Note: Descriptions are shown in the official language in which they were submitted.
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1 MULTI.-~YERED TEST CARD FOR THE DETERMINATION
OF SUBSTANCES IN LIQUIDS
Background of the Invention
This invention relates to a diagnostic test device for
determining the pre~ence of substances in liquid ~edia. More
specifically, it relates to the provision of a multi-layered test
device for determining the presence of immunological, biological,
enzymatic materials or other analytes in liquids especially
biological, industrial or agricultural liquids. Still more
specifically, it relates to the provision of a multi-layered
device for the detection of such substances in a rapid manner and
wherein the results thereof are determinable without a reading
~ instrument.
: General Description of the Prior Art
In the field of diagnostic testing, the art has evolved ~rom
the use of complex radioimmunoassays and enzyme immunoassays to
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the use of single card- ype devices. In general, the art has
~ought to both increase the reada~ility of such de~ices to
eliminate the need for an in~trument and to render the device
readable in a shorter period of time so that one may maXe a
determination of the analyte under field condition. ~he art has
also attempted ~o shorten the number of steps that are required
to conduct t~e test so that elements of simplicity and
convenience are introduced into the test system.
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1 The art-would indeed ~enefi't from a test device which is
portable, develops a visual color change almost immediately upon
the introduction of test sample, and is readable by the naked eye
in a remote location without the need for instrumentation. The
art would benefit ~rom a system which eliminates the need ~or
multiple steps in performing the test and spscifically, from the
eliminatîon of multiple wash steps and long incubation times so
that the time for xeading the test could be shortened and the
convenience could be amplified dramatically.
~ Objecl:s and Advantages of the Invention
It is an object of the invention to provide a test device
which can be employed at the ~ite and in the field and at the
immediate location where the ~ubstances are to be detected.
It is a further object of the invention to provide a
diagnostic kest device which following the addition of the sample
reguires little or no ~urther intervention prior to the
development of the r~action ~or the visualization of the end
result.
It is an additional ob~ect of the invention to provide a
test device having multi-layers and comprised of ~ilter type
planar m~mbranes which incorporate therein binding materials at
appropriate sites such as antibodies and/or antigens to
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1 facil~tate the r~action mechanism. Other obj~cts and advantages
will be apparent from the following specification and the
accompanying drawings.
- General Description of the Invention
The invention provldes a composite structure of porous
materials wherein, through the selection of different pore sizes
; in appropriate areas of the structure and the juxtaposition of
liquid barrier means, a liquid flow path is defined which results
in an unexpected liquid flow force sufficient to cause a
discernible cha~ge in an immunologic complex or in othe~
reactions when appropriate materials are selected.
A commonly encountered problem in the field of immunoassay
occurs ^~hen competi~ive or sandwich type binding assays are
parformed on a solid porous matrix. ~he immunological reaction
in such procedures often does not occur rapidly enough nor
conveniently visibly enough in the absence of several wash steps
or reagent additions. Even with 1:he additional steps, often
: times the reaction is not sufficiently visibl~ within a
convenient time to be useful.
In the typical competitive binding assay, an externally
supplied tagged antigen competes with the antigen of a sample to
react with an externally supplied antibody. tIf the analyte is
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1 an ~ntibody, the appropriate binding partners are chosen.~ Each
antigen molecule whether tagged or present in the sample has an
opportunity to react with the supplied antibody and the extent to
which they do react is a measure of the concentration of the
antigen in the original sample. Such a system lends itself well
to the present invention.
Theoretically, it is p~ssible to perform a competitive assay
by displacing tagged antigen from a tagged antigen/antibody
complex by contacting the complex with antigen from the sample.
Thus, if a tagged antigen/antibody complex were immobilized on a
solid substrate, and that complex contacted with a source of
untagged ant~gen, one might expect a displacement of the tagged
antigen to occur. Unfortunately, when the solid substrate is a
porous material, the mere contact as described above, does not
always result in a displacement which i~ suitable ~or commercial
analy~e detection. Applicant has discovered a structure and
technique which favors in a ~imple one step operation the
displacement of tagged antigen from a tayged antigen/antibody
complex by sample antigen (if the complex is originally
configured and supplied in this ~anner) or the ef~ective
competition between the sample analyte and externally supplied
tagged analyte for the immob~lized capture binding partner with
rapid xevelation of a visual indication of 6uch reaction.
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1 Other ~orms of immunoassay formats such as sandwich
r~actions and competitive binding using a detection system Por
antibodies as the analyte rather than antigens as described, may
also be employed as will be seen in the detailed description of
the invention.
Brie~ly, and without regard to the drawings at this point, a
structure is provided with a combination of various pore sizes
and li~uid ~low barriers so as to produce rapid wicking of a
sample in a pre-determined liquid flow direction to a reaction
lQ site at which the clesired re~ctinn takes place.
In its most generalized form, the present lnvention
contemplates a structure having a least two planar, flat porous
members in intimate contact with each other at their planar
surfaces. The multi layered.device is configured in such a way
that when a small amount, such as a drop or two, of a liquid test
sample is placed onto or into a sample receiving site provided at
the top member, the liquid is forced to travel transversely into
the bottom member owing to the presence of liquid flow barriers
placed or created adjacent to the sample receiving site. The
~ample receiving site may be a hole.in the top member, or may be
the top member itself appropriately shaped or confined in
consideration of the type of test to be performed.
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1 The device is con~igured in such a way as to Eorce the ~low
o~ sample liquid from the sample receiving port or site along a
pre-determined pathway to the bottom member and ultimately to a
reaction site on the bottom or top member depending upon the pre-
S determined path selected. ~he sample liquid thus ultimately
reaches the reaction site not by lateral flow ~rom the sample
receiving -eite, but rather by traversing the sample receiving
site in or on the top member in a transverse flow into the second
member and up into the top member. Usually, the reaction site is
placed on the top member. It may be placed at the bottom member
- under appropriate circumstances.
The characteristics of the members can be varied in such a
way as to create either a rapid pulling action from the top
~ember and/or a pumplng action due to di~ferential wicking
characteristics between the lower member and the top member.
Sample flow in the second member ~hich receives the sample from
the sample receiving site is also restricted by barrier means
which constrain the flow of liqui~ in the second member to a
de~ined space and a direction of that ~low up into the first
member.
; By this action there is facilitated a wide variety of
potential reactions and reaction ~ites. For example, the analyte
o~ the sampl~ dropped onto a top member in a manner in which the
sample is prevented from flowing laterally to any substantial
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1 degree across the top member but $s constrained to flow
transversely, can be ultimately transferrad to a reaction site
located on the top member. This reaction site (whether located
on the top member or on the bottom member) may either have
~ 5 xeagents deposited thereon or may be itself a receiving site for- additional reagents either directly applied or directed through
the same route as the original sample deposition. The present
- invention reguires the placement of a defined sample receiving
site juxtaposed with certain liquid flow barriers and a reaction
site in a pre-determined fashion 50 as to direct the flow of the
sample li~uid ~n the pre-selected fashion.
The versatility of the device of the present invention is
quite wide. For example, the members themselves may be ~ither
hydrophilic or hydrophobic depending on the test characteristics
and the analytes to be tested. For example, by appropriate
choice of structure and composition of members, analyses of
aqueous or non-aqueous samples may be performed. Non-aqueous
~amples containing organic solvenl:-based material may be employed
with hydrophobic members in appropriate circumstances. The
invention al50 permits the use of ~ydrophobic material with
~qu~ous samples should personal pref~rences dictate. ~xamples of
hydrophobic materials are glàss fiber, certain nylons, teflon,
and polyvinyl chloride polymers. Examples of hydrophilic
materials are paper, certain cellulose acetates, polyvinylidene
difluoride, cellulose nitrate, polypropylene, certain microfiber-
glass compositions and the like.
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1 A combination of member~ with di~erent porosities and
binding characteristics with the added capability of being either
hydrophilic or hydrophobic provides in the device a) the ability
to perfo~m on both aqueous and nonaqueous samples, b) the binding
of reactants on one or both of the ~embers and at different ~ites
in the flow path of sample or reagents, and c) through
impermeable barriers or slots, the ability to differentially
control flow rates.
In one form of an immunoassay, the sample is added to the
sample port to hydrate a reactant in the bottom or lower member.
The reactant may be a competing analyte bound to an indicator
~uch as enzyme or colloidal gold. A binding (capture) antibody
is in this case, located at the reaction site. The member may be
protein-binding, if desired to retain the capture anti~ody. The
lower member could be non-protein-binding in the region of the
placement of the indicator conjugate but could be protein-~inding
at the terminus past the reaction site to retain reactants and
prevent them from diffusing bacX to the reaction site.
In another assay, the bottom member could be initially
protein binding and during manufacture have adhered thereto one
or more enzymes for an as5ay to determine the presence of a
substrate in a ample solution. The sample would ~ove through
the lower member contaoting specific enz~mes in a determined
seguential manner to effect the production of a product which
could then ba observed at the reaction site.
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1 It will be obvious to those skilled in the art that numerous
modifications o~ immunoassay procedures can be performed on 'he
device~ These include ~ut are not limited to competitive assays
for small analytes, sandwich assays, and direct detection of
reactants in samples. It will also be obvious that other non~
immunologically based assays, such as substrate and product
detection, use of nucleic acid probes, lectins, or any other
ligand-receptor pairs with various indicator systems can be
performed on the device as well.
To illustrate further, when a liquid sample is applied to
the sample site in or on the upper member, the li~uid will wick
by capillary action into the lower member in either a rapid
fashion or slower fashion depending upon the characteristics of
the member selected. It is preferred under certain circumstances
that the lower member have a pore size su~stantially larger than
the pore size of the upper member to facilitate a pumping action
on the return flow fxom the bottom member to the top member.
Sample is prevented from movin~ laterally from the sample
receiving site because the edge of the sample site has been
r~ndered impermeable by the installed ~arrier means. These
barrier means may be compression sites, slots, discontinuitias in
the material, sonic or heat-generated barriers and the like and
are all within the skill cf the art to construct and place.
1 Once liciuid enters the lower member it ~oves laterally and
transversely until it contacts an impermeable barrier installed
in the lower member and juxtaposed in such a way as to permit
~low of liquid back up into the top member therein to come in
contact with reagents or the like at a reaction site. The
reaction site may contain the elements of an indicator system
useful in detecting the ~nalyte (or substrate) of interest. In
addition, if desired, the reaction site may be located in the
lower member with the top mem~er serving as the remote site for
accumulation of reactants and sample past the reaction site.
For example, in a preferred embodiment, wherein a
competitive immunoassay is performed, the reaction site may
conveniently contain antibodies (capture antibodies) to the
analyte of interest, which said antibodies are covalently bonded
or otherwise attached to the upper member. In this regard, if
desired, a protein-binding type of member may be selected as the
top member to ~acilitate the binding of the antibody. A
conjugate of an indicator molecule 2Ittached to the analyte of
interest is selected. A preferred c:onjugate is the analyte bound
directly to colloidal gold if feasible, or to a carrier molecule
if, for example, the analyte is unable or poorly able to bind to
the gold itself. Colloidal gold is a well-known reagent used in
diagnostic procedures because of its charaoteristic r~ddish
color. As carrier molecules ~here may be employed for example,
natural or synthetic proteins or other macromolecules such as
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1 BSA, poly L-Lysine, polysaccharides, histones, casein, horse-
radish peroxidase and the like. The conjugate may be admixed
with the sample prior to applying the sample to the receiving
~ite or may be installed in the device somewhere in the pre-
det~rmined liquid flow path prior to the reaction site. If the
analyte in the sample is homologous to the analyte adhered to the
colloidal gold, it will compete with the analyte-gold conjug~te
at the reaction site f or the capture antibody. Assuming
appropriate selection of antigen/analyte-gold conjugate
concentration in consideration of the conditions of the assay,
the analyte gold conjugate will lose in the ~ompetition to the
analyte in the sample, and no conjugate will remain bound to the
capture antibody. The reaction can then be traced by the absence
of the accumulation of gold at the reaction site. Thus, a
positive rea~tion is signified by a lack of change of color at
the reaction site (i.e., absence of conjugate). This reaction,
normally termed a competitive binding assay, is typical of the
ones that may be per~ormed with the device of the present
invention. Other formats may be used as well as will be
described hereinafter.
~he following is ~ brief description of the drawings
presented herein from a consideration of which the present
in~ention will be further understood.
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Figure i i~ A cross-sectional view taken across the plane A-
A of Figure 2 of a device of the present invention. Figure 2 is
an embodiment of the device of the invention shown in circular
form. Figure 3 is a device of the invention shown after it has
been acted upon by a test liquid operating sn the device of
Figure 1. Figure 4 is a top view of another embodiment of a
device of the present invention.
Detailed Description of the Drawings
Figure 1 shows a device of the present invention at 10
wherein a bilayered device is shown with two of the four reaction
sites 15 shown i~ the plane of dissection A-A of Figure 2. Upper
layer 20 is provided with a sample port 16 and vent ports 19, and
communicates with member 11 at interface 14. Bounding sample
port 16 are barriers 13. ~ember 20 and member 11 may be the same
or di~ferent material and may have the same or different
porosities andJor the same or different wicking actions. In a
preferred embodiment of the invention, the bottom layer has a
pore size about 5 to 10 times qreater than the pore size of the
upper member and the pore ~ize of the upper member is in the
~0 range of 0.2-0.75 microns.
Wîth regard to the varying pore sizes, it should be noted
that the larqer pore size should not be so large as to provide a
sink for the liquid, ~ince this will frustrate the pumping action
desired. What is highly d~sirable however is a pore size that
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1 re~ults in a "push" of the ~ample through the sample port into
the lower member followed by a "pull" (as will be described
later) of the liquid ~rom the lower member back into the first
member.
Members 11 and 20 are also equipped with barriers 13a and
13b which are generally incorporated during the manufacturing
process as by welding or by incorporation o* slots,
discontinuities or the liXe. Barriers 13b are incorporated to
proYide additional reservoir compartments and are optional
depending on the size of th~ reservoir desired. Barriers 13c are
also optional; in practice, the barrier at 13 is usually
sufficient. There may be some circumstances, however, where the
kinetics o~ the test and the device size are such that it would
be desirable to direct the upward flow of liquid into the top
member at a point somewha~ remote from the sample receiving site.
In such a case, barriers 13c may be provided and interface 14a
rendered impermeable or not, as desired.
Present on member 20 are reaction sites 15 containing
various reactants placed in accordance with and in consideration
o the ultimate test that is to be performed. As shown in Figure
2, these reaction ~ites may ~e ~ore than one in number and may be
for the s~me or di~ferent analytes coming from the same ~ample or
may be for a control. ~he device 10 may also have either
an~ibodies or antigens, but preferably in ~he discussion given
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1 herein, will have antibody 17 attached to the member 10 either ~y
covalent bonding or some other physical or chemlcal attachment.
~here is also provided in member 11, at 18, a conjugate shown as
Au ~ of colloidal gold or some other indicator system
conjugated to an antigen which is specific for or will react with
the antibody at 17. Thus, in its completed composite form ready
for use, there i5 a conjugate 18 of the gold (or other indicator
system) to the antigen incorporated into the flow path of the
device prior to the reaction site 15. Alternatively, the
conjugate may be admixed with ~ample instead o~ being
incorporated into the device, and the sample/conjugate mixture be
allowed to follow the flow path ~o reaction site 15.
Instead of gold, there may b~ employed any other detection
systems used in immunoassays such as enzymes, fluorescing agents,
latex beads, luciferases, chemilumniscent agents and the like
depending upon the best mode of reaction for the given analyte as
;~ determined by individual pre~erences.
In use, a liquid sample is dropped into sampl~ port 16 and
the sample allowed to diffuse into member 11 and mix ~ith
conjugate 18. Lateral flow is prevented by the barriers 13 which
direct flow of the sample in~o the second member. Upon reaching
the second member, the liquid is directed laterally until ~t
reaches a barrier (e.g. 13a) or until it is not permitted to go
any further and is l'pulled" into member 20 in admixture with
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1 conjugate 18. The mixture then migrates to the reaction site 15.
At the reaction site 15, if the sample contains analyte
corresponding to the antigen which is conjugated to the indicator
system, it will compete with the conjugate in reaction with
antibody 17. I~ the sample contains no such antigen, then the
only reactant at site 15 other than antibody 17 will be conjugate
which will then react and show ~ color change. If sample does
contain relevant antigen, the reaction at 15 will be almost
excl usively due to the presence of such antigen because ~he
concentration of con3ugate and antibody 17 selected in
constructing the device has deliberately been adjusted to favor
reaction from the normally encountered concentration of analyte
in the sample. Thus, a positive result is shown as no color
change. For example, in Figure 3 such a result is shown by an
"S" ~or sample being attached to the antibody 17. The
gold/antigen complex does not attach at reaction site 15 and i~
further removed ~rom that area by the flow of liquid continuing
past the reaction site and into the further reaches of members 20
and 11~ If desired, larger amounts of reservoir typs material
can be supplied simply by extendinc3 member 20 laterally or
providing additional reservoir space in member 11 depending upon
how ~uch res~rvoir space is needed as a function of the test that
is bein~ performed. Also, a reservoir of absorbent material may
be placed in ~u~taposition with member 20.
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1 Following ~ddition of the sample, it is desirable though not
necessary in many cases, to add a wash solution to further direct
the sample away from the reaction site so that any indicator
antigen complex adjacent to the reaction site is moved ~urther
away as is evidenced by Figure 3.
If the reaction device as shown in Figure 4 for example, is
equipped with viewing ports so that the mi~ration of the gold or
indicator system away from the reaction site 15 is obscured, then
a change in color ak the reaction site 15 is all that one needs
to observe. Sufficient wash matarial or sample may be applied to
parmit the visualization of the clear spot in the vent that
~ample does contain the suspected analyte.
Although Figure 2 has been shown as being circular in form,
it may be of any con~enient shape such as rectangular, cross-
shaped or the like. In addition, it need not be restricted toone reaction site or sample receiving port but may include a
variety and a plurality of either or both o~ those and may
include reactants for detection of various analytes.
With respect to the material that may be employed as tha
members, particular success has been observed with polyvinylidene
difluoride with pore sizes of about 0.2 to 0.75 microns for the
top member and from 3-5 microns for the lower ~ember. Various
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1 other materials may be employed ~uch as cellulose acetates,
cellulose nitrates, polypropylenes, certain microglass ~iber
compositions and the like.
Although the above description has been given with reference
to a competitive binding assay wherein the analyte to be detected
i6 an antigen and the immunoreagent at reactio~ site 15 is an
antibody and the indicator system at 18 is gold complexed to an
antigen which binds with the antibodyr the present invention is
also suitable for a competitive assay in which the analyte is an
lQ antibody to be determined instead of an antigen. Moreover, the
present in~ention is also suitable for the detection o~ an
antigen or antibody as an analyte wherein a sandwich technique is
employed for the reaction. For example, in an immunoassay for
human chorionic gonadotropin (h~G), gold conjugated to anti-beta
hCG is placed in the bottom member at 18 (Figure 1) and anti-
alpha hCG tcapture Ab) is covalent}y or otherwise attached to the
upper member at ~5. I~ sample cont:ains hCG it binds to the
gold/antibody conjugate and migrates to site 15 where the sample
hCG part of the complex binds to the capture antibody (17) and
yields a red color at site 15 indicating a positive result. If
hCG is absen from the sample, the gold-anti-beta hCG complex has
. no hCG bound to it. The oomplex would not bind to capture
antibody 17 ~ut would instead ~igrate pas~ the reaction site to
the remote reaches of member 20 and/or member 11 (e.g. to 12 on
member 20).
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