U.S. patent application number 10/035014 was filed with the patent office on 2003-06-26 for lateral flow assay devices and methods for conducting assays.
This patent application is currently assigned to Kimberly-Clark Worldwide, Inc.. Invention is credited to Song, Xuedong, Wei, Ning.
Application Number | 20030119203 10/035014 |
Document ID | / |
Family ID | 21880091 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030119203 |
Kind Code |
A1 |
Wei, Ning ; et al. |
June 26, 2003 |
Lateral flow assay devices and methods for conducting assays
Abstract
Disclosed is a method and apparatus for employing, in a lateral
flow assay, multiple control lines to assist in improving the
sensitivity of such an assay. Analytes of interest may be
quantified in a lateral flow assay by conducting internally derived
calibrations by quantifying the analyte and calibrating the assay
device, at essentially the same time, on the same device. That is,
calibration and sample testing may occur simultaneously, improving
sensitivity, and reducing errors that otherwise may be introduced
by comparing data produced in one assay with data or reference data
produced in a different assay. A multi-point calibration technique
may be employed. Visual spectrophotographic reading devices may be
employed to compare intensity of signals generated by probes
attached to the analyte with probes associated with control lines
upon a calibration zone.
Inventors: |
Wei, Ning; (Roswell, GA)
; Song, Xuedong; (Roswell, GA) |
Correspondence
Address: |
John E. Vick, Jr.
Dority & Manning
Attorneys at Law, P.A.
P.O. Box 1449
Greenville
SC
29602
US
|
Assignee: |
Kimberly-Clark Worldwide,
Inc.
|
Family ID: |
21880091 |
Appl. No.: |
10/035014 |
Filed: |
December 24, 2001 |
Current U.S.
Class: |
436/514 |
Current CPC
Class: |
G01N 33/54393 20130101;
G01N 33/558 20130101; G01N 33/54386 20130101 |
Class at
Publication: |
436/514 |
International
Class: |
G01N 033/558 |
Claims
What is claimed is:
1. A lateral flow assay for detecting the quantity of analyte
residing in a test solution, the assay comprising: (a) a plurality
of probes, the probes being configured for generating a detectable
signal, wherein the probes are capable of combining with analyte to
form a probe conjugate analyte complex; (b) a membrane, the
membrane being configured for mobilizing a test solution containing
both probes and probe conjugate analyte complexes, the membrane
comprising: (i) a detection zone, the detection zone having
deposited thereon an immobilized first capture reagent, wherein the
immobilized first capture reagent is configured for bonding with
probe-conjugate analyte complexes to form sandwich complexes that
generate signals; (ii) a calibration zone, the calibration zone
comprising at least first and second control lines, wherein the
first and second control lines each have applied thereon a
predetermined amount of a second capture reagent, the second
capture reagent being configured to immobilize probes upon the
first and second control lines to form control probe complexes
capable of generating a control signal.
2. The lateral flow assay of claim 1, further comprising: (c) a
comparison means for comparing the intensity of signals generated
by control probe complexes positioned upon said first and second
control lines with the intensity of signals generated by sandwich
complexes positioned upon the detection zone.
3. The lateral flow assay of claim 2, further wherein a calibration
curve is generated using signal intensity data generated by control
probe complexes positioned upon the control lines.
4. The lateral flow assay of claim 1 in which the probe comprises a
microparticle that is capable of generating a visual signal.
5. The lateral flow assay of claim 4 in which the microparticle is
capable of generating a color intensity.
6. The lateral flow assay of claim 1 in which the probe is capable
of generating a fluorescent signal.
7. The lateral flow assay of claim 1 in which the probe comprises a
microparticle.
8. The lateral flow assay of claim 7 in which the microparticle
exhibits a color intensity.
9. The lateral flow assay of claim 2 in which a detection device is
provided for detecting signals generated by control probe complexes
positioned upon respective control lines with the intensity of
signals generated by sandwich complexes positioned upon the
detection zone.
10. The lateral flow assay of claim 2 wherein the probe comprises a
microparticle, further wherein the comparison means comprises a
device adapted for comparing intensity of light signals generated
from the first and second control lines with the intensity of
signals generated by sandwich complexes positioned upon the
detection zone.
11. The lateral flow assay of claim 10, further wherein a
calibration curve is generated using signal intensity data from the
first and second control lines.
12. The lateral flow assay of claim 11 in which an automated system
of generating the curve and performing the comparison is
provided.
13. A method of detecting the quantity of an analyte present in a
test solution, the method comprising: (a) providing a membrane with
a test solution containing analyte; (b) providing a plurality of
probes and probe conjugates upon the membrane; (c) binding the
probe conjugates with the analyte to form probe analyte conjugates;
(d) wherein the membrane comprises a first end and a second end,
the membrane having a first capture reagent immobilized upon a
detection zone, the membrane being configured for mobilizing a test
solution containing probe-analyte conjugates from the first end to
the second end of the membrane; (e) capturing within a detection
zone probe-analyte conjugates, thereby forming immobilized signal
generating sandwich complexes within the detection zone; (f)
providing a calibration zone upon the membrane, the calibration
zone comprising at least first and second control lines having
predetermined amounts of a second capture reagent immobilized upon
said control lines; (g) capturing, with the second capture reagent,
probes upon the first and second control lines by forming control
probe complexes upon said control lines; (h) generating a first set
of control signals from the control probe complexes; (i) generating
a second set of measured signals from the sandwich complexes in the
detection zone; and (j) comparing the control signals to the
measured signals to determine the quantity of analyte present in
the test solution.
14. The method of claim 14, wherein prior to step (j), the method
further comprises the step of generating a signal intensity curve,
further wherein step (j) comprises comparing control signals to the
signal intensity curve to provide a determination of the quantity
of analyte present in a test solution.
15. The method of claim 14 in which the probe comprises a
microparticle that is capable of generating a signal.
16. The method of claim 15 in which the microparticle is capable of
generating a color intensity.
17. The method of claim 16 in which the microparticle is a latex
bead.
18. The method of claim 14 in which the probe is capable of
generating a fluorescent signal.
19. The method of claim 13 in which a third control line is
employed.
20. The method of claim 19 in which a fourth control line is
employed.
21. The method of claim 19 in which the probes comprise latex
beads.
22. The method of claim 21 in which latex beads further include
coloring agents for providing a visually detectable signal.
Description
BACKGROUND OF THE INVENTION
[0001] Membrane-based test devices, particularly devices used in
diagnostic medicine, employ a variety of internal and external
calibrators to provide a qualitative or a quantitative result for
an analyte of interest in a test solution. One type of
membrane-based test device is a lateral flow assay.
[0002] In general, lateral flow assays are membrane-based test
devices in which a sample that is suspected of containing the
analyte of interest is placed at or near one end of a membrane
strip. The sample is carried to the opposite end of the membrane
strip by a liquid phase that traverses the membrane strip by
capillary action. While traversing the membrane strip, the analyte
in the test sample, if any, encounters one or more "capture"
reagents with which it may react to produce a detectable
signal.
[0003] The early types of immunochromatography devices, such as
those taught in U.S. Pat. No. 4,366,241 (Tom et al.), lacked an
internal reference. Later devices, such as taught in U.S. Pat. No.
4,374,925 (Litman) do indeed employ an internal reference. In some
instances, the internal references used have required fairly
laborious cross-referencing to achieve results.
[0004] One known membrane-based test device is the dipstick. The
dipstick is a stick having a small reagent impregnated membrane
(stripped with capture reagents on different zones and wicking pads
on one end and the other) end for dipping into a test solution
either containing or suspected of containing the analyte of
interest. The dipstick membrane (hereinafter "dipstick") develops a
color that is proportional to the concentration of the analyte of
interest in the test sample. Typically, the user determines the
concentration of the analyte by comparing the color on the membrane
to the color on an external calibration, such as a series of
colored plates that are printed on a label. This is a subjective
determination.
[0005] External calibration of a dipstick, via colored plates,
provides several problems. First, it is difficult to accurately
match the color of the plates with the color on the dipstick.
Secondly, the color on the plates would not fade in proportion to
the adverse conditions affecting the color on the dipstick.
Further, the color on the plates would at best only be accurate for
a particular set of reaction conditions.
[0006] Many of such devices and methods rely upon calibration to
provide valid and meaningful results for semi-quantitative and
quantitative detections. Calibration methods are often critical to
provide accurate, reliable and reproducible results, especially
when the environments and conditions under which the measurements
are commenced are not carefully controlled. Two calibration
methods, external and internal calibrations, are commonly employed.
In the external calibration method, a standard curve is usually
obtained from standard samples containing a series of a known
amount of analyte, and the results obtained from the samples are
then compared with the standard curve to extract the information
regarding the presence and/or amount of the analyte in the sample.
External calibration methods are often subject to interference from
environmental and batch-to-batch variations, and sometimes are not
reliable. When an instrument or measuring device is used, it is
also subject to interference from the instability of the instrument
or device.
[0007] In general, lateral flow assay methods are limited in their
sensitivity by not using an internal calibration mechanism that
takes into account widely varying differences in temperature, flow
conditions, capillary action, pressure, and other factors that
affect movement or deposition of analyte on a membrane or support.
Any system that compares the migration characteristics of analyte
on a given test strip with references taken on another separate
strip at another time and place will not achieve maximum
sensitivity and accuracy.
[0008] What is needed in the industry is a lateral flow assay
system and method having improved sensitivity in relation to
existing methods and devices. The invention of this application is
directed to such an application.
SUMMARY OF THE INVENTION
[0009] A lateral flow assay system, apparatus, and method are
provided in the invention. The assay provides a method to detect
the quantity of analyte residing in a test solution. The assay
further comprises a probe. The probe may be of various types.
Probes are configured for generating a detectable signal. Probes
may be covalently reacted with antibody to form probe-conjugates
and this conjugate then may travel to react with analyte to form a
probe-conjugate analyte complex (or "sandwich"). Once this species
is immobilized upon a detection zone it is referred to as a
"sandwich complex". All these described species may be able to
migrate on a membrane and may be used for analyte detection.
[0010] A membrane may be configured to provide a sample pad,
conjugate pad, detection zone, calibration zone and wicking pad. On
the conjugate pad the probes and probe-conjugates may be dried down
upon the membrane and made available for analyte as the analyte
moves along the membrane from one end of the membrane to the other
end. When analyte molecules join probe conjugates, they form probe
conjugate analyte complexes, which are capable of becoming
mobilized, and moving to a detection zone.
[0011] Upon the detection zone, a first capture reagent may be
immobilized. The first capture reagent may be composed of any
ligand specific binder, thus, one example of such a capture reagent
is an antibody. This first capture reagent may immobilize such
probe-conjugate analyte complexes to form a "sandwich complex", or
"sandwich" upon the detection zone.
[0012] A calibration zone also may be provided. The calibration
zone comprises at least two control lines, however, in some
applications of the invention, three, four, or more control lines
may be provided. The control lines may have applied thereon a
predetermined amount of a second capture reagent. The second
capture reagent may be configured to immobilize probe-conjugates or
probes that migrate to the control lines without analyte, thereby
positioning them for generating a calibration or control
signal.
[0013] In general, each control line may have a predetermined
amount of the second capture reagent. Thus, commonly, the first
control line nearest the detection zone may have the least amount
of second capture reagent, while the last control line furthest
from the detection zone may have the greatest amount of second
capture reagent. In some applications, the control lines may vary
by predetermined amount from each other, so as supply a suitable
calibration curve, as shown in herein and described with reference
to FIG. 2.
[0014] Some applications of the invention utilize visual
comparisons. In other applications, reading devices such as
reflectometer or spectrophotometer may be employed to compare the
intensity of signals generated with reference standards that are
generated in the assay. Spectrophotometric methods may be employed
to compare the intensity of signals generated with reference
standards that are generated in the course of the assay.
[0015] In some applications, a calibration data curve may be
generated using signal intensity data generated from the control
lines. The curve may provide a "look-up table" that may be
automatically applied in an algorithm of an analytical
instrument.
[0016] The probe in some cases may comprise a microparticle that is
capable of generating a visual signal, such as a latex bead, for
example, that includes red or blue or another colored dye. In other
applications, the probe may generate fluorescent signals that are
detectable and are proportional to the amount of such species in a
given zone.
[0017] The invention may be directed to a method for detecting the
quantity of an analyte in a test solution. The method may include
applying a plurality of probes that are configured for migrating to
preselected locations and, when they become sandwich complexes,
generating a detectable signal.
[0018] Internal calibration methods are useful because such methods
may provide more accurate, more reliable and more reproducible
results than external calibration methods. Using internal
calibration methods, signals related to the analyte in the sample
are usually measured at the same time and/or upon the same membrane
device that generates the calibration signals. The simultaneous
measurements can eliminate some potential interference to provide
more consistent and sensitive detections.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] A full and enabling disclosure of this invention, including
the best mode shown to one of ordinary skill in the art, is set
forth in this specification. The following Figures illustrate the
invention:
[0020] FIG. 1 is a top view of one embodiment of the invention,
showing a lateral flow assay having three control lines in a
calibration zone;
[0021] FIG. 1A shows a perspective schematic view of the movement
of fluids and the formation of complexes upon the surface of the
membrane strip of a lateral flow assay, showing the membrane strip
after a test sample containing analyte has been applied to the
sample pad,
[0022] FIG. 1B shows the same schematic view of the membrane test
strip shown in FIG. 1A, but at a later time after migration of
fluids have occurred and complexes have formed; and
[0023] FIG. 2 shows a calibration curve that may be used in some
applications of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Reference now will be made to the embodiments of the
invention, one or more examples of which are set forth below. Each
example is provided by way of explanation of the invention, not as
a limitation of the invention. In fact, it will be apparent to
those skilled in the art that various modifications and variations
can be made in this invention without departing from the scope or
spirit of the invention.
[0025] The invention makes it possible to use multiple control
lines to quantify analytes of interest in a lateral flow assay
format. In particular, the method and apparatus of the invention
relate to conducting internal calibrations by: (1) quantifying the
analyte and (2) calibrating the assay device, at about the same
time, on the same membrane device. That is, calibration and sample
testing may occur on the same device, by affording a built-in
calibration data curve generated using the testing device. A
multi-point calibration technique may be employed in a lateral flow
assay format.
[0026] The method may be used for quantitative and
semi-quantitative detection. The probes used may reveal color
intensity, fluorescence intensity, as examples. The probes for
control lines may be microparticles such as latex beads, for
example, labeled with essentially any signal generating species.
Alternately, the probes may comprise labeled latex beads further
conjugated with antibodies, as further described herein. The
antibodies may be dried upon the conjugate zone of the
membrane.
[0027] Various amounts of predetermined capture reagents may be
provided on solid substrates, such as porous membranes, to form
multiple control lines for calibration purposes. In yet another
embodiment, the capture reagents may be antibodies. In yet another
embodiment, the capture reagents may be any molecules which are
capable of forming strong interactions with probes and/or probe
conjugates.
[0028] The membrane-based device of the invention comprises several
components, including a membrane, a sample pad, a conjugate pad and
a wicking pad, or a combination of these items. The membrane
typically includes at least two zones, that is, a detection zone
and a control zone. A sample pad contacts one end of the conjugate
pad.
[0029] One design of the assay device includes a liquid sample flow
direction through a sample pad, conjugate pad, detection zone of
the membrane, control zone of the membrane, and wicking pad. In
general, the wicking pad assists in promoting capillary action and
fluid flow one-way through the membrane of the device, and the
wicking pad "pulls" the liquid containing the analyte along the
membrane from one end of the membrane to another end of the
membrane.
[0030] Turning now to FIG. 1, a lateral flow assay 20 is provided
in top view. The lateral flow assay 20 comprises a membrane 23 as a
solid support, and includes a sample pad 21. The sample pad 21 is
configured to receive a liquid sample containing analyte 40 (seen
in FIG. 1A). A conjugate pad 22 is provided further "downstream" of
capillary movement direction 29, as shown by the arrow on the left
side of the FIG. 1.
[0031] Conjugate pad 22 typically contains probes 41 and probe
conjugates 42 (see FIG. 1A) in a form that makes the probe
conjugates available for bonding with the analyte 40 as the analyte
40 passes from the sample pad 21. A typical method employs
microparticles as probes 41, and their conjugate deposited on the
conjugate pad 22. Such particles may be comprised of latex, or
other suitable material, as further described herein. Latex
microparticles, when used as probes, may be colored with dyes that
are visible to the eye, or to detection apparatus. Sometimes a
probe 41 emits light (as in the case of fluorescence methods), or
the probe 41 may be detected by other techniques once it has
migrated and complexed, as further described herein.
[0032] A detection zone 31 is shown in FIG. 1. The detection zone
31 may comprise an immobilized capture reagent along detection line
24, as further described in connection with FIG. 1A. A calibration
zone 32 is shown with three control lines 25-27. A wicking pad 28
also is shown.
[0033] Referring to FIG. 1A, a membrane 23 is provided in which
molecules of the analyte 40 to be detected have been deposited upon
the sample pad 21. The analyte 40, which is fluidized, moves in the
direction of the arrow shown in FIG. 1A from one end of the
membrane to the other.
[0034] FIG. 1A shows a schematic view in which the components of
the assay 20 are enlarged for purposes of explanation. FIG. 1A
shows membrane 23 at a point when the test sample or test solution
has been applied to the sample pad 21 for only a short period of
time. Probes 41 are seen upon the conjugate pad 22. Typically,
probes 41 are dried or immobilized upon the conjugate pad 22. Probe
conjugate 42 also is immobilized upon the conjugate pad 22. Once
molecules of analyte 40 bind with probe conjugates 41-42, they
become probe conjugate analyte complexes (such as probe analyte
conjugate complex 49-50 shown in FIG. 1B) which are mobile along
the membrane 23.
[0035] The detection zone 24 is shown in FIG. 1A having several
capture reagents 43a-c immobilized upon the detection zone 44.
These capture reagents 43a-c serve as stationary binding sites for
the probe analyte conjugate complexes 49-50 which migrate to them,
as further shown in FIG. 1B. The chemical identity of capture
reagents 43a-c is further described herein.
[0036] The calibration zone 32 is shown near the end of the
membrane 23. The calibration zone 32 provides at least two or more
control lines, shown in this particular example as control lines
25-27. In many cases, the control lines are provided with a
"binder" which is used to bind probe 41 molecules which pass the
length of the membrane 23. The "binder" may include an antibody,
such as second antibody 47a-c shown in FIG. 1B. The control lines
25-27 have a certain and specific amount of second antibody 47a-c
provided thereon, so that in a saturated environment having large
amounts of probe 41 or probe conjugate 42, they will reveal a
specific, exact, and predetermined level of signal intensity. It
will be recognized that thousands of molecules are provided upon
the membrane 23, but the FIGS. 1A-1B show only a few molecules, for
purposes of illustration.
[0037] FIG. 1B shows the membrane 23 of FIG. 1A at a later point in
time after the solution has migrated as shown in the arrow of FIG.
1B. A probe conjugate complex 49 and a probe conjugate complex 50
may be seen migrating from the conjugate pad 22 to the detection
zone 44. Several sandwich complexes 45a, b and c have formed by the
union of probe conjugate complexes similar to that shown as probe
conjugate complex 49 with capture reagent 43a-c (FIG. 1A), forming
an immobilized sandwich complex 45a-c within the detection
zone.
[0038] Probes 41 and probe conjugates 42 which are not bound to
analyte, also become mobile through the detection line 24 (see for
example probe 52), and continue beyond the detection line 24 to the
calibration zone 32. The calibration zone 32 includes calibration
lines 25, 26, and 27. The calibration lines 25-27 may be pre-loaded
upon the membrane 23 with a second capture reagent, such as second
antibody 47, and thus an intensity of color is generated upon the
calibration lines 25-27 upon migration of probe 41 or probe
conjugates 42. A control probe complex 56 may be formed when a
probe 41 attaches. Likewise, a control probe conjugate complex 57
may be formed by attachment of a probe conjugate 42. Both probes 41
and probe conjugates 42 are available for binding in the detection
zone 32.
[0039] An excess of probe molecules, such as dyed microparticles,
can be employed in the assay 20, so that each calibration line
25-27 reaches its full and predetermined potential for signal
intensity. That is, the amount of probe 41 molecules that are
deposited upon calibration lines 25-27 are predetermined because
the amount of capture reagent employed on the calibration lines
25-27 is set at a predetermined and known level. A comparison may
be made between the intensity levels of the calibration lines 25-27
and the detection line 24 to calculate the amount of analyte 40
present in the sample or solution. This comparison step may occur
visually, or with the aid of a reading device (not shown). Wicking
pad 28 receives the fluid that has migrated through membrane
23.
[0040] The membrane 23, or solid support, which is employed in the
assay may be a cellulose ester. Nitrocellulose is known provides
good results in some applications. It should be understood that the
term "nitrocellulose" refers to nitric acid esters of cellulose,
which may be nitrocellulose alone, or a mixed ester of nitric acid
and other acids, in particular, aliphatic carboxylic acids having
from one to seven carbon atoms.
[0041] Although nitrocellulose may be a suitable material for
producing the membrane, it is to be understood that other materials
may also be employed for such solid supports including but not
limited to nylon, rayon, and the like.
[0042] In accordance with a particular preferred embodiment, the
pore size of the solid support is such that the probe, when bound
to the analyte remains on the surface of the membrane 23. Thus, for
example, good results have been obtained with nitrocellulose having
a pore size of from about 0.1 to 0.5 microns.
[0043] It is to be understood that the invention can be configured
for detecting a broad range of analytes, including therapeutic
drugs, drugs of abuse, hormones, vitamins, proteins (including
antibodies of all classes), peptides, steroids, bacteria, viruses,
parasites, components or products of bacteria, fungi, allergens of
all types, antigens of all types, products or components of normal
or malignant cells, and the like.
[0044] The following analytes are examples of analytes that may be
tested using the present invention: T.sub.4, T.sub.3, digoxin, hCG,
insulin, theophylline, luteinizing hormone, organisms causing or
associated with various disease states, such as streptococcus
pyogenes (group A), Herpes Simplex I and II, cytomegalovirus,
chlamydiae, and others known in the art.
[0045] U.S. Pat. No. 4,366,241 (Tom et al.) lists at columns 19-26
a variety of potential analytes of interest that are members of an
immunologic pair, including proteins, blood clofting factors,
hormones, microorganisms, pharmaceutical agents, and vitamins. Any
of these analytes are suitable for use as the analyte in present
invention.
[0046] Other examples of preferred ligands or analytes that may be
detected include the following: human bone alkaline phosphatase
antigen (HBAPAg); human chorionic gonadotropin (hCG); human
luteinizing hormone (hLH); human follicle stimulating hormone
(hFSH); creatine phosphokinase MB isoenzyme; ferritin;
carcinoembryonic antigen (CEA); prostate specific antigen (PSA);
CA-549 (a breast cancer antigen); hepatitis B surface antigen
(HBsAg); hepatitis B surface antibody (HBsAb); hepatitis B core
antigen (HBcAg); hepatitis B core antibody (HBcAb); hepatitis A
virus antibody; an antigen of human immunodeficiency virus HIV I,
such as gp 120, p66, p41, p31, p24 or p17; the p41 antigen of HIV
II; and the respective antiligand (preferably a monoclonal
antibody) to any one of the above ligands. The HIV antigens are
described more fully in U.S. Pat. No. 5,120,662 and in Gelderblood
et al., Virology 156:171-176 1987, both of which are incorporated
herein by reference.
[0047] As used herein, the term "probe-conjugate" refers to a
species that is capable of carrying an analyte in a lateral flow
assay to form a probe conjugate complex, which binds with a first
capture reagent in the detection zone 24 to become a "sandwich
complex" in detection area or detection zone 24.
[0048] As used herein, the term "microparticle" is a more specific
reference to a particular type of probe, and may include any beads
or probes to which an antibody may be bound, whether covalently, or
non-covalently such as by adsorption. An additional requirement for
some particles that are used in a quantitative assay is that the
particle contributes a signal, usually light absorption, which
would cause the zone in which the particles were located to have a
different signal than the rest of the membrane 23.
[0049] The microparticle employed typically must be capable of
being retained by the membrane 23. For example, when a
microparticle is subject to liquid flow, the microparticle must be
capable of remaining substantially immobilized. The microparticles
may be of any shape but are preferably spherical. The nature of the
microparticle may vary widely. It may be naturally occurring or
synthetic. It can be a single material, a few materials, or a
combination of a wide variety of materials. Naturally occurring
microparticles include nuclei, mycoplasma, plasmids, plastids,
mammalian cells (e.g., erythrocyte ghosts), unicellular
microorganisms (e.g., bacteria) and the like. Synthetic
microparticles may be prepared from synthetic or naturally
occurring materials, or combinations thereof.
[0050] For example, latex microparticles may be prepared from a
synthetic material such as styrene. Other microparticles may be
prepared from naturally occurring materials, such as
polysaccharides, e.g., agarose, or the like. (See, e.g., Gould, et
al., U.S. Pat. No. 4,837,168, which describes the use of a variety
of particles.) Preferred microparticles are microspheres of latex
(i.e., a natural or a synthetic polymer) or glass; more preferably
microspheres of latex. The microspheres of glass or latex are also
referred to in the art as "beads" or "microbeads."
[0051] A typical size for such beads is about 0.3 microns, but the
invention may employ microparticles having greater or lesser size.
For example, the mean diameter for the microparticle component of
the present invention is within the range from about 0.01 microns
to about 100 microns and more typically from about 0.1 microns to
about 75 microns. The mean diameter and type of the microparticle
chosen for a particular application will depend upon the pore size
of the membrane and/or its composition.
[0052] Latex microparticles for use in the present invention are
commercially available as polymeric microspheres of substantially
uniform diameter (hereinafter "polymeric microspheres"), such as
from Bangs Laboratories of Carmel, Ind., or Dow Chemical Co. of
Midland, Mich. Although any polymeric microsphere that is capable
of adsorbing or of being covalently bound to a binding partner may
be used in the present invention, the polymeric microspheres
typically are composed of one or more members of the group
consisting of polystyrene, butadiene styrenes, styreneacrylic-vinyl
terpolymer, polymethylmethacrylate, polyethylmethacrylate,
styrene-maleic anhydride copolymer, polyvinyl acetate,
polyvinylpyridine, polydivinylbenzene, polybutyleneterephthalate- ,
acrylonitrile, vinylchloride-acrylates and the like or an aldehyde,
carboxyl, amino, hydroxyl, or hydrazide derivative thereof.
[0053] The underivatized polymeric microspheres, such as
polystyrene, are hydrophobic and passively adsorb other hydrophobic
molecules, including most proteins and antibodies. Techniques for
adsorbing a protein or polypeptide on a hydrophobic particle are
provided in the publication by Cantarero, et al. "The Absorption
Characteristics of Proteins for Polystyrene and Their Significance
in Solid Phase Immunoassays," Analytical Biochemistry 105, 375-382
(1980); and Bangs, "Latex Immunoassays," J. Clin. Immunoassay, 13
127-131 (1980) both of which are incorporated herein by
reference.
[0054] Various procedures for adsorbing molecules on polymeric
microspheres are also described, in general terms, in Bangs, L. B.,
"Uniform Latex Particles," presented at a workshop at the 41st
National Meeting, Amer. Assoc. Clin. Chem., 1989, and available in
printed form from Seragen Diagnostics Inc., Indianapolis, Ind.; or
Galloway, R. J., "Development of Microparticle Tests and
Immunoassays," i.e., Seradyn Inc. of Indiana which is incorporated
herein by reference.
[0055] The covalent bonding of a binding partner to a microparticle
may be accomplished either directly, such as by reacting an
activated chemical functional group on the surface of a
microparticle with an appropriate chemical functional group on the
binding partner, or indirectly, such as by covalently binding the
binding partner to a spacer molecule that has been covalently bound
to the surface of the microparticle.
[0056] By the phrase "membrane" as used herein is meant a test
device that employs a membrane and one or more reagents to detect
the concentration of an analyte of interest in a test solution,
preferably an aqueous test solution. At least one of the reagents
associated with the membrane device is a binding partner of the
analyte of interest.
[0057] The calibration device and method of the present invention
is useful with essentially any membrane-based devices. A
particularly preferred use for the calibrator of the present
invention is as an internal calibrator. The choice and size of a
microparticle for the stabilized internal calibrator of a
membrane-based device is influenced by the choice of material for
the membrane. The internal calibrator of the present invention may
be affixed to the membrane by covalent or non-covalent bonding.
[0058] In the practice of the invention, calibration and sample
testing may be conducted under essentially exactly the same
conditions at the same time, thus providing reliable quantitative
results, with increased sensitivity.
[0059] The invention also may be employed for semi-quantitative
detection. As the multiple control lines provide a range of signal
intensities, the signal intensity of a given detection line can be
compared (i.e. such as for example, visually) with the intensity of
the control lines. Based upon the intensity range wherein the
detection line falls, the possible concentration range for the
analyte may be determined. The probes may be latex beads labeled
with any signal generating species or the labeled latex beads
further conjugated with antibodies.
[0060] The signal ratio between the detection lines and the control
lines may be plotted against the analyte concentrations for a range
of analyte concentrations to generate a calibration curve, such as
shown in FIG. 2 herein. To determine the quantity of an unknown
sample, the signal ratio may be converted to analyte concentration
according to the calibration curve.
EXAMPLE 1
[0061] Polyethyleneimine was used to demonstrate the invention. A
7.4% polyethyleneimine aqueous solution (stock solution)(1.times.),
a 10.times. dilution and a 100.times. dilution were stripped onto
Millipore SX membrane to form three control lines. The membrane was
dried for about 1 hour at about 37 degrees Centigrade. A wicking
pad was attached upon one end of the membrane. The other end of the
membrane was inserted into a suspension of blue latex beads or red
fluorescent latex beads containing 1.6% Tween 20 (a surfactant) or
antibody-conjugated latex beads with 1.6% Tween 20. Five minutes
later, the beads were captured on the lines where the
polyethyleneimine solution was stripped.
EXAMPLE 2
[0062] In another example, the membrane was stripped with three
different polyethyleneimine solutions (1.times., 10.times., 100-
dilution) on the lines of the calibration zone 32 and anti
C-reactive protein (CRP) monoclonal antibody (Mab A5804) was
immobilized on the detection zone. The membrane was dried for about
one hour at about 37 degrees Centigrade, and the wicking pad was
attached to the end of the membrane to form a half dipstick. The
other end of the half dipstick was inserted into a solution with
CRP antigen and anti CRP monoclonal antibody (Mab A5801 1)
conjugated to latex particles (blue). The solution flowed through
the detection and control zones, and then to the wicking pad. One
blue line on the detection zone and three blue lines on the control
zone were observed.
[0063] In the above examples, it was observed that the signal
intensities of the control lines were significantly different. The
control line stripped with 7.4% polyethyleneimine stock solution
exhibited the most signal intensity while the control line stripped
with 100.times. dilution solution exhibited the least signal
intensity. This observation was true for blue colored and red
fluorescent latex beads alone, as well as these beads further
conjugated with antibodies.
EXAMPLE 3
[0064] The membrane HF 09002 was stripped with 0.14% (calibration
#1), 0.64% (calibration #2) and 1.4% (calibration #3) of
polyethyleneimine solution on the calibration or calibration zone
32. On the detection line 24, anti CRP monoclonal antibody at 1
mg/ml (Mab A5804) was immobilized. The membrane was dried at
37.degree. C. for one hour and the wicking pad 28 was attached to
the end of the membrane to form the half stick. The half sticks
were inserted into the solutions containing the following
nano-grams of CRP antigen (0. 0.54, 5.4 and 54) with excess amount
of blue latex beads which are conjugated with anti CRP monoclonal
antibody (Mab58011). It was observed that three calibration lines
were formed with different intensities, where the line had 1.4%
polyethyleneimine concentration exhibits the highest line intensity
and the line had 0.14% polyethyleneimine concentration had the
least line intensity. The same experiments were carried out with a
mixture of blue latex beads and latex beads antibody conjugate and
the same results were observed. (Experiments with different
polyelectrolytes were also carried out, such that line 1, 2 and 3
may be totally different polymers).
[0065] Results from Example 3 are provided below in Table 1.
1TABLE 1 Signal Intensities of Calibration and Detection Lines
Calibration #1 A A A A Calibration #2 B B B B Calibration #3 C C C
C Detection None (0 ng) C (0.54 ng) B (5.4 ng) A (54 ng) Line
[0066] The results indicated that the intensity of calibration line
#3 represents 0.54 ng of analyte, calibration line #2 represents
5.4 ng of analyte, and calibration line #1 represents 54 ng of
analyte. When an unknown sample was tested, the analyte
concentration could be visually determined by comparing the
detection line intensity with the three calibration lines.
[0067] When the detection line intensity was less than calibration
line #3, the concentration of the analyte was determined to be less
than about 0.54 ng. When the detection line intensity was visually
determined to be between calibration line #3 and #2, the analyte
concentration was found to be between 0.54 and 5.4 ng. When the
detection line intensity was found to be higher than calibration
line #3, the analyte concentration was determined to be higher than
54 ng.
EXAMPLE 4
[0068] The same procedure as Example 3 was conducted, with the
exception that the concentration of the analyte was quantified by a
electronic reading device. In such diagnostic reading devices,
electronic routines make it possible to read automatically the
intensities of calibration and detection lines and provide a
readout or display for the analyte concentration.
[0069] The latex beads generate a detectable colored light signal,
from both the detection zone 31 and the control lines of the
calibration zone 32. The reading device provides a comparison means
for comparing the intensity of colored light signals generated by
latex beads positioned upon the control lines 25-27 with the
intensity of signals generated by microparticle-analyte conjugates
positioned upon the detection zone 31.
EXAMPLE 5
[0070] In yet another application of the invention, it is possible
to use fluorescence to determine the amount of analyte in a test
sample. In this manner, it is possible to use a probe or a
microparticle which itself is capable of exhibiting the property of
fluorescence, in which signals are generated from the probe or
microparticle once it has been deposited in either the calibration
zone 32 or the detection zone 31. A receiver or a receiving device
is capable of measuring the amount of signal generated in the
detection zone 31 and the calibration zone 32, and making the
appropriate comparisons to determine the quantity of analyte in a
given test sample.
[0071] It is understood by one of ordinary skill in the art that
the present discussion is a description of exemplary embodiments
only, and is not intended as limiting the broader aspects of the
present invention, which broader aspects are embodied in the
exemplary constructions. The invention is shown by example in the
appended claims.
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