U.S. patent application number 10/763466 was filed with the patent office on 2004-08-05 for enzyme substrate delivery and product registration in one step enzyme immunoassays.
Invention is credited to Nelson, Alan M., Pawlak, Jan W., Pronovost, Allan D..
Application Number | 20040152207 10/763466 |
Document ID | / |
Family ID | 25524910 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152207 |
Kind Code |
A1 |
Nelson, Alan M. ; et
al. |
August 5, 2004 |
Enzyme substrate delivery and product registration in one step
enzyme immunoassays
Abstract
One-step enzyme immunoassays in which enzyme-antibody conjugate
or label and enzyme substrate are separated until separation of
bound and free enzyme conjugate or label is complete. This
separation is accomplished by using variable flow paths,
immobilization of substrate at the test line, placement of
substrate in a sac or association with a particle label, enzyme
product chemical capture, delay zone dissolution and protected
enzyme substrates.
Inventors: |
Nelson, Alan M.; (San Diego,
CA) ; Pawlak, Jan W.; (San Jose, CA) ;
Pronovost, Allan D.; (San Diego, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
25524910 |
Appl. No.: |
10/763466 |
Filed: |
January 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10763466 |
Jan 22, 2004 |
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09943031 |
Aug 29, 2001 |
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6706539 |
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09943031 |
Aug 29, 2001 |
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08977183 |
Nov 24, 1997 |
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6306642 |
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Current U.S.
Class: |
436/514 |
Current CPC
Class: |
G01N 33/54366
20130101 |
Class at
Publication: |
436/514 |
International
Class: |
G01N 033/558 |
Claims
What is claimed is:
1. A lateral flow enzyme immunoassay device, comprising: a sample
pad comprising a slow lane and a fast lane separated by a
hydrophobic barrier, wherein said slow lane contains an enzyme
substrate and said fast lane contains an enzyme-antibody conjugate
having affinity for an analyte; a capture zone in fluid
communication with said sample pad, said capture zone having a
capture antibody incorporated therein having affinity for said
analyte; and an absorbent zone in fluid communication with said
capture zone.
2. The immunoassay device of claim 1, wherein said analyte is
selected from the group consisting of hormones, enzymes,
lipoproteins, bacterial or viral antigens, immunoglobulins,
lymphokines, cytokines, drugs and soluble cancer antigens.
3. The immunoassay device of claim 1, wherein said sample pad
comprises high density polyethylene.
4. A flow-through lateral flow enzyme immunoassay device,
comprising: a disk comprising inner and outer hydrophilic zones
separated by a hydrophobic barrier, said inner zone containing an
enzyme substrate and having a smaller pore size than said outer
zone, said outer zone containing an enzyme-antibody conjugate
having affinity for an analyte; a contact pad in fluid
communication with said disk; a capture zone in fluid communication
with said contact pad; and an absorbent zone in fluid communication
with said contact pad.
5. A lateral flow enzyme immunoassay device, comprising: a sample
pad; a label pad in fluid communication with said sample pad, said
label pad containing an enzyme-antibody conjugate having affinity
for an analyte; a capture zone in fluid communication with said
label pad, said capture zone containing a capture antibody having
affinity for said analyte and an enzyme substrate at a test
line.
6. The immunoassay device of claim 5, wherein said substrate is
chemically immobilized at the test line.
7. The immunoassay device of claim 5, wherein said substrate is
immobilized in a mordant under the test line.
8. The immunoassay device of claim 5, wherein said substrate is
immobilized in a mordant dispensed within the test line.
9 The immunoassay device of claim 5, wherein said capture zone
further comprises chemical groups incorporated therein, said
chemical groups capable of specifically reacting with the product
resulting from enzyme action on the substrate.
10. The immunoassay device of claim 9, wherein said chemical groups
comprise diazotized amines.
11. A lateral flow enzyme immunoassay device, comprising: a sample
pad; a label pad in fluid communication with said sample pad, said
label pad containing a substrate covalently attached to a particle
or imbibed within a sac, wherein said substrate-containing sac or
particle is attached to an antibody; a capture zone in fluid
communication with said label pad, said capture zone containing an
enzyme/mediator for releasing said substrate and a capture antibody
at a test line; and an absorbent zone in fluid communication with
said capture zone.
12. The immunoassay device of claim 11, wherein said sac comprises
a liposome.
13. The immunoassay device of claim 11, wherein said sac comprises
an erythrocyte ghost.
14. The immunoassay device of claim 11, wherein said particle label
comprises polyalkylcyanoacrylate polymer monosized colloids.
15. The immunoassay device of claim 11, wherein said
enzyme/mediator is immobilized in a mordant within or under the
test line.
16. The immunoassay device of claim 12, wherein said
enzyme/mediator is attached to the capture antibody.
17. An enzyme immunoassay device, comprising: a sample pad
comprising a first lane containing a first barrier zone and a
second lane containing a second barrier zone, wherein said first
lane contains an enzyme-antibody conjugate having affinity for an
analyte and said second lane contains an enzyme substrate, wherein
said first barrier zone dissolves before said second barrier zone;
a capture zone in fluid communication with said sample pad, said
capture zone containing a capture antibody incorporated therein
having affinity for said analyte; and an absorbent zone in fluid
communication with said capture zone.
18. The immunoassay device of claim 17, wherein said barrier zones
comprise structural hydrogel.
19. The immunoassay device of claim 17, wherein said barrier zones
comprise an enterosoluble coating.
20. The immunoassay device of claim 18, wherein said barrier zones
comprise a biodegradable phospholipid.
21. An enzyme immunoassay device, comprising: a sample pad
containing a first enzyme and a second enzyme, said second enzyme
conjugated to a first antibody having affinity for an analyte; a
label pad in fluid communication with said sample pad, said label
pad containing a substrate for said first enzyme, wherein said
substrate for said first enzyme is converted by said first enzyme
to a second substrate for said second enzyme; a capture zone in
fluid communication with said label pad, said capture zone
containing a second antibody having affinity for said analyte at a
test line, wherein said second substrate is converted by said first
antibody to an enzyme product; and an absorbent zone in fluid
communication with said capture zone.
22. The immunoassay of claim 21, wherein said first enzyme is
selected from the group consisting of alkaline phosphatase,
esterase, protease, sulfatase, chymotrypsin-like protease, creatine
amidinohydrolase and arginase.
23. The immunoassay of claim 21, wherein said second enzyme is
selected from the group consisting of .beta.-D-galactosidase,
N-acetylglucosamidase, .alpha.-L-arabinofuranosidase, exglucanase,
chitobiosidase, .alpha.-L-fucosidase, .beta.-D-glycosidase,
.alpha.-galactosidase, .beta.-glucosidase, glucansucrase,
.beta.-D-glucuronidase, .alpha.-amylase, .alpha.-mannosidase and
.beta.-mannosidase.
24. The immunoassay of claim 21, wherein said analyte is selected
from the group consisting of hormones, enzymes, lipoproteins,
bacterial or viral antigens, immunoglobulins, lymphokines,
cytokines, drugs and soluble cancer antigens.
25. A sample receiving layer for use in an enzyme immunoassay
device, comprising: a disk comprising inner and outer hydrophilic
zones separated by a hydrophobic barrier, said inner zone
containing an enzyme substrate and having a smaller pore size than
said outer zone, said outer zone containing an enzyme-antibody
conjugate having affinity for an analyte.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the detection of analytes
in biological fluids. More specifically, the invention relates to
enzyme substrate delivery and product registration in one step
enzyme immunoassays.
BACKGROUND OF THE INVENTION
[0002] Analyte-specific binding assays are important tools for
detecting and measuring environmental and biologically relevant
compounds, including hormones, metabolites, toxins and
pathogen-derived antigens. A convenient version of the binding
assay is an immunoassay which can be conducted in a "lateral flow"
format.
[0003] Devices useful for performing lateral flow assays typically
include several "zones" that are defined along a length of a
matrix. The matrix defines a flow path and provides fluid
connection between the various zones, including a sample receiving
zone, a labeling zone for specifically labeling the analyte, and a
capture (detection) zone located downstream from the sample
receiving zone and the labeling zone. An absorbent zone (sink)
typically is located downstream of the capture zone, and provides a
means for removing excess sample and unbound label from the
matrix.
[0004] In some applications the matrix of a lateral flow assay
device is a membrane capable of "non-bibulous lateral flow." In
these applications liquid flow occurs such that all of the
dissolved or dispersed components in the analyte-containing liquid
are carried at substantially equal rates and with relatively
unimpaired flow laterally through the membrane. This is
distinguished from a situation wherein preferential retention of
one or more components occurs, for example, in materials capable of
adsorbing or imbibing one or more of the components.
[0005] A principal advantage of the lateral flow immunoassay is the
ease with which the testing procedure is carried out. In this
procedure a fluid sample first contacts the matrix following
application to the sample receiving zone. Capillary action then
draws the liquid sample downstream into a labeling zone that
contains a means for indirectly labeling the target analyte. The
labeling me ans generally will be a labeled immunoglobulin; but
alternatively may be a non-immunoglobulin labeled compound which
specifically binds the target analyte. After flowing through the
labeling zone, the sample continues to flow into the capture zone
where it contacts an immobilized compound capable of specifically
binding the labeled target analyte or the complex formed by the
analyte and label. As a specific example, analyte-specific
immunoglobulins can be immobilized in the capture zone. Labeled
target analytes will bind the immobilized immunoglobulins upon
entering the capture zone and will be retained therein. The
presence of the labeled analyte in the sample typically will be
determined by visual detection of the label within the capture
zone. Finally, the procedure is complete when excess sample is
taken up by the material of the absorbent zone.
[0006] Lateral flow immunoassays typically employ test and
procedural control lines in the capture zone. The test line serves
to detect an analyte present in a test sample, while the procedural
control line conventionally serves to detect a ligand unrelated to
the analyte. Rather than being applied in the test sample, the
ligand unrelated to the analyte is disposed in the labeling zone of
the lateral flow immunoassay device. The test line ordinarily
employs specific competitive, sandwich or indirect binding
separation principles using a visual label. This requires the use
of a labeled detector antibody in the labeling pad of the labeling
zone and a capture antibody or ligand immobilized at the capture
test line.
[0007] The capture zone of lateral flow immunoassay devices may
also include a procedure control line useful for indicating that a
procedure has been performed. The procedure control line generally
is located downstream of the binding compound that is immobilized
in the capture zone at the test line where reaction occurs.
Retention of label by the procedural control line indicates that
liquid sample has flowed through the capture zone and contacted the
immobilized target-specific binding substance. The accumulation of
visible label may be assessed either visually or by optical
detection devices.
[0008] Another type of enzyme immunoassay utilizes a flow-through
device which is described in U.S. Pat. No. 4,632,901. This device
comprises a membrane or filter to which an antibody is bound. An
absorbent material in contact with the membrane or filter induces
flow therethrough when a fluid sample is added to the membrane cr
filter. A fluid sample is applied to the membrane and, if the
cognate antigen is present, is bound by the antibody. A solution of
labeled antibody against the antigen is then added followed by a
washing step to remove unbound labeled antibody. The presence of
labeled antibody on the membrane after washing indicates the
presence of the antigen in the sample being assayed.
[0009] In one step enzyme immunoassays (EIAs), whether they be
flow-through or lateral flow constructs, there is an inherent
limitation to the use of enzyme amplification wherein the enzyme
(as either enzyme-antibody conjugate or enzyme-label particulate)
must be kept separate from its substrate until separation of bound
and free enzyme conjugate or label is complete. The present
invention addresses methods for such separation.
SUMMARY OF THE INVENTION
[0010] One embodiment of the present invention is an enzyme
immunoassay device, comprising a sample pad comprising a slow lane
and a fast lane separated by a hydrophobic barrier, wherein the
slow lane contains an enzyme substrate and the fast lane contains
an enzyme-antibody conjugate having affinity for an analyte; a
capture zone in fluid communication with the sample pad, the
capture zone having a capture antibody incorporated therein having
affinity for said analyte; and an absorbent zone in fluid
communication with said capture zone. Preferably, the analyte is a
hormone, enzyme, lipoprotein, bacterial antigen, viral antigen,
immunoglobulin, lymphokine, cytokine, drug or soluble cancer
antigen. Advantageously, the sample pad comprises high density
polyethylene.
[0011] Another embodiment of the present invention is a
flow-through lateral flow enzyme immunoassay device, comprising a
disk comprising inner and outer hydrophilic; zones separated by a
hydrophobic barrier, the inner zone containing an enzyme substrate
and having a smaller pore size than the outer zone, the outer zone
containing an enzyme-antibody conjugate having affinity for an
analyte; a contact pad in fluid communication with the molded disk;
a capture zone in fluid communication with the contact pad; and an
adsorbent zone in fluid communication with the contact pad.
[0012] The present invention also provides a lateral flow enzyme
immunoassay device, comprising a sample pad; a label pad in fluid
communication with the sample pad, the label pad containing an
enzyme-antibody conjugate having affinity for an analyte; a capture
zone in fluid communication with the label pad, the capture zone
containing a capture antibody having affinity for the analyte; and
an enzyme substrate at a test line. In one aspect of this preferred
embodiment, the substrate is chemically immobilized at the test
line. Alternatively, the substrate is immobilized in a mordant
under the test line. Still alternatively, the substrate is
immobilized in a mordant dispensed within the test line.
Preferably, the capture zone further comprises chemical groups
incorporated therein, the chemical groups capable of specifically
reacting with the product resulting from enzyme action on the
substrate. Advantageously, the chemical groups comprise diazotized
amines.
[0013] Another embodiment of the invention is a lateral flow enzyme
immunoassay device, comprising a sample pad; a label pad in fluid
communication with the sample pad, the label pad containing a
substrate covalently attached to a particle or imbibed within a
sac, wherein the substrate-containing sac or particle is attached
to an antibody; a capture zone in fluid communication with the
label pad, the capture zone containing an enzyme/mediator for
releasing the substrate and a capture antibody at a test line; and
an absorbent zone in fluid communication with the capture zone.
Preferably, the sac comprises a liposome. Alternatively, the sac
comprises an erythrocyte ghost. Advantageously, the particle label
comprises polyalkylcyanoacrylate polymer monosized colloids. The
enzyme/mediator may immobilized in a mordant within or under the
test line, or may be attached to the capture antibody.
[0014] The present invention also provides an enzyme immunoassay
device, comprising a sample pad comprising a first lane containing
a first barrier zone and a second lane containing a second barrier
zone, wherein the first lane contains an enzyme-antibody conjugate
having affinity for an analyte and said second lane contains an
enzyme substrate, wherein the first barrier zone dissolves before
said second barrier zone; a capture zone in fluid communication
with the sample pad, the capture zone containing a capture antibody
incorporated therein having affinity for the analyte; and an
absorbent zone in fluid communication with the capture zone. The
barrier zones may comprise structural hydrogel, enterosoluble
coatings or biodegradable phospholipids.
[0015] Still another embodiment of the invention is an enzyme
immunoassay device, comprising a sample pad containing a first
enzyme and a second enzyme, the second enzyme conjugated to a
second antibody having affinity for an analyte; a label pad in
fluid communication with the sample pad, the label pad containing a
substrate for the first enzyme, wherein the substrate for the first
enzyme is converted by the first enzyme to a second substrate for
the second enzyme; and a capture zone in fluid communication with
the label pad, the capture zone containing a first antibody having
affinity for the analyte at a test line, wherein the second
substrate is converted by the second antibody to an enzyme product;
and an absorbent zone in fluid communication with the capture zone.
Preferably, the first enzyme is alkaline phosphatase, esterase,
protease, sulfatase, chymotrypsin-like protease, creatine
amidinohydrolase or arginase. Advantageously, the second enzyme is
.beta.-D-galactosidase, N-acetylglucosamidase,
.alpha.-L-arabinofuranosid- ase, exglucanase, chitobiosidase,
.alpha.-L-fucosidase, .beta.D-glycosidase, .alpha.-galactosidase,
.beta.-glucosidase, glucansucrase, .beta.-D-glucuronidase,
.alpha.-amylase, .alpha.-mannosidase or .beta.-mannosidase.
According to another aspect of this preferred embodiment, the
analyte is a hormone, enzyme, lipoprotein, bacterial antigen, viral
antigen, immunoglobulin, lymphokine, cytokine, drug or soluble
cancer antigen.
[0016] The present invention also provides a sample receiving layer
for use in an enzyme immunoassay device, comprising: a disk
comprising inner and outer hydrophilic zones separated by a
hydrophobic barrier, the inner zone containing an enzyme substrate
and having a smaller pore size than the outer zone, the outer zone
containing an enzyme-antibody conjugate having affinity for an
analyte.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a schematic diagram of a lateral flow assay strip
containing a variable flow path sample pad.
[0018] FIG. 2 is a schematic diagram of a lateral flow enzyme
immunoassay strip in which the substrate is covalently attached to
a particle label or imbibed inside a sac. X=analyte; SP=sample pad;
LP=label pad; Nitro=nitrocellulose; S=substrate;
Enz/Med=enzyme/mediator.
[0019] FIG. 3 is a schematic diagram of enzyme product chemical
capture. The analyte X is bound by both an enzyme (E)-conjugated
antibody and a capture antibody. The enzyme substrate (S) is
converted to product (P) which binds to chemical groups (R)
immobilized on the support.
[0020] FIG. 4 is a schematic diagram of an enzyme immunoassay test
strip for delayed enzyme substrate delivery using hydrogel barrier
zones.
[0021] FIG. 5 is a schematic diagram of an enzyme immunoassay test
strip for sequential delayed enzyme-antibody and enzyme substrate
release using hydrogel barrier zones.
[0022] FIG. 6 is a schematic diagram of an enzyme immunoassay test
strip for sequential delayed enzyme-antibody and enzyme substrate
release using enterosoluble barrier zones.
[0023] FIG. 7 is a schematic diagram of delayed release of enzyme
substrate using multi-enzyme systems and protected enzyme
substrates. X=analyte; E.sub.1=enzyme 1; E.sub.2=enzyme 2;
S=substrate; P=product.
[0024] FIG. 8 is a schematic diagram of delayed release of enzyme
substrate using the .beta.-D-galactosidase anti-analyte system.
[0025] FIG. 9 shows alternative "protected" substrates for the
.beta.-D-galactosidase-Ab conjugate shown in FIG. 8.
[0026] FIG. 10 shows "protected" substrates for urease as E1 in E1
Ab conjugates and the resulting products.
[0027] FIG. 11 is a top view of a molded disk comprising variable
flow paths for use as a top sample receiving layer in the
immunoassay device shown in FIG. 12.
[0028] FIG. 12 is a cross-sectional view of a flow-through lateral
flow enzyme immunoassay device.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0029] The present invention provides one step enzyme immunoassay
devices and methods in which enzyme and substrate are kept
separated until separation of bound from free enzyme conjugate or
label is complete. The invented assay devices comprise four
distinct zones. The device is designed so that the
analyte-containing sample is first applied to a sample receiving
zone, then flows through a labeling zone and into a capture zone.
The capture zone in turn is in contact with an absorbent zone which
provides a means for removing excess liquid sample. In conventional
immunoassay devices, the absorbent zone consists of an absorbent
such as filter paper or glass fiber filter.
[0030] As used herein, the term "pad" refers to the physical
material which corresponds to a zone or section of an immunoassay
strip. Thus, a sample pad is the material of the sample receiving
zone of an immunoassay strip. The labeling pad similarly refers to
the material of the labeling zone.
[0031] The invention generally concerns one-step lateral flow or
flow-through assays which are conducted on supports which may
conduct nonbibulous lateral flow of fluids. As defined herein
"nonbibulous" lateral flow refers to liquid flow in which all of
the dissolved or dispersed components of the liquid which are not
permanently entrapped or "filtered out" are carried at
substantially equal rates and with relatively unimpaired flow
laterally through the membrane or support. This is distinguished
from preferential retention of one or more components as would
occur, for example, in materials capable of absorbing or "imbibing"
one or more components as occurs in chromatographic separations.
"Bibulous" materials include untreated forms of paper,
nitrocellulose and the like which effect chromatographic separation
of components contained in liquids passed therethrough. Bibulous
materials can be converted to materials which exhibit nonbibulous
flow characteristics by the application of blocking agents. These
agents may be detergents or proteins which can obscure the
interactive forces that account of the bibulous nature of the
supports. Thus, nonbibulous materials include those which are
intrinsically capable of conducting nonbibulous flow, such as
porous polyethylene sheets or other inert materials or can be
comprised of bibulous materials which-have been blocked. Preferred
blocking agents include bovine serum albumin, either per se or in
methylated or succinylated form, whole animal sera, such as horse
serum or fetal calf serum, and other blood proteins. Other examples
of protein blocking agents include casein and nonfat dry milk.
Detergent-based blocking agents can also be used for rendering a
bibulous material capable of nonbibulous flow. The types of
detergents appropriate for this purpose are selected from nonionic,
cationic, anionic and amphoteric forms, and the selection is based
on the nature of the membrane that is being blocked.
[0032] To convert a bibulous support such as paper or
nitrocellulose to a support capable of effecting nonbibulous
lateral flow, the original support is treated with a solution of
the blocking agent in an effective concentration to dispose of
unwanted reactivities at the surface. In general, this treatment is
conducted with a blocking solution, such as a protein solution of
1-20 mg/ml protein at approximately room temperature for between
several minutes and several hours. The resulting coated material is
then permanently adsorbed to the surface by air-drying,
lyophilization, or other drying methods. Both the selection and
treatment of carrier porous materials used to construct immunoassay
strips of the sort described herein depend on the functional role
that each zone performs in the assay device.
[0033] The sample-receiving zone serves to begin the flow of
analyte-containing sample, and typically will be constructed of a
material that exhibits low analyte retention. One means for
imparting this property involves impregnating the sample receiving
zone with a neutral protein-blocking reagent, followed by treatment
to immobilize the blocking agent (e.g., lyophilization). An
additional advantage of this treatment is increased wetability and
wicking action which speeds transfer of the liquid sample into the
labeling zone. The sample-receiving zone may also function as a
mechanical filter, entrapping any undesirable particulates present
in the sample.
[0034] The labeling zone contains enzyme-antibody conjugate or
particulate moieties which may or may not be visible, which can be
detected if accumulated in the capture zone. The visible moieties
can be dyes or dyed polymers which are visible when present in
sufficient quantity, or can be, and are preferred to be particles
such as dyed latex beads, liposomes, or metallic, organic,
inorganic or dye solutions, dyed or colored cells or organisms, red
blood cells and the like. The enzyme-antibody conjugate or
particulate moieties used in the assay provide the means for
detection of the nature of and quantity of result, and accordingly,
their localization in the capture zone must be a function of the
analyte in the sample. In general, this can be accomplished by
coupling the enzyme-antibody conjugate or particulate moieties to a
ligand which binds specifically to the analyte, or which competes
with analyte for a capture reagent in the capture zone. In the
first approach, the conjugate or particulate moieties are coupled
to a specific binding partner which binds the analyte specifically.
For example, if the analyte is an antigen, an antibody specific for
this antigen may be used; immunologically reactive fragments of the
antibody, such as F(ab').sub.2, Fab or Fab' can also be used. These
conjugate or particulate moieties, or "test" moieties, then bind to
analyte in the sample as the sample passes through the labeling
zone and are carried into the capture zone by the liquid flow. When
the complex reaches the capture zone, it is captured by an
analyte-specific capture reagent, such as an antibody. Excess
liquid sample finally is taken up by the absorbent zone. In the
second approach, the conjugate or particulate moieties are coupled
to a ligand which is competitive with analyte for a capture reagent
in the capture zone, most typically, other molecules of the analyte
itself. Both the analyte from the sample and the competitor bound
to the conjugate or particulate moieties are then carried into the
capture zone. Both analyte and its competitor then react with the
capture reagent, which in this instance is also typically
specifically reactive with an analyte and its competitor. The
unlabeled analyte thus is able to reduce the quantity of
competitor-conjugated conjugate or particulate moieties which are
retained in the capture zone. This reduction in retention of the
conjugate or particulate moieties becomes a measure of the analyte
in the sample after enzyme turnover.
[0035] The labeling zone of immunoassay devices of the present
invention also may include a procedural control which comprises
visible moieties that do not contain the specific binding agent or
analyte competitor and that are also carried through to a control
area of the capture zone by the liquid flow. These visible moieties
are coupled to a control reagent which binds to a specific capture
partner and can then be captured in a separate procedural control
portion of the capture zone to verify that the flow of liquid is as
expected. The visible moieties used in the procedural control may
be the same or different color than those used for the test
moieties. If different colors are used, ease of reading the results
is enhanced.
[0036] The experimental results of a procedure conducted using an
immunoassay strip are read in the capture zone by noting the
presence or absence of a visible signal at the location of the
capture zone for the test line. The use of a procedural control
region is helpful for indicating the time when test results can be
read. Thus, when the expected color appears in the procedural
control region, the presence or absence of a color in the test
region can be noted. The use of different colors for test and
control regions aids in this process.
[0037] The use of a matrix which is bibulous inherently, but
convertible to a nonbibulous flow characteristic, is particularly
useful in the creation of the capture zone. Capture reagents can be
applied to the matrix before the application of blocking agents and
can be immobilized in situ. At this stage, the bibulous nature of
the matrix during the coupling of the capture reagents may be
advantageous. However, the blocking/washing treatment which
converts the bibulous membrane to nonbibulous support provides
unimpaired and speedy flow of all components of the system.
[0038] The extremely rapid nature of the assay, which typically
yields a result in less than one minute, provides in many instances
essentially an instantaneous result as the sample flows due to the
nonbibulous nature of the zones and of the short distance the
sample must traverse in each zone. Another factor which contributes
to the speed of the assay is the absorptive potential of the
material used to create the absorbent zone and the use of dessicant
as adsorbent.
[0039] Miniaturization of the diagnostic device also contributes to
the remarkable speed of the assay. Miniaturization permits
instantaneous results which are observable as soon as the sample
contacts the capture zone and which occur almost immediately or
within 60 seconds of the addition of the sample to the sample
receiving zone. The speed of appearance and intensity of the
positive visible reaction seen depends on the concentration of
analyte in the sample. The speed of appearance of the positive
visual reaction can be adjusted to provide the optimal visual
result with concentrations of analyte of clinical importance and
adjusted to suit the timing needs of the end-user.
[0040] Suitable analytes detectable by the invented immunoassay
devices are any for which a specific binding partner can be found.
In general, most analytes of medical and biological significance
can find specific binding partners in antibodies prepared against
them or fragments of these antibodies. Suitable analytes include
soluble analytes such as hormones, enzymes, lipoproteins, bacterial
or viral antigens, immunoglobulins, lymphokines, cytokines, drugs,
soluble cancer antigens, and the like. Also included as suitable
analytes are hormones such as human chorionic gonadotropin (hCG),
insulin, glucagon, relaxin, thyrotropin, somatotropin,
gonadotropin, follicle-stimulating hormone, gastrin, bradykinin,
vasopressin, and various releasing factors. A wide range of
antigenic polysaccharides can also be determined such as those from
Chlamydia, Neisseria gonorrheae, Pasteurella pestis. Shigella
dysentereae, and certain fungi such as Mycosporum and Aspergillus.
Another major group comprises oligonucleotide sequences which react
specifically with other oligonucleotides or protein targets. An
extensive list of soluble analytes determinable in the method of
the invention is found in U.S. Pat. No. 3,996,345, which is
incorporated herein by reference.
[0041] The invented immunoassay strips can be disposed within a
housing that is both protective and functional. In one preferred
embodiment the housing is adapted to have at least one port for
receiving a liquid sample and guiding fluid flow of the sample to
contact the immunoassay strip at the sample receiving zone. The
housing also can have windows which allow a user to view portions
of the immunoassay strip, including portions of the capture zone
and/or the absorbent zone.
[0042] Various enzyme substrate systems can be used in the one-step
enzyme immunoassays of the invention. These systems are summarized
in Table 1 but not limited thereto.
1TABLE 1 ENZYME 1) SOLUBLE PRODUCT 2) INSOLUBLE PRODUCT pH RANGE
Alkaline Phosphatase (p-Nitrophenyl BCIP, 3-IP 9-10.5 phosphate)
Azonaphtol phosphate (p-Aminophyenyl BCIP/tetrazolium salt
phosphate) 3-IP/tetrazolium salt Azonaphthol phosphate Naphthyl
phosphate Horseradish peroxidase DAB/MBTY 3-AEC 4-8 ABTS
4-Chloronaphthol TMB 4-Chloronaphthol/MBTH 4-Chloronapthol/4-AAP
DAB Glucose oxidase Tetrazolium salt/PMS Tetrazolium salt/PMS 6-8
Diaphorase Tetrazolium salt/ Tetrazolium salt/PMS 6-8
oxidoreductase NADH (NADPH) Galactosidase (Azonapthol (Indolyl 6-8
galactopyranoside) galactopyranoside (Aminophenyl (Azonaphthol
galactopyranoside) galactopyranoside) (Napthyl galactopyranoside)
(Nitrophenyl galactopyranoside) Urease Urea 6-8 Urea/pH indicator
Glucuronidase (Azonaphtol glucuronide) (Azonaphthol 6-8
(Nitrophenyl glucuronide) glucuronide) (Indolyl Naphthyl
glucuronide glucuronide) (Aminophenyl glucuronide) 1) Product of
Reaction of enzyme on substrate is water soluble. 2) Product of
Reaction of enzyme on substrate is water insoluble.
[0043] Various embodiments of enzyme-substrate delivery and product
registration in one-step EIAs are discussed below.
[0044] 1. Variable Flow Path
[0045] A lateral flow EIA device 2 is illustrated in FIG. 1.
Enzyme-antibody conjugate (E-Ab) 4 is lyophilized and placed in the
fast chromatographic lane 6 of a bi-phase solid support 8. The
enzyme substrate 10 is lyophilized and placed in the slow
chromatographic lane 12. The "fast" and "slow" lane designations
refer to the pore size of the high density polyethylene. The slow
lane contains smaller pore sizes than the fast lane. Accordingly,
the substrate 10 will move through the sample pad more slowly than
E-Ab 4. A volume of sample containing analyte is applied to solid
support 8 and contacts both substrate 10 and E-Ab 4 simultaneously.
The substrate 8 and E-Ab 4 then flow at variable rates to the
nitrocellulose detection zone 14 for sequential conjugate, then
substrate, reaction. An immunological separation occurs in the
detection zone 14 where analyte-Ab-E complex either binds to the
capture antibody at the test line 16 or Ab-E passes unbound to the
absorbent zone 18. Upon reaching the immobilized E-Ab, substrate is
converted to product which is either insoluble and precipitates at
the test line 16, or the soluble product is measured downstream by
conventional methods. A specific example of this embodiment is
provided in Example 1.
EXAMPLE 1
[0046] Substrate and Enzyme-Antibody Conjugate Delivery Using
Variable Flow Paths
[0047] Molded hydrophilic high density polyethylene (HDPE)
macroporous supports such as those custom manufactured by Interflo
Technologies (Brooklyn, N.Y.) were used to form the variable flow
paths. Two pieces of 2 mm thick hydrophilic HDPE (5 m mm.times.50
cm each) of different pore sizes (5-20 .mu.m and 20-80 .mu.m) were
separated by a hydrophobic non-porous material (2 mm.times.50 cm),
aligned along the length parallel to each other on the same plane,
then fused together. The resultant "two-lane" strips were cut into
12.times.20 mm pieces. To each two-lane strip was added a 5 .mu.l
aliquot of enzyme-antibody conjugate (3-6 mg/ml), spotted in the
center of the fast lane, and 5 .mu.l aliquots of the appropriate
enzyme substrate (5-12 mg/ml) supplemented with 10-20% (w/v) of
cyclodextrin were spotted in the center of the slow lane.
Cyclodextrin was added to increase the viscosity and further retard
movement of the substrate through the strip. The prepared materials
were lyophilized and assembled as intermediate zones into a lateral
"one-step" device. Subsequently, substrate and enzyme-antibody
conjugate delivery by use of a variable flow path were studied by
applying buffer solutions at different pH.
[0048] In a related embodiment, substrate and enzyme-antibody
conjugate were delivered using a variable flow path in a
flow-through lateral flow device. Referring to FIG. 11, a 20 mm
diameter molded disk of HDPE 100 was constructed by fusing a 9 mm
diameter inner disk 102 of 5-20 .mu.m pore size hydrophilic HDPE
with an outer 2 mm thick O-ring 104 of hydrophobic medium and an
outside 9 mm thick O-ring 106 of 20-280 .mu.m hydrophilic HDPE. The
inner disk 102 was saturated with the appropriate enzyme substrate
solution (0.2-2 mg/ml) containing 5-20% cyclodextrin and the
outside ring 106 was saturated with the corresponding
enzyme-antibody conjugate (5-25 .mu.g/ml) supplemented with 2-10
mg/ml bovine serum albumin (BSA). The use of any desired diameter
disk and O-ring thickness for forming the molded disk 100 is within
the scope of the invention. In addition, the use of hydrophilic
materials other than HDPE is also contempated. Such materials
include, for example, polypropylene and polyvinylchloride.
[0049] The resultant pieces were lyophilized and assembled as a
sample receiving top layer of the flow-through device 110 shown in
FIG. 12. The device has a top housing 111 and a bottom housing 113.
Disk 100, which functions as the label pad, is placed in sample
well 112 in contact with absorbent contact pad 114 which is in
fluid communication with capture zone 116 and absorbent pad 118.
Test line 120 containing a capture antibody is viewable through
view window 122.
[0050] A fluid sample containing an antigen of interest is applied
to the device 110 and, due to the larger pore size of O-ring 106 to
which the enzyme-antibody conjugate is applied, the
enzyme-antibody-antigen complex moves into the contact pad 114 and
capture zone 116 in fluid communication with disk 100 before the
enzyme substrate, thus keeping the enzyme and substrate separated
until antigen has bound to the antibody at the test line 120.
Unbound antigen and reagents flow into the absorbent pad 118.
[0051] 2. Substrate Delivery Through Immobilization at Test
Line
[0052] In this lateral flow embodiment, the substrate is
immobilized at the test line chemically, in a mordant under the
test line (e.g. protein, gel, etc.) or in a mordant dispensed
within the test line in the nitrocellulose membrane (e.g.
cyclodextrin, polyvinyl acetate, etc.). In a lateral flow assay
format, analyte reacts with enzyme-antibody conjugate localized in
a label pad, followed by complex formation in the presence of
antigen. The complex is captured by the capture antibody
immobilized at the test line or proceeds unbound to the absorbent
in the absence of analyte. The enzyme product is either insoluble
and precipitates on the test line or is soluble and measured
downstream.
[0053] 3. Substrate Delivery Vehicles
[0054] In one embodiment of this lateral flow EIA device 20 (FIG.
2), the substrate 22 is covalently attached to a particle label,
such as a latex particle bound to indoxyl phosphate moieties via
primary amine functionalities. Under the influence of alkaline
phosphatase (EC 3.1.3.1 from bovine and calf intestinal mucosa),
the phosphate ester is cleaved to liberate indol-3-ol which in turn
isoxidized by free oxygen in solution to blue indigo dye.
[0055] In another preferred embodiment, the substrate is imbibed
within a sac 24. One example of an enzymatic substrate imbibed
within a sac involves the use of liposomes to carry enzyme
substrates to the area where enzymes are immobilized, presumably at
the capture line. Techniques for incorporating substances into
liposomes are well known in the art. This technique requires
disruption of the liposome at the appropriate time to release the
substrate for the enzyme release factor.
[0056] Alternatively, an enzyme substrate is encapsulated in a red
blood cell (RBC) based on the fact that a RBC can be re-formed
after osmotic lysis under the proper conditions of osmolarity,
temperature and pH to trap any solutes present at the time of
reforming (D'Orazlo et al., Anal. Chem., 49:2083-2086, 1977),
hereby incorporated by reference). The resulting loaded erythrocyte
ghosts are functionalized with the antigen (or antibody) to an
antibody (or antigen) to be quantified. This may be accomplished by
mixing the antibody or antigen to be bound to the RBC surface with
a suspension of RBCs in the presence of tannic acid, chromium
chloride or a water-soluble carbodiimide. The functionalized RBC
sacs are then used to carry enzyme substrate to the enzyme at the
capture line. Upon capture of the erythrocyte ghost, the hemolysin
is brought into close proximity and is able to break down the
membrane wall, liberating the enzyme substrate. Also, release can
be accomplished by a variety of means, including enzymes,
surfactants, ionic strength shifts, complement formation and
opsonization. Thus, in FIG. 2, the components are as follows: 24,
substrate-loaded erythrocyte ghost; 22, antibody to target analyte
on RBC surface; 30, second antibody to analyte; 26, hemolysin
conjugated to second antibody.
[0057] In both embodiments mentioned above, an enzyme/mediator 26
is immobilized in a mordant within or under the test line 28, or
attached to the capture antibody 30. Analyte solution flows from
the sample pad 32 to the label pad 34 where the analyte binds to
antibody 36. Analyte-antibody conjugate then binds to capture
antibody 30 in a sandwich format in the detection zone 38. A
suitable enzyme/mediator (release-factor) 26 such as Phospholipase
C from C. perfringens (EC 3.1.4.3) or a surfactant degrades or
lyses the particles or sacs 24 to release substrate 22. The sac 24
may be, for example, a liposome prepared by procedures well known
in the art, or an erythrocyte ghost as described above. See, for
example, U.S. Pat. No. 4,342,826, published PCT Application No.
WO80/01515 and U.S. Pat. No. 4,703,017, all of which are hereby
incorporated by reference.
[0058] Other contemplated release factors include surfactants such
as octyl-.beta.-D-glucopyranoside which is stored as a mordant
under the capture line as opposed to attached or conjugated to the
capture antibody. An alternative system, as described above, is the
use of erythrocyte ghosts with enzyme substrates in which hemolysin
serves as the enzyme release factor.
[0059] When the particle or sac containing the substrate is
captured, enzyme substrate leaches from the sac 24 or is released
from the particle and enzyme product precipitates on the line or is
measured downstream upon enzyme turnover. Unbound antibodies and
analyte migrate to the absorbent zone 40.
EXAMPLE 2
Incorporation of Enzyme Substrate Within a Biodegradable Sac
[0060] To a 500 ml round-bottom rotary evaporator flask was added,
with mixing: 240 mg cholesterol, 520 mg distearoyl
phosphatidylcholine (20 mg/ml in CHCl.sub.3), 18.75 mg distearoyl
phosphatidylethanotaine-(p-male- imidophenyl) butyrate (2 mg/ml in
CHCl.sub.3); 30 mg of isopropyl ether and 5 ml methanol.
Subsequently, 25-100 ml of suitable enzyme substrate (2-50 mg/ml in
0.1 M sodium acetate, 0.1 M NaCl, pH 4.5) was added. The mixture
was mixed, emulsified by sonication and rotary evaporated at
45-55.degree. C. The warm liposomes were extruded sequentially
through 1.0 .mu.m, 0.7 .mu.m, 0.5 .mu.m, then 0.3 .mu.m, then 0.2
.mu.m nucleopore polycarbonate membranes. Following a series of
high-speed centrifugation (>50,000.times.g for. >30 minutes)
and wash steps (0.1 M sodium acetate buffer, 0.1 M NaCl buffer, pH
4.5), liposomes containing entrapped enzyme substrates were
resuspended in 10 mM Tris NaCl/EDTA buffer (pH 7.5-8.0 storage
buffer) of osmolarity ranging from 200-400 mOs/kg.
[0061] Subsequently, anti-human chorionic gonadotropin (hCG)
monoclonal antibody was derivatized using the SPDP/DTT procedure.
This method involves addition of a pyridyl disulfide group to the
anti hCG Mab which is to be added to the surface of the liposome
(Wong, S., CRC Chemistry of Protein Conjugation and Cross-Linking,
CRC Press, Inc., 1991; Liposomes: A Practical Approach, R.R.C. New,
Ed., IRL Press, 1990, both hereby incorporated by reference.).
Briefly, the antibody (about 6 mg/ml) was incubated with a 50-fold
molar excess of (N-succinimidyl-3)-[2-pyridyldit- hio]propionate)
(SPDP) previously dissolved in ethanol in 0,1 M sodium phosphate
buffer, pH 7.5, for 30 minutes at 25.degree. C. Pyridyl
disulfide-labeled antibody was isolated by gel filtration through a
Sephadex G-25 column. The pyridyl dithio-Mab solution was titrated
in citrate buffer to pH 5.5 by addition of 1 M HCl a solution of
2.5 m dithiothreitol (DTT, 380 mg/ml) in 0.2. M acetate buffer, pH
5.5 (165 mg sodium acetate/10 ml) was then prepared. To enable
formation of a stable thioether bond between the protein and
liposome, a 0.10 .mu.l aliquot of the freshly prepared DTT solution
was added to a mixture of the SPDP-labeled Mab and
maleimide-derivatized liposomes at pH 6.5 which was incubated
overnight at 25.degree. C.
[0062] The method described above resulted in the introduction of 1
to 6 SH groups per antibody as determined by the 5,5'
Dithio-(bis(nitrobenzoic acid) (DTNB) method (Deakin et al.,
Biochem. J, 89:296, 1963). In this method, unreacted --SH groups on
the protein are blocked with DTNB while unreacted maleimide groups
on the liposome are blocked with N-ethyl naleimide. The number of
thio groups introduced into the protein is assessed by monitoring
the absorbance of free thiopyridone groups at 343 nm that are
liberated as the labeling proceeds.
[0063] The substrate-loaded liposomes were sensitized on the outer
surface by reaction with SH-containing antibody for 2-3 hours at
25.degree. C. Liposomes were then applied to a Sepharose 6FF
(Pharmacia) size exclusion column equilibrated in storage buffer
containing 2-10 mg/ml BSA, 1.3% glycerol, 0.0005% dimethyl
sulfoxide (DMSO), 0.74% EDTA. Liposomes were diluted in storage
buffer that was supplemented with a 3:1 mixture of
sucrose:trehalose such that the final concentration of lipid in the
suspension was between 0.05 to 0.40 .mu.mole/ml and the final
concentration of total sugar was between 5 and 10 mg/ml. This was
then lyophilized into either polyester spunlace fabric or non-woven
rayon.
[0064] To determine substrate release kinetics from the liposomes
in a lateral flow one-step device, the lyophilized materials were
cut and assembled as intermediate zones into a device in a manner
described in International Publication No. WO92/12428. Capture zone
membranes were spotted with anti-hCG polyclonal antibody (1-5
mg/ml) conjugated to the corresponding enzyme or substrate
releasing factor. The substrate releasing factor was either
phospholipase (1-10 mg/ml), complement component C.sub.1q (0.1-4
mg/ml) or non-ionic polymeric detergents (0.1-0.4%) such as
polyoxyethylene alcohols, polyoxyethylene-p-t-octylphe- nols,
pblyoxyethylenenonyl phenols, polyoxyethylene sorbitol esters,
polyoxypropylene-polyoxyethylene esters of the Triton WR series.
Phospholipase may also be attached to the capture antibody
directly.
[0065] Substrate release from liposomes was studied by applying
urine samples with or without hCG to the sample receiving zone and
measuring enzyme product accumulation in the capture zone for
insoluble products or in the absorbent pad for soluble products. In
the case of a colored insoluble product, the optical density was
measured using a Umax 6SE flatbed scanner calibrated with a Kodak
paper gray scale. In the case of water-soluble products which would
precipitate out around the capture line, a densitometer (BioRad
Laboratories, Hercules, Calif.) was used which was also calibrated
with a Kodak paper gray scale. In both cases, a correlation was
observed between gray scale optical density units and analyte
concentration that can be used to estimate the analyte level in an
unknown specimen.
[0066] To assess substrate release from liposomes in a flow-through
assay format, the unsupported media containing lyophilized
liposomes were cut into 2 cm diameter circles and assembled as a
top layer of the device. Capture membranes were prepared similarly
as described for the lateral flow device. Substrate release from
liposomes was studied by applying urine samples and performing
similar measurements as for a lateral flow device.
EXAMPLE 3
Preparation of Biodegradable Substrate-Loaded Particles
[0067] Biodegradable synthetic polyalkylcyanoacrylate (PECA)
polymer monosize colloids able to sorb internally sufficient
quantities of desirable enzyme substrates were used as carriers for
controlled delayed substrate delivery. The general procedure for
the polymerization of colloidal particles for sustained drug
delivery is described in detail by Cicek et al. (in Biodegradable
Polymeric Biomaterials, E. Piskin, Ed., Marcel Dekker, New York,
1993, hereby incorporated by reference). This procedure was
modified to produce particles loaded with an appropriate enzyme
substrate in a desired particle size range for either lateral or
flow-through assembly and exhibiting adequate degradation kinetics
for a particular diagnostic assay format. To this end, the
concentrations of the components used in the polymerization process
were varied as described herein.
[0068] The PECA particles were prepared by polymerization of
monomers of 2-ethylcyanoacrylate (ECA) in an acidic aqueous medium
containing the desired enzyme substrate. Polymerizations were
performed using a copolymer of polyethylene oxide
(PEO)/polypropylene oxide (PPO) and a relatively high molecular
weight (10,000-50,000) dextran. The dispersion medium consisted of
an aqueous solution of hydrochloric acid (HCl) and phosphoric acid
(H.sub.3PO.sub.4). All components were of analytical reagent grade
and PEO/PPO copolymer was identical or similar to that commercially
available as F-88 Pluronic Polyol from BASF.
[0069] To produce monosize PECA enzyme substrate-loaded particles,
the ECA, PEO/PPO dextran, HCl, H.sub.3PO.sub.4 and enzyme substrate
concentrations were varied between 0.1-12.0% (v/v), 0.2-40 mg/ml,
0.3-100 mg/ml, 0.1-1.5 N, 0.1-15% (v/v), and 0.2-10 mg/ml,
respectively. En a typical polymerization process, 1 ml of ECA was
added dropwise to 100 ml of vigorously stirred (i.e., .gtoreq.1,000
rpm) solution containing PEO/PPO, dextran, phosphoric acid, enzyme
substrate and hydrochloric acid, at the concentration ranges
indicated above. The sealed vials containing the reaction mixture
were stirred at ambient temperature for between 6 and 64 hours and
the resultant reaction mixture was processed in a manner
conventional for the manufacture of monosize latex particles.
Following 2-3 routine washes of the particles with an acidic
(<pH 4.0) weak buffer solution, the particles were sized to
produce colloids loaded with an enzyme substrate within the desired
diameter size. Subsequently, the substrate-loaded PECA particles
were diluted in wash buffer to 0.1% to 5% solids. The suspension
was poured onto a nonbibulous support, abruptly frozen and
lyophilized. Alternatively, the supports containing
substrate-loaded particles were air dried overnight in a 45.degree.
C. oven or for 24 hours over P.sub.2O.sub.5 in vacuo.
[0070] To determine substrate release kinetics from the particles
in a lateral flow one-step device, the resulting materials were cut
into 10.times.4 mm rectangles and assembled as intermediate zones
into a device in a manner described in published PCT Application
WO92/12428, which is hereby incorporated by reference. Capture zone
membranes were prepared using appropriate enzyme for the particular
substrate-loaded PECA particles. Substrate release from PECA
particles in buffer solutions of different pH (1.5, 5.0, 6.0, 7.0,
7.4, 8.0, 9.6, 10.5) was studied by applying buffers to the sample
receiving zone and measuring color accumulation in the enzyme
capture zone for insoluble products or intensity of the color in
the absorbent pad for soluble products.
[0071] To determine substrate release kinetics from the particles
in a flow-through format, the unsupported media containing
substrate-loaded PECA particles were cut into 2 cm diameter circles
and assembled as a top layer of the device. Substrate release was
studied by applying buffer solutions as described above and
performing similar measurements as for a lateral flow one-step
device. The following observations were made: (1) The
substrate-loaded particles obtained with higher amounts of PEO/PPO
released substrate faster; (2) Decreasing HCl concentration in the
dispersion medium resulted in faster substrate release; and (3)
Particles degraded faster, releasing more efficiently with
increasing pH of the sample buffer medium.
[0072] 4. Enzyme Product Chemical Capture
[0073] In this embodiment, the enzyme product binds specifically to
the support which is coated or chemically derivatized with a
functional chemical group. Thus, the enzyme product is captured
specifically versus simply precipitating at the test line. This
technique is schematically diagrammed in FIG. 3. The capture
antibody 42 binds to the analyte-antibody-enzyme complex 44 at the
test line 46. Enzyme substrate (S) is then converted to product (P)
which binds to functional groups (R) at the test line 46 or
downstream to form a chemical bond with localization and
registration of enzyme by color or other means (i.e., fluorometric,
radiolabel, etc.).
EXAMPLE 4
Specific Chemical Capture of Enzyme Product
[0074] Compounds capable of reacting specifically with enzyme
products were incorporated into the capture zone. For the
substrates of alkaline phosphatase, galactosidase or glucuronidase,
diazotized amines were incorporated into the capture zone. Examples
of such compounds include diazonium salts ("fast salts") and
diazotized derivatives of polyamines, or natural polypeptides, such
as albumin, immunoglobulins and the like. After enzyme-mediated
formation of phenolic products, the diazotized amines react in situ
therewith to form stable colored azophenol compounds.
Alternatively, the chemical compounds were incorporated downstream
from the capture zone [i.e., either in the absorbent pad or in
additional intermediate zone(s)] to allow detection of enzyme
products away from the capture zone.
[0075] 5. Delay Zone Dissolution for Delivery of Substrate and/or
Conjugate
[0076] This lateral flow embodiment uses separate paths that employ
time-delayed dissolution of barrier zones for delivery of substrate
and/or conjugate reagents to the test area. This facilitates early
release and migration of antibody enzyme label (as conjugate),
which facilitates delayed release of substrate for catalysis.
Barrier 1 dissolves first to release enzyme-antibody conjugate,
while barrier 2 dissolves later to release substrate. The enzyme
product precipitates at the test line or is measured
downstream.
EXAMPLE 5
Delayed Enzyme Substrate Delivery Using Hydrogel Barrier Zones
[0077] Referring to FIG. 4, to construct delayed enzyme substrate
delivery using structural hydrogel barrier zones, the desired
enzyme substrate for the corresponding enzyme was dissolved in the
pH activity optimum buffer at 0.2-3 mg/ml and supplemented with 10
mg/ml BSA prepared in the same buffer. The enzyme substrate mixture
was then lyophilized in a nonbibulous support 50 such as polyester
or polyacrylic spunlace fabric. A printed hydrophobic polyurethane
barrier line 52 separates nonbibulous support 48 (no substrate)
from nonbibulous support 50 (containing substrate). The
enzyme-antibody label pad 58 was prepared by pouring the
enzyme-antibody conjugate (5-50 .mu.g/ml) supplemented with the
appropriate activators and stabilizers onto a similar support
followed by lyophilization. Capture zone nitrocellulose membranes
50 were spotted with anti-hCG polyclonal antibody at 1 to 5 mg/ml
and blocked with 10 mg/ml BSA or 0.2-2% (w/v) polyvinyl alcohol.
Fluid communication bridges 64 permit fluid flow between the sample
pad-label pad and label pad-capture zone.
[0078] In order to determine delayed enzyme substrate delivery in a
lateral flow "one-step" device, the materials described above were
cut and assembled as consecutive intermediate zones into a device
except that a 3-10 mm gap 54 was created between enzyme substrate
pad 50 and enzyme-antibody label pad 58. Subsequently, a strip of
Hypan TAU92 (Taupan) molecular hydrogel sponge was laid down
against the edge of the enzyme substrate pad, leaving a gap between
the enzyme-antibody label pad.
[0079] Structural (molecular) hydrogel barrier sponges 56 were
constructed using Hypan polymers (Hymedix International, Inc.,
Dayton, N.J.) which are hydrophilic acrylate derivatives with
multi-block copolymer structures of several sequences of amorphous
units with pendant hydrophilic groups derived from acrylic acid
(soft blocks) responsible swelling (flexibility) and several
sequences of organized, crystalline pendant polyacrylonitrile
structures (hard blocks) responsible for mechanical properties.
Particularly preferred polymers are highly swelling associative
polymers known as Hypan TN (Transient Network) Hydrogels, whose
hard blocks form reversible meltable transient clusters.
Subsequently, substrate release was studied by simultaneously
applying urine or serum samples with or without various levels of
hCG to zones 48 and 50 of a sample pad divided by a barrier zone 52
and measuring enzyme product accumulation in the capture zone 60
for insoluble products or in the absorbent pad 62 for soluble
products.
[0080] As shown in FIG. 5, sample is added concurrently to both
zones 48 and 50 of a sample pad that has-been divided by a barrier
zone 52 that-has been printed onto Sontara spunlace fabric. Zone 50
is loaded with enzyme substrate that has been lyophilized. Due to
hydrophobic barrier 52, half of the sample volume sits in zone 50
until the hydrogel barrier zone 56 imbibes enough fluid to expand
and close the gap 54 separating zone 50 from the label pad 58.
Meanwhile, the fluid in zone 48 has made progress forward such that
the enzyme-antibody label has been picked up and the immune complex
formed at the capture line 61 within the nitrocellulose membrane
60. At a later time, the substrate picked up in zone 50 arrives at
the capture line 61 which now has immobilized antibody associated
therewith. The delay imposed by the expansion time required by
Hypan to close the gap has allowed time for bound/free separation
of uncomplexed antibody and analyte to occur before arrival of the
substrate.
EXAMPLE 6
[0081] Sequential Delayed Enzyme-Antibody and Enzyme Substrate
Release Using Hydrogel Barrier Zones
[0082] Referring to FIG. 5, in a variation of Example 5, a 3-10 mm
gap 54 was also created between the enzyme-antibody zone 58 and the
capture zone 60, and a strip of Hypan sponge 66 was laid down
against the edge of the enzyme-antibody label pad 58, leaving a gap
54 between the capture zone 60 and the label pad 58. To achieve
sequential delayed release of enzyme-antibody label after initial
incubation with the sample (to increase immunological efficiency),
followed by delayed enzyme substrate release from the enzyme
substrate zone, different widths of sponge were used to facilitate
desired swelling time.
[0083] The performance of the device was tested by simultaneously
applying urine or serum samples without or with various levels of
hCG to the enzyme substrate zone 50 and the enzyme-antibody label
zone 58, and measuring enzyme product accumulation in the capture
zone 60 for insoluble enzyme products or in the absorbent pad 62
for soluble products.
[0084] The Hypan sponge 66 increases the dwell time of the sample
analyte within the label pad 58 to promote increased immunological
efficiency. As sponge 66 swells, it closes the gap between the
label pad 58 and capture zone 60, permitting fluid communication
between these sections. Hypan sponge 56 closes somewhat later such
that delivery of the enzyme substrate occurs after bound/free
separation of surplus enzyme conjugate is essentially complete.
EXAMPLE 7
[0085] Sequential Delayed Enzyme-Antibody and Enzyme Substrate
Release Using Enterosoluble Coating Barrier Zones
[0086] In a variation of the example just described, the
enterosoluble methacrylic acid copolymer coatings similar to those
used for solid pharmaceutical dosage (Eudragit S100; Rohm Tech.,
Inc., Malden, Mass.) were used to achieve sequential release of
enzyme-antibody label followed by enzyme substrate. For example, to
achieve sequential delayed release of alkaline phosphatase
(ALP)-labeled anti-hCG monoclonal antibody conjugate followed by
the ALP substrate, the conjugate (0.5-10 .mu.g/ml) was prepared in
50 mM Tris-HCl buffer (pH 8.5) containing 5 mg/ml BSA, 1 mM
MgCl.sub.2 and 0.1 mM ZnSO.sub.4, then lyophilized into a
nonbibulous support as described above. The desired ALP substrate
(i.e., 3-indoxylphosphate) was dissolved at 0.4-3 mg/ml in 0.1 M
2-amino-2-methyl-1-propanol (AMP) buffer (pH 10.5) and lyophilized
into a nonbibulous support as described above. Capture zone
membranes were spotted with anti-hCG monoclonal antibody at 1-5
mg/ml and blocked with 10 mg/ml BSA or 0.2-2% (w/v) polyvinyl
alcohol.
[0087] Subsequently, the materials were cut and assembled as
consecutive intermediate zones into a lateral flow one-step device
as shown in FIG. 6, except that 2-5 mm gaps were created between
the ALP substrate pad and the ALP-monoclonal antibody label pad as
well as between the label pad and capture zone. In order to create
the desired barrier zones, two anionic copolymer mixtures based on
methacrylic acid (MAA) and methylmethacrylate (MMA) were prepared
in methanol at 3-15% (w/v). Copolymer A comprised a MAA to MMA
ratio'of 12:88 and Copolymer B comprised a MAA to MMA ratio of
3:97. Copolymers A and B were dried separately in nonbibulous
supports 68 and 70, respectively. Subsequently, the gap between the
ALP substrate pad and the ALP-monoclonal antibody (MAb) label pad
(see FIG. 5) was closed by attaching rectangles of support 70
containing. Copolymer B with a 1 mm overlap on both sides.
Similarly, the gap between the label pad and capture zone (see FIG.
5) was closed using support 68 containing Copolymer A.
[0088] Copolymer B, having the lower ratio of MAA to MMA, will
dissolve more slowly than copolymer A and will enable delivery of
the enzyme substrate to the capture zone later than enzyme-antibody
conjugate. Due to the generally high pH (8-9.5) required by the ALP
enzyme reaction, the methacrylic acid units in the copolymer ionize
to bring about breakdown and dissolution of the copolymer.
Copolymer A, with its higher mole % MAA, dissolves relatively
quickly, allowing earlier delivery of enzyme-antibody conjugate to
the capture line, but increases the dwell time of the analyte
within the label pad sufficiently to enhance immunological
efficiency.
[0089] Performance of the device was assessed by simultaneously
applying urine samples, with or without hCG, to the ALP substrate
pad and ALP-MAb label pad and performing similar measurements as
described above.
EXAMPLE 8
[0090] Sequential Delayed Release of Enzyme-Antibody Label and
Enzyme Substrate Using Biodegradable Phospholipid Barrier Zones
[0091] In a variation of Example 7, biodegradable phospholipid
barriers were used to achieve sequential release of antibody-enzyme
label followed by enzyme substrate in the lateral flow one-step
device. ALP-MAb label pad and ALP substrate pad were prepared as
described in Example 7, except that the nonbibulous supports were
supplemented with various amounts of phospholipase (1-20 .mu.g/ml),
with the concentration in the substrate pad lower than that in the
label pad to allow dissolution of the enzyme-antibody barrier zone
prior to dissolution of the substrate barrier zone. Subsequently,
the pads and capture zone were assembled into a lateral flow
one-step device with 2-5 mm gaps left between the substrate and
label pad, as well as between the label pad and capture zone. To
create the desired barrier zones, liposomes were prepared as
described in Example 2 except that enzyme substrate was not
included and liposomes were not extruded through the membranes.
Liposomes were dried in a nonbibulous support and gaps were closed
using the liposome support.
[0092] Performance of the device was assessed by simultaneously
applying urine samples with or without hCG) to the ALP substrate
pad and ALP-Mab label pad, and performing similar measurements as
described in the previous example.
[0093] 6. Delayed Release of Enzyme Substrate Using Protected
Enzyme Substrate
[0094] This embodiment encompasses multi-enzyme systems wherein the
time-dependent production of the end product of the first enzyme
reaction is the substrate for the enzyme-antibody conjugate
reaction. Referring to EIA device 72 shown in FIG. 7, substrate
(S1) for the first enzyme (E1) is immobilized in excess in a sample
pad 74. After immunological separation upon lateral flow of a
solution containing an analyte (X), the time-dependent catalysis of
the substrate for the enzyme-antibody complex (E2-Ab2) begins with
E1 acting on S1 to produce substrate 2 (S2) in label pad 76. When
the concentration of S2 reaches an effective threshold
concentration for E2 to become active, the product (P) begins to
form in the capture zone 78. By this time, sufficient delay has
occurred so that most or all of the target analyte has been
recognized and captured at the test line 80. The product (P) is
then directly quantified by absorbance as a colored product,
electrochemically as an electroactive substance, fluorometrically,
luminescently or is free to participate in a secondary chemical
reaction so as to make possible one of these modalities of
detection either as a captured material or measured downstream as a
soluble material. Unbound analyte and antibodies flow to absorbent
zone 82. A specific example of this EIA is described in Example
9.
EXAMPLE 9
[0095] In this example, referring to FIG. 7,
E2-Ab2=.beta.-D-galactosidase- -anti-hCG; E1=ALP;
S1=o-nitrophenyl-.beta.-D-galactopyranoside-6-phosphate- ;
S2=o-nitrophenyl-.beta.-D-galactopyranoside; Ab1=goat-anti-hCG;
P=o-nitrophenol. The conjugate of E. coli .beta.-D-galactosidase
and an anti-hCG MAb was prepared in 50 mM HEPES, pH 7.5 containing
1 mM MgCl.sub.2 and diluted to 0.5-10 .mu.g/ml in the same buffer
supplemented with 5 mg/ml BSA (the conjugate diluent). ALP was
added to the conjugate solution to a final concentration of 10
.mu.g/ml to 1 mg/ml, and the final mixture was lyophilized into a
nonbibulous support as described above (the sample pad).
O-nitrophenyl-.beta.-D-galactopyranoside-6-phosph- ate
cyclohexylammonium salt (S1) (Sigma) was dissolved at 0.1-1.5 mg/ml
in the conjugate diluent and lyophilized into a nonbibulous support
as described above (the label pad). The capture zone was prepared
as previously described. Subsequently, the just-described materials
were cut and assembled as consecutive intermediate zones into a
lateral flow one-step device.
[0096] Performance of the device and delayed release of enzyme
substrate was assessed by applying urine samples (with or without
hCG) to the label pad and performing similar measurements as
previously described for a lateral flow one-step device. The
reaction scheme is summarized in FIG. 8 in which
S1=O-nitrophenyl-.beta.-D-galactopyranoside-6-phosphate;
S2=O-nitroophenyl-.beta.-D-galactopyranoside and P1=O-nitrophenol.
The product was measured colorimetrically by absorbance.
[0097] Alternative "protected" substrates for the
.beta.-D-galactosidase-E- 2 conjugate and corresponding enzymes E1
are shown in FIG. 9. Table 2 shows alternative "protected"
substrates for the E2-Ab conjugate when the .beta.-D-galactose
substrate core is replaced with other sugars, as well as the
corresponding enzymes E2. FIG. 10 shows "protected" substrates for
urease as E1 in the E1-Ab conjugate.
[0098] In each case in Table 2 below, the sugar group in the left
column would be protected by the presence of a phosphate group or
other protecting group as indicated in FIG. 9. The nitrophenyl,
bromophenyl, chlorophenyl, methoxyphenyl or aminophenyl groups are
removed by the corresponding enzyme in the right column and the
product quantified by the appropriate modality.
2 TABLE 2 Substitute for .beta.-D-galactase Enz.sub.2
2-acetamido-2-deoxy-.beta.-D- N-acetyl glucosaminidase (EC
glucosaminide 3.2.1.52) .alpha.-L-arabinofuranose
.alpha.-L-arabinofuranosidase III .beta.-D-cellobiose exglucanase
N,N'-diacetyl-.beta.-D-chitobiose chitobiosidase
.alpha.-L-fucopyranose .alpha.-L-fucosidase (EC 3.2.1.51)
.beta.-D-fucopyranose .beta.-D-glycosidase .alpha.-D-galactose
.alpha.-galactosidase (EC 3.2.1.23) .beta.-D-glucose
.beta.-glucosidase (EC 3.2.1.21) .alpha.-D-glucose glucansucrase
.beta.-D-glucopyranosiduronic acid .beta.-D-glucoronidase
.alpha.-D-maltoheptose .alpha.-amylase (EC 3.2.1.1)
.alpha.-D-maltohexose .alpha.-amylase (.alpha. or
.beta.)-mannopyranose (.alpha. or .beta.)-mannosidase
[0099] It should be noted that the present invention is not limited
to only those embodiments described in the Detailed Description.
Any embodiment which retains the spirit of the present invention
should be considered to be within its scope. However, the invention
is only limited by the scope of the following claims.
* * * * *