U.S. patent application number 16/636795 was filed with the patent office on 2020-11-26 for assay methods for improved analyte detection.
The applicant listed for this patent is ORASURE TECHNOLOGIES, INC.. Invention is credited to Michael REED, Graham YEARWOOD.
Application Number | 20200371100 16/636795 |
Document ID | / |
Family ID | 1000005077379 |
Filed Date | 2020-11-26 |
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United States Patent
Application |
20200371100 |
Kind Code |
A1 |
YEARWOOD; Graham ; et
al. |
November 26, 2020 |
ASSAY METHODS FOR IMPROVED ANALYTE DETECTION
Abstract
Disclosed herein are assay methods, lateral flow assay test
strips, and devices for improved analyte detection. Analyte binding
to target is performed both in solution phase and with a target
immobilized on a surface, resulting in improved analyte
detection.
Inventors: |
YEARWOOD; Graham;
(Bethlehem, PA) ; REED; Michael; (Bethlehem,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ORASURE TECHNOLOGIES, INC. |
Bethlehem |
PA |
US |
|
|
Family ID: |
1000005077379 |
Appl. No.: |
16/636795 |
Filed: |
August 8, 2018 |
PCT Filed: |
August 8, 2018 |
PCT NO: |
PCT/US2018/045749 |
371 Date: |
February 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62542612 |
Aug 8, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/536 20130101;
G01N 33/56988 20130101; G01N 2469/20 20130101; G01N 33/5302
20130101; C07K 16/1045 20130101; G01N 33/558 20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 33/53 20060101 G01N033/53; G01N 33/536 20060101
G01N033/536; G01N 33/558 20060101 G01N033/558 |
Claims
1. An assay method for detecting an analyte in a liquid biological
sample, comprising: a) contacting the liquid biological sample
suspected of containing an analyte with at least one ligand-target
conjugate in a liquid phase, wherein the ligand-target conjugate
binds to at least a fraction of the analyte to form a
ligand-target-analyte complex; b) contacting analyte in the
biological sample and the complex from step a) with a
receptor-label conjugate, to form an analyte-receptor-label
complex, and a ligand-target-analyte-receptor-label complex; c)
capturing the labeled complexes from step b) on a surface
comprising a capture agent capable of binding ligand, where a
fraction of the ligand binding sites of the capture agent are
occupied by ligand-target conjugates, to form
ligand-target-analyte-receptor-label complexes on the surface; and
d) detecting the ligand-target-analyte-receptor-label complexes
captured on the surface.
2. The assay method of claim 1, wherein the analyte is an antibody
and the target is an antigen.
3. The assay method of claim 2, wherein the analyte is an anti-HIV
antibody, selected from anti-HIV-1 and anti-HIV-2, and the antigen
is selected from the group consisting of HIV-1 peptide, HIV-2
peptide, rAg and mixtures thereof.
4. The assay method of any one of claims 1-3, wherein the ligand is
biotin.
5. The assay method of any one of claims 1-4, wherein the liquid
biological sample is selected from the group consisting of oral
fluid, whole blood and plasma.
6. The assay method of any one of claims 1-5, wherein the receptor
is protein A.
7. The assay method of any one of claims 1-6, wherein the label is
colloidal gold.
8. The assay method of any one of claims 1-7, wherein the capture
reagent is streptavidin.
9. The assay method of any one of claims 1-8, wherein the
ligand-target conjugate is selected from the group consisting of
biotinylated HIV-1 peptide, biotinylated HIV-2 peptide,
biotinylated rAg peptide or mixtures thereof.
10. The assay method of any one of claims 1-9, wherein the assay
method is performed on a lateral flow assay strip, further
comprising the step of contacting the lateral flow assay strip with
a developer solution, wherein, after contact, the developer
solution moves across the lateral flow assay strip.
11. The assay method of claim 10, wherein the liquid biological
sample is added to the developer solution.
12. The assay method of claim 10, wherein the liquid biological
sample is added to a sample receiving portion of the lateral flow
assay strip.
13. The assay method of claim 10, wherein the target-ligand
conjugate is added to the developer solution.
14. The assay method of claim 10, wherein the target-ligand
conjugate is eluted off a portion of the lateral flow assay strip
downstream of the sample receiving area to bind to the analyte.
15. A lateral flow assay strip for detecting an analyte from a
liquid biological sample comprising: a sample receiving area; a
blocker area downstream from the sample receiving area, optionally
containing ligand-target conjugates; a conjugate area downstream
from the blocker area, containing label-receptor conjugates; a test
zone downstream from the conjugate area comprising immobilized
capture agents capable of binding ligand, wherein a fraction of the
ligand binding sites is occupied with ligand-target conjugates;
optionally, a control zone on the lateral flow assay strip
downstream from the test zone to indicate assay completion; and
optionally, an absorbent pad in flow communication with the lateral
flow assay strip and located downstream from the control zone.
16. The lateral flow assay strip of claim 15, wherein the analyte
is an antibody and the target is an antigen.
17. The lateral flow assay strip of claim 16, wherein the analyte
is an anti-HIV antibody, selected from anti-HIV-1 and anti-HIV-2,
and the antigen is selected from the group consisting of HIV-1
peptide, HIV-2 peptide, rAg and mixtures thereof.
18. The lateral flow assay strip of any one of claims 15-17,
wherein the ligand is biotin.
19. The lateral flow assay strip of any one of claims 15-18,
wherein the liquid biological sample is selected from the group
consisting of oral fluid, whole blood and plasma.
20. The lateral flow assay strip of any one of claims 15-19,
wherein the receptor is protein A.
21. The lateral flow assay strip of any one of claims 15-20,
wherein the label is colloidal gold.
22. The lateral flow assay strip of any one of claims 15-21,
wherein the capture reagent is streptavidin.
23. The lateral flow assay strip of any one of claims 15-22,
wherein the control zone comprises anti-human antibodies.
24. The lateral flow assay strip of any one of claims 15-23,
wherein the ligand-target conjugate is selected from the group
consisting of biotinylated HIV-1 peptide, biotinylated HIV-2
peptide, biotinylated rAg peptide or mixtures thereof.
25. A kit comprising: a) a lateral flow assay strip for detecting
an analyte from a liquid biological sample comprising: a sample
receiving area; a blocker area downstream from the sample receiving
area, optionally containing ligand-target conjugates; a conjugate
area downstream from the blocker area, containing label-receptor
conjugates; a test zone downstream from the conjugate area
comprising immobilized capture agents capable of binding ligand,
wherein a fraction of the ligand binding sites is occupied with
ligand-target conjugates, optionally, a control zone on the lateral
flow assay strip downstream from the test zone to indicate assay
completion; and optionally, an absorbent pad in flow communication
with the lateral flow assay strip and located downstream from the
control zone; and b) a developer solution.
26. The method of claim 1, wherein the analyte is selected from the
group consisting of antibodies to HIV, antibodies to HPV,
antibodies to HCV, antibodies to Ebola, antibodies to Dengue,
antibodies to Zika, antibodies to Helicobacter pylori, antibodies
to hepatitis, antibodies to measles, hepatitis antigens, antibodies
to terponemes, antibodies to host or infections agents, cellular
markers of pathology including, but not limited to, cardiolipin,
lecithin, cholesterol, lipopolysaccharide and sialic acid,
antibodies to mumps, antibodies to rubella, cotinine, cocaine,
benzoylecgonine, benzodizazpines, tetrahydrocannabinol, nicotine,
ethanol theophylline, phenytoin, acetaminophen, lithium, diazepam,
nortriptyline, secobarbital, phenobarbital, theophylline,
testosterone, estradiol, 17-hydroxyprogesterone, progesterone,
thyroxine, thyroid stimulating hormone, follicle stimulating
hormone, luteinizing hormone, transforming growth factor alpha,
epidermal growth factor, insulin-like growth factors I and II,
growth hormone release inhibiting factor, IGA, sex hormone binding
globulin, glucose, cholesterol, caffeine, corticosteroid-binding
globulin, PSA, DHEA-binding glycoprotein, and combinations
thereof.
27. The lateral flow assay strip of claim 15, wherein the analyte
is selected from the group consisting of antibodies to HIV,
antibodies to HPV, antibodies to HCV, antibodies to Ebola,
antibodies to Dengue, antibodies to Zika, antibodies to
Helicobacter pylori, antibodies to hepatitis, antibodies to
measles, hepatitis antigens, antibodies to terponemes, antibodies
to host or infections agents, cellular markers of pathology
including, but not limited to, cardiolipin, lecithin, cholesterol,
lipopolysaccharide and sialic acid, antibodies to mumps, antibodies
to rubella, cotinine, cocaine, benzoylecgonine, benzodizazpines,
tetrahydrocannabinol, nicotine, ethanol theophylline, phenytoin,
acetaminophen, lithium, diazepam, nortriptyline, secobarbital,
phenobarbital, theophylline, testosterone, estradiol,
17-hydroxyprogesterone, progesterone, thyroxine, thyroid
stimulating hormone, follicle stimulating hormone, luteinizing
hormone, transforming growth factor alpha, epidermal growth factor,
insulin-like growth factors I and II, growth hormone release
inhibiting factor, IGA, sex hormone binding globulin, glucose,
cholesterol, caffeine, corticosteroid-binding globulin, PSA,
DHEA-binding glycoprotein, and combinations thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. application
62/542,612 filed Aug. 8, 2017; the disclosure of which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to assay methods, lateral flow assay
strips and devices for improved analyte detection.
BACKGROUND
[0003] Immunoassays are used to quantify molecules of biological
interest based on the specificity and selectivity of antibody
reagents generated. For point-of-care testing, immunoassays are
often used in a lateral flow assay format. See, e.g., U.S. Pat.
Nos. 7,192,555 and 6,303,081. Solid phase lateral flow devices
incorporate a solid support strip which binds a member of an
analyte-target pair. Porous materials such as nylon,
nitrocellulose, cellulose acetate, glass fibers, paper, and other
porous polymers are often employed as solid support strips. The
sample that may contain the analyte of interest flows along the
solid support across the assay. Several procedures may be utilized
including the analyte or its derivative becoming bound to the
immobilized target and the presence of the analyte or its
derivative being detected, or the analyte or its derivative may
react to form a product that is then detected.
[0004] Previous lateral flow assay strips have been used to detect
various analytes using capture of analytes in the sample by
orientated target molecules at a test line. For example, in a one
HIV-1/2 lateral flow assay strip, a test line was prepared by
conjugating Streptavidin (SA) to an anchor protein such as bovine
serum albumin (BSA), and striping the conjugate at the test line
location of the nitrocellulose. See e.g., U.S. Pat. Nos. 8,062,908,
7,192,555, 6,303,081, 7,541,194, and OraQuick ADVANCE.RTM. Rapid
HIV-1/2 Antibody Test package insert. The SA-BSA conjugates allowed
for a highly active and stable presentation of biotinylated
peptides on the test line. In that lateral flow assay strip, biotin
binding sites of the streptavidin were pre-loaded such that the
HIV-1 and HIV-2 peptides were already on the site on SA and that
captured anti-HIV antibodies in a positive patient sample as they
flowed on the line. As a specimen would migrate up the strip and
encounter the test line, if the specimen contained antibodies that
react with the synthetic peptide antigens immobilized on the
nitrocellulose membrane at the test line, a visible signal would
appear, qualitatively indicating the presence of antibodies to
HIV-1 and/or HIV-2 in the specimen. See id.
[0005] In typical lateral flow assay devices the specific capture
of the analytes at the test zone occurs only as the liquid test
sample containing analyte migrates through the test zone, and binds
to its targets at a solid/liquid interface. Such devices may be
improved with the addition of a binding step where the analyte
binds to its target in solution, which is the subject of this
invention.
SUMMARY OF THE INVENTION
[0006] This invention generally relates to assay methods, lateral
flow assays strips and devices for improved analyte detection.
Assay methods according to the invention detect analytes of
interest from a liquid biological sample. An assay method according
to the invention:
[0007] a) contacts the liquid biological sample suspected of
containing an analyte with at least one ligand-target conjugate in
a liquid phase, where the ligand-target conjugate binds to at least
a fraction of the analyte to form a ligand-target-analyte
complex;
[0008] b) contacts analyte in the biological sample and the complex
from step a) with a receptor-label conjugate, to form an
analyte-receptor-label complex, and a
ligand-target-analyte-receptor-label complex;
[0009] c) captures the labeled complexes from step b) on a surface
comprising a capture agent capable of binding ligand, where a
fraction of the ligand binding sites of the capture agent are
occupied by ligand-target conjugates, resulting in
ligand-target-analyte-receptor-label complexes on the surface;
and
[0010] d) detects the ligand-target-analyte-receptor-label
complexes captured on the surface. Thus, analyte binding to target
is performed both with a target in a solution phase and with a
target immobilized on a surface, resulting in improved analyte
detection.
[0011] Assay methods of the invention may be lateral flow assays
using lateral flow assay strips. Lateral flow assay strips
according to the invention are designed to detect an analyte of
interest from a liquid biological sample. A lateral flow strip
according to the invention comprises:
[0012] a sample receiving area;
[0013] a blocker area downstream from the sample receiving area,
optionally containing ligand-target conjugates;
[0014] a conjugate area downstream from the blocker area,
containing label-receptor conjugates;
[0015] a test zone downstream from the conjugate area comprising
immobilized capture agents capable of binding ligand, where a
fraction of the ligand binding sites are occupied with
ligand-target conjugates,
[0016] optionally, a control zone on the lateral flow assay strip
downstream from the test zone to indicate assay completion; and
[0017] optionally, an absorbent pad in flow communication with the
lateral flow assay strip and located downstream from the control
zone.
[0018] Lateral flow devices according to the invention comprise an
assay portion housing a lateral flow strips according to the
invention and an opening to view the test zone and optional control
zone.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows a representative device [100] housing a lateral
flow strip [102] according to the invention for improved detection
of antibodies, for purposes of illustration Anti-HIV antibodies,
prior to running the lateral flow assay. Anti-HIV antibodies from a
biological sample are shown on the flat pad sample collector [103].
The blocker pad [104] is shown to comprise biotinylated HIV-1
peptides, biotinylated HIV-2 peptides, and biotinylated rAg
peptides. The conjugate pad [105] is shown to comprise Protein A
conjugated to colloidal gold. The test line [107] on the cellulose
membrane [106] is shown with immobilized Bovine Serum Albumin and
streptavidin, with a fraction of the biotin binding sites on
streptavidin bound to biotinylated HIV-1 and biotinylated HIV-2
peptides. The control line [108] is shown with immobilized
anti-human antibodies.
[0020] FIG. 2 shows a representative device [100] housing a lateral
flow strip [102] according to the invention for improved detection
of Anti-HIV antibodies during the lateral flow assay, as the sample
reaches the test line [107]. As the sample containing Anti-HIV
antibodies moves to the blocker pad [104], a fraction of the
Anti-HIV antibodies bind to biotinylated HIV-1 peptides,
biotinylated HIV-2 peptides, and biotinylated rAg peptides. As the
sample moves to the conjugate pad [105], Anti-HIV antibodies bound
to biotinylated further bind to Protein A--colloidal gold
conjugates. Unbound Anti-HIV antibodies also bind to Protein
A--colloidal gold conjugates.
[0021] FIG. 3 shows a representative device [100] housing a lateral
flow strip[102] according to the invention for improved detection
of Anti-HIV antibodies after running the lateral flow assay as the
sample flows past the control line[108] and into the absorbent
pad[109]. At the test line [107], biotinylated HIV-1 and
biotinylated HIV-2 peptides complexed with streptavidin capture
anti-HIV antibodies complexed with Protein A-colloidal gold
conjugates. Also at the test line [107], streptavidin captures the
complex of biotinylated peptides complexed with Anti-HIV antibody
complexed with Protein A-colloidal gold conjugates. At the control
line [108], anti-human antibodies capture any human antibodies in
the biological sample. Anti-HIV antibodies complexed with Protein
A-colloidal gold conjugate are visible as they bind to the
anti-human antibodies on the control line [108].
DETAILED DESCRIPTION
[0022] This invention relates to assay methods, lateral flow assays
strips and devices for improved analyte detection. Improved analyte
detection is achieved by combining analyte binding to target in
solution phase with analyte binding to target on solid phase. An
assay method for detecting an analyte in a liquid biological sample
according to the invention:
[0023] a) contacts the liquid biological sample suspected of
containing an analyte with at least one ligand-target conjugate in
a liquid phase, where the ligand-target conjugate binds to at least
a fraction of the analyte, to form a ligand-target-analyte
complex;
[0024] b) contacts analyte in the biological sample and the complex
from step a) with a receptor-label conjugate, to form an
analyte-receptor-label complex, and a
ligand-target-analyte-receptor-label complex;
[0025] c) captures the labeled complexes from step b) on a surface
comprising a capture agent capable of binding ligand, where a
fraction of the ligand binding sites of the capture agent are
occupied by ligand-target conjugates, resulting in
ligand-target-analyte-receptor-label complexes on the surface;
and
[0026] d) detects the ligand-target-analyte-receptor-label
complexes captured on the surface.
[0027] Assay methods according to the invention afford two
mechanisms for binding the analyte to a target to bring it to a
surface: i) binding of the analyte in the sample to a surface
immobilized target; and ii) binding of the analyte to a
ligand-target conjugate in a liquid phase, followed by capture of
the analyte-target-ligand complex to a surface. In the latter, the
high affinity between ligand and a capture reagent on the surface
ensures that the analyte-target complexes are extracted from the
solution. This combination of mechanisms provides a highly active
and stable presentation of target immobilized on a surface to
capture the analyte, and also allows for advantageous solution
kinetics for binding in the liquid phase. By including an
additional binding step in solution, assay sensitivities improve
especially when slower forming complexes are given more time to
form.
[0028] In an assay method of the invention the assay is performed
on a lateral flow assay strip. The method further comprises
contacting a lateral flow assay strip with a developer solution.
Upon contact, the developer solution moves across the lateral flow
assay strip. In a method of the invention, the liquid biological
sample is placed in a developer solution, and the resulting
developer solution comprising the biological sample moves across
the assay strip to points downstream on the lateral flow assay
strip. In another method of the invention, the liquid biological
sample is placed on a sample receiving area of the lateral flow
assay strip, and a developer solution contacts the lateral flow
assay strip to facilitate the flow of the biological sample across
the assay strip from the sample receiving area to points
downstream.
[0029] The liquid biological sample taken for analysis using an
assay method of the invention may be any liquid biological sample,
such as a biological fluid, which may contain antibodies of
interest. Examples of biological fluids include, but are not
limited to, urine, blood, plasma, serum, oral fluids, sweat, semen,
stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid,
breast milk and the like. Oral fluid is the liquid present in the
oral cavity. Oral fluid is a mixture of saliva and oral mucosal
transudate. Saliva is produced by the salivary glands. Oral mucosal
transudate enters the mouth by crossing the buccal mucosa from the
capillaries. Oral fluids contain both pathogens and antibodies.
Biological fluid samples such as oral fluid, whole blood, blood
plasma, and blood serum are preferred types of samples useable in
an immunoassay of the invention. Each of these may be acquired
using means and techniques known in the art.
[0030] Viral infection, such as infection from the HIV virus, can
be detected in multiple matrices which may include liquid
biological samples such as whole blood (venous or fingerstick),
serum and plasma, oral fluid (e.g., saliva and oral mucosal
transudate, urine, seminal fluid and breast milk from patients with
clinical signs and symptoms and/or epidemiological risk factors.
For example, in a method directed to measuring an oral fluid
sample, a subject is handed the test device and instructed to swab
the upper and lower gums once to collect saliva with the flat pad
of the device. In a method of the invention directed to measuring
whole blood, an appropriate volume of blood is placed onto the
device by a healthcare worker. The amount of liquid biological
sample to be used can vary based on the liquid biological sample
and the assay format.
[0031] In an assay method of the invention, the analyte may be an
antibody. Viral infection, such as infection from the HIV virus,
leads to the production of anti-HIV antibodies in a patient. In a
preferred assay method, the antibody is an anti-HIV antibody. The
anti-HIV antibody may be an antibody against HIV-1 or HIV-2. While
the assay methods of the invention are described with regard to
HIV, an assay method of the invention may be used for
identification, determination and/or treatment of any
infection.
[0032] The assay devices, lateral flow assay strips and methods of
this invention can be used for the detection (positive or negative,
and/or quantification) of virtually any analyte in a biological
fluid sample. Moreover, the devices, lateral flow assay strips and
methods can be used to detect one or more analytes simultaneously.
Such analytes may include, but are not limited to, antibodies to
HIV, antibodies to HPV, antibodies to HCV, antibodies to Ebola,
antibodies to Dengue, antibodies to Zika, antibodies to
Helicobacter pylori, antibodies to hepatitis, antibodies to
measles, hepatitis antigens, antibodies to terponemes, antibodies
to host or infections agents, cellular markers of pathology
including, but not limited to, cardiolipin, lecithin, cholesterol,
lipopolysaccharide and sialic acid, antibodies to mumps, antibodies
to rubella, cotinine, cocaine, benzoylecgonine, benzodizazpines,
tetrahydrocannabinol, nicotine, ethanol theophylline, phenytoin,
acetaminophen, lithium, diazepam, nortriptyline, secobarbital,
phenobarbital, theophylline, testosterone, estradiol,
17-hydroxyprogesterone, progesterone, thyroxine, thyroid
stimulating hormone, follicle stimulating hormone, luteinizing
hormone, transforming growth factor alpha, epidermal growth factor,
insulin-like growth factors I and II, growth hormone release
inhibiting factor, IGA, sex hormone binding globulin, glucose,
cholesterol, caffeine, corticosteroid-binding globulin, PSA, and
DHEA-binding glycoprotein. Depending on the analyte or analytes of
interest, the targets and receptors to bind to the analyte may be
chosen from binding agents known in the art.
[0033] An antibody, as is known in the art, refers to a polypeptide
or complex of polypeptides, substantially encoded by an
immunoglobulin gene or immunoglobulin genes, or fragments thereof.
The recognized immunoglobulin genes include the kappa, lambda,
alpha, gamma, delta, epsilon, and mu constant regions, as well as
myriad immunoglobulin variable region genes. Light chains are
classified as either kappa or lambda. Heavy chains are classified
as gamma, mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes (isotypes), IgG, IgM, IgA, IgD, and IgE,
respectively. The constant domains of the heavy chains make up the
Fc region of an antibody. The Fc portion of the antibody determines
the antibody class.
[0034] Typically, an antibody is an immunoglobulin having an area
on its N-terminal surface or in a cavity that specifically binds to
and is thereby defined as complementary with a particular spatial
and polar organization of another molecule. The antibody can be
polyclonal or monoclonal. Antibodies may include a complete
immunoglobulin or fragments thereof. Fragments thereof may include
Fab, Fv and F(ab')2, Fab', and the like. Antibodies may also
include chimeric antibodies or fragment thereof made by recombinant
methods. An antigen is any compound capable of specifically binding
to an antibody of interest. Specific binding of between the antigen
and the antibody of interest means that the two molecules are
related such that their binding with each other is capable of
discriminating between binding other assay or sample
components.
[0035] The target refers to a component which binds to the analyte.
In assay methods of the invention the analyte specifically binds
the target. In an assay method of the invention, the target may be
an antigen. For example, when the analyte is an antibody, and the
target may be its cognate antigen. Alternatively, when the analyte
is an antigen the target may be an antibody that specifically binds
the antigen. Preferably, the target is a peptide. In a preferred
method of the invention, the target is selected from HIV-1 peptide,
HIV-2 peptide, rAg and mixtures thereof. Anti-HIV-1 antibodies
specifically bind to HIV-1 peptide, and to rAg. Anti-HIV-2
antibodies specifically bind to HIV-2 peptide. The HIV-1 and HIV-2
synthetic peptides may correspond to regions of envelope (gp120,
41) or p24 or p17 or other known proteins from the virus.
[0036] The term rAg refers to any antigenic HIV recombinant
polypeptide. U.S. Pat. Nos. 5,156,949, 5,217,861, 6,428,952,
5,830,641, 5,886,319, and 6,544,728, for example, describe
recombinant antigens useful for immunoassays for antibodies to HIV.
The nucleotide sequence shown in SEQ ID NO 1 encodes for the
recombinant antigen rAg, also known as ORA-1,
HIV-1(hSOD1-HIV1gp120-41). SEQ ID NO 2 is the amino acid sequence
for rAg. The mature envelope protein (Env) of HIV consists of a
homotrimer of non-covalently associated gp120-gp41 heterodimers.
The resulting complex protrudes from the virus surface as a spike.
Surface protein gp120 interacts with human CD4, CCR5 and CXCR4, to
form a P4HB/PDI-CD4-CXCR4-gp120 complex. This recombinant design of
rAg expands on and includes the HIV-1 peptide sequence. The rAg
antigen is recognized by anti-HIV-1 antibodies.
[0037] In methods of the invention, the target is conjugated to a
ligand, forming a ligand-target conjugate. Binding of the
ligand-target conjugate with the analyte results in the formation
of a ligand-target-analyte complex. In step a) in methods of the
invention some or all of the analyte binds to the ligand-target
conjugate. If all of the analyte does not bind to the ligand-target
conjugate, some of the analyte remains unbound.
[0038] The ligand serves to direct materials associated with the
ligand to a surface immobilized capture reagent. For example, the
ligand in the ligand-target-analyte complex serves to guide the
complex to the immobilized capture reagent. In a method according
to the invention, the ligand is biotin, and the ligand-target
conjugate is a biotinylated peptide. Biotinylation of peptides can
be performed using methods and reagents known in the art. For
example, NHS-biotin can be used to label primary amine groups of
peptides. The extent of biotinylation can be tested using
4'-hydroxyazobenzene-2-carboxylic acid reagent. In a method of the
invention, the ligand-target conjugate is selected from
biotinylated HIV-1 peptide, biotinylated HIV-2 peptide,
biotinylated rAg and mixtures thereof. In preferred methods of the
invention, the ligand-target-analyte complex is a complex of an
anti-HIV antibody bound to a biotinylated HIV-1 peptide, a
biotinylated HIV-2 peptide, or a biotinylated rAg. Biotin is
capable of strong non-covalent interaction with streptavidin. In
methods of the invention, streptavidin is a preferable capture
reagent.
[0039] The step of contacting the liquid biological sample with a
ligand-target conjugate occurs in a liquid phase. Preferably, the
ligand-target conjugate is present on the lateral flow assay strip,
on a blocker pad, and as liquid contacts the ligand-target
conjugate, it is eluted off the blocker pad. During preparation of
the lateral flow assay strip, the ligand-target conjugate may be
placed on the blocker pad and dried down upon the membrane. As the
analyte in the liquid phase moves across the blocker pad, the
ligand target conjugate is eluted off the blocker pad into the
liquid phase, is available for binding to the analyte. In a method
of the invention, the ligand-target conjugate is placed on the
blocker pad for liquid-phase binding. In another method of the
invention, the ligand-target conjugate is added to the developer
solution. In yet another method of the invention, the ligand-target
conjugate is placed on the blocker pad as well as in the developer
solution.
[0040] The label may be any substance which is visible itself or
capable of producing a signal that is detectable by visual or
instrumental means. The selection of a particular label is not
critical to the invention, but the label should be capable of
generating a detectable signal either by itself, or be
instrumentally detectable, or be detectable in conjunction with one
or more additional signal producing components, such as an
enzyme/substrate signal producing system.
[0041] As is known in the art the choice of the type of label
involves consideration of the analyte to be detected and the
desired means of detection. Detection of the detectable label will
depend on the chosen moiety or reagent, and can be done by any of
the methods known in the state of the art, for example, visual
inspection, ultraviolet and visible spectrophotometry, fluorimetry,
radioactivity counting or the like. Typically, a visually
detectable label is used. This allows for direct visual or
instrumental reading of the presence or amount of an analyte in the
sample without the need for additional signal producing components.
Various labels known in the art and suitable for use in the methods
of the invention include labels which are visible themselves or
which produce signals through either chemical or physical means.
Such labels can include enzymes and substrates, chromogens,
catalysts, fluorescent compounds, chemiluminescent compounds, and
radioactive labels. Other suitable labels known in the art include
particulate labels such as colloidal metallic particles such as
colloidal gold, colloidal non-metallic particles such as selenium
or tellurium, dyed or colored particles such as a dyed plastic or a
stained microorganism, organic polymer latex particles and
liposomes, colored beads, polymer microcapsules, sacs,
erythrocytes, erythrocyte ghosts, or other vesicles containing
directly visible substances, and the like. U.S. Pat. No. 4,313,734
describes the use of gold sols as labels for antibodies. In a
preferred assay method according to the invention, the detectable
label is a metal colloidal particle, such as gold microparticles or
gold nanoparticles, which can be seen by the human eye as well as
instrument read. In an assay method of the invention, colloidal
particles used as a detectable label have a particle size diameter
of between 20 nm and 80 nm. For an instrument read assay, the
concentration of the particles is measured by measuring Optical
Density (OD) at a specific wavelength using a spectrophotometer. OD
is then used as a measure of the total amount of labeled antigen.
In an assay method according to the invention, the amount of
labeled antigen should preferably be in excess of the antibody to
be detected to allow for complete labeling.
[0042] The receptor-label conjugate serves to bind to the analyte
in order to create a detectable complex. Depending on the analyte,
the receptor may be any molecule that specifically or
non-specifically binds to the analyte. In a preferred method of the
invention, the receptor is Protein A. Protein A can bind to the Fc
portion of several immunoglobulins. In an assay method of the
invention, the receptor-label conjugate is a Protein A-gold colloid
conjugate. Proteins may be attached to the colloidal gold through
techniques known in the art. Protein A-gold colloid conjugates are
commercially available, for example, P-6730, from Sigma
Aldrich.RTM.. Other examples of receptor-label conjugates include
gold conjugated antigen (Bridging assays) and secondary anti-human
heavy or light chain specific gold conjugates.
[0043] In assay methods of the invention, the receptor also binds
to the analyte. The receptor binds to the analyte irrespective of
whether the analyte is also bound to the target. Accordingly, the
receptor-label conjugate binds to the analyte to form an
analyte-receptor-label complex, and the receptor-label conjugate
also binds to the ligand-target-analyte complex to form a
ligand-target-analyte-receptor-label complex. The step of reacting
the receptor label with either the analyte or the
ligand-target-analyte complex occurs in liquid phase. In methods of
the invention using a lateral flow assay strip, preferably, the
ligand-target conjugate is present on a conjugate pad, and as
liquid contacts the receptor-label conjugate, it is eluted off the
conjugate pad. During preparation of the lateral flow assay strip,
the receptor-label conjugate may be placed on the conjugate pad and
dried down upon the membrane. As the analyte in the liquid phase
moves across the conjugate pad, the receptor-label conjugate is
eluted off the conjugate pad into the liquid phase, is available
for binding to the analyte and to the analyte-target-ligand
complex. In a preferred method according to the invention, the
ligand-target-analyte-receptor-label complex is a biotinylated
peptide bound, to an antibody, bound to a Protein A conjugated
colloidal gold.
[0044] In assay methods of the invention the capture reagent binds
to the ligand, and serves to bring labeled complexes comprising the
analyte onto a solid support for detection. Preferably the capture
reagent is immobilized on a solid support. Capture reagents may be
immobilized, or may be attached to other components which allow for
their separation from solution. The immobilization and attachment,
and placement of the capture reagent is dependent on the assay
format. For example, in some assay methods of the invention,
capture reagents may be immobilized on specific regions, zones, or
lines on a lateral flow assay test strip, in specific regions on
membranes, on solid supports of a chromatography column, or wells
in microtiter plates. In other assay methods according to the
invention, capture reagents may also be coated on beads or other
particles to enable separation from the rest of the sample in
solution. In assay methods of the invention a fraction of the
ligand binding sites on the capture reagent are bound to
target-ligand conjugate. In a preferred assay method of the
invention, the capture reagents are located at a test line on a
lateral flow assay strip, and a fraction of the capture reagents
are bound to target-ligand, to enable capture of analyte, and the
remaining ligand binding sites are free to capture ligand.
[0045] The affinity between the capture reagent and the ligand
allows for the ligand-target-analyte-receptor-label complex to be
captured by a capture reagent immobilized on a surface.
Additionally, the affinity between the analyte and target allows
for the analyte-receptor-label complex to bind to targets
immobilized on a surface. In a preferred assay method of the
invention the capture reagent is a biotin binding protein, for
example, avidin, neutravidin, streptavidin, and protein conjugates
thereof. Preferably, the capture reagent is streptaviding
conjugated to BSA, and is immobilized on a nitrocellulose surface.
In methods of the invention, biotinylated HIV-1 and HIV-2 peptides
are immobilized on a surface by being bound to streptavidin-BSA
immobilized on a surface, with a fraction of biotin binding sites
of the streptavidin unoccupied.
[0046] A preferred assay method according to the invention detects
anti-HIV-1 and anti-HIV-2 antibodies from a liquid biological
sample. The method: [0047] a) contacts a liquid biological sample,
suspected of containing anti-HIV-1 and anti-HIV-2 antibodies, with
biotinylated HIV-1, HIV-2 and/or rAg peptides in a liquid phase,
wherein at least a fraction of the anti-HIV-1 and anti-HIV-2
antibodies from the sample bind to the biotinylated peptides to
form antibody-biotinylated peptide complexes:
anti-HIV-1-biotinylated HIV-1 peptide, anti-HIV-1-biotinylated rAg
peptide, and/or anti-HIV-2-biotinylated HIV-2 peptide complexes;
[0048] b) contacts the unbound anti-HIV-1 and anti-HIV-2 antibodies
in the biological sample and the complexes formed in step a), with
Protein A-gold colloid conjugate to form labeled antibody complexes
and labeled antibody-biotinylated peptide complexes [0049] c)
captures the complexes from step b) with streptavidin immobilized
on a surface with a fraction of the biotin binding sites on
streptavidin are occupied by biotinylated HIV-1 and HIV-2 peptides
[0050] d) detects the gold colloid-Protein
A-antibody-peptide-biotin complexes from step c) captured on the
surface.
[0051] A preferred method according to the invention is a lateral
flow assay method. Lateral flow assays are performed on lateral
flow assay strips. In a lateral flow assay method of the invention,
a liquid biological sample is applied onto a lateral flow assay
strip, and the presence of analytes in the biological sample is
detected at a test line on the lateral flow assay strip.
[0052] A lateral flow assay strip refers to a strip utilized for
lateral flow chromatography. Lateral flow (chromatography) assays
typically involve the application of a liquid biological sample
suspected of containing an analyte to be detected to a sample
receiving area of a lateral flow (immunochromatographic) assay
strip. The assay strip comprises a matrix material (e.g., paper,
nitrocellulose, etc. See, e.g., U.S. Pat. No. 5,569,608) through
which the test fluid and analyte suspended or dissolved therein can
flow by capillary action from the sample receiving area to one or
more capture zones where a visible signal, or absence of such,
reveals the presence or absence of the analyte. A developer
solution facilitates the flow of the sample across the lateral flow
assay strip. Where the detection of the analyte utilizes an
antibody or antibody fragment, the assay may be referred to as a
lateral flow immunochromatography assay and the strip a lateral
flow immunochromatography strip.
[0053] The lateral flow strips according to the invention afford
two mechanisms of capture of the analyte of interest at the test
line: i) the capture of analytes, for example antibodies, in the
sample by orientated ligand-target conjugates, for example
biotinylated peptides, at the test line location; and ii) by
capture of ligand-target conjugates, for example biotinylated
peptides, that have complexed in the mobile solution phase with
analytes. The target-ligand conjugates are presented to the analyte
in the liquid phase, either by addition to a developer solution or
are eluted from pad component (e.g., blocker pad). In each case, it
is demonstrated below that significantly (>5.times.) lower
amounts of peptide result in a surprisingly similar reactive sample
response. In lateral flow strips according to the invention, a
mixture of partially occupied streptavidin-BSA (SA-BSA) leaves the
test line open to a capture function for biotinylated species
transported to the test line location with the sample. The test
line also contains biotinylated HIV-1, HIV-2 peptides, recombinant
HIV antigen(rAg) peptides, or combinations thereof, to capture
anti-HIV antibodies complexed to protein-A-gold conjugates.
[0054] Binding of analyte and target in solution allows for liquid
phase binding, where they are able to benefit from the test run
time to allow the targets to effectively incubate with the analytes
from the sample. For example, as a patient sample containing
anti-HIV antibodies is transported into the device by the developer
solution, which hydrates and elutes the pad components,
biotinylated peptides and recombinant proteins displaying HIV
epitopes can take advantage of solution kinetics and the flow time
to the test line to more completely complex with anti-HIV
antibodies in the sample. The high affinity biotin-streptavidin
binding then ensures these complexes are extracted from the
solution by the immobilized free biotin binding sites on
streptavidin at the test line.
[0055] A lateral flow assay strip for detecting an analyte from a
liquid biological sample according to the invention comprises:
[0056] a sample receiving area;
[0057] a blocker area downstream from the sample receiving area,
optionally containing ligand-target conjugates;
[0058] a conjugate area downstream from the blocker area,
containing label-receptor conjugates;
[0059] a test zone downstream from the conjugate area comprising
immobilized capture agents capable of binding ligand, wherein a
fraction of the ligand binding sites are occupied with
ligand-target conjugates,
[0060] optionally, a control zone on the lateral flow assay strip
downstream from the test zone to indicate assay completion; and
[0061] optionally, an absorbent pad in flow communication with the
lateral flow assay strip and located downstream from the control
zone. Preferably, a lateral flow strip of the invention is designed
with a liquid sample flow direction consecutively through a sample
receiving area, a blocker area, a conjugate area, a test line, a
control line and an absorbent pad. The absorbent pad assists in
promoting capillary action and fluid flow one-way through the
membrane of the strip, and pulls the liquid containing the analyte
along the membrane from one end of the strip to the other.
[0062] FIG. 1-3 illustrate a lateral flow device according to the
invention. The lateral flow device [100] has a housing [101]. The
device [100] contains a lateral flow assay strip [102] having a
flat pad [103] partially exposed outside the housing [101] which is
in flow communication with a blocker pad [104] and a conjugate pad
[105] disposed on a cellulose membrane [106] having a test line
[107] as its capture zone, and a control line [108] as its optional
control zone. The cellulose membrane 106 is in flow communication
with an optional absorbent pad [109]. The direction of flow is
indicated by the arrow. A drop of sample from a subject is applied
to the flat pad [103]. The sample is wicked across the lateral flow
strip [102] within the device housing [101], and flows from the
flat pad [103] to a conjugate pad [105] and across the cellulose
membrane [106]. As depicted here, the flat pad [103] acts as a wick
to deliver a liquid biological to the blocker pad [104].
[0063] The liquid biological sample analyzed using a lateral flow
strip of the invention may be any liquid biological sample, such as
a biological fluid, which may contain antibodies of interest.
Examples of biological fluids include, but are not limited to,
urine, blood, plasma, serum, oral fluids, sweat, semen, stool,
sputum, cerebral spinal fluid, tears, mucus, amniotic fluid, breast
milk and the like. Oral fluid is the liquid present in the oral
cavity. Oral fluid is a mixture of saliva and oral mucosal
transudate. Saliva is produced by the salivary glands. Oral mucosal
transudate enters the mouth by crossing the buccal mucosa from the
capillaries. Oral fluids contain both pathogens and antibodies.
Biological fluid samples such as oral fluids, whole blood, blood
plasma, and blood serum are preferred types of samples useable in
an immunoassay of the invention. Each of these may be acquired
using means and techniques known in the art. The amount of liquid
biological sample to be used can vary based on the liquid
biological sample, as known in the art.
[0064] A lateral flow strip of the invention may be used to detect
an analyte that is an antibody. Preferably, the antibody is an
anti-HIV antibody. The anti-HIV antibody may be an antibody against
HIV-1 or HIV-2. While the lateral flow strips of the invention are
described with regard to HIV, lateral flow strip of the invention
may be used for identification, determination and/or treatment of
any infection.
[0065] A "sample receiving area" of the lateral flow assay strip
refers to the area of the lateral flow assay test strip to which a
sample is first applied. In addition to receiving the sample, the
functions of the sample receiving area may include, for example: pH
control/modification and/or specific gravity control/modification
of the sample applied, removal or alteration of components of the
sample which may interfere or cause non-specific binding in the
assay, or to direct and control sample flow to the test region. The
filtering aspect, if present, allows an analyte of interest to
migrate through or across a lateral flow assay test strip in a
controlled fashion with few, if any, interfering substances. The
filtering aspect, if present, often provides for a test having a
higher probability of success and accuracy. In a lateral flow assay
test strip of the invention, the sample receiving area may also
incorporate reagents useful to avoid cross-reactivity with
non-target analytes that may exist in a liquid biological sample
and/or to condition the sample; depending on the particular
embodiment, these reagents may include anti-RBC reagents,
Tris-based buffers, EDTA, among others. When the use of whole blood
is contemplated, anti-RBC reagents are frequently utilized. In yet
another assay strip of the invention, the sample receiving area may
incorporate other reagents such as ancillary specific binding
members, fluid sample pretreatment reagents, and signal producing
reagents. In a lateral flow assay device according to the
invention, the flat pad [103] serves as a sample receiving
area.
[0066] In some lateral flow assay strips according to the
invention, the sample receiving area may be a sample pad and may be
made from any material capable of receiving the liquid biological
sample and absorbing the liquid sample when applied and of passing
the liquid sample to the blocker pad. The pad in the sample
receiving area can be constructed to act as a filter for cellular
components, hormones, particulate, and other certain substances
that may occur in the fluid sample. Sample pad materials suitable
for use in assay strips of the invention also include those
application pad materials disclosed in U.S. Pat. No. 5,075,078,
incorporated herein by reference. Suitable materials for the sample
application area include, but are not limited to, hydrophilic
polyethylene materials or pads, acrylic fiber, glass fiber, filter
paper or pads, desiccated paper, paper pulp, fabric, and the
like.
[0067] The sample receiving area may be comprised of a sample
application member (e.g., a wick or a flat pad). The sample
receiving zone can comprise a sample application pad as well as a
sample application member. Often the sample application member is
comprised of a material that readily absorbs any of a variety of
fluid samples contemplated herein, and remains robust in physical
form. Frequently, the sample application member is comprised of a
material such as white bonded polyester fiber. The sample
application member may also be treated with a hydrophilic finishing
agent. Moreover, the sample application member is positioned in
fluid-flow contact with the blocking pad of the lateral flow assay
strip. This fluid flow contact can comprise an overlapping,
abutting or interlaced type of contact. Often the sample
application member, if present, may contain similar reagents and be
comprised of similar materials to those utilized in exemplary
sample application pads. The liquid biological sample may be
applied to the flat pad [103] directly. For example, the flat pad
[103] may be inserted in the mouth of a subject, and be used as a
collection pad for oral fluids. Other fluids, for example blood or
serum may be directly placed on the flat pad [103]. The liquid
biological sample may also be diluted in a developer solution in a
vial, and the flat pad [103] may be placed in the vial.
[0068] The assay strips of this invention include a blocker area
which contains at least one target-ligand conjugate. In a lateral
flow strip of the invention, the blocker area is a blocker pad. As
shown in FIG. 1, the blocker pad [104] is downstream of the flat
pad [103]. As known in the art, a blocker pad contains reagents to
ensure minimal reactivity within the device to nonspecific or
interfering substances that are present in various sample matrices.
A blocker pad can be composed of a wide variety of materials as
long as they do not impede flow of oral fluid downstream to the
lateral flow assay test strip. Such materials include, but are not
limited to, paper, cellulose, nitrocellulose, polyester, glass
fiber, and the like. Materials may be selected to reduce or
eliminate backflow of reagents or oral fluid from the
chromatographic test strip to the capillary matrix. Buffering
reagents and salts present may be present in the blocker pad to
help adjust the pH and ionic strength for complexing the target
with analyte. A blocker pad can be impregnated with buffers to
adjust the sample pH of the liquid biological sample as it flows
and for compatibility with the lateral flow assay. A blocker pad
can also include one or more blocking reagents that reduce
non-specific binding of an analyte and/or reagents of the assay and
thereby reduce the occurrence of false positives. Exemplary
blocking reagents include, but are not limited to, bovine serum
albumin (BSA), methylated BSA, casein, nonfat dry milk,
deoxycholate, and n-lauroyl sarcosine. The blocking solution may
also contain surfactants, preservatives and other reagents to
enhance flow across the test strip, improve assay results and
protect sample integrity. Typically, a blocker pad is made by
applying an appropriate volume of blocking solution onto the pad
and drying before positioning it on the lateral flow assay
strip.
[0069] The blocker pad of a lateral flow assay test strip of the
invention optionally serves to maintain ligand-target conjugate
reagents in a stable state and to facilitate their rapid and
effective solubilization, mobilization and specific reaction with
analytes of interest potentially present in the liquid biological
sample. FIG. 1-3 illustrate a device housing [100] a lateral flow
strip according to the invention in which the blocker pad also
contains the target-ligand conjugate. As the developer solution
along with biological sample flows across the lateral flow strip
[102], target-ligand conjugate is eluted off the blocker pad [104]
and at least a fraction of the analyte present in the biological
sample binds to the target-ligand conjugate in the liquid phase. In
a lateral flow strip of the invention, the target is an antigen.
Preferably, the antigen is selected from the group consisting of
HIV-1 peptide, HIV-2 peptide, rAg and mixtures thereof, and the
ligand is biotin. Accordingly, preferred target-ligand conjugates
on the blocker pad include biotinylated HIV-1 peptide, biotinylated
HIV-2 peptide, biotinylated rAg, or mixtures thereof.
[0070] The conjugate area of a lateral flow assay test strip serves
to maintain label reagents and control reagents in a stable state
and to facilitate their rapid and effective solubilization,
mobilization and specific reaction with analytes of interest
potentially present in the liquid biological sample. In a lateral
flow assay test strip according to the invention, the conjugate
area can be a conjugate pad. A conjugate pad is positioned on a
lateral flow assay test strip such that the liquid biological
sample must pass across or though the conjugate pad in order to
migrate to the test zone or line. Alternatively, a conjugate area
can be woven into the lateral flow assay test strip or can be
placed in-line, in the same place, as the lateral flow assay test
strip. As with a blocker pad, a conjugate pad can be fashioned out
of any convenient material (e.g., nitrocellulose) that is
compatible with the assay and that does not substantially impede
flow of the oral fluid and reagents. Conjugate pad materials
suitable for use by the present invention include those
chromatographic materials disclosed in U.S. Pat. No. 5,075,078,
which is herein incorporated by reference.
[0071] In a lateral flow assay test strip according to the
invention, the conjugate pad carries the receptor-label conjugate
as well as release and stabilization agents to allow for detection
of antigen at a test zone or line. Depending on the analyte, the
receptor may be any molecule that specifically or non-specifically
binds to the analyte. In a preferred lateral flow assay strip of
the invention, the receptor is Protein A, and the label is a gold
colloid. As the developer solution along with the biological sample
flows across the lateral flow strip, receptor-label conjugate is
eluted off the conjugate pad, and the receptor-label binds to
analyte or to the analyte-target-ligand complex.
[0072] Assay strips according to the invention comprise a matrix
material (e.g., paper, nitrocellulose, etc., see, e.g., U.S. Pat.
No. 5,569,608) across which the test fluid and analyte suspended or
dissolved therein can flow by capillary action. Matrix materials
can include, but are not limited to, natural, synthetic, or
naturally occurring materials that are synthetically modified, such
as polysaccharides (for example, cellulose materials such as paper
and cellulose derivatives as cellulose acetate and nitrocellulose);
polyether sulfone; nylon; silica; inorganic materials, such as
deactivated alumina, diatomaceous earth, magnesium sulfate, or
other inorganic finely divided material uniformly dispersed in a
porous polymer matrix such as vinyl chloride, vinyl
chloride-propylene copolymer, and vinyl chloride-vinyl acetate
copolymer; cloth, both naturally occurring (for example, cotton)
and synthetic (for example, rayon); porous gels, such as silica
gel, agarose, dextran, and gelatin; polymeric films, such as
polyacrylamide, and the like. In a preferred assay strip of the
invention, the matrix material is a nitrocellulose membrane.
Different types of commercially available nitrocellulose membranes
(e.g., Millipore, HF90, GE FF120, Sartorius) possess different
absorbent capacity and capillary flow.
[0073] In a lateral flow assay test strip of the invention, the
matrix material may be adhered to a laminate backing. The backing
may be a plastic material, such as e.g. Mylar or PVC or
polystyrene. In preparing assay strips, the matrix material may be
laminated onto a plastic card using techniques known in the art.
Nitrocellulose membranes with laminate backings are commercially
available.
[0074] In a lateral flow assay test strip according to the
invention, a test zone or test line, downstream from the conjugate
area, serves to indicate the presence of analytes. The test zone or
line includes capture reagents and ligand-target conjugate
molecules immobilized on the lateral flow assay strip. The capture
reagent may be any compound capable of specifically binding to the
ligand. In a preferred lateral flow assay test strip according to
the invention, the immobilized capture reagent is streptavidin. The
test line also includes immobilized targets. The targets may be
biotinylated, and bound to streptavidin, and thereby immobilized on
the test zone. Accordingly, in a lateral flow assay strip of the
invention the test line comprises streptavidin molecules
immobilized on the strip where a fraction of the biotin binding
sites are occupied by biotinylated targets. In a preferred lateral
flow strip of the invention, the test zone includes, prior to any
addition of sample, streptavidin with available biotin binding
sites, and streptavidin bound to biotinylated HIV-1 peptide,
biotinylated HIV-2 peptide, biotinylated rAg, or mixtures thereof.
Attachment of proteins, such as streptavidin onto nitrocellulose is
known in the art. In a preferred lateral flow assay strip of the
invention, streptavidin-BSA is placed on the nitrocellulose at the
test line. As the sample moves to the test line, biotinylated
targets, biotinylated target-analyte complexes, and biotinylated
target-analyte-receptor-label complexes will bind to streptavidin
on the test zone, and analytes, and analyte-receptor-label
complexes will bind to the immobilized targets on the test line.
The presence of analyte is detected through visualization of the
label at the test line.
[0075] A control zone or control line may also be placed on the
lateral flow assay test strip downstream from the test zone. The
control line, binds labeled antigen not captured in the test zone.
A positive control line indicates that the device has functioned
appropriately. In a lateral flow assay strip according to the
invention testing for antibodies in the liquid biological sample
from a human, the control line on the nitrocellulose membrane is
striped with anti-human antibodies.
[0076] An adsorbent pad may be present downstream of the control
line in a lateral flow assay test device. The absorbent pad serves
not only as the end reservoir for device fluid but may also draw
the fluid across the lateral flow assay test strip. Absorbent pads
should have sufficient wicking characteristics to prevent backflow
of liquid upstream on the assay strip. The absorbent pad may be
made of materials known in the art.
[0077] Assay test strips according to the invention may be prepared
and assembled using techniques known in the art, as discussed
above. Assay test strips according to the invention may be in a
housing. An assay device according to the invention, comprises a
housing having an opening or window to view one or more capture
zones and control zones.
[0078] A lateral flow assay method according to the invention uses
a developer solution. One embodiment of the invention is a kit
comprising a lateral flow assay strip of the invention and a
developer solution. A developer solution facilitates the capillary
flow of the biological sample into the device and onto the assay
strip. In an assay method of the invention oral fluid may be
collected and serve as the biological sample. For oral fluid
collection, the sample application member or collection pad of the
device, is first used to receive or collected a biological liquid
sample. For example a collection pad of a device may be used to
collect an oral fluid sample from a subject's mouth, and then is
placed in a vial of developer solution. The collection pad wicks up
the developer solution carrying along with it the oral fluid sample
for the lateral flow assay test. For other biological samples, such
as whole blood, serum or plasma collection, the sample is added
directly into a vial containing developer solution or onto the
collection pad, and the collection pad is subsequently placed in
the developer vial to flow through the lateral fow assay test and
thereby transport the sample. In a particular assay method
according to the invention, whole blood is directly applied to the
sample application area, for example, a drop of whole blood drop (5
.mu.L to 50 .mu.L) obtained from a fingerstick or venipuncture or
needle without any type of prior dilution or treatment. A developer
solution is typically an aqueous solution of surfactants, salts,
preservatives, buffering agents, etc. as known in the art. Buffer
system such as phosphate, Tris-Cl borate, bicarbonate, etc., may be
used. Surfactants such as Tween 20, Triton X-100 or other non-ionic
detergents may be used. Preservatives, including anti-microbial and
anti-fungal substances, such as, e.g., sodium azides may be used in
the developer solution. The amount of developer solution used
should be sufficient to transport the sample but not so much as to
swamp the assay or dilute the results so they cannot be
determined.
EXAMPLES
Example 1
[0079] A lateral flow assay device was prepared. Striped
nitrocellulose, a blocker pad, a conjugate pad and an adsorbent pad
were assembled on an assay card backing.
[0080] Striped Nitrocellulose: FF120 nitrocellulose was line
striped with solutions of anti-human antibody (F(ab')2, Goat
anti-Human IgG) at the control line and SA-BSA, partially
pre-complexed with biotinylated HIV-1 and HIV-2 peptides at the
test line.
[0081] Blocker Pad: The biotinylated rAg was added to the blocker
pad solution along with biotinylated HIV-1 and HIV-2. The blocker
pad was treated with a common blocker base buffer and biotinylated
HIV-1 and HIV-2 peptides and biotinylated rAg.
[0082] Conjugate Pad: Colloid gold was formulated using the citrate
reduction process (Turkevich method), passively coated with protein
A. The conjugate pad was made by spraying formulated gold conjugate
solution onto the conjugate pad material. This conjugate pad was
allowed to dry appropriately before being positioned immediately
above the blocker pad on the assay card.
[0083] Once all major components of the assay card were available
(blocker pad, conjugate pad, nitrocellulose, laminate backing,
absorbent pad), the materials were assembled into an assay card on
a laminate backing card. Assay cards were cut into assay strips and
the assay strip was set in the device housing base and a collection
pad was placed on top of the assay strip in the device housing
base. The device housing top was pressed together to engage the
pins in the base with the sockets in the top to complete the
assembly. The assembled device was sealed upon assembly.
[0084] The assay method was performed by collection of the
biological sample, insertion of the sample into developer buffer,
followed by the insertion of the test device and interpretation of
the assay results after approximately 20 to 40 min. An oral fluid
specimen was collected using the flat sample collection pad of the
test device, followed by the insertion of the test device into the
vial of developer solution. The developer solution facilitated the
flow of the specimen into the device and onto the lateral flow
assay test strip.
Example 2
[0085] Seroconversion panels: Table 1 is a summary of the
performance of the seroconversion panels from the package insert
for the OraQuick ADVANCE.RTM. Rapid HIV-1/2 Antibody Test. Table 2
is a summary of seroconversion panels tested with a lateral flow
assay device according to the invention.
TABLE-US-00001 TABLE 1 Performance in Seroconversion Panels with
the OraQuick ADVANCE .RTM. Rapid HIV-1/2 Antibody Test device
Number Number Number of detected detected Average Average Number of
Concordant Earlier by earlier by Days Days Delay Panels Results
device EIA device EIA Days 23 7 3 13 36.46 34.99 1.48 (95% CI -0.1
to 3.1)
TABLE-US-00002 TABLE 2 Performance in Seroconversion Panels with a
test device according to the invention Number Number Number of
detected detected Average Average Number of Concordant Earlier by
earlier by Days Days Delay Panels Results device EIA device.sup.a
EIA.sup.a Days.sup.b 21 11 4 6 36.19 35.95 0.24 (95% CI: -1.34 to
1.81) .sup.aCalculated using MiniTab .RTM. Statistical Software.
.sup.bDelay Days are equivalent to the mean differential
sensitivity between the device and the U.S. FDA-approved EIA.
[0086] The package insert for the currently approved test device
indicates a delay of 1.48 days between when OraQuick detects
antibodies to HIV and when EIA (ELISA Immunoassay) detects the
antibodies, while the device according to the invention indicates a
delay of 0.24 days, essentially equivalent sensitivity to
laboratory based antibody tests. HIV is transmitted primarily
through sexual contact, contact with infected blood, blood products
or human tissue and from mother to child (Smith D K, Grohskopf L A,
Black R J, et al, MMWR Recomm. Rep. 54:1-20 2005; Kourtis A P, Lee
F K, Abrams E J, et al., Lancet Infect Dis. 6:726-732 2006.
Globally, most HIV transmission occurs as a result of unprotected
sexual contact. HIV transmission rates per coital act with an
infected partner are estimated to be relatively low, but the risk
can be increased substantially if the viral load in the source
patient is high (e.g. during acute HIV infection: Cohen M S,
Pilcher C D, J. Infect. Dis. 191:1391-1393 2005). Enhanced
sensitivity is important for detection for such individuals.
Example 3
[0087] The amount of antigen used in the assay method was evaluated
based on whether it was striped, eluted off a pad, or added to the
developer solution. For each condition below, 3 .mu.L of samples
(PM2=HIV-1 plasma sample; PM6=HIV-2 plasma sample;
PM10=HIV-negative plasma; 9012-8--seroconversion plasma sample) was
added to the liquid phase (Developer) and the device placed in this
mixture to initiate the test.
[0088] In Condition 1, as shown in Table 3, peptides were striped
on the nitrocellulose. The per-device level of each peptide was
estimated at 82.1 ng of biotinylated HIV-1 and 33.2 ng of
biotinylated HIV-2.
TABLE-US-00003 TABLE 3 Nitro/Condition Striped Modified avidin
complexed with HIV peptides/Condition 1 Developer Developer A (750
.mu.L) Blocker pad HIV blocker Sample Test Line Response PM 2
Reactive PM 6 Reactive
[0089] In Condition 2, shown in Table 4, peptides were added to the
developer solution. There were no biotinylated peptides pre-bound
on the test line. The per-device level of each peptide was
estimated at 13.3 ng of biotinylated HIV-1 and 5.3 ng of
biotinylated HIV-2.
TABLE-US-00004 TABLE 4 Nitro/Condition Striped biotin binding
protein/Condition 2 Developer Developer A (750 .mu.L) spiked with
100 ng HIV-1 and 40 ng HIV-2 biotinylated peptides Blocker pad HIV
blocker Sample Test Line Response PM 2 Reactive PM 6 Reactive PM 10
Non-Reactive
[0090] In Condition 3, shown in Table 5, the peptides were added to
the blocker pad. The per-device level of each peptide is estimated
at 14.4 ng of biotinylated HIV-1 and 5.8 ng of biotinylated
HIV-2.
TABLE-US-00005 TABLE 5 Nitro/Condition Striped biotin binding
protein/Condition 3 Developer Developer A (750 .mu.L) Blocker pad
Biotinylated HIV-1: 1800 ng/mL Biotinylated HIV-2: 720 ng/mL Sample
Test Line Response PM 2 Reactive PM 6 Reactive PM 10
Non-Reactive
[0091] In Conditions 2 and 3, the biotinylated peptides were
present in the liquid phase (developer solution) or eluted off from
the pad component, and in each case, it was demonstrated that
significantly (>5.times.) lower amounts of peptide result in a
surprisingly similar reactive sample response relative to when the
peptide was striped on the test line (Condition 1). Exploiting the
liquid phase kinetics of the lateral flow platform dramatically
reduced the amount of immunoreactive material required to secure a
device reactive response.
Example 5
[0092] Expanding the epitope coverage by use of a recombinant
protein also benefited from the solution phase versus solid phase
presentation. This was demonstrated through use of a Zeptomatrix
seroconversion sample (9012-8), a sample that has been
characterized as p24 and HIV EIA positive and is representative of
an acute infection.
[0093] In Condition 4, rAg was striped on the nitrocellulose strip.
The per-device level of the recombinant antigen (rAg) was estimated
at 189 ng of biotinylated rAg.
TABLE-US-00006 TABLE 6 Nitro/Condition 1 mg/mL rAg/Condition 4
Developer Developer A (750 .mu.L) Blocker pad HIV blocker Sample
Test Line Response PM2 Reactive 9012-8 Non-Reactive
[0094] In Condition 5, rAg was added to the developer buffer. BBP
was striped on the nitrocellulose, unbound with any biotinylated
peptides. The per-device level of the rAg was estimated at 50 ng of
biotinylated HIV-1 rAg. PM2 and seroconversion sample 9012-8 were
reactive on the device under this condition. By comparison, when
biotinylated peptide was added to the developer alone (Condition
6), 9012-8 was not reactive (Table 8).
TABLE-US-00007 TABLE 7 Nitro/Condition Striped biotin binding
protein/Condition 5 Developer Developer A (750 .mu.L) spiked with
50 ng biotinylated rAg Blocker Pad HIV blocker Sample Test Line
Response PM2 Reactive 9012-8 Reactive
TABLE-US-00008 TABLE 8 Nitro/Condition Striped biotin binding
protein (BBP)/Condition 6 Developer Developer A (750 .mu.L) spiked
with 50 ng biotinylated HIV-1 peptide Blocker pad HIV blocker
Sample Test Line Response PM2 Reactive 9012-8 Non-Reactive
Example 6
[0095] There is a surprising advantage to having a fraction of the
peptides striped on the nitrocellulose. Shown below in Table 9 are
a series of seroconversion samples, samples 9012-7, 9077-14, 965-4,
109-7, and 204-3, that are HIV EIA positive where reactivity is
absent when the peptides are only present in the blocker pad but
not pre-bound on the test line. As the proportion of peptides on
the nitrocellulose is increased, reactivity in these samples is
gained, while HIV negative samples remain negative.
TABLE-US-00009 TABLE 9 Nitrocellulose Striped BBP Striped BBP with
25% Peptides Striped BBP with 50% Peptides Developer HIV developer
Blocker pad HIV blocker with biotinylated HIV peptides and
biotinylated rAg Sample Test Line Response PM 2 Reactive Reactive
Reactive PM 6 Reactive Reactive Reactive PM 10 Non-Reactive
Non-Reactive Non-Reactive 9012-7 Reactive Reactive Reactive 9077-14
Non-Reactive Reactive Reactive 965-4 Non-Reactive Non-Reactive
Reactive 109-7 Non-Reactive Reactive Reactive 204-3 Non-Reactive
Reactive Reactive
Sequence CWU 1
1
21966DNAArtificial SequenceSynthetic 1atggcgacga aggccgtgtg
cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60ttcgagcaga aggaaagtaa
tggaccagtg aaggtgtggg gaagcattaa aggactgact 120gaaggcctgc
atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt
180gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga
tgaagagagg 240catgttggag acttgggcaa tgtgactgct gacaaagatg
gtgtggccga tgtgtctatt 300gaagattctg tgatctcact ctcaggagac
cattgcatca ttggccgcac actggtggtc 360catgaaaaag cagatgactt
gggcaaaggt ggaaatgaag aaagtacaaa gacaggaaac 420gctggaagtc
gtttggcttg tggtgtaatt gggatcgccc aagttttccg tcctggcggt
480ggcgatatga gagacaactg gagaagcgaa ttatacaaat acaaagtgat
taagattgaa 540ccattgggca ttgccccaac caaagcgaag cgtagagttg
tgcagcgcga aaaacgtcag 600gctagacaac tgttatctgg cattgttcaa
cagcaaaata acttgctgag agctatcgaa 660gcacagcaac atctgctgca
actgactgtg tggggtatca agcagttgca agctcgcgtc 720ctggcagtag
aacgttatct gcgtgatcag caactgttag gtatttgggg ctgtagcggt
780aaattgatct gcaccactgc cgttccgtgg aatgcgtctt ggtcaaacaa
gagtttagaa 840gatatttggg acaatatgac ctggatgcaa tgggaacgtg
aaattgacaa ctacacaaac 900acgatctaca cattattaga agaatcgcag
aaccagcagg aaaagaacga acaggaatta 960ttatag 9662321PRTArtificial
SequenceSynthetic 2Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp
Gly Pro Val Gln1 5 10 15Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn
Gly Pro Val Lys Val 20 25 30Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly
Leu His Gly Phe His Val 35 40 45His Glu Phe Gly Asp Asn Thr Ala Gly
Cys Thr Ser Ala Gly Pro His 50 55 60Phe Asn Pro Leu Ser Arg Lys His
Gly Gly Pro Lys Asp Glu Glu Arg65 70 75 80His Val Gly Asp Leu Gly
Asn Val Thr Ala Asp Lys Asp Gly Val Ala 85 90 95Asp Val Ser Ile Glu
Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys 100 105 110Ile Ile Gly
Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly 115 120 125Lys
Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg 130 135
140Leu Ala Cys Gly Val Ile Gly Ile Ala Gln Val Phe Arg Pro Gly
Gly145 150 155 160Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr
Lys Tyr Lys Val 165 170 175Ile Lys Ile Glu Pro Leu Gly Ile Ala Pro
Thr Lys Ala Lys Arg Arg 180 185 190Val Val Gln Arg Glu Lys Arg Gln
Ala Arg Gln Leu Leu Ser Gly Ile 195 200 205Val Gln Gln Gln Asn Asn
Leu Leu Arg Ala Ile Glu Ala Gln Gln His 210 215 220Leu Leu Gln Leu
Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val225 230 235 240Leu
Ala Val Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly Ile Trp 245 250
255Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala
260 265 270Ser Trp Ser Asn Lys Ser Leu Glu Asp Ile Trp Asp Asn Met
Thr Trp 275 280 285Met Gln Trp Glu Arg Glu Ile Asp Asn Tyr Thr Asn
Thr Ile Tyr Thr 290 295 300Leu Leu Glu Glu Ser Gln Asn Gln Gln Glu
Lys Asn Glu Gln Glu Leu305 310 315 320Leu
* * * * *