Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
Assay Method And Device For Detecting The Presence And
Concentration Of Analytes While Simultaneously Preventing
Analyte Adsorption To Solid Materials Used In The Assay
1. Field Of The Invention
This invention relates to chemical analysis tests, and
in particular, to a chromatographic assay device and methods
for detecting the presence and/or concentration of various
analytes in bodily fluids, such as saliva. The assay device
and the methods described in this disclosure allow increased
sensitivity of results by preventing analyte loss through
analyte adsorption to solid materials used in the assay.
2. Background
Chromatographic immunoassay with visual read-out has
often been used in detecting many analytes. See U. S.
Patent Nos. 5,559041; 4,943,522; 5,602,040; 5,5591,645,
5,037,736 and 4,098,876, and Wheeler, Prof. Nurse 14:571
(1999), which are incorporated herein by reference, as if
fully set forth. In a typical chromatographic immunoassay,
a sample containing the analyte to be tested is added to a
solid support. The solid support contains a movable labeled
specific antibody to the analyte, as well as an immobilized
binding reagent. Upon addition to the solid support, the
sample containing the analyte migrates to the zone
containing the movable labeled specific antibody. There,
the analyte binds to the antibody forming a labeled
antibody-analyte complex. Next, the labeled antibody-
analyte complex migrates to the zone containing the
immobilized binding reagent. Generally, with
chromatographic sandwich assays, the immobilized binding
reagent is an antibody to the analyte and is capable of
capturing the labeled antibody-analyte complex to form a
labeled antibody-analyte-immobilized antibody sandwich. On
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the other hand, with competitive chromatographic
immunoassays, the immobilized binding reagent is a binding
pair of the labeled antibody. This binding pair binds to
only labeled antibodies that are not bound by the analyte.
In other words, the binding pair of the labeled antibody
binds to unbound free labeled antibodies. Once binding is
complete, any unbound labeled antibody or labeled antibody-
analyte complex migrates further down the chromatographic
unit past the immobilized binding reagent zone. Detection
of the label within the immobilized binding reagent zone
shows the presence and or/quantity of the particular analyte
in the sample solution. See Dresser, D. W. Handbook of
Experimental Immunology, l: 8.1-8.21, (ed. Weir, D. M.
1986), Hall, B. J. et al., Analyt. Chem., 70: 1788-1796
(1999), Hurn, B. A. L. and Chantler, S.M., Meth. Enzymol.,
70: 104-41 (1980), Kiyai, K., Principles and Practice of
Immunoassay, 246-264 (eds. Price and Newman), (M. Stockton
Press, NY, 1991), Seabrook, R. and Atkimon, T, Principles
and Practice of Immunoassay, (eds. Price and Newman), (M.
Stockton Press, NY, 1991), Roth, K. D. W. et al., J.
Analytical Toxicology, 20: 291-300 (1996), Playfair, J. H.
L. et. al., Brit. Med. Bull., 30: 24-31 (1974), which are
incorporated herein by reference as if fully set forth.
Chromatographic immunoassays with visual read-out have
the advantage of being instrument free and rapid in terms of
obtaining assay results. It is because of this very
recognized advantage that a number of chromatographic assay
devices have become available for use over-the-counter
(OTC). For instance, the HCG test for pregnancy is commonly
available to consumers over the counter. See Ekins, R.P.
(1976), Hormone Assays and their Clinical Application. 4th
edition (eds. Galfre, G. and Milstein, C , and Meth.
Enzymol., 73:3-48 (1981). Furthermore, many chromatographic
immunoassay devices are being used for point-of-care (POC)
testing. Such chromatographic immunoassay POC tests include
the test for streptococcus and the one for
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tetrahydrocannabinol (THC), the active component of
marijuana.
Most chromatographic immunoassay devices use porous
materials at the sample addition area. These materials
cause some of the analyte to become adsorbed to the surface
of these porous materials before the analyte contacts the
assay reagents of the device. This adsorption often results
in poor assay sensitivity.
For instance, a number of POC tests for detecting~THC
constitute chromatographic immunoassay devices that use
porous materials at the sample addition area. The THC
analyte is adsorbed onto the porous surface before it
contacts the assay reagent. As such, most of the available
POC tests for THC have poor assay sensitivity and precision.
Summary Of The Invention
This invention involves a method and device for
prevention of loss of analytes through adsorption to solid
materials used in the analysis by adding an antibody to a
solution containing an analyte which will bind with the
analyte, and then applying the analyte-antibody complex in
the solution to a solid support. The solid support of the
assay consists of a separate chromatography unit having a
sample addition zone, a test zone, and an absorption zone.
In general, a sample mixed with a first antibody is added to
the sample addition zone, from where it migrates via
capillary flow onto the test zone. The test zone contains
either an immobilized analyte or analog of the analyte or an
immobilized second antibody. The immobilized analyte or
analog of the analyte binds to unbound labeled antibody,
whereas the immobilized second antibody binds to the first-
antibody-analyte complex in the solution. Any labeled
antibody that is not bound at the immobilized antibody or
immobilized antigen zone flows through onto the absorption
zone.
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Adding the antibody capable of binding to the
hydrophobic regions of the analyte in the sample containing
the analyte prior to applying the sample solution to the
chromatography unit decreases adsorption of the analyte to
the solid materials of the chromatography unit. This
increases sensitivity and enhances precision of the assay
measurement.
Brief Description Of The Drawings
Figure 1 shows a chromatographic assay strip 10 having
a sample addition zone 11, a test zone 12, and a receiving
zone 13.
Figure 2 shows a chromatography assay strip 20 having a
sample addition zone 21, a test zone 22, a receiving zone
23, and an antibody labeling zone 24. An antibody labeling
reagent is supported at zone 24.
Figure 3 depicts a container for the solubilizing
reagent.
Detailed Description Of The Preferred Embodiments
The assay materials of the invention comprise a
chromatographic assay device and a solubilizing reagent
containing an antibody specific for the analyte.
The chromatographic assay device of the invention
comprises a porous chromatographic strip with an upstream
end and a downstream end. At the upstream end of the porous
strip is a sample addition zone for receiving a sample
solution. A test zone having an immobilized binding reagent
is disposed downstream of the sample addition zone. In
addition to the test zone toward the downstream end of the
assay strip there is an absorption zone.
All zones of the porous strip are physically in flow
communication so that a sample solution applied to the
sample addition zone will flow through the test zone until
it reaches the absorption zone.
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The materials of the zones of the assay strip are
chosen from a group of materials that includes cellulose,
fiberglass, nylon, polyester, etc. The material for the
sample addition zone must be capable of receiving a
5 sufficient amount of sample solution. Fiberglass,
polyester, and cellulose paper are among the preferred
materials for the sample addition zone. The material for
the test zone must have the capability of immobilizing a
minimum of 10~,g/cm2 of protein. Nitrocellulose membrane is
one of the preferred materials for the test zone. The
material for the absorption zone is capable of~receiving the
sample solution with all unbound reagents at the test zone.
The preferred materials for the absorption zone and the
sample addition zone include cellulose paper.
The antibody of the solubilizing reagent is capable of
binding to the analyte molecule and blocking the hydrophobic
regions of the analyte, thus preventing the analyte from
being adsorbed to the surface of the sample addition zone of
the chromatographic assay strip. Many of the regions where
the antibody binds the analyte molecule are hydrophobic.
Without these hydrophobic regions of the analyte being
bound, the analyte would be more likely to be adsorbed to
hydrophobic regions of the chromatography strip. Such
adsorption would decrease the amount of analyte reaching the
test zone and reduce sensitivity and accuracy of the assay.
In one preferred embodiment of the invention, the assay
device and the solubilizing reagent are for a competitive
chromatographic immunoassay. The solubilizing reagent
consists of a labeled antibody. The binding reagent at the
test zone consists of the analyte or analogues of the
analyte, which are capable of binding antibodies to the
analyte. For example, a preferred binding reagent for a THC
test is THC-bovine serum albumin (BSA) conjugate. The
binding reagent is immobilized at the test zone by
adsorption or covalent binding. Nitrocellulose membrane is
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capable of binding large concentration of proteins by
passive adsorption.
Tn a different embodiment of the invention, which is
another competitive assay, the solublizing reagent consists
of an unlabled antibody. The binding reagent at the test
zone consists of the analyte or an analogue of the analyte,
capable of binding antibodies to the analyte. The solid
support or chromatograhic unit of this assay device further
comprises an antibody-labeling zone having an antibody-
labeling reagent. The antibody-labeling zone is positioned
downstream from the sample addition zone and upstream from
the test zone. A labeling reagent capable of binding the
antibody of the analyte is movably supported at the
antibody-labeling zone. The labeling reagent is a binding
protein of the antibody conjugated with a reporter substance
(label). The binding protein of the antibody is chosen from
a group of proteins including antibodies, protein A, and
protein G. The label of the labeling reagent is chosen from
a group of fluorescent, chemiluminescent, bioluminescent
compounds, radioactive isotopes, enzymes, and particular
color particles. Such particular particles include
colloidal gold, colloidal silver, carbon black, latex beads,
etc., which can be seen by the naked eye. The label is
coupled to a binding protein via various means including
covalent binding and physical adsorption, which have been
described in previous publications. See U.S. Patent Nos.
5,252,496, Kang et al.; 4,313,734, Zeuvering, et al.;
Hermanson, G., B.ioconjugate Techniques, Academic Press, San
Diego (1996), which are incorporated herein by reference as
if fully set forth., The solubilizing reagent to be used
with a device having an antibody-labeling zone consists of a
specific antibody of the analyte, which antibody can be
labeled by the labeling reagent at the antibody-labeling
zone.
In a different embodiment of the invention, the assay
device and reagent are for a sandwich chromatographic
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immunoassay. The binding reagent at the test zone is an
antibody of the analtye. The solubilizing assay reagent
consists of a labeled antibody that recognizes a different
epitope of the analtye than the antibody of the binding
reagent. Thus, both antibodies can bind the same analyte to
form a labeled antibody-analyte-binding antibody "sandwich".
In accordance with the present invention, a sample
solution containing the analyte is first exposed to the
solubilizing reagent. ' If the analyte is present in the
sample solution, the analyte binds to the antibody in the
solubilizing reagent to form an antibody-analyte complex.
The resulting solution is then applied to the sample
addition zone of the assay device. The labeled antibody-
analyte complex will migrate to the test zone. If the
antibody in the solubilizing reagent is unlabeled, the assay
device consists of an antibody-labeling zone. The antibody-
analyte complex is labeled by the antibody-labeling reagent
at the antibody labeling zone before the solution migrates
to the test zone. In either case, the antibody at the test
zone is a labeled antibody and the antibody-analyte complex
is a labeled antibody-analyte complex.
Tn a competitive chromatographic assay, the labeled
antibody binds to the binding reagent at the test zone, and
the labeled antibody-analyte complex flows through the test
zone. As a result, the tracer signal at the test zone is
inversely proportional to the concentration of analyte in
the sample solution. The presence or quantity of the
analyte in the sample solution is determined by measuring
the tracer signal at the test zone.
In a sandwich chromatographic assay, the labeled
antibody flows through the test zone and the labeled
antibody-analyte complex binds to the binding reagent of the
test zone. At the end, the tracer signal at the test zone
is proportional to the concentration of the analyte in the
assay sample. The presence or quantity of the analyte in
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the sample solution is determined by measuring the tracer
signal at the test zone.
Example 1: Moving of Fluorescent dye labeled THC on
Fiberglass Membrane:
09-Tetrahydrocannabinol was labeled with Cy5 dye. The
THC moiety of the labeled compound is hydrophobic. Our
study revealed that the THC-Cy5 dissolved in lOmM PBS is
easily absorbed onto the surface of plastic or glass
containers. Fiberglass or porous polyester membrane
materials have large surface areas, which can absorb even
more THC-Cy5.
A. Preparation of THC-Cy5 Solutions:
Control sample solution is lOmM PBS containing lOnM of
THC-CyS, 0.2o BSA, and 0.5~g/ml mouse IgG. A test sample
solution is lOmM PBS containing lOnM of THC-Cy5, 0.2o BSA,
and 0.5~.g/ml of mouse anti-THC monoclonal antibody.
B. Preparation of fiberglass and porous polyester
membrane strips:
1mm x 8mm x 50mm fiberglass and 1mm x 8mm x 50 mm
polyester membrane were laid on 8 mm x 30 mm vinyl strips
separately.
C. Migration of THC-Cy5 on fiberglass and polyester
membrane materials:
101 of THC-Cy5 control/test sample solution was spiked
in the center of each membrane strip at the point that is
l5mm to one end (end A) of the strip. 5 drops of 0. 2 o BSA
PBS solution was gradually applied onto each strip from end
A. When the solution migrates from end A to the other end,
end B, the strips were scanned with a fluorescence scanner.
Result:
On the control strips fluorescence was only detected at
the sampling area (7mm-15mm from end A) which suggests that
THC-Cy5 compound was absorbed on the membrane materials. On
the test strips 80% (750 - 920) of fluorescence was read at
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the half strip of end B. The result shows that the mouse
anti-THC monoclonal antibody prevents THC-Cy5 from being
adsorbed by the porous membrane materials.
Example 2: Chromatographic immunoassay of L-09-carboxy-
tetrahydrocannabinol (THCA) in urine.
A. Materials
L-09-THCA from Research Triangle, Institute, Cat#
5754-5 Id, mouse anti-THC monoclonal antibody from
Fitzgerald Industries International, Inc, THC-BSA conjugate
from LifePoint, Inc. #33, fiberglass and nitrocellulose
membrane from Schleicher & Schuell, Inc. were used in this
study. All other chemicals were purchased from
Sigma-Aldrich Corp.
B. Preparation of the Chromatographic Immunoassay
Strip for the Detection of THCA
A 5 x 30 mm plastic backed nitrocellulose strip and a 5
x 25 mm Whatman filter paper strip was aligned together on a
5 x 53 mm one-side glued vinyl strip. The plastic backed
nitrocellulose strip and the filter paper have a 2mm overlap
with each other.
A line of approximately 0.2~.g of THC-BSA conjugate was
placed on the nitrocellulose membrane at the point
approximately ZO mm from the overlap area,
C. Preparation of the soluble assay reagent
1 ml of colloidal gold solution (particle size: 20-
40~m) was adjusted to pH7.5. 0.02 mg of mouse anti-THC
antibody was mixed with the colloidal gold solution. 0.1 ml
of 0.01 mole/L sodium phosphate buffered saline containing
1o bovine serum albumin (BSA) was added into the antibody-
gold solution. The solution was centrifuged at 80008 for 20
minutes and the precipitated antibody-gold sol conjugate was
recovered after carefully removing the supernatant from the
centrifuge tube. The antibody-gold sol conjugate was
re-suspended in 1 ml of 0.01 mole/L sodium phosphate
buffered saline containing 0.1o BSA, 1o trehalose, and 0.050
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Tween 20. 0.01 ml aliquots of the antibody-gold sol
conjugate solution were dispensed into 2 ml glass vials and
freeze-dried.
D. Preparation of THC-urine samples
5 1-09-THCA was spiked into 1 ml urine (from a healthy
volunteer) aliquots to provide different drug concentrations
in the samples. .
E. Chromatographic assay of Z-D9-THCA in urine
0.2 ml of urine sample was added into a glass vial
having freeze-dried antibody-gold sol conjugate. The
antibody-gold sol conjugate was reconstituted by vortex
mixing. The nitrocellulose end of a chromatographic
immunoassay strip was dipped into the glass vial containing
urine sample mixed with antibody-gold sol conjugate.
15 The assay result was read after 10 minutes. The
presence of a red line at the test area of the assay strip
was interpreted as a negative test result. The absence of
red line at the test area of the assay strip was interpreted
as a positive test result.
20 Result
All ten samples spiked with 0-2ng/ml THCA tested
negative, and all ten samples spiked with >4.Ong/ml THCA
tested positive.
Example 3: Chromatographic immunoassay of 09-
tetrahydrocannabinol (THC)
Solutions of varied THC concentrations were made by
diluting THC into 0.2o BSA-PBS solution. The same device
and protocol for testing THCA in urine was used in testing
THC solutions.
Result:
All ten samples spiked with 0-l5ng/ml of THC tested
negative, and all ten samples spiked with > 25ng/ml of THC
tested positive.
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Example 4
A. Materials
L-~9-THCA from Research Triangle, Institute, Cat# 5754
Id, Goat anti-mouse IgG antibody from Sigma; THC-BSA
5 conjugate from LifePoint, Inc., and #33 fiberglass and
nitrocellulose membrane from Schleicher & Schuell, Inc were
used. All other chemicals used in this study were purchased
from Sigma-Aldrich Corp.
B. Preparation of the antibody labeling reagent
1 ml of colloidal gold solution (particle size: 20-40
um) was adjusted to pH9. 0.02 mg of goat anti-mouse IgG
antibody was mixed with the colloidal gold solution. 0.1 ml
of 0.01 mole/L sodium phosphate buffered saline containing
1o BSA was added into the antibody-gold solution. The
solution was centrifuged at 80008 for 20 minutes and the
precipitated antibody-gold sol conjugate was re-suspended in
1 ml of 0.01 mole/L sodium phosphate buffered saline
containing 0.1o BSA, to trehalose, and 0.05% Tween 20.
C. Preparation of the chromatographic immunoassay
strip for the detection of THC:
A 5 x 30 mm plastic backed nitrocellulose strip was
positioned on the middle area of a 5 x 76 mm one-sided glued
vinyl strip. A 5 x 25 Whatman filter paper strip and a 25
mm #33 fiberglass strip were separately positioned on the
ends of the vinyl strip. Both the filter paper and the
fiberglass have 2 mm overlapping with the nitrocellulose
strip. 10 u1 of goat anti-mouse IgG antibody-gold sol
conjugate solution was applied at one spot of the fiberglass
5 mm from the nitrocellulose-fiberglass overlap area.
A line of approximately 0.2 u8 of THC-BSA conjugate was
placed on the nitrocellulose membrane at the point
approximately 10 mm from the nitrocellulose-filter paper
overlap area.
D. Preparation of the soluble assay reagent
15 u1 of 0.01M sodium phosphate buffered saline
containing 0.01 ug/ml of mouse anti-THC antibody, 0.2o BSA,
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and 0.1o Tween 20 was added into each 2.5 ml polypropylene
vials as shown in figure 3. The solution was freeze dried.
E. Chromatographic assay of 1-O9 - THCA in urine:
0.2 ml of urine sample was added into a polypropylene
vial containing mouse anti-THC antibody solution. The
antibody solution was reconstituted by vortex mixing. The
nitrocellulose end of a chromatographic immunoassay strip
having an antibody labeling zone was dipped into the plastic
container containing the urine sample mixed with the
antibody solution.
The assay result was read after 10 minutes. The
presence of a red line at the test area of the assay strip
was interpreted as a negative test result. The absence of a
red line at the test area of the assay strip was interpreted
as a positive test result.
Result
All ten samples spiked with 0-5ng/ml of Z-~9-THCA
tested negative, and all ten samples spiked with > l0ng/ml
of L-~9-THCA tested positive.
