U.S. patent application number 10/643632 was filed with the patent office on 2004-03-18 for immunochromatographic assay method and apparatus.
Invention is credited to LaBorde, Ronald T..
Application Number | 20040053423 10/643632 |
Document ID | / |
Family ID | 24102976 |
Filed Date | 2004-03-18 |
United States Patent
Application |
20040053423 |
Kind Code |
A1 |
LaBorde, Ronald T. |
March 18, 2004 |
Immunochromatographic assay method and apparatus
Abstract
An immunochromatographic assay employing superparamagnetic
particles to lable the target analytes. An opaque cover prevents
misinterpretive readings in field situations and provides a
protective surface on the porous membrane. Additional features
include separability of the test strip from any backing or housing
which is configured to support the strip, and that quantitative
measurements of the target analytes are easily and accurately made
by means of an electromagnetic reader device.
Inventors: |
LaBorde, Ronald T.; (San
Diego, CA) |
Correspondence
Address: |
THE MAXHAM FIRM
750 "B" STREET, SUITE 3100
SAN DIEGO
CA
92101
US
|
Family ID: |
24102976 |
Appl. No.: |
10/643632 |
Filed: |
August 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10643632 |
Aug 18, 2003 |
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09527801 |
Mar 17, 2000 |
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6607922 |
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Current U.S.
Class: |
436/514 ;
435/287.2 |
Current CPC
Class: |
G01N 30/02 20130101;
G01N 33/558 20130101; B01D 15/3804 20130101; Y10S 436/807 20130101;
Y10S 435/97 20130101; G01N 33/585 20130101; G01N 30/02
20130101 |
Class at
Publication: |
436/514 ;
435/287.2 |
International
Class: |
C12M 001/34; G01N
033/558 |
Claims
What is claimed is:
1. A lateral flow assay device for quantitative detection of target
analytes in a sample, said device comprising: an assay support
member having a first end and a second end; a sample receiving
element at one end of said support member for introduction of the
sample to be analyzed to said device; and an immunoassay test strip
comprising: a porous analytical membrane removably mounted adjacent
to and generally parallel with said support member, said analytical
membrane having a first end and a second end; at least one capture
region in said analytical membrane intermediate said first and
second ends thereof, said at least one capture region being
configured to capture labeled analytes moving from said first end
of said analytical membrane toward said second end of said
analytical membrane; and a backing member between said analytical
membrane and said support member to facilitate emoval of said
analytical membrane from said support member for reading the assay
and for archiving said test strip.
2. The device recited in claim 1, and further comprising a
protective membrane covering said analytical membrane on the side
opposite to said support member, said protective membrane being
optically non-transparent.
3. The device recited in claim 2, wherein said protective membrane
is formed integrally with said porous membrane.
4. The device recited in claim 2, wherein said protective membrane
is formed pursuant to a surface treatment of said porous
membrane.
5. The device recited in claim 1, and further comprising a control
region in said porous membrane for collection of magnetic
conjugates that have passed the capture region to show that said
test strip has been used.
6. The device recited in claim 2, and further comprising at least
one magnetic calibration line printed on said protective
membrane.
7. The device recited in claim 2, wherein said protective membrane
is formed of material selected from the group consisting of
plastic, glass and paper.
8. The device recited in claim 1, and further comprising
superparamagnetic conjugate particles in said sample receiving
element, said particles being configured to bind with target
analytes in the sample.
9. A lateral flow assay device for quantitative detection of target
analytes in a sample, said device comprising: an assay support
member having a first end and a second end; a sample receiving
element near one end of said support member for introduction of the
sample to be analyzed to said device; and an immunoassay test strip
comprising: a porous analytical membrane removably mounted adjacent
to and generally parallel with said support member, said analytical
membrane having a first end and a second end; superparamagnetic
conjugate particles in said sample receiving element configured to
bind with the target analytes in the sample; a capture region in
said analytical membrane intermediate to said first and second ends
thereof, said capture region being configured to capture labeled
analytes moving from said first end of said analytical membrane
toward said second end of said analytical membrane; and a backing
member between said analytical membrane and said support member to
facilitate removal of said analytical membrane from said support
member for reading the assay and for archiving said test strip.
10. The device recited in claim 9, and further comprising a
protective membrane covering said analytical membrane on the side
opposite to said support member, said protective membrane being
optically non-transparent.
11. The device recited in claim 10, wherein said protective
membrane is formed integrally with said porous membrane.
12. An analytical immunoassay apparatus for quantitative detection
of target analytes in a sample, said apparatus comprising: an assay
support member having a first end and a second end; a sample
receiving element near one end of said support member for
introduction of the sample to be analyzed to said apparatus; an
immunoassay test strip comprising: a porous analytical membrane
removably mounted adjacent to and generally parallel with said
support member, said analytical membrane having a first end and a
second end; superparamagnetic conjugate particles in said sample
receiving element configured to bind with the target analytes in
the sample; a capture region in said analytical membrane
intermediate to said first and second ends of said analytical
membrane, said capture region being configured to capture labeled
analytes moving from said first end of said analytical membrane
toward said second end of said analytical membrane; and a backing
member between said analytical membrane and said support member to
facilitate removal of said analytical membrane from said support
member for selectively reading the assay and archiving said test
strip; and a magnetic reader device for determining the presence
and quantity of magnetic conjugate particle labeled target analytes
in said capture region, said reader device being shaped and
configured to receive said test strip after the lateral flow
process has been completed.
13. The apparatus recited in claim 12, and further comprising a
protective membrane covering said analytical membrane on the side
opposite to said support member, said protective membrane being
optically non-transparent.
14. The apparatus recited in claim 13, wherein said protective
membrane is formed integrally with said porous membrane.
15. A method for conducting a lateral flow immunoassay quantitative
detection of target analytes in a sample, a method comprising:
applying the sample to one end of the porous membrane of a lateral
flow test strip; coupling superparamagnetic conjugate particles
residing in the test strip at said one end, the superparamagnetic
particles being treated to bind with any target analyte in the
sample; capturing the bound complexes of analyte and
susperparamagnetic particles in the capture region of the porous
membrane as the sample and bound complexes move through the porous
membrane by capillary action; reading the quantity of labeled
analytes in the capture region; and providing an output
representative of the quantity of labeled analytes in the capture
region.
16. The method recited in claim 15, and further comprising removing
the test strip from the lateral flow assay device with the bound
complexes remaining available to be selectively stored and sensed
as to the quantity of the bound complexes in the capture region.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates generally to immunoassays, and more
specifically to an improved chromatographic assay, often referred
to as a lateral flow assay, having a test strip employing
susperparamagnetic particles as the labels for the analytes to be
detected, where, as an additional feature, the analytical strip is
removable for reading the quantity of analytes captured therein and
for archival purposes.
[0003] 2. Discussion of Related Art
[0004] Various chromatographic immunoassay techniques have been
available for many years. One common aspect of known devices,
particularly in the lateral flow technology is that the assay is
read visually, that is, by means of one or more optically readable
lines on a test strip, typically held in a carrier, which may have
various configurations. One end of the test strip is exposed to the
sample, normally a body fluid of some type, being tested for the
particular target analytes of interest. It is known that particular
analytes are indicative of particular biological, environmental,
and biohazard conditions, among others. For example, urine may be
tested for pregnancy or ovulation and if the target analytes are
present, the test is positive. Body fluids may be tested for the
presence of other analytes indicative of biological conditions or
they may be indicative of the presence of substances, such as
drugs. Another example would be for testing water for contaminates.
Examples of lateral flow assay methods and apparatuses, where the
reading is normally conducted optically, are shown in U.S. Pat.
Nos. 5,591,645; 5,798,273; 5,622,871; 5,602,040; 5,714,389;
5,879,951; 4,632,901; and 5,958,790.
[0005] A different technology is employed in other types of
biological technologies employing magnetic particles or micobeads,
sometimes more specifically termed superparamagnetic iron oxide
impregnated polymer beads. These beads are employed to bind with
the target analytes in the sample being tested and are then
typically isolated or separated out magnetically. Once isolation
has occurred, other testing may be conducted, including observing
particular images, whether directly optically or by means of a
camera. Examples of these technologies are disclosed in U.S. Pat.
Nos. 3,981,776; 5,395,498; 5,476,796; 5,817,526; and 5,922,284.
Another apparatus for detecting target molecules in a liquid phase
is shown in U.S. Pat. No. 5,981,297 where magnetizable particles
are employed and the output of magnetic field sensors indicates the
presence and concentration of target molecules in the sample being
tested. Other examples to sense magnetically using physical forces
are disclosed in U.S. Pat. Nos. 5,445,970; 5,981,297 and
5,925,573.
[0006] There are several limitations or disadvantages to the known
optically detected assays. Because they are optical, only surface
changes (coloration, typically) can be detected. The target
analytes may be in the sample solution but of such a low
concentration that only a relatively few are captured in the
capture zone in the porous membrane of the assay. This provides a
faint or even non-optically detectable line, and a resultant false
negative reading. Quantitative assessments are really only an
estimation based on color intensity of the detection line. Because
the prior art assays are optically read, they are subject to
contamination by exposure, and light-caused degradation. Optical
assays have a limited archival shelf life.
[0007] None of the known prior art employs magnetic particles in
conjunction with lateral flow assay technology.
SUMMARY OF THE INVENTION
[0008] Broadly speaking, the invention relates to lateral flow
immunoassay technology employing superparamagnetic particles as the
labels for the analytes to be detected. The bound complexes of
labeled particles and analytes are captured in predetermined areas
or regions on the test strip and the presence and quantity of
labeled analytes are then readable by magnetic means. An
advantageous additional feature of the invention is that the test
strip can be removable from the support member for archival
purposes or for reading by an appropriate magnetic sensing device,
or both.
[0009] A relatively standard lateral flow assay structure is
employed but the invention greatly improves the sensitivity of the
device over known lateral flow techniques. It provides a very rapid
(a few seconds) measurement of the analytical region in the test
strip. There are many advantages of using magnetic particles over
known colored particles or other optical indicators in the prior
art. These include linearity because magnetic detection is linear
with respect to the amount of magnetic material present over a wide
range, through at least four orders of magnitude. Time stability is
also significant because magnetic particles are stable. The
developed assay is available to be archived and retested as
necessary. Further, magnetic particles are generally inert to
biological systems and the environment so they not only remain
stable, they are environmentally and biologically safe. Further,
magnetic particles are already in widespread use throughout the
diagnostics industry with other technologies so they are readily
available. Other benefits of magnetic detection are that since the
particles are superparamagnetic, they are magnetic only when in a
magnetic field. This allows them to be freely manipulated in
solution without aggregating.
[0010] Another significant advantage over the prior art optical
lateral flow devices is that with this invention the total amount
of analytes in the capture region of the test strip is measured as
a single mass in one volumetric measurement by magnetic means. The
permeability of magnetic fields is such that any analyte contained
within the active region of the detector will be measured. This
contrasts with optical sensing techniques in which only
reporter-analyte interactions on or very near the surface are
detectable. In this invention the strength of the magnetic signal
increases directly with the mass of iron involved. This inherent
linearity of magnetic detection contributes to sensitivity,
accuracy and dynamic range. Finally, superparamagnetic particles
are physically similar to collodial gold with regard to size, and
may be easily adapted to a wide range of lateral flow assays. It is
noted that collodial gold, as well as fluorescent latex particles,
are typically employed in the prior art optically sensed
immunological assay techniques.
[0011] In lateral flow technology, at one end of the porous
membrane (the active part of the test strip) is the sample
introduction area conventionally comprising a sample pad and a
conjugate pad. In the prior art, the conjugate pad was the source
of freely moveable colored particles, typically gold sols from
collodial gold, or fluorescent latex particles. In the present
invention, the moveable particles are the superparamagnetic
particles which label the target analytes from the sample being
introduced through the sample pad. The sample, together with the
bound magnetic particle labels and target analytes, move with
capillary action along the porous membrane and are captured in a
predefined location called a capture region or capture zone. There
may be more than one capture zone to enable multiplexing, that is,
testing for more than one type of analyte at the same time in the
same test strip. Excess analytes and the carrying liquid continue
to move on through the capture zone to the other end of the porous
membrane, sometimes forming a control line or zone separate from
the capture zone. An added feature is that typically a wicking pad
is mounted on the far end of the porous membrane to enhance the
capillary action which drives the flow from the introduction at one
end of the porous membrane through the entire length of the
membrane.
[0012] The porous membrane typically is mounted on a relatively
rigid support or base member, but in an advantageous embodiment a
separation sheet, or adhesive layer, exists between the base member
and the porous membrane. This enables very easy removal of the test
strip, which normally would include the separation sheet, so that
the test strip is a very thin element which may be magnetically
sensed in an appropriate device. Since optical means are not
employed and color at the capture zone has no meaning, the top of
the porous member is preferably covered by another protective sheet
or membrane which is not transparent. It may be completely opaque.
This top sheet may also include pre-printed standards, which are
employed for calibrating purposes so that the magnetic detector can
be calibrated for each test to ensure complete accuracy. The
protective sheet may not be a separate element in some cases, but
may only be the upper surface of the membrane properly treated to
function as a protective sheet or surface.
BRIEF DESCRIPTION OF THE DRAWING
[0013] The objects, advantages and features of this invention will
be more clearly perceived from the following detailed description,
when read in conjunction with the accompanying drawing, is
which:
[0014] FIG. 1 is a schematic perspective, partially phantom, view
of an embodiment of the invention;
[0015] FIG. 2 is a schematic side view of the FIG. 1
embodiment;
[0016] FIG. 3 is a schematic side view of a preferred embodiment of
the invention;
[0017] FIG. 4 is a top view of the FIG. 3 embodiment;
[0018] FIG. 5 shows how the test strip of FIG. 3 is removed;
[0019] FIG. 6 is a perspective of a reader device used with the
assay apparatus of FIG. 1 or 3; and
[0020] FIG. 7 schematically shows how the assay strip is read by a
magnetic reader device.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0021] With reference now to the drawing, and more particularly to
FIGS. 1 and 2, there is shown an embodiment of the present
invention, a lateral flow test strip for an immunochromatographic
assay. A relatively rigid assay support membrane 11 serves as a
base and provides support for the test strip, which in this
embodiment is removable from the support. On top of the support
membrane is backing member 12 on which is mounted porous membrane
13. The backing member is removably adhered to the support, or it
may itself be an adhesive layer. Within the porous membrane is
capture zone 14. The capature zone is formed by striping with
antigens or antibodies, for example, as is well known in the art.
On top of the porous membrane is non-light transmissive cover or
surface 15. The cover or surface may be considered to be optically
opaque, at least to the extent that capture zone indicia would not
be visible through cover 15. The protective membrane may be made of
plastic, glass or paper, for example, or any practical combination
thereof. A printed standard or calibration line 16 is situated on
top of the cover and provides information utilized by the assay
reader after the test has been accomplished. This is contemplated
to be a magnetic stripe with the information the reader needs.
[0022] At the left end, as shown in FIGS. 1 and 2, is sample pad
17, through which an analyte-containing sample solution 20 is
administered to the porous membrane, with analytes 20A shown in the
sample pad. The sample pad may also include conjugate pad 19 which
is in communication with the porous membrane. Within the conjugate
pad are superparamagnetic beads or particles which are coupled with
antibodies, the combination of a bead and an antibody being
referred to as a conjugate, a plurality of them being labeled with
reference numeral 20B. These conjugates are configured to combine
with target analytes in the sample solution in a known manner to
create a sandwich assay, well known in the art, where the beads
provide labels for the target analytes. Competitive assay
techniques, also well known in the art, could also be employed.
[0023] The porous membrane will, as a feature of some embodiments,
have a wicking pad 18 at the opposite, or right, end of the test
strip. This is a conventional element.
[0024] The operation of the lateral flow assay is well known. The
labeled analytes (in a sandwich assay, for example) move by
capillary action from the left to the right as seen in FIG. 1.
Labeled analytes are captured in the capture zone where reading of
the assay results is accomplished. Wicking pad 18 enhances
capillary flow in the porous membrane by "pulling," or "driving"
the fluid through the porous membrane. A typical material from
which the porous membrane is made is nitrocellulose.
[0025] While the capillary action and the existence of a capture
zone are well known and conventional, the manner in which the
described embodiments of the invention detect the presence and the
quantity of the target analytes differs greatly from the prior art.
The known lateral flow assays depend upon color or fluorescence to
provide a visual or optical indication of the presence of target
analytes in the capture zone, but the ability of optical techniques
to detect the presence of the target analytes is limited. A
relatively low concentration of target analytes in the sample can
result in so few captured analytes as to be optically undetectable
on the surface of the porous membrane at the capture zone. Further,
the optical intensity of the capture zone with the captured
analytes is only a rough function of the quantity of target
analytes captured. However, there is no way to accurately measure
the total quantity of captured analytes within the capture zone
because only the surface is optically readable.
[0026] The present embodiment provides greatly enhanced sensitivity
and quantitative accuracy because the magnetic labeled analytes in
the capture zone are detectable by a suitable magnetic detector to
the extent of the target analytes within the entire volume of the
capture zone.
[0027] Additional features may be added, including additional
capture zones 14 (two are shown in FIGS. 3 and 5) and additional
calibration lines 16. In FIGS. 3-5 there are four calibration
lines, one before and one after each capture zone. There could be
several capture zones and equivalent calibration lines.
[0028] A significant aspect of an embodiment of the invention is
the means and manner of magnetically reading the assay. A magnetic
reader 21 of the type contemplated is shown in FIG. 6. This employs
the technology disclosed in PCT publication WO 99/27369, to
determine the presence of target analytes and their quantity. The
reader of the present invention is contemplated to be portable,
that is, approximately pocket size, so that it is easily employed
in the field. It will provide accurate assay readings even under
stressful conditions and in poor light. The apparatus of FIG. 6 has
a narrow slit opening 22 into which an assay strip can be inserted
for reading by electromagnetic means. The analyte quantity may be
shown in window 23, which could be an LED or an LCD screen, for
example. The manner in which the capture zone with analytes bonded
therein is read is shown schematically in FIG. 7. Lower
electromagnetic head 31 has a top surface 32 on which is mounted
detection coil 34 and across which strip 13/15 is positioned with
capture zone 14 centered over the detection coil. The strip is
shown in this example with surface or cover 15 of the strip
actually touching the surface of the detection coil. Cover 15 thus
protects the delicate structure of the porous membrane during the
reading process, as well as long term if the strip is archived. Top
magnet 33 is closely adjacent to the opposite side of the strip,
having stripping layer 12 thereon. The structure of the
electromagnetic head shown in FIG. 7 is generally equivalent to
that in reader 21.
[0029] To enable the reader of FIG. 6 to be employed to read the
analytes in the capture zone, the test strip is made readily
removable from support membrane 11, and from sample pad 17 and
wicking pad 18. As seen in FIGS. 3-5, pull tab 24 is secured to
cover 15, which is, in turn, secured to porous membrane 13. The
porous membrane is removably secured to the support by means of a
removable backing 12 membrane and secured to the bottom of the
porous membrane. It will be noted that the assay is essentially
inverted as shown here. The sample could be applied to sample pad
17 with the strip turned over from the position of FIGS. 3 and 5.
The conjugate pad 19 and the wicking pad 18 are positioned between
support membrane 11 and the peelable strip 12, 13, 15. Element 11A
may be a non-active polymer fill membrane. Sheet or adhesive 12 is
readily removable from the surfaces of elements 18, 19 and 11A.
[0030] It is contemplated that the test strip, primarily consisting
of the porous membrane and protective surfaces thereof, may be made
sufficiently rigid to not need a support membrane. Such a
configuration would not need to be stripped from anything. The
reader apparatus might have to be modified somewhat to handle the
different configuration of the test strip, but the principle of
magnetic reading shown in FIG. 7 still applies.
[0031] FIG. 5 shows how the test strip, comprised of the cover,
porous membrane and removable backing, is removed from the support
membrane. In actuality, this removed test strip is typically about
3-12 mm wide, and only about 150-500.mu. thick. This strip is
easily fed into reader 21 for a digital readout, which may be shown
on the screen or printed on paper in any desired form by reader 21.
The exposed test strip is stable and can be archived either before
or after being read. Since the superparamagnetic beads are
magnetized only during the reading process, the exposed test strip
is not subject to degradation. The analytes contained in the
capture zone remain there, labeled with the conjugate
combination.
[0032] Alternative features of the embodiment discussed above
contemplate the test strip being slid off the support membrane or
peeled off, either manner of removable being physically
possible.
[0033] The opaque surface or cover 15 has several positive
functions. Contrary to prior art optical lateral flow assays, where
very faint lines can easily be misinterpreted in the field,
especially in stressful situations or low light conditions, there
is no possibility of misinterpretation of test results with this
invention. Optically read assays, especially those visually read,
are also subject to operator bias. In the present invention the
reader reads the total number of labeled analytes in the capture
zone without inherent sources of error as mentioned above. Further,
the thickness of the opaque cover precisely positions the porous
membrane and thus, the capture zone, with respect to the magnet
head and detection coil 34. Since the test strip actually touches
the detection coil, without the protective surface the porous
nitrocellulose membrane would be damaged by rubbing across
detection coil 34, thereby possibly producing incorrect or
unreliable readings, or both. Although being very thin, in the
range of 30-50.mu. the cover protects against physical damage and
environmental contamination as well as providing precise
positioning for accurate electromagnetic readings.
[0034] While the present invention has been illustrated and
described by means of a specific embodiment, it is to be understood
that numerous changes and modifications can be made therein without
departing from the scope of the claims and equivalents thereto.
* * * * *