U.S. patent application number 10/838584 was filed with the patent office on 2004-11-04 for chromatographic assay system.
Invention is credited to Choi, Young Ho, Jung, Jaean.
Application Number | 20040219690 10/838584 |
Document ID | / |
Family ID | 33435108 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040219690 |
Kind Code |
A1 |
Choi, Young Ho ; et
al. |
November 4, 2004 |
Chromatographic assay system
Abstract
The present application discloses an analyte detection apparatus
having at least one reservoir area and a wicking membrane, wherein
a labeled specific binding partner is impregnated on the reservoir
area; and a region on the wicking membrane where at least one
chemical component is immobilized.
Inventors: |
Choi, Young Ho; (Princeton,
NJ) ; Jung, Jaean; (Monroe Twp., NJ) |
Correspondence
Address: |
Joseph Hyosuk Kim, Ph.D.
JHK Law
P.O. Box 1078
La Canada
CA
91012-1078
US
|
Family ID: |
33435108 |
Appl. No.: |
10/838584 |
Filed: |
May 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60467717 |
May 2, 2003 |
|
|
|
Current U.S.
Class: |
436/514 |
Current CPC
Class: |
G01N 33/54306 20130101;
G01N 33/558 20130101; G01N 2458/40 20130101; G01N 33/54366
20130101; Y10T 436/143333 20150115; G01N 33/533 20130101; G01N
21/8483 20130101 |
Class at
Publication: |
436/514 |
International
Class: |
G01N 033/558 |
Claims
We claim:
1. An analyte detection apparatus having at least one reservoir
area and a wicking membrane, wherein a labeled specific binding
partner is impregnated on the reservoir area; and a region on the
wicking membrane where at least one chemical component is
immobilized.
2. The apparatus according to claim 1, wherein the label is rare
earth chelate.
3. The apparatus according to claim 2, wherein the label is
lanthanide(III) chelate.
4. The apparatus according to claim 3, wherein the label is
europium(III), terbium(III), samarium(III), or dysprosium(III), or
a combination thereof.
5. The apparatus according to claim 1, wherein the analyte is
selected from the group consisting of an antigen, an antibody, a
nucleic acid and a hapten.
6. The apparatus according to claim 1, wherein the specific binding
partner is selected from the group consisting of an antigen, an
antibody, a nucleic acid, biotin or biotin analogue, streptavidin,
avidin and a hapten.
7. The apparatus according to claim 1, wherein at least one
chemical component is selected from a group consisting of an
antigen, an antibody, a nucleic acid, biotin or biotin analogue,
streptavidin, avidin and a hapten.
8. The apparatus according to claim 1, which is a lateral flow
assay format apparatus.
9. A method of determining the presence of an analyte in a sample
comprising applying an amount of the sample to the apparatus
according to claim 1, wherein if at least one analyte is present in
the sample, the sample migrates to the wicking membrane where a
chemical reaction occurs, wherein presence of a signal indicates
that the analyte is present in the sample.
10. The method according to claim 9, wherein the signal is
generated by a rare earth chelate.
11. The method according to claim 10, wherein the a rare earth
chelate is lanthanide(III) chelate.
12. The method according to claim 11, wherein the lanthanide(III)
is europium(III), terbium(III), samarium(III), or dysprosium(III),
or a combination thereof.
13. The method according to claim 9, wherein the sample is a
biological sample.
14. The method according to claim 13, wherein the sample is
selected from the group consisting of blood, serum, plasma, urine,
saliva, sweat and liquid media processed from a biological or
environmental sample.
15. The method according to claim 9, wherein the analyte is
selected from the group consisting of an antigen, an antibody, a
nucleic acid and a hapten.
16. The method according to claim 9, wherein the chemical reaction
is with a chemical selected from the group consisting of an
antigen, an antibody, a nucleic acid, biotin or biotin analogue,
streptavidin, avidin and a hapten.
17. The method according to claim 9, wherein a plurality of types
of analytes in a sample are detected.
18. The method according to claim 9, wherein the apparatus is a
lateral flow assay format apparatus.
19. The method according to claim 9, wherein the analyte is
specific to a pathogen.
20. A kit comprising a container which comprises the apparatus
according to claim 1 and instructions on using the kit.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to the field of high sensitivity
detection of molecules, especially biomolecules. The invention also
relates to using fluorescent labels.
[0003] 2. General Background and State of the Art
[0004] Ultrasensitive immunoassay methods are developed and used in
clinical diagnostics to measure extremely low concentrations of
specific compounds in highly complex samples. Although the
sensitivity, reliability, rapidity, simplicity, and cost of these
methods have steadily improved, further improvements are still
needed and possible (Hampl J, et al., Upconverting phosphor
reporters in chromatographic assays. Analytical Biochemistry,
2001;288, 176-187; Unger M. et al, Single-molecule fluorescence
observed with mercury lamp illumination. Biotechniques
1999;27:1008-1014; and Weiss S. Fluorescence spectroscopy of single
biomolecules. Science 1999;283:1676-1683). The current trend toward
miniaturized, multianalyte methods has introduced its own
challenges and requirements for immunoassay technology (Taylor J R,
et al., Probing specific sequences of single DNA molecules with
bioconjugated fluorescent nanoparticles. Anal Chem
2000;72:1979-1986; and Zijlmans, et al., Detection of cell and
tissue surface antigens using up-converting phosphors: a new
reporter technology. Anal Biochem 1999;267:30-36). The interest in
new label technologies has especially increased because none of the
commonly used direct or enzyme-amplified radioactive, calorimetric,
luminescent, or fluorescent reporters fulfills all of the
requirements for an ideal label, including specific activity, size,
nontoxicity, cost, stability, localization, and detection. Directly
detectable labels such as fluorophores suffer from limited
sensitivity, and enzyme-amplified or dissociation-enhanced methods
lose spatial information.
[0005] Recently, new detection methods based on high
specific-activity particulate labels, such as quantum dots,
luminescent inorganic crystals, up-converting phosphors,
fluorescent nanoparticles, and plasmon resonant particles, have
been introduced to respond to future demands for clinical
diagnostics and biological, genomic, and pharmaceutical research.
These submicrometer-sized labels are coupled to specific binding
reagents such as nucleic acid probes, receptors, lectins, enzymes,
and antibodies to detect specific molecules with sensitivities
equal to or better than the best conventional labels available. In
spite of the large molecular size and obvious stearic problems,
these particular labels have also been used successfully in
solid-phase immunoassays. It has been recognized, however, that the
production, colloidal stability, and nonspecific binding of
particle-protein bioconjugates may still require further
improvements.
[0006] Time-resolved fluorometry and lanthanide labels were
introduced for immunoassays 20 years ago. Since then, the
dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA.RTM.)
technology has been known as one of the most sensitive and reliable
immunoassay platforms. The research on intrinsically fluorescent,
inert, and stable lanthanide chelate and cryptate labels has led to
the development of novel homogeneous and heterogeneous assays that
are expected to be introduced into routine clinical diagnostics.
Moreover, an advanced dissociation-enhanced technology, based on
lanthanide cofluorescence, which amplifies the long-lifetime
fluorescence of europium(III), terbium(III), samarium(III), and
dysprosium(III), has been known. A unique feature of lanthanide
chelate fluorescence, the absence of self-quenching effects from
multiple labeling, makes them ideal and suitable for high-density
cluster labels such as dyed latex nanoparticles. The highly
fluorescent chelates used in the DELFIA technology can also be used
in fluorescent lanthanide(III) chelate nanoparticle because the
hydrophobic environment inside the latex protects the fluorescent
chelates from environmental effects, such as solvent quenching, and
stabilizes the kinetically weak complexes. The adaptation of
appropriate chelates for all four lanthanides would enable a
nanoparticle-based, quadruple-labeling technology with an extremely
low detection limit and a direct, surface readout measurement.
[0007] Detecting low levels of target marker in a sample using
classic fluorochrome is sometimes difficult and prone to errors
because specific fluorescence signals tend to be low and are
usually mixed with nonspecific signals. Furthermore,
autofluorescence produced from specimen can cause interference. The
fluorescence half-life of complex chelate of lanthanide
elements--e.g., europium (EU)--is as much as six orders of magitude
longer than conventional fluorescent labels. Consequently, the
emission from lanthanide chelate can be distinguished from
background fluorescence (which has a short decay half-life) by
using a time-resolved fluorometer with an appropriate delay,
counting, and cycle times. This unique dye confers luminescence
with a decay time of >500 .mu.s, far longer than that of
conventional fluorescent probes or autofluorescent samples,
typically having decay times of <50 ns. Thus, time-resolved
fluorometry can virtually eliminate autofluorescence.
[0008] These europium luminescent dyes feature long-wavelength
emissions (.about.610 nm) that are well separated from the
excitation peak (.about.365 nm). This unusually large Stokes shift
permits the use of filter combinations that effectively isolate the
desired luminescence signal (Harma et al., Europium nanoparticles
and time-resolved fluorescence of ultrasensitive detection of
prostate-specific antigen. 2001;47:561-568). In the DELFIA system,
lanthanide ions are dissociated from the chelating structure into
the fluorescence enhancement solution. This additional enhancing
step is required to provide an environment that effectively
eliminates quenching water and contains energy-absorbing chelating
compounds to transfer energy further to lanthanide ions. The
lanthanide phosphors can be detected directly without any enhancing
steps due to the water-protecting crystal structure. The
disadvantage of using the lanthanide phosphors is the lack of
light-absorbing groups that effectively transfer the absorbed
energy to the lanthanide ions. Frank and Sundberg realized, in the
late 1970s, that by combining these properties into a latex
particle, fluorescent particles with a very high specific activity
could be prepared. They prepared latex particles which contained a
thenoyltrifluoroacetone lanthanide chelate complexed with
tri-n-octylphosphine oxide (naphtoyltrifluoroacetone in the DELFIA
technology complexed with tri-n-octylphosphine oxide) providing
particles without quenching effects but having a light-absorbing
group inside the particle. The polymer shell efficiently removes
fluorescence-quenching water from the vicinity of the chelate by
producing a hydrophobic environment. Extremely sensitive assays can
be carried out using such particle labels (Hrm et al., 2001, Soukka
et al., 2001a). These nanosized polymer labels contain
30,000-2,000,000 europium molecules entrapped by .beta.-diketones,
which have one of the highest quantum yields of the known
lanthanide chelators (Harma et al., Europium nanoparticles and
time-resolved fluorescence of ultrasensitive detection of
prostate-specific antigen. 2001;47:561-568). This encapsulation has
no negative effect on the fluorescence efficiency. For a 100 nm
size europium particle, the fluorescence yield is equivalent to
about 3,000 molecules of fluorescein. Phycobiliprotein B-PE
(perhaps the most fluorescent substance known) has a fluorescence
yield equivalent to about 30 fluorescein molecules. Since a 100 nm
particle is about 10 times the diameter of phycobiliprotein B-PE
and a thousand times greater in volume/mass, these europium
particles are 100 times more fluorescent than B-PE on a molar
basis. This particle will give ultrasensitivity for assay because
of its great fluorescence, broad stoke shift and long-lived
luminescence. Encapsulation: 30,000-2,000,000 europium molecules in
a single particle.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to a highly sensitive
assay system, including but not limited to time-resolved
fluorescent dye used in a lateral flow assay format, which ensures
improved sensitivity while maintaining the beneficial aspects of
lateral flow assay.
[0010] In one respect, the present invention is directed to a
chromatographic test system that uses highly sensitive europium
particle as a label. Chromatographic assay can give rapid result,
conveniently (mostly one step) with reasonable sensitivity. In
another aspect, the invention is directed to a sensitive nucleic
acid detection device and system thereof. Application of a
fluorescent rare earth chelate incorporated into a matrix provides
a highly sensitive assay.
[0011] In another aspect, the invention is directed to a genetic
materials detection system for the presence of analytes such as RNA
virus, DNA virus, RNA or DNA of the cell components via either a
non-polymerase chain reaction or polymerase chain reaction nucleic
acid-based approach. This detection system is fast, easy to use and
has high sensitivity and specificity.
[0012] The advantages of the invention include the following:
[0013] This system is extremely sensitive, which is an essential
requirement for analytes detection.
[0014] The inventive product can be used on-site, reducing the need
for sample transportation to off-site premises.
[0015] The immediate alert of positive results within about 15 min
more or less after sample preparation is possible with the
inventive product. It is to be understood that while one of the
advantages of the inventive system is that rapidity of the
detection system, the invention is not limited to any particular
time of obtaining results. The results may be obtained in the
ballpark of 20 minutes, 30 minutes 40 minutes or somewhat longer,
depending on various conditions.
[0016] Assay procedure for this one-step method is simple to
perform and test result is reading within 15 minutes more or less
and does not require any additional steps.
[0017] The inventive product can be performed by personnel without
advanced education and professional skills.
[0018] The inventive product can be stored at room temperature
whereas other commercial products generally require
refrigeration.
[0019] In another aspect of the invention, the invention is
directed to a Point-of-Care Testing or various settings of testing
for the various analytes because it is 1) Simple step, 2)
Field-usable, 3) Utilizes stable reagents, 4) No special storage,
and 5) Rapid results.
[0020] The present invention is directed to an analyte detection
apparatus having at least one reservoir area and a wicking
membrane, wherein a labeled specific binding partner is impregnated
on the reservoir area; and a region on the wicking membrane where
at least one chemical component is immobilized. As used in the
apparatus, the label may be a rare earth chelate, in particular a
lanthanide(III) chelate, and further in particular, the label may
be europium(III), terbium(III), samarium(III), or dysprosium(III),
or a combination thereof.
[0021] In the apparatus, the analyte may be without limitation an
antigen, an antibody, a nucleic acid or a hapten. The specific
binding partner may be without limitation an antigen, an antibody,
a nucleic acid, biotin or biotin analogue, streptavidin, avidin or
a hapten. And the chemical component may be an antigen, an
antibody, a nucleic acid, biotin or biotin analogue, streptavidin,
avidin or a hapten.
[0022] In one aspect of the invention, the apparatus may be a
lateral flow assay format apparatus.
[0023] In another aspect of the invention, the invention is
directed to a method of determining the presence of at least one
type of an analyte in a sample comprising applying an amount of the
sample to the apparatus described above, wherein if at least one
type of analyte is present in the sample, the sample migrates to
the wicking membrane where a chemical reaction occurs, wherein
presence of a signal indicates that the analyte is present in the
sample. In the practice of this invention, the signal may be
generated by a rare earth chelate, in particular a lanthanide(III)
chelate, and further in particular, the label may be europium(III),
terbium(III), samarium(III), or dysprosium(III), or a combination
thereof.
[0024] In this method, the sample may be a biological sample.
Further, the sample may be without limitation blood, serum, plasma,
urine, saliva, sweat and liquid media processed from a biological
or environmental sample. The analyte detected may be an antigen, an
antibody, a nucleic acid or a hapten. And the chemical reaction may
be with an antigen, an antibody, a nucleic acid, biotin or biotin
analogue, streptavidin, avidin or a hapten. A plurality of analytes
in a sample may be detected. The apparatus used in the method may
be without limitation a lateral flow assay format apparatus. And
the analyte may be specific to a pathogen.
[0025] The present invention is also directed to a kit comprising a
compartment that contains the apparatus described above, and
instructions for using the apparatus as described above.
[0026] The present invention is also directed to high sensitivity
nucleic acid detection system. The nucleic acid based detection
system of the invention is able to amplify a signal from a low
concentration of specific genomic DNA or RNA sequences from
biological samples. This method is also unique, specific, simple,
and the amplifying system is easy to operate in a field-deployable
detection module.
[0027] Time-resolved fluorometry technology is a system based on
europium embedded micro particles conjugated with oligonucleotides,
peptide nucleic acid (PNA), antigens or antibodies.
[0028] Any disease markers or environmental substances that require
highly sensitive tools for their detection may make use of this
technology.
[0029] These and other objects of the invention will be more fully
understood from the following description of the invention, the
referenced drawings attached hereto and the claims appended
hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The present invention will become more fully understood from
the detailed description given herein below, and the accompanying
drawings which are given by way of illustration only, and thus are
not limitative of the present invention, and wherein;
[0031] FIG. 1 shows a schematic depiction of the inventive
device.
[0032] FIG. 2 shows the inventive device for detecting a plurality
of analytes.
[0033] FIG. 3 shows picture of a test device.
[0034] FIG. 4 shows a configuration of rapid chromatographic
detection system.
[0035] FIGS. 5A and 5B show (A) Image result of colloidal gold
antibody conjugate, (B) Image result of europium particle.
[0036] FIG. 6 shows a configuration of rapid nucleic acid detection
system.
[0037] FIG. 7 shows a diagram of assay principle for detection of a
specific DNA sequence in a sample, such as from a biological
pathogen.
[0038] FIG. 8 shows an instrument for measuring time-resolved
lanthanide emission (Xiao, M and Selvin, P R. An improved
instrument for measuring time-resolved lanthanide emission and
resonance energy transfer. Review of scientific instruments 1999;
70(10):3877-81).
[0039] FIG. 9 shows picture of a test device.
[0040] FIG. 10 shows an embodiment of the test strip.
[0041] FIG. 11 shows an embodiment of the test strip.
[0042] FIG. 12 shows an embodiment of the test strip.
[0043] FIG. 13 shows an embodiment of the test strip.
[0044] FIG. 14 shows an embodiment of the test strip.
[0045] FIG. 15 shows an embodiment of the test strip.
[0046] FIG. 16 shows an embodiment of the test strip.
[0047] FIG. 17 shows an embodiment of the test strip.
[0048] FIG. 18 shows an embodiment of the test strip.
[0049] FIG. 19 shows an embodiment of the test strip.
DETAILED DESCRIPTION OF THE INVENTION
[0050] In the present application, "a" and "an" are used to refer
to both single and a plurality of objects.
[0051] As used herein, "base member" refers to a solid material,
which provides support and unity for the strip. This support may be
constructed from a thin sheet of glass, paper or plastic which has
been cut to a size appropriate to include entire assay contents
while providing convenience to the user.
[0052] As used herein, "dry porous carrier" or "wicking membrane"
refers to a substance that is porous enough to allow migration of
liquid and contiguous to filter element. Typical materials for use
in a dry porous carrier include, but are not limited to, nylon,
cellulose, polysulfone, polyvinylidene difluoride, cellulose
acetate, polyurethane, fiberglass, and nitrocellulose.
[0053] As used herein, "filter" may be fashioned from any number of
filter materials. Typical filter materials for use may include, but
are not limited to, cellulose, polyesters, polyurethanes, nylon and
fiberglass. Such a filter area may include a reservoir pad and
absorbent pad.
[0054] As used herein, "fluorescent rare earth chelate" may be
described in U.S. Pat. Nos. 4,259,313 and 4,283,382, which patents
are incorporated herein by reference in their entirety. Thus, this
invention describes method of utilizing long-lived fluorescent
compositions prepared by incorporating chelates of the rare earth
metals, preferably europium and terbium, into polymeric matrix such
as latex particles. The chelating agent strongly absorbs light and
efficiently transfers energy to the metal. The latex configuration
confers aqueous stability to fluorescent rare earth chelates, which
in the past have been subject to quenching in aqueous liquids. The
polymeric beads derived from the latex and having the rare earth
chelate incorporated therein can then be used as fluorescent labels
to form labeled reagents by adsorbing or covalently binding
antigens, antibodies, plant lectins, carbohydrates or other such
proteinaceous compounds, lipids and nucleic acids to the surface of
the polymeric latex beads.
[0055] As used herein, "impregnated" refers to reagents which are
incorporated in the assay system, wherein they are either dried or
lyophilized onto or into the assay system.
[0056] As used herein, "polymeric particle" refers to a spherical
or near-spherical polymer particle at various sizes. Preferably,
the size is about 0.05-0.5 .mu.m in diameter. However, it is
understood that the invention is not limited to the use of any
particular type of polymeric particle. In its broadest sense, any
substance or particle that can encapsulate the fluorescent dye is
encompassed by the present invention.
[0057] As used herein, "specific binding reagent" or "specific
binder" or "specific binding partner" includes, but is not limited
to, antibody, antigen, hapten, hapten-macromolecule (e.g. bovine
serum albumin) conjugate, avidin, streptavidin, biotin,
biotin-macromolecule (e.g. bovine serum albumin) conjugate,
oligonucleotide, peptide nucleic acid and nucleic acid genetic
material.
[0058] As used herein, "tag" refers to a substance that is labeled
to the specific binding reagent, which is labeled with
time-resolved fluorescent dye. The tag reacts specifically to
specific binder immobilized on solid phase. In particular, the tag
may be biotin when the specific binder immobilized on solid phase
is avidin or streptavidin. The tag may be a hapten when the
specific binder immobilized on solid phase is antibody that is
specific to the hapten of the tag.
[0059] As used herein, "time-resolved fluorometer" refers to a tool
for measuring time dependence of fluorescence intensity after a
short excitation pulse which can also be made as a function of
emission wavelength.
[0060] Chromatographic Amplification
[0061] In one aspect, the present invention is directed to a
apparatus that is suitable for rapid chromatographic tests.
[0062] The inventive test may be performed on-site by a lay person
with minimal training while rapid results are obtained after adding
one or two drops of sample to a disposable test device or card or
strip. The results may be read visually without any further
intervention. In one aspect of the invention, the procedure for the
proposed test may be as follows. For discussion purposes only, the
test may be discussed in terms of an antigen/antibody reaction.
However, it is to be understood that the assay is not limited an
antigen/antibody complex. For any molecule of interest for which a
specific binding partner is known, the specific binder may be used
and impregnated into the apparatus to assay for the presence of the
molecule of interest. Such molecule of interest and its specific
binding partner may include without limitation, antigen/antibody,
ligand/receptor, nucleic acid/nucleic acid, nucleic acid/antibody,
lipid/specific binding partner such as an antibody,
carbohydrate/specific binding partner such as an antibody and so
on. For purposes of illustration only, in the following
discussions, antigen/antibody and nucleic acid/nucleic acid
interactions are primarily discussed, with the understanding that
the principles of using specific binding partners is applicable to
any type of molecule from any sample source.
[0063] A liquid sample is added to the test device or card or
strip. As the fluid wicks/moves across the card the target antigens
react with labeled specific antibodies embedded in the dye pad. The
material flows into a membrane where a set of unlabeled antibodies
are immobilized in at least one distinct zone(s). The
antigen-labeled antibody complex is retained/captured creating (a)
defined line(S) for reading. This line can be detected by a reader
equipped with time-resolved fluorescence or any other suitable
detection mechanism. In a separate control zone, the excess-labeled
antibody in the test reacts with embedded non-specific antibody to
provide assurance that the key test component is functioning
properly, thereby being useful as a positive control. A
non-limiting principle of the test is further seen in FIG. 1.
[0064] A liquid sample is added to the test device or card or
strip. As the fluid wicks/moves across the card the target antigens
react with labeled specific antibodies embedded in the dye pad. The
material flows into a membrane where a set of unlabeled antibodies
are immobilized in at least one distinct zone(s). The
antigen-labeled antibody complex is retained/captured creating (a)
defined line(S) for reading. This line can be detected by a reader
equipped with time-resolved fluorescence or any other suitable
detection mechanism.
[0065] Chromatographic assay in the form of a lateral flow assay,
for instance, is an amplification system. The target antigens in a
liquid sample are steadily concentrated by the high-affinity
antibodies immobilized on a membrane when they are moving through
the membrane by capillary power. As a result, even though the
antigen may be at a very low concentration in the actual sample,
the concentration of the antigen captured at the test line or test
zone is much higher than in the sample. Also, capillary migration
supplies target antigens continuously to the immobilized
antibody.
[0066] Usually, when the capture antibody forms a complex with an
antigen as in a conventional assay, there is a decrease of
micro-environmental antigen concentration surrounding the capture
antibody immobilized on the solid phase. In general, this depletion
of antigen around the antibody immobilized on the solid phase is
one of the major problems in assay sensitivity, especially with
microplate assays and chip-based assay, since this causes an actual
decrease of antigen concentration around the antibody.
[0067] In an alternative embodiment, the three-dimensional
structure of a bibulous membrane (e.g. nitrocellulose) provides
more surface area for antibody binding to solid phase. This allows
the chromatographic assay to have significantly more capturing
capacity than other two dimensional assay systems (e.g. microplate
assay).
[0068] The invention is also directed to multiple analyte detection
system, which is conducted with only a single sampling and no
further procedural steps with the inventive chromatographic assay.
If various antibody-dye conjugates are embedded in the dye pad area
and antibody specific for each antigen is respectively immobilized
in a separate zone on the membrane, each test line provides
distinctive information for each specific antigen for the specific
agent (FIG. 2). It is also contemplated that the device may detect
more than one type of analyte. For instance, by way of example,
referring to FIG. 2, Test zone 1 may be impregnated with an
antibody to detect an antigen; Test zone 2 may be impregnated with
a receptor to detect a ligand; and Test 3 may be impregnated with a
nucleic acid for sequence specific binding.
[0069] In one aspect, the invention is directed to a signal
amplification system using fluorescent europium particles that
contain about 30,0000-1,000,000 europium atoms in a single
particle. This system may be an improved chromatographic assay with
advantageous features such as 1) Simple testing procedure, 2)
Field-usable, 3) Utilizes stable reagents, 4) No special storage
required, and 5) Rapid results. The amplified signal can be
detected by a small, portable reader that provides quantitative or
qualitative results.
[0070] The test strip may be enclosed within a case, which is
preferably a disposable plastic case, but may be made with any
substance, which is able to contain the contents safely. The test
kit may contain a plurality of windows, preferably at least two
windows or openings, at least one to view the control and/or test
lines or test bands, and the other providing a well to receive the
sample (FIG. 3). Referring to FIG. 4, inside the device may be a
test strip with preferably two pads. The first pad, the reservoir
area 1 or sample well may be used for the uptake of the sample and
the removal of interfering materials from sample. In addition, the
first pad may contain europium particle-specific binding partner
conjugates 3 in a specially formulated buffer system. The labeled
specific binding reagent impregnated into the reservoir area may
bind an analyte in the sample and the analyte/specific binding
partner complex is wicked through a wicking membrane 6 until a test
band 4 is formed, where another specific binder of the analyte
captures the analyte/specific binding partner complex thus forming
a test band. The sample is further wicked and encounters a binding
partner to the specific binding partner impregnated in the wicking
membrane, and a control band 5 is established. The second pad 7,
absorbent pad, may be used for removing excess fluid that has
already passed through the reaction membrane. The first pad, second
pad and wicking membrane are connected to a plastic plate 2.
[0071] Two types of reagents may be separately immobilized on the
membrane as thin lines or bands. The antibody that is specific to
the conjugate antibody may be immobilized in the control window,
while the antibody that is specific to an analyte such as a
biothreat agent or any agent that is desired to be detected is
immobilized in the test window (FIGS. 3 and 4).
[0072] Chromatographic Assay Principle
[0073] The inventive test kit is designed to be a self-performing
device. It may contain all of the reagents and components in
precise quantities to generate test results after sample addition,
as shown in FIG. 1. The sample passes first through the reservoir
pad that contains various materials. It may contain (a) buffer(s)
to optimize the pH of the sample, (a) detergent(s) to suspend all
components in the sample, and (a) porous filter(s) to generate
proper flow through the device. The sample subsequently flows, by
capillary action or diffusion, to the dye area or dye pad, where
the test agents react with the labeled specific binder, which is
preferably labeled with an europium particle-label, which is
specific to the agent. The reaction complex then migrates through
the wicking membrane where the non-reacted binding sites of the
agent react with immobilized specific binder, and generating a line
or band in the test window. The depleted sample and remainder of
the unbound dye complex continue to migrate to the control window
where antibody specific to the conjugate antibody is immobilized to
retain the dye complex and form a control line. It is also
contemplated that the reaction complex may encounter the control
region before reaching the test region.
[0074] In one aspect of the invention, the result is a solid-phase
chromatographic assay for the qualitative or quantitative detection
of a bio agent. In the test procedure, about 60 .mu.L of liquid
sample may be added to the sample application area and the result
may be provided by the instrument within about 15 minutes more or
less. It is to be understood that while the rapidity of the assay
is an advantageous feature of the inventive system, the exact time
to obtain the result may vary depending conditions and sample.
Accordingly, the present invention is not limited by any particular
time of assay.
EMBODIMENT 1
[0075] In one embodiment of the detection apparatus, the apparatus
includes the following features:
[0076] At least one filter element 1 having impregnated one or more
specific binding reagent(s) labeled with fluorescent label
comprising a fluorescent rare earth chelate incorporated into a
polymeric particle 3; and
[0077] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to
filter element, wherein
[0078] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 10).
EMBODIMENT 2
[0079] In another embodiment of the detection apparatus, the
apparatus includes the following features:
[0080] At least one first filter element 1 having impregnated one
or more specific binding reagent(s) labeled with fluorescent label
comprising a fluorescent rare earth chelate incorporated into a
polymeric particle 3;
[0081] At least one second filter element 11 which is interposed
between the first filter element and dry porous carrier and which
is porous enough to allow migration of liquid; and
[0082] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to the
second filter element, wherein
[0083] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 11).
EMBODIMENT 3
[0084] In another embodiment of the detection apparatus, the
apparatus includes the following features:
[0085] At least one first filter element 1 which is interposed
between the second filter element 11 and dry porous carrier 6, and
which is porous enough to allow migration of liquid, and which has
impregnated one or more specific binding reagent(s) labeled with
fluorescent label comprising a fluorescent rare earth chelate
incorporated into a polymeric particle 3;
[0086] At least one second filter element 11 which is contiguous to
the first filter element 1 and which is porous enough to allow
migration of liquid; and
[0087] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to the
first filter element, wherein
[0088] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 12).
EMBODIMENT 4
[0089] In another embodiment of the detection apparatus, the
apparatus includes the following features:
[0090] A base member 2;
[0091] An array disposed on base member 2, array comprising:
[0092] At least one filter element 1 having impregnated one or more
specific binding reagent(s) labeled with fluorescent label
comprising a fluorescent rare earth chelate incorporated into a
polymeric particle 3; and
[0093] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to
filter element, wherein
[0094] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 13).
EMBODIMENT 5
[0095] In another embodiment of the detection apparatus, the
apparatus includes the following features:
[0096] A base member 2;
[0097] An array disposed on base member 2, array comprising:
[0098] At least one first filter element 1 having impregnated one
or more specific binding reagent(s) labeled with fluorescent label
comprising a fluorescent rare earth chelate incorporated into a
polymeric particle 3;
[0099] At least one second filter element 11 which is interposed
between the first filter element 1 and dry porous carrier 6 and
which is porous enough to allow migration of liquid; and
[0100] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to the
second filter element, wherein
[0101] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 14).
EMBODIMENT 6
[0102] In another embodiment of the detection apparatus, the
apparatus includes the following features:
[0103] A base member 2;
[0104] An array disposed on base member 2, array comprising:
[0105] At least one first filter element 1 which is interposed
between the second filter element 11, and which is porous enough to
allow migration of liquid, and which has impregnated one or more
specific binding reagent(s) labeled with fluorescent label
comprising a fluorescent rare earth chelate incorporated into a
polymeric particle 3;
[0106] At least one second filter element 11 which is contiguous to
the first filter element 1 and which is porous enough to allow
migration of liquid; and
[0107] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to the
first filter element 1, wherein
[0108] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 15).
EMBODIMENT 7
[0109] In another embodiment of the detection apparatus, the
apparatus includes the following features:
[0110] At least one first filter element 1 having impregnated one
or more specific binding reagent(s) labeled with fluorescent label
comprising a fluorescent rare earth chelate incorporated into a
polymeric particle 3;
[0111] At least one second filter element 11 which has impregnated
one or more specific binding reagent(s) labeled with tag that is
specifically reactive to the specific binding reagent immobilized
in dry porous carrier 6, and which is interposed between the first
filter element 1 and dry porous carrier 6 and which is porous
enough to allow migration of liquid; and
[0112] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to the
second filter element, wherein
[0113] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 16).
EMBODIMENT 8
[0114] In another embodiment of the detection apparatus, the
apparatus includes the following features:
[0115] At least one first filter element 1 which is interposed
between the second filter element 11, and which is porous enough to
allow migration of liquid, and which has impregnated one or more
specific binding reagent(s) labeled with fluorescent label
comprising a fluorescent rare earth chelate incorporated into a
polymeric particle 3;
[0116] At least one second filter element 11 which has impregnated
one or more specific binding reagent(s) labeled with tag 3 that is
specifically reactive to the specific binding reagent immobilized
in dry porous carrier 6, and which is contiguous to the first
filter element 1 and which is porous enough to allow migration of
liquid; and
[0117] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to the
first filter element, wherein
[0118] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 17).
EMBODIMENT 9
[0119] In another embodiment of the detection apparatus, the
apparatus includes the following features:
[0120] A base member 2;
[0121] An array disposed on base member, array comprising:
[0122] At least one first filter element 1 having impregnated one
or more specific binding reagent(s) labeled with fluorescent label
comprising a fluorescent rare earth chelate incorporated into a
polymeric particle 3;
[0123] At least one second filter element 11 which has impregnated
one or more specific binding reagent(s) labeled with tag that is
specifically reactive to the specific binding reagent immobilized
in dry porous carrier 6, and which is interposed between the first
filter element 1 and dry porous carrier 6 and which is porous
enough to allow migration of liquid; and
[0124] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to the
second filter element, wherein
[0125] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 18).
EMBODIMENT 10
[0126] In another embodiment of the detection apparatus, the
apparatus includes the following features:
[0127] A base member 2;
[0128] An array disposed on base member 2, array comprising:
[0129] At least one first filter element 1 which is interposed
between the second filter element 11, and which is porous enough to
allow migration of liquid, and which has impregnated one or more
specific binding reagent(s) labeled with fluorescent label
comprising a fluorescent rare earth chelate incorporated into a
polymeric particle 3;
[0130] At least one second filter element 11 which has impregnated
one or more specific binding reagent(s) labeled with tag that is
specifically reactive to the specific binding reagent immobilized
in dry porous carrier 6, and which is contiguous to the first
filter element 1 and which is porous enough to allow migration of
liquid; and
[0131] A dry porous carrier 6 (e.g. nitrocellulose membrane) which
is porous enough to allow migration of liquid and contiguous to the
first filter element 1, wherein
[0132] At least one specific binding reagent is immobilized in at
least one zone of the dry porous carrier (FIG. 19).
[0133] Sandwich Assays
[0134] Antibody-Antibody Sandwich
[0135] Specific binder immobilized on solid phase: monoclonal
antibody and/or polyclonal antibody. Specific binding reagent
labeled with fluorescent dye: monoclonal antibody and/or polyclonal
antibody labeled with fluorescent dye.
[0136] Antibody-Antigen Sandwich
[0137] Antibody-Antigen Sandwich 1
[0138] Specific binder immobilized on solid phase: antigen specific
to analyte antibody in sample (e.g. HIV-1 gp41 antigen that is
specifically reactive to human anti-HIV-1 gp41 antibody)
[0139] Specific binding reagent labeled with fluorescent dye:
secondary monoclonal antibody and/or polyclonal antibody labeled
with fluorescent dye. This antibody is specific to the primary
antibodies in samples.
[0140] Antibody-Antigen Sandwich 2
[0141] Specific binder immobilized on solid phase: secondary
monoclonal antibody and/or polyclonal antibody. This antibody is
specific to the primary antibodies in samples.
[0142] Specific binding reagent labeled with fluorescent dye:
antigen (labeled with fluorescent dye) specific to analyte antibody
in sample (e.g. HIV-1 gp41 antigen that is specifically reactive to
human anti-HIV-1 gp41 antibody)
[0143] Antigen-Antigen Sandwich
[0144] Specific binder immobilized on solid phase: antigen specific
to analyte antibody in sample (e.g. HIV-1 gp41 antigen that is
specifically reactive to human anti-HIV-1 gp41 antibody)
[0145] Specific binding reagent labeled with fluorescent dye:
antigen (labeled with fluorescent dye) specific to analyte antibody
in sample (e.g. HIV-1 gp41 antigen that is specifically reactive to
human anti-HIV-1 gp41 antibody)
[0146] This format comprising:
[0147] Specific binder (e.g. avidin or streptavidin) immobilized on
solid phase; specific binding reagent labeled with tag(s) (e.g.
biotin), which reacts specifically to specific binder immobilized
on solid phase; specific binding reagent labeled with fluorescent
dye.
[0148] This format is applicable to all assay formats described
above with; replacing the specific binder immobilized on solid
phase to the specific binder (e.g. avidin or streptavidin)
immobilized on solid phase and the specific binding reagent labeled
with tag.
[0149] Competition Assay
[0150] Format 1
[0151] Specific binding reagent (e.g. antibody specific to hapten)
labeled with fluorescent dye: capable of binding to analyte of
interest in sample to form reaction complex.
[0152] Specific binder (e.g. hapten) immobilized on solid phase:
capable of reacting with free specific binding reagent labeled with
fluorescent dye and capable of competitively displacing analyte
from the reaction complex and reacting with specific binding
reagent labeled with fluorescent dye.
[0153] Format 2
[0154] Specific binding reagent (e.g. antibody specific to hapten)
immobilized on solid phase: capable of competitively binding to
analyte of interest in sample or specific binder (e.g. hapten)
labeled with fluorescent dye.
[0155] Specific binder (e.g. hapten) labeled with fluorescent dye:
capable of competing with analyte of interest in sample.
[0156] Format 3
[0157] This format comprising:
[0158] Specific binder (e.g. avidin or streptavidin) immobilized on
solid phase; specific binding reagent labeled with tag(s) (e.g.
biotin), which reacts specifically to specific binder immobilized
on solid phase; specific binding reagent (hapten) labeled with
fluorescent dye.
[0159] This format is applicable to all assay formats described
above with; replacing the specific binder immobilized on solid
phase to the specific binder (e.g. avidin or streptavidin)
immobilized on solid phase and the specific binding reagent labeled
with tag.
[0160] Detection of Multiple Analytes
[0161] Another embodiment of the present invention permits the
detection of multiple analytes in a single fluid sample by the
presence of more than one specific type of labeled reagent and the
same number of types of corresponding immobilized reagent. The
device can be set up unidirectionally with multiple labeled
reagents impregnated throughout the filter elements and multiple
corresponding immobilized substances defined in several assay
indicia zones on the dry porous carrier. In the bidirectional or
multidirectional embodiment, more than one set of components such
as the filter elements and dry porous carrier are associated with a
common reservoir.
[0162] Kit and Instructions for Using the Kit
[0163] The present invention includes a kit for using the inventive
detection apparatus. The kit may comprise a container made of
cardboard, plastic or any other solid object or plastic bag, which
may house the detection apparatus. Included in the kit may be
instructions on how to use the kit. Such instructions may be
written on the container or in written form placed inside the
container such as a paper instruction sheet. Instructions for using
the kit and the detection apparatus may also be in electronic
format, on a website, in addition or in lieu of paper format.
Instructions may also be placed in a catalog for selling the
kit.
EXAMPLES
Example 1
Preparation of Troponin I Test Strip Using Europium Chelate
Nanoparticle
[0164] Preparation of Anti-Troponin I Coated Nanoparticle
[0165] The carboxyl group of europium chelate nanoparticles were
activated with 10 mmol/L
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and 100 mmol/L
N-hydroxysulfosuccinimide for 30 min. The activated particle washed
once with 50 mM MES buffer, pH 6.1. 20 mM/L anti-troponin I
antibody was added. After 2 hour incubation, the antibody coated
particles were washed three times with 50 mM MES buffer, pH
6.1.
[0166] Preparation of Dye Pad
[0167] The glass fiber filter was prepared by impregnating with a
solution containing anti-troponin I antibody coated nanoparticle,
0.2% tween-20, 0.25% bovine serum albumin, 0.5% sucrose, 10 mM
sodium phosphate, pH 7.5 to rectangular piece of glass fiber filter
measuring 8 mm.times.305 mm and dried under constant vacuum in a
lyophilizer. The pad was stored dry in a desiccator until use.
[0168] Preparation of Filter Pad
[0169] The glass fiber filter was treated with a solution of 0.05%
Tween-20, 2% of sucrose, 1% of BSA and 100 mM sodium phosphate, pH
7.4, and then air dried at room temperature.
[0170] Immobilization of Antibody on Membrane
[0171] A double sided transparent tape (305 mm.times.25 mm size)
was attached to 20 mm from the bottom of the thin plastic plate
(305.times.60 mm). A nitrocellulose membrane was cut to 305
mm.times.25 mm size and attached directly on the top of the double
sided tape. An assay indicia zone of immobilized test line for
Troponin I was defined on the 9 mm from the bottom of the membrane
by spraying 30 micro liter of solution of 1 mg/ml goat
anti-troponin I antibody in 10 mM PBS, pH 7.5. For control band, 1
mg/ml of polyclonal anti-mouse IgG antibody was defined on the 13
mm from the bottom of the membrane by spraying. After spraying, the
membrane was dried at ambient temperature for approximately 12
hours. The base and wicking membrane was stored in a desiccator
until further processed.
[0172] Strip Construction
[0173] Dye pad was attached to plastic base right below the bottom
of nitrocelluolse membrane and the filter pad is attached adjacent
to the dye pad. The plastic plate then was cut into a plurality of
strips 60 mm in length and 4 mm in width so that each contains a
linear array of nitrocellulose membrane, dye pad and filter
pad.
[0174] Assay Method and Result:
[0175] When 70 micro liter sample was added into filter pad, a
detectable signal began to appear in the assay indicia zone after
incubation when the strip was exposed to UV light.
Example 2
Preparation of Troponin I Test Strip Using Colloidal Gold
[0176] Preparation of Gold Sol
[0177] 1400 ml of deionized water was brought to a boil. Hydroauric
acid (299 to 305 mg) was added, and boiling continued for 5
minutes. Sodium citrate (440 mg) dissolved in 10 ml of distilled
water was poured into the gold solution and the solution boiled for
another 10 minutes. The solution was allowed to cool to ambient
room temperature.
[0178] Preparation of Label
[0179] pH of gold sol was adjusted to 6.8 with 40 mM potassium
carbonate. Same monoclonal antibody used in Example 1 was added to
50 ml of gold solution which was stirred vigorously for 30 min at
ambient temperature. 1 ml of 15% bovine serum albumin was added,
and the solution was continuously stirred for approximately 15 min
at ambient temperature. Colloidal gold-monoclonal antibody
conjugate was recovered by centrifugation at 10,000 rpm in GSA
rotor for 1 hr, discarding the supernatant and suspending the
resultant pellet in 25 ml of 2% bovine serum albumin in 10 mM
sodium phosphate, pH 7.5. The suspension was then spun down at
10,000 rpm for 1 hr in GSA rotor. The supernatant once again was
discarded and the pellet suspended in 6 ml of 2% bovine serum
albumin in 10 mM sodium phosphate, pH 7.5.
[0180] Preparation of Dye Pad
[0181] The glass fiber filter was prepared by impregnating with a
solution containing anti-troponin I antibody coated colloidal gold,
0.2% tween-20, 0.25% bovine serum albumin, 0.5% sucrose, 10 mM
sodium phosphate, pH 7.5 to rectangular piece of glass fiber filter
measuring 8 mm.times.305 mm and dried under constant vacuum in a
lyophilizer. The pad was stored dry in a desiccator until use.
[0182] Preparation of Filter Pad
[0183] The glass fiber filter was treated with a solution of 0.05%
Tween-20, 2% of sucrose, 1% of BSA and 100 mM sodium phosphate, pH
7.4, and then air dried at room temperature.
[0184] Immobilization of Antibody on Membrane
[0185] A double sided transparent tape (305 mm.times.25 mm size)
was attached to 20 mm from the bottom of the thin plastic plate
(305.times.60 mm). A nitrocellulose membrane was cut to 305
mm.times.25 mm size and attached directly on the top of the double
sided tape. An assay indicia zone of immobilized test line for
Troponin I was defined on the 9 mm from the bottom of the membrane
by spraying 30 micro liter of solution of 1 mg/ml goat
anti-troponin I antibody in 10 mM PBS, pH 7.5. For control band, 1
mg/ml of polyclonal anti-mouse IgG antibody was defined on the 13
mm from the bottom of the membrane by spraying. After spraying, the
membrane was dried at ambient temperature for approximately 12
hours. The base and wicking membrane were stored in a desiccator
until further processed.
[0186] Strip Construction
[0187] Dye pad was attached to plastic base right below the bottom
of nitrocelluolse membrane and the filter pad was attached adjacent
to the dye pad. The plastic plate then was cut into a plurality of
strips 60 mm in length and 4 mm in width so that each contains a
linear array of nitrocellulose membrane, dye pad and filter
pad.
[0188] Assay Method and Result:
[0189] When 70 micro liter sample was added into filter pad, a
detectable signal began to appear in the assay indicia zone after
incubation.
Example 3
Comparison of the Sensitivity of the Strips Prepared Using Each
Method
[0190] The strip prepared in Example 1 and the strip prepared in
Example 2 were tested to compare the sensitivity. The strip
prepared in Example 1 showed 0.025 nanogram/ml of sensitivity while
the strip prepared in Example 2 showed 0.5 nanogram/ml of
sensitivity. The strip prepared using europium nanoparticle showed
about 20 times more sensitive result than the strip prepared using
colloidal gold.
Example 4
Preparation of hCG Test Strip Using Europium Nanoparticle
[0191] Preparation of Anti-hCG Coated Nanoparticle
[0192] The carboxyl group of europium chelate nanoparticles were
activated with 10 mmol/L
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and 100 mmol/L
N-hydroxysulfosuccinimide for 30 min. The activated particle washed
once with 50 mM MES buffer, pH 6.1. 20 mM/L anti-hCG antibody was
added. After 2 hour incubation, the antibody coated particles were
washed three times with 50 mM MES buffer, pH 6.1.
[0193] Preparation of Dye Pad
[0194] The glass fiber filter was prepared by impregnating with a
solution containing anti-hCG antibody coated nanoparticle, 0.2%
tween-20, 0.25% bovine serum albumin, 0.5% sucrose, 10 mM sodium
phosphate, pH 7.5 to rectangular piece of glass fiber filter
measuring 8 mm.times.305 mm and dried under constant vacuum in a
lyophilizer. The pad was stored dry in a desiccator until use.
[0195] Preparation of Filter Pad
[0196] The glass fiber filter was treated with a solution of 0.05%
Tween-20, 2% of sucrose, 1% of BSA and 100 mM sodium phosphate, pH
7.4, and then air dried at room temperature.
[0197] Immobilization of Antibody on Membrane
[0198] A double sided transparent tape (305 mm.times.25 mm size)
was attached to 20 mm from the bottom of the thin plastic plate
(305.times.60 mm). A nitrocellulose membrane was cut to 305
mm.times.25 mm size and attached directly on the top of the double
sided tape. An assay indicia zone of immobilized test line for hCG
was defined on the 9 mm from the bottom of the membrane by spraying
30 micro liter of solution of 1 mg/ml monoclonal anti-hCG antibody
in 10 mM PBS, pH 7.5. For control band, 1 mg/ml of polyclonal
anti-mouse IgG antibody was defined on the 13 mm from the bottom of
the membrane by spraying. After spraying, the membrane was dried at
ambient temperature for approximately 12 hours. The base and
wicking membrane was stored in a desiccator until further
processed.
[0199] Strip Construction
[0200] Dye pad was attached to plastic base right below the bottom
of nitrocelluolse membrane and the filter pad is attached adjacent
to the dye pad. The plastic plate then was cut into a plurality of
strips 60 mm in length and 4 mm in width so that each contains a
linear array of nitrocellulose membrane, dye pad and filter
pad.
[0201] Assay Method and Result:
[0202] When 70 micro liter sample was added into filter pad, a
detectable signal began to appear in the assay indicia zone after
incubation when the strip was exposed to UV light.
Example 5
Preparation of hCG Test Strip Using Colloidal Gold
[0203] Preparation of Gold Sol
[0204] 1400 ml of deionized water was brought to a boil. Hydroauric
acid (299 to 305 mg) was added, and boiling continued for 5
minutes. Sodium citrate (440 mg) dissolved in 10 ml of distilled
water was poured into the gold solution and the solution boiled for
another 10 minutes. The solution was allowed to cool to ambient
room temperature.
[0205] Preparation of Label
[0206] pH of gold sol was adjusted to 6.8 with 40 mM potassium
carbonate. Same monoclonal antibody used in Example 4 was added to
50 ml of gold solution which was stirred vigorously for 30 min at
ambient temperature. 1 ml of 15% bovine serum albumin was added,
and the solution was continuously stirred for approximately 15 min
at ambient temperature. Colloidal gold-monoclonal antibody
conjugate was recovered by centrifugation at 10,000 rpm in GSA
rotor for 1 hr, discarding the supernatant and suspending the
resultant pellet in 25 ml of 2% bovine serum albumin in 10 mM
sodium phosphate, pH 7.5. The suspension was then spun down at
10,000 rpm for 1 hr in GSA rotor. The supernatant once again was
discarded and the pellet suspended in 6 ml of 2% bovine serum
albumin in 10 mM sodium phosphate, pH 7.5.
[0207] Preparation of Dye Pad
[0208] The glass fiber filter was prepared by impregnating with a
solution containing anti-hCG antibody coated colloidal gold, 0.2%
tween-20, 0.25% bovine serum albumin, 0.5% sucrose, 10 mM sodium
phosphate, pH 7.5 to rectangular piece of glass fiber filter
measuring 8 mm.times.305 mm and dried under constant vacuum in a
lyophilizer. The pad was stored dry in a desiccator until use.
[0209] Preparation of Filter Pad
[0210] The glass fiber filter was treated with a solution of 0.05%
Tween-20, 2% of sucrose, 1% of BSA and 100 mM sodium phosphate, pH
7.4, and then air dried at room temperature.
[0211] Immobilization of Antibody on Membrane
[0212] A double sided transparent tape (305 mm.times.25 mm size)
was attached to 20 mm from the bottom of the thin plastic plate
(305.times.60 mm). A nitrocellulose membrane was cut to 305
mm.times.25 mm size and attached directly on the top of the double
sided tape. An assay indicia zone of immobilized test line for hCG
was defined on the 9 mm from the bottom of the membrane by spraying
30 micro liter of solution of 1 mg/ml goat anti-troponin I antibody
in 10 mM PBS, pH 7.5. For control band, 1 mg/ml of polyclonal
anti-mouse IgG antibody was defined on the 13 mm from the bottom of
the membrane by spraying. After spraying, the membrane was dried at
ambient temperature for approximately 12 hours. The base and
wicking membrane were stored in a desiccator until further
processed.
[0213] Strip Construction
[0214] Dye pad was attached to plastic base right below the bottom
of nitrocelluolse membrane and the filter pad was attached adjacent
to the dye pad. The plastic plate then was cut into a plurality of
strips 60 mm in length and 4 mm in width so that each contains a
linear array of nitrocellulose membrane, dye pad and filter
pad.
[0215] Assay Method and Result:
[0216] When 70 micro liter sample was added into filter pad, a
detectable signal began to appear in the assay indicia zone after
incubation.
Example 6
Comparison of the Sensitivity of the Strips Prepared Using Each
Method
[0217] The strip prepared in Example 4 and the strip prepared in
Example 5 were tested to compare the sensitivity. The strip
prepared in Example 4 showed 1.5 mIU/ml of sensitivity while the
strip prepared in Example 5 showed 15 mIU/ml of sensitivity. The
strip prepared using europium nanoparticle showed about 10 times
more sensitive result than the strip prepared using colloidal
gold.
Example 7
Preparation of Dengue Virus Test Strip Using Europium Chelate
Nanoparticle
[0218] Preparation of Oligonucleotides Coated Nanoparticle
[0219] The carboxyl group of europium chelate nanoparticles were
activated with 10 mmol/L
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and 100 mmol/L
N-hydroxysulfosuccinimide for 30 min. The activated particle washed
once with 50 mM MES buffer, pH 6.1. 20 mM/L oligonucleotide with
carriers such as bovine serum album was added. After 2 hour
incubation, the oligonucleotide coated particles were washed three
times with 50 mM MES buffer, pH 6.1.
[0220] Preparation of Dye Pad
[0221] The glass fiber filter was prepared by impregnating with a
solution containing oligonucleotides coated nanoparticle, 0.2%
tween-20, 0.25% bovine serum albumin, 0.5% sucrose, 10 mM sodium
phosphate, pH 7.5 to rectangular piece of glass fiber filter
measuring 8 mm.times.305 mm and dried under constant vacuum in a
lyophilizer. The pad was stored dry in a desiccator until use.
[0222] Preparation of Filter Pad
[0223] The glass fiber filter was treated with a solution of 0.05%
Tween-20, 2% of sucrose, 1% of BSA and 100 mM sodium phosphate, pH
7.4, and then air dried at room temperature.
[0224] Immobilization of Oligonucleotide on Membrane
[0225] A double sided transparent tape (305 mm.times.25 mm size)
was attached to 20 mm from the bottom of the thin plastic plate
(305.times.60 mm). A nitrocellulose membrane was cut to 305
mm.times.25 mm size and attached directly on the top of the double
sided tape. An assay indicia zone of immobilized test line for
dengue virus specific oligonucleotide was defined on the 9 mm from
the bottom of the membrane by spraying 30 micro liter of solution
of 1 mg/ml oligonucleotide-BSA conjugate in 10 mM PBS, pH 7.5.
After spraying, the membrane was dried at ambient temperature for
approximately 12 hours. The base and wicking membrane was stored in
a desiccator until further processed.
[0226] Strip Construction
[0227] Dye pad was attached to plastic base right below the bottom
of nitrocelluolse membrane and the filter pad is attached adjacent
to the dye pad. The plastic plate then was cut into a plurality of
strips 60 mm in length and 4 mm in width so that each contains a
linear array of nitrocellulose membrane, dye pad and filter
pad.
[0228] Assay Method and Result:
[0229] When 70 micro liter sample was added into filter pad, a
detectable signal began to appear in the assay indicia zone after
incubation when the strip was exposed to UV light.
Example 8
Preparation of Dengue Virus Test Strip Using Europium Chelate
Nanoparticle with Polymerase Chain Reaction
[0230] Preparation of Streptavidin Coated Nanoparticle
[0231] The carboxyl group of europium chelate nanoparticles were
activated with 10 mmol/L
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and 100 mmol/L
N-hydroxysulfosuccinimide for 30 min. The activated particle washed
once with 50 mM MES buffer, pH 6.1. 20 mM/L strepatavidine was
added. After 2 hour incubation, the streptavidin coated particles
were washed three times with 50 mM MES buffer, pH 6.1.
[0232] Preparation of Dye Pad
[0233] The glass fiber filter was prepared by impregnating with a
solution containing streptavidin coated nanoparticle, 0.2%
tween-20, 0.25% bovine serum albumin, 0.5% sucrose, 10 mM sodium
phosphate, pH 7.5 to rectangular piece of glass fiber filter
measuring 8 mm.times.305 mm and dried under constant vacuum in a
lyophilizer. The pad was stored dry in a desiccator until use.
[0234] Preparation of Filter Pad
[0235] The glass fiber filter was treated with a solution of 0.05%
Tween-20, 2% of sucrose, 1% of BSA and 100 mM sodium phosphate, pH
7.4, and then air dried at room temperature.
[0236] Immobilization of Anti-Hapten Antibodies on Membrane
[0237] A double sided transparent tape (305 mm.times.25 mm size)
was attached to 20 mm from the bottom of the thin plastic plate
(305.times.60 mm). A nitrocellulose membrane was cut to 305
mm.times.25 mm size and attached directly on the top of the double
sided tape. An assay indicia zone of immobilized test line for
anti-hapten antibody was defined on the 9 mm from the bottom of the
membrane by spraying 30 micro liter of solution of 1 mg/ml
streptavidin oligonucleotide-BSA conjugate in 10 mM PBS, pH 7.5.
After spraying, the membrane was dried at ambient temperature for
approximately 12 hours. The base and wicking membrane was stored in
a desiccator until further processed.
[0238] Strip Construction
[0239] Dye pad was attached to plastic base right below the bottom
of nitrocelluolse membrane and the filter pad is attached adjacent
to the dye pad. The plastic plate then was cut into a plurality of
strips 60 mm in length and 4 mm in width so that each contains a
linear array of nitrocellulose membrane, dye pad and filter
pad.
[0240] Assay Method and Result:
[0241] When 2 micro liter sample and 70 micro liter of develop
solution were added into the filter pad, a detectable signal began
to appear in the assay indicia zone after incubation when the strip
was exposed to UV light.
[0242] All of the references cited herein are incorporated by
reference in their entirety.
[0243] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention
specifically described herein.
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