sample under normal or routine assay conditions.
[0068] As used herein, the term "specifically binds" means that an
agent binds with a dissociation constant of 1 .mu.M or less. An
agent that "specifically binds" an analyte will preferentially bind
that analyte in the presence of a large number of non-related
molecules. With regard to binding agents, it will commonly be the
situation that both a reversibly immobilized binding reagent and an
immobilized binding reagent will specifically and essentially
exclusively bind the analyte of interest. However, the reversibly
immobilized labeled reagent and the immobilized reagent need not
necessarily have specificity for binding only to the analyte of
interest. For example, one can use one reagent that binds a class
of analytes, for example, on the basis of a structural similarity
shared by members of the class, and a second reagent that binds the
analyte of interest.
[0069] As used herein, the term "spatially distinct" means that the
given region is sufficiently separated from another region as to be
discerned as separate and non-overlapping by eye. Generally, as the
term is used herein with regard to regions on a detection zone, two
regions are "spatially distinct" if they are greater than or equal
to 0.2 mm apart, e.g., 0.2 mm, 0.3 mm, 0.5 mm, 1.0 mm, etc.
[0070] As used herein, the terms "wider" and "longer" mean that a
given piece of material, e.g., a sheet of non-porous material, is
at least 1 mm greater, and preferably more (e.g., 2 mm, 3 mm,
etc.), in width and length than another to which it is being
compared.
[0071] As used herein, the term "midstream sample application"
refers to the application of liquid sample by holding the assay
device in the flow of a liquid, e.g., in the flow of urine during
urination.
[0072] As used herein, the term "sample reservoir" refers to an
element of an assay device as described herein that can hold liquid
sample at least sufficient to permit an assay device to provide a
test result. A sample reservoir will generally be located at the
proximal end of the assay device, such that application of liquid
sample to the reservoir will result in application of liquid sample
to the non-porous solid surface near the reversible immobilization
or mobilization zone.
[0073] As used herein, a "housing" is a casing surrounding at least
the detection zone of an assay device described herein--the housing
will have at least an aperture for the addition of sample, and for
the observation of a test result. An aperture to permit
evaporation, e.g., from a sink, can also be present. It is
preferred where a housing is used, that the housing be an active
member in the assay device, e.g., by providing as a surface for
reversible mobilization and/or the immobilization zone.
[0074] As used herein, the term "detected above background" means
that a given signal is greater than the amount of signal one would
obtain in an assay run in the absence of analyte.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] The drawings are not to scale.
[0076] Drawing 1 shows one embodiment of the devices described
herein. Drawing 1A shows the assembled device and Drawing 1B shows
the unassembled device.
[0077] Drawing 2A shows an embodiment of the reservoir in the
sampling-end of a device according to one aspect of the invention.
Drawings 2B, 2C, 2D and 2E show several alternative design options
for the said reservoir.
[0078] Drawing 3 shows an embodiment comprising an enlarged
non-porous surface. Drawing 3A shows the assembled device, and 3B
shows the unassembled unattached device.
[0079] Drawing 4A shows the one embodiment of the construction of
the housing according to one aspect of the invention. Drawing 4B
shows the same housing from the top and Drawing 4C shows the same
housing from the bottom. Drawing 4D shows the same housing with no
optional components and with an alternative placement of the
labeled reagent.
[0080] Drawing 5 shows examples the accessory devices and housings
according to several aspects of the invention.
[0081] Drawing 6 shows schematic diagrams of several configurations
of single step assay devices as described herein which, in addition
to a non-porous surface having a reversibly immobilized reagent and
a porous material having an immobilized reagent, further comprise a
porous sample receiving element. The reversibly immobilized reagent
(1) is on the non-porous surface (2) and the irreversibly
immobilized reagent(s) are in the porous material (3). Liquid
sample is received by liquid sample receiving element (4).
[0082] Drawing 7A-7D shows schematic diagrams of a quantitative
assay device embodying one aspect of the invention. That portion of
the test strip labeled (a) contains an immobilized detection
reagent (the detection zone), while (b) does not.
[0083] Drawing 8 shows a side view of a quantitative assay
embodiment as described in Example 11.
DETAILED DESCRIPTION OF THE INVENTION
[0084] The invention provides a new assay platform that comprise a
hybrid test strip comprising a non-porous solid surface and a
porous carrier. The use of a non-porous material for the reversible
immobilization of labeled reagent, which must be readily dispersed
or solubilized by liquid sample application if the assay device is
to function, avoids non-specific interactions that frequently
interfere with such dispersal or solubilization. While not limited
in usefulness to assays using particulate labels, the non-porous
character of the material used for reversible immobilization avoids
problems caused by particles becoming trapped within the pores of
materials commonly used for reversible immobilization and the
increased binding surface area of the porous media enables a higher
irreversible immobilization therefore a higher signal intensity.
The use of minimal number of device components decreases production
related complications and the cost. The use of non-capillary liquid
transport means reduces the residual liquid volume not contributing
to the assay chemical reaction and the back-flow. The devices
described herein are well suited for single step analyte detection,
i.e., one need only apply liquid sample to the device in order to
obtain a test result.
[0085] The present invention also provides improvements in
quantitative, dipstick and mid-stream test device designs.
[0086] In one aspect, the reversible immobilization zone is open
not only on the ends into which (inlet) and out of which (outlet)
liquid sample flows, but also on one or more sides and potentially
on the top. This design enhances the relative ease of manufacture.
Thus, rather than being a closed system, the immobilization zone is
open on at least three sides (inlet, outlet, and one side),
preferably four sides (inlet, outlet and both sides), and
potentially even five sides (inlet, outlet, both sides and the top,
i.e., no second surface on top of the reversible immobilization
zone. Further, the entire device can be open from the sides, as
well as from the inlet and after the detection zone or optional
sink. In addition to ease of manufacture, the openness of, for
example, the distal end of the device, can permit evaporation from
that end which drives the continued flow of liquid. Thus, in one
aspect, the device is open on three (inlet, outlet and one side) or
four (inlet, outlet and both sides) sides.
[0087] In one aspect, a single step analytical test device is
provided, which produces a relevant signal depending upon the
presence of a specific analyte in a liquid sample. The device
comprises a non-porous solid surface and a porous carrier attached
to it in fluid communication. The non-porous solid surface
comprises a reversibly immobilized labeled reagent that
specifically binds the analyte, located in a designated
mobilization zone. The porous carrier comprises an immobilized
reagent that specifically binds the analyte, located in a detection
zone. The liquid sample taken into the device can initiate the
mobilization of the labeled reagent, and the labeled reagent+sample
mix can move to the detection zone through the porous carrier to
produce the relevant signal.
[0088] In operation, the labeled reagent is not mobilized by the
sample liquid passing through the porous carrier, but rather the
labeled reagent dissolves or is dispersed into the liquid sample as
the sample passes the non-porous surface of the mobilization zone,
and is then carried into or onto the porous carrier by the liquid
sample.
[0089] As noted, in the single step assay devices described herein,
when liquid sample is applied to the devices described herein, that
is all that is necessary to generate an assay result. However, the
device can be modified to permit multi-step assay systems, such as
the pre-mix of the sample and labeled conjugate before application.
Furthermore, following the application of liquid sample to the
device, the flow of liquid is essentially continuous. That is, the
flow does not substantially stop to permit an incubation before the
flow is resumed. The rate of flow can change, for example, as
liquid sample moves from the non-porous solid material to the
porous carrier material, but there are no impediments designed to
substantially arrest the flow of the sample.
[0090] The lack of such designed impediments enables simplified and
low cost production and provides an assay platform which can
deliver results faster. It may also provide less complications for
test performance.
[0091] In one aspect, the surface of the reversible immobilization
zone is substantially smooth, i.e., the surface is not grooved or
etched in order to, or in a manner that, provides increased surface
area or changes the dynamics of liquid flow. A substantially smooth
non-porous surface tends to release labeled reagent more
efficiently than does an irregular surface containing grooves or
etchings. With regard to surface area, in one aspect, the
non-porous surface area occupied by the reversibly immobilized
labeled reagent is minimized. While other arrangements can provide
satisfactory results, in this aspect, the "footprint" of the
reversibly immobilized labeled reagent is kept as small as
possible. This minimizes the potential for nonspecific interaction
of the labeled reagent with the non-porous surface, and can provide
for a more concentrated mobile label front. It can also be
advantageous to locate the reversibly immobilized reagent on the
non-porous surface very close to the porous carrier material.
Preferably, the labeled reagent may be lyophilized or dried under
vacuum in the reversible immobilization zone however, air drying of
the labeled reagent is most preferred.
[0092] The devices and methods involving the novel hybrid phase
(non-porous/porous) labeled reagent mobilization/detection assembly
described herein can include aspects of other lateral flow assay
devices well known to those of skill in the art, e.g., mobile
and/or immobile control reagents, competitive assay format,
etc.
[0093] In these and other aspects of the invention, the device can
be contained in any housing comprising an orifice or aperture, to
which a liquid sample can be introduced to initiate the assay. Such
housings are known and commonly produced from water-impermeable
thermoform or thermoplastic materials. As discussed elsewhere
herein, the housing can play an integral role in the function of
the devices in some aspects.
[0094] Another aspect of the invention relates to a reservoir
structure for dip-stick tests, wherein the test strip comprises two
parallel solid layers on each wide side of the strip sandwiching
the porous carrier, and the said layers, by coming together, form a
sample reservoir in the sampling-end of the said strip.
[0095] Another aspect of the invention relates to a device designed
for mid-stream sample loading, wherein the test device comprises an
enlarged outer surface with a sampling aperture. The enlarged outer
surface protects the dry parts of the device from undesired wetting
during the mid-stream sampling, and provides means for improved
handling. A displaceable cap or shroud can optionally be included,
to cover the sample application element and prevent unwanted
evaporation from the sample application end of the device or to
avoid damage to surfaces when the device is set down after sample
loading.
[0096] In another aspect of the invention the device may be
modified to perform a competition-type specific binding assay.
Competition assay techniques are widely known in the prior art.
Specifically the mobilizable reagent competes with the reagent
(e.g. hCG antigen) in the sample for binding in the detection zone,
therefore if a detectable line is present, the test is negative for
the reagent (e.g. hCG antigen).
[0097] In another aspect, the devices of the invention can comprise
a housing, wherein at least one element of the housing is also at
least part of the non-porous solid surface comprising the
mobilization zone, and the non-porous solid surface is in liquid
contact with the porous carrier.
[0098] The housing according to this aspect or others described
herein can be made of a single piece, or multiple pieces. The
housing can be in any practical size and shape and can be made of
any non-porous and non-water absorbing solid material with low
affinity to the labeled reagent, including the known housings
commonly produced from water impermeable thermoform or
thermoplastic materials. In another aspect of the present invention
the housing may be made of porous materials such as paper that is
treated to become non-porous in critical areas. A porous housing
can be used advantageously to absorb excess sample or can be used
as a sink, in addition to adding structural integrity to the
device.
[0099] In another aspect, the invention relates to accessory
devices and shell housings wherein, the device in its immediate
housing as described herein, can also be contained in accessory
devices or shell housings.
[0100] In one aspect, the invention provides an analytical test
device, which is capable of producing a relevant signal dependent
upon the presence of a specific analyte in a liquid sample. Drawing
1 shows the basic construction of such a device. Drawing 1A shows
the assembled device and Drawing 1B shows the unassembled device.
The said device comprises a non-porous solid surface (1) and a
porous carrier (2) attached to it. The said non-porous solid
surface comprises reversibly immobilized labeled reagent specific
for the analyte in a designated mobilization zone (3), and the
porous carrier comprises an irreversibly immobilized reagent
against the analyte in a designated immobilization zone (4).
Optional control zone (5) also contains an irreversibly immobilized
reagent.
[0101] The proximal-end (6), i.e., the wet-end, or sampling-end of
the device and the distal-end (7), i.e., the dry-end of the device
are also shown.
[0102] The liquid sample, introduced to the device from the
proximal end can initiate, before contacting the porous carrier
(2), the mobilization of the reversibly immobilized labeled reagent
in the mobilization zone (3) and the label reagent-sample mix can
migrate on the solid surface via porous carrier (2) to the
immobilization zone through the incubation zone (9) to produce the
signal and further migrate to the optional control zone (5),
producing the control signal, and finally migrate to the sink zone
(10).
[0103] The non-porous surface (1), can be made of any low protein
binding, non-water absorbing, solid material such as thermoform or
thermoplastic polymers (e.g. polystyrene, polyethylene,
polycarbonate, polypropylene, fluoropolymer, or polyester, or a
combination) or glass or metals or ceramics or composite materials
well known to those skilled in art. The non-porous surface can also
be made of any material which is surface coated with low protein
binding, non-water absorbing material e.g. Teflon.RTM.
fluoropolymer resins or Mylar.RTM. polyester film coated cellulose.
Furthermore, the non-porous surface can be created using any
material (porous or otherwise) which is capable of being compressed
and or surface treated), so long as the treated surface properties
are such that they do not permit liquid sample and/or the assay
reagents to enter into or pass through the surface under normal
assay conditions.
[0104] The porous carrier (2) can be chosen from a wide variety of
commercially available porous materials, according to its flow
rate, pore size, protein binding capacity, blocking requirements,
suitability for a specific analyte and label, thickness and backing
layer, all within the knowledge of those skilled in art. The most
common types of porous carrier are nitrocellulose and porous
plastics. If necessary to prevent non-specific binding or, for
example, to aid in wetting properties, the porous carrier can be
pre-treated with blocking agents, e.g. bovine serum albumin, or
surfactants, or alternatively can be obtained pre-treated from
commercial sources.
[0105] The mobilization zone (3) is essentially coated with the
labeled reagent to be mobilized upon wetting. The coating can be
done with a number of techniques known to those skilled in art e.g.
spraying or dispensing.
[0106] Where desired, the non-porous surface, the porous surface,
or both can be treated with agents (e.g., non-ionic surfactants,
etc.) that modify the flow characteristics of applied liquid
sample.
[0107] The immobilization zone (4) is essentially coated with the
unlabeled capture reagent against the same specific analyte. The
coating can be done with number of techniques known to those
skilled in art e.g. spraying or dispensing. Depending upon the
methods used, the coating will result in varying degrees of
penetration of the immobilized reagent into the porous carrier.
Such penetration is neither required for nor detrimental to the
function of the devices described herein. That is, there is
generally no negative impact of such penetration, although it can
be desirable, e.g., to provide a higher concentration of
immobilized reagent in a smaller area. The most common method of
permanently binding the capture reagent to the immobilization zone,
is passive absorption e.g. dispensing/spraying, incubating, and
drying an aqueous reagent solution on the porous carrier.
[0108] Optionally, the immobilization zone of the porous carrier
can be compressed, to slow down the liquid flow and increase the
immuno-concentration, thus increasing the signal strength. The
compression of the porous carrier in the immobilization zone can be
performed prior to or simultaneously with the application of the
capture reagent.
[0109] The porous carrier (2) has two basic functions, to regulate
the migration of the liquid sample+the labeled reagent mix, and to
comprise the immobilization zone (4) and optionally the control
zone (5).
[0110] Incubation between the analyte (if present) and the labeled
reagent takes place partly in the mobilization zone (3) and partly
through the migration between mobilization and immobilization
zones. Therefore, liquid passage time between the mobilization zone
(3) and the immobilization zone (4) through the incubation zone (9)
has an effect on the sensitivity of the device. It is known that by
adjusting the length of the incubation zone (9) and the flow rate
of the porous carrier, the sensitivity of the device can be
altered.
[0111] The distal portion of the porous carrier after the optional
control zone (5) acts as a liquid sink (10).
[0112] In a preferred embodiment, the device also comprises a
sample reception zone (8) on the non-porous solid surface (1),
comprising sample treatment reagents, either to be mobilized
instantly when wet (e.g. surfactants, salts, pH agents, blockers)
or binding agents for molecules to be blocked from the test system.
The treatment agents can additionally, or in the alternative, be
applied to the porous carrier.
[0113] In a preferred embodiment, the device also comprises an
optional zone (13) and an optional attachment zone (21) on the
non-porous carrier, which can be used for production and assembly
purposes. The optional zone (13) can also comprise wetting agents,
such as non-ionic surfactants to promote the liquid migration to
within the porous carrier.
[0114] In a preferred embodiment, the device comprises a second
non-porous surface (11) which is attached parallel to the first
non-porous surface (1), to enhance the sample application and/or
the liquid sample transport from the optional sample reception zone
(8) to the proximal end (14) of the porous carrier and/or to avoid
the surface transport over the porous carrier (2) and to avoid
premature drying of the liquid sample and/or to improve the
robustness of the device.
[0115] In a preferred embodiment, the second non-porous surface
(11) is shorter than the first non-porous surface (1) to expose the
optional sample application zone (8) and to create guidance to the
sample application, in the proximal-end of the device.
[0116] In a preferred embodiment, the second non-porous surface
(11) or the first non-porous surface (1) is shorter than the other
one in the distal-end (7) of the device subject to the invention.
Thus exposing a part of the liquid sink (10) to the atmosphere,
therefore creating an evaporation surface, enhancing the liquid
absorption capacity of the sink (10) and inhibiting back-flow of
the liquid sample.
[0117] In a preferred embodiment, the inner surface (communicating
with the porous carrier) of the second non-porous surface (11) also
comprises a mobilization zone (16) together with the mobilization
zone (3) on the first non-porous surface (1) or instead of the zone
on the first surface. The same is possible between the sample
reception zone (15) on the second non-porous surface (11) and the
sample reception zone (8) on first non-porous surface (1). It is
additionally preferred that when the second non-porous surface
bears the mobilization zone, the second non-porous surface is above
the first non-porous surface during operation of the device, such
that gravity also aids in mobilizing the labeled reagent into the
solution.
[0118] In a preferred embodiment, the test device as shown in
Drawing 1 is assembled into a housing made of various materials
such as thermoformed or molded plastic materials. Such housing
materials and designs are well known to those skilled in art.
[0119] Another aspect of the invention concerns a reservoir
structure for dip-stick tests, wherein the test strip comprises two
parallel solid layers on each wide side of the strip sandwiching
the porous carrier and the said layers, that, by coming together,
form a sample reservoir in the sampling-end of the said strip.
[0120] As shown in Drawing 2A, both non-porous surfaces (1 and 11)
are attached to the each other or are the continuation of the each
other or overlap each other, at the proximal-end (6) of the device
subject to the invention, therefore creating a reservoir (17) for
the sample. The reservoir (17) can collect the required amount of
the liquid sample instantly, thus eliminating the need to dip the
device for a measured time into a liquid sample. The reservoir (17)
can be designed in any of a number of shapes, from bulbous to
angular; some alternatives are shown in un-scaled side views in
Drawings 2B, 2C, 2D and 2E. The reservoir (17) can also comprise
substances similar to that of the optional sample reception zone
(8).
[0121] In a preferred embodiment, the reservoir (17) comprises an
orifice (18) on either of the non-porous solid surfaces, which can
provide means for sample dispensing and ventilation to release the
liquid sample from the reservoir (17).
[0122] Another aspect concerns a device optimized for midstream
sample collection, wherein the test device comprises an enlarged
surface with a sampling orifice. The said enlarged surface protects
the dry parts of the device from undesired wetting during the
mid-stream sampling and providing means for improved handling.
[0123] As shown in Drawing 3, as an assembled device in Drawing 3A
and as an unassembled device in 3B, the second non-porous surface
is sized wider and longer (19) than the first non-porous surface
(1), thus providing improved handling and protection from undesired
wetting in mid-stream sample application. In such a device design,
the sample is taken to the sample reception zone (8) through the
orifice (20) on the enlarged non-porous surface (19).
[0124] In a preferred embodiment, the enlarged second non-porous
surface (19) can be attached to the optional attachment zone (21)
in the distal end of the first non-porous surface and to the porous
carrier (2) on the upper surface of the porous carrier. An optional
spacer (22) can be used to maintain an even distance between the
surfaces through the sample reception zone (8).
[0125] In a preferred embodiment, a third layer can be attached to
a device with normal sized non-porous surfaces (Drawing 1 and 2) in
the same manner.
[0126] Another aspect, concerns a housing, wherein at least one
element of the housing is also part of the non-porous solid surface
comprising the mobilization zone. In this aspect, as in the first
aspect, the said solid surface is in liquid contact with the porous
carrier.
[0127] The housing subject to invention can be made of a single
piece or multiple pieces. The housing can be in any practical size
and shape and can be made of any non-porous and non-water absorbing
solid material with low affinity to the labeled reagent including
the known housings commonly produced from water impermeable
thermoform or thermoplastic materials. However, it is further
possible to have a porous housing made non-porous in critical areas
such that the porous nature of the housing can advantageously be
used to absorb the excess applied sample or can be used as a
sink.
[0128] The device with housing shown in see-through side view in
Drawing 4A is composed of a top (23) and a bottom (24) housing
parts and a porous carrier (2). The inner surfaces (25 and 26) of
both or either parts comprise the mobilization zone (3). The liquid
sample is taken into the device through the intake orifice (27).
The excess of liquid taken into the device is drained out through
the optional drain orifice (28). The inner surfaces (25 and 26)
between the intake orifice (27) and the mobilization zone (3) are
the sample reception zone. There can be an optional evaporation
grill (29) on the distal bottom end of the device, to assist the
sink function. The porous media can, optionally be folded over
itself (30), to create a shorter device design, without decreasing
the sink capacity.
[0129] Drawing 413 shows the same device from the top and Drawing
4C shows the same device from the bottom. Drawing 4D shows in
see-through side view, the same housing without optional drain
orifice (28), and other optional components, e.g. folded porous
carrier (30) or evaporation grill (29). Also shown in Drawing 4D is
an alternative placement of the mobilization zone (3).
[0130] Another aspect concerns accessory devices and shell housings
wherein the device in immediate housing or in other words, a
primary housing, subject to the invention can also be contained in
accessory devices or shell housings, in other words, secondary
housings.
[0131] Drawing 5 shows examples of such an adaptation, wherein the
device with an immediate housing (Drawing 4) is contained in a
molded plastic shell for home use (32) or on/in a dip stick
extender (34) or in a bench top device (33) or used as it is (31).
Adaptation can be achieved by a simple receptacle slot (35) in the
accessory device as an example shown (35) in cut-away view from
inside of any of these accessory devices.
[0132] For all aforementioned devices, it will be apparent to those
skilled in art that, to enable signal observation, either or both
sides of the signal producing zones should be transparent or should
comprise means to enable the observation of the test result e.g. a
window, and all above mentioned solid parts, non-porous or porous
can be attached to each other by known techniques such as
ultrasonic welding, hot laminating, adhesives or mechanical
means.
Examples
1. Non-Porous Solid Surface Material:
[0133] The non-porous solid surface can be chosen from wide variety
of materials e.g. polystyrene, polyethylene, polycarbonate,
polypropylene, or polyester, or a combination, available from
numerous commercial sources e.g. General Electric, USA in sheets in
a variety of forms and thicknesses.
2. Porous Carrier:
[0134] The porous carrier can be chosen from a wide variety of
chromatographic materials e.g. cellulose, nitrocellulose or porous
plastic, available from numerous commercial sources e.g. Whatman,
UK; Schleicher & Schuell, Germany and Porex, USA. Non-limiting
examples include hydrophilic porous polyethylene membrane, for
example with pore size between 2 and 8 micrometer, or lateral flow
test nitrocellulose membrane.
3. Label Material:
[0135] The label can be chosen from wide variety of materials
commonly known to those skilled in art, e.g. metals, dyes,
polymers, enzymes and fluorophores. Use of colloidal gold and latex
polymer particles are the most common.
[0136] Colloidal gold can be obtained from commercial sources e.g:
BBI, UK. The gold particles can also be produced, for example,
according to U.S. Pat. No. 4,313,734. As an example, colloidal gold
particles of approximately 40 nm in diameter can be used.
[0137] Polymer particles can be obtained from numerous commercial
sources commonly known to those skilled in art e.g. Bangs
Laboratories, USA.
4. Binding Reagents:
[0138] The binding reagents may be antigens, antibodies or other
binding partner arrangements (Avidin-Biotin). Antibodies may be
useful as binding reagents, and can be monoclonal or polyclonal
obtained from numerous commercial sources, commonly known to those
skilled in art. Alternatively, antibodies can be raised to a
desired antigen according to methods well known in the art.
[0139] Labeling and capture antibodies can be obtained, for example
from Medix, Finland (clone codes: 5006 and 6601) for hCG in case of
a pregnancy test.
[0140] A control antibody against the labeling antibody, for
example, can be obtained from Dako, Denmark (code: Z 0109).
[0141] It should be understood that while antibodies (or, for
example, antigen-binding fragments of antibodies) are preferred
binding reagents, the binding reagents can be selected from any
reagent that binds or specifically binds the analyte of interest.
Thus, agents such as nucleic acids, including, but not limited to
aptamers, as well as specific binding partners, e.g., a polypeptide
with a complementary binding domain for a target protein, such as a
leucine zipper or SH2 domain, are candidates for use in devices and
methods as described.
5. Conjugation of Antibody to a Label:
[0142] Conjugation of antibody to a label is a common practice to
those skilled in art described, e.g. in U.S. Pat. No. 4,313,734 or
U.S. Pat. No. 5,571,726. Gold conjugates can be prepared by the
adsorption of antibody to the gold surface within minutes through
charge attraction, hydrophobic attraction, or sulfur binding.
[0143] Polymer conjugates can be prepared by adsorption or covalent
binding of antibodies to the particles according to known methods
e.g. Bangs Laboratories TechNotes 204 (adsorption to microspheres),
205 (covalent coupling), and-303 (lateral flow tests).
[0144] Alternatively pre-conjugated antibodies can be obtained from
numerous commercial sources e.g. Arista, USA, and British Biocell,
UK.
6. Reversible Immobilization of Labeled Antibody to the Non-Porous
Solid Surface:
[0145] Reversible immobilization solution was obtained from British
Biocell, UK, and contained 40 nm colloidal gold coated with anti
hCG antibodies at optical density 10. If desired, solubilizing
agents such as trehalose (Sigma, T-5251) can be added to the
reversible immobilization solution.
[0146] 1 microliter of this solution was dispensed per mm width of
the solid surface, to the designated mobilization zone (3) on
non-porous surface. Drying was done in room temperature under 20%
relative humidity, over night.
[0147] Stabilizing systems or buffers for mobilizable dried
reagents are known (see, e.g., Brooks et al., 1986) and are also
commercially available (e.g., from Surmodics, U.S.A.). The use of
such buffer systems can enhance the stability of labeled reagents
in the dry state and can improve the mobilization characteristics
of such reagents.
[0148] The non-porous surface material used was a transparent
polycarbonate sheet of 200 micrometer thickness. The sheet was cut
into strips of 5 mm width in varying lengths.
7. Immobilization of Unlabeled Antibodies onto the Porous
Carrier:
[0149] Monoclonal antibody solution against the alpha-subunit of
hCG at 5 mg/ml was dispensed at 0.2 microliter/per mm width of the
porous carrier onto the designated immobilization zone (4) in
surfactant-treated porous carrier and dried at room temperature
under 20% relative humidity, overnight.
[0150] Anti-mouse control antibody was also immobilized using the
same protocol.
[0151] The porous member can be treated with surfactants or other
wetting, blocking or optimizing agents if desired.
8. Assembly:
[0152] The transparent non-porous solid surface was cut in the
shape of a rectangular strip of 55 mm.times.5 mm. From the proximal
end, at the 10th millimeter to 55th mm the porous carrier
(5.times.45 mm) was attached using water-resistant double-sided
transparent adhesive tape. A second non-porous solid surface was
cut into a strip of 5.times.60 mm and attached to the porous
carrier, parallel to the first non-porous surface, using adhesive
tape.
[0153] Thus, the structures shown in Drawing 1 were achieved.
[0154] In another assembly format, the transparent non-porous solid
surface was in the shape of a rectangular strip of 121 mm to 5 mm.
From the distal end to the 45th millimeter the porous carrier
similar to the previous assembly was attached using water-resistant
double-sided adhesive tape. Between the 60th and 61st millimeters
the solid surface was bent twice at 90 degrees and was attached to
the porous carrier using water-resistant double-sided adhesive
tape.
[0155] Thus, the structure similar to what is shown in Drawing 2A
was achieved.
[0156] In another assembly format, the transparent non-porous solid
surface was cut in the shape of a rectangular strip of 70 mm by 5
mm. From the proximal end, 25th millimeter to 70th millimeter, the
coated porous carrier was attached using water-resistant
double-sided adhesive tape.
[0157] A second transparent non-porous solid surface, made of the
same material, was cut in a rectangular shape of 120 mm by 50 mm.
From the proximal end, a hole of 3 mm diameter was punched between
the 12th and 15th millimeters on the longitudinal axis. Using
double-sided tape over the porous carrier and on the proximal 5 mm
of the aforementioned strip structure, the strip structure was
attached to the under side of the rectangular sheet, in such a way
that the sample receiving zone of the strip was exposed through the
hole.
[0158] Thus, the structure shown in Drawing 3 was achieved.
9. Assay for hCG:
[0159] To test the function and sensitivity of the assay kits as
disclosed herein, a lyophilized hCG standard (calibrated against
World Health Organization International Standard 75/537) was
diluted in PBS. 20 to 80 microliters of freshly-prepared hCG
dilutions (20IU hCG/l, 100 IU/l, 20000 IU/l, and 20000 IU/l) were
pipetted to the sample reception zone of the device for each of 96
identical devices (total of 384 devices used for hCG dilutions). As
a control, 80 microliters of PBS were pipetted to another 20
devices.
[0160] All devices with PBS and 20 IU hCG/l gave no visible
positive signal.
[0161] All devices with 100 IU hCG/l or higher gave visible
positive signal.
[0162] It is noted that the devices described herein have
sensitivity effective for diagnosis of pregnancy at its earliest
stages.
10. Devices Including a Porous Sample Receiving Element.
[0163] In one aspect, devices embodying the non-porous/porous
hybrid phase assembly described herein can include a porous
sample-receiving element as shown in Drawing 6. Referring to the
thawing, in one embodiment, the assembly includes a non-porous
surface (2) upon which labeled reagent (1) is reversibly
immobilized, adjacent to a porous carrier material (3) that can be
attached to the non-porous surface (2). A porous sample-receiving
material (4), e.g., filter paper, such as Whatman paper or a porous
plastic material or other materials commonly used as sample
application pad materials for lateral flow assay devices, is placed
such that it overlaps or abuts with the non-porous surface. Several
different arrangements with regard to the placement of the
sample-receiving element and the reversibly immobilized reagent are
shown in panels A-D of Drawing 6. As shown in Drawing 6A, the
porous sample-receiving material can overlap the reversibly
immobilized labeled reagent (1). Alternatively, as shown in Drawing
6B, the porous sample-receiving element (4) can overlap both the
reversibly immobilized labeled reagent (1) and the porous carrier
material (3). In another alternative, shown in Drawing 6C, labeled
reagent (1) is reversibly immobilized on the non-porous surface
(2), the porous carrier material (3) overlaps the reversibly
immobilized labeled reagent (1), and the porous sample-receiving
element (4) overlaps the porous carrier material (3). In yet
another alternative, depicted in Drawing 6D, the labeled reagent
(1) is reversibly immobilized on the non-porous surface (2)
adjacent to, and preferably, abutting or in contact with the porous
sample receiving element (4) on one (upstream) side and the porous
carrier material (3) on the other (downstream) side.
11. Quantitative Assay Format:
[0164] In another aspect, a quantitative lateral flow assay is
provided. In this aspect, rather than immobilizing a capture
reagent in a relatively narrow discrete line perpendicular to the
flow of the liquid sample, the non-reversibly immobilized reagent
is applied in a larger detection area, longitudinal and parallel to
the direction of liquid flow. Examples are depicted in Drawing 7,
in which the detection zone, comprising the non-reversibly
immobilized capture reagent is applied essentially to the length of
a porous material, which is one option (it is important to note
that there is no requirement, according to this aspect, that the
detection area span the entire length of the porous material).
[0165] The detection area is coated with non-labeled reagents as
used in the detection zones of qualitative assay devices as
described herein, but preferably in lesser concentration and over a
larger area, i.e., not in a narrow line. The labeled reagents
useful in this assay format are the same as would be used in the
qualitative assay formats.
[0166] The detection area can cover the entire width of the
material, as shown in Drawing 7B, or, alternatively, just a strip
on the material, as shown in Drawing 7A, (a). Where the arrangement
of Drawing 7A is desired, it can be achieved, for example, by
making the remainder of the membrane (b) hydrophobic or non-porous,
which thereby directs the flow of liquid sample into a narrow
strip. This arrangement can increase the flow, and thus, the
immuno-concentration on the longitudinal (strip-like) detection
area.
[0167] The detection arrangement described above and depicted in
Drawings 7A-7D can be incorporated into an assay device comprising
a reversibly immobilized, labeled reagent either as described
herein (reversibly immobilized on a non-porous surface) or as is
known in the art (for example, reversible immobilization on the
chromatographic material, e.g., through use of a glazing material,
e.g., a sugar, protein or polymer, or reversible immobilization in
a material such as a glass fiber filter or porous plastic which
releases particulate label more readily than the chromatographic
material used in the detection area, among others). While
particulate labels are preferred, devices according to this aspect,
like the others described herein, are not limited to the use of
particulate labels. Alternative labels can include, for example,
dyes, such as fluorescent dyes, enzymes, isotopes, etc.
[0168] In use, following the application of liquid sample which
mobilizes reversibly immobilized labeled reagent (e.g., particle
labeled antibody against the analyte), the labeled reagent,
complexed with analyte, if present, migrates onto the detection
area. Labeled reagent-analyte conjugate will be trapped by the
immobilized detection reagent (e.g., a second antibody that binds
the analyte) as the conjugate migrates over or through the
detection zone. The conjugate will tend to bind the immobilized
detection reagent in the proximal region of the detection area
(that region first coming in contact with the liquid sample
carrying the conjugate) first, giving a detectable signal.
Quantitation is achieved based on how far distal to that first
proximal region the signal is detected above background. That is,
as the immobilized reagent in the proximal detection area is
saturated for binding, unbound conjugate will continue along the
detection zone until free immobilized detection reagent is
encountered. The more analyte present, the further along the
detection area (distally) the signal will be detected. This is
depicted schematically, for example, in Drawing 8. As an example,
gold-conjugated anti-hCG, bound to hCG present in the sample, is
trapped by proximal capture antibodies first, and how far distally
the color signal goes will depend purely on the concentration of
the analyte in the sample.
[0169] To aid in interpretation, the detection area for the
quantitative embodiments can be, for example, graduated, as shown
in Drawing 7C, or it can contain a cut-off point as shown in
Drawing 7D. Alternatively, or in addition, the detection area can
be arranged as successive parallel lines of unlabeled binding
reagent, each separated by a small gap, such that the detection
area is graduated, permitting quantitation of analyte based upon
the number of lines showing bound label. Assays in the quantitative
format can, of course, also contain a control reagent zone as used
in other lateral flow assay devices.
[0170] Additional aspects of the devices described herein enhance
the ability to obtain quantitative results. For example, the
reservoir designs discussed herein above can provide a fixed volume
of sample. The fixed volume permits uniform sampling, which aids in
reliable quantitation of analyte. Without a fixed volume of sample,
quantitation can be, at best, semi-quantitative. In contrast,
measurement of a fixed volume permits true quantitation.
[0171] Further, because the non-porous surface does not retain a
dead volume of sample, essentially all of the applied sample (e.g.,
essentially all of a fixed volume of sample) is transferred to the
porous carrier material wherein detection of analyte can occur.
Where there is a dead volume of liquid sample, for example in kits
which comprise a porous sample application element or a porous
conjugate release element, the amount of liquid sample retained,
and thus not measured for analyte, can vary from assay to assay, in
a manner that does not occur when a reservoir as described herein
is employed. It is noted that the space between upper and lower
sheets of non-porous material as employed in several embodiments
described herein is also a fixed volume reservoir, i.e., the device
does not necessarily have to have a reservoir formed by bending one
non-porous surface over on itself as also described.
[0172] In addition, the reservoir designs, coupled with the
non-porous nature of the zone in which the labeled reagent is
temporarily immobilized, also provide a fixed volume for labeled
reagent release, resulting in a fixed concentration of labeled
reagent that does not result in assays that lack such a fixed
volume reservoir. This design further enhances the ability to
obtain reliable quantitative results.
[0173] The reservoir designs described herein provide a further
benefit with regard to quantitative assays in that the filling of
the reservoir, e.g., by dipping vertically into a liquid, does not
necessarily start the assay. In one aspect, for example, the
reservoir can be designed such that the surface of the liquid, when
the reservoir is full and the device is in the vertical position,
is separated from the porous carrier strip and/or from the
reversible immobilization zone. In this aspect, only when the
device is laid horizontal does the liquid sample migrate across the
reversible immobilization zone and enter the porous carrier strip.
Thus, such a reservoir design can permit closer control of the
timing of the assay when this is critical, because the assay does
not begin until the device is laid horizontal.
[0174] It is to be understood that the measurement of analytes in
addition to hCG are encompassed by the devices and methods
described herein, including, but not limited to other hormones
(e.g., luteinizing hormone (LH), follicle stimulating hormone
(FSH), thyroid stimulating hormone (TSR), insulin, pancreatic
glucagon and its fragment peptides, parathyroid hormone,
calcitonin, adrenocorticoid hormones, growth hormone, etc.),
proteins (e.g., viral, fungal or bacterial antigens, antibodies,
alphafetoprotein, carcinoembryonic protein, enzymes, PSA, etc.) and
chemical analytes such as clinical chemistry analytes and drugs
(including, but not limited to drugs of abuse, e.g., cocaine,
heroin, other narcotics, and steroids) and drug metabolites,
toxins, etc. That is, the devices described herein can be adapted
by one of skill in the art, given specific binding reagents for the
particular analyte, for the identification of such analytes in
liquid samples.
[0175] All patents, patent applications, and published references
cited herein are hereby incorporated by reference in their
entirety. While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *