U.S. patent application number 09/924309 was filed with the patent office on 2003-11-06 for analytical test device and method of use.
This patent application is currently assigned to SYNTRON BIORESEARCH, INC.. Invention is credited to Po Lee, Jin.
Application Number | 20030207466 09/924309 |
Document ID | / |
Family ID | 22561015 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207466 |
Kind Code |
A1 |
Po Lee, Jin |
November 6, 2003 |
Analytical test device and method of use
Abstract
The present invention provides an analytical test device for
conducting assays of biological fluids. Methods for carrying out
the assays with the disclosed analytical test device are also
provided.
Inventors: |
Po Lee, Jin; (Poway,
CA) |
Correspondence
Address: |
CAMPBELL & FLORES LLP
4370 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122
US
|
Assignee: |
SYNTRON BIORESEARCH, INC.
|
Family ID: |
22561015 |
Appl. No.: |
09/924309 |
Filed: |
August 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09924309 |
Aug 6, 2001 |
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09649387 |
Aug 28, 2000 |
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09649387 |
Aug 28, 2000 |
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09156770 |
Sep 18, 1998 |
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6140136 |
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Current U.S.
Class: |
436/514 |
Current CPC
Class: |
G01N 33/54366 20130101;
G01N 33/558 20130101; Y10S 435/97 20130101; Y10S 436/807
20130101 |
Class at
Publication: |
436/514 |
International
Class: |
G01N 033/558 |
Claims
I claim:
1. An analytical test device, comprising: (a) a top having one or
more display ports and, optionally, a receiving port and a vertical
bar; (b) a bottom, optionally having the receiving port, an upper
plane, a slope and a lower plane; (c) a strip comprising an
absorbent membrane, a reaction membrane having one or more reagents
that form a reaction complex with an analyte in a fluid sample and,
optionally, a collecting pad; (d) an absorbent sample pad in a
container; and (e) a stopping means, wherein the absorbent
membrane, the reaction membrane and the collecting pad, when
present, are in fluid flow contact with one another; the strip is
placed on the bottom such that the reaction membrane is visible
through the display port and the absorbent membrane is towards the
receiving port; the top or the bottom have the receiving port or
together the top and the bottom form the receiving port; and in the
assembled top and bottom the absorbent sample pad is slidably
insertable through the receiving port until stopped by the stopping
means, which places the absorbent sample pad in fluid flow contact
with the absorbent membrane.
2. The analytical test device of claim 1, further comprising the
top having the optional vertical bar, wherein the vertical bar
deflects the absorbent membrane to follow the contour of the slope
and further comprising the bottom having the optional upper plane,
slope and lower plane.
3. The analytical test device of claim 1, wherein the stopping
means is the container contacting the outside of the assembled top
and bottom.
4. The analytical test device of claim 1, further comprising a
bottom having a horizontal bar, wherein the horizontal bar is
inside the assembled top and bottom and is the stopping means.
5. The analytical test device of claim 4, wherein the stopping
means is the horizontal bar contacting the container.
6. The analytical test device of claim 1, wherein the edge of the
absorbent sample pad that contacts the absorbent membrane is
orthogonal to the absorbent membrane.
7. The analytical test device of claim 1, wherein the one or more
reagents is a diffusively bound labeled reagent bound to the
absorbent membrane at a point spatially separated from the point at
which the absorbent sample pad first contacts the absorbent
membrane.
8. The analytical test device of claim 7, wherein bound to the
reaction membrane is: (a) a non-diffusively bound reagent
complementary to an analyte in the fluid sample at a point beneath
the display port; and (b) a non-diffusively bound control reagent
complementary to the diffusively bound labeled reagent at a point
beneath the display port spatially separated from the point at
which the non-diffusively bound reagent is bound.
9. The analytical test device according to claim 8, further
comprising a first display port and a second display port, wherein
the non-diffusively bound reagent is positioned beneath the first
display port and the non-diffusively bound control reagent is
positioned beneath the second display port.
10. The analytical test device of claim 7, wherein bound to the
reaction membrane at a point beneath the display port is a
non-diffusively bound reagent, wherein the analyte and the
diffusively bound labeled reagent compete for binding to the
non-diffusively bound reagent.
11. A method for detecting an analyte in a fluid sample, comprising
the steps of: (a) adding fluid sample to the absorbent sample pad
of the analytical test device of claim 1; (b) inserting the
absorbent sample pad into the receiving port until the absorbent
sample pad is stopped by the stopping means; (c) detecting the
analyte by observing the reaction complex through the display
port.
12. The method according to claim 11, wherein the reaction complex
is visible.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a novel analytical test device for
analysis of biological fluids such as urine. The invention further
relates to methods for detecting analytes in fluids using the test
device.
BACKGROUND INFORMATION
[0002] The sampling and testing of biological fluids such as urine
for the presence of analytes provide important information
regarding various health-related matters, including pregnancy and
conception.
[0003] In recent years, test devices have undergone nearly
continuous refinement in an effort to simplify and speed the
process of detecting selected ligand in fluids. As a result of this
work, current test devices use an immunoassay for determining
pregnancy or conception. In these devices a reagent, such as an
antibody, specifically reacts with an analyte to form a complex,
which can usually be detected by the unaided eye.
[0004] Current pregnancy test devices assay for hormones associated
with pregnancy, such as, for example, chorionic gonadotrophin
(hereinafter "hCG") since the presence of hCG in urine is usually
an indicator that a woman is pregnant. Such test devices obtain
qualitative results indicating either the presence or absence of
hCG. Typically, a pregnancy immunoassay contains an antibody
directed against hCG. The reaction complex can then be viewed by
the user.
[0005] Conception test devices also assay for hormones associated
with the ovarian cycle, such as, for example, luteinizing hormone
(hereinafter "LH"). LH is present normally in urine but its
concentration increases markedly during ovulation, the time at
which a woman is most likely to conceive. Thus, the probability
that a woman can conceive a child varies directly with LH
concentration. Such test devices obtain semi-quantitative results
regarding the relative concentration of LH in the urine. Typically,
a conception immunoassay contains an antibody directed against LH
and a separate detection antibody.
[0006] In known devices, the fluid to be tested wicks up through an
absorbent membrane that is in fluid flow contact with the reagents
that detect an analyte in a fluid. A major problem with this type
of device is that as the fluid progresses through the membrane, the
front edge of the fluid is uneven. The uneven fluid front will
cause the results to be smeared, produce false negatives and make
the results difficult to detect. These artifacts are called
"leading edge effects." An uneven fluid front can arise for a
variety of reasons, for example, because the sample is not
uniformly applied to the absorbent membrane.
[0007] Therefore, a need exists for a device that does not produce
an uneven fluid front and associated leading edge effects. The
present invention provides a device that eliminates the uneven
fluid front problem and related methods of using such a device.
SUMMARY OF THE INVENTION
[0008] The present invention provides an analytical test device for
analyzing biological fluids, for example, urine. The device has a
top with one or more display ports and, optionally, a receiving
port and a vertical bar. The device also has a bottom and,
optionally, a receiving port, an upper plane, a slope and a lower
plane. The device further has a strip made up of an absorbent
membrane, a reaction membrane with one or more reagents that form a
reaction complex with an analyte in a fluid sample and, optionally,
a collecting pad. The device also has an absorbent sample pad in a
container and a stopping means.
[0009] The absorbent membrane, the reaction membrane and the
collecting pad, when present, are in fluid flow contact with one
another. The strip is placed on the bottom such that the reaction
membrane is visible through the display port and the absorbent
membrane is toward the receiving port. The top or the bottom has
the receiving port or together the top and the bottom form the
receiving port and in the assembled top and bottom the absorbent
sample pad is slidably insertable through the receiving port until
stopped by the stopping means, which places the absorbent sample
pad in fluid flow contact with the absorbent membrane.
[0010] In one embodiment, the analytical test device also has the
top with the optional vertical bar. The vertical bar deflects the
absorbent membrane to follow the contour of the slope. The bottom
has the optional lower plane, upper plane and slope and also a
stopping means.
[0011] The present invention also provides methods for detecting an
analyte in a fluid sample, comprising adding fluid sample to the
absorbent sample pad, inserting the absorbent sample pad into the
receiving port until the absorbent sample pad is stopped by the
stopping means and detecting the analyte by observing the reaction
complex through the display port. In one method the reaction
complex is visible.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a perspective view of the analytical test device
containing an absorbent sample pad in a container.
[0013] FIG. 2 is an exploded view showing the components of the
analytical test device of FIG. 1.
[0014] FIG. 3 is a perspective view of an embodiment of an
analytical test device with an absorbent sample pad inside a
container.
[0015] FIG. 4 is an exploded view showing the components of the
analytical test device of FIG. 3.
[0016] FIG. 5 is an enlarged sectional view along line 3-3 of the
analytical test device of FIG. 3 showing that the absorbent sample
pad is not in fluid flow contact with the absorbent membrane.
[0017] FIG. 6 is an enlarged sectional view along 3-3 of the
analytical test device of FIG. 3 showing that the absorbent sample
pad is in fluid flow contact with the absorbent membrane.
[0018] FIG. 7 is a perspective view of an embodiment of an
analytical test device containing a slidably insertable absorbent
sample pad in a container.
[0019] FIG. 8 is an exploded view showing the components of the
analytical test device of FIG. 7.
[0020] FIG. 9 is an enlarged sectional view along line 3-3 of the
analytical test device of FIG. 7 showing initial placement of the
absorbent sample pad not in fluid flow contact with the absorbent
membrane.
[0021] FIG. 10 is an enlarged sectional view along line 3-3 of the
analytical test device of FIG. 7 showing placement of the absorbent
sample pad in fluid flow contact with the absorbent membrane.
[0022] FIG. 11 is a perspective view of the absorbent and reaction
membranes of the analytical test device employing a sandwich assay
system showing a positive assay result.
[0023] FIG. 12 is a perspective view of the absorbent and reaction
membranes of the analytical test device employing a sandwich assay
system showing a negative assay result.
[0024] FIG. 13 is a perspective view of the absorbent and reaction
membranes of the analytical test device employing a sandwich assay
system showing an inconclusive assay result.
[0025] FIG. 14 is a perspective view of the absorbent and reaction
membranes of an embodiment of the analytical test device employing
a competitive assay system showing a positive assay result.
[0026] FIG. 15 is a perspective view of the absorbent and reaction
membranes of an embodiment of the analytical test device employing
a competitive assay system showing a negative assay result.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention is directed to an analytical test
device that eliminates the uneven fluid front and leading edge
effects associated with prior devices. The analytical test device
ensures that fluid sample comes into fluid flow contact with the
membrane that contains the reagents to detect the analyte, evenly
and at the same height across the membrane, thereby producing an
even fluid front.
[0028] Fluid sample is collected in an absorbent sample pad that is
initially not in fluid flow contact with the absorbent membrane.
Then the absorbent sample pad is contacted to the absorbent
membrane so that the fluid sample comes into fluid flow contact
with the absorbent membrane. The edge of the absorbent sample pad
contacts the absorbent membrane orthogonal to the absorbent
membrane, ensuring that the fluid sample will contact the absorbent
membrane evenly and at the same height across the membrane.
[0029] A further advantage of the analytical test device is that
the user can control when the assay starts. Thus, if desired, the
user can add more than one fluid sample to the absorbent sample pad
before the assay starts. This feature is especially important where
the user only wishes to know whether an analyte is present in more
than one fluid sample. In prior devices, once the fluid sample is
added, the assay proceeds and the reagents are depleted before a
second sample can be added. Thus, the user is not given the time to
test additional samples.
[0030] The analytical test device of the present invention includes
a top and a bottom. The top has a display port that allows the user
to see the results. Preferably, the top will have a first display
port and a second display port that are in close proximity for easy
comparison of results. The bottom has a receiving port for an
absorbent sample pad. Inside the device is a strip formed by an
absorbent membrane, a reaction membrane and, optionally, a
collecting pad. The absorbent membrane and the reaction membrane
contain the reagents to detect an analyte in the fluid sample. When
the top is placed on the bottom in the assembled device, the
absorbent membrane will be positioned on the bottom toward the
receiving port and the reaction membrane will be positioned on the
bottom underneath the display port.
[0031] The absorbent sample pad is held in a container. In one
embodiment, the absorbent sample pad will be protruding from the
container. Fluid sample is deposited on the absorbent sample pad
and, if desired, more than one fluid sample can be applied or
contacted with the absorbent sample pad before the test is allowed
to proceed. At this point the absorbent sample pad is not in fluid
flow contact with the strip. The absorbent sample pad is then
inserted into the receiving port until the absorbent sample pad is
in fluid flow contact with the absorbent membrane. The analytical
test device also has a stopping means that prevents the absorbent
sample pad from reaching far enough into the assembled top and
bottom to by-pass the absorbent membrane and contact the reaction
membrane.
[0032] The bottom of the analytical test device of the present
invention can also be divided into an upper plane and a lower plane
by a slope. As used herein, the term "slope" refers to a surface or
plane of the bottom of the device that is at such an angle in
relation to the other planes of the bottom that it slows the rate
of fluid sample wicking through the absorbent membrane. Thus, the
slope prevents an excess amount of fluid sample migrating through
the absorbent membrane, which would otherwise and compromise the
assay.
[0033] When the slope is present, the absorbent membrane is along
the lower plane and the slope and the reaction membrane is on the
upper plane. Also, the analytical test device has a vertical bar
extending downward from the top. As used herein, the term "vertical
bar" refers to a part of the top of the device extending downward
from the top at a point just in front of the slope that holds or
deflects the absorbent membrane to follow the contour of the slope.
Optionally, the vertical bar can extend downward far enough to only
allow fluid sample to proceed further into the device by wicking
through the absorbent membrane.
[0034] In one embodiment, the absorbent sample pad is inside a
container that can be slidably inserted through the receiving port
until it comes into fluid flow contact with the absorbent membrane.
In a further embodiment, the absorbent sample pad is in a container
separate from the assembled top and bottom. The absorbent sample
pad protrudes from the container and can be inserted into the
receiving port. In another embodiment, the absorbent sample pad is
in a container that is disposed between the assembled top and
bottom of the analytical test device, and partially protruding
therefrom. A knob attached to the container allows the user to
slide the container inside the device until the stopping means
stops the container and the absorbent sample pad contacts the
absorbent membrane.
[0035] An amount of fluid sample, such as urine, which is suspected
of containing, for example hCG, is applied by dropping or pouring
(from a pipette or other container) the sample onto the absorbent
sample pad. Alternatively, in one embodiment, the absorbent sample
pad can be dipped into a fluid sample. In addition, the absorbent
sample pad may be wetted by carefully holding in a stream of urine.
The absorbent sample pad is then contacted with the absorbent
membrane by inserting the absorbent sample pad through the
receiving port until the container is stopped by the stopping
means.
[0036] The fluid sample then proceeds by capillary action (i.e.,
wicking) through the absorbent membrane. Bound evenly and at the
same height extending from side to side of the absorbent membrane,
at a downstream point spatially separated from the point where the
absorbent sample pad contacts the absorbent membrane and downstream
from the vertical bar is a diffusively bound labeled reagent. The
spatial separation ensures that the fluid sample wicks up through
the absorbent membrane to contact the diffusively bound labeled
reagent. Thus, the diffusively bound labeled reagent stays within
the absorbent membrane and does not migrate into the fluid sample,
which would otherwise destroy the performance of the assay.
[0037] If analyte is present in the fluid sample it will complex
with the diffusively bound labeled reagent. Fluid sample containing
either diffusively bound labeled reagent alone or a diffusively
bound labeled reagent/analyte complex (i.e., "first complex") will
move by wicking up to a "test region" in the reaction membrane.
Bound evenly and at the same height extending from side to side of
the reaction membrane in the test region is a non-diffusively bound
reagent capable of complexing the first complex. The
non-diffusively bound reagent may be non-diffusively bound to the
membrane by means known in the art, including covalent bonding or
attachment to an insoluble protein-coated surface (see, for
example, U.S. Pat. No. 4,200,690, which is incorporated herein by
reference). Preferably, the non-diffusively bound reagent will be
bound in a bar shape extending from side to side of the reaction
membrane in a manner similar to the attachment of the diffusively
bound labeled reagent to the absorbent membrane.
[0038] If a first complex is present in the fluid sample, the
analyte portion of the first complex will bind to the
non-diffusively bound reagent creating a detectable signal,
preferably a visible straight line or bar extending from side to
side of the reaction membrane at the first display port. As a
control, fluid sample will continue its migration past the first
display port to a "control region" portion of the reaction
membrane.
[0039] The control region is so called because bound evenly and at
the same height extending from side to side of the reaction
membrane is a non-diffusively bound control reagent. The
diffusively bound labeled reagent, complexed with analyte or not,
will bind to the non-diffusively bound control reagent present in
the control region. This binding will create a detectable signal,
preferably a visible straight line or bar extending from side to
side of the reaction membrane at the second display port. For ease
of comparison, the shape and orientation of the non-diffusively
bound control reagent should be similar to the shape and
orientation of the non-diffusively bound reagent. In addition, the
non-diffusively bound control reagent may be non-diffusively bound
in the manner similar to that of the non-diffusively bound reagent,
described above. When the top is aligned with the bottom in the
assembled device the diffusively bound labeled reagent will be
bound so as to be spatially separated from the point where the
absorbent sample pad contacts the absorbent membrane, the
non-diffusively bound reagent will be bound beneath first display
port and the non-diffusively bound control reagent will be bound
beneath second display port.
[0040] The results can then be interpreted. If a signal appears at
the first display port and a signal appears at the second display
port, the test is positive for the presence of analyte. If there is
no signal detectable at the first display port compared to the
signal at the second display port, the test is negative for the
presence of analyte. If there is no signal at the second display
port, the user is alerted that the test results are compromised and
that the test should be repeated.
[0041] Alternatively, the analytical test device can employ a
competitive assay system. A diffusively bound labeled reagent
capable of competing with the analyte for a non-diffusively bound
reagent is applied evenly and at the same height extending from
side to side of the absorbent membrane. It is applied at a
downstream point spatially separated from the point where the
absorbent sample pad contacts the absorbent membrane and downstream
from the vertical bar. The diffusively bound labeled reagent here
does not complex with the analyte. A non-diffusively bound reagent
that can bind either the analyte or the diffusively bound reagent
is applied evenly and at the same height extending from side to
side of the reaction membrane at a point beneath the display port.
In this embodiment of the analytical test device, the top need only
have one display port, as no comparison is required. However, if
desired, a second display port may be incorporated to demonstrate
the integrity of the reagents and device. In this embodiment, the
presence of a visible signal, such as a straight line or bar across
the reaction membrane in the display port is a validation of the
assay and indicates that no analyte is present in the fluid sample.
No signal in the display port indicates the presence of analyte in
the fluid sample.
[0042] The analytical test device allows the assay to be performed
in simple steps. The user only has to apply the fluid sample, slide
the absorbent sample pad into the receiving port to contact the
absorbent sample pad with the absorbent membrane and then observe
the results.
[0043] The analytical test device is assembled by placing the
absorbent membrane and the reaction membrane on the bottom and then
the top is then placed on the bottom to ensure a tight fit. One
skilled in the art would understand that any suitable means to
ensure a tight fit between the top and bottom can be used. For
example, the top can be fitted on the bottom with snaps or glue. In
addition, once the absorbent sample pad is inserted into the
receiving port, the device becomes substantially fluid-tight and
evaporation or leakage of the fluid sample is minimized. To further
prevent such leakage, the top and bottom are preferably made of a
nonporous plastic, such as the commercially available plastic "ABS"
supplied by the Monsanto Company of St. Louis, Mo.
[0044] Several features of the analytical test device assist in
avoiding compromised results. First, simultaneous migration of an
excessive amount of fluid sample is limited by requiring the fluid
sample to ascend the slope from the absorbent membrane to the
reaction membrane. Second, the spatial separation of the
diffusively bound labeled reagent on the absorbent membrane from
the point at which the fluid sample first contacts the absorbent
membrane ensures that the assay is not compromised by reagent
leaking out into the fluid sample. Third, the impact of leading
edge effects caused by depletion of bound reagents or labels are
minimized by physically separating the test and control regions, by
applying the fluid sample evenly and at the same height across the
absorbent membrane and by using the presence of a reaction, rather
than the appearance of a given shape (such as a "+" or a "-") to
signify a positive or negative result. In addition, the entire
process is simplified by having the label bound to a substrate
placed within the analytical test device, thus avoiding any need
for the user to add a reagent or mix it with fluid sample.
[0045] As described above, the present invention is also ideally
suited to the application of more than one fluid sample to a single
analytical test device. In prior devices, such as the one described
in May et al., U.S. Pat. No. 5,602,040, issued Feb. 11, 1997, the
device (hereinafter the '040 device) is typically held in a stream
of urine. The protruding sample aperture in the '040 device is in
contact with the dry porous carrier inside the hollow casing of the
device. Thus, the test proceeds immediately upon application of the
sample. Therefore, the '040 device is suited for the testing of
only one sample at a time because once the first sample is applied,
the test proceeds, thereby depleting the reagents.
[0046] Moreover, since the user of the '040 device typically holds
the protruding bibulous member in a urine stream, the sample is
unevenly applied to the membrane. Thus, the '040 device suffers
from leading edge effects created by an uneven solvent front along
the membrane. Accordingly, reagents and bound labels could be
depleted and the results could be misleading. In contrast, the user
of the present analytical test device is assured that the solvent
front formed by the fluid sample will be uniform since the fluid
sample is applied evenly and at the same height across the
absorbent membrane. Therefore, the present analytical test device
provides more reliable results.
[0047] As used herein, the terms "ligand" and "control reagent" are
used interchangeably and refer to a molecule to which another
molecule will bind. A ligand or control reagent can be human,
animal or plant in origin. For purposes of this invention, these
may include LH, hCG or other naturally occurring biological control
reagents in serum, plasma, urine or other fluids and will
preferably include analytes. Specifically, it will be appreciated
by those skilled in the art that the control reagent or analyte may
be a protein, peptide, amino acid, nucleic acid, sugar, hormone,
steroid, vitamin, toxin in the sample fluid, pathogenic
microorganism and metabolites from analogs of such analytes, or
antibodies to these substances. They may also include hormones,
haptens, immunoglobulin, polynucleotides, drugs and infectious
disease agents (bacterial or viral) such as Streptoccus, Neisseria,
Chlamydia, Gonorrhea and HIV.
[0048] As used herein, the term "reagent" refers to receptor
molecules which bind to a control reagent. Reagents may, in this
context, include any naturally occurring or synthetic biological
receptor and will preferably include antibodies. The antibodies may
be polyclonal or monoclonal. For simplicity, the terms antibody and
analyte will be used interchangeably with reagent and control
reagent, respectively, throughout this disclosure. It will be
appreciated by those skilled in the art, however, that the
invention is not limited to use with analytes and antibodies.
[0049] Ligand-reagent pairs useful in the present invention include
specific binding pairs such as antigens and antibodies, or
fragments of antibodies, both polyclonal and monoclonal, lectins
and carbohydrates, hormones and hormone receptors, enzymes and
enzyme substrates, biotin and avidin, vitamins and vitamin binding
proteins, complementary polynucleotide sequences, drugs and
receptors, enzymes and inhibitors, apoproteins and cofactors,
growth factors and receptors, and the like. Biotin and avidin
derivatives may also be used, including biotin analogs/avidin,
biotin/streptavidin, and biotin analogs/streptavidin. Members of
the complex may be "engineered," that is, made by synthetic means.
Such techniques are well known in the art, and include techniques
for chimeric and humanized antibodies and fragments thereof,
synthetic peptides, and synthetic RNA and DNA oligonucleotides.
[0050] Any known reagent can be used in any known format such as,
for example, sandwich and competitive binding formats, to
specifically detect an analyte in a fluid sample. Examples of such
reagents are those disclosed in: H. J. Friesen, U.S. Pat. No.
4,861,711, issued Aug. 29, 1989; J. Bunting, U.S. Pat. No.
4,271,140, issued Jun. 2, 1981; May et al., U.S. Pat. No.
5,622,871, issued Apr. 22, 1997; May et al., U.S. Pat. No.
5,656,503, issued Aug. 12, 1997; May et al., U.S. Pat. No.
5,602,040, issued Feb. 11, 1997; and R. Rosenstein, U.S. Pat. No.
5,591,645, issued Jan. 7, 1997 (each of which is incorporated by
reference herein). Such reagents can form a detectable complex with
such control reagents as listed above.
[0051] Preferred reagents include antibodies to a hormone or
infectious disease agent. Preferred antibodies include anti-hCG
antibodies and anti-human LH antibodies, especially of the IgG
class, and even more so murine monoclonal antibodies and especially
those that have been affinity purified. One skilled in the art
would recognize, however, that polyclonal antibodies can also be
used in the present invention.
[0052] By the term "non-diffusively bound" is meant covalent or
non-covalent attachment to the absorbent membrane or reaction
membrane such that the advancing fluid sample does not cause
movement of the non-diffusively bound reagent or non-diffusively
bound control reagent from the place it is applied on these
membranes. Conversely, by the term "diffusively bound" is meant
placement on the absorbent membrane or reaction membrane such that
the advancing fluid sample does cause movement of the diffusively
bound labeled reagent from the place it is applied on these
membranes.
[0053] As used herein, the term "fluid sample" refers to a material
suspected of containing an analyte. The fluid sample can be used
directly as obtained, for example, from any biological source. The
fluid sample can also be obtained from an organism and the relevant
portion extracted or dissolved into a solution. For example, the
fluid sample can be a physiological fluid, such as, for example,
saliva, ocular lens fluid, cerebral spinal fluid, sweat, blood,
pus, mucus, serum, urine, milk, ascites fluid, synovial fluid,
peritoneal fluid, amniotic fluid, and the like. In addition, the
fluid sample fluid can be extracted from throat swabs, feces, or
from other biological samples.
[0054] As used herein, the term "label" refers to a molecule that
directly or indirectly mediates the production of a signal (such as
a color change) which is used in assay procedures to indicate the
presence or absence of analyte in a fluid sample. Labels may
include enzymes, fluorescent molecules and will preferably include
metal sols. Labels include colored latex spheres and colloidal
metals. Labels include those disclosed by D. Yost et al., U.S. Pat.
No. 4,954,452, issued Sep. 4, 1990; J. Leuvering, U.S. Pat. No.
4,313,734, issued Feb. 2, 1982; P. Tarcha et al., U.S. Pat. No.
5,252,459, issued Oct. 12, 1993; T. Gribnau et al., U.S. Pat. No.
4,373,932, issued Feb. 15, 1983; and R. Campbell, U.S. Pat. No.
4,703,013, issued Oct. 27, 1987 (each of which is incorporated by
reference herein).
[0055] Alternatively, the label can be colored latex particles (see
Campbell, U.S. Pat. No. 4,703,017, issued Oct. 27, 1987, which is
incorporated by reference herein) or can be an enzyme that has
reacted with a colorless substrate to give a colored product and is
encapsulated, for example, in a liposome (see E. Soini, U.S. Pat.
No. 5,518,883, issued May 21, 1996, which is incorporated herein by
reference). The label may also be an inducible property of the
particles, such as colorable latex particles (see Gribnau et al.,
U.S. Pat. No. 4,373,932, issued Feb. 15, 1983, and de Jaeger et
al., U.S. Pat. No. 4,837,168, issued Jun. 6, 1989, both of which
are incorporated by reference herein).
[0056] Alternatively, the label can be fluorescent molecules, such
as the rhodamine, fluorescein, or umbelliferone series, employed by
themselves or with a quencher molecule (see, for example, Ullman et
al., U.S. Pat. No. 3,996,345, issued Dec. 7, 1976 and Tom et al.,
U.S. Pat. No. 4,366,241, issued Dec. 28, 1982, both of which are
incorporated herein by reference). Chemiluminescent molecules, such
as luminol, luciferin, lucigenin, or oxalyl chloride can be used as
a signal means (see, for example, Maier, U.S. Pat. No. 4,104,029,
issued Aug. 1, 1978, which is incorporated herein by reference).
Finally, enzymatic systems that react with a colorless substrate to
give a colored product, such as horseradish peroxidase, alkaline
phosphatase, indoxyl phosphate and aminoethylcarbazole may be used
as labels.
[0057] Diffusively bound complexed antibodies can be impregnated
into the assay and within the absorbent membrane. The diffusively
bound complexed antibodies are located upstream from a zone on the
reaction membrane of the assay that contains immobilized
antibodies. The diffusively bound label complexed antibodies bind
to the analyte and are carried to the zone containing the
immobilized antibodies where a sandwich antibody-hormone complex is
formed and detected. However, if desired, both the diffusively
bound and non-diffusively bound antibodies may be disposed on the
same membrane.
[0058] In one embodiment, the diffusively bound labeled reagent
will be labeled according to means known in the art. For purposes
of producing a clearly visible reaction, labels of metal sols are
preferred, with labels of colloidal gold or selenium being most
preferred. An example of a suitable product is colloidal gold.
These colloidal metals will produce colored reactions without
addition of further reagents. However, fluorescent compounds (such
as fluorescein and phycobiliprotein) and enzymes (such as those
identified in U.S. Pat. No. 4,275,149, which is incorporated by
reference herein), may also be used. To maximize contact of analyte
with labeled reagent, the latter should be bound to the membrane
across its face; i.e., from one side to the other. In addition, the
labeled reagent should be bound to the membrane so that it is
spatially separated from the absorbent sample pad when the
absorbent sample pad is contacted with the absorbent membrane (see
bar 125 shown in phantom on FIGS. 9, 10, 11, 12 and 13).
[0059] As used herein, the term "metal label" includes labels of
metal sols; i.e., metal or metal compounds such as metal oxides,
metal hydroxides, metal salts or polymer nuclei coated with a metal
or metal compound. These metal labels may include dry forms of any
of the above-named metals or metal compounds, and will preferably
include colloidal gold in dry form. For example, the metal label
can be composed of a metal sol, a selenium sol or a carbon sol
(see, for example, Leuvering et al., U.S. Pat. No. 4,313,734,
issued Feb. 2, 1982; Moremans et al., U.S. Pat. No. 4,775,636,
issued Oct. 4, 1988; Yost et al., U.S. Pat. No. 4,954,452, issued
Sep. 4, 1990; and Kang et al., U.S. Pat. No. 5,559,041, issued Sep.
24, 1996, each of which is incorporated by reference herein).
[0060] Depending on the context in which it is used, "reaction
complex" or "complex" shall mean an a complex of analyte and
diffusively bound labeled reagent complex first produced in the
assay ("first complex"), a complex of the first complex and the
non-diffusively bound reagent produced second in the assay ("second
complex"), or the second complex and the non-diffusively bound
control reagent produced third in the assay ("third complex"). In
another embodiment of the analytical test device, "complex" shall
mean a complex of analyte and non-diffusively bound reagent or a
complex of the diffusively bound labeled reagent and
non-diffusively bound reagent. The complex can be made up of a
ligand-reagent pair that has spatial and/or polar features which
permit it to bind specifically to each other. By "first member" of
the complex is meant the member bound, covalently or
non-covalently, at least to a sub-population of the particles. The
"second member" of a complex refers to the corresponding binding
member of the pair non-diffusively bound in the control region.
[0061] The term "display port" refers to any means whereby visual
access to the reaction membrane can be gained. In one embodiment, a
display port will be an aperture on the top positioned over the
reaction membrane. The term also encompasses all or any part of the
top that is made of a clear or transparent material so that the
results can be seen. Thus, if the entire top is made of clear or
transparent material, then the entire top can be referred to as the
display port. In addition, there may be a clear or transparent
membrane placed between the reaction membrane and the aperture of
the display port so as to prevent evaporation of fluid sample, to
prevent fluid sample or other fluid from entering the display port,
or to prevent disruption of the membranes by touching, all of which
can affect the performance of the assay.
[0062] The term "absorbent sample pad" refers to any material
capable of containing the fluid sample and when contacted with the
absorbent membrane produces an even fluid front along the absorbent
membrane. For example, the edge of the absorbent sample pad that
comes into contact with the absorbent membrane should be orthogonal
to the absorbent membrane, ensuring that fluid sample will be
applied evenly and at the same height across the membrane, thereby
producing an even fluid front. The absorbent sample pad can be made
from any bibulous, porous or fibrous material capable of absorbing
fluid rapidly. The porosity of the material can be unidirectional
or multidirectional. Porous plastic materials, such a
polypropylene, polyethylene (preferably of high molecular weight),
polyvinylidene fluoride, ethylene vinylacetate, acrylonitrile and
polytetrafluoroethylene can be used. It can be advantageous to
pre-treat the pad with a surface-active agent during manufacture to
reduce any inherent hydrophobicity in the pad and, therefore,
enhance its ability to take up and deliver a fluid sample rapidly
and efficiently. The absorbent sample pad can also be made from
paper or other cellulosic materials, such as nitro-cellulose.
Preferably the material comprising the absorbent sample pad should
be chosen such that the pad can be saturated with fluid sample
within a matter of seconds. Also preferably, the material remains
robust when moist, and for this reason, paper and similar materials
are less preferred. In addition, by preferably providing a tight
fit between the absorbent sample pad and the assembled top and
bottom, the application of fluid sample to the absorbent sample pad
will not result in fluid sample entering the device directly and
by-passing the absorbent sample pad.
[0063] Also, the absorbent sample pad of the present invention is
movable or "slidably insertable." As used herein, the term
"slidably insertable" refers to the ability of the absorbent sample
pad to be moved inside the device. In one embodiment of the device,
the absorbent sample pad is disposed between the top and the bottom
and can be slid inside the assembled top and bottom by the user
(see FIG. 1). The absorbent sample pad will be in an container
disposed between the top and bottom in the assembled device. The
container is preferably made out of the same non-porous material as
the top and bottom. In another embodiment of the device, the
absorbent sample pad will be initially separate from the assembled
top and bottom but insertable into the receiving port. The
absorbent sample pad will preferably be complementary in shape for
the receiving port such that when the absorbent sample pad is
inserted into the receiving port and contacts the absorbent
membrane, the analytical test device becomes substantially
fluid-tight.
[0064] As used herein, the term "receiving port" refers to an
aperture in the top, or the bottom or optionally an aperture in the
top and the bottom together that allows the absorbent sample pad to
enter the assembled top and bottom and contact the absorbent
membrane. Preferably, the receiving port will be complementary in
size to the absorbent sample pad to ensure a tight fit.
[0065] As used herein the term "container" refers to a material
capable of supporting the absorbent sample pad. The container is
preferably made of any suitable material that prevents the user
from coming into direct contact with the fluid sample, which would
otherwise contaminate the assay and make its use unpleasant.
Specifically, the container in one embodiment of the invention is
made of a non-porous material such as the commercially available
"ABS" plastic (Monsanto Co., St. Louis, Mo.) comprising a top and a
bottom (see FIGS. 1, 2, 3 and 4). It is also understood that the
container could be in the shape of a stick, rod or tongue
depressor.
[0066] Also, as used herein the term "stopping means" refers to a
structure capable of stopping the movement of the container so that
the absorbent sample pad is in direct fluid flow contact with the
absorbent membrane but does not allow the absorbent sample pad to
come into direct fluid flow contact with the reaction membrane. In
one embodiment, the assembled top and bottom are the stopping means
for the container. In another embodiment a horizontal bar on the
bottom is the stopping means for the container.
[0067] The terms "absorbent membrane" and "reaction membrane" refer
to any bibulous, porous or fibrous material capable of rapidly
absorbing an aqueous fluid and conducting the fluid via capillary
attraction. Suitable materials are described, for example, in H. J.
Friesen, U.S. Pat. No. 4,861,711, issued Aug. 29, 1989; J. Bunting,
U.S. Pat. No. 4,271,140, issued Jun. 2, 1981; May et al., U.S. Pat.
No. 5,622,871, issued Apr. 22, 1997; May et al., U.S. Pat. No.
5,656,503, issued Aug. 12, 1997; May et al., U.S. Pat. No.
5,602,040, issued Feb. 11, 1997; and R. Rosenstein, U.S. Pat. No.
5,591,645, issued Jan. 7, 1997 (each of which is incorporated by
reference herein). The preferred material for the absorbent
membrane is a fiberglass product such as that marketed as
"MANNIWEB" or "MANNIGLAS" by Lydall, Inc., Manchester, Conn. Other
suitable materials include polyethylene or nitrocellulose pads and
strips. The means for binding reagents to these materials are
well-known in the art. Preferred porous materials include
nitrocellulose, nylon, paper and silica gel. An advantage of a
nitrocellulose membrane is that an immobilized antibody described
above can be attached without prior chemical treatment. However,
antibodies can be immobilized on other materials such as filter
paper using well known chemical coupling methods such as, for
example, CNBr, carbonyldimidazole or tresyl chloride. The reaction
membrane will preferably be a chromatographic strip coated with
gelatin to enhance the life of the strip and clarity of any visible
reactions produced in the test.
[0068] The present invention also provides methods of using the
analytical test device for detecting an analyte in a fluid sample,
comprising adding fluid sample to the absorbent sample pad of the
analytical test device, contacting the absorbent sample pad with
the absorbent membrane and observing the reaction complex through
the display port. Preferably, the reaction complex will be visible.
Also preferably, there will be a first display port and a second
display port.
[0069] FIG. 1 shows a perspective view of one embodiment of an
analytical test device for performing assays in accord with the
invention. FIG. 1, therefore, depicts a housing 106 of rectangular
shape, although any appropriate shape may be employed. Housing 106
is composed of two parts. The first is top 100, in which one
opening appears, and the second is bottom 112. The opening through
housing top 100 is display port 102 through which the test result
can be viewed and compared. An opening through bottom 112 is
receiving port 118. Absorbent sample pad 108 is in container 126.
Container 126 is preferably composed of two parts, top 128 and
bottom 130.
[0070] FIG. 2 is an exploded view of the analytical test device of
FIG. 1. In this view, it can be seen that bottom 112 contains floor
143, sidewalls 145, snaps 128 and receiving port 118. Contained
within housing 106 will be membranes containing the reagents for
carrying out the assay. In one embodiment there will be absorbent
membrane 124 and reaction membrane 122. Absorbent membrane 124 and
reaction membrane 122 will be in contact with, but need not be
affixed, to each other. In addition, they will be disposed along
floor 143 in the assembled device with absorbent membrane 124
toward receiving port 118 and reaction membrane 122 beneath display
port 102. Absorbent sample pad 108 forms a separate part of the
device which can be contacted with fluid sample, and then inserted
through receiving port 118 to be in fluid flow contact with
absorbent membrane 124.
[0071] FIG. 3 shows a perspective view of another embodiment of the
analytical test device for performing assays in accord with the
invention. To that end housing 207 is composed of top 208, bottom
216, and has a first display port 210 and second display port 212
positioned on top 208, preferably in close proximity for ease of
comparison of results. In addition, housing 208 has receiving port
214. Inside container 202, will be absorbent sample pad 204.
Container 202 is preferably composed of top 200 and bottom 206.
[0072] FIG. 4 is an exploded view of the analytical test device
shown in FIG. 3, and shows the components, top 208, bottom 216,
lower plane 221, upper plane 240, slope 234, sidewalls 223, snaps
218, locating points 232, 242, 228 and 238, fluid gully 224,
receiving port 214, and membranes containing the reagents for
carrying out the assay, as described below. To that end, it can be
seen that contained within housing 207 is absorbent membrane 224,
reaction membrane 222 and collecting pad 220. Absorbent membrane
224 will be placed along lower plane 221 and along slope 234.
Preferably, absorbent membrane 224 will be affixed to a nonporous
substrate 226 such as vinyl coated paper or plastic coated paper by
an adhesive or other suitable means. Substrate 226 will extend
along upper plane 240 to form a surface to which reaction membrane
222 will be affixed. Absorbent membrane 224 and reaction membrane
222 will be in contact with, but need not be affixed, to each
other. In another embodiment, absorbent membrane 224 will be split
at its point of attachment to substrate 226 to extend above and
below the substrate. This configuration serves to regulate the flow
of fluid sample from absorbent membrane 224 to reaction membrane
222. Flow control can also be enhanced by placement of a well or
fluid gully 224 below the junction between membranes 224 and 222.
FIG. 4 also shows the placement of absorbent sample pad 204 in
container 202 between top 200 and bottom 206, which are preferably
held together by snaps 218. In addition, ridges 236 hold absorbent
sample pad 204 in place.
[0073] FIG. 5 is an enlarged sectional view along line 3-3 of the
analytical test device shown in FIG. 3. In this view the
orientation of absorbent sample pad 204 can be seen. Vertical bar
250 can be seen holding absorbent membrane 224 down to follow the
contour of slope 234. Absorbent sample pad 204 is not in fluid flow
contact with absorbent membrane 224 at this point. Thus, the user
has the opportunity to add additional samples or reagents if
desired.
[0074] FIG. 6 is another enlarged sectional view along line 3-3 of
the analytical test device shown in FIG. 3. In this view absorbent
sample pad 204 is in fluid flow contact with absorbent membrane 224
after having been contacted with fluid sample, and then inserted
into receiving port 214. Contact with absorbent membrane 324 will
be indicated by a resistance. In this embodiment of the analytical
test device, assembled top 208 and bottom 216 serve as stopping
means for absorbent sample pad 204. Vertical bar 250 assures that
absorbent membrane 224 is held down so that it can contact
absorbent sample pad 204.
[0075] FIG. 7 shows a perspective view of another embodiment of the
analytical test device. To that end, housing 306 is composed of top
300, bottom 312 and has first display port 304 and second display
port 302 positioned on top 300, preferably in close proximity for
ease of comparison of results. Housing 306 also has knob 314 that
can be slid along groove 316 to stop point 315. Knob 314 is
connected to and controls the movement of container 310 containing
absorbent sample pad 308.
[0076] FIG. 8 is an exploded view of the analytical test device
shown in FIG. 7 and shows the components, bottom 312, sidewalls
336, snaps 328, slope 342, upper plane 346, lower plane 343,
receiving port 318, stopping means 332, locating points 334, 336,
338 and 340, fluid gully 330 and membranes containing the reagents
for carrying out the assay, as described below. To that end it can
be seen that contained within housing 306 is absorbent membrane
324, reaction membrane 322 and collecting pad 320. Absorbent
membrane 324 will be placed along lower plane 343 and along slope
342. Preferably, absorbent membrane 324 will be affixed to a
nonporous substrate 326 such as vinyl coated paper or plastic
coated paper by an adhesive or other suitable means. Substrate 326
will extend along upper plane 346 to form a surface to which
reaction membrane 322 will be affixed. Absorbent membrane 324 and
reaction membrane 322 will be in contact with, but need not be
affixed, to each other. In another embodiment, absorbent membrane
324 will be split at its point of attachment to substrate 326 to
extend above and below the substrate. This configuration serves to
regulate flow of fluid sample from absorbent membrane 324 to
reaction membrane 322. Flow control can also be enhanced by
placement of a well or fluid gully 330 below the junction between
membranes 324 and 322.
[0077] FIG. 9 is an enlarged sectional view along line 3-3 of the
analytical test device shown in FIG. 7. In this view the
orientation of absorbent sample container 310, absorbent sample pad
308 and absorbent membrane 324 can be seen. Vertical bar 350
extends downward from the top and holds absorbent membrane 324
along slope 342. Stopping means 332 can also be seen. Fluid sample
is added to absorbent sample pad 308. It can be seen that absorbent
sample pad 308 is not in fluid flow contact with absorbent membrane
324 at this point. Thus, the user has the opportunity to add
additional samples or reagents if desired.
[0078] FIG. 10 is another enlarged sectional view along line 3-3 of
the analytical test device shown in FIG. 7. It can be seen that
absorbent sample pad 308 is in fluid flow contact with absorbent
membrane 324 at this point, having been stopped by stopping means
332.
[0079] In FIGS. 11, 12 and 13, analyte in the fluid sample, if
present, will bind to the diffusively bound labeled reagent at
point 425 on absorbent membrane 424 to form a first complex. If
desired, further fluid sample may be added up to the maximum
binding capacity of unlabeled reagent and absorbency of the
collecting pad material 420. The first complex and unbound first
reagent, if any, will be carried along with the fluid sample by
capillary action up to the reaction membrane 422. The position of
the non-diffusively bound labeled reagent and first complex, if
any, are indicated as 425 on FIGS. 11, 12 and 13.
[0080] All first complexes carried with the fluid sample will
contact the reaction membrane 422. Fluid sample will pass through
the reaction membrane 422, bringing the first complex, if any, into
contact with the non-diffusively bound reagent to the reaction
membrane 422 to bind therewith to form a second complex composed of
first complex/non-diffusively bound reagent. If second complexes
are formed, they will appear in the test region 427 (FIG. 12).
[0081] Fluid sample will continue its migration by wicking through
reaction membrane 422 past the non-diffusively bound reagent to the
non-diffusively bound control reagent. Diffusively bound labeled
reagent not complexed to analyte will bind with the non-diffusively
bound control reagent to form a third complex. The third complex
will appear as display 421 in the control region (FIGS. 11, 12 and
13). Substrate 426 is also shown.
[0082] Formation of all complexes in the assay method described
herein may be by sandwich or competitive immunoassay techniques
well known to those skilled in the art. Within a predetermined time
period, any second complex present and the third complex will
produce a detectable signal mediated by the label. In one
embodiment, the detectable signal will be a color change. This
color change will signal either a positive (analyte present) or
negative (analyte not present) reaction by providing two separate
reactive regions within those portions of reaction membrane 422
visible to the user through the display ports.
[0083] Other control or comparative result signals may be provided,
including signals that indicate whether an invalid result is
obtained, by similar means known to those skilled in the art (see,
for example, the signal system described in Brown et al., U.S. Pat.
No. 5,160,701, issued Nov. 3, 1992, which is incorporated by
reference herein).
[0084] As shown in FIG. 11, a positive result is indicated when
color changes forming substantially similar shapes (in this
instance, horizontal bars 421 and 423) appear. In contrast, as
shown in FIG. 12, a negative result is indicated when a color
change forming a distinguishable shape only appears, for example,
as a horizontal bar 421. Finally, an inconclusive result is shown
in FIG. 13, where color changes appear in both regions having
substantially dissimilar shapes (i.e., horizontal bar 421 and
smudge 429). In this latter instance, the user would be alerted
that the test should be repeated. While due to the decreased impact
of leading edge effects and similar phenomena in this assay it is
not expected that inconclusive results will be frequent, the assay
requires such a small application of fluid sample that repetition
should not be problematic in most applications.
[0085] FIG. 14 and FIG. 15 show perspective views of the absorbent
and reaction membranes wherein a competitive assay is employed.
FIG. 14 shows a positive assay result, as indicated by no signal in
the display port. FIG. 15 shows a negative assay result, as
indicated by the presence of bar 452 in the display port.
EXAMPLE I
Analytical Test Device with Separate Absorbent Sample Pad
[0086] This example describes the method and analytical test device
depicted in FIG. 3 containing a separate absorbent sample pad that
is inserted into the receiving port.
[0087] This assay procedure is performed using anti-hCG antibody to
form the second complex and an anti-hCG-colloidal gold compound to
form the first complex. A fluid sample can be collected at any time
of day, but for best results, it is best to test the first urine of
the morning because it contains the highest concentration of hCG.
The fluid sample is 1 cubic centimeter (hereinafter "cc") of urine
suspected of containing hCG; the assay is performed at ambient
temperatures not below 15.degree. C. or exceeding 30.degree. C.
Absorbent sample pad 204 is placed into contact with a sample of
urine, either by dipping it into a container containing at least 1
cc of urine, or by holding container 202 containing absorbent
sample pad 204 into a stream of urine. Absorbent sample pad 204 is
then inserted into receiving port 214 so that absorbent sample pad
contacts absorbent membrane 224. Within 3 minutes visible color
changes, (from pure white to pink) through substantially the center
of display ports 210 and 212 on reaction membrane 222, are faintly
visible. By the end of five minutes, a vividly pink bar symbol has
appeared through substantially the center of each display port,
indicating the presence of hCG in the fluid sample. All other
visible areas of the reaction membrane visible through the second
display port remain white, which is the normally visible color of
the reaction membrane. This assay may be performed at any time
after a suspected conception to determine the presence of hCG and,
therefore, pregnancy.
EXAMPLE II
Analytical Test Device with Container Disposed Between Top and
Bottom of Device
[0088] This example describes the method and analytical test device
depicted in FIG. 7 containing a absorbent sample pad and container
disposed between the top and bottom of the device.
[0089] The assay procedure is performed using anti-hCG antibody to
form the second complex and an anti-hCG-colloidal gold compound to
form the first complex. A fluid sample can be collected at any time
of day, but for best results, it is best to test the first urine of
the morning because it contains the highest concentration of hCG.
The fluid sample is at least 1 cubic centimeter (hereinafter "cc")
of urine suspected of containing hCG; the assay is performed at
ambient temperatures not below 15.degree. C. or exceeding
30.degree. C. The 1 cc of urine is added to absorbent sample pad
308 by pipette. The container 310 is slid using knob 314 along
groove 316 to its stop position 315. Within 3 minutes visible color
changes, (from pure white to pink) through substantially the center
of display ports 304 and 302 on reaction membrane 322, are faintly
visible. By the end of five minutes, a vividly pink bar symbol has
appeared through substantially the center of each display port,
indicating the presence of hCG in the sample. All other visible
areas of the reaction membrane visible through the second display
port remain white, which is the normally visible color of the
reaction membrane. This assay may be performed at any time after a
suspected conception to determine the presence of hCG and,
therefore, pregnancy.
[0090] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
* * * * *