U.S. patent application number 13/585509 was filed with the patent office on 2012-12-06 for detection of early stages and late stages hpv infection.
Invention is credited to Shuling CHENG.
Application Number | 20120309079 13/585509 |
Document ID | / |
Family ID | 41415135 |
Filed Date | 2012-12-06 |
United States Patent
Application |
20120309079 |
Kind Code |
A1 |
CHENG; Shuling |
December 6, 2012 |
DETECTION OF EARLY STAGES AND LATE STAGES HPV INFECTION
Abstract
Embodiments of the invention provide methods, monoclonal
antibodies, polyclonal antibodies, assays, and kits for detecting
HPV infection and HPV related cancer diagnosis, including infection
by various HPV genotypes, early and/or late stage HPV-associated or
HPV-specific cancers. Various specific or pan monoclonal antibodies
recognizing specific epitope for specific HPV protein or HPV type,
or common epitope for various HPV proteins or HPV types are
obtained. The invention also provides one or more solid surface to
coat the testing cell lysate. Also, the anti-HPV antibody can be
coated on the solid surface of the invention to capture HPV
proteins and detect HPV infection.
Inventors: |
CHENG; Shuling; (Fremont,
CA) |
Family ID: |
41415135 |
Appl. No.: |
13/585509 |
Filed: |
August 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12456076 |
Jun 10, 2009 |
8278056 |
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13585509 |
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61131991 |
Jun 13, 2008 |
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61192912 |
Sep 22, 2008 |
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Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
C07K 16/084 20130101;
G01N 33/571 20130101; G01N 33/56983 20130101; C12Q 1/708 20130101;
G01N 2469/10 20130101; G01N 2333/025 20130101 |
Class at
Publication: |
435/287.2 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Claims
1. A lateral flow through device for detecting a papillomavirus
protein from one or more papillomavirus types present in a clinical
sample being processed into a cell lysate solution, comprising: a
first solid surface on one end of a strip having a first anti-HPV
antibody immobilized, wherein the first anti-HPV antibody is
capable of binding to a papillomavirus protein from the one or more
papillomavirus types such that the first anti-HPV antibody is able
to capture the papillomavirus protein present in the cell lysate
solution onto the solid surface; and a second solid surface on the
other end of the strip having a second anti-HPV antibody
immobilized to react with the cell lysate solution flowing
laterally from the first solid surface of the strip to form into a
complex with second anti-HPV antibody on the second solid surface
of the strip, wherein the second anti-HPV antibody is capable of
binding to a papillomavirus protein such that the second anti-HPV
antibody is able to bind and detect the papillomavirus protein
present in the clinical sample.
2. The lateral flow rapid test device of claim 1, wherein the
second anti-HPV antibody is pre-labeled with a gold particle such
that a colormetric complex of the gold particle-labeled second
anti-HPV antibody and the papillomavirus protein can be visualized
on the solid surface.
3. A vertical flow-through rapid test device for detecting a
papillomavirus protein from one or more papillomavirus types
present in a clinical sample being processed into a cell lysate
solution, comprising: a solid surface of a membrane having a first
anti-HPV antibody immobilized, wherein the first anti-HPV antibody
is generated against one or more first recombinant proteins encoded
by one or more first papillomavirus genes such that the first
anti-HPV antibody is able to react with the cell lysate solution
and capture the one or more papillomavirus proteins in the cell
lysate solution onto the solid surface; and a second anti-HPV
antibody to be added with the cell lysate solution onto the solid
surface of the membrane for flowing vertically through the solid
surface and forming into a complex with first anti-HPV antibody on
the solid surface of the membrane, wherein the second anti-HPV
antibody is generated against one or more second recombinant
proteins encoded by one or more second papillomavirus genes such
that the second anti-HPV antibody is able to bind and detect the
one or more papillomavirus proteins in the cell lysate
solution.
4. The vertical flow-through rapid test device of claim 3, further
comprising an pre-labeled antibody which is capable of binding to
the second anti-HPV antibody, the pre-labeled antibody is labeled
with an detection agent selected from a group comprising horse
radish peroxidase conjugate, biotin, gold particle, and
combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This instant application is a divisional application of U.S.
patent application Ser. No. 12/456,076 filed on Jun. 10, 2009 and
entitled "Detection of early Stages and Late Stages HPV Infection",
which claims benefit of U.S. provisional patent application Ser.
No. 61/131,991, filed Jun. 13, 2008, and U.S. provisional patent
application Ser. No. 61/192,912, filed Sep. 22, 2008. Each of the
aforementioned related patent applications is herein incorporated
by reference.
BACKGROUND OF THE INVENTION
[0002] Infection by human papillomaviruses (HPV) at specific
epithelium cells to induce epithelial proliferations plays an
important role for cervical carcinogenesis. About 99 percent of
confirmed cervical cancer cases are found to be associated with HPV
infection with biopsy-confirmed squamous intraepithelial lesions
(SIL) or cervical intraepithelial neoplasia (CIN). The incidence of
HPV infection, primarily transmitted through sexual contact, is
highest among young women and about 20 million sexually active men
and women worldwide are currently infected. Approximately 1% of the
population has genital warts and 4% of women have cervical
precancerous lesions, such as low grade of squamous intraepithelial
lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL)
or atyptical squamous cells of undetermined significance
(ASCUS).
[0003] These lesions, preferentially observed in women aged 35-40
yrs, have a high risk of progression toward invasive cervical
cancer. It is generally thought that persistent infection of human
papillomavirus (HPV) is essential for developing precancerous
epithelival lesions. Infection of high-risk types of HPV in women
with LSIL may or may not progress to HSIL. In fact, remission
occurs in the majority of LSIL human subjects while some progress
to HSIL. Although 99.7% of cervical cancers are HPV positive,
integration of viral genome into the host genome is required to
facilitate the necessary genetic expression for developing into
HSIL or cancer. In fact, only one in every 10 women with persistent
HPV infection may develop higher grades of CIN lesions, such as
cervical intraepithelial neoplasia (CIN) grade 2 and grade 3 (CIN2,
and CIN3, respectively), and a portion of these epithelival lesion
cases may ultimately progress into cervical cancer.
[0004] In the past, screening for cervical cancer has been based on
conventional cytology screening tests, e.g., obtaining papanicolaou
(Pap) smears for cytological staining tests, and suspicious smears
are followed up with colposcopy, and/or histological biopsy. The
use of these cytological screening tests contributes to a reduction
in the mortality of cervical cancer. However, due to subjective
test criteria, there are various drawbacks for pap smear tests:
difficulty in obtaining samples, poor inter- and intra-observer
agreement, high rates of false negatives and false positives, the
requirement of specialized labs staffed with highly trained
personnel, and inability to identify the majority of HPV-infected
human subjects. More reproducible assays are needed to improve the
current screening tests to avoid unnecessary medical intervention
and psychological distress for the affected women. The current
conventional cervical cytology screening tests have sensitivity
varied from about 30% to about 87%.
[0005] Nucleic acid tests, such as "DNA Hybrid Capture", have been
developed with high assay sensitivity, but these tests are still
not ideal, due to not only their high cost, assay operation
procedures, the requirements for facility, equipment, and highly
trained personnel, but also their very low positive predictive
value (PPV) in cervical intraepithelial neoplasia (CIN) testing
samples. Assays like PreTect HPV-Proofer.RTM. provide the detection
of E6/E7 mRNA with sensitivity equivalent to HPV Hybrid Capture
tests with higher positive predictive value; but cannot directly
detect E6/E7 oncoprtoeins in situ. In addition, DNA testing can not
differentiate disease stages after HPV infection nor can it
diagnose different cell lesions (e.g., cannot differentiate LSIL
from HSIL, nor CIN lesions from non-transforming latent or
remissive viral infection). What is needed is a low cost, simple,
sensitive and specific assay that can be routinely performed on in
a clinical lab or doctor office and is capable of detecting early
stages of epithelial lesions, distinguishing LSIL from HSIL, or
predicting the risk of progression into cervical cancer.
[0006] Known protocols for producing monoclonal antibodies are
generally unsuitable for the production of anti-HPV monoclonal
antibodies and cannot be used in immunocytochemical diagnostic
tests performed on human subjects of the general population. This
is because antibodies produced by these protocols will not
necessarily react with the naturally occurring HPV viral proteins
in infected human cells. It is thought that the epitopes recognized
by antibodies if generated by conventional protocols will not
necessarily be those epitopes which are resistant to the harsh
procedures involved in standard sampling, fixing and storing of
clinical specimens. In addition, three problems exist in clinical
HPV detection. One is that HPV proteins in clinical samples are
present in very small quantities. Secondly, there are too many HPV
types and most HPV types present in clinical samples are not known
or systemically identified due to the lack of available antibodies.
Third, HPV virus can not be cultured in labs by standard tissue
culture techniques. Thus, there are no available HPV proteins
purified to large quantities as immunogens for generating anti-HPV
antibodies, and there are no available HPV proteins or purified
anti-HPV antibodies to recognize anti-viral antibodies or viral
proteins present in clinical samples for clinical HPV
detection.
[0007] Only 15 out of more than 100 available types of HPV
infection are at high risk of developing into cervical
intraepithelial neoplasia (CIN) or cervical cancer. Among them,
around 70% of reported cervical cancer cases and 50% of reported
CIN 2 and CIN 3 cases are caused by two high risk HPV types, HPV
type-16 and HPV type-18. However, some progressive cervical cancer
cases are reported to be infected by low risk HPV types, while
infection of some high risk HPV types will never progress into
cervical cancer. Infections by these two prevailing high risk HPV
types do not correlate with tumor development or cancer
progression. It seems important to identify those HPV-infected
human subjects that express particular oncogenic proteins rather
than just identify HPV infection by high risk types.
[0008] Thus, there is a need to detect the expression of
HPV-related oncoproteins in clinical samples as these oncoprotiens
may serve as cervical cancer biomarkers to better predict the risk
of developing into high grade cell lesions or cervical
cancer-related diseases. There is also a need to develop anti-HPV
antibodies and appropriate HPV immunoassays to detect the presence
of invasive cervical cancer and/or HPV-related oncoproteins as
cervical cancer biomarkers and predict the risk for malignant
transformation of epithelial lesions into cervical cancer.
SUMMARY OF THE INVENTION
[0009] Embodiments of the invention provide various solid surface
immunoassays for the detection of HPV proteins using various
anti-HPV antibodies against recombinant HPV proteins such that
infection by high risk and/or low risk HPV types can be detected by
a single specific monoclonal antibody and/or a general pan
antibody. The invention also provides HPV blot membrane assays,
protein chip microarray assays, HPV beads assays, HPV lateral flow
rapid tests, HPV vertical flow-through rapid tests, HPV
microfluidic rapid tests, direct enzyme immunoassays (EIA), and
enzyme linked immunoabsorbant assays (ELISA) to detect the presence
of HPV proteins in a biological sample, such as cervical cells or
cervical tissues. In addition, kits and devices for performing
these assays are also provided.
[0010] In one embodiment, a method is provided to detect one or
more papillomavirus proteins and includes providing one anti-HPV
antibody capable of binding to the one or more papillomavirus
proteins from one or more papillomavirus types and present in a
clinical sample, providing a solid surface having coated thereon
the anti-HPV antibody or the various proteins present in the cell
lysate solution, and processing the clinical sample into a cell
lysate solution containing various proteins including the one or
more papillomavirus proteins. The method further includes reacting
the anti-HPV antibody with the cell lysate solution to form a
complex of the one or more papillomavirus proteins with the
anti-HPV antibody on the solid surface and detecting the complex on
the solid surface to confirm the presence of the one or more
papillomavirus proteins present in the clinical sample.
[0011] In another embodiment, a method is provided for detecting
the presence of one or more papillomavirus proteins from one or
more papillomavirus types in a biological sample and includes
providing a biological sample processed into a cell lysate
solution, providing a first anti-HPV antibody immobilized on a
solid surface to react with the cell lysate solution, and reacting
the cell lysate solution with a second anti-HPV antibody. Both
antibodies are capable of binding to one or more papillomavirus
proteins from one or more papillomavirus types. The method further
includes forming a complex of the one or more papillomavirus
proteins with the first and the second anti-HPV antibody on the
solid surface, and detecting the formation of the complex on the
solid surface for the presence of the one or more papillomavirus
proteins in the biological sample.
[0012] In addition, a lateral flow through device for detecting the
presence of one or more papillomavirus proteins from one or more
papillomavirus types in a biological sample is provided according
to one embodiment of the invention. The lateral flow through device
includes a first solid surface of a strip having a first anti-HPV
antibody immobilized on one end of the strip and a second solid
surface of a strip having a second anti-HPV antibody on the other
end of the strip able to react with a cell lysate solution
processed from the biological sample for flowing laterally on the
solid surface of the strip to form into a complex with the first
anti-HPV antibody. In another embodiment, a vertical flow-through
rapid test device is provided for detecting the presence of one or
more papillomavirus proteins from one or more papillomavirus types
in a biological sample and includes a solid surface of a membrane
having a first anti-HPV antibody immobilized thereon to react with
a cell lysate solution processed from the biological sample, and a
second anti-HPV antibody to be added with the cell lysate solution
onto the solid surface of the membrane for flowing vertically
through the solid surface and forming into a complex with the first
anti-HPV antibody on the solid surface of the membrane.
[0013] In one aspect, the first anti-HPV antibody is generated
against one or more first recombinant proteins encoded by one or
more first papillomavirus genes such that the first anti-HPV
antibody is able to capture the one or more papillomavirus proteins
in the cell lysate solution onto the solid surface. In another
aspect, the second anti-HPV antibody is generated against the same
first recombinant proteins encoded by the same papillomavirus genes
such that the second anti-HPV antibody is able to bind and detect
the one or more papillomavirus proteins in the cell lysate
solution.
SUMMARY OF DRAWING
[0014] FIG. 1 shows the results of a dot blot assay to detect HPV
L1 proteins using an anti-HPV L1 mouse monoclonal antibody
according to one embodiment of the invention.
[0015] FIG. 2 show the results of another dot blot to detect HPV L1
proteins using the same mouse monoclonal anti-HPV L1 antibody as
used in FIG. 1.
[0016] FIG. 3 shows the results of using the same blot as shown in
FIG. 1 to detect HPV E6 proteins using an anti-HPV E6 mouse
monoclonal antibody according to another embodiment of the
invention.
[0017] FIG. 4 shows the results of using the same dot blot shown in
FIG. 2 to detect HPV E6 proteins using the same anti-HPV E6 mouse
monoclonal antibody as the one used in FIG. 3 according to another
embodiment of the invention.
[0018] FIG. 5 shows the results of the same dot blot shown in FIG.
1 and FIG. 3 botting with an anti-HPV E7 mouse monoclonal antibody
to detect HPV E7 proteins according to another embodiment of the
invention.
[0019] FIG. 6 is a graph showing the average fluorescent intensity
results of an antibody microarray assay performed on cell lysate
from 10 cervical scrape samples to detect various HPV proteins and
various cellular endogenous proteins according to another
embodiment of the invention.
[0020] FIG. 7 is another graph showing the fluorescent intensity
results of the antibody microarray assay for each of the 10 cell
lysate samples as shown in FIG. 6 to detect HPV L1 proteins
according to another embodiment of the invention.
[0021] FIG. 8 is another graph showing the fluorescent intensity
results of the antibody microarray assay for each of the 10 cell
lysate samples as shown in FIG. 6 to detect HPV E7 proteins and
cellular p16 INK4a proteins according to another embodiment of the
invention.
[0022] FIG. 9 is another graph showing the fluorescent intensity
results of the antibody microarray assay for each of the 10 cell
lysate samples as shown in FIG. 6 to detect HPV L1 proteins and
cellular p53 proteins according to another embodiment of the
invention.
[0023] FIG. 10 is another graph showing the fluorescent intensity
results of the antibody microarray assay for each of the 10 cell
lysate samples as shown in FIG. 6 to detect HPV E7 proteins and
cellular phosphate form of Rb proteins according to another
embodiment of the invention.
[0024] FIG. 11 is another graph showing the fluorescent intensity
results of the antibody microarray assay for sample S2.
[0025] FIG. 12 is another graph showing the fluorescent intensity
results of the antibody microarray assay for each of the 10 cell
lysate samples as shown in FIG. 6 to detect cellular p21 WAF1 and
p53 proteins according to another embodiment of the invention.
[0026] FIG. 13 shows the results of an ELISA assay to detect HPV
E6, E7 and L1 proteins in human serum samples diagnosed as SCC
(squamous cell carcinoma) or HPV positive (by PCR) as compared to a
HPV negative serum sample (by PCR) using anti-HPV rabbit polyclonal
antibody for coating and detection according to another embodiment
of the invention.
[0027] FIG. 14 shows the results of a sandwich beads assay by FACS
to detect recombinant HPV16 L1 protein using an anti-HPV16 L1 mouse
monoclonal antibody as coating antibody and an anti-HPV16 L1 rabbit
polyclonal antibody as detecting antibody.
[0028] FIG. 15 shows the results of a sandwich beads assay by FACS
to detect recombinant HPV16 E6 protein using an anti-HPV16 E6 mouse
monoclonal antibody as coating antibody and an anti-HPV16 E6 rabbit
polyclonal antibody as detecting antibody.
[0029] FIG. 16 shows the results of a sandwich beads assay by FACS
to detect recombinant HPV18 E6 protein using an anti-HPV18 E6 mouse
monoclonal antibody as coating antibody and an anti-HPV18 E6 rabbit
polyclonal antibody as detecting antibody.
[0030] FIG. 17 shows the results of a sandwich beads assay by FACS
to detect recombinant HPV16 E7 protein using an anti-HPV16 E7 mouse
monoclonal antibody as coating antibody and an anti-HPV16 E7 rabbit
polyclonal antibody as detecting antibody.
[0031] FIG. 18 shows the results of a sandwich beads assay by FACS
to detect recombinant HPV16 E6 protein using an anti-HPV16 E6 mouse
monoclonal antibody as detecting antibody and an anti-HPV16 E6
rabbit polyclonal antibody as coating antibody.
[0032] FIG. 19 shows the results of a sandwich beads assay by FACS
to detect recombinant HPV18 E6 protein using an anti-HPV18 E6 mouse
monoclonal antibody as detecting antibody and an anti-HPV18 E6
rabbit polyclonal antibody as coating antibody.
[0033] FIG. 20A shows the results of a one-step lateral flow
through assay to detect a HPV L1 recombinant protein (from left to
right: 875, 435, 0 ug/ml) using a rabbit anti-HPV L1 polyclonal
antibody coated on membrane, and conjugated with gold particle (as
the detection agent).
[0034] FIG. 20B shows the results of a one-step lateral flow
through detecting HPV L1 protein in human serum sample from SCC
(squamous cell carcinoma) patient (left) compared to a normal
(known HPV negative) serum (right) using the same strips shown in
FIG. 9A.
[0035] FIG. 20C shows the results of a one-step lateral flow
through detecting HPV E6 recombinant protein (from left to right:
10, 2, 0 ug/ml) using a mouse anti-HPV E6 monoclonal antibody
coated on membrane, and gold conjugate for detection.
[0036] FIG. 20D shows the results of a one-step lateral flow
through detecting HPV L1 protein in human serum sample from SCC
(squamous cell carcinoma) partient (1.sup.st and 2.sup.nd from the
left) compared to a normal (known HPV negative) serum (right) using
the same strips shown in FIG. 9C.
[0037] FIG. 20E shows the results of a one-step lateral flow
through detecting HPV E6 recombinant protein (from left to right:
875, 435, 0 ug/ml) using another mouse anti-HPV E6 monoclonal
antibody coated on membrane, and gold conjugate for detection.
[0038] FIG. 20F shows the results of a one-step lateral flow
through detecting HPV E7 recombinant protein (from left to right:
0, 660, 66, 6.6, 0.66, ug/ml) using a mouse anti-HPV E7 monoclonal
antibody coated on membrane, and gold conjugate for detection.
[0039] FIG. 20G shows the results of a one-step lateral flow
through detecting HPV E7 protein from a known HPV positive human
serum sample (left) compared to a known HPV negative serum (right)
using the same strips shown in FIG. 20F.
DETAILED DESCRIPTION
[0040] Embodiments of the invention provide various immunological
assays, methods, detection devices, kits, polypeptides, recombinant
proteins, nucleic acids, and monoclonal antibodies against HPV
viral proteins useful for detecting HPV infection, including
general HPV infection as well as infection by various HPV
genotypes, high risk HPVs and low risk HPVs.
[0041] In one embodiment, a method is provided for detecting the
presence of one or more papillomavirus proteins from one or more
papillomavirus types in a biological sample processed into a cell
lysate solution to react with an anti-HPV antibody. The anti-HPV
antibody is generated against one or more recombinant proteins
encoded by one or more papillomavirus genes such that the anti-HPV
antibody is able to bind and detect the one or more papillomavirus
proteins in the cell lysate solution. As an example, the one or
more papillomavirus proteins detected by the methods of the
invention may include papillomavirus E6 proteins, papillomavirus E7
proteins, and papillomavirus L1 proteins.
[0042] The method further includes providing a solid surface and at
least one protein coated on the solid surface. Suitable proteins
that can be coated on the solid surface of the invention include,
but are not limited to, one or more anti-HPV antibodies, one or
more HPV proteins, and various proteins present in a clinical
sample. In addition, the anti-HPV antibody is reacted with the cell
lysate solution to form a complex of the one or more papillomavirus
proteins with the anti-HPV antibody on the solid surface. For
example, various proteins present in the clinical sample, including
the one or more papillomavirus proteins present in the cell lysate
solution, are coated on the solid surface. As another example, the
anti-HPV antibody is coated on the solid surface.
[0043] The solid surface may include, but is not limited to, a
surface of a bead, a surface of a strip, a surface of a rapid test
strip, a surface of a membrane, a membrane surface of a vertical
flow through device, a surface of a microfluidic device, a surface
of a blot membrane, a surface of a protein chip, a glass surface,
and a bottom surface of a microtiterplate, among others. For
example, the solid surface can be the surfaces of one or more beads
and the anti-HPV antibody can be coated thereon to capture one or
more papillomavirus proteins on the surfaces of one or more beads
and the anti-HPV antibody-papillomavirus protein complexes formed
on the surfaces of the beads can be detected by a FACS
(Fluorescence-activated cell sorting) detection instrument.
[0044] As another example, the solid surface can be a strip of a
rapid test device and the one or more papillomavirus proteins bound
by the anti-HPV antibody can be detected on the strip of the rapid
test device. The anti-HPV antibody can be a first anti-HPV antibody
immobilized on one end of the strip, and a second anti-HPV antibody
is provided to be added to the other end of the strip. The cell
lysate solution for testing the presence of the one or more
papillomavirus proteins is also added to the other end of the strip
to be recognized by the second anti-HPV antibody. Thus, the one or
more papillomavirus proteins present in the cell lysate solution
recognized and bound by the second anti-HPV antibody flow laterally
on the solid surface of the strip of the rapid test device in order
to form into a complex with the first anti-HPV antibody.
[0045] As still another example, the solid surface can be a surface
of a rapid test strip, such as a membrane surface of a vertical
flow through device. One or more anti-HPV antibodies can be
coated/immobilized on the solid surface of the membrane of a
vertical flow-through rapid test device to detect the complex of
the anti-HPV antibodies with the one or more papillomavirus
proteins present in the cell lysate solution. Alternatively, two
anti-HPV antibodies can be used with a first anti-HPV antibody
coated on the solid surface of the membrane before adding the cell
lysate solution and a second anti-HPV antibody onto the membrane of
the vertical flow rapid test device to form a complex with the
first anti-HPV antibody.
[0046] As still another example, the solid surface can be inside a
surface of a microfluidic device. Various testing samples in
fluidic cell lysate solutions are designed to flow inside one or
more channels of the microfludic device. One or more anti-HPV
antibodies can be coated/immobilized on the solid surface inside
the channels of the microfludic device to bind to the one or more
papillomavirus proteins present in the fludic cell lysate testing
solution and form a complex. Also, two anti-HPV antibodies can be
used in a microfluidic device to bind to the one or more
papillomavirus proteins present in the cell lysate solution of a
testing sample and be circulated inside the channels of the
microfluidic device.
[0047] In one aspect, one or more papillomavirus proteins present
in the cell lysate solution are coated on the solid surface before
reacting with the anti-HPV antibody. For example, direct detection
of the papillomavirus proteins can be obtained by adding a
pre-labeled anti-HPV antibody capable of binding to the one or more
papillomavirus proteins to detect the formation of the anti-HPV
antibody-papillomavirus protein complex. The pre-labeled anti-HPV
antibody can be labeled with a detection agent, including but not
limited to, horse radish peroxidase conjugate, biotin, gold
particle, fluorescent, and combinations thereof. Alternatively, the
anti-HPV antibody-papillomavirus protein complex can be recognized
by adding a pre-labeled secondary antibody capable of binding to
the anti-HPV antibody. The secondary antibody can be labeled with a
detection agent to detect the formation of the anti-HPV
antibody-papillomavirus protein complex by binding to the anti-HPV
antibody. In one example, proteins from cell lysate solution are
coated on the solid surface of a membrane blot or a microtiter
plate device before reacting with the anti-HPV antibody.
[0048] In another aspect, the anti-HPV antibody is coated on the
solid surface before reacting with the one or more papillomavirus
proteins present in the cell lysate solution to detect
papillomavirus infection of the human subject. For example, direct
detection of the papillomavirus proteins can be obtained by adding
an anti-HPV antibody pre-coated on the solid surface and
pre-labeled with a detection agent to detect the formation of the
anti-HPV antibody-papillomavirus protein complex. Alternatively,
the anti-HPV antibody can be pre-coated on the solid surface
without pre-labeling and a secondary antibody capable of binding to
the anti-HPV antibody can be added later to detect the formation of
the anti-HPV antibody-papillomavirus protein complex.
[0049] In another aspect, the anti-cellular protein antibody is
coated on the solid surface before reacting with one or more
cellular proteins present in the cell lysate solution of the
clinical sample to detect the presence of the cellular protein.
These cellular proteins may include any of the HPV related cellular
proteins that are affected inside a human subject after HPV
infection, such as p16INK4a, p53, p63, Rb, pRb, p21WAF1, ki67
(MIB-1), MYC cellular oncogene, cyclin proteins (e.g., cyclin A,
cyclin B, cyclin E), CDKN2A/p16INK4a, telomerase (e.g., TERC),
replication complex proteins, MCM5, CDC6, topoisomerase II alpha
(TOP2A), MCM2, survivine, minichromosome maintenance proteins
(e.g., minichromosome maintenance protein 2, minichromosome
maintenance protein 4, and minichromosome maintenance protein 5),
and combinations thereof.
[0050] The expression levels of the cellular proteins and the
expression level of the papillomavirus viral proteins in the
clinical sample of the human subject are compared to detect the
presence of the HPV infection and/or a disease stage of HPV
infection (early infection, late infection, etc.). For example, in
high grade CIN lesions, E6 and E7 are strongly expressed in host
basal epithelial cells and interfere substantially with the cell
cycle of its host cells. Expression of HPV oncoproteins interfers
with G1-S-Phase regulation in host cells. The HPV E6 and E7
proteins target a plethora of cellular interactions, such as the
inactivation of pRB by E7 and the degradation of p53 by E6. High
levels of HPV E7 proteins inactivate pRB and lead to disruption of
E2F-Rb binding. Usually, binding of pRB to E2F blocks E2F driven
cell cycle activation. In replicating cells, E2F is regulated by
phosphorylation of RB. Rb phosphorylation is normally mediated by
cyclin dependent kinases (CDK4, CDK6) that are controlled by
several kinase inhibitors (INKs).
[0051] As a result of the loss of Rb/E2F repression and the strong
activation by free E2F, the expression of a host cell protein,
p16INK4a, is strongly overexpressed. In addition, S-phase genes are
continuously activated since the p16INK4a mediated repression of
Cdk4/6 has no downstream effect on pRb host cell protein. Since
E7-dependent E2F release is not mediated by phosphorylation of pRb,
the counter-regulatory p16INK4a expression has no effect on the
activated cell cycle. Under physiological conditions p16INK4a is
expressed when cells undergo a genomic stress situation such as
substantial shortening of telomeres in ageing tissues. Also,
apoptosis is abrogated by HPV E6 mediated degradation of p53. The
overexpression of the cyclin dependent kinase (CDK) inhibitor,
p16INK4a, is a direct consequence of deregulated HPV oncogene
expression.
[0052] In addition, host cell proteins important for proliferation
and host cell genome replication may be overexpressed as a result
of HPV infection. These host cell proteins include, ki67 (MIB-1),
MYC cellular oncogene, Cyclin proteins (e.g., cyclin A, B, E,
etc.), CDKN2A/p16INK4a, telomerase (e.g., TERC), replication
complex proteins (e.g., MCM5, CDC6, topoisomerase II alpha (TOP2A),
MCM2, minchromosome maintenance proteins 2, 4, and 5, etc.).
[0053] Other host cell proteins affected by HPV infection may
include host cellular stress and invasion proteins, such as heat
shock protein (e.g., HSP.sub.40, HSP.sub.60, HSP.sub.70), carbonic
anhydrase (e.g, CA9/MN antigen). Also, host cell proteins that
enhance viral oncogene activity can be affected by HPV infection
and these proteins include TSLC1, DAPK1, RARB, TWIST1, brn-3s
transcription factor, among others. In addition, survivin protein
which is involved in cell cycle and apoptosis regulation can be
affected by HPV infection. The expression of VEGF can be
upregulated by HPV E6 protein, which is independent from E6
mediated p53 degradation.
[0054] Accordingly, examples of host cell proteins whose expression
levels may be altered by HPV infection include, but are not limited
to, p16INK4a, cyclin dependent kinase inhibitors, pRB, p53, E2F,
E2F activated cell cycle proteins, cyclin dependent kinases, CDK4,
CDK6, S-phase genes, Ki-67 (MIB-1), MYC protein, cyclin-A,
cyclin-B, cyclin-E, telomerase-TERC, MCM2 protein, TOP2A protein,
heat shock protein 40 (HSP.sub.40), heat shock protein 60
(HSP.sub.60), heat shock protein 70 (HSP.sub.70), CA9/MN protein,
laminin 5, laminin proteins, brn-3a, CDK N2 protein, topoisomerase
2A, microsome maintenance proteins-2, microsome maintenance
proteins-4, microsome maintenance proteins-5, survivin protein,
VEGF protein, p27 (kip1) protein, p21 (waf) protein, and
combinations thereof.
[0055] Changes in the expression levels of among these proteins
affected by HPV infection (e.g., E6, E7, p53, Rb, p16INK4a, among
others) serve as a signature for high risk of contracting cervical
cancer. Elevated levels of HPV-associated viral proteins, viral
antigens, and host cells proteins (e.g., E6 proteins, E7 proteins,
p16INK4a, E2F, Ki-67 (MIB-1), MYC protein, CDK4, cyclin-A,
cyclin-B, cyclin-E, telomerase-TERC, MCM2 protein, TOP2A protein,
heat shock protein 40 (HSP.sub.40), heat shock protein 60
(HSP.sub.60), heat shock protein 70 (HSP.sub.70), CA9/MN protein,
laminin 5, laminin proteins, brn-3a, CDK N2 protein, topoisomerase
2A, microsome maintenance proteins-2, microsome maintenance
proteins-4, microsome maintenance proteins-5, survivin protein,
VEGF protein), and reduced levels of host cell proteins (e.g., p53,
RB, p27(kip1), and p21 (waf), etc.) confirm not just HPV infection
but also that the subjects are at high risk of contracting cervical
cancer. On the contrary, unchanged levels of p53 and RB in the
human subjects with elevated levels of HPV-associated viral
proteins or antigens may indicate a general HPV infection and
cervical cancer not yet progressed.
[0056] As an example, the immunological assays for detection of HPV
protein, such as E6, E7, L1, etc., or immune response thereof due
to HPV infection can be performed in high throughput ELISA
screening assays, rapid immunological screening assays, and
additional multiplexed protein chip assays, etc., and combinations
thereof to assay the expression levels of the HPV viral proteins
and/or many of the HPV interfering cellular proteins.
[0057] The anti-HPV antibody or the anti-cellular protein antibody
can be coated on any of the solid surface of any immunological
detecting devices/kits as described herein to be used in an
immunological assay for detecting the presence of the HPV viral
proteins or the cellular proteins in a testing sample. For example,
the anti-HPV antibody can be coated on the surface of a bead, the
surface of a strip, the surface of a rapid test strip, the surface
of a membrane, the membrane surface of a vertical flow through
device, the surface of a microfluidic device, the surface of a blot
membrane, the surface of a protein chip, a glass surface, and the
bottom surface of a microtiterplate. For example, the anti-HPV
antibody can be coated on the solid surface of a protein chip
device before reacting with the cell lysate solution. The anti-HPV
antibody or a secondary antibody capable of binding to the anti-HPV
antibody is pre-labeled with a detection agent in order to detect
the complex of the anti-HPV antibody and the papillomavirus
proteins present in the cell lysate solution on the solid surface
of a protein chip device. As another example, the anti-HPV antibody
can be coated on the solid surface of a microtiter plate or a rapid
test device before reacting with the cell lysate solution. The
rapid test device may be a lateral flow rapid test device, a
vertical flow-through rapid test device, a microfluidic rapid test
device, or any other rapid test devices.
[0058] In another embodiment, more than one anti-HPV antibodies can
be used in a sandwiched format to detect the presence of one or
more papillomavirus proteins from one or more papillomavirus types
present in a biological sample that has been processed into a cell
lysate solution. The detection of the one or more papillomavirus
proteins is obtained by forming a complex of a sandwich of two or
more anti-HPV antibodies with the testing papillomavirus proteins.
A first anti-HPV antibody and a second anti-HPV antibody are used,
both are generated against recombinant HPV proteins, and used to
react with the cell lysate solution such that the second anti-HPV
antibody is able to bind and detect one or more papillomavirus
proteins in the cell lysate solution captured by the first anti-HPV
antibody. In one aspect, the cell lysate solution is added to react
with the first anti-HPV antibody before reacting with the second
anti-HPV antibody. In another aspect, the cell lysate solution is
pre-mixed with the second anti-HPV antibody before reacting with
the first anti-HPV antibody.
[0059] In one aspect, the second anti-HPV antibody is pre-labeled
with a detection agent, which may be, for example, a horse radish
peroxidase conjugant, biotin, gold particle, and fluorescent
agents. In another aspect, the cell lysate solution is pre-mixed
with the second anti-HPV antibody and the second anti-HPV antibody
is pre-labeled with a detection agent. In another aspect, a
secondary antibody capable of binding to the first or the second
anti-HPV antibody is pre-labeled with a detection agent.
[0060] Accordingly, a complex of one or more papillomavirus
proteins with the first and the second anti-HPV antibody formed on
the solid surface can be detected for the presence of the one or
more papillomavirus proteins in the biological sample. As an
example, the complex of anti-HPV antibodies-papillomavirus proteins
is detected on the solid surface of one or more beads and the
complex is detected by FACS (Fluorescence-activated cell sorting).
As another example, the complex is detected on the solid surface of
a strip of a rapid test device with the first anti-HPV antibody
immobilized on one end of the strip, where the second anti-HPV
antibody and the cell lysate solution are added onto the other end
of the strip before flowing laterally across the solid surface of
the strip of the rapid test device to form a complex with first
anti-HPV antibody. As yet another example, the complex is detected
on the solid surface of a membrane of a vertical flow-through rapid
test device with the first anti-HPV antibody immobilized thereon,
and wherein the cell lysate solution and the second anti-HPV
antibody are added sequentially onto the solid surface of the
membrane of the vertical flow rapid test device to form a complex
with first anti-HPV antibody. As another example, the complex can
be detected on the solid surface of a microfluidic device, and the
complex is flowing through the microfluidic device. As another
example, the complex is detected on the solid surface of the bottom
of a microtiter plate.
[0061] Various formats can be used to detect the anti-HPV
antibody-papillomavirus proteins complex by using a pre-labeled
detection agent. For example, a detection agent, such as a horse
radish peroxidase conjugant, biotin, gold particle, and fluorescent
agents, can be pre-labeled on various agents, proteins and
antibodies. For example, the detection agent can be pre-labeled on
the anti-HPV antibody, the various proteins present in the cell
lysate solution, a secondary antibody that binds to the anti-HPV
antibody, or a second anti-HPV antibody different from the anti-HPV
antibody to be detected. The secondary antibody can be an
anti-mouse antibody if the primary anti-HPV antibody is a mouse
antibody. The secondary antibody may also be an anti-rabbit
antibody if the primary anti-HPV antibody is a rabbit antibody. The
secondary antibody can be an anti-goat antibody if the primary
anti-HPV antibody is a goat antibody.
[0062] The presence or levels of the detection agent prelabeled on
proteins or antibodies as described herein can be qualitatively or
quantitatively measured. For example, a direct qualitative
visualization of the color change of a detection agent prelabeled
on a anti-HPV antibody or proteins present in cell lysate solution
can be performed in a lateral or vertical rapid test assays. A
pre-labeled detection agent can also be used to react with its
suitable substrate reagents such that positive reaction between the
detecting agent and its substrates can be detected through readout
by an ELISA reader. Another example of a detection agent suitable
for pre-labeling is a detection agent capable of being detected by
a microarray scanner after a positive binding reaction when the
solid surface is the glass or membrane of a protein chip
device.
[0063] Both polyclonal and monoclonal anti-HPV antibodies can be
used in the immunological assays having a solid surface for coating
an anti-HPV antibody or various testing samples. Useful monoclonal
antibodies obtained include specific monoclonal antibodies with
binding reactivity for a single HPV protein, and general pan
antibodies with binding reactivity for more than one HPV proteins
or more than one HPV type (high risk and/or low risk).
[0064] The antibodies as developed herein lend themselves to the
high quality and properly purified recombinant proteins encoded by
HPV early and late genes. They are useful in immunological assays
to generate very high sensitivity and specificity for screening HPV
infection and cervical cancer detection. The purified recombinant
papillomavirus proteins may include, but are not limited to,
papillomavirus E6 protein, papillomavirus E7 protein,
papillomavirus L1 protein, and combinations thereof. The
recombinant papillomavirus proteins include, but are not limited
to, recombinant HPV-16 E6 proteins, recombinant HPV-16 E7 proteins,
recombinant HPV-18 E6 proteins, recombinant HPV-18 E7 proteins,
recombinant HPV-16 L1 proteins, and recombinant HPV-18 L1
proteins.
[0065] Various monoclonal antibodies against HPV viral proteins are
provided such that infection by high risk and low risk HPV types
can be detected by a single monoclonal antibody. The invention also
provides HPV type specific monoclonal antibodies for detecting only
the high risk HPV types. The one or more papillomavirus types
include high risk HPV types, low risk HPV types, HPV-16, HPV-18,
HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56,
HPV-58, HPV-59, and HPV-68, HPV-6, HPV-11, HPV-42, HPV-43, HPV-44,
HPV-53, HPV-54, HPV-55, and HPV-56, and combinations thereof.
[0066] One embodiment of the invention provides one or more
anti-HPV antibodies capable of binding to two or more HPV viral
proteins from the same HPV type. Another embodiment, of the
invention provides one or more anti-HPV antibody is antibodies
capable of binding to two or more HPV viral proteins from different
HPV types. For example, the invention provides a monoclonal
antibody capable of recognizing a common epitope on E6 protein from
two different HPV types, both HPV16 and HPV18 by screening
antibody-producing hybridoma cells with a purified HPV16 E6
recombinant protein and a purified HPV18 E6 recombinant protein.
Another example provides a monoclonal antibody that recognizes a
common epitope on HPV16 E7 and HPV18 E7 proteins. Still another
embodiment of the invention provides a monoclonal antibody that
recognizes a common epitope on HPV16 E6, HPV16 E7, HPV16 L1, HPV18
E6, and HPV18 E7 proteins.
[0067] As another example, a monoclonal antibody capable of
recognizing a specific epitope on only one HPV viral protein, but
not another HPV viral protein, is obtained by screening
antibody-producing hybridoma cells with a first purified
recombinant papillomavirus protein from a first HPV type and a
second purified recombinant papillomavirus protein from a second
HPV type, wherein the first and second viral proteins correspond to
the first and the second purified recombinant papillomavirus
proteins of the first and second HPV types.
[0068] In one embodiment, a method of detecting papillomavirus
infection in a human subject includes obtaining a clinical or
biological sample from the human subject, and conducting one or
more immunological assays on the clinical sample from the human
subject using various HPV recombinant proteins and lab-generated
antibodies specific for HPV oncoproteins in order to detect and
screen for the presence of HPV infection from the presence of HPV
proteins and HPV antibodies in the human subject. The biological
sample includes, but is not limited to, cervical cells, cervical
tissues, cervical swabs, body fluids, serum, blood, tumors, cell
cultures, biopsies, and combination thereof, wherein the biological
sample is obtained from the general population for routine
screening of cervical cancer.
[0069] In another embodiment, the HPV proteins in the human subject
are detected using one or more antibodies raised against HPV
recombinant proteins, including, but not limited to, various
polyclonal and monoclonal antibodies against various HPV early and
late proteins. One antibody among the one or more antibodies is
capable of recognizing a common epitope present on two or more
papillomavirus proteins. The two or more papillomavirus proteins
may include a papillomavirus early protein and a late
papillomavirus protein and the antibody is capable of recognizing
both the papillomavirus early protein and the late papillomavirus
protein. The antibodies obtained include antibodies recognizing HPV
E6 proteins, antibodies recognizing HPV E7 proteins, antibodies
recognizing HPV E6 and E7 proteins, antibodies recognizing HPV L1
proteins, antibodies recognizing HPV E6 proteins from different HPV
types, antibodies recognizing HPV E7 proteins from different HPV
types, antibodies recognizing HPV L1 proteins from different HPV
types, antibodies recognizing HPV E6 and E7 proteins of the same
HPV types, antibodies recognizing HPV E6 and E7 proteins of
different HPV types, and antibodies recognizing HPV E6, E7, and L1
proteins.
[0070] The antibodies can be used for one or more immunological
assays, including, but not limited to, ELISA (enzyme linked
immunoabsorbant assays), antigen assays for papillomavirus
proteins, antibody assays for antibodies against papillomavirus
proteins, assays for papillomavirus immunocomplexes, protein chip
assays, radioimmunoprecipitation assays, rapid membrane
immunochromatographic assays, rapid stick immunochromatographic
assays, immunohistochmistry for tissues and/or cervical cells, and
immunocytological assays followed by flow cytolmetry, among others.
In one embodiment, the one or more immunological assays may be
non-invasive with minimal or no additional instrument required.
[0071] A cytological papanicolaou smear (Pap smear) assay may also
be performed on a clinical sample to compare the results of the
cytological papanicolaou smear assay with the results of the
immunocytological assays. In addition, nucleic acid hybridization
assays can also be performed on the clinical sample to detect the
presence of a papillomavirus genome in the clinical sample from the
human subject. The nucleic acid hybridization assays may include,
but are not limited to polymerase chain reactions, nucleic acid
hybridization assays, DNA chip assays, radioactive nucleic acid
hybridization and detection assays, and non-radioactive nucleic
acid hybridization and detection assays.
[0072] In another embodiment, the immunological assay is used to
detect a disease stage caused by HPV infection. The disease stage
may be, for example, an early stage HPV infection, a late stage HPV
infection, an early stage cervical cell lesion, a late stage
cervical cell lesion, low grade of squamous intraepithelial lesion
(LSIL), high grade of squamous intraepithelial lesion (HSIL),
atypical squamous cells of undetermined significance (ASCUS),
cervical intraneoplasm stage 1, 2, 3 (CIN1, CIN2, CIN3,
respectively), developed cervical cancer, adenocarcinoma, or
squamous cell carcinoma (SCC).
[0073] The basic techniques for conducting the immunological assays
can be found in "Antibodies: A Laboratory Manual", Harlow and Lane,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989;
"Molecular Cloning", A Laboratory Manual, eds. Sambrook, Fritsch
and Maniatis, Cold Spring Harbor Laboratory Press, 1989, and others
books and manuals known in the art. This application is
cross-related to U.S. Pat. No. 7,732,166 filed on Nov. 13, 2006 and
entitled "Detection Method for Human Papillomavirus (HPV) and Its
Application in Cervical Cancer"; U.S. Pat. No. 7,972,776 filed on
Apr. 14, 2008 and entitled "Protein Chips for HPV Detection"; U.S.
Patent App. Ser. No. 61/131,991, filed Jun. 13, 2008 titled
"Antibodies and assays for HPV detection"; Ser. No. 61/192,912
Filed on Sep. 22, 2008, titled "Novel monoclonal antibodies against
HPV proteins useful for early stage and late stage detection,
screening, and diagnosis of HPV related cervical cancer"; Ser. No.
12/456,053, filed on Jun. 10, 2009, titled "Novel monoclonal
antibodies against HPV proteins"; serial number 12,456/054, filed
on Jun. 10, 2009, titled "in situ detection of early stages and
late stages HPV infection"; Ser. No. 12/456,055, filed on Jun. 10,
2009, titled "in situ detection of early stages and late stages HPV
infection". All of the above referenced applications are herein
incorporated by reference.
[0074] The invention also provides various methods, detection
assays, and kits, polyclonal and monoclonal antibodies,
polypeptides, recombinant proteins, and nucleic acids useful for
detecting general HPV infection as well as infection by various HPV
genotypes, high risk HPVs and low risk HPVs. In addition, the
assays or sample formats in detecting the presence of HPV proteins
are not limited and can be used for cervical tissues, cervical
cells, cervical scrapes, serum, body fluids, etc. The useful
screening or diagnosing assay can be IHC, ICC, flow cytometry,
antibodies coupled to beads, rapid tests, protein chips, dot blots,
slots, as well a conventional ELISA assay. HPV proteins can be
detected by the antibodies of the invention to be present in
epithelium tissue as evidenced by IHC staining after scoring by a
pathologist.
[0075] For example, a lateral flow through rapid test device having
a solid surface of a strip membrane is provided. The solid surface
may include a first anti-HPV antibody immobilized on one end of the
strip and a second anti-HPV antibody parked on the other end of the
strip able to react with a cell lysate solution processed from a
biological sample. The device may be conveniently performed in one
step for detecting the presence of one or more papillomavirus
proteins from one or more papillomavirus types in the biological
sample. The second anti-HPV antibody parked on the other end of the
strip will form into a complex with HPV proteins present in the
cell lysate and then flow across the surface of the strip on the
rapid test device. As the complex containing the HPV proteins flow
across toward the other end of the strip, it can then be captured
by the first anti-HPV antibody on the surface of the strip.
[0076] In operation, as an example, clinical samples collected from
liquid based solution were processed, washed, centrifuged, and
lysed with lysis buffer to obtain cell lysate containing the one or
more papillomavirus proteins. The cell lysate was added onto the
top of the strip device from one end containing an anti-HPV
detecting antibody, conjugated with gold particles to react with
the one or more papillomavirus, and flowed through to the other end
of the strip device, wherein an anti-HPV pre-coated on the surface
captured the immunocomplex of the flow through containing the one
or more papillomavirus proteins bound to the anti-HPV detecting
antibody, and an agent showed a pink band when the complex was
captured and retained on the surface, demonstrating a positive
reaction of the one-step lateral flow through assay for detection
of HPV proteins. A test control was also included in the device.
The test control includes coating one protein on the surface of one
end, which is for example, 0.5 cm away from the surface where the
anti-HPV antibody is coated, to capture its binding protein which
is pre-labeled with agent and flows through with the testing
sample. For the test control protein selected, there is no cross
binding activity or interference with the testing protein for
binding to anti-HPV antibody. Therefore, there are two lines
showing pink bands to demonstrate positive results, and there is
only one line showing a pink band on the test control position to
demonstrate negative results of the assay.
[0077] As another example, a vertical flow-through rapid test
device for detecting the presence of one or more papillomavirus
proteins from one or more papillomavirus types in a biological
sample is also provided. The vertical flow-through rapid test
device may include a solid surface of a membrane, a first anti-HPV
antibody, and a second anti-HPV antibody. The first anti-HPV
antibody is immobilized on the solid surface to react with a cell
lysate solution processed from the biological sample. The second
anti-HPV antibody and the cell lysate solution are added onto the
surface of the membrane, flow vertically through the solid surface,
and form a complex with first anti-HPV antibody on the solid
surface of the membrane. The first and the second anti-HPV
antibodies are generated against recombinant proteins encoded by
one or more papillomavirus genes such that the anti-HPV antibodies
are able to bind and detect the one or more papillomavirus proteins
in the cell lysate solution.
[0078] The surface of the flow through device can be coated with
one or more strips or spots of one, two, or more anti-HPV
antibodies to detect one, two, or more HPV proteins present in the
biological samples on the same device. This flow through assay may
take multiple steps including the adding of testing protein, adding
of detecting antibody and substrate, and washing between each step,
or it may take two steps by premixing the testing protein with the
detecting antibody which is prelabeled with agent, followed by
reacting with colometric substrate to show results. It may take
only one step as in a lateral flow through assay format but in a
device for vertical flowthrough. The dot blot assay described in
this invention may be formatted to fit into the vertical flow
through device. The assay gives results within about 10 min, about
30 min, or less than an hour, and thus is suitable for use of point
of care in a doctor's office. The second anti-HPV antibody may be
pre-labeled with gold particles such that the complex captured on
the surface of the device can be detected by its color. High
intensity of color indicates the presence of the one or more
papillomavirus proteins in the biological samples and can be read
out by naked eye or by an instrument.
[0079] As still another example, a microfludic rapid test device
for detecting the presence of one or more papillomavirus proteins
from one or more papillomavirus types in a biological sample is
also provided. The microfludic rapid test device may include a
solid surface bound with a first anti-HPV antibody and a second
anti-HPV antibody. A microfluidic device has one or more channels
with at least one dimension less than 1 mm. Common fluids used in
microfluidic devices include whole blood samples, bacterial cell
suspensions, protein or antibody solutions and various buffers. The
use of microfluidic devices to conduct biomedical research and
create clinically useful technologies has a number of significant
advantages. First, because the volume of fluids within these
channels is very small, usually several nanoliters, the amount of
reagents and analytes used is quite small. This is especially
significant for expensive reagents. The fabrication techniques used
to construct microfluidic devices are relatively inexpensive and
are very amenable both to highly elaborate, multiplexed devices and
also to mass production. In a manner similar to that for
microelectronics, microfluidic technologies enable the fabrication
of highly integrated devices for performing several different
functions on the same substrate chip. Ultimately, the microfluidics
device is to create integrated, portable clinical diagnostic
devices for home and bedside use, thereby eliminating time
consuming laboratory analysis procedures.
[0080] Another example is a device for performing a beads
immunological assay. The device may include beads coated with
antibody that bind and capture the protein of interest followed by
a detecting antibody with a labeled agent able to be detected by
FACS. It may also include a lysis buffer to lyse the cells to
obtain protein to bind on the beads and react with the detecting
antibody.
[0081] Still another example is a device for performing a dot blot
immunological assay. The device may include a membrane with an
absorbing pad underneath, similar to vertical flow through device,
where the cell lysate containing testing protein can be spotted
directly onto a very small area of the surface to allow highly
concentrated protein to be retained on the surface, followed by
binding detecting antibody to HPV protein on the surface of the
membrane. A colormetric substrate reacts with the detecting
antibody and shows a color spot demonstrating a positive reaction
of the assay.
[0082] Yet another example is a device for performing a protein
chip assay. The device may include a slide with a glass or membrane
surface pre-spotted with various antibodies specific to various
proteins of interest including HPV proteins and host cellular
proteins. It may also include lysis buffer to lyse the cells to
obtain the proteins of interest. It may also include labeling
reagent to directly label the cell lysate, or label a second
antibody detecting the proteins of interest.
[0083] In operation for a protein chip assay, the surface on which
proteins are coated/bound may be, for example, a surface-chemistry
treated glass or membrane, which can covalently or non-covalently
bind with capture agents or proteins thereto. A spotting machine
with fine pins dipped with capture agents, such as the recombinant
proteins, antigens, antibodies, or other proteins in suitable
buffers is generally used to facilitate binding of such proteins or
antibodies to the treated surface. Like other surfaces described in
the microtiter plate format, the spotted and thus captured proteins
or antibodies bind strongly to the surface-chemistry treated
surface of a protein chip and remain on the treated surface to
allow the interaction and specific binding of the captured proteins
with target proteins, antibodies, or antigens, even after several
washings of removing non-specific binding, to be detected with a
detection system conjugated with Cy3 or Cy5. The detection of
specific interactions is obtained and measured by the fluorescent
intensities of the spotted/dipped images via a microarray
scanner.
[0084] As an example, antibody microarray can be used as the
protein chips assay format for detection of HPV protein and other
cellular proteins. First, cells, samples or cultured cells to be
tested were collected, centrifuged, washed, and lysed to generate
cell lysate as analyte. The protein in the cell lysate was
quantitated and labeled with biotin or Cy3, Cy5 or any other
chromagen for subsequent detection of the binding of the labeled
protein on the surface of prespotted antibody. The surface of the
protein chips for the protein chip assays can be a membrane or
glass for different analysis and quantification techniques.
[0085] Various novel monoclonal antibodies against HPV proteins,
identified as useful biomarkers and useful tools for detecting HPV
viral proteins, HPV oncoproteins, early screening of cervical
cancer, and diagnosing CIN and/or invasive cervical and other
cancers, are provided. The tools of the inventions can also be used
in early clinical screening for HPV infection and general diagnosis
for cervical cancer and other cancers, specific detection of
invasive cervical cancer, detection of other HPV related cancers,
early stage precancerous lesions as well as late stage cancer
progression.
[0086] The antibodies described in this invention provide a tool to
detect HPV proteins present in various sources of biological
samples. The biological samples includes, but are not limited to,
cervical cells, cervical tissues, cervical swabs, body fluids,
serum, blood, tumors, cell cultures, biopsies, and combinations
thereof, wherein the biological sample is obtained from a group of
people from the general population for routine screening of
cervical cancer.
[0087] As an example, the antibodies described herein can be used
as a capture antibody to coat on microtiterplate and/or used as a
detection antibody in a sandwisch format of ELISA (Enzyme Linked
Immuno Sandwich Assay). Antibodies can be selected based on the
specificity described herein of a monoclonal antibody to particular
HPV proteins or HPV types, or in combinations thereof. The
detection antibody with selected specificity to the monoclonal
antibodies described herein can be directly conjugated with a label
like biotin, alkaline phosphatase, HRP, fluorescent, etc., followed
by colormetric, chemiluminescent or fluorescent substrate for
readout. The detection antibody can also be a polyclonal antibody
described herein and be followed by a secondary antibody conjugated
with a label like biotin, alkaline phosphatase, HRP, fluorescent,
etc. A combination of using polyclonal and monoclonal antibodies
for the sandwich ELISA as capture and detection antibodies or vice
versa, increases assay sensitivity by incoporating secondary
antibody to amplify the signal for detection. For direct EIA
(Enzyme Immuno Assay), cells, samples or cultured cells to be
tested were collected and lysed to generate cell lysate as analyte.
The protein in the cell lysate was quantitated and coated to a
microtiterplate using the same amount of protein in each well
followed by the detection antibody with specificity described in
this invention.
[0088] Detection of HPV DNAs, genomes, early viral proteins, late
viral proteins, oncoproteins, and/or capsid proteins from various
HPV genotypes can be performed by various in vitro and in vivo
methods and detection assays according to "Antibodies: A Laboratory
Manual", Harlow and Lane, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. 1989; "Molecular Cloning", A Laboratory Manual,
eds. Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory
Press, 1989, and others books and manuals and can be very useful in
general clinical screening for HPV infection.
[0089] Detection of HPV antibodies and/or oncoproteins by
immunological assays can be used in early clinical screening for
HPV infection and general diagnosis for cervical cancer and can be
performed in a single rapid test or in a multiplexed test.
Comparative detection of altered levels of HPV proteins and host
proteins can be performed in the same or different assays. It can
also be used in diagnosing HPV-associated carcinomas of the uterine
cervix, as well as those cases associated with epithelial cell
abnormalities induced by HPV infection, pre-malignant and malignant
HPV-associated epithelial cell lesions, and those at risk of
developing HPV-associated cervical carcinoma and adenocarcinoma.
The methods as described herein can be used independently or as an
adjunct screening tool to convention cytological papanicolaou smear
tests or histological tests and the results thereof can be compared
for follow-up patient management.
EXAMPLES
[0090] A. Detecting HPV Proteins from Biological Samples Using One
Anti-HPV Antibody 1. Direct EIA: one or more HPV proteins coated on
microtiterplate to be detected by one or more anti-HPV
antibodies
[0091] Clinical samples from cervical scrapes were obtained for
detection of HPV E6, E7 or L1 proteins on direct EIA. Cervical
cells from various sample sources included cervical scrape cells in
liquid based cytology solution, cervical scrape cells in transport
medium (used for HPV DNA test sample), or cervical scrape cells in
lysis buffer. To perform the direct EIA described herein, specimens
were processed, centrifuged, washed, and lysed to generate cell
lysate as analyte. The proteins in the cell lysate were quantitated
and coated to a microtiterplate with the same amount of protein in
each well. The plate was blocked, and detected by each HPV
monoclonal antibody followed by HRP-conjugated secondary antibody
(anti-mouse IgG or anti-rabbit IgG for example). TMB substrate was
added followed by a stopping solution. OD at 450 nm was taken by an
ELISA plate reader.
[0092] As an example, cervical cells from various stages of
cervical neoplasm collected in liquid based solution were processed
to obtain the cell lysate for detection of HPV DNA and HPV
proteins. For HPV DNA detection, touchdown PCR protocol was used.
For HPV protein detection by direct EIA, cell lysate was coated
directly on the microtiterplate, blocked, then specific HPV
antibodies were used followed by a secondary antibody conjugated
with HRP. OD450 was taken from a microtiter plate coated with cell
lysate with or without adding the primary HPV antibody followed by
the secondary antibody. Since various proteins from the cell lysate
were coated on the microplate, OD from each sample with no primary
HPV antibody was considered to be the non-specific binding of the
cell lysate with the secondary antibody. To obtain OD for specific
binding of HPV protein with anti-HPV antibody, net OD for each
sample obtained by subtraction of the OD from its non-specific
binding of the secondary antibody was considered to be the specific
binding of HPV protein with the primary anti-HPV antibody. Net OD
from each PCR negative sample was obtained. Mean OD from PCR
negative samples was used as the baseline of the assay. Samples
with net OD over two folds of average OD from PCR negative samples
were considered positive, otherwise they were negative for the EIA
test described herein.
TABLE-US-00001 TABLE 1 Detection of HPV DNA and HPV protein from
liquid based cervical scrapes Liquid based cervical scrapes Sample
Dx or HPV DNA by Direct EIA by No. pap smear results PCR poly
anti-E7 1 ASCUS pos pos 2 ASC-H pos pos 3 ASCUS pos pos 4 ASCUS neg
pos 5 ASCUS neg neg 6 CIN1 pos pos 7 CIN1 pos pos 8 CIN1 pos pos 9
CIN1 pos neg 10 CIN1/ASCUS neg pos 11 CIN1 neg neg 12 CIN2 pos neg
13 CIN2 pos neg 14 CIN2 neg neg 15 CIN2 neg neg 16 CIN3 pos pos 17
CIN3 pos pos 18 CIN3 pos pos 19 CIN3 neg neg 20 CIN3 neg neg 21
CIN3 neg neg 22 SCC pos pos 23 SCC pos pos 24 AD neg neg
[0093] As an example, clinical samples diagnosed by histology or
pap smear staining including abnormal cells, ASCUS, CIN1, CIN2,
CIN3, SCC and Adenocarcinoma are obtained and processed into cell
lysate for direct coating on a microtiterplate. Detection of HPV
DNA by PCR and detection of HPV proteins by EIA are shown as Table
1 and compared with the clinical diagnosis and pap smear results.
Data show 79% (19 out of 24) correlation of HPV DNA with HPV
proteins detected by polyclonal anti-E7 antibody. For those HPV DNA
and HPV proteins not in correlation (21%; 5 out of 24), 3 are PCR
pos, EIA negative (case No. 9/CIN1, No. 12/CIN2, and No. 13/CIN2)
indicating HPV infection with no expression or non-detectable E7
oncoproteins. For those that are PCR negative, EIA positive like
case No. 4 (ASCUS), and No. 10 (CIN1/ASCUS), it could be false
negative of PCR, or E7 oncoproteins could be expressed with loss of
HPV DNA.
[0094] For those HPV DNA positive but HPV EIA negative samples, the
HPV DNA assay may be false positive, or there may be positive HPV
DNA detection with no expression of HPV oncogenic proteins. These
data indicate that HPV EIA described herein has relevance for the
screening of cervical cancer. It's important to detect the HPV
oncoporteins to follow up if progression of dysplasia HSIL occurs.
These data indicate that the HPV oncoproteins are good biomarkers
for screening and early detection of cervical cancers, and other
HPV associated cancers. For cases that are both PCR and EIA
negative but diagnosed ASCUS, or CIN, the loss of HPV DNA and HPV
oncoprotein detection is possibly due to the sampling of cervical
scrape cells or treatment of the patients, or false positive of the
pap smear results. However, more samples should be tested.
2. Dot Blot Assay: Spotting Cell Lysate on Membrane to Detect HPV
Proteins from Biological Samples Using One or More Anti-HPV
Antibodies
[0095] To develop a rapid test showing results with no instrument
required for the read out, a dot blot assay demonstrates the
feasibility of detecting HPV proteins from cell lysate on a
membrane with visual results followed by colormetric substrate. As
an example, cervical cells from various stage of cervical neoplasm
collected in liquid based solution are processed to obtain the cell
lysate to be spotted on a membrane. The membrane was air dried
prior to blocking the blot with blocking solution. An anti-HPV
antibody was added to react with the blot followed by a secondary
antibody capable of binding to the anti-HPV antibody. The blot was
washed between each step to avoid non-specific binding of the
antibody onto the membrane. In the final step, TMB substrate was
added to the blot and the appearance of a blue dot indicated a
positive reaction of the cell lysate binding with the anti-HPV
antibody used in this dot blot assay. Recombinant HPV proteins were
also spotted on the membrane to be used as positive or negative
control.
[0096] FIG. 1 shows the results of a dot blot detecting HPV L1
proteins using a mouse monoclonal anti-HPV L1 antibody. As
indicated, dots from the first row are cell lysate from various SCC
cervical scrapes in liquid based solution and dots from the second
row are recombinant HPV16 L1 protein at concentrations of 20, 2,
0.2 and 0 .mu.g/ml from left to the right as indicated A, B, C, D
respectively. As the results indicated in the second row of the
blot, recombinant HPV L1 proteins reacts highly positive to 0.2
.mu.g/ml or lower of purified recombinant proteins with the mouse
monoclonal anti-HPV L1 antibody used in FIG. 1. These data
demonstrate that HPV L1 proteins from both recombinant HPV 16 L1
and cell lysate can be detected by dot blot assay using a mouse
monoclonal anti-HPV L1 antibody as shown in FIG. 1.
[0097] FIG. 2 show results of another dot blot detecting HPV L1
proteins using the same mouse monoclonal anti-HPV L1 antibody shown
in FIG. 1. As indicated, dots from the first and second row are
cell lysate from various SCC cervical scrapes in liquid based
solution. In the third row of the blot, recombinant HPV16 E6, HPV18
E6, HPV16 E7, HPV18 E7, HPV16 L1 proteins are spotted from left to
right as indicated A, B, C, D, and E, respectively. As the results
in the third row of the blot indicate, recombinant HPV L1 protein,
spotted on 3E, reacts positively with the mouse monoclonal anti-HPV
L1 antibody used in the assay, compared to HPV16 E7 and HPV16 E6
with no detectable spot or HPV18 E7 and HPV18 E6 with very weak
spot shown on the third row of the blot. The weak spots were likely
due to the non-specific binding of the assay. Thus when considering
them as the background of the assay, clinical samples 2C and 2E
have equally strong spot signal compared to that from HPV16 L1
recombinant protein. These data indicate that HPV L1 proteins from
both recombinant HPV 16 L1 and cell lysate can be detected by dot
blot assay using a mouse monoclonal anti-HPV L1 antibody
demonstrated in FIG. 1.
[0098] To detect HPV E6 protein on dot blot assay, FIG. 3 shows
results of a dot blot with a mouse monoclonal anti-HPV E6 antibody.
As indicated, dots from the first row are cell lysate from various
SCC cervical scrapes (same as the first row of FIG. 1) in liquid
based solution and dots from the second row are recombinant HPV16
E6 protein at concentrations of 20, 2, 0.2 and 0 .mu.g/ml from left
to the right as indicated A, B, C, D respectively. As the results
in the second row of the blot indicate, recombinant HPV E6 proteins
react positively to 20, and 2 .mu.g/ml, and weakly to 0.2 .mu.g/ml
or lower of purified recombinant proteins with the mouse monoclonal
anti-HPV E6 antibody used in FIG. 3. These data indicate that HPV
E6 proteins from both recombinant HPV 16 E6 and cell lysate can be
detected by dot blot assay using a mouse monoclonal anti-HPV E6
antibody demonstrated in FIG. 3.
[0099] The same spotting blot shown in FIG. 2 was also used to
demonstrate detection of HPV E6 protein on dot blot assay. FIG. 4
shows results of a dot blot detecting HPV E6 proteins using the
same mouse monoclonal anti-HPV E6 antibody shown in FIG. 3. As the
results in the third row of the blot indicate, recombinant HPV16 E6
proteins on spot 3A react positively with the mouse monoclonal
anti-HPV16 E6 antibody used in the assay, compared to HPV 18 E6
with very weak spot and other recombinant proteins with no
detectable spots shown on the third row of FIG. 2. These results
demonstrate the specificity of HPV E6 proteins with the mouse
monoclonal anti-HPV E6 antibody, with no cross reacting with HPV L1
or HPV E7 proteins. Using the weak spot corresponding to background
or cross-reactive binding of HPV 18 E6 in the assay, samples 2C and
2E show very strong spots compared to others with moderate spots
and 2D with no detectable spot. These data indicate 70% (7 out of
10) clinical samples containing HPV E6 proteins can be detected by
dot blot assay using a mouse monoclonal anti-HPV16 E6 antibody
demonstrated in FIG. 4.
[0100] To demonstrate detection of HPV E7 proteins on dot blot
assay, the same spotting blot shown in FIG. 2 and FIG. 4 was also
used to blot with a mouse monoclonal anti-HPV E7 antibody detecting
HPV E7 proteins as shown in FIG. 5. As the results in the third row
of the blot in FIG. 5 indicate, recombinant HPV18 E7 proteins,
spotted on 3D, react positively with the mouse monoclonal
anti-HPV18 E7 antibody used in the assay, compared to other HPV
recombinant proteins with no detectable spots, or a very weak spot
with HPV16 L1 on spot 3E shown on the third row of FIG. 2. These
results demonstrate specificity of HPV E7 proteins with the mouse
monoclonal anti-HPV E7 antibody and has no cross reaction with HPV
L1, or HPV E6 proteins. Using the weak spot corresponding to
background or cross-reactive binding of HPV 18 E7 in the assay,
samples 2C and 2E show very strong spots compared to others with no
detectable spots. These data indicate samples 2C and 2E containing
HPV18 E7 proteins can be detected by dot blot assay using a mouse
monoclonal anti-HPV18 E7 antibody as demonstrated in FIG. 5.
3. Antibody Microarray: Spotting Antibodies on a Protein Chip to
Detect HPV Proteins and Cellular Endogenous Proteins in a Labeled
Cell Lysate from Biological Sample
[0101] For example, in a protein chip assay, the surface for
proteins to be coated/bound to may be, for example, a glass or
membrane surface that is chemistry treated, which can covalently or
non-covalently bind or coat with capture agents or proteins
thereto. A spotting machine with fine pins dipped with capture
agents, such as the recombinant proteins, antigens, antibodies, or
other proteins in suitable buffers is generally used to facilitate
the binding of such proteins or antibodies to the treated surface.
Like other surfaces described in the microtiter plate format, the
spotted and thus captured proteins or antibodies bind strongly to
the chemistry treated surface of a protein chip and remain on the
treated surface to allow the interaction and specific binding of
the capured proteins with target proteins, antibodies, or antigens,
even after several washings of removing non-specific binding, to be
detected with a detection system conjugated with Cy3 or Cy5. The
detection of specific interactions is obtained and measured by the
fluorescent intensities of the spotted/dipped images via a
microarray scanner.
[0102] As an example, an antibody microarray can be used in the
protein chips assay format for detection of HPV protein and other
cellular proteins. First, cells, samples or cultured cells to be
tested were collected, centrifuged, washed, and lysed to generate
cell lysate as anyalyte. The protein in the cell lysate was
quantitated and labeled with biotin or Cy3, Cy5 or any other
chromagen for subsequent binding and detection of the labeled
protein on the surface of prespotted antibody. The surface of the
protein chips for the protein chip assays can be a membrane or
glass for different analysis and quantification techniques.
[0103] Table 2 shows the results of protein chip assays for
detecting the presence of various HPV proteins and various host
cellular proteins. An antibody array pre-spotted with antibodies
against HPV and various cellular proteins was used to detect the
presence of these HPV proteins and host cellular proteins in a
human cervical scrape clinical sample. A total of 10 samples of
cervical scrapes (labeled as S1-S10 shown on Table 2) diagnosed as
keratinizing squamous cell carcinoma (grade 2 or grade 3) in liquid
based solution was processed and lysed to generate protein lysate
with proper labeling (such as biotin label) for subsequent
detection (followed by strepavidin-Cy3, for example). The
fluorescent intensity indicating binding of the proteins from the
human sample with prespotted antibody against proteins including,
but not limited to, HPV-16 E7, HPV-16 L1, p63, p53, p21WAF1,
p16INK4a, phosphorylated Rb, and unphosphorylated Rb, was obtained
and is shown in Table 2. Fluorescent intensity for the binding of
the specific protein with the specific antibody prespotted on the
microarray indicated changes in the expression levels of these
proteins affected by HPV infection and is shown in FIG. 6 to FIG.
12.
TABLE-US-00002 TABLE 2 Fluorecsent intensity (after background
subtraction) of individual spots shows binding of specific antibody
with proteins expressed in cell lysate from cervical cancer
patients on antibody microarray Ab spotted S1 S2 S3 S4 S5 S6 S7 S8
S9 S10 HPV16 E7 849 1407 422 355 443 403 316 337 383 267 HPV 16
1309 236 1477 418 620 1206 251 205 700 3407 p63 398 128 205 51 167
146 215 230 427 174 p53 325 102 86 161 119 83 226 242 465 335
P21WAF1 594 100 130 92 167 54 177 178 493 250 p16INK4a 164 549 97
107 116 87 72 128 87 174 Retinoblastoma 753 170 140 185 109 70 219
247 448 317 Rb (phosph) 491 236 269 143 238 245 156 224 310 171
[0104] To compare the expression of each protein from the 10 SCC
samples tested, the average of the fluorescent intensity for a
specific protein from each sample was obtained to demonstrate the
protein expression level with standard deviation bar for the graphs
in FIG. 6. The results indicate various HPV proteins and various
cellular endogenous proteins from the cell lysates of the 10
cervical scrape samples can be detected on the antibody microarray
assay described herein. As indicated in FIG. 6, HPV 16 and HPV16E7
are over expressed compared to other cellular proteins.
[0105] To demonstrate variation of HPV16 in different samples, FIG.
7 shows the fluorescent intensity of each sample for detecting HPV
L1 proteins in cell lysate from the cervical scrape cells shown in
FIG. 6. The results demonstrate binding of HPV16 antibody (anti-HPV
L1 antibody) with HPV proteins, particularly the L1 viral protein
expressed in cell lysate from cervical cancer patients, on antibody
microarray. HPV16 L1 proteins are expressed predominantly in sample
S1, S3, S6, and S10, medium for sample S4, S5, and S9 while
expression is low in sample S2, S7, and S8 which might be due to a
different type of HPV not recognized by the HPV16 antibody used in
this assay.
[0106] HPV E6 and E7 proteins play critical roles in the
oncogenesis of HPV in cervical cancer. To study the interaction of
the HPV E6 E7 oncoproteins with cellular proteins, the antibody
microarray assay described herein provides tools for simultaneous
detection of HPV proteins and cellular proteins such as p53, or Rb
which directly interact with HPV oncoproteins or cellular proteins
such as p16, p21, etc., affected by HPV infection. P16INK4a has
been commonly used as a surrogate for detection of cervical cancer.
To demonstrate HPV viral proteins, such as E6 or E7 oncoprotein,
can serve as a better biomarker for detecting cervical cancer, the
antibody microarray (protein chip assay) described herein
demonstrates detection of multiple proteins including various HPV
proteins and various cellular proteins simultaneously. FIG. 8 shows
detection and comparison of HPV E7 and p16 protein expression in
the 10 SCC samples. Fluorescent intensity from each individual
sample (1 through 10) demonstrates binding of HPV16E7 antibody and
p16INK4a antibody with proteins expressed in cell lysate from
cervical cancer patients on antibody microarray. For the 10 samples
tested, each sample shows higher fluorescent intensity with HPV E7
antibody than p16 antibody, indicating more HPVE7 protein expressed
compared to p16. These data suggest HPVE7 serves as a better marker
for detecting cervical cancer.
[0107] To demonstrate p53 affected by HPV in cervical cancer, FIG.
9 shows fluorescent intensity for both HPV16 and p53 antibody,
indicating overexpression of HPV16 with p53 suppression in clinical
samples with HPV infection. Comparing expression of HPV16 and p53,
the results indicate p53 is expressed at much lower level with high
level of HPV16 expression in most clinical samples except clinical
samples S7 and S8, which might be a different type of HPV other
than HPV 16 infection. However, low p53 expression in all clinical
samples indicates most p53 proteins are degraded by HPV E6
oncoproteins during cervical cancer development,
[0108] To demonstrate interaction of E7 and retinoblastoma (Rb)
protein and phosphorylated Rb affected by HPV in cervical cancer,
FIG. 10 shows fluorescent intensity for HPV16E7 and pRb antibody,
indicating HPV16 E7 expression at higher level while Rb is
inactivated, which is recognized by anti-Rb-phosphate specific
antibody at lower level. In sample S2, there is overexpression of
HPV16 E7 and suppression of phosphorylated Rb. Data indicate
inactivation of Rb (low in reacting with Rb-phosphate antibody) by
E7 pathway causes malignant transformation developing cervical
cancer.
[0109] To demonstrate expression profiling of protein chip assay
for cervical cancer as an example, FIG. 11 shows expression
profiling of the selected HPV proteins and cellular proteins for
sample S2. Results indicate HPV E7 and p16 were overexpressed,
while other cellular proteins were suppressed. All together with
results from FIG. 8, FIG. 10, and FIG. 11 suggest overexpression of
HPV E7 protein in sample #2 inactivates Rb and induces p16INK4a
expression, resulting in malignant transformation and the
development of cervical cancer. Sample 51 with high HPV-E7
expression level may undergo pathway independent from Rb, thus
didn't express a high level of p16INK4a.
[0110] To demonstrate another cellular protein p21 WAF1 expression
in cervical cancer and its correlation with p53, FIG. 12 shows
fluorescent intensity of each sample for detecting cellular p21
WAF1 and p53 proteins using cell lysate from cervical scrape cells.
As data in FIG. 12 shows, 9 out of 10 samples (except sample 51)
p21WAF1 that is well correlated with expression of p53. These data
demonstrate degradation of tumor suppressor p53 by HPV-E6 pathway
through p21WAF1 inhibition causes malignant transformation for
cervical cancer development.
[0111] Protein chip assays provide tools to detect HPV proteins as
well as cellular protein induced and/or inhibited by HPV infection
in malignant of cancer development. Study results using protein
chip assay described in the invention indicate that patients with
cervical cancer in this study adopt various pathways, thus progress
differently. This technology applies to other HPV associated
cancers to predict the pathways involved in malignance. An
algorithm can be developed to predict the signature of all proteins
involved in the pathways during cancer development. Thus,
recommendation of specific treatment for personalized medicine can
be provided.
B. Detection of HPV Proteins in Biological Samples Using a First
Anti-HPV Antibody and a Second Anti-HPV Antibody as the Capture
Antibody and the Detection Antibody in a Sandwich Assay
[0112] As an example, an antigen sandwich assay involves coating a
first antibody, such as a capture antibody or a spotting antibody,
having an affinity for binding to an antigen of interest, on a
surface, such as bottom surfaces of a protein chip, a membrane
and/or a microtiter plate, etc. The antigen of interest may be, for
example, a papillomarivus protein, an oncoprotein, a capsid
protein, which may be encoded by a HPV viral gene, e.g., an early
gene or a late gene, etc. After blocking unbound portions on the
surface, the clinical sample to be analyzed can be applied to bind
with the capture antibody to form an immunocomplex, which can be
detected by a second antibody or a detection antibody by binding to
the antigen of interest. Hence, the first and the second antibodies
or the pair of the capture antibody and the detection antibody
interact with the antigen of interest, much like a sandwich. The
capture or spotting antibody can be the same or different antibody
as the detection antibody, as long as the two antibodies can
specifically bind to the antigen of interest, e.g., a HPV viral
protein, a HPV oncoprotein, a capsid protein, among others.
[0113] Next, the sandwiched bound antibody-antigen complex can be
detected by a secondary antibody, which has an affinity for the
detection antibody and facilitates measurement by a standard
immunological complex detection system using colormetric,
chemilluminescent, fluorescent and many different kinds of
substrates. The final readouts or visualizations can be performed
by an instrument with appropriate light absorbance readers or
directly visualized by eye and compared to a control sample.
Positive results indicate binding of the antigen of interest to the
primary antibodies, the capture antibody, and the detection
antibody, and thus the presence of the antigen of interest in the
clinical sample. On the contrary, negative results indicate no
binding of the antigen of interest to the primary antibodies and
thus the absence of the antigen of interest in the clinical
sample.
1. ELISA: Coating First Anti-HPV Antibody on Microtiterplate to
Detect HPV Proteins by a Second Anti-HPV Antibody
[0114] To demonstrate a sandwich ELISA on microtiter plate, serum
diagnosed with SCC or HPV PCR pos or HPV PCR neg was diluted and
used as the analyte for the detection of HPV E6, E7, or L1 protein
present in the serum sample. The assay format is demonstrated
herein as the ELISA sandwich assay using rabbit polyclonal antibody
for E6, E7, or L1 protein as the coating antibody or the first
anti-HPV antibody followed by analyte (serum) and its corresponding
second anti-HPV antibody detected by another antibody conjugated
with HRP. After incubation with substrate and stopper, OD 450 was
taken by a microtiter plate reader. As an example shown in FIG. 13,
the results demonstrate an ELISA detecting HPV E6, E7 and L1
protein in human serum sample diagnosed with SCC (squamous cell
carcinoma) or HPV positive (by PCR) compared to a HPV negative
serum (by PCR) using rabbit polyclonal anti-HPV antibody for
coating and detection.
[0115] FIG. 13 demonstrates the presence of the E6, E7
oncoproteins, and L1 viral proteins can be detected in serum from
patients diagnosed with SCC or HPV PCR positive samples using serum
from HPV PCR negative samples as a control. The data indicate that
E7 is the predominant protein detected from serum compared to E6,
or L1. Both SCC and HPV PCR pos samples have predominant E6 and E7
protein expression compared to the serum from PCR neg sample. It's
noted that SCC has predominant L1 detection compared to HPV PCR neg
serum, while HPV PCR pos sample did not have predominant L1
expression compared to HPV PCR neg sample. However, expression of
L1 protein is not as predominant as E6, or E7 in either case. These
data indicate expression of L1 may be present or absent in the
serum depending on the stage and/or cycles of viral infection.
However, detection of oncoproteins E6, or E7 in serum from both SCC
and HPV PCR pos sample suggests that E6 or E7 represents a better
marker for HPV detection in serum. This is the first report for
detection of E6, E7 oncoproteins from serum. More serum samples
needed to be analyzed.
2. Flow Beads Assay: Coating First Anti-HPV Antibody on Beads to
Detect HPV Proteins by a Second Anti-HPV Antibody
[0116] As an example, a first anti-HPV antibody coated on the
surface of the beads reacts with HPV proteins in cell lysate of
biological sample, forming a complex on the surface of the beads to
capture a second anti-HPV antibody. The complex can be detected
directly when the second anti-HPV antibody is pre-labeled, or can
be detected by adding a pre-labeled antibody capable of binding to
the second anti-HPV antibody. The pre-labeled antibody can be
labeled with a detection agent including, but not limited to, horse
radish peroxidase conjugate, biotin, gold particle, fluorescent,
and combinations thereof. As an example, the complex present on the
solid surface of beads can be detected by FACS
(Fluorescence-activated cell sorting) using anti-mouse or
anti-rabbit PE as the secondary antibody. When multiple HPV
proteins are captured on the beads, multiple second anti-HPV
antibodies labeled by different fluorescent dye can be detected
simultaneously by the FACS. Thus, the beads assay by FACS provides
powerful multiplex assay for detection of one or more HPV proteins
from biological samples.
[0117] To demonstrate the beads assay described herein can be used
for detection of various HPV proteins, various antibodies against
HPV E6, HPV E7 and HPV L1 were used as the coating and detecting
antibody to detect HPV E6, HPV E7, and HPV L1 proteins. FIG.
14-FIG. 17 show results of beads assay detecting HPV 16 L1, HPV
16E6, HPV 18 E6, and HPV 16E7 protein, respectively, by FACS. As an
example, FIG. 14 shows results of a sandwich beads assay by FACS to
detect recombinant HPV16 L1 protein using a rabbit polyclonal
anti-HPV16 L1 antibody as coating antibody and a mouse monoclonal
anti-HPV16 L1 antibody as detecting antibody followed by a
secondary antibody, anti-mouse conjugated with PE agent. As data
indicated, sample containing purified recombinant HPV 16 L1
protein, which is captured on the surface of the beads to be
detected by FACS, shows a discrete peak with higher fluorescent PE
(the peak on the right in FIG. 14) than sample containing buffer as
a negative control of the assay (the peak on the left in FIG. 14).
Results indicate about 250 fold differences in fluorescence between
the sample containing the specific detecting protein (geometric
mean about 2958) and the control sample (geometric mean about 12)
with no detecting protein present. These data suggest this beads
format provides dynamic range of assays allowed to detect various
amount of HPV L1 proteins present in the clinical samples.
[0118] As an another example, FIG. 15 shows results of a sandwich
beads assay by FACS to detect recombinant HPV16E6 protein using a
rabbit polyclonal anti-HPV16 E6 antibody as coating antibody and a
mouse monoclonal anti-HPV16 E6 antibody as detecting antibody
followed by a secondary antibody, anti-mouse conjugated with PE
agent. As data has indicated, sample containing purified
recombinant HPV 16 E6 protein which is captured on the surface of
the beads to be detected by FACS shows a discrete peak with higher
fluorescent PE (the peak on the right in FIG. 15) than sample
containing buffer as a negative control of the assay (the peak on
the left in FIG. 15). Results indicate about a 7 fold difference in
fluorescence between the sample containing the specific detecting
protein (geometric mean about 114) and the control sample
(geometric mean about 17) with no detecting protein present. These
data suggest this beads format provides dynamic range of assays
allowed to detect various amounts of HPV E6 proteins present in the
clinical samples.
[0119] FIG. 16 shows results of a sandwich beads assay by FACS to
detect recombinant HPV16 L1 protein using a rabbit polyclonal
anti-HPV18 E6 antibody as coating antibody and a mouse monoclonal
anti-HPV18 E6 antibody as detecting antibody followed by a
secondary antibody, anti-mouse conjugated with PE agent. As the
data has indicated, sample containing purified recombinant HPV 18
E6 protein which is captured on the surface of the beads to be
detected by FACS shows a discrete peak with higher fluorescent PE
(the peak on the right in FIG. 16) than sample containing buffer as
a negative control of the assay (the peak on the left in FIG. 16).
Results indicate about a 15 fold difference in fluorescence between
the sample containing the specific detecting protein (geometric
mean about 2294) and the control sample (geometric mean about 148)
with no detecting protein present. These data suggest this beads
format provides dynamic range of assays allowed to detect various
amounts of HPV 18E6 proteins present in the clinical samples.
[0120] FIG. 17 shows results of a sandwich beads assay by FACS to
detect recombinant HPV16 E7 protein using a rabbit polyclonal
anti-HPV16 E7 antibody as coating antibody and a mouse monoclonal
anti-HPV16 E7 antibody as detecting antibody followed by a
secondary antibody, anti-mouse conjugated with PE agent. As the
data has indicated, sample containing purified recombinant HPV 16
E7 protein which is captured on the surface of the beads to be
detected by FACS shows a discrete peak with higher fluorescent PE
(the peak on the right in FIG. 17) from sample containing buffer as
a negative control of the assay (the peak on the left in FIG. 17).
Results indicate about a 122 fold difference in fluorescence
between the sample containing the specific detecting protein
(geometric mean about 673) and the control sample (geometric mean
about 5.5) with no detecting protein present. These data suggest
this beads format provides dynamic range of assays allowed to
detect various amounts of HPV16 E7 proteins present in the clinical
samples.
[0121] To demonstrate assay performance varies from different assay
format by changing the antibody from coating to detecting antibody,
FIG. 18-FIG. 19 show different assays for detecting of HPV 16 E6,
and HPV 18 E6 compared to FIG. 15 and FIG. 16, respectively. As an
example, FIG. 18 shows results of a sandwich beads assay by FACS to
detect recombinant HPV16 E6 protein using a rabbit polyclonal
anti-HPV16 E6 antibody as detecting antibody and a mouse monoclonal
anti-HPV16 L1 antibody as coating antibody followed by a secondary
antibody, anti-rabbit conjugated with PE agent. As the data has
indicated, sample containing purified recombinant HPV 16 E6 protein
which is captured on the surface of the beads to be detected by
FACS shows a discrete peak with higher fluorescent PE (the peak on
the right in FIG. 18) from sample containing buffer as a negative
control of the assay (the peak on the left in FIG. 18). Results
indicate only about a 2 fold difference in fluorescence between the
sample containing the specific detecting protein (geometric mean
about 2755) and the control sample (geometric mean about 1223) with
no detecting protein present. Comparing FIG. 18 to FIG. 15 for
detection of HPV 16 E6 protein on beads assay, data suggest that
the beads format shown in FIG. 15 provides better dynamic range for
assays to allow detecting various amounts of HPV E6 proteins
present in the clinical samples.
[0122] As an another example, FIG. 19 shows results of a sandwich
bads assay by FACS to detect recombinant HPV18E6 protein using a
rabbit polyclonal anti-HPV18 E6 antibody as coating antibody and a
mouse monoclonal anti-HPV 18E6 antibody as detecting antibody
followed by a secondary antibody, anti-mouse conjugated with PE
agent. As the data has indicated, sample containing purified
recombinant HPV 18E6 protein which is captured on the surface of
the beads to be detected by FACS shows a discrete peak with higher
fluorescent PE (the peak on the right in FIG. 19) from sample
containing buffer as a negative control of the assay (the peak on
the left in FIG. 19). Results indicate about a 5 fold difference in
fluorescence between the sample containing the specific detecting
protein (geometric mean about 3803) and the control sample
(geometric mean about 787) with no detecting protein present.
Comparing FIG. 19 to FIG. 16 for detection of HPV 18 E6 protein on
beads assay, data suggest the beads format shown in FIG. 16
provides better dynamic range for assays to allow detecting various
amount of HPV E6 proteins present in the clinical samples.
3. Rapid flow through assay for detecting HPV infection: The rapid
immunological assay can be performed vertically on a membrane or
laterally in a strip. The lateral flow-through or diffusion
one-step rapid immunological assays may also be referred to as
immunochromatographic strip tests that would take about 5-15
minutes to obtain results and is easy to use, requiring limited
training and no instrumentation. The basic principles of the assay
include a solid phase nitrocellulose membrane or strip containing
the capture agent to react with a swab sample from a Pap smear. If
the patient sample contains the target agent, then the capture
agent in the nitrocellulose membrane reacts with the target agent,
and a complex is formed and migrates in the nitrocellulose membrane
through diffusion or capillary action.
[0123] The membrane or stick can also be administered to the test
human subject during sample collection and/or combined with the
cotton swabs, independently or together, to allow the designed
immunological reactions to start and thus obtain the test results
instantly, for example, right after insertion of a speculum and the
swab into the endocervix of the test human subject. Thus, the
one-step rapid immunological assay can serve as a primary screening
test. The one-step rapid immunological assay can be performed
before additional HPV confirmatory tests, including pap smear
cytological tests, the immunological assays and nucleic acid
hybridization assays as described herein, or combinations
thereof.
[0124] The vertical rapid immunological test is conducted in a
device having a membrane as a capturing/binding surface for coating
or spotting a capture agent thereon. The device further contains a
pad underneath the membrane to allow the samples and assay reagent
to flow through the membrane. Any target proteins, antibodies, or
antigens that are contained in the samples and specifically
interact and bind to the capture agent will not flow through and
will be captured and be retained on the surface of the membrane,
even after several washings to remove non-specific binding. A
secondary antibody conjugated with HRP or others enzyme that can be
applied on the surface for detecting any protein-antibody complexes
retained on the surface and be visualized by colormetric
substrates.
[0125] The one-step rapid immunological assay as provided herein is
a non-invasive and easy to run assay, similar to the types of
over-the-counter pregnancy tests without the need of any particular
test instrument. The one-step rapid immunological assay can be an
in vitro immunochromatographic assay for direct, qualitative
detection of common HPV antigens, specific antigens for high risk
HPV types, or HPV associated antibodies. The one-step rapid
immunological assay can be used as an adjunct test to Pap smear
examination, as point-of-care diagnosis, and/or small clinic
laboratory testing. The one-step rapid immunological assay is
suitable for testing at room temperature conditions by simply
adding an obtained sample, with or without dilution, waiting for a
reaction time period for the designed reactions to occur, and
scoring the results, for example, visualization of the results.
[0126] The lateral rapid immunological test is a one-step test
using a membrane strip with the capture proteins or antibodies
already applied/coated to designated positions on the surface
thereof. The only step the test requires is to combine obtained
samples having the target proteins or antibodies with a detecting
antibody conjugated with collateral gold particles and directly
apply the combined mixtures to the membrane strip for the sample
fluid to laterally flow through the membrane strip up to the
designated positions of the surface of the membrane strip. The
capture-target-detecting protein-antibody immuno-complexes can be
formed and retained on the designated positions where the capture
proteins or antibodies are coated. Positive results can be
visualized at these designated positions and no washing or
separation is required, thus this is called a one-step method. The
whole procedure for the test takes only minutes, for example, less
than 15 minutes, and thus the test is also referred to as a
one-step rapid test.
[0127] The one-step rapid immunochromatographic assay is a simple,
fast, and easy to operate assay, which can be conveniently
developed for point-of-care use. In general, there is simply mixing
of a sample to be tested with a detection antibody as developed
herein. The mixture can be applied The mixture can be applied onto
a surface (e.g., a nenvrabe it a glass) that can have the capture
antibody already fixed on the surface for a pre-determined reaction
time (e.g., in minutes, etc.) at optimized incubation temperature,
such as at room temperature. The reaction can be optimized to be
short for convenience depending on the quality of the detection
antibody used and the assay reaction conditions. Thus, a rapid
immunological test with short waiting time period can be performed
and the assay results are generally designed to be visually scored
without the need of any detection instruments.
4. One-Step HPV Lateral Flow Through Assay: Coating First Anti-HPV
Antibody on Membrane to Detect HPV Proteins by a Second Anti-HPV
Antibody Conjugated with Collateral Gold Particles
[0128] The one-step rapid immunological assay may be performed on a
membrane or stick test coated with a capture agent, e.g., purified
HPV antibodies, recombinant proteins, or HPV-associated antibodies
and proteins, etc., as described herein to capture a target agent,
e.g., HPV-associated antibodies and HPV-associated proteins, etc.,
in the clinical sample, followed by an immunoassay detection
system.
[0129] As an example, FIGS. 20A-20G show detection of HPV proteins
using antibodies described in this invention on one-step lateral
flow through. FIG. 20A shows the results of a one-step lateral flow
through for detection of HPV L1 recombinant protein using a rabbit
anti-L1 polyclonal antibody coated on membrane, and gold particle
conjugated for detection. The Test Control (TC) is shown the top
line. The second line from the top (arrowed) indicates positive
detection of the assay on the left, while the arrow on the right
with no visible band shows the negative control of the assay. The
concentration of the L1 recombinant protein from left to right is
6, 3, 1.5, 0.75, 0.375, 0 ug/ml. These data demonstrate the
one-step HPV lateral flow through assay can detect HPV L1
recombinant protein at concentration of 375 ng/ml or lower.
[0130] As an example to demonstrate the lateral flow through rapid
test can detect HPV L1 protein in clinical samples, FIG. 20B show
the results of a one-step lateral flow through for detection of HPV
L1 protein in serum sample using the same rabbit anti-HPV L1
polyclonal antibody coated on membrane shown in FIG. 20A. The Test
Control (TC) is shown on the top line. The second line from the top
(arrowed) indicates positive detection of L1 protein from serum
samples of SCC patient (on the left). Serum from a known HPV
negative by PCR with no visible band is used as the negative
control of the assay (on the right). These data demonstrate the
one-step HPV lateral flow through assay can detect HPV L1 protein
from a SCC serum sample compared to a HPV negative.serum.
[0131] To demonstrate detection of HPV E6 proteins using the
one-step lateral flow through assay, FIG. 20C shows the results of
a one-step lateral flow through assay for detection of HPV E6
recombinant protein using a mouse anti-HPV E6 monoclonal antibody
coated on membrane, and gold conjugate for detection. The Test
Control (TC) is shown on the top line. The second line from the top
(arrowed) indicates positive detection of the assay on the left,
while on the right it showed no visible band indicating the
negative control of the assay. The concentration of the E6
recombinant protein from left to right is 10, 2, 0 .mu.g/ml. These
data demonstrate the one-step HPV lateral flow through assay can
detect HPV E6 recombinant protein at concentration of 2 .mu.g/ml or
lower.
[0132] To further demonstrate the lateral flow through device can
be used for detecting HPV E6 protein in clinical sample, FIG. 20D
shows the results of a one-step lateral flow through assay for
detection of HPV E6 protein in serum sample using the same mouse
anti-E6 monoclonal antibody coated on membrane shown in FIG. 20C.
The Test Control (TC) is shown on the top line. The second line
from the top (arrowed) indicates positive detection of E6 protein
from SCC serum samples (1.sup.st and 2.sup.nd from the left). Serum
from a known HPV negative by PCR with no visible band is used as
the negative control of the assay (on the right). These data
demonstrate the one-step HPV lateral flow through assay can detect
HPV E6 protein from a SCC serum sample compared to a HPV negative
serum.
[0133] FIG. 20E shows the results of a one-step lateral flow
through assay for detection of HPV E6 recombinant protein using
another mouse anti-HPV E6 monoclonal antibody coated on membrane
and gold conjugate for detection. The Test Control (TC) is shown on
the top line. The second line from the top (arrowed) indicates
positive detection of the assay on the left, while on the right it
showed no visible band indicating that the negative control of the
assay. The concentration of the E6 recombinant protein from left to
right is 875, 438, 0 .mu.g/ml. These data demonstrate this one-step
HPV lateral flow through assay can detect HPV E6 recombinant
protein at concentration of 435 .mu.g/ml or lower.
[0134] To demonstrate detection of HPV E7 protein using lateral
flow through assay, FIG. 20F shows the results of a one-step
lateral flow through assay for detection of HPV E7 recombinant
protein using a mouse anti-HPV E7 monoclonal antibody coated on
membrane, and gold conjugated for detection. The Test Control (TC)
is shown on the top line. The second line from the top (arrowed)
indicates positive detection of the assay on the right, while on
the left it showed no visible band indicating that the negative
control of the assay. The concentration of the E7 recombinant
protein from left to right is 0, 660, 66, 6.6, 0.66, .mu.g/ml.
These data demonstrate this one-step HPV lateral flow through assay
can detect HPV E7 recombinant protein at a concentration of 660
ng/ml or lower.
[0135] To further demonstrate the lateral flow through device can
be used for detecting HPV E7 protein in linical sample, FIG. 200
show the results of a one-step lateral flow through assay for
detection of HPV E7 protein in serum sample using the mouse
anti-HPV E7 monoclonal antibody coated on membrane shown in FIG.
20F. The Test Control (TC) is shown the top line. The second line
from the top (arrow) indicates positive detection of E7 protein
from serum samples of a known HPV positive by PCR (on the left).
Serum from a known HPV negative by PCR with no visible band is used
as the negative control of the assay (on the right). These data
demonstrate the one-step HPV lateral flow through assay can detect
HPV E7 protein from a known HPV positive serum sample compared to a
HPV negative serum.
5. Expression, Purification, and Preparation of HPV Recombinant
Protein Used as Immunogens for Generating Antiserum, and Screening
for Monoclonal Antibody from Hybridoma Cell Lines
[0136] The method described in this Example can be applied to HPV
recombinant proteins from any kinds of HPV proteins, HPV proteins
of early genes or late genes, including, but not limited to, E2,
E6, E7, L1, L2 and can be from various HPV types. One aspect of the
invention provides recombinant proteins, such as recombinant hybrid
proteins containing a partial sequence or a full length sequence of
HPV oncogenic proteins. Examples include full-length E6, E7, and L1
polypeptide sequences, which have been found very difficult to
obtain and purify due to undesirable aggregation during protein
purification, protein instability, low levels of expression, and
low immunogenic responses of purified proteins. For example, many
early E6 oncoproteins contain many cysteine amino acids and thus
the correct topography of the E6 oncoproteins requires formation of
many disulfide bonds properly. In addition, it was known that
certain immunological assays using small peptides of early E6 and
E7 proteins results in extremely low assay specificity and
sensitivity and thus are unsuitable as tools for clinical in vitro
diagnostics.
[0137] 1). Cloning and production of various recombinant proteins
encoded by HPV16 E6 and HPV18 E6 gene. Cloning of an exemplary
oncogenic E6 early gene from an exemplary HPV type, HPV-16
described herein a 474 base pair (b.p.) DNA fragment containing the
157 amino acid coding region of the HPV-16 E6 gene was obtained by
polymerase chain reaction (PCR) amplification. The DNA sequence of
the isolated DNA fragment was confirmed by comparing with the
sequence from Gene Bank database. All cloning procedures were
carried out according to the protocols described in "Molecular
Cloning", A Laboratory Manual, eds. Sambrook, Fritsch and Maniatis,
Cold Spring Harbor Laboratory Press, 1989. In addition, HPV18E6,
fragments were also cloned and sequence confirmed.
[0138] 2). Cloning and production of various recombinant proteins
encoded by HPV16 E7 and HPV18 E7 gene. Cloning of an exemplary
oncogenic E7 early gene from an exemplary HPV type, HPV-16, is
described herein. A 294 base pair (b.p.) DNA fragment containing
the 99 amino acid coding region of the HPV-16 E7 gene was obtained
by polymerase chain reaction (PCR) amplification. The DNA sequence
of the isolated DNA fragment was confirmed by comparing with the
sequence from Gene Bank database. In addition, E7 DNA fragments
from different strains of HPV-16 were also cloned from different
clinical samples or sources.
[0139] The one or more recombinant proteins as described herein
were expressed in various suitable systems, such as bacterial
expression systems, viral expression systems, yeast expression
systems, mammalian expression systems, e.g., in E coli, yeast,
baculovirus, and/or mammalian cell cultures, generally known in the
field. Although the polypeptides have been obtained by other means,
embodiments of the instant invention provide one or more
recombinant proteins mostly in (or close to) their native forms
with a desirable conformation for binding with antibodies from
tissues of human subjects with HPV infection in an immunological
assay.
[0140] For example, GST, MBP, or His tagged-HPV16-E6, HPV18 E6,
HPV16 E7, HPV18 E7, HPV16 L1, and HPV18 L1 recombinant proteins
were expressed in E. coli BL21(DE3) using IPTG driven induction.
After induction of protein expression, tagged-HPV recombinant
proteins were obtained from soluble fraction after lysis of the
cultured cells and purified to a final concentration of about 0.1
to 1 mg/ml or higher. The purity of the recombinant HPV proteins
was estimated to be >90% based on PAGE analysis. Recombinant HPV
proteins were used to detect the presence of HPV antibody on
clinical samples and were also used as immunogens for production of
polyclonal antiserum and monoclonal antibodies.
[0141] The cell culture containing various recombinant
papillomavirus proteins in various expression vectors as described
herein were then scaled up to 1 liter or 10 liter, or 100 liters or
higher to obtain high quantity of soluable recombinant protein for
purification. The soluble fraction was passed through various
chromatography columns with appropriate system to bind to the tag
expressed along with the HPV recombinant proteins. The tag-HPV
recombinant proteins were then eluted from the column and
concentrated down to 100 ml or 10 ml to 1 ml. The purified soluble
recombinant HPV proteins were further concentrated and dialyzed
with buffers at neutral pH or PBS buffers to be used as immunogen
to generate antiserum against the HPV proteins. The soluble
recombinant HPV proteins were thus purified from soluble fractions
and folded close to their native folding states as in vivo natural
conditions.
[0142] Obtaining high quality purified recombinant HPV proteins is
critical in generating various types of monoclonal antibodies that
recognizing common epitopes or specific epitopes for detecting HPV
infection. The purified recombinant HPV proteins were tested to
confirm its binding to the HPV antibody from the HPV infected
clinical samples. Thus, such purified recombinant HPV proteins are
suitable for use as immunogen to raise antiserum producing antibody
recognizing the natural HPV proteins in vivo.
6. HPV Monoclonal Antibody Development:
[0143] Recombinant HPV E6, E7 or L1 proteins expressed in E coli
was purified, concentrated, and dialyzed with PBS to be used as
immunogen. Immunization of mice was performed by following the
standard procedure. Titer of serum was tested by ELISA followed by
periodical boosting and bleeding. When the titer reaches optimal,
fusion was done using standard procedure.
[0144] 1). Hybridoma screening: To obtain hybridoma cell line
producing HPV monoclonal antibody with specificity described in
this invention, fusion clones were screened against not only the
immunogen but also related or unrelated proteins as well. Two or
more purified HPV recombinant proteins were used to screen against
each hybridoma clones to obtain the specificity of each monoclonal
antibody described herein.
[0145] As an example of hybridoma screening, a monoclonal antibody
was obtained by screening antibody-producing hybridoma cells with
two or more purified recombinant human papillomavirus proteins such
that the monoclonal antibody is capable of reacting with the two or
more purified recombinant human papillomavirus proteins. The two or
more purified recombinant human papillomavirus proteins described
herein consists of HPV 16 E6 protein, HPV 16 E7 protein, HPV 16 L1
protein, HPV 18 E6 protein, HPV18 E7 protein, HPV 18 L1 protein,
and combinations thereof.
[0146] The two or more purified recombinant human papillomavirus
viral proteins are HPV early proteins such that the monoclonal
antibody is capable of reacting with the two or more human
papillomavirus early proteins. For example, the selected hybridoma
cell line produced a monoclonal antibody recognizing a common
epitope on both HPV16 E6 and HPV16 E7 proteins. As another example,
the selected hybridoma cell line produced a monoclonal antibody
recognizing a common epitope on both HPV18 E6 and HPV18 E7
proteins.
[0147] As another example, the two or more purified recombinant
human papillomavirus proteins comprise a purified recombinant human
papillomavirus early protein and a purified recombinant human
papillomavirus late protein such that the monoclonal antibody is
capable of reacting with a common epitope on the purified
recombinant human papillomavirus early protein and the purified
recombinant human papillomavirus late protein. The purified
recombinant human papillomavirus early protein consists of HPV 16
E6 protein, HPV 16 E7 protein, HPV 18 E6 protein, HPV18 E7 protein,
and combinations thereof, and the purified recombinant human
papillomavirus late protein consists of HPV 16 L1 protein, HPV 18
L1 protein, and combinations thereof.
[0148] For example, the selected hybridoma cell lines produced a
monoclonal antibody recognizing a common epitope on HPV16 E6, HPV16
E7, and HPV16 L1 proteins or a common epitope on HPV16 E6 and HPV18
E6 proteins or a common epitope on HPV16 E7 and HPV18 E7 proteins
or a common epitope on HPV16 E6, HPV16 E7, HPV16 L1, HPV18 E6, and
HPV18 E7 proteins as described in the drawings of this
invention.
[0149] As another example of hybridoma screening described in this
invention, a monoclonal antibody was obtained by screening
antibody-producing hybridoma cells with a first purified
recombinant human papillomavirus protein from a first HPV type and
a second purified recombinant human papillomavirus protein from a
second HPV type such that the monoclonal antibody is capable of
reacting with a common epitope on human papillomavirus proteins
from two or more different HPV types. The first and the second HPV
types are selected from the group consisting of HPV 16, and HPV 18.
The two or more different HPV types can also be selected from the
group consisting of high risk HPV types, low risk HPV types,
HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51,
HPV-52, HPV-56, HPV-58, HPV-59, and HPV-68, HPV-6, HPV-11, HPV-42,
HPV-43, HPV-44, HPV-53, HPV-54, HPV-55, and HPV-56, and
combinations thereof. As an example, the first and the second
purified recombinant human papillomavirus proteins consists of HPV
16 E6 protein, HPV 16 E7 protein, HPV 16 L1 protein, HPV 18 E6
protein, HPV18 E7 protein, HPV 18 L1 protein, and combinations
thereof.
[0150] As another example of hybridoma screening described in this
invention, a monoclonal antibody was obtained by screening
antibody-producing hybridoma cells with a first purified
recombinant human papillomavirus protein from a first HPV type and
a second purified recombinant human papillomavirus protein from a
second HPV type such that the monoclonal antibody is capable of
reacting with a specific epitope on only one of the first and the
second purified recombinant human papillomavirus proteins and not
the other purified recombinant human papillomavirus protein. The
first and the second purified recombinant human papillomavirus
proteins consists of HPV 16 E6 protein, HPV 16 E7 protein, HPV 16
L1 protein, HPV 18 E6 protein, HPV18 E7 protein, HPV 18 L1 protein,
and combinations thereof.
[0151] 2). Hybridoma cell line stocks: positive clones with desired
reactivity on ELISA were selected and cloned down to single cell.
Each single clone was then grown up by tissue culture. When the
cell numbers reach millions of cell per ml, the cells were frozen
down and kept at -80 C or in liquid nitrogen as stock for each cell
line.
[0152] 3). Ascites production: each cell line was grown in tissue
culture and injected to mice for ascites production. Ascites were
collected and processed for Ig purification by protein G column.
Purified Ig from each cell line was isotyped and used for HPV
immunoassays.
7. The Specificity of Anti-HPV Antibodies.
[0153] One or more immunological assays can be used to test the
specificity of the monoclonal antibodies generated by screening the
hybridoma cell lines with two or more HPV recombinant proteins. EIA
(Enzyme Immuno Assay) and/or Western blots were used as the assay
format to test the specificity of the HPV antibodies described
herein. Various purified recombinant HPV proteins, including the
original screening proteins used for obtaining the anti-HPV
antibodies and other proteins not used for screening, were used to
coat on the microtiter plate to test the specificity of the
obtained anti-HPV antibodies on EIA. Proteins in cell lysate from
cervical cancer cell lines (with or without HPV infection) were
also used to test the specificity of the anti-HPV antibodies by
western blot. To confirm the binding and reacitivity of the HPV
antibodies with proteins from HPV infected cell lines, western blot
is very useful to demonstrate specific protein bands corresponding
to the proteins present in the HPV-infected cell lines. The protein
bands from Western blots were compared to recombinant HPV proteins
at their expected molecular weight positions on SDS-PAGE gels. Cell
lysate from cervical cancer cell lines, including Hela cell line
(HPV18 positive), SiHa cell line (HPV16 postivie) and C33A cell
line (non-HPV infected) were used to demonstrate detection of HPV
E6, E7, or L1 by the HPV monoclonal antibody on western blot.
[0154] To demonstrate a monoclonal antibody capable of binding to
two or more HPV viral proteins from the same HPV type as described
in this invention, FIG. 21A and FIG. 21B show the specificity of a
monoclonal antibody, capable of reacting with both recombinant
HPV16 E6 and HPV16 E7 proteins on EIA. These data demonstrate the
monoclonal antibody described herein reacts specifically to HPV16
E6 and HPV16 E7, but not reactive to HPV16 L1, HPV18 E6 or HPV18E7.
FIG. 21A and FIG. 21B represent two different clones of hybridoma
cells, with each clone being capable of producing a monoclonal
antibody recognizing a common epitope on both HPV16 E6 and HPV16 E7
proteins.
[0155] To demonstrate a monoclonal antibody capable of binding to
an early HPV viral protein and a late HPV viral proteins from the
same HPV type as described in this invention, FIG. 22A shows the
specificity of a monoclonal antibody capable of reacting with
recombinant HPV E6, HPV E7 and HPV L1 proteins on EIA. The
recombinant protein coated on microtiter plate to be detected by
the antibody described herein is in native form. These data
demonstrate the monoclonal antibody described herein reacts
strongly to the native forms of recombinant HPV16 E6 and L1
proteins and weakly to the native form of recombinant HPV16 E7
protein, but is non-reactive to native form of recombinant HPV18 E6
or HPV18 E7. These data indicate that this antibody recognizes an
HPV 16 common epitope on the native form of HPV16E6, HPV16E7 and
HPV16 L1 protein.
[0156] FIG. 22B shows the results of a Western blot analysis of a
monoclonal antibody capable of reacting with recombinant HPV E6,
HPV E7 and HPV L1 proteins. The recombinant protein detected by
Western blot using the antibody described herein demonstrates the
detection of HPV E6 (about 18-20 kDa) and HPV L1 (about 55 kDa)
proteins. The bands from each recombinant protein shown with
expected molecular weight indicate the monoclonal antibody
described herein reacts strongly to denatured HPV16 E6 and HPV18E6
and weakly to denatured HPV L1 proteins on Western blot, and there
is no detectable reactivity to HPV16 E7 nor HPV18 E7. Comparing the
results as shown in FIG. 22A and FIG. 22B, these data indicate that
this anti-HPV monoclonal antibody recognizes an HPV common epitope
on the native forms of HPV16 E6, HPV16 E7 and HPV16 L1 protein as
well as denatured forms of HPV18 E6 recombinant protein.
[0157] FIG. 22C shows the results of a Western blot using cell
lysate from various cervical cancer cell lines to react with the
same monoclonal antibody tested in FIG. 22B binding with
recombinant HPV E6, HPV E7 and HPV L1 proteins. Both the cell
lysate and recombinant proteins in their denatured forms are tested
and shown here (the same monoclonal antibodies as shown in FIG.
22B). The double bands as detected by the monoclonal antibody
around the standard molecular weight marker of 17 kDa demonstrate
the detection of HPV E6 protein (about 18 kDa) and HPV E7 (about 15
kDa) protein from cervical cancer cell line in HeLa (HPV18) and
SiHa (HPV16) cell lines, but not C33A (non-HPV infection) cell
line. The bands on the recombinant protein lanes shown with
expected molecular weight indicate that the monoclonal antibody
reacts strongly to denatured HPV16 E6 and HPV18E6 recombinant
proteins, but weakly to denatured HPV L1 recombinant proteins on
western blot, and there is no detectable binding to HPV16E7 nor
HPV18E7 recombinant proteins.
[0158] To demonstrate a monoclonal antibody capable of binding to
an early HPV viral protein and a late HPV viral protein from
different HPV types as described in this invention, FIG. 23A shows
the specificity of a monoclonal antibody capable of reacting with
recombinant E6, E7 and L1 proteins from both HPV16 and HPV 18 on
EIA. These data demonstrate this monoclonal antibody reacts
specifically to all of the recombinant E6, E7 and L1 proteins of
HPV16, and the recombinant E6 and E7 proteins of HPV18, but not to
its common his-tag peptide. These data indicate that this antibody
recognizes a common epitope shared by HPV16 and HPV18, as evidenced
by its ability to bind to all of the recombinant HPV16 E6, HPV16
E7, HPV16 L1, HPV18 E6, and HPV18 E7 proteins.
[0159] FIG. 23B shows the results of a Western blot using a
monoclonal antibody that recognized a common epitope and is capable
of binding to the recombinant E6, E7 and L1 proteins of HPV16 and
HPV18. The reactivity of this monoclonal antibody to these
recombinant proteins demonstrate that the monoclonal antibody is
capable of recognizing E6 (about 18 kDa), E7 (About 15 kDa) and L1
(about 55 kDa) proteins. The resulting bands from each recombinant
protein lane of the Western blot analysis showed up at the expected
molecular weight position and indicated that this monoclonal
antibody reacts strongly to denatured E6 and E7 proteins from both
HPV 16 and HPV18, and weakly to denatured L1 proteins on Western
blot. The results of FIG. 23A and FIG. 23B indicate that this
monoclonal antibody recognizes an HPV common epitope and is capable
of binding to the native and denatured form of HPV16 E6, HPV16 E7,
HPV16 L1, HPV18 E7 and HPV18 E6 proteins.
[0160] FIG. 23C shows the results of a Western blot using cell
lysate from various cervical cancer cell lines to react with the
same monoclonal antibody tested in FIG. 23B binding with
recombinant HPV E6, HPV E7 and HPV L1 proteins. Both the cell lyate
and the recombinant proteins in their dentured forms were tested
and shown here (the same monoclonal antibodies as shown in FIG.
23B). The double bands as detected by the monoclonal antibody
around the standard molecular weight marker of 17 kDa demonstrate
the detection of HPV E6 protein (about 18 kDa) and HPV E7 (about 15
kDa) protein from cervical cancer cell line in HeLa (HPV18) and
SiHa (HPV16) cell lines, but not C33A (non-HPV infection) cell
line. The bands on the recombinant protein lanes shown with
expected molecular weight indicate that the monoclonal antibody
reacts strongly to denatured HPV16 E6, HPV18 E6, HPV18 E7
recombinant proteins but weakly to denatured HPV L1 recombinant
proteins, and there is no detectable binding to HPV16E7 recombinant
proteins on the Western blot.
[0161] To demonstrate a monoclonal antibody capable of binding to
two or more HPV viral proteins from different HPV types as
described in this invention, a monoclonal antibody capable of
reacting with recombinant E6 proteins of HPV 16 and HPV18 was also
the obtained. FIG. 24A shows the specificity of a monoclonal
antibody that recognizes the common epitode and is capable of
binding to recombinant HPV16 E6 and HPV18E6 proteins on EIA. The
recombinant protein coated on microtiter plate to be detected by
the antibody described herein is in its native form. These data
demonstrate that the monoclonal antibody reacts strongly to the
native form of recombinant HPV16 E6 and HPV18E6 proteins, but does
not react with the native form of either recombinant HPV E7 or
recombinant HPV L1 proteins. These data indicate that this antibody
recognizes an HPV E6 common epitope and is capable of binding to
the native form of recombinant HPV16 E6, and HPV18 E6 proteins.
[0162] FIG. 24B shows the results of a Western blot using cell
lysate from various cervical cancer cell lines to react with the
same monoclonal antibody tested in FIG. 24A binding with
recombinant E6 proteins of HPV 16 and HPV18. Both the cell lyate
and the recombinant proteins in their dentured forms were tested
and shown here (the same monoclonal antibodies as shown in FIG.
24A). The single band as detected by the monoclonal antibody around
the standard molecular weight marker of 17 kDa demonstrate the
detection of HPV E6 protein (about 18 kDa) from cervical cancer
cell line in HeLa (HPV18), but not C33A (non-HPV infection) cell
line. The bands on the recombinant protein lanes shown with
expected molecular weight indicate that the monoclonal antibody
reacts strongly to denatured HPV18E6 recombinant proteins.
[0163] As another example to demonstrate a monoclonal antibody
capable of binding to two or more HPV viral proteins from different
HPV types as described in this invention, FIG. 25 shows the
specificity of a monoclonal antibody capable of reacting with both
recombinant HPV16 E7 and HPV18E7 protein on EIA. The recombinant
protein coated on microtiter plate detected by the antibody
described herein is in its native form. These data demonstrate the
monoclonal antibody described herein reacts strongly to native form
of recombinant HPV16 E7 and HPV18 E7 proteins, but non-reactive to
native form of recombinant HPV E6 nor HPV L1 proteins. These data
indicate that this antibody recognizes an HPV E7 common epitope and
is capable of binding to the native form of HPV16 E7 and HPV18 E7
proteins.
[0164] To demonstrate a monoclonal antibody capable of binding to
only a first HPV viral protein, but not a second HPV viral protein
different from the first HPV viral protein, FIG. 26 shows the
specificity of a monoclonal antibody capable of reacting with
recombinant HPV16 E6 but not with other recombinant HPV proteins on
EIA. Data indicate the specificity of this monoclonal antibody that
recognizes a specific epitope and is capable of binding to HPV16 E6
only, and not to HPV18 E6 or other recombinant HPV proteins on EIA.
The recombinant protein coated on microtiter plate detected by the
antibody described herein is in native form. These data demonstrate
the monoclonal antibody described herein reacts strongly to the
native form of recombinant HPV16 E6 proteins but is non-reactive to
the native form of recombinant HPV E7 or L1 proteins. These data
also indicate that this antibody recognizes an HPV16 E6-specific
epitope and is capable of binding to HPV16 E6 protein only.
[0165] As an another example, FIG. 27 shows the specificity of a
monoclonal antibody capable of reacting with recombinant HPV18 E6
protein but not with other recombinant HPV proteins on EIA. Data
indicate the specificity of this monoclonal antibody that
recognizes a specific epitope and is capable of binding to HPV18 E6
only, but not to HPV16 E6 or other recombinant HPV proteins. The
recombinant protein coated on microtiter plate detected by the
antibody described herein is in native form. These data demonstrate
the monoclonal antibody described herein reacts strongly to the
native form of recombinant HPV18 E6 proteins but is non-reactive to
the native form of recombinant HPV E7 or HPV L1 proteins. These
data indicate that this antibody recognizes an HPV18 E6-specific
epitope and is capable of binding to HPV18 E6 protein only.
[0166] FIG. 27B shows the results of a Western blot using cell
lysate from various cervical cancer cell lines to react with the
same monoclonal antibody tested in FIG. 27A binding with
recombinant HPV 18 E6 proteins. Both the cell lyate and the
recombinant proteins in their dentured forms were tested and shown
here (the same monoclonal antibodies as shown in FIG. 27A). The
single band as detected by the monoclonal antibody around the
standard molecular weight marker of 17 kDa demonstrate the
detection of HPV E6 protein (about 18 kDa) from cervical cancer
cell line in HeLa (HPV18), but not C33A (non-HPV infection) cell
line. The bands on the recombinant protein lanes shown with
expected molecular weight indicate that this monoclonal antibody
reacts strongly to denatured HPV18E6 recombinant proteins only.
[0167] As another example, FIG. 28 shows the specificity of a
monoclonal antibody capable of reacting with recombinant HPV16 E7
but not with other recombinant HPV proteins on EIA. Data indicate
the specificity of the monoclonal antibody that recognizes specific
epitope and is capable of binding to HPV16 E7 only, but not to
HPV18 E7 or other recombinant HPV proteins on EIA. The recombinant
protein coated on microtiter plate detected by the antibody
described herein is in its native form. These data demonstrate the
monoclonal antibody described herein reacts strongly to the native
form of recombinant HPV16 E7 proteins, but no detectable binding to
the native form of recombinant HPV E6 or L1 proteins. These data
indicate that this antibody recognizes an HPV16 E7-specific epitope
and is capable of binding to HPV16 E7 protein only.
[0168] As an another example to demonstrate a monoclonal antibody
capable of binding to only a first HPV viral protein, but not a
second HPV viral protein different from the first HPV viral
protein, FIG. 29 shows the specificity of a monoclonal antibody
capable of reacting with recombinant HPV18 E7 but not with other
recombinant HPV proteins on EIA. Data indicate the specificity of
the monoclonal antibody that recognizes a specific epitope and is
capable of binding to HPV18 E7 only, and not to HPV16 E7 or other
recombinant HPV proteins. The recombinant protein coated on
microtiter plate detected by the antibody described herein is in
native form. These data demonstrate the monoclonal antibody
described herein reacts strongly to the native form of recombinant
HPV18 E7 proteins but is non-reactive to native form of recombinant
HPV E6 or HPV L1 proteins. These data indicate that this antibody
recognizes an HPV18 E7-specific epitope and is capable of binding
to HPV18 E7 protein only.
[0169] As an another example to demonstrate a monoclonal antibody
capable of binding to only a first HPV viral protein, but not a
second HPV viral protein different from the first HPV viral
protein, FIG. 30 shows the specificity of a monoclonal antibody
capable of reacting with recombinant HPV16 L1-N terminal but not
with other recombinant HPV proteins on EIA. Data indicate the
specificity of the monoclonal antibody that recognizes a specific
epitope and is capable of binding to HPV16 L1 N-terminal only, but
does not corssed react with HPV16 L1 or other recombinant HPV
proteins on EIA. The recombinant protein coated on microtiter plate
detected by the antibody described herein is in its native form.
These data demonstrate the monoclonal antibody described herein
reacts strongly to the native form of recombinant HPV16 L1-N
terminal proteins but is non-reactive to the native form of
recombinant HPV E6 or HPV E7 proteins. These data indicate that
this antibody recognizes an HPV16 L1 N-terminal specific epitope
and is capable of binding to HPV16 L1 N-terminal protein only. FIG.
30A and FIG. 30B represent two different hybridoma clones of cell
line producing antibody specific to HPV L1 proteins.
[0170] As an another example, FIG. 31 shows the specificity of a
monoclonal antibody capable of reacting with recombinant HPV16 L1
and HPV16 L1-N terminal but not with other recombinant HPV proteins
on EIA. Data indicate the specificity of the monoclonal antibody
that recognizes a specific epitope and is capable of binding to
HPV16 L1 and HPV16 L1 N-terminal only, and not to other recombinant
HPV proteins on EIA. The recombinant protein coated on microtiter
plate detected by the antibody described herein is in native form.
These data demonstrate the monoclonal antibody described herein
reacts strongly to the native form of recombinant HPV 16 L1 and
HPV16 L1-N terminal proteins, but is non-reactive to the native
form of recombinant HPV E6 or HPV E7 proteins. These data indicate
that this antibody recognizes an HPV16 L1 N-terminal-specific
epitope and is capable of binding to HPV16 L1 and HPV16 L1
N-terminal protein.
[0171] To demonstrate the antibodies described in this invention
can be used in various immunoassay, a sandwich ELISA was performed.
The assay includes 1). Coating first anti-HPV antibody on the
microtiter plate, 2). Adding HPV protein to react with the first
antibody, thus to be captured on the surface of microtiter plate,
3). Adding a second anti-HPV antibody which is conjugated with HRP,
followed by TMB substrate to report the binding activity by ELISA
reader. The sandwich assay provides specific binding of the protein
with the first and the second antibody and thus differentiates the
antibody specificity for the proteins on the surface to be detected
by the ELISA reader. Various antibodies described in this invention
were applied in the coating and detection to demonstrate the
antibody specificity. As an example, Table 3 shows the experiment
design and result of an ELISA (Enzyme linked Immuno Sandwich Assay)
to detect the presence of HPV18 E6 recombinant protein. The results
show HPV18E6 recombinant protein can be detected in the assay when
the coating and detecting antibody are capable of reacting with
HPV18E6, while HPV16E6 recombinant protein can't be detected if the
coating antibody is capable of binding to HPV16E6 but the detecting
antibody reacts with HPV18E6 only. Similar results were obtained
when using an HPV18 E6-specific antibody as a coating followed by a
detecting antibody capable of binding to both HPV16E6 and HPV18E6.
Data demonstrate the specificity of the antibody recognizes HPV18
E6 when HPV18 E6 recombinant protein is used as the testing protein
in the sandwich assay but is non-reactive to recombinant HPV16 E6
protein as the antigen of the sandwich assay. The assay format
described herein can be used to detect HPV18 E6 proteins present in
biological samples, including but not limited to cell lysate from
cervical cancer cell lines, cervical scrape samples, tissue, body
fluid, serum, etc. This specific sandwich assay provides type
specific assay for HPV 18, and thus excludes the binding of HPV16
E6.
TABLE-US-00003 TABLE 3 Sandwich ELISA for detecting of HPV E6
protein coating antibody anti- anti- anti-HPV18E6 anti-HPV18E6
HPV16&18E6 HPV16&18E6 testing protein recombinant
recombinant recombinant recombinant HPV18E6 HPV16E6 HPV18E6 HPV16E6
detecting antibody anti-HPV18E6 anti-HPV18E6 anti- anti-
HPV16&18E6 HPV16&18E6 ELISA results 1.5 0.05 1.45 0.05
(OD450)
[0172] As an another example to demonstrate the antibodies
described in this invention can be used to detect HPV E7 protein,
Table 4 shows the result of ELISA to detect the presence of HPV18
E7 recombinant protein using a monoclonal antibody against HPV 18
E7 (recognizing HPV 18 E7) for both the coating and the detecting
antibody. Data demonstrate that the specificity of the antibody
recognizes HPV18E7 using HPV18E7 recombinant protein as the antigen
of the sandwich assay but is non-reactive using HPV16E7 as the
antigen of the sandwich assay. The assay format described herein
can be used to detect HPV E7 proteins present in biological
samples, including but not limited to cell lysate from cervical
cancer cell lines, cervical scrape samples, tissue, body fluid,
serum, etc. This sandwich assay provides an E7-specific assay for
HPV 18, and thus is useful for the screening of HPV infection and
the detecting of HPV E7 oncogenic proteins.
TABLE-US-00004 TABLE 4 Sandwich ELISA for detecting of HPV E7
protein coating antibody anti-HPV18E7 anti-HPV18E7 testing protein
recombinant recombinant HPV18E7 HPV16E7 detecting antibody
anti-HPV18E7 anti-HPV18E6 ELISA results 1.25 0.04 (OD450)
[0173] 8. Application of the Anti-HPV Antibodies.
[0174] The HPV antibodies described in this invention can be used
in various immunoassays for detecting general HPV infection as well
as infection by various specific HPV genotypes, high risk HPVs and
low risk HPVs. The samples to be used in detecting the presence of
HPV proteins can be obtained from, but are not limited to, cervical
tissues, cervical cells, cervical scrapes, serum, and body fluids.
The immunoassays useful for screening or diagnosing cervical cancer
or HPV infection include IHC assays, ICC assays, flow cytometry
assays, assays using antibodies coupled to beads, rapid tests,
protein chip assays, immunoassays with dot blots, immunoassays with
slots, as well a conventional ELISA assay. As a screening test, the
HPV antibodies can be used to detect HPV proteins in situ present
in epithelium cells of cervical scrape from general population in
cervical cancer screening as evidenced by ICC staining scored by
certified cytologists. As a confirming test, the HPV antibodies can
also be used to detect HPV proteins in situ present in epithelium
tissue as evidenced by IHC staining scored by certified
pathologists.
[0175] 1). The reactivity of the purified anti-HPV Antibodies with
HPV Proteins found in Biological Samples. To confirm the binding
activity of the HPV antibodies with HPV proteins, purified HPV
recombinant proteins and/or HPV containing cell lysate from
biological samples can be tested on ELISA or direct EIA. Biological
samples include, but are not limited to, cells from cultured cell
lines or from clinical samples. As an example, as data shown on
Table 5, monoclonal antibodies specific to HPV E6, HPV E7 or HPV L1
proteins were able to react specifically with cell lysate from
various cervical cancer cell lines in a direct EIA format while
using HEC-1A as negative control. Cell lysate from cervical cancer
cell lines, including Caski, Siha, Cxca, Hela, and endometrial
cancer cell line like HEC-1A (non-HPV infected) were used to
demonstrate detection of HPV E6, E7, or L1 by the HPV monoclonal
antibody specific to HPV E6, HPV E7, and HPV L1 respectively as
shown in Table 5.
TABLE-US-00005 TABLE 5 EIA detection of E6, E7, and L1 proteins in
cervical cancer cell lines. Anti-HPV16 Anti- Anti- Anti- Anti- E6,
HPV18 HPV18 E6 HPV18 E7 HPV16 E7 HPV16 L1 OD E6 antibody antibody
antibody antibody antibody Caski 0.392 0.48 0.442 0.464 0.355
(HPV16.sup.+) SiHa 1.165 1.314 1.162 1.202 1.115 (HPV16.sup.+) CxCa
1.126 1.047 0.802 0.825 0.724 (HPV16.sup.+) Hela 0.779 0.762 0.734
0.654 0.652 (HPV18.sup.+) HEC-1A 0.173 0.206 0.219 0.186 0.173 (no
HPV)
[0176] Cultured cell lines tested and described herein, include,
but not limited to, cervical cancer cells such as Caski (HPV16
positive), Siha (HPV16 positive), Cxca, Hela (HPV18 positive), and
endometrial cancer cell line like HEC-1A (no HPV infection). For
direct EIA, cells were collected, centrifuged, washed, and lysed to
generate cell lysate as anyalyte. The protein in the cell lysate
was quantitated and coated to microtiter plate using the same
amount of protein for coating of each sample in each well. The
plate was blocked, and detected by each of the HPV monoclonal
antibody as indicated followed by HRP conjugated anti-mouse IgG.
TMB substrate was added followed by a standard reaction stopping
solution. OD.sub.450 was taken by an ELISA plate reader.
[0177] 2). The reactivity of the purified anti-HPV Antibodies with
HPV Proteins found in clinical samples. Clinical samples to be
tested and described herein include, but not limited to, cells from
cervical scrapes, body fluid, or serum samples Clinical specimens
from cervical scrapes were also obtained for detection of HPV E6,
E7 or L1 protens on EIA.
[0178] Cell lysate from various sample source including cervical
scrape cells in liquid based solution, culture medium (used for HPV
DNA test sample), or pap smear sample demonstrate detection of HPV
E6, E7, or L1 from clinical samples on EIA format using various HPV
monoclonal antibody described in this invention. To perform the
direct EIA described herein, specimens were processed, centrifuged,
washed, and lysed to generate cell late as anyalyte. The proteins
in the cell lysate was quantitated and coated to microtiterplate
with the same amount of proteins for coating in each well. The
plate was blocked, and detected by each HPV monoclonal antibody
followed by HRP conjugated anti-mouse IgG. TMB substrate was added
followed by a stopping solution. OD.sub.450 was taken by an ELISA
plate reader.
[0179] Results shown in Table 6 indicate that each monoclonal
antibody detects HPV E6, E7, or L1 protein respectively from SCC
samples using pap smear normal (HPV neg) as neg control of the
assay. For samples from high-grade HPV DNA pos, one out of three is
positive on the E6, E7, and L1 by EIA. These data indicate that E6,
E7, or L1 proteins from SCC lysate can be detected by EIA using the
monoclonal antibodies described herein, while high-grade HPV DNA
positive samples (CIN1/2) may or may not contain detectable HPV E6,
E7, or L1 proteins. The high-grade HPV DNA test used in this study
was hc2, the only FDA approved HPV DNA test. For those HPV DNA
positive but HPV EIA negative samples, it is possible false
positive of the HPV DNA assay, or positive HPV DNA detection with
no expression of HPV oncogenic proteins. These data indicate that
HPV EIA assay described herein provides additional clinical
relevance for screening of cervical cancer.
TABLE-US-00006 TABLE 6 EIA detection of E6, E7, and L1 proteins in
cervical scrapes samples. Anti- Anti- Anti HPV18 E6 HPV16 E7 HPV L1
Samples Dx antibody antibody antibody Squamous cell carcinoma (SCC)
+++ +++ +++ Squamous cell carcinoma (SCC) +++ +++ +++ high grade
HPV DNA test positive - - - high grade HPV DNA test positive + + +
high grade HPV DNA test positive - - - pap smear normal, PCR
negative - - - pap smear normal, PCR negative - - - pap smear
normal, PCR negative - - -
[0180] 3). The reactivity of the purified anti-HPV Antibodies with
HPV Proteins in situ by Immunohistochemisty (IHC): Paraffin tissue
blocks sectioned into 4 microns were placed on slide and baked at
60.degree. C. for overnight. Deparaffin/hydrate sections were
unmasked followed by standard IHC staining procedures. Purified
monoclonal antibodies against HPV proteins as described in this
invention were diluted to use as the primary antibody. Staining
procedure was followed by secondary antibody solution and washing,
then followed by appropriate substrate reagent to each section. As
soon as the sections developed, slides were immersed in distilled
water, sections were counterstained with hematoxylin and
dehydrated, and the coverslips were mounted.
[0181] As an example, various cervical tissues from various stages
of CIN were prepared to perform IHC assay using rabbit polyclonal
anti-HPV E7 antibodies described herein. As another examples, a
number of cervical biopsy samples were tested in an
immunohistohistochemistry (IHC) assay concurrently as a tissue
microarray format using a monoclonal antibody to detect HPV
proteins from a variety of HPV types (as confirmed by HPV DNA
genotyping). Using a monoclonal antibody against HPV viral proteins
and/or oncoproteins, this invention provides antibodies to detect
the presence of HPV L1 viral proteins and E6, E7 oncoproteins in
clinical samples having either single HPV infection or multiple HPV
infections. A single anti-HPV monoclonal antibody as described
herein can detect single HPV infection by at least HPV-6, HPV-16,
HPV-18, HPV-31, HPV-33, HPV-52, etc, which are cancer-related HPV
types (either high risk HPV types or low risk HPV types). A single
anti-HPV monoclonal antibody can detect HPV infection by two or
more HPV types, such as the combination of HPV-16, HPV-18, HPV-52,
HPV-58, HPV-44, HPV-51, HPV-39, HPV-59, etc., which include high
risk, low risk, and non-oncogenic .alpha.-papillomaviruses.
[0182] As an example, the HPV antibodies described in this
invention can be applied in clinical utility. The results of the
IHC assay demonstrate detection of the HPV E7 protein present in
situ from various stages of cervical tissues using a mouse
monoclonal anti-HPV E7 antibody. As another example, the antibodies
described herein were also used in ICC assay using various cervical
tissues from various stages of CIN. As another examples, results of
IHC staining using a mouse monoclonal anti-HPV E6 antibody
demonstrate detecting the HPV E6 protein present in situ from
various stages of CIN tissues. These results indicate that HPV E6
and HPV E7 oncoproteins over-expressed in the dysplasia cells can
be specificly detected by the IHC staining using the specific HPV
antibodies.
[0183] As an example, FIGS. 12A-12D show IHC staining of CIN tissue
demonstrated by a mouse monoclonal anti-HPV E6 antibody. Results
indicate expression of E6 oncoprotein can be detected early in the
precancerous stage of CIN2. Solid Black arrows indicate the
specific staining of E6 protein in dysplasia cells, while empty
clear arrows indicate the normal cells with no stain. Highly
magnified images indicate localization of the E6 proteins expressed
early in the nuclear of dysplasia cells.
[0184] FIG. 32A shows the representative image of the dysplasia
cells of CIN2 tissues stained by immunohistocytostaining (IHC)
using an anti-E6 monoclonal antibody. FIG. 32B shows the
representative image of the adjacent normal epithelium from the
dysplasia tissue of the CIN2 sample of FIG. 32A. FIG. 32C-32D shows
the representative image of the dysplasia epithelium of two CIN3
samples stained by IHC using the same anti-E6 monoclonal antibody.
These data suggest the IHC staining by E6 monoclonal antibody is
specific in the nuclear and cytoplasm of dysplasia cells.
[0185] As an another example, FIGS. 13A-13D show IHC staining of
squamous cell carcinoma demonstrated by mouse monoclonal HPV E7
antibody. Results indicate expression of E7 oncoprotein can be
detected in the tumor cells of SCC tissue. Solid Black arrows
indicate the specific staining of E7 protein in dysplasia cells,
while empty clear arrows indicate the normal cells with no stain.
Highly magnified images indicate localization of the E7 proteins
expressed in the cytoplasm of tumor cells, but not in the normal
epithelium, or stroma cells. These data suggest the IHC staining by
E7 monoclonal antibody is specific in the cytoplasm of tumor cells.
FIG. 33A shows the representative image of the squamocarcinoma
(SCC) tissue from tissue microarray stained by IHC using an anti-E7
monoclonal antibody. FIG. 33B shows the representative image of the
normal epithelium (15 mm away from the tumor tissue) of the SCC
subject from FIG. 33A. FIG. 33C shows the representative image of
another SCC sample from tissue microarray stained by IHC using the
same anti-E7 monoclonal antibody. FIG. 33D shows the magnified
representative image of the tumor cells stained in cytoplasm from
FIG. 33C.
[0186] 9. The Reactivity of the Purified Anti-HPV Antibodies with
HPV Proteins in Situ by Immunocytochemistry (ICC):
[0187] Cervical scrapes collected by Liquid based solution were
processed according to the manufacture instruction. The cell
preparation was divided into two parts, one for conventional
papsmear, the other one for immunostaining. Monolayer of cervical
cells on slide was processed by cytospin or thin prep techniques.
The cells were then fixed and stained followed by immunostaining
protocol. Stained cells are visualized under microscope.
[0188] As an example, FIG. 34A-34C demonstrate immunocytochemistry
assay using anti-HPV antibody. FIG. 34A shows the representative
image of cervical cells from a CIN2 cervical scrape sample prepared
by thin prep and stained by ICC using a mouse monoclonal anti-HPV
E7 antibody. FIG. 34B shows the representative image of cervical
cells from a CIN3 cervical scrape sample prepared by thin prep and
stained by ICC using a mouse monoclonal anti-E 6 antibody. FIG. 34C
shows the representative image of cervical cells from an
adenocarcinoma (ADC) cervical scrape sample prepared by thin prep
and stained by ICC using the same anti-E6 antibody shown in FIG.
34B.
[0189] The one or more immunological assays using antibodies and
purified recombinants proteins derived from HPV early and/or late
genes as obtained herein serve as reliable indicators whether HPV
infection has occurred. In addition, HPV associated malignancy or
pre-malignant cell transformation can be assayed. One of the most
useful aspects of the invention is in diagnosing cervical
carcinoma, both squamous cell carcinoma and adenocarcinoma as well
as any epithelial cell abnormality associated with oncogenic HPV
infection including koilocytosis; hyperkerotosis; precancerous
conditions encompasssing intraepithelial neoplasias or
intraepithelial lesion; high-grade dysplasias; and invasive or
malignant cancers.
[0190] In high grade CIN lesions, E6 and E7 are strongly expressed
in host basal epithelial cells and interfere substantially with
cell cycle control of these replication competent host cells.
Expression of HPV oncoproteins interfers with .beta.1-S-Phase
regulation in host cells. The HPV E6 and E7 proteins target a
plethora of cellular interactions, such as the inactivation of pRB
by E7 and the degradation of p53 by E6. High level of HPV E7
proteins inactivates pRB and leads to disruption of E2F-Rb binding.
Usually, binding of pRB to E2F blocks E2F driven cell cycle
activation. In replicating cells, E2F is regulated by
phosphorylation of RB. Rb phosphorylation is normally mediated by
cyclin dependent kinases (CDK4, CDK6) that are controlled by
several kinase inhibitors (INKs).
[0191] FIG. 21A shows the specificity of a monoclonal antibody
capable of reacting with both HPV16 E6 and HPV16 E7 recombinant
proteins (different HPV proterins from the same HPV type) and
recognizing a common epitope on the different HPV16 E6 and HPV16 E7
proteins from the same HPV 16 type as assayed on EIA (enzyme immuno
assays) according to one embodiment of the invention.
[0192] FIG. 21B shows the specificity of another monoclonal
antibody capable of reacting with both HPV16 E6 and HPV16 E7
recombinant proteins and recognizing a common epitope on the HPV 16
E6 and HPV16 E7 proteins as assayed on EIA according to one
embodiment of the invention.
[0193] FIG. 22A shows the specificity of a monoclonal antibody
capable of reacting with HPV16 E6, E7, L1 & L1 N-terminal
recombinant proteins (different HPV proteins from the same HPV
type) and recognizing a common epitope on the different E6, E7, L1,
and L1 N-terminal proteins from the same HPV 16 type as assayed on
EIA according to another embodiment of the invention.
[0194] FIG. 22B shows a western blot of the monoclonal antibody as
shown in FIG. 22A, confirming its binding to all of the HPV16 E6,
E7 and L1 recombinant proteins.
[0195] FIG. 22C shows the results of a western blot of cell lysate
from cervical cancer cell lines using the monoclonal antibody as
shown in FIG. 22A, confirming its binding to all of the HPV16 E6,
E7 and L1 viral proteins present in these cervical cancer cell
lines.
[0196] FIG. 23A shows the specificity of a monoclonal antibody
capable of binding to all of the recombinant HPV16 E6, E7, and L1
N-terminal proteins as well as HPV18 E6 and E7 proteins (HPV
proteins from different HPV types) and recognizing a common epitope
on the E6, E7, L1 N-terminal proteins from HPV16 and HPV18 as
assayed on EIA according to another embodiment of the
invention.
[0197] FIG. 23B shows the results of a western blot using the
monoclonal antibody as shown in FIG. 3A, confirming its binding to
the different recombinant proteins and recognizing a common epitope
on the different E6, E7 and L1 proteins from the two different HPV
types HPV16 and HPV18.
[0198] FIG. 23C shows a western blot cell lysate from cervical
cancer cell lines using the monoclonal antibody as shown in FIG.
3A, confirming its binding to the--HPV16 E6, E7 and L1 proteins as
well as HPV18 E6, E7 and L1 viral proteins present in these
cervical cancer cell lines.
[0199] FIG. 24A shows the specificity of a monoclonal antibody
capable of binding to two E6 recombinant proteins (HPV16 E6 and
HPV18 E6, E6 proteins from different HPV types) and recognizing a
common epitope on the two E6 proteins from different HPV types as
assayed on EIA according to another embodiment of the
invention.
[0200] FIG. 24B shows the results of a western blot analyzing the
cell lysate from cervical cancer cell lines using the monoclonal
antibody as shown in FIG. 24A, confirming its binding to HPV16 E6
as well as HPV18 E6 viral proteins present in these cervical cancer
cell lines.
[0201] FIG. 25 shows the specificity of a monoclonal antibody
capable of reacting with two recombinant HPV16 E7 and HPV18 E7
proteins (E7 proteins from different HPV types) and recognizing a
common epitope on the two E7 proteins from different HPV types as
assayed on EIA.
[0202] FIG. 26 shows the specificity of a monoclonal antibody
capable of reacting with only HPV16 E6 recombinant protein but not
with any other HPV recombinant proteins on EIA according to one
embodiment of the invention.
[0203] FIG. 27A shows the specificity of a monoclonal antibody
capable of reacting specifically with only HPV18 E6 recombinant
protein, but not with any other HPV16 or HPV18 recombinant proteins
as assayed on EIA according to another embodiment of the
invention.
[0204] FIG. 27B shows the results of a western blot analyzing the
cell lysate from different cervical cancer cell lines using the
monoclonal antibody as shown in FIG. 27A, confirming its binding to
the--HPV18 E6 viral protein but not HPV E7 viral protein that are
present in Hela cell line.
[0205] FIG. 28 shows the specificity of a monoclonal antibody
capable of reacting specifically with an HPV16 E7 recombinant
protein, but not with any other HPV recombinant proteins as assayed
on EIA according to another embodiment of the invention.
[0206] FIG. 29 shows the specificity of a monoclonal antibody
capable of reacting specifically with a recombinant HPV18 E7
recombinant protein, but not with any other HPV recombinant
proteins as assayed on EIA according to another embodiment of the
invention.
[0207] FIG. 30A shows the specificity of a monoclonal antibody
capable of reacting specifically with an HPV16 L1 N-terminal
recombinant protein, but not with any other HPV recombinant
proteins as assayed on EIA according to another embodiment of the
invention.
[0208] FIG. 30B shows the specificity of another monoclonal
antibody capable of reacting specifically with an HPV16 L1
N-terminal recombinant protein, but not with any other HPV
recombinant proteins as assayed on EIA.
[0209] FIG. 31 shows the specificity of a monoclonal antibody
capable of reacting specifically with only the HPV16 L1 & L1
N-terminal recombinant proteins, but not with any other HPV
recombinant proteins as assayed on EIA according to another
embodiment of the invention.
[0210] FIG. 32A shows the representative staining image of the
dysplasia cells of CIN2 tissues using an anti-E6 monoclonal
antibody in an immunohistocytostaining (IHC) assay.
[0211] FIG. 32B shows the representative staining image of the
normal epithelium adjacent to the dysplasia tissue of the CIN2
sample in FIG. 32A.
[0212] FIG. 32C shows the representative staining image of the
dysplasia epithelium of a CIN3 sample stained by the same anti-E6
monoclonal antibody as used in FIG. 32A in an IHC assay,
demonstrating specific IHC staining in the nuclear and cytoplasm of
dysplasia cells by the anti-E6 monoclonal antibody.
[0213] FIG. 32D shows the representative staining image of the
dysplasia epithelium of another CIN3 sample stained by the same
anti-E6 monoclonal antibody as used in FIG. 32A in an IHC
assay.
[0214] FIG. 33A shows the representative staining image of the
squamocarcinoma (SCC) tissue from tissue microarray using an
anti-E7 monoclonal antibody in an immunohistocytostaining (IHC)
assay.
[0215] FIG. 33B shows the representative staining image of the
normal epithelium (about 15 mm away from the tumor tissue) adjacent
the SCC tissue of FIG. 33A.
[0216] FIG. 33C shows the representative staining image of another
SCC sample stained by the same anti-E7 monoclonal antibody as used
in FIG. 33A in an IHC assay, demonstrating specific IHC staining in
the tumor cells by the anti-E7 monoclonal antibody.
[0217] FIG. 33D shows the magnified representative image of the
tumor cells from FIG. 33C to view the staining of the cytoplasm of
the tumor cells.
[0218] FIG. 34A shows the representative staining image of cervical
cells from a CIN2 cervical scrape sample prepared by thin prep and
stained by a mouse monoclonal anti-HPV E7 antibody in an
immunocytochemistry (ICC) assay.
[0219] FIG. 34B shows the representative staining image of cervical
cells from a CIN3 cervical scrape sample prepared by thin prep and
stained by a mouse monoclonal anti-E6 antibody in an ICC assay.
[0220] FIG. 34C shows the representative image of cervical cells
from an adenocarcinoma (ADC) cervical scrape sample prepared by
thin prep and stained by the same anti-E6 antibody shown in FIG.
34B in an ICC assay.
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