U.S. patent application number 16/532855 was filed with the patent office on 2019-12-12 for method and device for combined detection of viral and bacterial infections.
The applicant listed for this patent is Rapid Pathogen Screening, Inc.. Invention is credited to Uma Mahesh Babu, Peter Condon, Robert P. Sambursky, Robert W. VanDine.
Application Number | 20190376970 16/532855 |
Document ID | / |
Family ID | 48870536 |
Filed Date | 2019-12-12 |
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United States Patent
Application |
20190376970 |
Kind Code |
A1 |
Sambursky; Robert P. ; et
al. |
December 12, 2019 |
Method and Device for Combined Detection of Viral and Bacterial
Infections
Abstract
A lateral flow assay is capable of detecting and differentiating
viral and bacterial infections. A combined point of care diagnostic
device tests markers for viral infection and markers for bacterial
infection, to effectively assist in the rapid differentiation of
viral and bacterial infections. In some preferred embodiments,
bimodal methods and devices determine if an infection is bacterial
and/or viral. A dual use two strip sample analysis device includes
a first lateral flow chromatographic test strip to detect MxA and a
low level of C-reactive protein and a second lateral flow
chromatographic test strip to detect high levels of C-reactive
protein. In some preferred embodiments, the sample is a fingerstick
blood sample.
Inventors: |
Sambursky; Robert P.;
(Lakewood Ranch, FL) ; VanDine; Robert W.;
(Montoursville, PA) ; Babu; Uma Mahesh;
(Bradenton, FL) ; Condon; Peter; (Tierra Verde,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rapid Pathogen Screening, Inc. |
Sarasota |
FL |
US |
|
|
Family ID: |
48870536 |
Appl. No.: |
16/532855 |
Filed: |
August 6, 2019 |
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14956956 |
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10408835 |
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16532855 |
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13790125 |
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12782162 |
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12469207 |
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12782162 |
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PCT/US2009/057775 |
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12782162 |
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12481631 |
Jun 10, 2009 |
8470608 |
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61179059 |
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61071833 |
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61060258 |
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61266641 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 2219/00743
20130101; B82Y 30/00 20130101; B01J 2219/00576 20130101; G01N
33/54386 20130101; G01N 2333/4737 20130101; G01N 33/54346 20130101;
G01N 33/56983 20130101; G01N 2333/914 20130101; B01J 2219/00648
20130101; B01J 2219/00725 20130101; G01N 33/56911 20130101; G01N
2469/00 20130101; G01N 2333/4703 20130101; B01J 2219/0074 20130101;
G01N 33/569 20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 33/543 20060101 G01N033/543 |
Claims
1.-45. (canceled)
46. A method to simultaneously detect at least one extracellular
analyte and at least one intracellular analyte, comprising the
steps of: a) collecting a sample; b) transferring the sample to a
sample analysis device; c) lysing the sample; and d) simultaneously
detecting the extracellular analyte and the intracellular analyte
on the sample analysis device.
47. The method of claim 46, wherein the extracellular analyte is
C-reactive protein and the intracellular analyte is MxA
protein.
48. The method of claim 46, wherein the sample is a blood
sample.
49. A method of detecting MxA protein and C-reactive protein in a
sample, comprising the steps of: a) adding the sample to a mixture
of an antibody to MxA protein conjugated to a first label and an
antibody to C-reactive protein conjugated to a second label
different from the first label; b) detecting a presence of MxA
protein by determining whether the antibody to MxA protein has
agglutinated; and c) detecting a presence of C-reactive protein by
determining whether the antibody to C-reactive protein has
agglutinated.
50. The method of claim 49, further comprising, before step a), the
steps of: d) conjugating the antibody to MxA protein to the first
label; e) conjugating the antibody to C-reactive protein to the
second label.
51. A method of detecting the presence of an unknown viral
infection, comprising the steps of: a) collecting a sample; b)
transferring the sample to a sample application zone of a sample
analysis device comprising: i) a conjugate zone comprising a sialic
acid nanomicelle comprising a label inside the nanomicelle; and ii)
a detection zone laterally downstream from the sample application
zone, comprising a sialic acid homolog nanoparticle; c) analyzing
the sample for a positive result in the detection zone.
52. A method of detecting the presence of a specific viral
infection, comprising the steps of: a) collecting a sample; b)
transferring the sample to a sample application zone of a sample
analysis device comprising: i) a conjugate zone comprising a
molecule selected from the group consisting of: a nanomicelle
comprising a binding partner for a specific virus that causes the
viral infection and a label; and a sialic acid homolog nanomicelle
comprising a label inside the nanomicelle; and ii) a detection zone
laterally downstream from the sample application zone, comprising a
nanoparticle specific for the virus that causes the viral
infection. c) analyzing the sample for a positive result in the
detection zone.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional patent application of
co-pending application Ser. No. 13/790,125, filed Mar. 8, 2013,
entitled "METHOD AND DEVICE FOR COMBINED DETECTION OF VIRAL AND
BACTERIAL INFECTIONS", which is a continuation-in-part patent
application of:
[0002] Co-pending application Ser. No. 12/782,162, filed May 18,
2010, entitled "METHOD AND DEVICE FOR COMBINED DETECTION OF VIRAL
AND BACTERIAL INFECTIONS", which claims one or more inventions
which were disclosed in Provisional Application No. 61/179,059,
filed May 18, 2009, entitled "METHOD AND DEVICE FOR COMBINED
DETECTION OF VIRAL AND BACTERIAL INFECTIONS" and is also a
continuation-in-part application of application Ser. No.
12/469,207, filed May 20, 2009, entitled "NANOPARTICLES IN
DIAGNOSTIC TESTS", which claimed priority from Provisional
Application No. 61/071,833, filed May 20, 2008, entitled
"NANOPARTICLES IN DIAGNOSTIC TESTS", and is also a
continuation-in-part of PCT application Serial Number
PCT/US2009/057775, filed Sep. 22, 2009, entitled "METHOD AND DEVICE
FOR COMBINED DETECTION OF VIRAL AND BACTERIAL INFECTIONS";
[0003] application Ser. No. 12/481,631, filed Jun. 10, 2009,
entitled "COMBINED VISUAL/FLUORESCENCE ANALYTE DETECTION TEST", now
U.S. Pat. No. 8,470,608, issued Jun. 25, 2013, which claimed
priority from Provisional Application No. 61/060,258, filed Jun.
10, 2008, entitled "COMBINED VISUAL/FLUORESCENCE ANALYTE DETECTION
TEST";
[0004] application Ser. No. 12/502,626, filed Jul. 14, 2009,
entitled "LATERAL FLOW NUCLEIC ACID DETECTOR", now U.S. Pat. No.
8,669,052, issued Mar. 11, 2014, which claimed priority from
Provisional Application No. 61/080,879, filed Jul. 15, 2008,
entitled "LATERAL FLOW NUCLEIC ACID DETECTOR";
[0005] application Ser. No. 12/502,662, filed Jul. 14, 2009,
entitled "IN SITU LYSIS OF CELLS IN LATERAL FLOW IMMUNOASSAYS", now
U.S. Pat. No. 8,614,101, issued Dec. 24, 2013, which claimed
priority from Provisional Application No. 61/098,935, filed Sep.
22, 2008, entitled "IN SITU LYSIS OF CELLS IN LATERAL FLOW
IMMUNOASSAYS";
[0006] application Ser. No. 12/958,454, filed Dec. 2, 2010,
entitled "MULTIPLANAR LATERAL FLOW ASSAY WITH SAMPLE COMPRESSOR",
now U.S. Pat. No. 8,609,433, issued Dec. 17, 2013, which claimed
priority from Provisional Application No. 61/266,641, filed Dec. 4,
2009, entitled "LATERAL FLOW NUCLEIC ACID DETECTOR", Provisional
Application No. 61/331,966, filed May 6, 2010, entitled
"MULTIPLANAR LATERAL FLOW ASSAY WITH SAMPLE COMPRESSOR",
Provisional Application No. 61/352,093, filed Jun. 7, 2010,
entitled "LATERAL FLOW ASSAYS", and Provisional Application No.
61/392,981, filed Oct. 14, 2010, entitled "MULTIPLANAR LATERAL FLOW
ASSAY WITH SAMPLE COMPRESSOR"; and
[0007] application Ser. No. 13/788,616, filed Mar. 7, 2013,
entitled "MULTIPLANAR LATERAL FLOW ASSAY WITH DIVERTING ZONE", now
U.S. Pat. No. 8,815,609, issued Aug. 26, 2014.
[0008] The benefit under 35 USC .sctn. 119(e) of the United States
provisional applications are hereby claimed, and the aforementioned
applications are hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0009] The invention pertains to the field of lateral flow
immunoassays. More particularly, the invention pertains to a
lateral flow immunoassay that rapidly detects viral and bacterial
infection.
Description of Related Art
[0010] Fever is a common cause of childhood visits to urgent care
centers for both family practice and pediatric offices. Most
commonly, this relates to either a respiratory infection or
gastroenteritis. The high incidence of fever in children and the
precautious administration of unnecessary antibiotics is reason to
develop a rapid screening test for the biomarkers that indicate
viral and/or bacterial infection.
[0011] It is often challenging to differentiate viral from
bacterial infections. This is especially true in young children
that cannot verbalize their symptoms and in the outpatient setting
where access to laboratory diagnostics is expensive, time
consuming, and requires several days to produce a result. More
recently, many new diagnostic markers have been identified. Several
of these markers show great promise to differentiate viral from
bacterial infections. Two such proteins include MxA and C-Reactive
Protein (CRP). Most respiratory infections are related to
pharyngitis of which 40% are caused by viruses and 25-50% by group
A beta hemolytic streptococcus. The lesser causes are acute
bronchiolitis and pneumonia.
[0012] Severe community-acquired pneumonia is caused by bacterial
infections in around 60% of cases, requiring admission to an
intensive care unit (ICU) for about 10% of patients. The remaining
30% are related to respiratory viruses.
[0013] About 80% of all antimicrobials are prescribed in primary
care, and up to 80% of these are for respiratory tract indications.
Respiratory tract infections are by far the most common cause of
cough in primary care. Broad spectrum antibiotics are often
prescribed for cough, including acute bronchitis, and many of these
prescriptions will benefit patients only marginally if at all, and
may cause side effects and promote antibiotic resistance. Factors
that urge physicians to give antibiotics include the absence of an
adequate diagnostic marker of bacterial infections, the concern
about lack of patient follow-up, and the time pressure.
[0014] Mx proteins are members of the superfamily of high molecular
weight GTPases. Accordingly, these GTPases are upregulated by type
I alpha/beta or type II interferons (IFN). The Mx GTPases are
expressed exclusively in IFN alpha/beta but not IFN gamma treated
cells. Type I interferons play important roles in innate immune
responses and have immunomodulatory, antiproliferative, and
antiviral functions. Human MxA, a 78 kDa protein, accumulates in
the cytoplasm of IFN treated cells and inhibits the replication of
a wide range of viruses. MxA protein may offer certain advantages
as a marker for viral infection over the other induced proteins
such as 2', 5'-oligoadenylate synthetase, because of its lower
basal concentration, longer half-life (2.3 days) and fast
induction. MxA mRNA is detectable in isolated peripheral blood
white blood cells stimulated with IFN within 1 to 2 h of IFN
induction, and MxA protein begins to accumulate shortly
thereafter.
[0015] Studies have shown that MxA protein expression in peripheral
blood is a sensitive and specific marker for viral infection. The
higher MxA levels in the viral infection group compared with the
bacterial infection group can be explained by the fact that the MxA
protein is induced exclusively by type I IFN and not by IFN-gamma,
IL-1, TNF-alpha, or any of the other cyotokines by bacterial
infection. Serum type I IFN levels remain within normal limits,
even in patients with severe bacterial infections.
[0016] Similarly, most viral infections have been reported to cause
little acute phase response, and low C-Reactive Protein (CRP)
concentrations have been used to distinguish illnesses of viral
origin from those of bacterial etiology. Because the plasma
concentration of CRP increases rapidly after stimulation and
decreases rapidly with a short half-life, CRP can be a very useful
tool in diagnosing and monitoring infections and inflammatory
diseases. In Scandinavia, point of care CRP testing is part of the
routine evaluation of patients with respiratory infections in
general practice, and its use has proved cost-effective. In general
practice, CRP is found valuable in the diagnosis of bacterial
diseases and in the differentiation between bacterial and viral
infections. Often the diagnostic value of CRP is found superior to
that of the erythrocyte sedimentation rate (ESR) and superior or
equal to that of the white blood cell count (WBC).
[0017] Clinically, it can be challenging to differentiate certain
systemic viral and bacterial infections. Bacterial cultures are
usually performed in cases of severe infection such as pneumonia,
or when the consequence of missing a diagnosis can lead to severe
complications, such as with Strep throat. Often times, cultures are
difficult to obtain. Unfortunately, viral cultures are not
routinely performed due to the significant time delay in receiving
results. New viral screening PCR panels are useful but they are
expensive and do not provide information at the point of care.
Thus, there remains a need for a simple, easy to use diagnostic
test that is capable of differentiating viral and bacterial
infections.
SUMMARY OF THE INVENTION
[0018] The present invention provides a lateral flow assay that is
capable of detecting and differentiating viral and bacterial
infections. A combined point of care diagnostic device tests
markers for viral infection and markers for bacterial infection, to
effectively assist in the rapid differentiation of viral and
bacterial infections. In one preferred embodiment, the bacterial
marker is CRP. In another preferred embodiment, the viral marker is
MxA. In some embodiments of the invention, it is unnecessary to
lyse the cells in the sample prior to applying it to the
device.
[0019] In one preferred embodiment, a method determines if an
infection is bacterial and/or viral by first collecting a sample.
The sample is then transferred to a dual use two strip sample
analysis device. The sample analysis device includes a first
lateral flow chromatographic test strip with a first reagent zone
and a second reagent zone. The first reagent zone includes at least
one first reagent specific to a low level of C-reactive protein
such that, when the sample contacts the first reagent, a first
labeled complex forms if the low level of C-reactive protein is
present in the sample. The second reagent zone includes at least
one second reagent specific to MxA such that, when the sample
contacts the second reagent, a second labeled complex forms if MxA
is present in the sample. The first lateral flow chromatographic
test strip also includes a first detection zone comprising a first
binding partner which binds to the first labeled complex; and a
second binding partner which binds to the second labeled complex.
The two strip lateral flow assay device also includes a second
lateral flow chromatographic test strip parallel in a lateral flow
direction to the first lateral flow chromatographic test strip. The
second lateral flow chromatographic test strip includes at least
one third reagent zone including at least one third reagent
specific to a high level of C-reactive protein, such that, when the
sample contacts the third reagent, a third labeled complex forms if
the high level of C-reactive protein is present in the sample. The
third reagent on the second lateral flow chromatographic test strip
only detects a level of C-reactive protein that is higher than the
level of C-reactive protein detected by the second reagent on the
first lateral flow chromatographic test strip. The second lateral
flow chromatographic test strip also includes a second detection
zone with a third binding partner which binds to the third labeled
complex. The sample is also analyzed for a presence of the low
level of C-reactive protein, MxA, and the high level of C-reactive
protein.
[0020] In another preferred embodiment, a dual use two strip
lateral flow assay device detects a bacterial and/or viral marker
in a sample. The device includes a first lateral flow
chromatographic test strip with a first reagent zone and a second
reagent zone. The first reagent zone includes at least one first
reagent specific to a low level of C-reactive protein such that,
when the sample contacts the first reagent, a first labeled complex
forms if the low level of C-reactive protein is present in the
sample. The second reagent zone includes at least one second
reagent specific to MxA such that, when the sample contacts the
second reagent, a second labeled complex forms if MxA is present in
the sample. The first lateral flow chromatographic test strip also
includes a first detection zone comprising a first binding partner
which binds to the first labeled complex; and a second binding
partner which binds to the second labeled complex. The two strip
lateral flow assay device also includes a second lateral flow
chromatographic test strip parallel in a lateral flow direction to
the first lateral flow chromatographic test strip. The second
lateral flow chromatographic test strip includes at least one third
reagent zone comprising at least one third reagent specific to a
high level of C-reactive protein, such that, when the sample
contacts the third reagent, a third labeled complex forms if the
high level of C-reactive protein is present in the sample. The
third reagent on the second lateral flow chromatographic test strip
only detects a level of C-reactive protein that is higher than the
level of C-reactive protein detected by the second reagent on the
first lateral flow chromatographic test strip. The second lateral
flow chromatographic test strip also includes a second detection
zone with a third binding partner which binds to the third labeled
complex.
[0021] Another preferred embodiment is a method for determining if
an infection is bacterial and/or viral, and includes the step of
collecting a sample. The sample is then transferred to a sample
analysis device. The sample analysis device includes a sample
compressor with a first reagent zone including at least one first
reagent specific to a low level of C-reactive protein such that,
when the sample contacts the first reagent, a first labeled complex
forms if the low level of C-reactive protein is present in the
sample, and at least one second reagent specific to MxA such that,
when the sample contacts the second reagent, a second labeled
complex forms if MxA is present in the sample, and a second reagent
zone including at least one third reagent specific to a high level
of C-reactive protein, where the third reagent only detects a level
of C-reactive protein that is higher than the level of C-reactive
protein detected by the second reagent, such that, when the sample
contacts the third reagent, a third labeled complex forms if the
high level of C-reactive protein is present in the sample. The
device also includes a first lateral flow chromatographic test
strip that includes a first detection zone including a first
binding partner which binds to the first labeled complex, a second
binding partner which binds to the second labeled complex and a
first diverting zone located upstream of the first detection zone
on the lateral flow chromatographic test strip. The first diverting
zone interrupts lateral flow on the first lateral flow
chromatographic test strip. The device also includes a second
lateral flow chromatographic test strip parallel in a lateral flow
direction to the first lateral flow chromatographic test strip. The
second lateral flow chromatographic test strip includes a second
detection zone including a third binding partner which binds to the
third labeled complex and a second diverting zone located upstream
of the first detection zone on the lateral flow chromatographic
test strip. The second diverting zone interrupts lateral flow on
the second lateral flow chromatographic test strip. The device also
includes a first sample application zone where sample is placed on
the sample analysis device. The first sample application zone is
located in a location selected from the group consisting of: i) on
the first lateral flow chromatographic test strip upstream of the
detection zone and ii) on the first reagent zone of the sample
compressor. The device also includes a second sample application
zone where sample is placed on the sample analysis device. The
second sample application zone is located in a location selected
from the group consisting of: i) on the second lateral flow
chromatographic test strip upstream of the detection zone and ii)
on the second reagent zone of the sample compressor. The sample
compressor is in a different plane than the first lateral flow
chromatographic test strip and the second lateral flow
chromatographic test strip. The first reagent zone of the sample
compressor creates a bridge over the first diverting zone and the
second reagent zone of the sample compressor creates a bridge over
the second diverting zone, diverting flow onto the sample
compressor and returning flow to the first chromatographic test
strip and the second chromatographic test strips at the end of the
first diverting zone and the second diverting zone. The sample is
analyzed for a presence of the low level of C-reactive protein,
MxA, and the high level of C-reactive protein.
[0022] Another preferred embodiment is a lateral flow device for
detecting an analyte in a sample. The device includes a sample
compressor with a first reagent zone including at least one first
reagent specific to a low level of C-reactive protein such that,
when the sample contacts the first reagent, a first labeled complex
forms if the low level of C-reactive protein is present in the
sample, and at least one second reagent specific to MxA such that,
when the sample contacts the second reagent, a second labeled
complex forms if MxA is present in the sample, and a second reagent
zone including at least one third reagent specific to a high level
of C-reactive protein, where the third reagent only detects a level
of C-reactive protein that is higher than the level of C-reactive
protein detected by the second reagent, such that, when the sample
contacts the third reagent, a third labeled complex forms if the
high level of C-reactive protein is present in the sample. The
device also includes a first lateral flow chromatographic test
strip that includes a first detection zone including a first
binding partner which binds to the first labeled complex, a second
binding partner which binds to the second labeled complex and a
first diverting zone located upstream of the first detection zone
on the first lateral flow chromatographic test strip. The first
diverting zone interrupts lateral flow on the first lateral flow
chromatographic test strip. The device also includes a second
lateral flow chromatographic test strip parallel in a lateral flow
direction to the first lateral flow chromatographic test strip. The
second lateral flow chromatographic test strip includes a second
detection zone comprising a third binding partner which binds to
the third labeled complex and a second diverting zone located
upstream of the first detection zone on the lateral flow
chromatographic test strip. The second diverting zone interrupts
lateral flow on the second lateral flow chromatographic test strip.
The device also includes a first sample application zone where
sample is placed on the sample analysis device. The first sample
application zone is located in a location selected from the group
consisting of: i) on the first lateral flow chromatographic test
strip upstream of the detection zone and ii) on the first reagent
zone of the sample compressor. The device also includes a second
sample application zone where sample is placed on the sample
analysis device. The second sample application zone is located in a
location selected from the group consisting of: i) on the second
lateral flow chromatographic test strip upstream of the detection
zone and ii) on the second reagent zone of the sample compressor.
The sample compressor is in a different plane than the first
lateral flow chromatographic test strip and the second lateral flow
chromatographic test strip. The first reagent zone of the sample
compressor creates a bridge over the first diverting zone and the
second reagent zone of the sample compressor creates a bridge over
the second diverting zone, diverting flow onto the sample
compressor and returning flow to the first chromatographic test
strip and the second chromatographic test strips at the end of the
first diverting zone and the second diverting zone.
[0023] In another preferred embodiment, a method simultaneously
detects at least one extracellular analyte and at least one
intracellular analyte, by collecting a sample and transferring the
sample to a sample analysis device. The sample is also lysed and
the extracellular analyte and the intracellular analyte are
simultaneously detected on the same sample analysis device. In one
preferred embodiment, the extracellular analyte is C-reactive
protein and the intracellular analyte is MxA protein.
[0024] In another preferred embodiment, a method of detecting MxA
protein and C-reactive protein in a sample includes the steps of
adding the sample to a mixture of an antibody to MxA protein
conjugated to a first label and an antibody to C-reactive protein
conjugated to a second label different from the first label,
detecting a presence of MxA protein by determining whether the
antibody to MxA protein has agglutinated, and detecting a presence
of C-reactive protein by determining whether the antibody to
C-reactive protein has agglutinated.
[0025] In another preferred embodiment, a method of detecting the
presence of an unknown viral infection in a sample first collects
the sample. The sample is then transferred to a sample application
zone of a sample analysis device. The sample analysis device
includes a conjugate zone including a sialic acid nanomicelle with
a label inside the nanomicelle and a detection zone laterally
downstream from the sample application zone, which includes a
sialic acid homolog nanoparticle. The sample is analyzed for a
positive result in the detection zone.
[0026] In another preferred embodiment, a method of detecting the
presence of an unknown viral infection in a sample first collects
the sample. The sample is then transferred to a sample application
zone of a sample analysis device. The sample analysis device
includes a conjugate zone with a molecule selected from the group
consisting of: a nanomicelle including a binding partner for a
specific virus that causes the viral infection and a label and a
sialic acid homolog nanomicelle including a label inside the
nanomicelle. The sample analysis device also includes a detection
zone laterally downstream from the sample application zone, with a
nanoparticle specific for the virus that causes the viral
infection. The sample is analyzed for a positive result in the
detection zone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows rapid screening test window visual test results
to distinguish viral and bacterial infections and an interpretation
of those results.
[0028] FIG. 2 shows three cassettes with different colored test
lines.
[0029] FIG. 3 shows a comparison of a two line detector, where both
lines are the same color, and an extra sensitive two line detector,
where the two lines are different colors.
[0030] FIG. 4A shows a device with a test line corresponding to the
presence of a viral marker and a second, separate test line that
detects the presence of a bacterial marker in an embodiment of the
present invention.
[0031] FIG. 4B shows a device with a test line corresponding to the
presence of a viral marker and a second, separate test line that
detects the presence of a bacterial marker in another embodiment of
the present invention.
[0032] FIG. 5A shows a sample analysis device including a lysis
zone located between a sample application zone and a reagent zone
in an embodiment of the present invention.
[0033] FIG. 5B shows a sample analysis device including a lysis
zone overlapping a sample application zone in an embodiment of the
present invention.
[0034] FIG. 5C shows a sample analysis device including a lysis
zone overlapping a reagent zone in an embodiment of the present
invention.
[0035] FIG. 5D shows a sample analysis device including a lysis
zone overlapping a sample application zone and a reagent zone in an
embodiment of the present invention.
[0036] FIG. 6A shows a device with a test line corresponding to the
presence of a bacterial marker such as high CRP levels in an
embodiment of the present invention.
[0037] FIG. 6B shows a device with a test line corresponding to the
presence of a bacterial marker such as high CRP levels in another
embodiment of the present invention.
[0038] FIG. 7A shows a sample analysis device including a lysis
zone located between a sample application zone and a reagent zone
in an embodiment of the present invention.
[0039] FIG. 7B shows a sample analysis device including a lysis
zone overlapping a sample application zone in an embodiment of the
present invention.
[0040] FIG. 7C shows a sample analysis device including a lysis
zone overlapping a reagent zone in an embodiment of the present
invention.
[0041] FIG. 7D shows a sample analysis device including a lysis
zone overlapping a sample application zone and a reagent zone in an
embodiment of the present invention.
[0042] FIG. 8A shows a fully open sample analysis device with dual
test strips, as well as a conjugate zone and a sample application
zone on a sample compressor in a plane separate from the test
strips in an embodiment of the present invention.
[0043] FIG. 8B shows the sample analysis device of FIG. 8A with
part of the housing closed, but the conjugate zone still visible on
the left side of the device.
[0044] FIG. 8C shows the sample analysis device of FIG. 8A after
the test has been initiated.
[0045] FIG. 9A shows a test result negative for both MxA and CRP in
an embodiment of the present invention.
[0046] FIG. 9B shows a test result positive for MxA in an
embodiment of the present invention.
[0047] FIG. 9C shows a test result positive for MxA in an
embodiment of the present invention.
[0048] FIG. 9D shows a test result positive for CRP in an
embodiment of the present invention.
[0049] FIG. 9E shows a test result positive for CRP in an
embodiment of the present invention.
[0050] FIG. 9F shows a test result positive for both CRP and MxA,
indicating co-infection, in an embodiment of the present
invention.
[0051] FIG. 10A shows a fully open sample analysis device with dual
test strips and a conjugate zone on a sample compressor in a plane
separate from the test strips in an embodiment of the present
invention.
[0052] FIG. 10B shows the sample analysis device of FIG. 10A with
part of the housing closed, but the conjugate zone still visible on
the left side of the device.
[0053] FIG. 10C shows the sample analysis device of FIG. 10A after
the test has been initiated.
[0054] FIG. 11 shows a kit for sample analysis using a sample
analysis device in an embodiment of the present invention.
[0055] FIG. 12 shows a sample analysis device with dual test strips
in another embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0056] The present invention provides a lateral flow assay that is
capable of differentiating between viral and bacterial infections.
Instead of testing for analytes specific to a particular bacterial
or viral infection, the lateral flow assays described herein test
for diagnostic markers that are specifically produced in a host in
response to general, unspecified bacterial infection and general,
unspecified viral infection. The diagnostic markers are preferably
markers of an unspecified and/or unknown illness of bacterial or
viral origin. In preferred embodiments, the diagnostic markers are
specific markers for an immune response to an unspecified and/or
unknown bacterial and/or viral infection.
[0057] A combined point of care diagnostic device tests markers for
both viral and bacterial infection and can effectively assist in
the rapid differentiation of viral and bacterial infections, for
example at the outpatient office or during an urgent care visit.
This ability can dramatically reduce health care costs by limiting
misdiagnosis and the subsequent overuse of antibiotics. Such a
practice may limit antibiotic allergies, adverse events, and
antibiotic resistance. The rapid result obtained from the test also
permits a diagnosis while the patient is still being examined by
the practitioner. In a preferred embodiment, the test result is
obtained in under 10 minutes after applying the sample to the
device, and it is preferably read at approximately 10 minutes. In
samples that are highly positive, the test line is visible within
approximately 1-5 minutes.
[0058] In a preferred embodiment of the present invention, the
lateral flow immunoassay device of the present invention includes a
sample-transporting liquid, which can be a buffer, and a
chromatographic test strip containing one or several fleece
materials or membranes with capillary properties through which
sample flows. Some preferred materials and membranes for the test
strip include, but are not limited to, Polyethylene terephthalate
(PET) fibers, such as Dacron.RTM. fibers, nitrocellulose,
polyester, nylon, cellulose acetate, polypropylene, glass fibers,
and combinations of these materials and their backings. In some
embodiments of the invention, it is unnecessary to lyse the cells
in the sample or treat the sample in any way prior to applying it
to the test strip.
[0059] One preferred method of the present invention uses a sample
analysis device, for example a chromatographic test strip, to
determine if an infection is bacterial or viral. In this method, a
sample is collected, and transferred to the chromatographic test
strip. In a preferred embodiment, the sample is a sample including
leukocytes. The test strip includes a reagent zone. The reagent
zone preferably includes at least one first reagent specific to a
bacterial marker such that, when the bacterial marker present in
the sample contacts the first reagent, a first labeled complex
forms. The reagent zone also preferably includes at least one
second reagent specific to a viral marker such that, when the viral
marker present in the sample contacts the second reagent, a second
labeled complex forms. A detection zone includes both a bacterial
marker binding partner which binds to the first labeled complex and
a viral marker binding partner which binds to the second labeled
complex. The sample is then analyzed for the presence of the viral
marker and/or the bacterial marker.
[0060] A preferred embodiment of a device of the present invention
includes a sample application zone. The device also includes a
reagent zone, which includes at least one first reagent specific to
a bacterial marker such that, when a bacterial marker present in
the sample contacts the first reagent, a first labeled complex
forms and at least one second reagent specific to a viral marker
such that, when a viral marker present in the sample contacts the
second reagent, a second labeled complex forms. A detection zone on
the device includes a bacterial marker binding partner which binds
to the first labeled complex and a viral marker binding partner
which binds to the second labeled complex. One example of a device
that could be used is a chromatographic test strip. In other
preferred embodiments, some of the zones of the device are on one
or more chromatographic test strips, while other zones (for
example, the reagent zone, the sample application zone, and/or the
control binding partner) are on a sample compressor, separate from
and in a different plane than the chromatographic test strip.
[0061] In a preferred embodiment, the presence of the viral marker
or the bacterial marker is indicated by a test line visible to the
naked eye. The presence of the viral marker may be indicated by a
first test line while the presence of the bacterial marker is
indicated by a second test line. In some embodiments, the first
test line displays a first color when positive and the second test
line displays a second color different from the first color when
positive. In embodiments where both the first test line and the
second test line are located in the same space on the sample
analysis device, a third color is preferably formed when both the
first test line and the second test line are positive. In other
embodiments, the two test lines are spatially separate from each
other on the device.
[0062] Viral and bacterial infections are highly contagious and
difficult to clinically differentiate due to a significant overlap
in signs and symptoms, which often leads to the over prescription
of systemic antibiotics and fosters antibiotic resistance. In
developed countries, acute respiratory infections are the leading
cause of morbidity, accounting for: 20% of medical consultations,
30% of absences from work, and 75% of all antibiotic prescriptions.
In the U.S., there are approximately 76 million physician office
visits annually for acute respiratory infection. The ability to
detect an immune response to an infection aids in the clinical
diagnostic ability to differentiate infections resulting from a
viral and/or bacterial etiology.
[0063] In one preferred embodiment, the bacterial marker is CRP. In
another preferred embodiment, the viral marker is MxA. In some
preferred embodiments, the detection zone also includes a control
line that is visible to the naked eye when the device is
working.
[0064] In one preferred embodiment, the marker for viral infection
is MxA and the marker for bacterial infection is C-reactive protein
(CRP). High MxA protein levels are strongly correlated with
systemic viral infection and increased CRP is more associated with
bacterial infections. The present invention includes a rapid
infectious screening test for identifying MxA and CRP in samples.
MxA is present in leukocytes (white blood cells). Therefore, the
sample can be taken anywhere leukocytes are available, for example
in a peripheral blood sample, nasopharyngeal aspirates, tears,
spinal fluid, and middle ear aspirates.
[0065] In some preferred embodiments with a single test strip
containing CRP and MxA, the threshold concentration of CRP in a
sample needed to elicit a positive result is approximately 6-15
mg/L. In other preferred embodiments, the threshold concentration
of MxA in a sample to elicit a positive result may be as low as
approximately 15 ng/ml; however, the threshold concentration may by
higher, in a range from approximately 20 ng/ml to approximately 250
ng/ml. The threshold concentration may depend on the size of the
sample being applied to the test strip, as well as its dilution, if
applicable.
[0066] In some embodiments, the devices and methods described
herein allow for the rapid, visual, qualitative in vitro detection
of both MxA and CRP directly from peripheral whole blood. In one
preferred embodiment, the test measures an immune response to a
suspected viral and/or bacterial infection in patients older than
one year that present within seven days of onset of a fever, with
respiratory symptoms consistent with respiratory disease, and with
a suspected diagnosis of acute pharyngitis or community acquired
pneumonia. Negative results do not necessarily preclude respiratory
infection and should not be used as the sole basis for diagnosis,
treatment, or other management decisions. In some embodiments, the
use of additional laboratory testing (e.g., bacterial and viral
culture, immunofluorescence, viral polymerase chain reaction, and
radiography) and clinical presentation is preferably additionally
used to confirm whether a specific lower respiratory or pharyngeal
pathogen exists.
[0067] In addition, there are some conditions that lead to
erroneous false positives or negatives. These include, but are not
limited to, current use of immunosuppressive drugs by the patient
providing the sample, current use of oral anti-infective drugs by
the patient providing the sample, current use of interferon therapy
(e.g. for multiple sclerosis, HIV, HBV, HCV) by the patient
providing the sample and live viral immunization within the last 30
days by the patient providing the sample. Both false negatives and
false positives are possible since the levels can fluctuate due to
therapy.
[0068] In preferred embodiments, the devices and methods are
intended for professional use in an outpatient office or urgent
care clinic and should be used in conjunction with other clinical
(laboratory or radiographic) and epidemiological information.
[0069] In preferred embodiments, a dual-use dual chromatographic
test strip assay detects the body's immune response to viral and/or
bacterial infections in patients using a multiplexed pattern of
results. In one specific preferred embodiment, the assay tests for
Myxovirus resistance A (MxA), low levels of C-reactive Protein
("low" CRP), and high levels of C-reactive Protein ("high" CRP).
Two test strips are preferably used. In some embodiments, a sample
compressor in a different plane from the chromatographic test
strips is also used. The first test strip assays for MxA and low
levels of C-reactive Protein, and the second test strip is an assay
for high levels of C-reactive Protein. The first test strip and/or
the sample compressor include reagents to detect MxA protein and a
low level of C-reactive protein. The second test strip and/or the
sample compressor include reagents to detect a high level of
C-reactive protein. The two test strips are preferably run
side-by-side, and each strip also preferably includes a control
line. The control reagents are preferably either on the test strips
or on the sample compressor. These tests detect and classify
biological infections as viral, bacterial, or a co-infection of
virus and bacteria. In some preferred embodiments, the dual-use
dual chromatographic test strip assay is used to detect samples
from patients with a febrile respiratory illness.
[0070] In some preferred embodiments with two test strips, on the
first test strip, a threshold concentration of CRP ("low" CRP
level) of approximately 6-15 mg/L (serum cut-off value) in the
sample is needed to elicit a positive result and a threshold
concentration of at least 15 ng/ml MxA in a sample is needed to
elicit a positive result. In other preferred embodiments, the
threshold concentration for MxA may be in a range from
approximately 15 ng/ml to approximately 250 ng/ml to elicit a
positive result. The threshold concentration may depend on the size
of the sample being applied to the test strip, as well as its
dilution, if applicable. In one preferred embodiment, the threshold
concentration of low CRP, for example in extracellular serum from a
blood sample, is 7 mg/L for a fingerstick cut-off value, which is
equivalent to 10 mg/L for a serum cut-off value. In one preferred
embodiment, the threshold concentration of MxA, for example in
peripheral blood mononuclear cells from a blood sample, is 40 ng/ml
for a fingerstick cut off value, which is equivalent to a 40 ng/ml
venous blood cut-off value. On the second test strip, a threshold
concentration of CRP ("high" CRP level) of approximately 60-100
mg/L in the sample is needed to elicit a positive result in some
preferred embodiments. In one particularly preferred embodiment, a
threshold concentration of high CRP on the second test strip is
approximately 80 mg/L on a fingerstick cut-off value.
[0071] In other embodiments, other markers for viral infection
and/or bacterial infection may be used. For example, approximately
12% of host genes alter their expression after Lymphocytic
Choriomeningitis Virus (LCMV) infection, and a subset of these
genes can discriminate between virulent and nonvirulent LCMV
infection. Major transcription changes have been given preliminary
confirmation by quantitative PCR and protein studies and are
potentially valuable candidates as biomarkers for arenavirus
disease. Other markers for bacterial infection include, but are not
limited to, procalcitonin, urinary trypsin inhibitor (uTi),
lipopolysaccharide, IL-1, IL-6, IL-8, IL-10, ESR and an elevated
WBC count (increased bands), Lactate, Troponin, vascular
endothelial growth factor, platelet derived growth factor,
cortisol, proadrenomedullin, macrophage migratory inhibitory
marker, activated protein C, CD 4,8,13,14, or 64, caspase, placenta
derived growth factor, calcitonin gene-related peptide, high
mobility group 1, copeptin, naturietic peptides, lipopolysaccharide
binding protein, tumor necrosis factor alpha, circulating
endothelial progenitor cells, complement 3a, and triggering
receptor expresssed on myeloid cells (trem-1).
[0072] In one embodiment, the infections being distinguished are
respiratory infections. In other embodiments, other types of
infections, which can be bacterial or viral, are differentiated
using the system of the present invention. Some examples include,
but are not limited to, encephalitis, meningitis, gastroenteritis,
febrile respiratory illness (including bronchitis, pharyngitis,
pneumonia), sinusitis, otitis media, urinary tract infections, and
conjunctivitis.
[0073] Lateral flow devices are known, and are described in, e.g.,
U.S. Published Patent Application Nos. 2005/0175992 and
2007/0059682. The contents of both of these applications are
incorporated herein by reference. Other lateral flow devices known
in the art could alternatively be used with the systems and methods
of the present invention.
[0074] U.S. Published Patent Application No. 2007/0059682 discloses
detecting an analyte and a sample which can also contain one or
more interfering substances. This publication teaches separating
the analyte from the interfering substances by capturing the
interfering substances on the chromatographic carrier, and
detecting the analyte on the carrier separated from the interfering
substances.
[0075] U.S. Published Patent Application No. 2005/0175992 discloses
a method for detecting targets, such as pathogens and/or
allergy-associated components, in a human body fluid where the body
fluid sample is collected by a collection device, such as a swab
member. The samples are transferred from the swab member to a
sample analysis device, on which an analysis of the targets can
occur by immunochemical or enzymatic means. The test result is
capable of being displayed within a very short period of time and
can be directly read out by the user. This enables point-of-care
testing with results available during a patient visit. The
inventions disclosed in this copending application are particularly
advantageous for the diagnosis of conjunctivitis.
[0076] In a method of the invention, the sample to be analyzed is
applied to a chromatographic carrier. The carrier can be made of
one single chromatographic material, or preferably several
capillary active materials made of the same or different materials
and fixed on a carrier backing. These materials are in close
contact with each other so as to form a transport path along which
a liquid driven by capillary forces flows from an application zone,
passing a reagent zone, towards one or more detection zones and
optionally a waste zone at the other end of the carrier. In other
embodiments, the liquid passes the reagent zone prior to flowing
into the sample application zone. In an especially preferred
embodiment, the carrier is a chromatographic test strip. In other
preferred embodiments, the sample may be applied to a sample
compressor in a different plane from the chromatographic test
strip, and then transferred to the chromatographic test strip by
the sample compressor.
[0077] In some embodiments, the sample is directly applied to the
carrier by dipping the carrier's application zone into the sample.
Alternatively, application of the sample to the carrier may be
carried out by collecting the sample with a dry or wetted wiping
element from which the sample can be transferred, optionally after
moistening, to the carrier's application zone. Usually, the wiping
element is sterile and may be dry or pretreated with a fluid before
the collection step. Materials suitable for wiping elements
according to the invention may comprise synthetic materials, woven
fabrics or fibrous webs. Some examples of such wiping elements are
described in German Patents DE 44 39 429 and DE 196 22 503, which
are hereby incorporated by reference. In other embodiments, the
sample may be collected by a collection receptacle, such as a
pipette, and transferred directly to the carrier.
[0078] Depending on the type of detection method, different
reagents are present in the carrier's reagent zone, which, in some
embodiments, is preferably located between the application zone and
the detection zone or, in other embodiments, is preferably located
before the application zone. In yet other embodiments, the reagents
may be on a sample compressor separate from and in a different
plane than the carrier including the detection zone.
[0079] In a sandwich immunoassay, it is preferred to have a
labeled, non-immobilized reagent in the reagent zone that is
specific to each bacterial and viral marker that is being detected.
Thus, when a viral or bacterial marker present in the sample
contacts the corresponding labeled viral or bacterial reagent
present in the reagent zone, a labeled complex is formed between
the marker and the corresponding labeled reagent. The labeled
complex in turn is capable of forming a further complex with an
immobilized viral or bacterial marker binding partner at a test
line in the detection zone. In a competitive immunoassay, the
reagent zone preferably contains a labeled, non-immobilized marker
analogue which competes with the marker for the immobilized marker
binding partner in the detection zone. The marker binding partners
in the reagent zone and in the detection zone are preferably
monoclonal, polyclonal or recombinant antibodies or fragments of
antibodies capable of specific binding to the corresponding
marker.
[0080] In a preferred embodiment, the present invention provides
for the reduction of interfering substances that might be present
in the sample to be tested. Since an interfering substance, e.g. a
human anti-mouse antibody (HAMA), may also be capable of forming a
complex with the labeled, non-immobilized reagent of the reagent
zone and the immobilized binding partner of the detection zone,
thus indicating a positive test result in the immunoassay, the
carrier may further include at least one capturing zone. Each
capturing zone contains an immobilized capturing reagent
specifically binding to a certain interfering substance, thereby
immobilizing the interfering substance in the capturing zone. As
the capturing zone is separated from the detection zone by space,
and the sample starts to migrate over the reagent zone and the
capturing zone before reaching the carrier's detection zone, the
method allows a separation of the interfering substance or
substances from the analyte or analytes of interest. Preferably,
the capturing zone is located between the reagent zone and the
detection zone. However, the capturing zone may also be located
between the application zone and the reagent zone.
[0081] Detection of the marker may be achieved in the detection
zone. The binding molecule immobilizes the labeled complex or the
labeled marker-analogue by immune reaction or other reaction in the
detection zone, thus building up a visible test line in the
detection zone during the process. Preferably, the label is an
optically detectable label. Forming a complex at the test line
concentrates and immobilizes the label and the test line becomes
visible for the naked eye, indicating a positive test result.
Particularly preferred are direct labels, and more particularly
gold labels which can be best recognized by the naked eye.
Additionally, an electronic read out device (e.g. on the basis of a
photometrical, acoustic, impedimetrical, potentiometric and/or
amperometric transducer) can be used to obtain more precise results
and a semi-quantification of the analyte. Other labels may be
latex, fluorophores or phosphorophores.
[0082] In one embodiment, the sensitivity of visually read lateral
flow immunoassay tests is enhanced by adding a small quantity of
fluorescing dye or fluorescing latex bead conjugates to the initial
conjugate material. When the visible spectrum test line is visibly
present, the test result is observed and recorded. However, in the
case of weak positives that do not give rise to a distinct visual
test line, a light of an appropriate spectrum, such as a UV
spectrum, is cast on the test line to excite and fluorescent the
fluorescing latex beads which are bound in the test line to enhance
the visible color at the test line.
[0083] In a preferred embodiment, the reagents are configured such
that the visible test line corresponding to the presence of the
viral marker will be separate from the test line corresponding to
the presence of the bacterial marker. Therefore, it can be readily
determined whether the sample contained bacterial or viral markers
(or both) simply by the location of the development of the test
lines in the detection zone. In another preferred embodiment, the
reagents may be chosen such that differently colored test lines are
developed. That is, the presence of a viral marker will cause the
development of a differently colored line than that developed by
the presence of a bacterial marker. For example, the label
corresponding to the reagent recognizing the viral marker may be
red, whereas the label corresponding to the reagent recognizing the
bacterial marker may be green. Differently colored labels that may
be attached to the non-immobilized reagents are well known. Some
examples include, but are not limited to, colloidal gold, colloidal
selenium, colloidal carbon, latex beads, paramagnetic beads,
fluorescent and chemiluminescent labels and mixtures thereof.
[0084] FIGS. 4A and 4B show a chromatographic test strip (400) with
a test line (402) corresponding to the presence of a viral marker
and a second, separate test line (403) that detects the presence of
a bacterial marker. The sample is applied to the application zone
(401) of the chromatographic test strip (400). As shown in FIG. 4A,
the sample then passes a reagent zone (460) containing at least one
labeled viral binding partner and at least one labeled bacterial
binding partner that is eluted by and then able to migrate with a
sample transport liquid (e.g. a buffer solution). Alternatively, as
shown in FIG. 4B, the reagent zone (460) is located upstream of the
sample application zone (401) such that the labeled binding
partners in the reagent zone are eluted by the sample transport
liquid and travel to the sample. The labeled viral binding partner
is capable of specifically binding to a viral marker of interest to
form a complex which in turn is capable of specifically binding to
another specific reagent or binding partner in the detection zone.
The labeled bacterial binding partner is capable of specifically
binding to a bacterial marker of interest to form a complex which
in turn is capable of specifically binding to another specific
reagent or binding partner in the detection zone. Although not
shown in these Figures, an absorbent pad, as well as other known
lateral flow immunoassay components including, but not limited to,
a waste zone, a carrier backing, a housing, and an opening in the
housing for result read out, may optionally also be a component of
the test strip (400) in these embodiments.
[0085] The test strip (400) also includes a detection zone (405)
containing at least one first section for detection of a viral
marker, e.g. a test line (402), including an immobilized specific
binding partner, complementary to the viral reagent complex formed
by the viral marker and its labeled binding partner. Thus, at the
test line (402), detection zone binding partners trap the labeled
viral binding partners from the reagent zone (460) along with their
bound viral markers. This localization of the viral marker with its
labeled binding partners gives rise to an indication at the test
line (402). At the test line (402), the presence of the viral
marker is determined by qualitative and/or quantitative readout of
the test line (402) indication resulting from the accumulation of
labeled binding partners.
[0086] The detection zone (405) also includes at least one second
section for detection of a bacterial marker, e.g. a test line
(403), including an immobilized specific binding partner,
complementary to the bacterial reagent complex formed by the
bacterial marker and its labeled binding partner. Thus, at the test
line (403), detection zone binding partners trap the labeled
bacterial binding partners from the reagent zone (460) along with
their bound bacterial markers. This localization of the bacterial
marker with its labeled binding partners gives rise to an
indication at the test line (403). At the test line (403), the
presence of the bacterial marker is determined by qualitative
and/or quantitative readout of the test line (403) indication
resulting from the accumulation of labeled binding partners. While
test line (402) is upstream of test line (403) relative to the
direction of flow (408) in the figures, in alternative embodiments,
test line (403) is upstream of test line (402). In still other
embodiments, test lines (402) and (403) are located in the same
location on the test strip.
[0087] Optionally, the detection zone (405) may contain further
test lines to detect other viral and/or bacterial markers, as well
as a control line (404). The control line (404) indicates that the
labeled specific binding partner traveled through the length of the
assay, even though it may not have bound any viral or bacterial
markers, thus confirming proper operation of the assay. As shown in
FIGS. 4A through 4B, the control zone (404) is preferably
downstream of the test lines (402) and (403). However, in other
embodiments, the control zone (404) may be located upstream of
either or both of the test lines (402) and (403).
[0088] In a preferred embodiment, the control line (404) includes
an antibody or other recombinant protein which binds to a component
of the elution medium or other composition being used in the test.
In embodiments where nucleic acids are the targets, the control
line (404) preferably includes a nucleic acid complementary to the
labeled nucleic acid being used as a binding partner for the target
nucleic acid.
[0089] Although only one test line is shown in the figures for each
of the viral and bacterial markers, multiple test lines for both or
either of the viral and bacterial markers may be used within the
spirit of the invention. In some embodiments where there are
multiple bacterial and/or viral targets, the presence of each
target preferably corresponds to a separate test line (402) or
(403). In other embodiments, both the bacterial marker and the
viral marker are detected on a single test line. In these
embodiments, the presence of both a bacterial marker and a viral
marker on the same test line has different characteristics than the
presence of either a bacterial or viral marker alone. For example,
the presence of both a bacterial marker and a viral marker on the
same test line may be visually indicated by a different color than
the presence of either a bacterial marker or a viral marker
alone.
[0090] Fresh whole blood samples of patients showing symptoms of
viral infections (flu like symptoms and fever of >100.5.degree.
F.) were tested to determine what levels of MxA in the blood could
be detected with the lateral flow tests described herein. The
lateral flow assays used in these experiments had a similar
configuration as the device shown in FIG. 4B described above,
without a second test line for the presence of a bacterial marker.
More specifically, the test strip included a reagent zone upstream
of a sample application zone. The reagent zone included mobilizable
antibodies to MxA (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan)
labeled with colloidal gold. The test strip also included a test
line in a detection zone. The test line included an immobilized
antibody for MxA (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan). The
control line in the detection zone included rabbit anti-chicken
antibody plus rabbit Ig (for an extra stabilizing effect), which
binds to mobilized chicken IgY labeled with blue latex beads.
[0091] The whole blood samples were collected with EDTA as the
anticoagulant. In these tests, the amount of MxA protein in the
blood samples was determined using an MxA Protein ELISA Test kit
(Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan). The blood was lysed
1:10 with lysing solution provided in the kit, prior to being
applied to the test strip. 100 .mu.l of lysed blood was tested in
the ELISA test. 10 .mu.l of lysed blood was used as the sample in
the MxA lateral flow test.
[0092] The lysed blood samples were applied to the application zone
of the test strip. The labeled MxA antibodies in the reagent were
eluted by the sample transport liquid and travelled to the blood
samples. At the test line, the immobilized MxA antibody trapped any
labeled MxA antibody from the reagent zone bound to MxA. This
localization of the MxA with its labeled antibody gave rise to a
red visual indication at the test line if there was a sufficient
concentration of MxA.
TABLE-US-00001 TABLE 1 Calibrator Lateral Concentration Flow MxA
(ng/ml) OD Test 24 2.223 + 12 1.259 Shadow 6 0.700 Not tested 3
0.391 Not tested 1.5 0.220 Not tested .75 0.140 Not tested 0.38
0.102 Not tested
[0093] Table 1 shows the MxA ELISA kit standards run per the test
instructions. As shown in Table 1, an MxA concentration of 24 ng/ml
produced a positive result in the lateral flow test. The kit
standard was used to generate the standard curve from which the MxA
concentrations were determined.
[0094] Table 2 shows the results of clinical fresh whole blood
samples of patients showing symptoms of viral infections (flu like
symptoms and fever of >100.5.degree. F.)).
TABLE-US-00002 TABLE 2 Concentration .times. Lateral Concentration
dilution Flow MxA Sample OD (ng/ml) (10x) (ng/ml) Test A 0.008 0 0
- B 0.123 0.591 5.911 - C 1.125 10.489 104.894 + D 0.111 0.487
4.872 - E 0.068 0.121 1.211 - F 0.300 2.177 21.77 + G 0.027 0 0
-
[0095] The OD (optical density) values were used in combination
with the standard curve from the kit's standard in order to
determine the MxA concentration in the samples. The concentration
(ng/ml) column was the concentration as diluted with the lysing
agent. The concentration.times.dilution (10.times.) (ng/ml) column
was the actual concentration in the whole blood sample. As shown in
the table, the lateral flow test produced a positive result for MxA
in samples C and F, which had approximately 105 ng/ml of MxA and
approximately 22 ng/ml of MxA, respectively, in the samples.
[0096] Table 3 shows the results of frozen whole blood samples from
normal individuals from the Tennessee blood bank. None of the blood
samples had any discernible amounts of MxA, and all of them were
negative in the lateral flow test.
TABLE-US-00003 TABLE 3 CONCEN- CONCENTRATION .times. Lateral
TRATION DILUTION Flow MxA Sample OD (ng/ml) (10x)(ng/ml) Test 1 0.0
0 0 - 2 0.0 0 0 - 3 0.0 0 0 - 4 0.0 0 0 - 5 0.0 0 0 - 6 0.0 0 0 - 7
(0. 0 0 - 8 (0. 0 0 - 9 0.0 0 0 - 10 0.0 0 0 - 11 0.0 0 0 - 12 0.0
0 0 - 13 0.0 0 0 - 14 0.0 0 0 - 15 0.0 0 0 - 16 0.0 0 0 - 17 0.0 0
0 - 18 0.0 0 0 - 19 0.0 0 0 - 20 0.0 0 0 - 21 0.0 0 0 - 22 0.1
1.163 11.631 - 23 0.0 0 0 - 24 0.0 0 0 - 25 0.0 0 0 -
[0097] Table 4 shows freshly frozen whole blood samples from
BioReclamation (BioReclamation, Hicksville, N.Y.) of patients
showing apparent symptoms of viral infections (flu like symptoms
and fever of >100.5.degree. F.)). None of these patients had ODs
that corresponded to MxA levels higher than approximately 8 ng/ml.
These samples were all negative in the lateral flow test.
TABLE-US-00004 TABLE 4 Concentration .times. Lateral Concentration
dilution Flow MxA Sample OD (ng/ml) (10x) (ng/ml) Test 26 0.029 0 0
- 27 0.026 0 0 - 28 0.018 0 0 - 29 0.146 0.792 7.92 - 30 0.004 0 0
- 31 0.128 0.635 6.35 -
[0098] The results of these tests indicate that the lateral flow
tests described herein can detect MxA levels at least as low as
approximately 20 ng/ml in a 10 .mu.l sample (diluted 1:10).
[0099] One example of a rapid screening test for distinguishing
viral and bacterial infection is shown in FIG. 1. As discussed
above, MxA is a diagnostic marker for viral infection, while CRP is
a diagnostic marker for bacterial infection. In this example, a
blue line ("control line" in A-D of the Figure) represents the
control. A green line represents a C-reactive protein (CRP) level
>15 mg/L ("CRP test" in A-D of the figure). A red line
represents an MxA level >20 ng/ml ("MxA test" in A-D of the
figure). A positive result for the MxA protein, with a negative
result for the CRP protein indicates only a viral infection (Visual
Test Result A). A positive result for the (CRP) with a negative
result for the MxA protein indicates only a bacterial infection
(Visual Test Result B). A positive result for both MxA and CRP
indicates co-infection (infection with both a bacteria and a virus)
(Visual Test Result C). No bacterial or viral infection is
indicated by a negative result for both MxA and CRP (Visual Test
Result D). While particular color lines are discussed in this
example, other colors, or the same colors at different locations on
the test strip to indicate viral or bacterial markers, are within
the spirit of the present invention.
[0100] When development of different colored lines is utilized, the
lines may or may not be separated by space. In the latter instance,
the labels are chosen such that the color seen when both markers
are present is different from the colors seen when the individual
markers are present. For example, the presence of the viral marker
may be indicated by a red line; the presence of the bacterial
marker by a blue line; and the presence of both by a purple line
(combined red and blue).
[0101] The use of two colors to distinguish acute and chronic
infection is shown in FIG. 2. In the first cassette, only IgM
antibodies are present, which indicates an acute infection. In this
cassette, the test line is red. In the second cassette, the test
line is blue because the immunoglobulins are IgG. The third
cassette shows an intermediate case, where both IgM and IgG
antibodies are present. Consequently, the test line is purple.
While this example is shown to test for IgMs and IgGs, the same
concept is alternatively used with a single line which detects both
viral and bacterial markers for infection.
[0102] In another preferred embodiment, the test strip may also
include a control section which indicates the functionality of the
test strip. FIG. 1 shows a control line. FIG. 2 shows an example
where there is a control section for all three cassettes. If
present, the control section can be designed to convey a signal to
the user that the device has worked. For example, the control
section may contain a reagent (e.g., an antibody) that will bind to
the labeled reagents from the reagent zone. In one preferred
embodiment, rabbit anti chicken is used as the control line and
chicken IgY conjugated to a label, for example blue latex beads, is
the control conjugate. Alternatively, the control section may
contain an anhydrous reagent that, when moistened, produces a color
change or color formation, e.g. anhydrous copper sulphate which
will turn blue when moistened by an aqueous sample. As a further
alternative, the control section could contain immobilized viral
and bacterial markers which will react with excess labeled reagent
from the reagent zone. The control section may be located upstream
or downstream from the detection zone. A positive control indicator
tells the user that the sample has permeated the required distance
through the test device.
[0103] FIG. 3 compares two test strips, the "Adeno 1" and the
"Adeno HS", which both include control lines. In the Adeno 1, both
the control (upper line on each cassette) and test (lower line on
each cassette) lines are red. In the Adeno HS, the control line is
blue and the test line is red. In embodiments where the control
line is a different color than the test line, it is easier to
distinguish between the two lines, and to ensure that the test is
working.
[0104] In some preferred embodiments, the devices and methods of
the present invention include a lysis zone to help differentiate
viral and bacterial infections. In these embodiments, the sample
that has been collected is not lysed prior to collection and
transfer to the sample analysis device. This decreases the number
of steps needed to collect and prepare the sample for analysis. One
situation where a lysis agent improves assay efficiency is in
assaying for the presence of MxA. As discussed herein, the presence
of this protein can help to distinguish between bacterial and viral
infection in febrile children. In situ lysis using a combination of
1% to 6% weight/volume CHAPS and 0.5% to 2% weight/volume NP40 as
the lysis agent improves detection of MxA in fresh or frozen whole
blood.
[0105] In the embodiments utilizing a lysis agent, following sample
loading, the sample traveling with the transport liquid (buffer)
will encounter the lysis agent. The lysis agent will have
preferably been pre-loaded onto the test strip and is eluted by the
transport liquid. In some preferred embodiments the lysis agent has
been dried into the test strip. Alternatively, the lysis agent may
be pre-dried by freeze drying or lyophilizing and then pre-loaded
into the test strip. In other embodiments, the lysis agent may be
absorbed, adsorbed, embedded or trapped on the test strip. The
initially dried lysis agent is preferably localized between the
sample application zone and a reagent zone. In embodiments where
the reagent zone is upstream of the sample application zone, the
lysis zone is downstream of the sample application zone. The lysing
agent is preferably soluble in the sample transport liquid, and the
lysing agent is solubilized and activated upon contact with the
sample transport liquid. The sample transport liquid then contains
both lysing agent in solution or suspension and sample components
in suspension. Any lysis-susceptible components in a sample, then
being exposed in suspension to the lysing agent, are themselves
lysed in situ. The running buffer then carries the analyte,
including any lysis-freed components, to the detection zone.
[0106] The location where the lysis agent is pre-loaded and dried
can be varied as needed. In order to maximize the time that the
sample has to interact with the lysis agent as well as to minimize
the amount of lysis agent reaching the detection zone, the dried,
absorbed, adsorbed, embedded, or trapped lysis agent may be located
in or just downstream of the sample application zone. Or, in order
to minimize the distance along which the lysis product must travel
before reaching the reagent zone, the dried lysis agent may be
located closer to the reagent zone. In other embodiments, the lysis
agent may be included in the running buffer.
[0107] The concentration of lysis agent pre-loaded onto a test
strip is preferably between 0.001% and 5% weight/volume. The volume
to be pre-loaded depends on where the lysis agent is pre-loaded.
Appropriate ranges are 1 to 10 microliters when pre-loaded into the
sample collector fleece (the sample application zone) or 5 to 50
microliters when pre-loaded into the absorbent pad or into other
locations within the test strip. Ideally, the amount pre-loaded
should be approximately 3 microliters pre-loaded into the sample
collector fleece or approximately 10 microliters pre-loaded into
the absorbent pad or into other locations within the test
strip.
[0108] Selection of a specific lysing environment and agent will
depend on the viral and bacterial markers and the assay. The pH and
ionic strength are key to the lysing environment. As to pH
established by the lysis agent, a pH below 4.0 tends to precipitate
materials, especially proteins. Higher pH, above approximately
10.0, tends to lyse materials such as proteins and cells walls.
Therefore, a pH of approximately 10.0 or above is preferable for
many applications. Alternatively, lower pH may be preferred for
nucleic acid targets.
[0109] As to ionic strength established by the lysis agent, both
the high and low ionic strength may be used to lyse. For example, a
lower ionic strength (hypotonic) tends to break up erythrocytes.
For example, water by itself can lyse erythrocytes. Higher ionic
strength environments may be used to rupture certain cell walls and
membranes.
[0110] As to specific lysis agents, they may be grouped and
selected based on their properties: salts, amphoteric and cationic
agents, ionic and non-ionic detergents. The salt, Ammonium Chloride
(NH.sub.4Cl), lyses erythrocytes. Other salts, including, but not
limited to, high concentrations of Sodium Chloride (NaCl) and
Potassium Chloride (KCl), may rupture certain cell walls and
membranes. Other lysis agents are amphoteric agents including, but
not limited to, Lyso PC, CHAPS, and Zwittergent. Alternatively,
cationic agents including, but not limited to, C16 TAB and
Benzalkonium Chloride may be used as a lysis agent. Both ionic and
non-ionic detergents are often used to break or lyse the cell wall
or cell membrane components such as lipoproteins and glycoproteins.
Common ionic detergents include, but are not limited to, SDS,
Cholate, and Deoxycholate. Ionic detergents are good solubilizing
agents. Antibodies retain their activity in 0.1% SDS or less.
Common non-ionic detergents include, but are not limited to,
Octylglucoside, Digitonin, C12E8, Lubrol, Triton X-100, Noniodet
P-40, Tween 20, and Tween 80. Non-ionic and mild ionic detergents
are weaker denaturants and often are used to solubilize membrane
proteins such as viral surface proteins. Additional lysis agents
include, but are not limited to, urea and enzymes. Combinations of
different lysis agents may be used to optimize the lysing
environment.
[0111] Surfactants are generally wetting agents and lower the
surface tension of a liquid. This then allows easier spreading by
lowering the interfacial tension between liquids. So, surfactants
can interfere with the natural binding of antigen and antibody or
ligand and receptors. The concentrations are, therefore,
experimentally chosen for each class of lysis agent. Once lysis
occurs, it is important that the desired binding reactions not be
hindered. Generally, 0.001% lysis agent concentration is considered
the lower limit, and the upper limit is approximately 1%. There is
an additive or synergistic effect when combinations of lysis agents
are used. This expands the working range of concentration to run
from approximately 0.001% to 1%. Finally, some undesirable
non-specific binding may be prevented at a Tween 20 concentration
of 5%. In all cases, the total amount of lysis agent pre-loaded
onto all locations of an individual test strip must be sufficient
to lyse barriers to immunodetection, permitting practical operation
of the test strip.
[0112] The lysis agent itself should not interfere with any other
assay detector or indicator agents and thus does not interfere with
any other assay interactions and reactions to such an extent as to
prevent practical operation of the assay. A lysis agent should have
sufficient shelf life to allow manufacture, distribution and
storage before use of a test strip in point-of-care testing.
[0113] In preferred embodiments where MxA is the viral marker, in
situ lysis using a combination of 1% to 6% weight/volume CHAPS and
0.5% to 2% weight/volume NP40 as the lysis agent is preferably
used. As a more specific example, 2 microliters of 100 mM HEPES
buffer (pH 8.0) containing 5% CHAPS and 2% NP-40 with 150 mM Sodium
Chloride, 0.1% BSA, and 0.1% Sodium Azide (all percentages
weight/volume) are dried onto a lysis zone of a test strip.
[0114] In a preferred embodiment, as shown in FIGS. 5A through 5D,
the sample is applied to the application zone (201) on a
chromatographic test strip (200). The sample passes a lysis zone
(250), where a lysis agent will have preferably been pre-loaded
onto the test strip and is eluted by the transport liquid. The
lysis agent lyses any lysis-susceptible components in the sample in
situ.
[0115] The chromatographic test strip contains a sample application
zone (201), a lysis zone (250) containing a lysis agent, and a
reagent zone (260) containing at least one labeled binding partner
that binds to a viral marker and at least one labeled binding
partner that binds to a bacterial marker that are eluted by and
then able to migrate with a sample transport liquid (e.g. a buffer
solution). While the reagent zone (260) is shown downstream of the
sample application zone in these figures, in alternative
embodiments, the reagent zone (260) could be upstream of the sample
application zone (see FIG. 4B), as long as the reagents encounter
the sample at some point after the sample reaches the lysis zone
and is effectively lysed. The labeled binding partners are capable
of specifically binding to a viral or bacterial marker of interest
to form a complex which in turn is capable of specifically binding
to another specific reagent or binding partner in the detection
zone. Although not shown in these Figures, an absorbent pad, as
well as other known lateral flow immunoassay components including,
but not limited to, a waste zone, a carrier backing, a housing, and
an opening in the housing for result read out, may optionally also
be a component of the test strip (200) in these embodiments.
[0116] In a preferred embodiment, the lysis agent is localized in
the lysis zone (250) between the sample application zone (201) and
the reagent zone (260). The lysis agent is preferably soluble or
miscible in the sample transport liquid, and the lysis agent is
solubilized and activated upon contact with the sample transport
liquid. The sample transport liquid then contains both lysis agent
in solution or suspension and sample components in suspension. Any
lysis-susceptible components in a sample, then being exposed in
suspension to the lysis agent, are themselves lysed in situ. The
running buffer then carries the sample, including any lysis-freed
components, to the detection zone (205).
[0117] The lysis zone (250) is preferably located between the
sample application zone (201) and the reagent zone (260), as shown
in FIG. 5A. In other embodiments, the lysis zone (250) overlaps the
sample application zone (201), the reagent zone (260) or both the
sample application zone (201) and the reagent zone (260) as shown
in FIGS. 5B, 5C, and 5D, respectively. Note that the figures are
schematic, and are not drawn to scale. The amount of overlap
between the different zones (as shown in FIGS. 5B through 5D) may
be highly variable.
[0118] The test strip (200) also includes a detection zone (205)
containing a first section for detection of at least one bacterial
marker, e.g. a test line (203), including an immobilized specific
binding partner, complementary to the bacterial conjugate formed by
the bacterial marker and its labeled binding partner. Thus, at the
test line (203), detection zone binding partners trap the bacterial
labeled binding partners from the reagent zone (260) along with
their bound bacterial markers. This localization of the bacterial
markers with their labeled binding partners gives rise to an
indication at the test line (203). At the test line (203), the
presence of a bacterial marker is determined by qualitative and/or
quantitative readout of the test line (203) indication resulting
from the accumulation of labeled binding partners.
[0119] The detection zone (205) also includes a second section for
detection of at least one viral marker, e.g. a test line (202),
including an immobilized specific binding partner, complementary to
the viral conjugate formed by the viral marker and its labeled
binding partner. Thus, at the test line (202), detection zone
binding partners trap the viral labeled binding partners from the
reagent zone (260) along with their bound viral markers. This
localization of the viral markers with their labeled binding
partners gives rise to an indication at the test line (202). At the
test line (202), the presence of a viral marker is determined by
qualitative and/or quantitative readout of the test line (202)
indication resulting from the accumulation of labeled binding
partners. While test line (203) is upstream of test line (202)
relative to the direction of flow (208) in the figures, in
alternative embodiments, test line (202) is upstream of test line
(203). In still other embodiments, test lines (202) and (203) are
located in the same location on the test strip.
[0120] Optionally, the detection zone (205) may contain further
test lines to detect other bacterial and/or viral markers, as well
as a control line (204). The control line (204) indicates that the
labeled specific binding partner traveled through the length of the
assay, even though it may not have bound any markers, thus
confirming proper operation of the assay. As shown in FIGS. 5A
through 5D, the control zone (204) is preferably downstream of the
test lines (203) and (202). However, in other embodiments, the
control zone (204) may be located upstream of either or both of the
test lines (203) and (202).
[0121] In a preferred embodiment, the control line (204) includes
an antibody or other recombinant protein which binds to a component
of the elution medium or other composition being used in the test.
In embodiments where nucleic acids are the targets, the control
line (204) preferably includes a nucleic acid complementary to the
labeled nucleic acid being used as a binding partner for the target
nucleic acid.
[0122] Although only one test line is shown in the figures,
multiple test lines are within the spirit of the invention. In some
embodiments where there are multiple targets, the presence of each
target preferably corresponds to a separate test line (202). In
other embodiments where there are multiple targets, the presence of
multiple targets may be indicated on the same test line such that
the presence of more than one target has different characteristics
than the presence of a single target. For example, the presence of
multiple targets on the same test line may be visually indicated by
a different color than the presence of each of the targets
alone.
[0123] In other embodiments, it is possible to have one or more
mild lysis agents in the running buffer itself. In these
embodiments, there is no adverse effect on the reagent zone which
will be downstream and the sample can either be upstream or
downstream of the reagent zone. A lysing enzyme in the running
buffer can "target" its substrate and cut it to open up the cell
membrane or cell wall. As an example, penicillin can excise or
"punch a hole" in a susceptible bacteria. In other embodiments,
when the lysis agent is applied to the sample collection material,
then the reagent zone may be upstream of the sample application
zone.
[0124] As an example, one or more lysis agents are dried onto the
sample application zone of a lateral flow strip. On a per strip
basis, the lysis agent is made of approximately 2 microliters of
100 mM HEPES buffer (pH 8.0) containing 5% CHAPS and 2% NP-40 with
150 mM Sodium Chloride, 0.1% BSA, and 0.1% Sodium Azide (all
percentages weight/volume). Up to 10 microliters of whole blood are
then added to the sample application zone to be lysed in situ. MxA
protein is released from inside white blood cells to react with an
MxA monoclonal antibody on a visual tag (colloidal gold or visible
latex beads). This complex traverses with a running buffer
containing Triton X-100 and is captured by MxA monoclonal
antibodies immobilized at the test line of the nitrocellulose
membrane. This binding at the test line gives rise to a visible
indication.
Sample Analysis Device with Bimodal Dual Test Strips
[0125] MxA is a derivative of interferon alpha/beta cells that
becomes elevated in the presence of viral infections but is not
specific for a particular type of virus. MxA protein expression in
peripheral blood is a sensitive and specific marker for viral
infection.
[0126] MxA inhibits the replication of a wide range of viruses. MxA
has a low basal concentration [less than 50 ng/ml] and a fast
induction [1-2 hours]. It peaks at 16 hours and remains elevated in
the presence of elevated interferon. MxA also has a long half-life
[2.3 days] and constant titres in presence of interferonemia. Viral
infections elevate MxA levels while only having a modest increase
in CRP levels.
[0127] In one prospective clinical trial using ELISA (Towbin H et
al. J Interferon Res 1992; 12:67-74, herein incorporated by
reference), the trial enrolled 87 normal healthy adults. The MxA
levels were measured to be <5 ng/ml in 66% of the adults,
between 5-50 ng/ml in 29% of the adults, and above 50 ng/ml in 5%
of the adults.
[0128] Another prospective clinical trial using ELISA (Chieux V et
al., J Virol Methods 1998; 70:183-191, herein incorporated by
reference) enrolled 174 children. 45 of these children had acute
fever (respiratory infection and/or gastroenteritis) and there were
30 age-matched controls. The MxA values were 7 ng/ml .+-.7 ng/ml in
the 30 age-matched controls. The MxA values were 10 ng/ml.+-.6
ng/ml in 13 confirmed bacterial infections.
[0129] Another prospective clinical trial using ELISA enrolled 60
patients (Kawamura M et al. J Clin Lab Anal 2012; 26:174-183,
herein incorporated by reference). 42 of the patients had acute
fever (respiratory infection and/or gastroenteritis) and there were
18 age matched controls. The median MxA value was 110.0 ng/ml in 31
confirmed viral infections. The median MxA value was 10.6 ng/ml in
11 confirmed bacterial infections. The median MxA value was 2.0
ng/ml in the 18 age matched controls. The ELISA test had a
sensitivity of 87.1% and a specificity of 90.9% for differentiating
viral from bacterial infection, but only tested MxA values to make
this determination. Patients with viral infection were sharply
distinguished from the healthy controls with 100% sensitivity and
specificity. A cut-off of 36.7 ng/ml MxA was used to determine
viral infection by ELISA in this study.
[0130] Another prospective clinical trial using ELISA enrolled 174
children (Nakabayashi M et al., Pediatr Res 2006; 60:770-774,
herein incorporated by reference, data corrected for recalibrated
ELISA standard). 122 of the children had acute fever (respiratory
infection and/or gastroenteritis) and there were 52 age-matched
controls. The mean MxA value was 123.7 ng/ml.+-.83.0 ng/ml in 95
confirmed viral infections. The mean MxA value was 12.3
ng/ml.+-.10.0 ng/ml in 27 confirmed bacterial infections. The mean
MxA value was 14.5 ng/ml.+-.11.0 ng/ml in the 52 age matched
controls. The test showed a 92.6% specificity and a positive
likelihood ratio of 13.1 for accurately identifying viral
infection. A cut-off of 36.7 ng/ml MxA was used to determine viral
infection by ELISA in this study.
[0131] The cut-off in the ELISA test is artificial and is picked to
discriminate between positive and negative. Therefore, it is
preferable to routinely assign10% CV from this cut-off. In a point
of care test, 100% of the people can visibly see the test line at
>40 ng/ml, but some people can see a positive result at lower
levels.
[0132] CRP becomes elevated in the presence of bacterial infections
but is not specific for a particular type of bacteria. CRP is a
nonspecific indicator for the presence of acute inflammation and is
elevated in the presence of bacterial infections. CRP is an
acute-phase protein synthesized by the liver. IL-6 is the primary
mediator of CRP production. Bacterial infection is a potent
stimulus of marked CRP elevation. Following antibiotic treatment,
CRP levels fall rapidly. Bacterial infections dramatically elevate
CRP levels while MxA levels remain low. Bacterial infection is a
potent stimulus of CRP with marked elevation in serum CRP levels
occurring within a few hours. CRP levels elevate within 4-6 hours
after stimulation and peak after 36 hours. The serum concentration
of CRP is normally less than 3 mg/L. With severe infection or
inflammation, CRP can rise above 500 mg/L.
[0133] Pneumonia has elevated serum CRP levels (>10 mg/L). The
serum CRP levels are typically greater than 100 mg/L for severe
pneumonia. 32% of patients with pneumococcal bacteremia had serum
CRP less than 60 mg/L. Serum CRP is not usually elevated above 10
mg/L in viral infection. Invasive Adenovirus and Influenza can
raise CRP to 10-80 mg/L. Very infrequently, the CRP levels exceed
60 mg/L in viral infections.
[0134] Meta analysis of ten studies (Aouifi et al., Crit care Med.
2000, 28:3171-6; Hatherill et al., Arch Dis Child 1999: 81: 417-21;
Muller et al., Crit Care Med. 2000, 28: 977-83; Penel et al., Rev
Med Interne 2001:22: 706-714; Rothenberger et al., Clin Chem Lab
Med, 1999, 37: 275-9; Schwarz et al., Crit Care Med 2000, 28:
1828-32; Selberg et al., Crit Care Med 2000, 28: 2793-8; Suprin et
al., Intensive Care Med 2000, 26: 1232-8; Ugarte et al., Crit Care
Med 1999, 27: 498-504; Viallon et al., Intensive Care Med 2000, 26:
1082-8, all herein incorporated by reference) that looked at a
single value for serum CRP to be used as a cut-off for bacterial
disease resulted in a bimodal outcome. Three of the studies (Aouifi
et al., Crit care Med. 2000, 28:3171-6; Penel et al., Rev Med
Interne 2001:22: 706-714; Schwarz et al., Crit Care Med 2000, 28:
1828-32) recommended that the CRP cut-off value be set at 6-15
mg/L, while the other seven studies (Hatherill et al., Arch Dis
Child 1999: 81: 417-21; Muller et al., Crit Care Med. 2000, 28:
977-83; Rothenberger et al., Clin Chem Lab Med, 1999, 37: 275-9;
Selberg et al., Crit Care Med 2000, 28: 2793-8; Suprin et al.,
Intensive Care Med 2000, 26: 1232-8; Ugarte et al., Crit Care Med
1999, 27: 498-504; Viallon et al., Intensive Care Med 2000, 26:
1082-8) recommended a cut-off of 60-100 mg/L.
[0135] In isolation, neither MxA nor CRP alone is sensitive or
specific at identifying both viral and bacterial infection. Low
cut-off values of CRP show high sensitivity and low specificity for
detecting bacterial infection. High cut-off values of CRP show low
sensitivity and high specificity for detecting bacterial infection.
MxA is specific to identify viral infection, but it is not
sensitive for bacterial infection. A multiplexed pattern of results
including medical decision points reflected cut-off levels of low
CRP, high CRP, and MxA together provide a sensitive and specific
way to identify an immune response to a viral and/or bacterial
infection.
[0136] In one preferred embodiment of a multiplexed lateral flow
immunoassay, the fingerstick blood pattern of test results shows a
positive result with a serum equivalence to a low CRP level cut-off
of approximately 10 mg/L, a serum equivalence to a high CRP level
cut-off of approximately 80 mg/L, and a MxA cut-off of
approximately 40 ng/ml. These preferred values are shown in Table
5.
TABLE-US-00005 TABLE 5 Biomarker Location Fingerstick Cut-off value
MxA Intracellular 40 ng/ml (Peripheral Blood Mononuclear Cells)
CRP-low Extracellular 7 mg/L (Serum) CRP-high Extracellular 80 mg/L
(Serum)
[0137] The specificity of the test is further enhanced by
restricting the intended use. For example, in preferred
embodiments, only certain ages of the patient population are tested
(preferably one year of age or older) and/or patients with specific
underlying conditions that may lead to confounding factors are
preferably not given this test.
[0138] A rapid, point-of-care MxA immunoassay was developed and
compared to the MxA ELISA in 25 peripheral blood samples from
patients with a febrile respiratory illness, as shown in Table 6.
Table 7 sorts the same data from lowest to highest amounts of MxA
in the ELISA test.
[0139] The MxA ELISA cut-off value was 36.7 ng/ml (+/-10%CV=33
ng/ml to 40/5 ng/ml). Patient 19 had a positive result in the MxA
rapid point of care test, even though the ELISA results were much
less than 40 ng/ml (15 ng/ml). However, the MxA immunoassay
demonstrated 100% (9/9) sensitivity and 94% (15/16)
specificity.
TABLE-US-00006 TABLE 6 Number MxA rapid test (40 ng/ml) MxA EIA 1
Negative 32.9 2 Negative 5.9 3 Negative 18.8 4 Negative 5.9 5
Negative 19.2 6 Negative 0.0 7 Negative 6.0 8 Negative 4.0 9
Negative 7.0 10 Positive 42.0 11 Negative 21.3 12 Positive 49.0 13
Positive 58.1 14 Negative 7.3 15 Negative 3.8 16 Negative 15.0 17
Negative 0.0 18 Positive 47.2 19 Positive 15.9 20 Positive 89.5 21
Positive 67.4 22 Negative 19.4 23 Positive 36.0 24 Positive 57.0 25
Positive 47.6
TABLE-US-00007 TABLE 7 Number MxA rapid test (40 ng/ml) MxA EIA
15/16 Negative 0.0 Negative 0.0 Negative 3.8 Negative 4.0 Negative
5.9 Negative 5.9 Negative 6.0 Negative 7.0 Negative 7.3 Positive
15.0 Negative 15.9 Negative 18.8 Negative 19.2 Negative 19.4
Negative 21.3 Negative 32.9 9/9 Positive 36.0 Positive 42.0
Positive 47.2 Positive 47.6 Positive 49.0 Positive 57.0 Positive
58.1 Positive 67.4 Positive 89.5
[0140] Rapid, point-of-care low-CRP level and high-CRP level
immunoassays were developed and compared to the CRP ELISA in 25
peripheral blood samples from patients with a febrile respiratory
illness. These patients are the same patients that were tested for
MxA in Tables 6 and 7. The results are shown in Table 8.
TABLE-US-00008 TABLE 8 High CRP Low CRP Rapid Test Rapid Test
Number (80 mg/L) (10 mg/L) CRP EIA 1 Negative Positive 10.8 2
Negative Positive 45.1 3 Negative Positive 56.0 4 Negative Positive
37.6 5 Negative Positive 27.8 6 Positive Positive 67.0 7 Negative
Positive 16.2 8 Positive Positive 59.0 9 Negative Negative 40.7 10
Negative Positive 43.0 11 Negative Positive 13.3 12 Negative
Positive 6.2 13 Negative Positive 16.1 14 Negative Positive 36.2 15
Negative Positive 28.4 16 Negative Positive 11.4 17 Positive
Positive 110.0 18 Negative Positive 43.7 19 Negative Positive 15.3
20 Positive Positive 110.0 21 Negative Positive 44.0 22 Negative
Positive 20.0 23 Negative Positive 80.0 24 Negative Positive 28.0
25 Negative Positive 32.0
[0141] Patient number 6 and 8 showed a positive CRP result even
though the ELISA results were below 80 mg/L. Patient 23 showed a
negative result even though the ELISA results were exactly 80 mg/L.
Patient number 9 showed a negative low CRP test result even though
the ELISA results for that patient were above 10 mg/L. But,
overall, the CRP values correlated well with the CRP ELISA at both
cut-off values.
[0142] Using an MxA and CRP ELISA, RPS analyzed 25 healthy, normal
blood bank samples for the presence or absence of elevated MxA and
CRP. The average CRP concentration in plasma was shown to be 1.6
mg/L. CRP levels were shown to range from 0.1 to 3.7 mg/L. The
results are shown in Table 9.
TABLE-US-00009 TABLE 9 MxA ELISA CRP ELISA Concentration
Concentration Sample (ng/ml) (mg/L) MxA Rapid Test 1 0 1.8 - 2 0
1.5 - 3 0 1.0 - 4 0 1.8 - 5 0 3.0 - 6 0 0.8 - 7 0 2.9 - 8 0 0.8 - 9
0 1.5 - 10 0 0.2 - 11 0 1.5 - 12 0 1.2 - 13 0 3.7 - 14 0 0.1 - 15 0
0.4 - 16 0 2.3 - 17 0 2.4 - 18 0 3.1 - 19 0 0.9 - 20 0 0.5 - 21 0
2.3 - 22 11.631 1.8 - 23 0 1.7 - 24 0 3.2 - 25 0 0.5 -
[0143] The bimodal dual test strips can be used to differentiate
bacterial and viral infection in humans, but also may be used in
veterinary applications for animals. Since CRP differs depending
upon the species, there are not common antibodies to CRP between
species. Therefore, the veterinary tests need to include CRP
specific to the particular species being tested. MxA is well
conserved among species, so it is possible to use human MxA in
veterinary tests. However, MxA to a particular species could
alternatively be used to try to further increase specificity.
Veterinary tests using the bimodal dual test strips described
herein may be developed for a specific species, including, but not
limited to, cats, dogs, rabbits, pigs, sheep, horses, cows,
monkeys, chimpanzees, baboons, and orangutans.
[0144] The strip with MxA and low CRP could be made with any
configuration, for example the configurations shown in FIGS. 4A and
4B, or FIGS. 5A through 5D, where MxA is the viral marker being
detected and relatively low levels of CRP is the bacterial marker
being detected. In other embodiments, the MxA test line and the CRP
test line could overlap, or be in the same location on the test
strip. In these embodiments, the presence of low CRP and MxA on the
same test line has different characteristics than the presence of
either a bacterial or viral marker alone. For example, the presence
of both low CRP and MxA on the same test line may be visually
indicated by a different color than the presence of either MxA or
low CRP alone. In these embodiments, a positive result for MxA
would give a different color or indication than a positive result
for low CRP, so that the person reading the assay could distinguish
between a completely negative result, a positive result for MxA, a
positive result for low CRP, and a positive result for both MxA and
low CRP. For example, a positive result for MxA could result in a
red test line, and a positive result for low CRP could result in a
blue test line. So, when a sample is positive for both MxA and low
CRP, the line is visibly purple.
[0145] Some embodiments for lateral flow assay devices to detect
high levels of CRP are shown in FIGS. 6A-6B and 7A-7D. These
configurations are similar to the configurations shown in FIGS.
4A-4B and 5A-5D, without a test line for a viral marker, and the
same reference numerals are used for the same components of the
strip (600), (700).
[0146] FIGS. 6A and 6B show a chromatographic test strip (600) with
a test line (623) that detects the presence of a bacterial marker,
such as high levels of CRP. The sample is applied to the
application zone (401) of the chromatographic test strip (600). As
shown in FIG. 6A, the sample then passes a reagent zone (660)
containing at least one labeled bacterial binding partner that is
eluted by and then able to migrate with a sample transport liquid
(e.g. a buffer solution). Alternatively, as shown in FIG. 6B, the
reagent zone (660) is located upstream of the sample application
zone (401) such that the labeled binding partners in the reagent
zone are eluted by the sample transport liquid and travel to the
sample. The labeled bacterial binding partner is capable of
specifically binding to a bacterial marker of interest, for example
high levels of CRP, to form a complex which in turn is capable of
specifically binding to another specific reagent or binding partner
in the detection zone. Although not shown in these Figures, an
absorbent pad, as well as other known lateral flow immunoassay
components including, but not limited to, a waste zone, a carrier
backing, a housing, and an opening in the housing for result read
out, may optionally also be a component of the test strip (600) in
these embodiments.
[0147] The test strip (600) also includes a detection zone (605)
containing a section for detection of a bacterial marker, e.g. a
test line (623), including an immobilized specific binding partner,
complementary to the bacterial reagent complex formed by the
bacterial marker and its labeled binding partner. Thus, at the test
line (623), detection zone binding partners trap the labeled
bacterial binding partners from the reagent zone (660) along with
their bound bacterial markers. This localization of the bacterial
marker with its labeled binding partners gives rise to an
indication at the test line (623). At the test line (623), the
presence of the bacterial marker is determined by qualitative
and/or quantitative readout of the test line (623) indication
resulting from the accumulation of labeled binding partners.
[0148] Optionally, the detection zone (605) may contain further
test lines to detect other bacterial and/or viral markers, as well
as a control line (404). The control line (404) indicates that the
labeled specific binding partner traveled through the length of the
assay, even though it may not have bound any bacterial markers,
thus confirming proper operation of the assay. As shown in FIGS. 6A
through 6B, the control zone (404) is preferably downstream of the
test line (623). However, in other embodiments, the control zone
(404) may be located upstream of the test line (623).
[0149] In a preferred embodiment, the control line (404) includes
an antibody or other recombinant protein which binds to a component
of the elution medium or other composition being used in the test.
In embodiments where nucleic acids are the targets, the control
line (404) preferably includes a nucleic acid complementary to the
labeled nucleic acid being used as a binding partner for the target
nucleic acid.
[0150] In other preferred embodiments to test for a bacterial
marker, such as high CRP levels, as shown in FIGS. 7A through 7D,
the sample passes a lysis zone (250), where a lysis agent will have
preferably been pre-loaded onto the test strip and is eluted by the
transport liquid. The lysis agent lyses any lysis-susceptible
components in the sample in situ.
[0151] The chromatographic test strip (700) contains a sample
application zone (201), a lysis zone (250) containing a lysis
agent, and a reagent zone (760) containing at least one labeled
binding partner that binds to a bacterial marker, for example high
levels of CRP, that is eluted by and then able to migrate with a
sample transport liquid (e.g. a buffer solution). While the reagent
zone (760) is shown downstream of the sample application zone in
these figures, in alternative embodiments, the reagent zone (760)
could be upstream of the sample application zone (see FIG. 6B), as
long as the reagents encounter the sample at some point after the
sample reaches the lysis zone and is effectively lysed. The labeled
binding partner is capable of specifically binding to a bacterial
marker of interest, for example high levels of CRP, to form a
complex which in turn is capable of specifically binding to another
specific reagent or binding partner in the detection zone. Although
not shown in these Figures, an absorbent pad, as well as other
known lateral flow immunoassay components including, but not
limited to, a waste zone, a carrier backing, a housing, and an
opening in the housing for result read out, may optionally also be
a component of the test strip (700) in these embodiments.
[0152] In a preferred embodiment, the lysis agent is localized in
the lysis zone (250) between the sample application zone (201) and
the reagent zone (760). The lysis agent is preferably soluble or
miscible in the sample transport liquid, and the lysis agent is
solubilized and activated upon contact with the sample transport
liquid. The sample transport liquid then contains both lysis agent
in solution or suspension and sample components in suspension. Any
lysis-susceptible components in a sample, then being exposed in
suspension to the lysis agent, are themselves lysed in situ. The
running buffer then carries the sample, including any lysis-freed
components, to the detection zone (705).
[0153] The lysis zone (250) is preferably located between the
sample application zone (201) and the reagent zone (760), as shown
in FIG. 7A. In other embodiments, the lysis zone (250) overlaps the
sample application zone (201), the reagent zone (760) or both the
sample application zone (201) and the reagent zone (260) as shown
in FIGS. 7B, 7C, and 7D, respectively. Note that the figures are
schematic, and are not drawn to scale. The amount of overlap
between the different zones (as shown in FIGS. 7B through 7D) may
be highly variable.
[0154] The test strip (700) also includes a detection zone (705)
containing a section for detection of at least one bacterial
marker, e.g. a test line (723), including an immobilized specific
binding partner, for example, a specific binding partner for a high
level of CRP, complementary to the bacterial conjugate formed by
the bacterial marker and its labeled binding partner. Thus, at the
test line (723), detection zone binding partners trap the bacterial
labeled binding partners from the reagent zone (760) along with
their bound bacterial markers. This localization of the bacterial
markers with their labeled binding partners gives rise to an
indication at the test line (723). At the test line (723), the
presence of a bacterial marker is determined by qualitative and/or
quantitative readout of the test line (723) indication resulting
from the accumulation of labeled binding partners.
[0155] Optionally, the detection zone (705) may contain further
test lines to detect other bacterial and/or viral markers, as well
as a control line (204). The control line (204) indicates that the
labeled specific binding partner traveled through the length of the
assay, even though it may not have bound any markers, thus
confirming proper operation of the assay. As shown in FIGS. 7A
through 7D, the control zone (204) is preferably downstream of the
test line (723). However, in other embodiments, the control zone
(204) may be located upstream of the test line (723).
[0156] In a preferred embodiment, the control line (204) includes
an antibody or other recombinant protein which binds to a component
of the elution medium or other composition being used in the test.
In embodiments where nucleic acids are the targets, the control
line (204) preferably includes a nucleic acid complementary to the
labeled nucleic acid being used as a binding partner for the target
nucleic acid.
[0157] One preferred configuration for a bimodal dual test strip
sample analysis device is shown in FIGS. 8A through 8C. The sample
analysis device or test card (800) includes a closable housing
(835) with two sides (836), (837) and a spine or hinged portion
(831). In one preferred embodiment, the test card (800) is
approximately 11.5 cm long (L).times.7 cm wide (W) when the two
sides (836), (837) are closed. However, any size test card (800)
that accommodates all of the components may be used. Within the
first side (836) of the housing (835), there are two test strips
(815), (825), each including a receiving pad (845), a diverting
zone (850), a transfer pad (855) and a detection zone (805). The
first side (836) also includes an absorbent pad (840) and
preferably a waste pad (860). The first test strip (815) preferably
includes a detection zone (805) with an MxA test line (802), a low
CRP test line (803) and a control line (804). The second test strip
(825) preferably includes a detection zone (805) with a high CRP
test line (823) and a control line (804). All of the test lines are
visible through the windows (865) on the second side (837) of the
housing (835) when the housing (835) is closed. The absorbent pad
(840) is preferably a single pad that the running buffer is added
to to start lateral flow. Similarly, the waste pad (860) is
preferably a single pad that collects excess running buffer at the
end of the test. However, in other embodiments, each strip could
have a separate absorbent pad (840) and/or waste pad (860).
[0158] The second side (837) of the housing (835) includes three
separate sections (838), (839) and (870). The middle portion, a
sample compressor or flap (870), preferably includes two conjugate
zones (872), (874), each including a labeled binding partner for at
least one analyte, and a labeled control. A window (843) is located
in the lower portion (838) of the second side (837) of the housing
so that the buffer can be added to the absorbent pad (840) when the
housing (835) is closed. The viewing windows (865) for the
detection zones (805) are on the upper portion (839) of the second
side (837) of the housing (835).
[0159] The upper portion (839) and the lower portion (838) of the
second side (837) of the housing (835) also preferably each include
at least one knob, peg or protrusion (875) that mates with one or
more holes (895) so that the upper and lower portions (838), (839)
may be easily fastened onto the first side (836) of the housing
(835). In a preferred embodiment, there are two pegs (875) on the
lower portion (838) that mate with two holes (895) flanking the
absorbent pad (840) on the first side (836) of the housing (835)
and two pegs (875) on the upper portion (839) that mate with two
holes (895) flanking the waste pad (860) on the first side (836) of
the housing (835). In other embodiments, the holes (895) are on the
second side (837) of the housing (835) and the pegs (875) are on
the first side (836) of the housing (835). In yet other
embodiments, other reversible fastening mechanisms could be used to
secure the upper portion (838) and/or lower portion (839) of the
second side (837) of the housing (835) to the first side (836) of
the housing (835). In other embodiments, the upper and lower
sections (838), (839) are permanently closed, for example using an
adhesive, before use.
[0160] The flap (870), also known as a sample compressor, on the
second side (837) of the housing includes two conjugate zones
(872), (874) and two sample application zones (873), (876), and can
be easily opened and closed. The flap (870) also preferably
includes at least one knob, peg or protrusion (875) that mates with
one or more holes (895) so that the flap (870) is easily correctly
closed onto the first side (836) of the housing (835) after sample
has been added to the sample application zones (873), (876). In
other embodiments, the holes (895) are on the second side (837) of
the housing (835) and the pegs (875) are on the first side (836) of
the housing (835). In yet other embodiments, other reversible
fastening mechanisms could be used to secure the flap (870) to the
first side (836) of the housing (835).
[0161] The conjugate zones (872), (874) and the sample application
zones (873), (876) preferably overlap. In preferred embodiments,
the conjugate zones (872), (874) are colored due to the dyes in the
sample conjugates and control conjugates, and the sample is placed
directly on the colored portion of the flap (870). In one preferred
embodiment, the conjugate zone (872) that is used for the first
test strip (815) contains an MxA binding partner that is labeled
with a red dye, a low CRP binding partner that is labeled with a
black dye, and a control binding partner that is labeled with a
blue dye. In this embodiment, the conjugate zone (872) appears
purplish. The other conjugate zone (874) contains a high CRP
binding partner that is labeled with a black dye and a control
binding partner that is labeled with a blue dye. In this
embodiment, the conjugate zone (874) appears bluish.
[0162] The diverting zone (850) preferably includes a gap or
barrier that interrupts lateral flow, diverting the running buffer
up into the flap (870) that includes the conjugate zones (872),
(874) and the sample application zones (873), (876).
[0163] In operation, the upper and lower portions (838), (839) of
the second side (837) of the housing (835) are preferably snapped
closed before use by securing the pegs (875) to the holes (895).
The sample analysis device, or test card (800) is preferably placed
on a flat surface. If the flap (870) is not already open, the user
opens it to access the sample application zones (873), (876). A
blood sample to be tested is taken from the patient. The sample may
be taken by any procedure known in the art. In a preferred
embodiment, a sample of 5 .mu.l of blood is added to each of the
sample application zones (873), (876) and then the flap (870) is
closed. Each of the 5 .mu.l samples is preferably collected
independently of each other. The blood samples are preferably added
directly to the device (800), without any pretreatment.
[0164] To ensure that the sample compressor or flap (870) has been
closed correctly, pressure is preferably applied to the housing
(835) above the pegs (875) to snap the pegs (875) closed. The top
of the flap (870) needs to be flush with the top of the rest of the
second side (837) of the housing (835) for the test to run
properly. Running buffer is added to the absorbent pad (840), which
initiates lateral flow (885). In preferred embodiments, the running
buffer includes one or more lysis agents, for example detergents,
to lyse the blood sample and expose the intracellular MxA in the
sample. When the running buffer reaches the diverting zone (850),
it is diverted up into the flap (870). It travels through the
conjugate zones (872), (874), collecting any complexes formed
between the MxA binding partner and MxA in the sample, the low CRP
binding partner and low levels of CRP in the sample, the high CRP
binding partner and high levels of CRP in the sample, as well as
the control conjugate.
[0165] Since the conjugate zones (872), (874) bridge the diverting
zone (850) on the lateral flow test strips (815), (825), the
running buffer, which now contains sample, conjugate, and the
complexes described above, then travels into the transfer pad
(855), and to the detection zones (805) on each of the test strips
(815), (825). If MxA is present in the sample, the MxA test line
(802) on the first test strip (815) will be red. If a threshold low
level of CRP is present in the sample, the low CRP test line (803)
on the first test strip (815) will be black. If a threshold high
level of CRP is present in the sample, the high CRP test line (823)
on the second test strip (825) will be black. If the test is run
correctly, the control lines (804) on both the first strip (815)
and the second test strip (825) will be blue. In preferred
embodiments, the levels of detection are 40 ng/ml for MxA, 10 mg/L
for low CRP on the first test strip (815) and 80 mg/L for high CRP
on the second test strip (825). The results of the test should be
visible after approximately 5-20 minutes, preferably within about
10 minutes.
[0166] Since the control binding partner is on the sample
compressor or flap (870) and not on either of the test strips
(815), (825), there is a true procedural control to this
configuration. If the flap (870) is not closed properly, nothing
will show up in the detection zone (805), indicating that the test
was run improperly.
[0167] FIGS. 9A through 9F show test results using the device (800)
shown in FIGS. 8A through 8C, with two test strips (815), (825)
side by side, where a first test strip (815) tests for the presence
of both MxA and low levels of CRP and the second test strip (825)
tests for high levels of CRP.
[0168] FIG. 9A shows a negative result at the MxA test line (802)
and a negative result at the low CRP test line (803) on the first
test strip (815), as well as a negative result at the high CRP test
line (823) on the second test strip (825). More specifically, the
only visible lines in the detection zone (805) of the lateral flow
assay (800) are the two blue control lines (804). This result
indicates that the sample is negative for both viral and bacterial
infection.
[0169] FIGS. 9B and 9C are positive for viral infection. In FIG.
9B, the presence of two blue control lines (804) and a red MxA line
(802) indicate a viral infection. In FIG. 8C, the presence of two
blue control lines (804) and a red MxA line (802) indicate a viral
infection. Since there is also a black low CRP line (803) in FIG.
9C, there is a possibility of bacterial co-infection, although
there is an absence of a high CRP line (823).
[0170] FIGS. 9D and 9E are positive for bacterial infection. In
FIG. 9D, the presence of two blue control lines (804) and a black
low CRP line (803) indicates a bacterial infection. In FIG. 9E, the
presence of two blue control lines (804), a black low CRP line
(803), and a black high CRP line (823) also indicates a bacterial
infection. The MxA line is absent in both FIGS. 9D and 9E,
indicating an absence of a viral infection.
[0171] FIG. 9F indicates co-infection (both bacterial and viral
infection). The presence of two blue control lines (804), a red MxA
line (802), a black low CRP line (803), and a black high CRP line
(823) indicates the presence of both viral and bacterial
infection.
[0172] Another preferred configuration for a bimodal dual test
strip sample analysis device (1000) is shown in FIGS. 10A through
10C. This configuration is similar to the configuration (800) shown
in FIGS. 8A through 8C, but the sample application zones (1073),
(1076) are located on each of the test strips (1015), (1025),
downstream of the diverting zone (850). The sample analysis device
or test card (1000) includes a closable housing (835) with two
sides (836), (837) and a spine or hinged portion (831). In one
preferred embodiment, the test card (1000) is approximately 11.5 cm
long (L).times.7 cm wide (W) when the two sides (836), (837) are
closed. However, any size test card (1000) that accommodates all of
the components may be used. Within the first side (836) of the
housing (835), there are two test strips (1015), (1025), each
including a receiving pad (845), a diverting zone (850), a transfer
pad (1055) and a detection zone (805). The first side (836) also
includes an absorbent pad (840) and preferably a waste pad (860).
The first test strip (1015) preferably includes a detection zone
(805) with an MxA test line (802), a low CRP test line (803) and a
control line (804). The second test strip (1025) preferably
includes a detection zone (805) with a high CRP test line (823) and
a control line (804). All of the test lines are visible through the
windows (865) on the second side (837) of the housing (835) when
the housing (835) is closed. The absorbent pad (840) is preferably
a single pad to which the running buffer is added to start lateral
flow. Similarly, the waste pad (860) is preferably a single pad
that collects excess running buffer at the end of the test.
However, in other embodiments, each strip could have a separate
absorbent pad (840) and/or waste pad (860).
[0173] The second side (837) of the housing (835) includes three
separate sections (838), (839) and (1070). The middle portion, or
flap (1070), also known as a sample compressor, preferably includes
two conjugate zones (872), (874), each including a labeled binding
partner for at least one analyte, and a labeled control. A window
(843) is located in the lower portion (838) of the second side
(837) of the housing so that the buffer can be added when the
housing (835) is closed. The viewing windows (865) for the
detection zones (805) are on the upper portion (839) of the second
side (837) of the housing (835).
[0174] The upper portion (839) and the lower portion (838) of the
second side (837) of the housing (835) also preferably each include
at least one knob, peg or protrusion (875) that mates with one or
more holes (895) so that the upper and lower portions (838), (839)
may be easily fastened onto the first side (836) of the housing
(835). In a preferred embodiment, there are two pegs (875) on the
lower portion (838) that mate with two holes (895) flanking the
absorbent pad (840) on the first side (836) of the housing (835)
and two pegs (875) on the upper portion (839) that mate with two
holes (895) flanking the waste pad (860) on the first side (836) of
the housing (835). In other embodiments, the holes (895) are on the
second side (837) of the housing (835) and the pegs (875) are on
the first side (836) of the housing (835). In yet other
embodiments, other reversible fastening mechanisms could be used to
secure the upper portion (838) and/or lower portion (839) of the
second side (837) of the housing (835) to the first side (836) of
the housing (835). In other embodiments, the upper and lower
sections (838), (839) are permanently closed, for example using an
adhesive, before use.
[0175] The flap (1070) on the second side (837) of the housing
includes two conjugate zones (872), (874) and can be easily opened
and closed. The flap (1070) also preferably includes at least one
knob, peg or protrusion (875) that mates with one or more holes
(895) so that the flap (1070) is easily correctly closed onto the
first side (836) of the housing (835) after sample has been added
to the sample application zones (1073), (1076) on the test strips
(1015), (1025). In other embodiments, the holes (895) are on the
second side (837) of the housing (835) and the pegs (875) are on
the first side (836) of the housing (835). In yet other
embodiments, other reversible fastening mechanisms could be used to
secure the flap (1070) to the first side (836) of the housing
(835).
[0176] In preferred embodiments, the conjugate zones (872), (874)
are colored due to the dyes in the sample conjugates and control
conjugates. In one preferred embodiment, the conjugate zone (872)
that is used for the first test strip (1015) contains an MxA
binding partner that is labeled with a red dye, a low CRP binding
partner that is labeled with a black dye, and a control binding
partner that is labeled with a blue dye. In this embodiment, the
conjugate zone (872) appears purplish. The other conjugate zone
(874) contains a high CRP binding partner that is labeled with a
black dye and a control binding partner that is labeled with a blue
dye. In this embodiment, the conjugate zone (874) appears
bluish.
[0177] The diverting zone (850), which preferably includes a gap or
barrier, interrupts lateral flow, diverting the running buffer up
into the flap (1070) that includes the conjugate zones (872),
(874).
[0178] In operation, the upper and lower portions (838), (839) of
the second side (837) of the housing (835) are preferably snapped
closed before use by securing the pegs (875) to the holes (895).
The sample analysis device, or test card (1000) is preferably
placed on a flat surface. If the flap (1070) is not already open,
the user opens it to access the sample application zones (1073),
(1076). The sample application zones (1073), (1076) may be located
in any portion of the transfer pad (1055). A blood sample to be
tested is taken from the patient. The sample may be taken by any
procedure known in the art. In a preferred embodiment, a sample of
5 .mu.l of blood is added to each of the sample application zones
(1073), (1076) zones and then the flap (1070) is closed. Each of
the 5 .mu.l samples is preferably collected independently of each
other. The blood is preferably added directly to the device (1000),
without any pretreatment. In preferred embodiments, an arrow (1002)
or other indication (shown in FIG. 10A), for example the words "add
sample here" shows the user where to place the sample on the test
strips (1015), (1025).
[0179] To ensure that the flap (1070) has been closed correctly,
pressure is preferably applied to the housing (835) above the pegs
(875) to snap the pegs (875) closed. The top of the flap (1070)
needs to be flush with the top of the rest of the second side (837)
of the housing (835) for the test to run properly. Running buffer
is added to the absorbent pad (840), which initiates lateral flow
(885). In preferred embodiments, the running buffer includes one or
more lysis agents, for example detergents, to lyse the blood sample
and expose the intracellular MxA in the sample. When the running
buffer reaches the diverting zone (850), it is diverted up into the
flap (1070). It travels through the conjugate zones (872), (874),
collecting the MxA binding partners, the low CRP binding partners,
and the high CRP binding partners, as well as the control
conjugate.
[0180] Since the conjugate zones (872), (874) bridge the diverting
zone (850) on the lateral flow test strips (1015), (1025), the
running buffer, which now contains conjugate, then travels into the
transfer pad (1055), which includes the sample application zones
(1073), (1076), and to the detection zones (805) on each of the
test strips (1015), (1025). If MxA is present in the sample, the
MxA test line (802) on the first test strip (1015) will be red. If
a threshold low level of CRP is present in the sample, the low CRP
test line (803) on the first test strip (1015) will be black. If a
threshold high level of CRP is present in the sample, the high CRP
test line (823) on the second test strip (1025) will be black. In
preferred embodiments, the levels of detection are 40 ng/ml for
MxA, 10 mg/L for low CRP on the first test strip (1015) and 80 mg/L
for high CRP on the second test strip (1025). The results of the
test should be visible after approximately 5-20 minutes, preferably
within about 10 minutes. If the test was run correctly, the control
lines (804) on both the first strip (815) and the second test strip
(825) will be blue.
[0181] Since the control binding partner is on the flap (1070) and
not on either of the test strips (1015), (1025), there is a true
procedural control to this configuration. If the flap (1070) is not
closed properly, nothing will show up in the detection zone (805),
indicating that the test was run improperly.
[0182] In an alternative embodiment, the sample application zones
(1073), (1076) are located on the receiving pad (845), before the
diverting zone (850). In this embodiment, the running buffer
travels through the sample application zones (1073), (1076), and
then is diverted into the flap (1070).
[0183] In preferred embodiments of the configurations shown in
FIGS. 8A through 8C and 10A through 10C, greater than approximately
1.2 ml of running buffer is placed on the absorbent pad (840). If
less than 1.0 ml is added in embodiments where the diverting zone
(850) is a gap, the buffer gets stalled at the gap because the gap
holds approximately 1.0 ml.
[0184] As shown in FIG. 11, in one preferred embodiment, a kit
(1100) includes the sample analysis device (800), (1000), a lancet
(1102), one or more pipettes (1101), and a running buffer (1103).
The lancet (1102) is used to make a skin puncture and one or more
pipettes (1101) are used to collect the blood from the puncture
site. In a preferred embodiment, 5 .mu.l of blood is transferred
from a first pipette (1101) to the first conjugate zone (872) and
another 5 .mu.l of blood is transferred from a second pipette
(1101) and added to the second conjugate zone (874). The flap (870)
is closed, and the running buffer (1103) is added to the absorbent
pad (840), as described in the description of FIGS. 8A through 8C
and 10A through 10C.
[0185] The diverting zone (850) preferably includes at least one
feature that interrupts flow in the plane in which flow is
occurring. The diverting zone may include a barrier, a gap, a
ditch, or any combination of these features. The barrier is
preferably an impermeable membrane (or substantially impermeable
membrane) that may be made of any material that prevents the flow
of liquid from continuing to flow in the same plane. Some materials
for the barrier include, but are not limited to, inert materials,
semi-permeable materials, plastics, hydrocarbons, metal,
hydrophobic materials, Sephadex, Sepharose, cellulose acetate, a
hygroscopic material (for example CaCl.sub.2, CaSO.sub.4 or silica
gel), or hydrogels. The gap or ditch is any break in the plane of
the lateral flow test strip that extends to a depth sufficient to
stop flow. In one preferred embodiment, the gap is preferably at
least approximately 0.1 mm deep.
[0186] The diverting zone (850) in FIGS. 8A through 8C and 10A
through 10C delays or completely stops flow until the sample
compressor/flap (870), (1070) is brought into contact with the rest
of the device, and creates a bridge along which the fluid can flow.
The sample compressor (870), (1070) acts as a bridge and redirects
flow into a different plane. Flow is diverted into the sample
compressor (870), (1070). This increases collection of the reagents
on the sample compressor (870), (1070). For example, in embodiments
where the conjugate is on the sample compressor (870), (1070),
collection of the conjugate increases in devices with a diverting
zone (850). In embodiments where both the sample application zones
(873), (876), (1073), (1076) and the conjugate are on the sample
compressor (870), (1070), the sample and conjugate both encounter
the running buffer when it is diverted into the sample compressor
(870), (1070), and a 1/2 sandwich or full sandwich (depending upon
where the second binding partner for the analyte is located on the
sample analysis device) is formed before the running buffer is
diverted back to the test strips if the analyte is present in the
sample. Embodiments with a diverting zone (850) and a sample
compressor (870), (1070) increase speed, allow for better
interactions between the conjugate and the sample, and allow for
more sensitivity because more conjugate is placed into the fluid.
In these embodiments, all of the fluid preferably interacts with
the conjugate. This is a significant improvement over compressor
embodiments without redirection, where approximately 20-30% of the
fluid interacts with the conjugate.
[0187] Another preferred configuration for a bimodal dual test
strip sample analysis device (1200) is shown in FIG. 12. This
configuration is similar to the configurations (800), (1000) shown
in FIGS. 8A through 8C and FIGS. 10A through 10C, without a second
section (837) of the housing (1235) or a diverting zone (850).
Instead, all of the components of the test are located in the same
plane and flow proceeds laterally from the absorbent pad (840) to
the waste pad (860). Note that this embodiment could also include a
housing with a window to facilitate application of the buffer to
the absorbent pad (840), a window located above each sample
application zone (1273), (1276) for applying sample to the device
(1200), and viewing windows for the detection zone (805). In one
preferred embodiment, the sample analysis device (1200) is
approximately 11.5 cm long (L).times.7 cm wide (W). However, any
size test card (1200) that accommodates all of the components may
be used. There are two test strips (1215), (1225), each including a
receiving pad (845), a conjugate zone (1272), (1274), a transfer
pad (1240) containing a sample application zone (1273), (1276), a
detection zone (805) and a waste pad (860). The device (1200) also
preferably includes an absorbent pad (840) and a waste pad (860).
While the conjugate zones (1272), (1274) are shown upstream of the
sample application zones (1273), (1276) in this figure, in other
embodiments, one or both of the conjugate zones (1272), (1274) are
located downstream of the sample application zones (1273), (1276).
The detection zone (805) of the first test strip (1215) preferably
includes an MxA test line (802), a low CRP test line (803) and a
control line (804). The detection zone (805) on the second test
strip (1225) also preferably includes a high CRP test line (823)
and a control line (804). The absorbent pad (840) is preferably a
single pad that the running buffer is added to to start lateral
flow. Similarly, the waste pad (860) is preferably a single pad
that collects excess running buffer at the end of the test.
However, in other embodiments, each strip could have a separate
absorbent pad (840) and/or waste pad (860).
[0188] In preferred embodiments, the conjugate zones (1272), (1274)
are colored due to the dyes in the sample conjugates and control
conjugates. In one preferred embodiment, the conjugate zone (1272)
that is used for the first test strip (1215) contains an MxA
binding partner that is labeled with a red dye, a low CRP binding
partner that is labeled with a black dye, and a control binding
partner that is labeled with a blue dye. In this embodiment, the
conjugate zone (1272) appears purplish. The other conjugate zone
(1274) contains a high CRP binding partner that is labeled with a
black dye and a control binding partner that is labeled with a blue
dye. In this embodiment, the conjugate zone (1274) appears
bluish.
[0189] In operation, a blood sample to be tested is taken from the
patient. The sample may be taken by any procedure known in the art.
In a preferred embodiment, a sample of 5 .mu.l of blood is added to
each of the sample application zones (1273), (1276). Each of the 5
.mu.l samples is preferably collected independently of each other.
In preferred embodiments, an arrow (1002) or other indication
(shown in FIG. 10A), for example the words "add sample here" shows
the user where to place the sample on the test strips (1215),
(1225).
[0190] The blood is preferably added directly to the device (1200),
without any pretreatment. Running buffer is added to the absorbent
pad (840), which initiates lateral flow (1285). In preferred
embodiments, the running buffer includes one or more lysis agents,
for example detergents, to lyse the blood sample and expose the
intracellular MxA in the sample. It travels through the conjugate
zones (1272), (1274), collecting the MxA binding partners, the low
CRP binding partners, the high CRP binding partners, as well as the
control conjugate.
[0191] The running buffer, which now contains conjugate, then
travels into the transfer pad (1255), which includes the sample
application zones (1273), (1276), and to the detection zones (805)
on each of the test strips (1215), (1225). If MxA is present in the
sample, the MxA test line (802) on the first test strip (1215) will
be red. If a threshold low level of CRP is present in the sample,
the low CRP test line (803) on the first test strip (1215) will be
black. If a threshold high level of CRP is present in the sample,
the high CRP test line (823) on the second test strip (1225) will
be black. In preferred embodiments, the levels of detection are 40
ng/ml for MxA, 10 mg/L for low CRP on the first test strip (1215)
and 80 mg/L for high CRP on the second test strip (1225). The
results of the test should be visible after approximately 5-20
minutes, preferably within about 10 minutes. If the test was run
correctly, the control lines (804) on both the first strip (1215)
and the second test strip (1225) will be blue.
[0192] In an alternative embodiment, the sample application zones
(1273), (1276) are located upstream of the conjugate zones (1272),
(1274). In this embodiment, the running buffer travels through the
sample application zones (1273), (1276), and then to the conjugate
zones (1272), (1274). In still other embodiments, the conjugate
zones (1272), (1274) overlap the sample application zones (1273),
(1276). In still other embodiments, the conjugate zones (1272),
(1274), and/or the sample application zones (1273), (1276) may be
located in the receiving pad (845).
[0193] In preferred embodiments of the configurations shown in
FIGS. 4A through 8C, 10A through 10C and 12, the control is rabbit
anti-chicken and the control conjugate is blue latex beads coupled
to chicken IgY, In other preferred embodiments, there is at