U.S. patent application number 12/400629 was filed with the patent office on 2009-07-16 for kits for detecting cervical dysplasia.
Invention is credited to Magnus Von Knebel Doeberitz, Matthias Herkert, Anja Reichert, Rudiger Ridder, Wolfgang Rudy, Marcus Trunk-Gehmacher.
Application Number | 20090181406 12/400629 |
Document ID | / |
Family ID | 30129176 |
Filed Date | 2009-07-16 |
United States Patent
Application |
20090181406 |
Kind Code |
A1 |
Ridder; Rudiger ; et
al. |
July 16, 2009 |
KITS FOR DETECTING CERVICAL DYSPLASIA
Abstract
This invention provides methods and kits for improved diagnosis
of medically relevant conditions by solution based biochemical
testing procedures performed in solutions of test samples. The
invention provides a method to substitute the cell based
morphological information contained within the cytological and/or
histological data of the test sample by molecular information
obtainable from the solution, wherein the original test sample is
dissolved and thus enables for accurate and reproducible assessment
of medically relevant diagnosis from dissolved test samples. The
method according to the invention comprises the steps of
determining the levels of one or more disease markers associated
with the condition to be diagnosed, determining the level of one or
more normalization markers suitable to substitute the information
related to morphological aspects of the sample, comparing and/or
combining the data of the disease and normalization markers, and
assessing diagnosis of a medically relevant condition.
Inventors: |
Ridder; Rudiger;
(Schriesheim, DE) ; Rudy; Wolfgang; (Bretten,
DE) ; Herkert; Matthias; (Heidelberg, DE) ;
Trunk-Gehmacher; Marcus; (Heidelberg, DE) ; Reichert;
Anja; (Nussloch, DE) ; Doeberitz; Magnus Von
Knebel; (Ziegelhausen, DE) |
Correspondence
Address: |
HOWREY LLP-CA
C/O IP DOCKETING DEPARTMENT, 2941 FAIRVIEW PARK DRIVE, SUITE 200
FALLS CHURCH
VA
22042-2924
US
|
Family ID: |
30129176 |
Appl. No.: |
12/400629 |
Filed: |
March 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10633484 |
Jul 31, 2003 |
7517662 |
|
|
12400629 |
|
|
|
|
Current U.S.
Class: |
435/7.1 ;
435/29 |
Current CPC
Class: |
G01N 33/57411 20130101;
G01N 33/5091 20130101; G01N 2333/82 20130101; G01N 33/5076
20130101; G01N 33/574 20130101 |
Class at
Publication: |
435/7.1 ;
435/29 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12Q 1/02 20060101 C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 1, 2002 |
EP |
EP 02017313.4 |
Claims
1. A test kit for detecting cervical dysplasia, comprising: (a) a
first reagent for detecting a first marker molecule characteristic
for cervical dysplasias selected from the group consisting of
p16.sup.INK4a, p14ARF, cyclin E, cyclin A, cyclin B, MN, her2/neu,
mdm-2, bcl-2, EGF-Receptor, mcm-2, mcm-5, claudin-1, markers
indicative for human papilloma virus infection, pRb, and p53; and
(b) a second reagent for detecting a normalization marker molecular
characteristic for the presence or absence of epithelial cells
selected from the group consisting of CK8, Ep-Cam, CK13, CK8, CK8,
E-Cadherin, alpha-Catenin, beta-Catenin, gamma-Catenin, and
involucrin.
2. The test kit according to claim 1, wherein at least one of the
first reagent and the second reagent is fixed to a solid phase.
3. The test kit according to claim 1, furthermore comprising at
least one of the following components: (a) the first marker
molecular, (b) the normalization marker molecular, and (c) reagents
and buffers commonly used for carrying out the detection
reaction.
4. The test kit according to claim 1, wherein the reagents for
detecting the first marker molecule and the normal marker molecule
comprise binding agents specific for said marker molecules.
5. The test kit according to claim 4, wherein the binding agents
are an antibody, a miniantibody, or a peptidomimetic comprising an
antigen binding epitope.
6. The test kit according to claim 1, wherein the test kit is a
diagnostic test kit, an in-vitro diagnostic device, a research kit,
or an analytical kit.
7. The test kit according to claim 1, wherein the first marker
molecule is p16.sup.INK4a, and the normalization marker molecule is
gamma-Catenin.
8. The test kit according to claim 7, further comprising a reagent
for the detection of Ep-Cam.
9. The test kit according to claim 7, further comprising a buffer
for sample lysis.
10. The test kit according to claim 7, wherein the reagents for
detecting p16.sup.INK4a and gamma-Catenin are fixed to solid
phases.
11. The test kit according to claim 7, wherein the test kit is an
in-vitro diagnostic device.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 10/633,484, filed Jul. 31, 2003, which claims benefit to a
foreign application, EP 02017313.4, filed Aug. 1, 2002. The
contents of the above applications are incorporated herein by
reference in their entirety.
REFERENCE TO ELECTRONIC SEQUENCE LISTING FILE
[0002] This application includes a sequence listing submitted
electronically herewith as an ASCII text file named "sequence.txt",
which is 53 kB in size and was created Mar. 9, 2009; the electronic
sequence listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] This invention relates to methods for performing diagnosis
of medically relevant conditions by detecting the levels of
relevant markers characteristic for the medically relevant
condition and the levels of normalization markers. The methods
pertain to characterization of the sample in a solution phase,
without relying on morphological cell based information.
BACKGROUND OF THE INVENTION
[0004] The diagnosis of a large number of medically relevant
conditions is currently performed using molecular markers as tools.
The molecular tools are generally used as one aspect in a complex
examination, taking into account a series of different parameters
characterizing the samples to be examined.
[0005] In medically relevant analysis, the morphological
examination of samples by cytological or histological means is in
common use. Such methods based on morphological characterization of
cell based samples are applicable for example in analysis of
clinical samples such as body fluids, blood, surgical resections,
secretions, swabs or lavages.
[0006] In screening for cervical cancer, for example, swabs are
used for detection of neoplastic lesions of the cervix uteri. In
the screening procedure, lesions of different origin have to be
distinguished. Causes for lesions may for example be inflammations
(due to infectious agents or physical or chemical damage) or
preneoplastic and neoplastic changes. In morphological examinations
the lesions of different characteristics are sophisticated to
distinguish. Thus, for examination of swabs, cytologists and
pathologists have to be especially trained and even experienced
examiners have a high inter- and intra-observer variance in the
assessment of a diagnosis based on cytological specimens. In
general the result of the examination is based upon the subjective
interpretation of diagnostic criteria by the examining
pathologist/cytologist. As a result the rate of false positive and
false negative results in the screening tests remains unsatisfying
high.
[0007] Therefore, in many cases these cytological or histological
examination procedures are supported by the use of molecular
markers. Such markers are often used in immuno-histochemical
staining reactions, or in the course of in-situ hybridization
reactions. In the prior art combinations of morphological
examinations and immuno-histochemical staining reactions based on
marker molecules, characteristic for different medically relevant
states of tissues or cells, may lead to enhanced results. The
morphologic examination remains laborious and time consuming and
thus expensive, even when supported by the molecular methods, that
make the results more reliable. Additionally, the diagnosis on a
morphologically cell based level is, even when supported by
molecular parameters, subject to individual perception of the
morphology by individual examiners. Thus the diagnosis is dependent
on the person, that performs the examinations.
[0008] Only in very few cases, molecular markers may be used as
diagnostic tools without further support by cell based
morphological examinations. This is especially the case, if markers
are to be detected in an environment, where they do only occur
under exactly defined conditions. So the methods for diagnosis of
conditions on a molecular level only, without the support of cell
based information, are restricted to cases, where there are
suitable markers, that are non-ambiguously specific for the
condition to be characterized. For example, detection of viral
infections may be carried out in solutions of samples, because the
markers characteristic for the presence of viruses in tissues do
not occur in unaffected human tissues.
[0009] The reproducibility of the results of examination can be
enhanced by the use of supporting molecular tools. However, the
problem with the preservation and preparation of the samples may
not be overcome by just additionally using molecular markers.
[0010] When using molecular tools in cytological or histological
examinations, strict precautions in preserving the samples have to
be taken to prevent artefacts and improper results of the tests.
This is in part due to the instability of the cell based
morphological information and in part to the instability of the
molecular markers to be detected during the tests. If the samples
are not prepared, transported or stored in the appropriate manner,
the cell based information, or even the molecular information may
get lost, or may be altered. So the diagnosis may be impossible, or
may be prone to artefacts. For example, the interpretation of
biopsies or cytological preparations is frequently made difficult
or impossible because of damaged (physically or biochemically)
cells. Regarding tissue samples or biopsies, the preservation of
molecular constituents of the samples, which are subject to a rapid
turnover, seems sophisticated due to the time elapsed until
penetration of the total sample by appropriate preservatives.
[0011] The morphologically supported diagnostic methods performed
routinely in the art show two major disadvantages. First, the
methods are highly dependent on individual perception of the
examiners. Secondly the morphological information is quite
sensitive to decay processes and thus may cause artefacts after
preparation of the samples. Both aspects contribute to improper
reproducibility of the results.
[0012] For improved diagnosis of medically relevant conditions,
methods that do not depend on cell based morphological information
would be desirable.
SUMMARY OF THE INVENTION
[0013] The present invention is directed to a method for diagnosing
a medically relevant condition of a patient. The method comprises
the steps of: obtaining a raw sample containing cells or cell
debris from a patient; preparing a sample solution from the raw
sample; detecting the levels of one or more relevant markers
characteristic for said medically relevant condition in said sample
solution; detecting the levels of one or more normalization
markers; normalizing the detected level of the relevant markers
with respect to said normalization parameters; and diagnosing the
medically relevant condition from the normalized levels of said
relevant markers within the sample solution. The normalization
markers are characteristic for at least one of the following
normalization parameters: the presence or absence of a particular
cell type among the cells represented within the sample solution,
the presence or absence of a particular differentiation pattern in
the cells represented within the sample solution, and the presence
or absence of particular proliferation properties of the cells
represented within the sample solution.
[0014] In one embodiment of the invention, the medically relevant
condition is a cell proliferative disorder, cancer or a precursory
lesion.
[0015] The present invention is also directed to a test kit for
diagnosing a medically relevant condition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows the specific immunohistochemical staining of
endocervical and ectocervical epithelial cells in cervical
sections. FIG. 1A shows a positive reaction detected in columnar
epithelium of the endocervix using an antibody directed against
cytokeratin 18 (CK18). FIG. 1B shows no specific staining in
columnar epithelium of the ectocervix using an antibody directed
against cytokeratin 18 (CK18). FIG. 1C shows no specific staining
in columnar epithelium of the endocervix using an antibody directed
against cytokeratin 10/13 (CK10/13). FIG. 1D shows a strong
staining of the squamous epithelium of the ectocervix using an
antibody directed against cytokeratin 10/13 (CK10/13).
[0017] FIG. 2 shows the Western Blot analysis of solubilized
samples from cervical swabs. The numbers 1 to 4 refer to samples
(cervical swabs) obtained from individual patients
[0018] FIG. 3 shows the Western blot and ELISA analysis to
demonstrate sample adequacy. Samples of four patients with
high-grade cervical dysplasias (see Diagnosis) were analysed using
western blot analysis (upper panel of figure). The lower panel of
this figure shows the results of ELISA analysis.
DETAILED DESCRIPTION OF THE INVENTION
[0019] This invention provides methods for improved diagnosis of
medically relevant conditions by solution-based biochemical testing
procedures performed in solutions of test samples. The invention
provides a method to substitute the cell based morphological
information contained within the cytological and/or histological
data of the test sample by molecular information obtainable from
the solution, wherein the original test sample is dissolved and
thus enables for accurate and reproducible assessment of medically
relevant diagnosis from dissolved test samples. The method
according to the invention comprises the steps of determining the
levels of one or more markers associated with the condition to be
diagnosed, determining the level of a set of normalization markers
suitable to substitute the information related to morphological
aspects of the sample, that would have enabled or supported
diagnosis in a cell based test system, comparing and/or combining
the data concerning the levels of said markers and assessing
diagnosis of a medically relevant condition.
[0020] The present invention discovers that diagnosis of
conditions, which is normally (in cell based diagnostic systems)
enabled and/or supported by histological and/or cytological
examination procedures, may be performed in solutions from raw
samples containing various cell types of different characteristics,
by a method comprising the steps of obtaining a raw sample,
dissolving the sample in an appropriate solute, detecting the level
of one or more markers associated with the condition to be
diagnosed and additionally one or more normalization markers within
the sample solution, normalizing the data correlating to the
markers associated with said condition with respect to the data
correlating to the normalization markers and diagnosing the
presence or absence of a condition in the sample.
[0021] The method according to the present invention may for
example be applied as a primary screening test in cases, where a
cytological, histological or pathological examination is normally
performed. Using the present invention one may discriminate, if the
condition to be diagnosed may be present in the sample. If the
solution based diagnosis gives a negative result concerning a
particular condition, a further examination may be omissible. In
case of positive results, ascertaining by classically applicable
methods may follow. Thus, expensive and time consuming microscopic
or other examinations could be avoided by means of an inexpensive
rapid primary screening test.
[0022] One aspect of the present invention is a method for enhanced
diagnosis of medically relevant conditions, wherein the assessment
of diagnosis is performed using solutions of lysed raw tissue- or
cell-samples. The method for diagnosis disclosed according to the
present invention does not rely on morphological parameters but
enables for a diagnosis by means of biochemical analysis.
[0023] A second aspect of the present invention is a method for
characterizing a complex sample in solution by means of molecular
markers characteristic for the parameters of interest, thus
substituting information, which could otherwise be obtainable from
cytological or histological examinations.
[0024] A third aspect of the present invention is to provide
suitable combinations of markers for the diagnosis of particular
conditions of medical relevance in complex samples. The markers for
normalization are chosen such that parameters included within the
raw sample, that enable or support the diagnosis, which are lost by
the dissolution of the sample, may be substituted.
[0025] A fourth aspect of the present invention are test kits for
performing diagnostic or research studies according to the present
invention.
[0026] The present invention enables for a rapid and easy assay for
diagnosing of conditions in raw samples such as body fluids, swabs,
ravages (e.g. bronchio-alveolar lavages, breast ductal lavages,
etc.), aspirates (needle-apirates, fine-needle-aspirates) or
complex cell- or tissue samples. In general, a problem with raw
materials is the presence of a number of different cell-types
within the sample and the presence of particular microorganisms and
extracellular substances. Thus the raw material contains a mixture
of cells and compositions, that is prone to give artefacts as
results. The presence of different cell types with different
proliferative characteristics, of organisms and substances within
the raw sample gives rise to multiple factors, that may contribute
to the particular level of a marker molecule. Detecting solely the
level of one single molecular marker may thus only lead to a
diagnostically useful information, if further (morphological)
parameters concerning the raw sample are provided. All morphologic
data obtainable from the raw sample are lost due to lysis in
solution. Yet there are suitable molecular markers corresponding to
particular morphologic or other parameters obtainable by
histological, cytological methods.
[0027] For example, the information about the single constituents
within the raw sample may be classically obtained by microscopic
examination. Morphologic inspection gives hints about the
differentiation, the localization of cells, as well as about the
environment, in which the cells appear. In cytological preparations
of cervical-swabs, for example, the particular cells may be
identified as epithelial cells and further categorized as e.g.
endocervical or ectocervical epithelial cells. Even the presence of
non-cervical cells such as endometrial cells may be ascertained
easily by microscopic inspection.
[0028] According to the present invention, raw materials may
directly be dissolved in an appropriate solvent without further
preparation or characterization independent of the homogeneous or
heterogeneous character of the sample material. Data, which are
lost through lysis of the material are contained within the sample
solution encoded by the levels of a series of marker molecules and
may thus be reconstructed using said molecular data for
normalization to the respective morphologic characteristics. This
is achieved by employing a suitable set of molecular markers for
each of the characteristic parameters needed for unambiguous
diagnosis. By detecting a suitable array of markers one may assess
the relevant parameters characterizing the raw sample and thus
overcome the disadvantage of loss of information through lysis of
the sample.
[0029] The testing procedure according to the present invention
includes detecting the levels of markers characteristic for cell
conditions in question and of markers for normalizing the data with
respect to parameters characterizing the particular environment in
the test sample. The markers suitable for the present invention may
be of various origin. The expression pattern of a marker, that is
suitable for the detection of conditions in question, may be
dependent on the proliferative status of cells, on the
differentiation status, on the cell type or on the organism.
Examples for appropriate markers are set forth below.
[0030] The term diagnosis as used herein generally comprises any
kind of assessment of the presence of absence of a medically
relevant condition. Diagnosis thus comprises processes such as
screening for the predisposition for a medically relevant
condition, screening for the precursor of a medically relevant
condition, screening for a medically relevant condition, clinical
or pathological diagnosis of a medically relevant condition, etc.
Diagnosis of medically relevant conditions as used herein may
comprise examination of any condition, that is detectable on a
cytological, histological, biochemical or molecular biological
level, that may be useful in respect to the human health and/or
body. Such examinations may comprise e.g. medically diagnostic
methods and research studies in life sciences. In one embodiment of
the invention, the method is used for diagnosis of medically
relevant conditions such as e.g. diseases. Such diseases may for
example comprise disorders characterized by non-wild type
proliferation of cells or tissues.
[0031] In one embodiment, the diagnosis pertains to diagnosis of
cancers and their precursory stages, to monitoring of the disease
course in cancers, to assessment of prognosis in cancers and to
detection of disseminated tumor cells e.g. in the course of minimal
residual disease diagnosis. The method according to the present
invention may for example be used in the course of clinical or
pathological diagnosis of cancers and their precursory stages or in
routine screening tests as performed for particular cancers such as
for example for examination of swabs e.g. in screening tests for
cervical lesions, of bronchial lavages for lung cancer or of stool
for lesions of the gastrointestinal tract, e.g. colorectal
lesions.
[0032] The method according to the present invention is applicable
to all kinds of medically relevant conditions.
[0033] Medically relevant conditions as used according to the
present invention may for example be compositions of tissues, body
fluids, secretions, washes or swabs. Such conditions may for
example comprise the cellular composition of body fluids, such as
the composition of blood, the composition of liquor or the
composition of semen. In this context the compositions shall be for
example the presence or absence of particular cell types (e.g.
pathogens, such as, viruses etc., preneoplastic, neoplastic and/or
dysplastic cells etc.), the presence or absence of differentiation
patterns of particular cell types, the total number of a particular
cell types (e.g. erythrocytes, leucocytes, sperm, etc.), the total
number of all cells of any cell types or the fraction of cells of
particular other characteristics present or absent in the
sample.
[0034] Furthermore, medically relevant conditions may also comprise
disorders related to cells, or tissues. The conditions to be
diagnosed may comprise parameters related to cells in cytological
or histological tissue samples. The conditions may comprise a
differentiation pattern of cells in a tissue sample, such as
surgical resection samples, biopsies, swabs, ravages etc. Such
conditions may comprise e.g. congenital disorders, inflammatory
disorders, mechanical disorders, traumatic disorders, vascular
disorders, degenerative disorders, growth disorders, benign
neoplasms, malignant neoplasms. Another aspect of the conditions
according to the present invention may comprise conditions
characterized by the presence or absence of proliferative
characteristics. Conditions characterized by the presence or
absence of proliferative characteristics may be for example cell
proliferative disorders.
[0035] Cell proliferative disorders according to the present
invention comprise diseases characterized by abnormal growth
properties of cells or tissues compared to the growth properties of
normal control cells or tissues. The growth of the cells or tissues
may be for example abnormally accelerated, decelerated or may be
regulated abnormally. Abnormal regulation as used above may
comprise any form of presence or absence of non wild-type responses
of the cells or tissues to naturally occurring growth regulating
influences. The abnormalities in growth of the cells or tissues may
be for example neoplastic or hyperplastic.
[0036] In one embodiment, the cell proliferative disorders are
tumors. Tumors may comprise tumors of the head and the neck, tumors
of the respiratory tract, tumors of the anogenital tract, tumors of
the gastrointestinal tract, tumors of the urinary system, tumors of
the reproductive system, tumors of the endocrine system, tumors of
the central and peripheral nervous system, tumors of the skin and
its appendages, tumors of the soft tissues and bones, tumors of the
lymphopoietic and hematopoietic system, etc. Tumors may comprise
for example neoplasms such as benign and malignant tumors,
carcinomas, sarcomas, leukemias, lymphomas or dysplasias. In a
particular embodiment, the tumor is for example cancer of the head
and the neck, cancer of the respiratory tract, cancer of the
anogenital tract, cancer of the gastrointestinal tract, cancer of
the skin and its appendages, cancer of the central and peripheral
nervous system, cancer of the urinary system, cancer of the
reproductive system, cancer of the endocrine system, cancer of the
soft tissues and bone, cancer of the hematopoietic and
lymphopoietic system.
[0037] Tumors of the anogenital tract may comprise cancer of the
perineal, the perinanal and the scrotal skin, cervical cancer,
cancer of the vulva, cancer of the vagina, caner of the penis,
cancer of the anus, etc. Cervical cancer may comprise squamous
lesions, glandular lesions or other epithelial tumors. Squamous
lesions comprise, e.g., cervical intraepithelial neoplasias (mild,
moderate and severe dysplasia), carcinoma in-situ, squamous cell
carcinoma (e.g., keratinizing, nonkeratinizing, verrucous, warty,
papillary, lymphoepithelioma-like). Glandular lesions may comprise
atypical hyperplasias, adenocarcinoma in-situ, andenocarcinoma
(such as, e.g., mucinous, endometrioid, clear cell, adenoma
malignum, papillary, serous or mesonephric adenocarcinoma). Other
epithelial tumors may comprise adenosquamous carcinoma, glassy cell
carcinoma, adenoid cystic carcinoma, adenoid basal carcinoma,
carcinoid tumor, small cell carcinoma and undifferentiated
carcinoma. For more detailed information, confer "Kurman, R.,
Norris, H., et al., Tumors of the Cervix, Vagina, and Vulva, Atlas
of Tumor Pathology, 1992, AFIP," the contents of which shall be
incorporated herein by reference.
[0038] Gastrointestinal tumors may comprise colon cancer, cancer of
the colon ascendens, of the colon descendens, of the colon
transversum, of the sigmoidum, of the rectum, cancer of the small
intestine, cancer of the jejunum, cancer of the duodenum, gastric
cancer, oesophageal cancer, liver cancer, cancer of the bile,
cancer of the biliary system, pancreatic cancer, etc. A
comprehensive overview over gastrointestinal lesions is given in
"Hamilton Sr, Aaltonen L A (Eds.): World Health Organization
Classification of Tumours, Pathology and Genetics of Tumors of the
Digestive System, IARC Press: Lyon 2000," which shall be
incorporated herein by reference.
[0039] Tumors of the respiratory tract may comprise any malignant
condition of the respiratory tract such as, e.g., cancer of the
lung, the alveoles, the bronchioles, the bronchial tree and the
broncus, the nasopharyngeal space, the oral cavity, the pharynx,
the nasal cavity and the paranasal sinus. Lung cancer such as small
cell lung cancer, non-small cell lung cancer, squamous cell lung
carcinoma, small cell lung carcinoma, adenocarcinoma of the lung,
large cell lung carcinoma, adeno-squamous lung carcinoma, carcinoid
tumor of the lung, broncheal gland tumor or (malignant)
mesothelioma. An overview over tumors of the respiratory tract may
be found in Colby T V, et al.: Tumors of the Lower Respiratory
Tract, Atlas of Tumor Pathology, Third Series, Fascicle 13, AFIP:
Washington 1995," which shall be incorporated herein by
reference.
[0040] Tumors of the urinary system may comprise bladder cancer,
cancer of the kidney, renal pelvis, cancer of the ureters and
cancer of the urethra, etc. Tumors of the reproductive system may
comprise cancer and precursory stages thereof of the ovary, the
uterus, the testis, the prostate, the epididymis, etc.
[0041] In all cases, the methods according to the present invention
also apply to precursor stages of the lesions, tumors or
cancers.
[0042] In one embodiment, the method according to the present
invention pertains to the detection of disseminated tumor cells or
metastases.
[0043] In one embodiment of the invention, the carcinoma is e.g.
cervical cancer, colon cancer, gastric cancer, breast cancer,
bladder cancer, lung cancer, cancer of the oral cavity etc.
[0044] The present invention provides a number of robust, fast and
easy ways to preserve molecular properties of samples, whereby the
morphological information of samples is lost. Samples may be e.g.
prepared in a reproducible and easy to store and transport form by
dissolving the cellular components of the raw sample in a suitable
solvent immediately after or even during obtaining the sample. Body
fluids may directly be transferred from the body of an individual
to a solution containing suitable detergents and preservative
substances. Furthermore, tissue samples may immediately be
transferred to denaturing lysis conditions (eventually supported by
physical forces) and be thus preserved. Using appropriate
ingredients in the solvent, the molecular components of the
original sample may be preserved, and no degradation may occur. The
degradation by enzymatic activities may, for example, be minimized
by the use of enzyme inhibitors. Thus, a solution of test samples
may easily represent the molecular properties of a test sample at
the time of dissolution, without requiring additional preservative
precautions.
[0045] Raw samples may comprise clinical samples, such as e.g.
secretions, swabs, lavages, body fluids, blood, urine, semen,
stool, bile, liquor, bone marrow, biopsies, cell- and
tissue-samples. Biopsies as used in the context of the present
invention may comprise e.g. resection samples of tumors, tissue
samples prepared by endoscopic means or punch- or needle-biopsies
of organs. Furthermore, any sample potentially containing the
marker molecules to be detected may be a sample according to the
present invention. In one embodiment of the invention, the sample
comprises cervical swabs, bronchial lavages, stool etc. Raw sample
as used in the context of the present invention may comprise fixed
or preserved cell or tissue samples. E.g. cells preserved in
suitable solutions (alcohols etc.) or fixed tissue samples may be
used as raw samples in the methods according to the present
invention.
[0046] A raw sample according to the method of the present
invention includes any sample comprising cells or cell debris. The
cells may for example be prokaryotic or eukaryotic cells. When the
present invention is applied for the detection of infectious
diseases, the cells to be determined may be cells of microorganisms
such as chlamydia, E. coli, candida, etc.
[0047] According to the present invention, all or part of the
molecular components of the raw samples are solubilized in a
suitable lysis buffer comprising e.g. solvents. Such solvents may
for example be aqueous solutions of chaotropic agents such as e.g.
urea, GuaSCN, Formamid, of detergents such as anionic detergents
(e.g. SDS, N-lauryl sarcosine, sodium deoxycholate, alkyl-aryl
sulphonates, long chain (fatty) alcohol sulphates, olefine
sulphates and sulphonates, alpha olefine sulphates and sulphonates,
sulphated monoglycerides, sulphated ethers, sulphosuccinates,
alkane sulphonates, phosphate esters, alkyl isethionates, sucrose
esters), cationic detergents (e.g. cetyl trimethylammonium
chloride), non-ionic detergents (e.g. TWEEN.RTM.-20, polyethylene
glycol sorbitan monolaurate; nonidet P-40, TRITON.RTM. X-100,
t-octylphenoxypolyethoxyethanol; NP-40, IGEPAL.RTM. CA 630, nonidet
P 40; N-Octyl-Glucosid) or amphoteric detergents (e.g. CHAPS,
3-Dodecyl-dimethylammonio-propane-1-sulfonate, Lauryldimethylamine
oxide) and/or of alkali hydroxides such as e.g. NaOH or KOH. The
solvent is designed, so that cells, cell debris, nucleic acids,
polypeptides, lipids and other biomolecules potentially present in
the raw sample are dissolved. The solution for dissolving the raw
samples according to the present invention may furthermore comprise
one or more agents that prevent the degradation of components
within the raw samples. Such components may for example comprise
enzyme inhibitors such as proteinase inhibitors, RNAse inhibitors,
DNAse inhibitors etc. In one embodiment of the present invention
the sample is lysed directly in the form it is obtainable from the
test-individuals. In another embodiment of the present invention
the sample may be further purified before being lysed. Such
purification procedures may for example comprise washing away of
contaminants such as mucus or the like, separation or concentration
of cellular components, preserving and transporting of the cells.
Thus the cellular components of the raw samples are included in a
single sample solution.
[0048] The preparation of a sample for use in a method as disclosed
herein may also comprise several steps of further preparations of
the sample, such as separation of insoluble components, isolation
of polypeptides or nucleic acids, preparation of solid phase fixed
peptides or nucleic acids or preparation of beads, membranes or
slides to which the molecules to be determined are coupled
covalently or non-covalently.
[0049] According to the present invention, the detection of the
marker molecules is performed directly from this solution. The
detection may be carried out in solution or using reagents fixed to
a solid phase. In certain embodiments of the present invention the
detection of the marker molecules is performed from a solution of
dissolved body samples. Therefore detection may be carried out in
solution or using reagents fixed to a solid phase. A solid phase as
used in the context of the present invention may comprise various
embodiments of solid substances such as planar surfaces, particles
(including micro-, nano-particles or even smaller particles). In
certain embodiments particles may be provided as beads, colloids or
the like. The fixation of reagents to the solid phase in a test kit
or an in-vitro diagnostic device may be effected via direct
fixation or via indirect fixation. Direct fixation may e.g. be
effected by covalent or non-covalent binding or association to
surfaces. Indirect fixation may be effected through binding of the
reagents (e.g. antibodies, probes etc.) to agents which themselves
are directly fixed to solid phases. Such agents may comprise
antibodies or other binding agents like avidin, streptavidin,
biotin or the like. The detection of one or more molecular markers
may be performed in a single reaction mixture or in two or more
separate reaction mixtures. The detection reactions for several
marker molecules may for example be performed simultaneously in
multi-well reaction vessels or as the case may be on one single or
two or more separate test strips. The markers characteristic for
the cell proliferative disorders may be detected using reagents
that specifically recognise these molecules. Simultaneously the
normalization markers may be detected using reagents, that
specifically recognize them. The detection reaction for each class
of markers may comprise one or more further reactions with
detecting agents either recognizing the initial marker molecules or
preferably recognizing the prior molecules (e.g. primary
antibodies) used to recognize the initial markers. The detection
reaction further may comprise a reporter reaction indicating the
level of the markers characteristic for cell proliferative
disorders or the normalization markers.
[0050] The terms "marker" or "marker molecule" in all their
grammatical forms as used in the context of the present invention
refers to nucleic acid as well as polypeptide molecules. Marker or
marker molecule thus comprises e.g. RNA (mRNA, hnRNA, etc.), DNA
(cDNA, genomic DNA, etc.), proteins, polypeptides, proteoglycans,
glycoproteins and the respective fragments of these molecules. The
term "relevant marker" shall refer to marker molecules
characteristic for a medically relevant condition. The term
normalization marker shall refer to marker molecules used for
normalization purposes.
[0051] A level of a marker molecule as used herein refers to a
semiquantitave as well as a quantitative value regarding the amount
of the respective marker present in a sample. A quantitative value
may e.g. be represented in terms of a concentration. A
semiquantitative value may be expressed in terms of a scale of
levels e.g. undetectable levels, low levels, intermediate levels,
high levels or any other suitable mode. The level of a marker may
also be represented in terms of a dependent parameter such as the
intensity of a signal generated in an assay format in response to
the presence of a marker molecule.
[0052] A probe for the detection of the marker molecules as used in
the context of the present invention shall be any molecule, that
specifically binds to said marker molecules. The probe may for
example be an antigen binding agent such as antibodies (monoclonal
or polyclonal), antibody fragments or artificial molecules
comprising antigen binding epitopes, DNA or RNA binding molecules
such as proteins or nucleic acids. Nucleic acids binding to other
nucleic acids may for example be peptide nucleic acids (PNAs) or
oligonucleotides (RNA, DNA, PNA, artificial nucleic acids, etc.)
for detection purposes or primers.
[0053] A molecule is said to recognize another molecules if it
specifically interacts with that molecule. Specific interaction may
for example be specific binding to or of the other molecule.
[0054] The reporter reaction may be for example a reaction
producing a colored compound. In one embodiment of the present
invention the reporter substances correlated to the particular
markers develop different colors. In another embodiment, the
normalization marker specific reporter may be a molecule quenching
the signal produced by the reporter molecule specific for the
marker, characteristic for the medically relevant condition, in
dependence on the level of the normalization marker present in the
sample. In yet another embodiment the reporter reactions may
produce fluorescent dyes with differing wavelength characteristics.
In a further embodiment of the present invention the reporter
reaction may comprise light emitting reactions with different
wavelength characteristics for the reporter substances specific for
either marker to be detected. In another embodiment of the present
invention the reporter reaction may comprise the emission of
radioactive radiation and additional methods for visualizing or
quantifying the radiation. In one embodiment, the different marker
molecules may be recognized by agents, that bear radio-nuclides
emitting radiation with different energetic properties, so that the
signals referring to marker molecules could be distinguished.
[0055] Applicable formats for the detection reaction according to
the present invention may be blotting techniques, such as
Western-Blot, Southern-blot, Northern-blot. The blotting techniques
are known to those of ordinary skill in the art and may be
performed for example as electro-blots, semidry-blots, vacuum-blots
or dot-blots. Furthermore immunological methods for detection of
molecules may be applied, such as for example immunoprecipitation
or immunological assays, such as EIA, ELISA, RIA, lateral flow
assays, flow through assays, immunochromatographic strips, etc.
immunoassays for use in the invention may comprise competitive as
well as non-competitive immunoassays such as sandwich assays.
[0056] In certain embodiments of the invention immunochemical or
nucleic acid based testing may be performed using a testing device
for clinical laboratories. Such testing device may comprise any
device suitable for immunochemical or nucleic acid based testing
including any format such as e.g. Point of care testing devices as
well as bench top or laboratory devices. The devices may be e.g.
provided as open or closed platform systems. The system may be
based on any suitable methodology such as e.g. employing microtiter
plates, multiwell plates, flow through or lateral flow systems,
microchip or array based systems or bead or membrane based systems.
The detection methods employed may comprise any methods known to
those of skill in the art useful for immunochemical or nucleic
acids based detection reactions. Such detection systems may be e.g.
luminescence systems (electroluminescence, bioluminescence,
photoluminescence, radioluminescence, chemiluminescence,
electrochemoluminescence), fluorescence based systems, conductivity
based detection systems, radiation (light, UV, X-ray, gamma etc.)
or any other known method.
[0057] The method for detection of the level of the marker
molecules in one embodiment of the present invention is any method,
which is suited to detect even very small amounts of specific
molecules in biological samples. Furthermore any method for
detection of the marker molecules irrespective of the sensitivity
may be applied. The detection reaction according to the present
invention may comprise for example detection reactions on the level
of nucleic acids and/or detection reactions on the level of
polypeptides. In one embodiment of the invention, the detection of
the marker molecules may comprise the detection of particular
splicing variants. In another embodiment of the present invention,
the detection method may comprise the detection of modifications of
marker molecules such as phosphorylation or glycosylation etc of
polypeptides or the methylation of nucleic acid molecules in
samples.
[0058] In one embodiment of the invention, the detection of the
level of marker molecules is carried out by detection of the level
of nucleic acids coding for the marker molecules or fragments
thereof present in the sample. The means for detection of nucleic
acid molecules are known to those skilled in the art. The procedure
for the detection of nucleic acids can for example be carried out
by a binding reaction of the molecule to be detected to
complementary nucleic acid probes, proteins with binding
specificity for the nucleic acids or any other entities
specifically recognizing and binding to said nucleic acids. This
method can be performed as well in vitro as directly in-situ for
example in the course of a detecting staining reaction. Another way
of detecting the marker molecules in a sample on the level of
nucleic acids performed in the method according to the present
invention is an amplification reaction of nucleic acids, which can
be carried out in a quantitative manner such as for example the
polymerase chain reaction. In one embodiment of the present
invention e.g. real time RT PCR may be used to quantify the level
of marker RNA in samples of cell proliferative disorders.
[0059] In another embodiment of the invention, the detection of the
level of marker molecules is carried out by determining the level
of expression of a protein. The determination of the marker
molecules on the protein level may for example be carried out in a
reaction comprising a binding agent specific for the detection of
the marker molecules. These binding agents may comprise for example
antibodies and antigen-binding fragments, bifunctional hybrid
antibodies, peptidomimetics containing minimal antigen-binding
epitopes etc. The binding agents may be used in many different
detection techniques for example in western-blot, ELISA, RIA, EIA,
flow through assay, lateral flow assay, latex-agglutination,
immunochromatographic strips or immuno-precipitation. Generally
binding agent based detection may be carried out as well in vitro
as directly in situ for example in the course of an
immunocytochemical staining reaction. Any other method suitable for
determining the amount of particular polypeptides in solutions of
biological samples can be used according to the present
invention.
[0060] Methods for the detection of the modified states of nucleic
acid molecules and/or polypeptides are known to those of ordinary
skill in the art.
[0061] Methods for detection of methylation of nucleic acids are
known to those of skill in the art and may comprise for example
methods employing chemical pre-treatment of nucleic acids with e.g.
sodium bisulphite, permanganate or hydrazine, and subsequent
detection of the modification by means of specific restriction
endonucleases or by means of specific probes e.g. in the course of
an amplification reaction. The detection of methylation may
furthermore be performed using methylation specific restriction
endonucleases. Methods for the detection of methylation states in
nucleic acids are e.g. disclosed in patent application
EP02010272.9, U.S. Pat. No. 5,856,094, WO0031294, U.S. Pat. No.
6,331,393 etc. The cited documents are incorporated herein by
reference.
[0062] Detection of modified states of polypeptides may for example
comprise binding agents specifically recognizing modified or
unmodified states of polypeptides. Alternatively enzymes such as
phosphatases or glycosylases may be used to remove modifications in
molecules. The presence or absence of modifications can thus be
detected by determination of mass or charge of the molecules by
means of electrophoresis, chromatography, mass spectrometry etc.
prior and subsequent to the incubation with a respective
enzyme.
[0063] In a further embodiment of the present invention, the
detection of a series of marker molecules is carried out on the
level of polypeptides and simultaneously the detection of a further
series of marker molecules and/or of all or some of the same marker
molecules is carried out on the level of nucleic acids.
[0064] Markers associated with medically relevant cellular
conditions may e.g. be molecules which influence and/or reflect the
proliferation and/or differentiation characteristics of cells
and/or tissues. Such molecules may comprise for example cell cycle
regulatory proteins, proteins associated with the DNA replication,
transmembrane proteins, receptor proteins, signal transducing
proteins, calcium binding proteins, proteins containing DNA-binding
domains, metalloproteinases, kinases, kinase inhibitors,
chaperones, embryogenesis proteins, heat shock proteins or enzymes
which modify other proteins posttranslationally thus regulating
their activity, or nucleic acids coding for the named proteins.
Also mRNA coding for the named proteins may be marker molecules
useful according to the present invention. In one embodiment the
marker associated with the cell proliferative disorder may be for
example uniquely expressed in cells affected by the disorder, may
be not expressed in said cells or may be overexpressed in said
cells.
[0065] Marker molecules for use according to the present invention
may comprise one or more markers chosen from p13.5, p14, p15, p16
(also referred to p16.sup.INK4a), p19, p21, p27, p53, pRb, p14ARF,
cyclin A, cyclin B, cyclin E, MDM-2, MCM2, MCM5, MCM6, CDC2, CDC6,
Id1, osteopontine, GRP, renal dipeptidase, her2/neu, TGF.beta.II
receptor, HPV associated markers e.g. derived from HPV genes L1,
L2, E1, E2, E4, E5, E6 or E7, etc. A selection of markers useful in
one embodiment of the present invention for the detection of
medically relevant conditions is shown below in Table 1.
[0066] In one embodiment the marker for a medically relevant
condition may be a marker for tumors (tumor markers). The marker
molecules characteristic for tumors may e.g. be proteins, that are
expressed in a non-wild type manner in tumors compared to normal
control tissue. Non-wild type expression as used herein may
comprise increased or decreased levels of expression or lack of
expression or expression of non-wild type forms of the respective
molecules. Expression of non-wild type forms of a protein may
comprise expression of mutated forms of proteins, arising by
insertion, deletion, substitution, or frameshift mutations or any
other known types of mutations in proteins or nucleic acids. In all
cases of the expression of non-wild type proteins or non-wild type
levels of proteins the proteins, polypeptides or fragments thereof
or nucleic acids encoding these proteins or polypeptides or
fragments of these nucleic acids may be used as molecular markers
associated with tumors and may thus be understood under the term
"tumor marker" as used in the context of the present invention.
Proteins that show non-wild type expression in association with
tumors are disclosed for example in the documents WO9904265A2,
WO0149716A2, WO0055633A2 and WO142792A2, which shall be
incorporated by reference herein.
[0067] In one embodiment of the invention, the marker
characteristic for the medically relevant condition may be a cell
cycle regulatory protein such as for example a cyclin, a cyclin
dependent kinase or a cyclin dependent kinase inhibitor. In a
further embodiment of the invention the marker characteristic for
the medically relevant condition may be a marker associated with a
transient or a persistent viral infection. The viral infection may
comprise an infection by a human papilloma virus (HPV) such as high
risk or low risk HPV. The high risk HPV may comprise HPV subtypes
such as e.g. HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56 and 58. The
markers for HPV infection may e.g. comprise HPV expression products
of HPV genes L1, L2, E2, E4, E5, E6 or E7. In a third embodiment of
the invention a marker characteristic for a viral infection may be
used in combination with any other marker for a medically relevant
condition such as e.g. in combination with a cell cycle regulatory
protein. Combinations of marker molecules, which may be of special
interest with respect to HPV association are e.g. disclosed in
WO0208764 which document shall be incorporated herein by
reference.
[0068] In one embodiment, cell cycle regulatory proteins for use in
combination with HPV markers may for example be chosen from a group
comprising pRb, p53, p14 ARF, cyclin dependent kinase inhibitors.
In one special embodiment for example p16.sup.INK4a may be used in
combination with markers for HPV infection (e.g. L1, L2, E2, E4,
E5, E6 or E7).
[0069] It must be understood, that as the case may be markers
useful as markers for medically relevant conditions in certain
embodiments may serve as markers for normalization in certain other
embodiments and vice versa. However, in each single embodiment, a
marker can only serve either as marker for the medically relevant
condition or as marker for normalization. For example, Ki67 as a
marker for cell proliferation may be useful as a normalization
marker in certain embodiments (e.g. in combination with p16,
p14ARF, claudin-1 or others as markers for medically relevant
condition). In other embodiments, Ki67 may serve as a marker for
medically relevant condition (e.g. as a marker for cervical
dysplasia or other dysplastic diseases) in combination with
suitable normalization markers (e.g. cytokeratins, catenins or
others). Various other markers may likewise serve either as a
marker for medically relevant conditions or for a normalization
depending on the particular embodiment of application.
[0070] Normalization markers according to the present invention may
comprise for example housekeeping genes, such as actin, gapdh,
histone proteins, phospholipase, .beta.2-microglobulin, proteins
associated with active cell proliferation such as e.g. Ki67, PCNA
or statin, or proteins characteristic for particular cell types
such as for example CK20 in epithelial cells or any cell specific
cell-surface antigens. In addition, carbohydrate structures present
on glycoproteins, proteoglycans, lectin receptors such as the
concanavalin A receptor, mucins and enzymes which are involved in
the biosynthesis of these molecules such as GalNac transferases and
oligosaccharyltransferases might also serve as normalization
markers. The type of marker protein has to be chosen according to
the information, which shall be provided by the marker. Principally
the markers useful for particular medically relevant conditions may
under certain circumstances be useful as normalization markers. A
selection of markers useful in performing the methods according to
the present invention are given in Table 1.
[0071] As well concerning markers for medically relevant conditions
as well as concerning normalization markers modified states of
molecules (such as polypeptides and nucleic acids) may be used as
markers in the method according to the present invention. For
example phosphorylated, glycosylated or otherwise modified
polypeptides or methylated nucleic acids may be addressed as
markers in the method according to the present invention.
[0072] Normalization as used according to the present invention
shall comprise any method suitable for relating the detected levels
of markers to parameters valuable for the assessment of the
diagnosis. One aspect of this normalization may be a reconstruction
of the relevant cytological and histological information contained
within the raw sample by means of suitable molecular markers
detectable in the sample solutions. Normalization may comprise for
example the detection of the total number of cells present in the
sample, of the presence or absence of a particular cell types in a
sample, of the presence or absence of an organism or of cells of an
organism in a sample, of the number of cells of a particular cell
type or organism present in the sample, of the proliferative
characteristics of cells present in the sample or of the
differentiation pattern of the cells present in the sample.
[0073] In certain embodiments normalization may also comprise
proving the adequacy of the test, wherein as the case may be
inadequate test results may be discarded or classified as invalid.
Therefore, normalization as used in the context of the present
invention may comprise qualitative or semi-quantitative methods for
normalization. In certain embodiments, semi-quantitative
normalization may comprise determining a threshold value for a
normalization marker. In one embodiment, semi-quantitative
normalization may be applied e.g. as follows: the level determined
for the relevant marker may be regarded as a valid test result only
if the level of the normalization marker exceeds a defined
threshold value; in case the threshold value is not reached the
test result for the relevant marker is regarded as invalid;
diagnosis may not be assessed on the basis of the test. In other
embodiments a threshold may be set that may not be exceeded. In
certain embodiments, qualitative normalization may be performed
with respect to the presence or absence of a normalization marker.
In those cases, e.g. the value determined for the relevant marker
is compared to the presence or absence of a normalization marker.
As predefined, the value is valid only in case the normalization
parameter (presence or absence of a detectable level of the
normalization marker) is met.
TABLE-US-00001 TABLE 1 marker for cell type antigen Antibody
supplier Literature cell type epithelial human epithelial HEA125
Research Kommoss et al., cells cell surface IgG1 (W, Diagnostics
Hum Pathol. 2000 glycoprotein IHC, ICC, Inc. Sep; 31(9): 1055-61
IF) Human epithelial AUA-1 Research Gottschalk et al, proliferation
40 kD IgG1 Diagnostics Pathol Res Pract. protein (from (Elisa) Inc.
1992 Feb; 188(1-2): LoVo) 182-90 Human epithelial Ber-EP4, Dako
Latza U et al., J Clin antigen (34 + 39 kD) IgG1 Pathol. 1990 (IHC,
Elisa) Mar; 43(3): 213-9 Human epithelial AUA-1 Research Epenetos,
A et al., proliferating (Elisa, W, Diagnostics Lancet. 1982 Nov
antigen (40 kD) IHC) Inc. 6; 2(8306): 1004-6 endocervix Cytokeratin
18 RGE 53, Research Smedts F et al., Am columnar (45 kD) IgG1
Diagnostics J Pathol. 1990 cells (W, IHC, IF) Inc. Mar; 136(3):
657-68 Cytokeratin 18 RCK 106 Research Smedts F et al., Am (45 kD)
(W, IHF, Diagnostics J Pathol. 1990 IHC) Inc Mar; 136(3): 657-68
Cytokeratin 8 CAM 5.2 BD Smedts F et al., Am (52.5 kD) (W, IHC)
PharMingen J Pathol. 1990 Mar; 136(3): 657-68 Endocervical Mucin
Antigens DF3 Centocor Tashiro et al., Hum columnar (Tn, STn, MUC1,
Pathol. 1994 cells MUC2 Apr; 25(4): 364-72 Endocervic Concanavalin
A Herckenrode et al., Columnar receptor Br J Cancer. 1988 cells
Mar; 57(3): 293-4; Koch et al., Br J Cancer. 1986 Jan; 53(1): 13-22
Endocervix GalNacTransferase Chilton et l.,
Oligosaccharyltransferase Endocrinology. 1988 Sep; 123(3): 1237-44
Endocervic/ Lectins (ConA, Di Loretto et al., Ectocervix WGA, PNA,
Basic Appl UEA I, DBA, Histochem. SBA, SNA 1987; 31(2): 143-52;
Versura et al., Basic Appl Histochem. 1988; 32(2): 219-27
ectocervix Plakophilin (80.5 kD PP1-5C2, Research Heid, HW,
squamous IgG1 Diagnostics Differentiation. cells (W, Elisa, Inc.
1994 Dec; 58(2): 113-31 IHC, IF) endometrial Vimentin VIM 3B4,
Research Smedts F et al., Am cells IgG1, (W, Diagnostics J Pathol.
1990 ELISA, IF, Inc. Mar; 136(3): 657-68 IHC) Erythrocytes
Haemoglobin RDI-CBL63, Research Smith et al., J. IgM Diagnostics
Histochem. (RIA, EIA) Inc. Cytochem. 1998 Inflammation neutrophilic
CD16(NK, DJ130-c, DIANOVA Grundhoever D and granulocytes Macro,
Gran) IgG1 Patterson BK, NK-cells (IHC) Cytometry Macrophages 2001;
46: 340-344 CD56(NK) clone Ki-M6 Hermann et al., J. Clin. Immunol.
1990 CD68(Macro) (antiCD68) Cavayal et al., Eur. J. Immunol:
1998(6)1991-2002 (CD56) B-cells CD19 clone AE 1, DIANOVA Harrada et
al., Blood (CD20) FACS 1993; 81: 2658-63 (CD19) Mason et al., Am J.
Pathol 1990; 136: 12 15-22(CD20) T-cells CD3 (panTcell) clone CRIS-
DIANOVA Jones et al., J (CD4); (CD8) 7 (antiCD3); Immunol 1993; IF,
IHC, WB 150: 5429-35 dysplastic p16.sup.INK4a E6H4, D7D7 MTM Klaes
R., et. al. Int J and Cancer. 2001 Apr neoplastic 15; 92(2): 276-84
cervical cells tumor different P53 (mutations) Mendoza-Rodriguez
cells cancer cell CA, et al., Rev types Invest Clin 2001 May-Jun;
53(3): 266-73 adeno- CEA Mistretta et al., carcinoma Experientia.
1974 cells Oct 15; 30(10): 1209-10; Rogers et al., Eur J Cancer
Clin Oncol. 1984 Oct; 20(10): 1279-86 bladder NMP22, BTA van der
Poel HG et cancer cells al., Curr Opin Urol, 11, 503-509, 2001 lung
cancer PreproGRP Hamid et al., cells Cancer, 63, 266-271, 1989,
Pagani et al., Int. J. Cancer 47, 371-375, 1991 Proliferation all
PCNA Pc10, IgG2a Zymed Waseem NH, Lane proliferating Ki67 DP, J
Cell Sci cells 1990: 96: 121 (PCNA) Cattoretti et al., J Pathol
1992: 168: 357-63(Ki67) Infectious HPV 16 E6 BF 7, IgG1 Research
Iftner et al., J Virol. agent (IHC and in Diagnostics 1988
diagnostic Inc. Oct; 62(10): 3655-61 kits for cervical swabs) L1
CamVir-1, Research Browne L et al., J IgG2a Diagnostics Gen Virol.
1988 (IP, W, IF, Inc. Jun; 69 (Pt 6): 1263-73 IHC) HPV 18 L1
RDI-HPV18- Research Iftner et al., J Virol. 5A3, IgG1 Diagnostics
1988 (W, IHC) Inc. Oct; 62(10): 3655-61 HPV 6, 11, 18 RDI-HPVX-
Research Iftner et al., J Virol. 4C4 Diagnostics 1988 Inc. Oct;
62(10): 3655-61 Gouillou et al., Am. J. Surg. Pathol., 1991
[0074] According to the present invention the normalization may
comprise the determination of the presence of a number of (human)
cells in question in a sample. This is a crucial aspect of the
invention. In particular, embodiments false (especially false
negative) results of tests can only be avoided, if the testing
procedure verifies, that the test sample contains the materials
(e.g. cells, tissues organisms etc.), that are necessary for
performing the particular test. In various tests this will comprise
ensuring, that the sample contains cells. In a wide range of
embodiments of the invention the verification of the adequacy of
the sample will not just comprise ensuring of the presence of
cells, but will include the detection of the presence of cells of a
distinct origin or of a special cell type.
[0075] Thus normalization may also comprise the determination of
cells of particular origin such as e.g. cells from a particular
organ or of a particular histological localization such as for
example the detection of cells of distinct regions of epithelia, or
of cells of connective tissue, cells originating from the basal
lamina of a tissue or of cells of a heterologous origin, such as
metastatic cells. This may be necessary in particular cases,
because there might be cells, that under certain circumstances do
express a marker, which might be used for the detection of a
medically relevant condition, such as e.g. neoplasia or dysplasia,
under certain normal conditions. Normalization as used according to
the present invention may comprise the detection of the presence or
absence and/or the level of any cell-types, that may possibly
contribute to the total level of a particular marker selected to
diagnose a medically relevant condition.
[0076] In one embodiment, the method may be applied for the
detection of cervical lesions. Cervical lesion may comprise any
kind of cervical dysplasia such as cervical cancers as defined
above and its precursory stages. Markers and combinations thereof
useful for this detection purpose are for example disclosed in
WO0208764 and EP1217377, which documents shall be incorporated
herein by reference. In this embodiment the test may be performed
using any suitable sample of cervical origin. The sample may for
example comprise biopsies or microbiopsies of the cervix or swabs
taken from the cervical region. Cervical swabs as used herein are
samples that may for example be obtained using a suitable device
such as a brush, a tampon, a spatula or the like, which is
contacted with the uterine cervix during the sampling procedure.
The sampling device may be any suitable device, which may be used
in conventional testing performed by a physician or a self sampling
device.
[0077] Promising molecular markers for enhancing the evaluation of
cervical swabs are e.g. p16.sup.INK4a, p14ARF, cyclin E, cyclin A,
cyclin B, MN, her2/neu, mdm-2, bcl-2, EGF-Receptor, mcm-2, mcm-5,
claudin-1, Markers indicative for Human papilloma virus infection,
pRb, p53 etc. which might be used to detect dysplastic and
neoplastic cells. Normalization according to the present invention
for the purpose of analysis of cervical swabs may comprise the
detection of the presence of human cells at all, the detection of
cells of the cervical epithelium, the detection of the presence of
endocervical as well as ectocervical cells and the detection of
cells of endometrial origin. The endocervical epithelium is a
glandular columnar epithelium. Cells originating from the
endocervix may thus be identified by markers that are selectively
expressed by columnar epithelial cells or by cells in glandular
epithelia. The ectocervical epithelium is a squamous epithelium.
Identification of ectocervical cells thus may be achieved by
detection of markers characteristic for squamous epithelial cells.
In certain embodiments, the detection of epithelial cells
(comprising squamous as well as columnar epithelia) may be
sufficient. In other embodiments, the differentiation of especially
endocervical cells may be crucial. It is a crucial step to ensure
the presence of ecto- and endocervical cells within the sample to
ensure, that the specimen was taken at the cervical transformation
zone, where most dysplasias and neoplasias arise. If there are no
such cells, the sample is not adequate for the testing procedure,
for it is prone to give false negative results. As p16.sup.INK4a
may be expressed in normal endometrial cells normalization of the
p.sub.16.sup.INK4a expression level in regard to the number of
endometrial cells might be necessary.
[0078] To enable for reliable diagnosis the normalization
furthermore may comprise the detection of the presence or absence
of the named cellular components within the sample, and
additionally the detection of the total level of a particular cell
type or of the fraction, that a particular cell type contributes to
the total number of cells within the sample.
[0079] Thus, in one embodiment the detected level of the
p16.sup.INK4a protein may be normalized to the cytological
conditions represented by the particular sample, so that one may
state, if the detected level of the p16.sup.INK4a protein is
indicative for cervical cells overexpressing p16.sup.INK4a, or if
there is an abundant number of endometrial cells present in the
sample, thus mimicking the overexpression of p16.sup.INK4a. In this
respect normalization may comprise the determination of the
quantity of endometrial cells within a cervical sample on the basis
of a molecular marker. Comparing the level of e.g. p16.sup.INK4a as
a marker for a medically relevant condition determined in a
cervical sample to the quantity of endometrial cells assessed by
means of molecular markers, one may state, whether the total amount
of p16 may arise only from the endometrial cells present within the
sample solution. Thus, false positive results in diagnosing
cervical dysplasias overexpressing p16.sup.INK4a attributable to
the presence of high levels of endometrial cells may be excluded. A
quantity as used in the context of the present invention may refer
to a quantitative or semi-quantitative assessment. This may e.g.
comprise the assessment of a total number of cells or the
assessment of a fraction with respect to the total number of cells.
In certain embodiments of the invention the determination of a
quantity may refer to the assessment of the fraction of an overall
marker level that is contributed by a particular type of cells.
[0080] For the purpose of providing a normalization marker for the
evaluation of cervical specimens, several normalization markers
appear to be useful and may e.g. be selected from the following:
Cytokeratins, E-Cadherins, Involucrin, Urokinase-like
Plasminogen-activator, SCCA (Squamous cell carcinoma antigen),
Catenins, (e.g. alpha-catenin, beta-Catenin, gamma-Catenin
(Plakoglobin)), Ep-Cam.
[0081] Several candidates for normalization markers have been
examined for their properties in characterization of cervical
specimens. The results are given in Table 2 and Table 3.
TABLE-US-00002 TABLE 2 Clinical/Biochemical Name Histology/Cytology
Data Literature UPA-1 Cervical tissue .uparw. In cervix CA Horn LC
Aust N Z J (Urokinase-type Normal epithelium Obstet Gynaecol, 2002
Plasminogen- showed presence of both Larsson G Thromb. Activator;
Swissprot t-PA and u-PA Haemost. 1987 Accession P00749;
immunoreactivity only also known as EC in the superficial
3.4.21.73, U- cellular layer, whereas plasminogen activator in
preinvasive lesions uPA) they were present in all layers. PAI-1
Cervical tissue .uparw. In cervix CA Horn LC Aust N Z J
(Plasminogen- Normal epithelium positive prognostic marker Obstet
Gynaecol, 2002 Activator Inhibitor 1; showed presence of both
Larsson G Thromb. Swissprot Accession t-PA and u-PA Haemost. 1987
P05121; also known as immunoreactivity only PAI-1 in the
superficial Endothelial cellular layer, whereas plasminogen
activator in preinvasive lesions inhibitor they were present in all
PAI; Isoforms: PAI-2 layers. P05120 and PAI-3 P05154) Involucrin
Only squamous Involucrin expression is Shirley A, Human (Swissprot
Accession epithelia, no columnar abnormal in squamous cell
Pathology, 2001 P07476) cells; immature and carcinomas and de Boer
et al., 1999, SEQ ID NO: 7 mature squamous premalignant lesions,
and is Am J of Pathol, metaplastic cells. reduced in severe
dysplasias 155: 505-515 In normal epidermis, it of the larynx and
cervix. Nair SA, Pathobiology, is first expressed in the Marker for
terminal 1996 upper spinous layers, differentiation. and in
keratinocyte cultures it is expressed by all cells that have left
the basal layer. gamma-Catenin Squamous epithelia High in normal
cervical de Boer et al., 1999, (Swissprot Accession epithelium at
cell-cell- Am J of Pathol, Q86W21; also known boundaries. Moderate
155: 505-515 as Plakoglobin; e.g. reduction in high grade Epitope:
C-Terminus; SILS AA553-738) SEQ ID NO: 1 Alpha-1 Catenin Squamous
epithelia High in normal cervical de Boer et al., 1999, Am
(Swissprot Accession epithelium at cell-cell- J of Pathol, 155:
505-515 P35221; also known boundaries as Cadherin-associated Strong
reduction in high protein grade SILS Alpha E-Catenin) SEQ ID NO: 4
Alpha-2 Catenin (Swissprot Accession P26232; also known as
Alpha-Catenin related protein Alpha N-Catenin) SEQ ID NO: 5
beta-Catenin Squamous epithelia High in normal cervical de Boer et
al., 1999, Am (Swissprot Accession epithelium at cell-cell- J of
Pathol, 155: 505-515 P35222 also known as boundaries PRO2286)
Strong reduction in high SEQ ID NO: 6 grade SILS Desmoplakin
stratified epithelia, .dwnarw. in HSIL area de Boer et al., 1999,
Am (Swissprot Accession simple epithelia, J of Pathol, 155: 505-515
P15924; also known as including glands, DP urothelium, thymic
250/210 kDa reticular epithelium, paraneoplastic hepatocytes,
pemphigus antigen) intercalated disks of myocardium and arachnoid
cells of meninges suprabasal layers of cervix (Superficial cells
largely negative) .dwnarw.: down regulated; .uparw.: up
regulated;
TABLE-US-00003 TABLE 3 Western Blot Analysis (clinical samples were
freshly lysed with Marker Histological testing Cytological testing
MTM buffer) E-Cadherin Squamous epithelia, Parabasal, intermediate
Only weak signal for HT- (Swissprot Accession (Parabasal, cells, no
columnar cells 29. All clinical samples P12830; also known as
intermediate cells) are negative Uvomorulin, no columnar epithelia
Cadherin-1, CAM 120/80; e.g.epitope: C- Terminus; AA735- 883) SEQ
ID NO: 3 p120 Squamous epithelia, Very strong staining of Only
negative control Swissprot Accession (Parabasal, parabasal,
intermediate (lymphocytes) and O60716; p120 catenin, intermediate
cells) cells positive control (C4.1) p120(ctn), Cadherin- also very
strong in strong columnar cells positive associated Src columnar
epithelia substrate, CAS, p120(cas); e.g.epitope: C-Terminus;
AA790- 911) SEQ ID NO: 8 gamma-Catenin Squamous epithelia, Very
strong staining of Double bands (82/95 kD) Swissprot Accession
(Parabasal, parabasal, intermediate in 60% of samples Q86W21; also
known intermediate cells) no cells (9/15); after acetone as
Plakoglobin; e.g. columnar epithelia, no columnar cells
precipitation of 150 .mu.l of epitope: C-Terminus; total epithelium
is samples: 87% (13/15) AA553-738) stained indysplasia positive
Ep-Cam strong columnar strong columnar cells, (Tumor-associated
epithelia, at very high calcium signal at very high concentrations
rather transducer 1, concentrations rather unspecific Swissprot
Accession unspecific (cytoplasmic) staining P16422; also known as
(cytoplasmic) staining of squamous epithelia, Major
gastrointestinal of squamous epithelia, (Parabasal,
tumor-associated (Parabasal, Intermediate Cells) protein, GA733-2,
Intermediate Cells) Epithelial cell surface antigen, Epithelial
glycoproteins, EGP, Adenocarcinoma- associated antigen KSA KS 1/4
antigen Cell surface glycoprotein Trop-1) SEQ ID NO: 2 Involucrin
Strong staining of All cells and (Swissprot Accession squamous
epithelia, structures positive P07476) (Parabasal, Intermediate
Cells) and columnar epithelia; unspecific staining of stromal
cells;
[0082] The markers for normalization may for example be applied as
markers indicative of the presence of specific cell differentiation
patterns such as e.g. terminal differentiation or differentiation
as specific epithelial cells. In certain embodiments, normalization
markers may be marker molecules characteristic for squamous
epithelial cells e.g. indicative for the presence of ectocervical
cells in a cervical sample. Suitable markers may comprise e.g.
CK13, E-Cadherin, gamma-Catenin, or Involucrin. In another
embodiment the markers may be characteristic for the presence of
columnar epithelial cells indicating the presence of endocervical
cells in the specimen. Suitable markers comprise: Ep-Cam, CK18,
CK8.
[0083] In certain embodiments, normalization may comprise the
detection of epithelial cells generally; in these cases any marker
suitable for the detection of epithelial cells may be employed.
Markers may be for example those given in Tables 2 and 3.
[0084] In yet another embodiment of the invention, the method
disclosed herein may be used for the detection of disorders of the
respiratory tract. In the diagnosis of small cell lung cancer
detection of neuron specific enolase (NSE) is one of the employed
markers. Samples of tumor specimens are yielded by bronchoscopy
with collection of cells by means of brushes or bronchoalveolar
ravages. Since NSE is also expressed in few normal cells within the
lung, the level of NSE expression detected in the dissolved sample
has to be set in relation to the normalisation maker (for example
actin) for detection of the amount of cells present within the
sample.
[0085] A third embodiment of the present invention is the detection
of lesions of the gastrointestinal tract, e.g. colorectal lesions
from stool samples. In this case the origin of indicative nucleic
acids and/or polypeptides detectable in stool samples may be
crucial for the assessment of diagnosis. According to the present
invention, it is possible to determine the origin (cell
types/organism) of the employed marker molecules. Thus false
results based e.g. on the detection of marker molecules originating
from foodstuff ingested by individuals rather than from lesion of
the mucosa of the gastrointestinal tract may be eliminated.
Furthermore artefacts produced by the presence of traces of markers
from the blood circulation, or originating from swallowed sputum
etc. may be eliminated using the methods disclosed herein.
[0086] Another aspect of the present invention is a testing kit for
performing the method according to the present invention. The kit
may be for example a diagnostic kit, an analytical kit or a
research kit.
[0087] The term kit as used according to the present invention may
comprise kits as well as diagnostic devices. The kits or devices
may e.g. be designed for ELISA (e.g. sandwich, competitive,
non-competitive, etc.), EIA (competitive, non-competitive, etc.)
RIA tests, bead based test systems, lateral flow assays, flow
through assays, strip test assays, dip stick assays, or any other
known laboratory-, bench top- or point of care-testing format. A
kit according to the present invention may in certain embodiments
comprise in-vitro diagnostic devices for performing diagnostic
tests. In vitro-diagnostic devices may e.g. be ELISA devices of any
kind known to those of skill in the art. These devices comprise
devices for sandwich ELISA formats, for competitive ELISA formats
and any other ELISA formats. In another embodiment the in-vitro
diagnostic device may be a lateral flow assay device, or a flow
through assay device e.g. employing at least one reagent binding to
a marker characteristic for a medically relevant condition and one
reagent binding to a normalization marker, both fixed to a solid
phase. Such devices may employ various mechanisms for visualization
of the test result. In certain embodiments the tests may employ
secondary detection reagents directed against the marker molecules
coupled to detectable moieties. The detectable moieties may
comprise colloidal gold, (coloured) latex particles and others.
[0088] In yet another embodiment the in-vitro diagnostic test
device may be a flow through assay device based on capillaries or
on porous members (such as membranes, beads or other three
dimensional arrangements of porous substances). Depending on the
embodiment the size of pores or capillaries need to be adjusted to
ensure optimal flow conditions.
[0089] A kit according to present invention may comprise
[0090] a) reagents for the detection of the marker molecules,
[0091] b) the reagents and buffers commonly used for carrying out
the detection reaction, such as buffers, detection-markers, carrier
substances and others,
[0092] c) one or more markers and/or samples representative for
medically relevant conditions to be diagnosed for carrying out
positive and/or control reactions, and
[0093] d) one or more normalization marker samples for carrying out
a positive and/or control reaction.
The test kit may optionally include a lysis buffer for
solublization of the raw sample. Generally the lysis buffer may be
any suitable solvent known to those of skill in the art. The lysis
buffer for use in the kit may for example be aqueous solutions of
chaotropic agents such as e.g. urea, GuaSCN, Formamid, of
detergents such as anionic detergents (e.g. SDS, N-lauryl
sarcosine, sodium deoxycholate, alkyl-aryl sulphonates, long chain
(fatty) alcohol sulphates, olefine sulphates and sulphonates, alpha
olefine sulphates and sulphonates, sulphated monoglycerides,
sulphated ethers, sulphosuccinates, alkane sulphonates, phosphate
esters, alkyl isethionates, sucrose esters), cationic detergents
(e.g. cetyl trimethylammonium chloride), non-ionic detergents (e.g.
TWEEN.RTM.-20, nonidet P-40, TRITON.RTM. X-100, NP-40, IGEPAL.RTM.
CA 630, N-Octyl-Glucosid) or amphoteric detergents (e.g. CHAPS,
3-Dodecyl-dimethylammonio-propane-1-sulfonate, Lauryldimethylamine
oxide) and/or of alkali hydroxides such as e.g. NaOH or KOH.
Examples of Lysis Buffers are given in Table 4.
TABLE-US-00004 TABLE 4 Solubilization of p16.sup.INK4a in
compatibility Lysis buffer Western blot with Elisa Detergents:
0.1-1% SDS + +/- 0.2-3% SDS + <0.5% 0.2-3% DOC ++ +/- 0.1-1%
n-Octylglycoside + yes 0.1-3% TRITON .RTM. X-100 + yes 0.1-1% Chaps
+ nd Detergent-Mix: RIPA (1% NP40, 0.5% DOC, ++ yes 0.1% SDS, PBS)
40-100% SOX (0.5% DOC, 0.5% n- + yes Octylglycoside) 40-100% mtm
lysis buffer (3% TRITON .RTM. ++ yes X-100, 0.4% SDS, PBS)
Commerical lysis buffers: Dynal (Dynal, Oslo, Norway) ++ yes
M-PER/B-PER (Pierce, Rockford, ++ yes IL) Miscellaneous: 0.5-8 M
urea in PBS +++ Compatible < 2 M Lammli sample buffer +++ no
10-80% DMSO +++ no 10-80% Formamide nd no 50-70% formic acid ++ no
PBS +/- yes Citrate buffer pH 6.0 +/- yes 500 mM NaCl in Phosphate
buffer +/- yes nd: not determined; +/-: poor; +: good; ++: very
good; +++: excellent;
The lysis buffer may furthermore comprise one or more agents that
prevent the degradation of components within the raw samples. Such
components may for example comprise enzyme inhibitors such as
proteinase inhibitors, RNAse inhibitors, DNAse inhibitors etc. The
inhibitors may e.g. comprise proteinase inhibitors selected from
the compositions given in Table 5. In certain embodiments the lysis
buffer by the way of providing an inhibitor of degradation enables
for detection of p16 in the sample. In certain embodiments the
cyclin dependent kinase inhibitor p16 is degraded in the
solubilized samples and may thus not be detected. This is
especially true, if the samples are directly transferred to a
lysing medium and stored therein for a certain period of time.
TABLE-US-00005 TABLE 5 Class of Solubility stability inhibited in
in Inhibitor proteinase concentration water water Aprotinin Serine
0.6-2 .mu.g/ml Very good good Benzamidine Serine 0.5-4 mM good good
Bestatin Aminopeptidases 1-10 .mu.M good good Calpeptin Cysteine
0.3-1 .mu.M good good Cystatin Cysteine 1 .mu.M good good E-64
Cysteine 1-10 .mu.M good good EDTA Metallo 0.5-5 mM good good
Elastatinal Serine 0.5-2 .mu.g/ml poor good EST Cysteine 20-50
.mu.g/ml bad poor Fetal calf all classes 10% good good serum
Leupeptin Serine/Cysteine 10-100 .mu.M good good a2- all classes 1
.mu.M good good Macroglobulin NCO-700 Cysteine 0.5-100 mM poor poor
Pefabloc = Serine 0.2-10 .mu.M good very AEBSF poor Pepstatin A
Aspartic 1 .mu.M bad poor PMSF Serine 0.2-10 .mu.M bad very poor o-
Metallo 1-10 mM bad poor Phenanthroline
[0094] For stabilization purpose the lysis buffer may also comprise
bulk protein (e.g. albumin such as bovine serum albumin or calf
serum albumin or other bulk proteins) to compete in degradation
with the sample proteins. The bulk proteins may e.g. be present in
combination with proteinase inhibitors or may be added instead of
proteinase inhibitors. In one embodiment the solvent may be
selected to be compatible with the performance of the test (EIA,
ELISA or strip test performance), so that solubilized samples may
directly be applied to the test. Test as used in the context may
comprise any procedure for detecting the presence or absence and/or
the level of marker molecules.
[0095] The reagent for the detection of the marker molecules may
include any agent capable of binding to the marker molecule. Such
reagents may include proteins, (poly)peptides, nucleic acids,
peptide nucleic acids (PNAs), glycoproteins, proteoglycans,
polysaccharids or lipids.
[0096] The markers characteristic for medically relevant conditions
and/or normalization marker samples for carrying out positive
and/or negative controls may comprise for example nucleic acids in
applicable form such as solution or salt, peptides in applicable
form, tissue section samples, microorganisms or positive or
negative cell-lines.
[0097] In one embodiment of the invention, the detection of the
marker molecules is carried out on the level of polypeptides. In
this embodiment the binding agent may be for example an antibody
specific for the marker molecules or a fragments thereof.
Furthermore binding agents may comprise antigen-binding fragments
such as Fab fragments, single chain antibodies, bifunctional hybrid
antibodies, peptidomimetics containing minimal antigen-binding
epitopes etc. Moreover the binding agent might be a lectin binding
to a specific carbohydrate structure on the marker molecule.
[0098] In another embodiment of the test kit the detection of the
marker molecules is carried out on the nucleic acid level. In this
embodiment of the invention the reagent for the detection may be
for example a nucleic acid probe or a primer reverse-complementary
to said marker nucleic acid.
[0099] The following examples are given for the purpose of
illustration only and are not intended to limit the scope of the
invention disclosed herein.
EXAMPLES
Example 1
Specific Immunohistochemical Detection of Endocervical and
Ectocervical Epithelial Cells in Cervical Sections
[0100] In order to evaluate markers indicating the adequacy of
cervical swabs, cervical sections (fixed in 4% formaldehyde
solution and paraffin-embedded) were stained with antibodies
directed against Cytokeratin 18 (marker for endocervical columnar
epithelia) and Cytokeratin 10/13 (marker for ectocervical squamous
epithelia). FIG. 1 shows specific staining of endocervical
epithelia with anti-Cytokeratin 18 antibody and specific staining
of ectocervical epithelia with anti-Cytokeratin 10/13 antibody. The
experiment was performed as follows:
[0101] Formalin-fixed, paraffin-embedded sections were
deparaffinized in xylene bath for 5 min (step was repeated once),
excess liquid was tapped off and slides were placed in 95-96%
ethanol for 3 (.+-.1) min, in 70% ethanol for 3 (.+-.1) min (step
was repeated once) and finally in distilled water for a minimum of
30 sec. For epitope retrieval, slides were placed in a Coplin jar
and boiled for 40 min at 95-99.degree. C. in 10 mM Citrate buffer
pH 6.0. Slides were allowed to cool down for 20 min (.+-.1 min) at
RT in this buffer. Slides were covered with Peroxidase-Blocking
Reagent (3% H.sub.2O.sub.2; NaN.sub.3 15 mM) and incubated for 5
(.+-.1) min at RT. After 5 min washing in washing buffer, slides
were incubated with primary antibodies (CK 10/13: DE-K13, 1:50,
DAKO; CK 18: K18.7, 1 .mu.g/ml, dianova) for 30 min. Thereafter,
slides were rinsed with wash buffer and washed in wash buffer for 5
min at RT. Following 30 min incubation with EnVision (ready to use
anti-mouse horseradish peroxidase-complex; DAKO), slides were
washed 3.times.5 min and incubated in DAB substrate for 10 min,
counterstained with hematoxylin and mounted with Faramount mounting
medium.
[0102] Using an antibody directed against cytokeratin 18 (CK18) in
an immunohistochemical staining procedure, a positive reaction was
detected in columnar epithelium of the endocervix (FIG. 1A),
whereas the squamous epithelium of the ectocervix showed no
specific staining (FIG. 1B). Immunohistochemical staining with an
antibody directed against cytokeratin 10/13 (CK 10/13) showed no
staining in the columnar epithelium of the endocervix (FIG. 1C),
whereas there is a strong staining of the squamous epithelium of
the ectocervix (FIG. 1D). So CK18 might be used as a specific
marker for the detection of columnar epithelial cells of the
endocervix and CK10/13 as a specific marker for squamous epithelial
cells of the ectocervix.
Example 2
Western Blot Analysis of Solubilized Samples from Cervical
Swabs
[0103] In order to evaluate, whether western blot analysis of
solubilized samples allows assessing diagnosis of cervical lesions,
clinical samples with known diagnosis were subjected to an
immuno-chemical analysis on the basis of marker molecules after
lysis of the sample material.
[0104] The clinical material (cervical swabs) samples were analyzed
by Standard Western Analysis as follows.
[0105] In brief, the clinical material was in a first step
solubilzed by boiling (5 min, 95.degree. C.) in Lammli Protein
Sample buffer (100 mM Tris pH.6.8, 2% SDS, 200 mM DTT, 0.05% BpB)
prior to sonification. In a second step, protein samples were
resolved on a SDS-PAGE (12% Acrylamide) and subsequently
transferred on a nitrocellulose membrane by tank blotting (Towbin
et al., 1979, Proc Natl Acad Sci: 76:4350-4354). In a further step,
the membranes were: blocked to prevent unspecific antibody binding
(10% non fat dry milk in PBS) and subsequently incubated with the
specific monoclonal mouse antibody (CK 8: 35.beta.H11, 1:100, DAKO;
p16.sup.INK4a: D7D7, 1:140, MTM Laboratories). The binding of the
specific antibody was visualized by Horseradish Peroxidase
conjugated secondary reagents (binding to the marker specific
antibody) catalyzing photon emitting substrates.
[0106] Cytokeratin 8 (CK 8) was used as an endocervical cell
specific marker, indicating the adequacy of the sample collection
in the present experiments. The cyclin dependent kinase inhibitor
p16.sup.INK4a was used as specific disease related marker.
[0107] The results of the present experiment are given in FIG. 2.
The numbers 1 to 4 refer to samples (cervical swabs) obtained from
individual patients. Immunoblot detection was performed using
specific antibodies directed against cytokeratin 8 (CK8) and
specific antibodies directed against p16.sup.INK4a (p16). The
samples of patient 1, 2, and 3 show no signal for p16.sup.INK4a.
This indicates that no dysplastic cervical cells were present in
these samples. The sample of patient 4 shows a strong signal for
p16.sup.INK4a. This indicates that dysplastic cervical cells were
present in the sample. The upper bands show the specific signals
for cytokeratin 8. In sample 1,3, and 4 cytokeratin 8 can be
detected, whereas in for sample 2 no signal can be seen. This
indicates that endocervical columnar cells were present in samples
1, 3, and 4, and absent in sample 2. As the presence of
endocervical columnar epithelial cells is one of the parameters for
the adequacy of cervical swabs, sample 2 is considered inadaequat
and no diagnostic conclusions can be drawn from the negative result
of the p16.sup.INK4a detection. Samples 1, 3, 4 are considered
adequate. So based on the negative signal of sample 1 and 3 for
p16.sup.INK4a it could be concluded, that these patients had no
cervical dysplasia. Sample 4 showed a positive signal for
p16.sup.INK4a, indicating the presence of a dysplastic cervical
lesion in this patient.
The parallel cytological analysis of the swabs indicated a normal
cellular composition for woman 1 and 3. In women 2, no diagnosis
due to sparse cellular material could be obtained. In woman 4, a
high-grade dysplasia was diagnosed. Note that the upper band (CIK
8) refers to the endocervical cell specific normalization marker
Cytokeratin 8, indicating the adequacy of the sample collection.
The lower band indicates the specific disease related marker
p16.sup.INK4a. The blot shows for patient 4 a positive signal for
p16.sup.INK4a consistent with a high-grade cervical dysplasia.
Samples of patient 1 and 3 show only the CK 8 specific band,
indicating proper sample collection, but no disease related marker
(p16.sup.INK4a) consistent with a normal, healthy cervical
epithelium. The sample of patient 2 shows no CK 8 signal,
consistent with the low cell number in this sample, so no
diagnostic conclusion can be drawn from the negative signal for
p16.sup.INK4a.
Example 3
Western Blot and ELISA Analysis to Demonstrate Sample Adequacy
[0108] To evaluate, whether results of solution based analysis
differing from diagnosis of samples may be due to inadequacy of
sample, Western blot analysis of cervical swabs of four different
patients with ascertained diagnosis (high-grade cervical
intraepithelial neoplasia according to the cytological diagnosis of
Pap IVa and Pap IVb) was performed. Antibody against p16.sup.INK4a
was used to indicate presence of dysplastic cells, whereas
antibodies against CK18 and CK10/13 were used to demonstrate
adequacy of the sample.
[0109] Western blot analysis was performed as follows: Patient
samples were collected with a cervical brush and directly lysed in
Laemmli Sample Buffer (2% SDS, 60 mM Tris pH.6.8, 0.01%, 100 mM
DTT) for 5 min at 95.degree. C. (1.times.10.sup.7 cells/ml) with
subsequent sonification (5.times.5 sec pulses, maximum intensity).
Lysates were centrifuged for 12 min at 16,600.times.g) in a
microcentrifuge and supernatant was transferred into a new tube.
Precast 4-20% linear gradient acrylamide gels (Criterion System,
Bio-Rad) were loaded with 10 .mu.l (10.sup.5 cells) of whole cell
extracts and proteins were separated at 25 mA constant current for
45 min. Proteins were transferred from the gel to HYBOND.RTM.
(Hydrophobic polyvinylidene difluoride membrane) ECL Nitrocellulose
membrane (Amersham) by standard tank blotting using the Bio Rad
Criterion Blotter (15 min at constant 100 Volt and subsequently 45
min at constant 50 Volt). Nitrocellulose-membrane was stained for 5
min in Ponceau S solution to assure protein transfer. Ponceau S
solution was removed by 2.times.10 min washes in PBS. For
immunodetection, blots were blocked over night in blocking buffer
(10% milk powder in PBS with 0.1% TWEEN-20.RTM.). Primary
antibodies were incubated at dilutions according to the
manufacturer in blocking buffer for 1 h at RT with agitation (CK18:
MAB 3236), 1:1000, CHEMICON; CK 10/13: DE-K13, 1:500, DAKO,
p16.sup.INK4a: D7D7, 1:140, MTM Laboratories). After 6 washes for
10 min with PBS/0.1% TWEEN-20.RTM., blots were incubated with
rabbit anti mouse-HRP, (DAKO, diluted 1:5,000 in blocking buffer)
for 1 h at RT. After 6 washes for 10 min with PBS/0.1% Tween-20,
membranes were incubated for 5 min in substrate solution (Super
Signal West Femto Maximum Substrate, Pierce), wrapped in a plastic
envelope and exposed to an x-ray film for 1-5 min. Finally, x-ray
films were developed, fixed, dried and documented with an imaging
system (Bio-Rad). The same samples were used to perform ELISA
analysis for p16.sup.INK4a, CK 10/13, CK18. The detected signals
and results were the similar to the Western blot analysis and the
same conclusions were drawn.
[0110] The ELISA analysis was performed as follows: Flat bottom 96
well plates (MAXISORB.RTM., chemical product; Nunc) were coated
with capture antibody (p16.sup.INK4a: MTM-E6H4, 2 .mu.g/ml in PBS,
MTM Laboratories; CK10: MS481P1ABX, 2 .mu.g/ml, dianova; CK18:
K18.7, 2 .mu.g/ml, dianova; 50 .mu.l/well) over night at 4.degree.
C. Plates were washed 6.times. with PBS/0.1% TWEEN.RTM.-20 and
blocked with SUPERBLOCK.RTM. buffer (chemical reagent, Pierce).
Solubilized protein extract from cervical swabs were dissolved in
incubation buffer (PBS, 3% SUPERBLOCK.RTM. (Chemical reagent), 0.1%
TWEEN.RTM.-20), and added in triplicates to each well. After 1 h
incubation at RT, plates were washed 6.times. with PBS/0.1%
TWEEN.RTM.-20 and incubated with biotinylated detection antibody
(p16.sup.INK4a: MTM-D7D7 (0.2 .mu.g/ml, MTM Laboratories, CK10:
MS481-BO, 200 .mu.g/ml, dianova; CK18: MS142-BO, 200 .mu.g/ml,
dianova; in incubation buffer) for 1 h at RT. Following 6.times.
washes with PBS/0.1% TWEEN.RTM.-20 TMB, 50 .mu.l of
streptavidin-coated alkaline phosphatase (1:1000 dilution; Dianova)
was added for 30 min. Thereafter, plates were washed 6.times. with
PBS/0.1% TWEEN.RTM.-20 and 100 .mu.l of p-nitrophenyl phosphate
substrate (PnPP; dissolved in diethanol amine buffer) were added to
each well. OD 405 nm (620 nm reference wavelength) was measured
with an ELISA reader (TECAN.RTM.) after 30 min, 1 h and 2 hrs. The
present example shows, that the sandwich ELISA format exhibits
sensitivity, which is suitable for the use in the methods according
to the present invention. For use in the method disclosed herein
the sandwich ELISA format as described in this example may be
applied to multiple marker molecules, such as markers for
normalization/adequacy and markers characteristic for medically
relevant conditions.
[0111] Samples of four patients with high-grade cervical dysplasias
(see Diagnosis) were analysed using western blot analysis (upper
panel of figure). For the left blot immunoblot detection was
performed using antibodies specific for .beta.-actin and
p16.sup.INK4a, for the middle blot antibodies specific for
cytokeratin 10/13 and for the right blot antibodies specific for
cytokeratin 18 were used. .beta.-actin, CK18, and CK10/13 were used
as markers demonstrating the adequacy of the sample. .beta.-actin
indicates the presence of any cells, CK10/13 the presence of
ectocervical squamous cells, CK 18 the presence of endocervical
columnar cells.
[0112] As shown in FIG. 3, for the samples of patient 1 and 2, the
immunoblot detections show positive signals for all the applied
adequacy markers (CK10/13, CK 18, .beta.-actin) and for the marker
(p16.sup.INK4a) indicative of dysplastic cells. Samples 3 and 4
were negative for p16.sup.INK4a bands in Western blot. However, in
these cases the .beta.-actin and the two cytokeratin markers showed
an extremely weak (patient 3, .beta.-actin) or negative (patient 4,
all markers; patient 3, CK markers) signal in the Western blot
analysis. So no diagnostic conclusion can be drawn from the
negative signal for p16.sup.INK4a.
[0113] The lower panel of this figure shows the results of ELISA
analysis. Positive signals for the adequacy markers (CK10/13, CK
18) were detected for the sample of patient 1 and 2, whereas for
the samples of patients 3 and 4 no signals for CK10/13 and CK 18
was seen. So the ELISA analysis results resemble the Western blot
analysis results and the same conclusions can be drawn.
Example 4
Western Blot Analysis of Different Samples of Pulmonary Origin
[0114] In order to evaluate, whether Western blot analysis of
solubilized samples allows to assess diagnosis of pulmonary
lesions, clinical samples with known diagnosis were solubilized and
subjected to an immuno-chemical analysis on the basis of marker and
normalization molecules.
[0115] The clinical samples (cells collected by brushing or
bronchoalveolar lavage) were analyzed by Standard Western Analysis
as follows. Cells from bronchoalveolar lavage were pelleted by
centrifugation (5 min, 1000 rpm) and the pellet was dissolved in
Lammli Protein Sample buffer (100 mM Tris pH.6.8, 2% SDS, 200 mM
DTT, 0.05% BpB). Cells obtained by brushing were dissolved directly
in Lammli Protein Sample buffer (100 mM Tris pH.6.8, 2% SDS, 200 mM
DTT, 0.05% BpB). The material was boiled (5 min, 95.degree. C.)
prior to sonification. In a second step aliquots of the protein
samples were resolved in duplicates on a SDS-PAGE (12% acrylamide)
and subsequently transferred on a nitrocellulose membrane by tank
blotting (Towbin et al., 1979, Proc Natl Acad Sci; 76:4350-4354).
In a further step the membranes were blocked to prevent unspecific
antibody binding (10% non fat dry milk in PBS) and subsequently one
membrane was incubated with specific monoclonal mouse antibodies
against NSE (DAKO Germany, clone BSS/NC/V1-H14, mouse monoclonal,
dilution 1:1000;) and one membrane was incubated with the
normalization marker actin (ICN, USA, clone C4, mouse monoclonal,
dilution 1:400). The binding of the specific antibody was
visualized by Horseradish Peroxidase conjugated secondary reagents
(binding to the marker specific antibody) catalyzing photon
emitting substrates.
[0116] In the bronchoalveolar lavages of patients with known small
cell lung cancers high levels of NSE in comparison with the
expression levels of actin was detected, whereas in patients
without tumor hardly any NSE could be detected, the actin level
however was comparable to the level of the cancer patients. (Data
not shown)
[0117] The results indicate, that a normalization of the solution
based testing procedure according to the method presented herein
enables for assessing diagnosis of diseases without relying on
morphological information
Example 5
Detection of Cervical Intraepithelial Neoplasia in an ELISA Test
Format
[0118] 34 cervical swabs provided in lysis buffer have been
subjected to ELISA based detection of overexpression of cyclin
dependent kinase inhibitor p16.sup.INK4a in solutions prepared from
the cells contained in the swabs. The ELISA testing was performed
as follows:
(A) Cell Lysis
[0119] Cervical swab brushes are given into 15 ml vessels,
containing 2 ml of mtm lysis buffer. Cervical cells present in the
brush are lysed for at least 20 h. The lysates of the cervical swab
samples are then transferred in 2 ml tubes and are centrifuged at
4.degree. C. (15 min at 28.000.times.g (16.600 rpm High-speed
Centrifuge JEC Multi RF)); Supernatant is transferred to a fresh
tube. As the case may be the supernatant may be stored at
-20.degree. C.
(B) Performing the ELISA
Coating of ELISA-Plates
[0120] Stock-solutions of p16.sup.INK4a-specific antibody clone mtm
E6H4, Ep-Cam specific antibody Ber-Ep4 and gamma-Catenin specific
antibody clone 15 are diluted in PBS to give ready-to-use coating
solution.
[0121] 50 .mu.l of each ready-to-use capture antibody coating
solution is added to ELISA plates.
[0122] For coating, the plates are incubated overnight at 4.degree.
C.
[0123] Coating solutions are removed from the ELISA plates and the
plates are rinsed using an automated ELISA washer as follows:
[0124] 7.times.250 .mu.l washing buffer (0.1% TWEEN.RTM.-20 (v/v)
in PBS)
[0125] after removing remnants of the washing buffer, 300 .mu.l
blocking buffer (2% BSA in PBS) is added to each well. Plates are
incubated for 1 h on a rocking device at ambient temperature.
Incubation with Samples
[0126] After removing the blocking buffer 100 .mu.l of the lysed
cell sample is added to each well. Lysates of HeLa-cells are used
as positive control for antibodies specifically detecting
p16.sup.INK4a and gamma-Catenin; Lysates of HT29-cells are used as
positive control for antibodies specifically detecting Ep-Cam;
[0127] For purpose of calibration of the test, different
concentrations of recombinant p16 protein, recombinant
gamma-Catenin and Ep-Cam (0 pg/ml, 50 pg/ml, 100 pg/ml, 200 pg/ml,
400 pg/ml, 800 pg/ml) are included in the test.
[0128] Samples are incubated for 1 h at room temperature.
[0129] Thereafter washing is performed on an automated ELISA washer
as follows [0130] 7.times.250 .mu.l washing buffer. The remaining
buffer is removed. Incubation with Detection Antibody
[0131] Working solutions of biotinylated secondary antibodies
(clone mtm D7D7 specific for p16.sup.INK4a, clone A5B4 for Ep-Cam
and clone MAB 2083 specific for gamma-Catenin) are prepared by
dilution of stock solutions.
[0132] 100 .mu.l of working solutions of biotinylated secondary
antibodies are added to wells incubated with corresponding antigen
and capture antibody. After incubation for 1 h at RT, antibody
solutions are removed and ELISA plates are washed by an automated
ELISA washer [0133] 7.times. with 250 .mu.l washing buffer.
Detection
[0134] Streptavidin-HRP-polymers (1 mg/ml) are pre-diluted 1:10 (4
.mu.l+36 .mu.l incubation buffer); Final incubation solution is
prepared by dilution 1:300 in incubation buffer (0.1% BSA in PBS)
to a final concentration of 0.33 .mu.g/ml.
[0135] 100 .mu.l of this solution are added to each well and
incubated for 1 h at RT.
[0136] Thereafter, the buffer is removed and the plates are washed
manually with 200 .mu.l washing buffer per well 5 times.
[0137] Substrate Incubation
[0138] TMB-substrate is equilibrated to 25.degree. C. for 1 h in
the dark.
[0139] 100 .mu.l of substrate solution is added to each well.
[0140] The ELISA plates are incubated at 25.degree. C. for exactly
15 min in the dark. Then the reaction is stopped by addition of 80
.mu.l 2.5M H.sub.2SO.sub.4.
[0141] Within 5 min. after stopping the reaction OD 450 nm is
determined. After evaluation of the results, each sample returns a
value for the OD.
Evaluation of Results
[0142] For sample adequacy, OD values of all samples for
gamma-Catenin have to exceed a defined threshold value to prove
proper sampling of a minimum of cells. Furthermore to ensure proper
sampling a threshold for the OD value of Ep-Cam indicating the
presence of endocervical cells has to be exceeded.
[0143] For detection of dysplastic cells, OD values for
p16.sup.INK4a have to exceed a defined threshold value to prove the
presence of a minimum of p16-positive dysplastic cells.
[0144] Results of this experiment are given in Table 6.
TABLE-US-00006 TABLE 6 No. of Gamma- samples P16.sup.INK4a Catenin
Conclusion 3 + + Sample is adequate; p16.sup.INK4a indicates the
presence of dysplastic cells 30 - + Sample is adequate; absence of
detectable p16.sup.INK4a indicates absence of dysplastic cells 1 -
- Sample is inadequate; re- sampling necessary
[0145] Comparison of OD values for p16.sup.INK4a and gamma-Catenin
of 34 samples with corresponding threshold values revealed that 33
samples were adequate and could be further evaluated. From theses
33 samples, 30 samples were negative for p16.sup.INK4a and 3 were
positive.
[0146] The ELISA results were compared to the diagnostic results of
a Papanicolaou test (PAP test, cervical cytology) from the same
patients. The cervical cytology were evaluated according to the
Munich Classification II (1990). Pap II encompasses benign cells,
cervicitis and metaplasia, Pap IV encompasses severe dysplasia and
carcinoma in situ. It turned out that samples returning an OD for
p16.sup.INK4a of greater than 0.9 in the ELISA correspond to
samples, that are classified as dysplastic by the conventional
cytological PAP test.
[0147] Applying OD 0.9 as threshold for the evaluation of the
samples the ELISA results may be reported as follows.
TABLE-US-00007 TABLE 7 Diagnosis/ ELISA ELISA ELISA positive for
negative results p16.sup.INK4a for p16.sup.INK4a Pap II 0 30 Pap IV
3 0 Not enough cells 0 1
[0148] The ELISA test is positive in all 3 samples (100%) from
women having severe dysplasia and is negative in all 30 samples
(100%) of women having no dysplasia. One sample only contained very
few cells and therefore was excluded from evaluation, since
sampling was inadequate.
[0149] The normalization of p16.sup.INK4a protein levels in
solubilized patient samples with respect to a normalization marker
characteristic for the presence of epithelial cells allows to
assess diagnosis of dysplasias from the samples. The normalization
in the present case allows especially to avoid false negative
results due to inadequate sampling (for example total amount of
patient material not sufficient to perform analysis, or the patient
material is not taken at the correct anatomical location). The
normalization is carried out in the testing format by applying a
threshold value for the OD for the gamma-Catenin normalization
marker determined in the ELISA above which the sample is to be
classified as adequate. Below a certain threshold (corresponding to
200.000 squamous ectocervical cells) the sample does not contain an
adequate amount of patient material. The use of a second
normalization marker indicating the presence of endocervical cells
provides further information about the adequacy of the sample. The
normalization is carried out in the testing format by applying a
threshold value for the OD for the Ep-Cam normalization marker
determined in the ELISA above which the sample is to be classified
as adequate. Below a certain threshold (corresponding to 2000
columnar endocervical cells) the sample does not contain an
adequate amount of endocervical cells. (It must be understood that
the threshold value applied in this example are adjusted to the
particular reaction conditions. The value for the cells as well for
OD may vary depending on the reaction conditions. Thus the values
herein are intended to exemplify the conditions and not to limit
the scope of the invention. Those of skill in the art know how an
appropriate threshold value for a particular test format may be
established.) The presence of endocervical cells provides the
information that the swab or brush has had contact with the
columnar epithelium of the endocervix and thus hints to a contact
of the swab or brush with the transformation zone, where cervical
dysplasia usually originates. In particular the detection of a
certain amount of ectocervical cells (gamma-catenin) together with
a certain amount of endocervical cells (Ep-Cam) provides with a
high probability the information that the patient material was
taken at the correct anatomical location (cervical transformation
zone).
[0150] Using the threshold values evaluated in these experiments,
cytological specimens of 300 patients were tested in the presented
ELISA testing format. In this experiments the specimens identified
as being dysplastic by cytological examination may also be
identified as being dysplastic in the ELISA testing format.
Example 6
Detection of Cervical Intraepithelial Neoplasia in an ELISA Test
Format
[0151] The 34 cervical swabs as already used in Example 5 provided
in lysis buffer have been subjected to ELISA based detection of
overexpression of HPV E7 Protein and one adequacy marker in
solutions prepared from the cells contained in the swabs. The ELISA
testing was performed as follows:
(A) Cell Lysis
[0152] Cervical swab brushes are given into 15 ml vessels,
containing 2 ml of mtm lysis buffer. Cervical cells present in the
brush are lysed for at least 20 h. The lysates of the cervical swab
samples are then transferred in 2 ml tubes and are centrifuged at
4.degree. C. (15 min at 28.000.times.g (16.600 rpm High-speed
Centrifuge JEC Multi RF)); Supernatant is transferred to a fresh
tube. As the case may be the supernatant may be stored at
-20.degree. C.
(B) Performing the ELISA
[0153] Coating of ELISA-Plates
[0154] Stock-solutions of E7-specific antibody clone NM2 and
gamma-Catenin specific antibody clone 15 are diluted in PBS to give
ready-to-use coating solution.
[0155] 50 .mu.l of each ready-to-use capture antibody coating
solution is added to ELISA plates.
[0156] For coating, the plates are incubated overnight at 4.degree.
C.
[0157] Coating solutions are removed from the ELISA plates and the
plates are rinsed using an automated ELISA washer as follows:
[0158] 7.times.250 .mu.l washing buffer (0.1% TWEEN.RTM.-20 (v/v)
in PBS)
[0159] after removing remnants of the washing buffer, 300 .mu.l
blocking buffer (2% BSA in PBS) is added to each well. Plates are
incubated for 1 h on a rocking device at ambient temperature.
Incubation with Samples
[0160] After removing the blocking buffer 100 .mu.l of the lysed
cell sample is added to each well. Lysates of HeLa-cells are used
as positive control for antibodies specifically detecting
gamma-Catenin; For purpose of calibration of the test, different
concentrations of recombinant HPV 16 E7-protein, recombinant
gamma-Catenin (0 pg/ml, 50 pg/ml, 100 pg/ml, 200 pg/ml, 400 pg/ml,
800 pg/ml) are included in the test.
[0161] Samples are incubated for 1 h at room temperature.
[0162] Thereafter washing is performed on an automated ELISA washer
as follows [0163] 7.times.250 .mu.l washing buffer. The remaining
buffer is removed.
[0164] Incubation with Detection Antibody
[0165] Working solutions of biotinylated secondary antibodies
(clone NM13 specific for HPV16 E7 protein and clone MAB 2083
specific for gamma-Catenin) are prepared by dilution of stock
solutions.
[0166] 100 .mu.l of working solutions of biotinylated secondary
antibodies are added to wells incubated with corresponding antigen
and capture antibody. After incubation for 1 h at RT, antibody
solutions are removed and ELISA plates are washed by an automated
ELISA washer [0167] 7.times. with 250 .mu.l washing buffer.
Detection
[0168] Streptavidin-HRP-polymers (1 mg/ml) are pre-diluted 1:10. (4
.mu.l+36 .mu.l incubation buffer); Final incubation solution is
prepared by dilution 1:300 in incubation buffer (0.1% BSA in PBS)
to a final concentration of 0.33 .mu.g/ml.
[0169] 100 .mu.l of this solution are added to each well and
incubated for 1 h at RT.
[0170] Thereafter, the buffer is removed and the plates are washed
manually with 200 .mu.l washing buffer per well 5 times.
Substrate Incubation
[0171] TMB-substrate is equilibrated to 25.degree. C. for 1 h in
the dark.
[0172] 100 .mu.l of substrate solution is added to each well.
[0173] The ELISA plates are incubated at 25.degree. C. for exactly
15 min in the dark. Then the reaction is stopped by addition of 80
.mu.l 2.5M H.sub.2SO.sub.4.
[0174] Within 5 min. after stopping the reaction OD 450 nm is
determined. After evaluation of the results, each sample returns a
value for the OD.
Evaluation of Results
[0175] For sample adequacy, OD values of all samples for
gamma-Catenin have to exceed a defined threshold value to prove
presence of a minimum of epithelial cells. (cf. Example 5)
[0176] For detection of dysplastic cells, OD values for HPV 16 E7
have to exceed a defined threshold value to prove the presence of a
minimum of transformed cells. The threshold depends on the ELISA
conditions applied and was set as OD 0.7 in our test format.
[0177] Comparison of OD values for HPV 16 E7, gamma-Catenin of 34
samples with threshold values revealed that the 33 samples proven
to contain epithelial cells by means of detection of
gamma-Catenin.
[0178] Although the invention has been described with reference to
the presently preferred embodiments, it should be understood that
various modifications could be made without departing from the
scope of the invention.
Sequence CWU 1
1
81745PRTHomo sapiensmisc_feature(1)..(745)gamma-Catenin, Swissprot
Accession Q86W21 1Met Glu Val Met Asn Leu Met Glu Gln Pro Ile Lys
Val Thr Glu Trp1 5 10 15Gln Gln Thr Tyr Thr Tyr Asp Ser Gly Ile His
Ser Gly Ala Asn Thr20 25 30Cys Val Pro Ser Val Ser Ser Lys Gly Ile
Met Glu Glu Asp Glu Ala35 40 45Cys Gly Arg Gln Tyr Thr Leu Lys Lys
Thr Thr Thr Tyr Thr Gln Gly50 55 60Val Pro Pro Ser Gln Gly Asp Leu
Glu Tyr Gln Met Ser Thr Thr Ala65 70 75 80Arg Ala Lys Arg Val Arg
Glu Ala Met Cys Ser Gly Val Ser Gly Glu85 90 95Asp Ser Ser Leu Leu
Leu Ala Thr Gln Val Glu Gly Gln Ala Thr Asn100 105 110Leu Gln Arg
Leu Ala Glu Pro Ser Gln Leu Leu Lys Ser Ala Ile Val115 120 125His
Leu Ile Asn Tyr Gln Asp Asp Ala Glu Leu Ala Thr Arg Ala Leu130 135
140Pro Glu Leu Thr Lys Leu Leu Asn Asp Glu Asp Pro Val Val Val
Thr145 150 155 160Lys Ala Ala Met Ile Val Asn Gln Leu Ser Lys Lys
Glu Ala Ser Arg165 170 175Arg Ala Leu Met Gly Ser Pro Gln Leu Val
Ala Ala Val Val Arg Thr180 185 190Met Gln Asn Thr Ser Asp Leu Asp
Thr Ala Arg Cys Thr Thr Ser Ile195 200 205Leu His Asn Leu Ser His
His Arg Glu Gly Leu Leu Ala Ile Phe Lys210 215 220Ser Gly Gly Ile
Pro Ala Leu Val Arg Met Leu Ser Ser Pro Val Glu225 230 235 240Ser
Val Leu Phe Tyr Ala Ile Thr Thr Leu His Asn Leu Leu Leu Tyr245 250
255Gln Glu Gly Ala Lys Met Ala Val Arg Leu Ala Asp Gly Leu Gln
Lys260 265 270Met Val Pro Leu Leu Asn Lys Asn Asn Pro Lys Phe Leu
Ala Ile Thr275 280 285Thr Asp Cys Leu Gln Leu Leu Ala Tyr Gly Asn
Gln Glu Ser Lys Leu290 295 300Ile Ile Leu Ala Asn Gly Gly Pro Gln
Ala Leu Val Gln Ile Met Arg305 310 315 320Asn Tyr Ser Tyr Glu Lys
Leu Leu Trp Thr Thr Ser Arg Val Leu Lys325 330 335Val Leu Ser Val
Cys Pro Ser Asn Lys Pro Ala Ile Val Glu Ala Gly340 345 350Gly Met
Gln Ala Leu Gly Lys His Leu Thr Ser Asn Ser Pro Arg Leu355 360
365Val Gln Asn Cys Leu Trp Thr Leu Arg Asn Leu Ser Asp Val Ala
Thr370 375 380Lys Gln Glu Gly Leu Glu Ser Val Leu Lys Ile Leu Val
Asn Gln Leu385 390 395 400Ser Val Asp Asp Val Asn Val Leu Thr Cys
Ala Thr Gly Thr Leu Ser405 410 415Asn Leu Thr Cys Asn Asn Ser Lys
Asn Lys Thr Leu Val Thr Gln Asn420 425 430Ser Gly Val Glu Ala Leu
Ile His Ala Ile Leu Arg Ala Gly Asp Lys435 440 445Asp Asp Ile Thr
Glu Pro Ala Val Cys Ala Leu Arg His Leu Thr Ser450 455 460Arg His
Pro Glu Ala Glu Met Ala Gln Asn Ser Val Arg Leu Asn Tyr465 470 475
480Gly Ile Pro Ala Ile Val Lys Leu Leu Asn Gln Pro Asn Gln Trp
Pro485 490 495Leu Val Lys Ala Thr Ile Gly Leu Ile Arg Asn Leu Ala
Leu Cys Pro500 505 510Ala Asn His Ala Pro Leu Gln Glu Ala Ala Val
Ile Pro Arg Leu Val515 520 525Gln Leu Leu Val Lys Ala His Gln Asp
Ala Gln Arg His Val Ala Ala530 535 540Gly Thr Gln Gln Pro Tyr Thr
Asp Gly Val Arg Met Glu Glu Ile Val545 550 555 560Glu Gly Cys Thr
Gly Ala Leu His Ile Leu Ala Arg Asp Pro Met Asn565 570 575Arg Met
Glu Ile Phe Arg Leu Asn Thr Ile Pro Leu Phe Val Gln Leu580 585
590Leu Tyr Ser Ser Val Glu Asn Ile Gln Arg Val Ala Ala Gly Val
Leu595 600 605Cys Glu Leu Ala Gln Asp Lys Glu Ala Ala Asp Ala Ile
Asp Ala Glu610 615 620Gly Ala Ser Ala Pro Leu Met Glu Leu Leu His
Ser Arg Asn Glu Gly625 630 635 640Thr Ala Thr Tyr Ala Ala Ala Val
Leu Phe Arg Ile Ser Glu Asp Lys645 650 655Asn Pro Asp Tyr Arg Lys
Arg Val Ser Val Glu Leu Thr Asn Ser Leu660 665 670Phe Lys His Asp
Pro Ala Ala Trp Glu Ala Ala Gln Ser Met Ile Pro675 680 685Ile Asn
Glu Pro Tyr Gly Asp Asp Leu Asp Ala Thr Tyr Arg Pro Met690 695
700Tyr Ser Ser Asp Val Pro Leu Asp Pro Leu Glu Met His Met Asp
Met705 710 715 720Asp Gly Asp Tyr Pro Ile Asp Thr Tyr Ser Asp Gly
Leu Arg Pro Pro725 730 735Tyr Pro Thr Ala Asp His Met Leu Ala740
7452314PRTHomo sapiensmisc_feature(1)..(314)Ep-Cam, Swissprot
Accession P16422 2Met Ala Pro Pro Gln Val Leu Ala Phe Gly Leu Leu
Leu Ala Ala Ala1 5 10 15Thr Ala Thr Phe Ala Ala Ala Gln Glu Glu Cys
Val Cys Glu Asn Tyr20 25 30Lys Leu Ala Val Asn Cys Phe Val Asn Asn
Asn Arg Gln Cys Gln Cys35 40 45Thr Ser Val Gly Ala Gln Asn Thr Val
Ile Cys Ser Lys Leu Ala Ala50 55 60Lys Cys Leu Val Met Lys Ala Glu
Met Asn Gly Ser Lys Leu Gly Arg65 70 75 80Arg Ala Lys Pro Glu Gly
Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp85 90 95Pro Asp Cys Asp Glu
Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly100 105 110Thr Ser Thr
Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp115 120 125Lys
Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile130 135
140Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser
Lys145 150 155 160Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr
Arg Tyr Gln Leu165 170 175Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr
Glu Asn Asn Val Ile Thr180 185 190Ile Asp Leu Val Gln Asn Ser Ser
Gln Lys Thr Gln Asn Asp Val Asp195 200 205Ile Ala Asp Val Ala Tyr
Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser210 215 220Leu Phe His Ser
Lys Lys Met Asp Leu Thr Val Asn Gly Glu Gln Leu225 230 235 240Asp
Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala245 250
255Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val
Ile260 265 270Val Val Val Val Met Ala Val Val Ala Gly Ile Val Val
Leu Val Ile275 280 285Ser Arg Lys Lys Arg Met Ala Lys Tyr Glu Lys
Ala Glu Ile Lys Glu290 295 300Met Gly Glu Met His Arg Glu Leu Asn
Ala305 3103882PRTHomo sapiensmisc_feature(1)..(882)E-Cadherin,
Swissprot Accession P12830 3Met Gly Pro Trp Ser Arg Ser Leu Ser Ala
Leu Leu Leu Leu Leu Gln1 5 10 15Val Ser Ser Trp Leu Cys Gln Glu Pro
Glu Pro Cys His Pro Gly Phe20 25 30Asp Ala Glu Ser Tyr Thr Phe Thr
Val Pro Arg Arg His Leu Glu Arg35 40 45Gly Arg Val Leu Gly Arg Val
Asn Phe Glu Asp Cys Thr Gly Arg Gln50 55 60Arg Thr Ala Tyr Phe Ser
Leu Asp Thr Arg Phe Lys Val Gly Thr Asp65 70 75 80Gly Val Ile Thr
Val Lys Arg Pro Leu Arg Phe His Asn Pro Gln Ile85 90 95His Phe Leu
Val Tyr Ala Trp Asp Ser Thr Tyr Arg Lys Phe Ser Thr100 105 110Lys
Val Thr Leu Asn Thr Val Gly His His His Arg Pro Pro Pro His115 120
125Gln Ala Ser Val Ser Gly Ile Gln Ala Glu Leu Leu Thr Phe Pro
Asn130 135 140Ser Ser Pro Gly Leu Arg Arg Gln Lys Arg Asp Trp Val
Ile Pro Pro145 150 155 160Ile Ser Cys Pro Glu Asn Glu Lys Gly Pro
Phe Pro Lys Asn Leu Val165 170 175Gln Ile Lys Ser Asn Lys Asp Lys
Glu Gly Lys Val Phe Tyr Ser Ile180 185 190Thr Gly Gln Gly Ala Asp
Thr Pro Pro Val Gly Val Phe Ile Ile Glu195 200 205Arg Glu Thr Gly
Trp Leu Lys Val Thr Glu Pro Leu Asp Arg Glu Arg210 215 220Ile Ala
Thr Tyr Thr Leu Phe Ser His Ala Val Ser Ser Asn Gly Asn225 230 235
240Ala Val Glu Asp Pro Met Glu Ile Leu Ile Thr Val Thr Asp Gln
Asn245 250 255Asp Asn Lys Pro Glu Phe Thr Gln Glu Val Phe Lys Gly
Ser Val Met260 265 270Glu Gly Ala Leu Pro Gly Thr Ser Val Met Glu
Val Thr Ala Thr Asp275 280 285Ala Asp Asp Asp Val Asn Thr Tyr Asn
Ala Ala Ile Ala Tyr Thr Ile290 295 300Leu Ser Gln Asp Pro Glu Leu
Pro Asp Lys Asn Met Phe Thr Ile Asn305 310 315 320Arg Asn Thr Gly
Val Ile Ser Val Val Thr Thr Gly Leu Asp Arg Glu325 330 335Ser Phe
Pro Thr Tyr Thr Leu Val Val Gln Ala Ala Asp Leu Gln Gly340 345
350Glu Gly Leu Ser Thr Thr Ala Thr Ala Val Ile Thr Val Thr Asp
Thr355 360 365Asn Asp Asn Pro Pro Ile Phe Asn Pro Thr Thr Tyr Lys
Gly Gln Val370 375 380Pro Glu Asn Glu Ala Asn Val Val Ile Thr Thr
Leu Lys Val Thr Asp385 390 395 400Ala Asp Ala Pro Asn Thr Pro Ala
Trp Glu Ala Val Tyr Thr Ile Leu405 410 415Asn Asp Asp Gly Gly Gln
Phe Val Val Thr Thr Asn Pro Val Asn Asn420 425 430Asp Gly Ile Leu
Lys Thr Ala Lys Gly Leu Asp Phe Glu Ala Lys Gln435 440 445Gln Tyr
Ile Leu His Val Ala Val Thr Asn Val Val Pro Phe Glu Val450 455
460Ser Leu Thr Thr Ser Thr Ala Thr Val Thr Val Asp Val Leu Asp
Val465 470 475 480Asn Glu Ala Pro Ile Phe Val Pro Pro Glu Lys Arg
Val Glu Val Ser485 490 495Glu Asp Phe Gly Val Gly Gln Glu Ile Thr
Ser Tyr Thr Ala Gln Glu500 505 510Pro Asp Thr Phe Met Glu Gln Lys
Ile Thr Tyr Arg Ile Trp Arg Asp515 520 525Thr Ala Asn Trp Leu Glu
Ile Asn Pro Asp Thr Gly Ala Ile Ser Thr530 535 540Arg Ala Glu Leu
Asp Arg Glu Asp Phe Glu His Val Lys Asn Ser Thr545 550 555 560Tyr
Thr Ala Leu Ile Ile Ala Thr Asp Asn Gly Ser Pro Val Ala Thr565 570
575Gly Thr Gly Thr Leu Leu Leu Ile Leu Ser Asp Val Asn Asp Asn
Ala580 585 590Pro Ile Pro Glu Pro Arg Thr Ile Phe Phe Cys Glu Arg
Asn Pro Lys595 600 605Pro Gln Val Ile Asn Ile Ile Asp Ala Asp Leu
Pro Pro Asn Thr Ser610 615 620Pro Phe Thr Ala Glu Leu Thr His Gly
Ala Ser Ala Asn Trp Thr Ile625 630 635 640Gln Tyr Asn Asp Pro Thr
Gln Glu Ser Ile Ile Leu Lys Pro Lys Met645 650 655Ala Leu Glu Val
Gly Asp Tyr Lys Ile Asn Leu Lys Leu Met Asp Asn660 665 670Gln Asn
Lys Asp Gln Val Thr Thr Leu Glu Val Ser Val Cys Asp Cys675 680
685Glu Gly Ala Ala Gly Val Cys Arg Lys Ala Gln Pro Val Glu Ala
Gly690 695 700Leu Gln Ile Pro Ala Ile Leu Gly Ile Leu Gly Gly Ile
Leu Ala Leu705 710 715 720Leu Ile Leu Ile Leu Leu Leu Leu Leu Phe
Leu Arg Arg Arg Ala Val725 730 735Val Lys Glu Pro Leu Leu Pro Pro
Glu Asp Asp Thr Arg Asp Asn Val740 745 750Tyr Tyr Tyr Asp Glu Glu
Gly Gly Gly Glu Glu Asp Gln Asp Phe Asp755 760 765Leu Ser Gln Leu
His Arg Gly Leu Asp Ala Arg Pro Glu Val Thr Arg770 775 780Asn Asp
Val Ala Pro Thr Leu Met Ser Val Pro Arg Tyr Leu Pro Arg785 790 795
800Pro Ala Asn Pro Asp Glu Ile Gly Asn Phe Ile Asp Glu Asn Leu
Lys805 810 815Ala Ala Asp Thr Asp Pro Thr Ala Pro Pro Tyr Asp Ser
Leu Leu Val820 825 830Phe Asp Tyr Glu Gly Ser Gly Ser Glu Ala Ala
Ser Leu Ser Ser Leu835 840 845Asn Ser Ser Glu Ser Asp Lys Asp Gln
Asp Tyr Asp Tyr Leu Asn Glu850 855 860Trp Gly Asn Arg Phe Lys Lys
Leu Ala Asp Met Tyr Gly Gly Gly Glu865 870 875 880Asp
Asp4906PRTHomo sapiensmisc_feature(1)..(906)Alpha-1 Catenin,
Swissprot Accession P35221 4Met Thr Ala Val His Ala Gly Asn Ile Asn
Phe Lys Trp Asp Pro Lys1 5 10 15Ser Leu Glu Ile Arg Thr Leu Ala Val
Glu Arg Leu Leu Glu Pro Leu20 25 30Val Thr Gln Val Thr Thr Leu Val
Asn Thr Asn Ser Lys Gly Pro Ser35 40 45Asn Lys Lys Arg Gly Arg Ser
Lys Lys Ala His Val Leu Ala Ala Ser50 55 60Val Glu Gln Ala Thr Glu
Asn Phe Leu Glu Lys Gly Asp Lys Ile Ala65 70 75 80Lys Glu Ser Gln
Phe Leu Lys Glu Glu Leu Val Ala Ala Val Glu Asp85 90 95Val Arg Lys
Gln Gly Asp Leu Met Lys Ala Ala Ala Gly Glu Phe Ala100 105 110Asp
Asp Pro Cys Ser Ser Val Lys Arg Gly Asn Met Val Arg Ala Ala115 120
125Arg Ala Leu Leu Ser Ala Val Thr Arg Leu Leu Ile Leu Ala Asp
Met130 135 140Ala Asp Val Tyr Lys Leu Leu Val Gln Leu Lys Val Val
Glu Asp Gly145 150 155 160Ile Leu Lys Leu Arg Asn Ala Gly Asn Glu
Gln Asp Leu Gly Ile Gln165 170 175Tyr Lys Ala Leu Lys Pro Glu Val
Asp Lys Leu Asn Ile Met Ala Ala180 185 190Lys Arg Gln Gln Glu Leu
Lys Asp Val Gly His Arg Asp Gln Met Ala195 200 205Ala Ala Arg Gly
Ile Leu Gln Lys Asn Val Pro Ile Leu Tyr Thr Ala210 215 220Ser Gln
Ala Cys Leu Gln His Pro Asp Val Ala Ala Tyr Lys Ala Asn225 230 235
240Arg Asp Leu Ile Tyr Lys Gln Leu Gln Gln Ala Val Thr Gly Ile
Ser245 250 255Asn Ala Ala Gln Ala Thr Ala Ser Asp Asp Ala Ser Gln
His Gln Gly260 265 270Gly Gly Gly Gly Glu Leu Ala Tyr Ala Leu Asn
Asn Phe Asp Lys Gln275 280 285Ile Ile Val Asp Pro Leu Ser Phe Ser
Glu Glu Arg Phe Arg Pro Ser290 295 300Leu Glu Glu Arg Leu Glu Ser
Ile Ile Ser Gly Ala Ala Leu Met Ala305 310 315 320Asp Ser Ser Cys
Thr Arg Asp Asp Arg Arg Glu Arg Ile Val Ala Glu325 330 335Cys Asn
Ala Val Arg Gln Ala Leu Gln Asp Leu Leu Ser Glu Tyr Met340 345
350Gly Asn Ala Gly Arg Lys Glu Arg Ser Asp Ala Leu Asn Ser Ala
Ile355 360 365Asp Lys Met Thr Lys Lys Thr Arg Asp Leu Arg Arg Gln
Leu Arg Lys370 375 380Ala Val Met Asp His Val Ser Asp Ser Phe Leu
Glu Thr Asn Val Pro385 390 395 400Leu Leu Val Leu Ile Glu Ala Ala
Lys Asn Gly Asn Glu Lys Glu Val405 410 415Lys Glu Tyr Ala Gln Val
Phe Arg Glu His Ala Asn Lys Leu Ile Glu420 425 430Val Ala Asn Leu
Ala Cys Ser Ile Ser Asn Asn Glu Glu Gly Val Lys435 440 445Leu Val
Arg Met Ser Ala Ser Gln Leu Glu Ala Leu Cys Pro Gln Val450 455
460Ile Asn Ala Ala Leu Ala Leu Ala Ala Lys Pro Gln Ser Lys Leu
Ala465 470 475 480Gln Glu Asn Met Asp Leu Phe Lys Glu Gln Trp Glu
Lys Gln Val Arg485 490 495Val Leu Thr Asp Ala Val Asp Asp Ile Thr
Ser Ile Asp Asp Phe Leu500 505 510Ala Val Ser Glu Asn His Ile Leu
Glu Asp Val Asn Lys Cys Val Ile515 520 525Ala Leu Gln Glu Lys Asp
Val Asp Gly Leu Asp Arg Thr Ala Gly Ala530 535 540Ile Arg Gly Arg
Ala Ala Arg Val Ile His Val Val Thr Ser Glu Met545 550 555 560Asp
Asn Tyr Glu Pro Gly Val Tyr Thr Glu Lys Val Leu Glu Ala Thr565 570
575Lys Leu Leu Ser Asn Thr Val Met Pro Arg Phe Thr Glu Gln Val
Glu580 585 590Ala Ala Val Glu Ala Leu Ser Ser Asp Pro Ala Gln Pro
Met Asp Glu595 600 605Asn Glu Phe Ile Asp Ala Ser Arg Leu Val Tyr
Asp Gly Ile Arg Asp610 615 620Ile Arg Lys Ala Val Leu Met Ile Arg
Thr Pro Glu Glu Leu Asp Asp625 630 635 640Ser Asp Phe Glu Thr Glu
Asp Phe Asp Val Arg Ser Arg Thr Ser Val645 650 655Gln Thr Glu Asp
Asp Gln Leu Ile Ala Gly Gln Ser Ala Arg
Ala Ile660 665 670Met Ala Gln Leu Pro Gln Glu Gln Lys Ala Lys Ile
Ala Glu Gln Val675 680 685Ala Ser Phe Gln Glu Glu Lys Ser Lys Leu
Asp Ala Glu Val Ser Lys690 695 700Trp Asp Asp Ser Gly Asn Asp Ile
Ile Val Leu Ala Lys Gln Met Cys705 710 715 720Met Ile Met Met Glu
Met Thr Asp Phe Thr Arg Gly Lys Gly Pro Leu725 730 735Lys Asn Thr
Ser Asp Val Ile Ser Ala Ala Lys Lys Ile Ala Glu Ala740 745 750Gly
Ser Arg Met Asp Lys Leu Gly Arg Thr Ile Ala Asp His Cys Pro755 760
765Asp Ser Ala Cys Lys Gln Asp Leu Leu Ala Tyr Leu Gln Arg Ile
Ala770 775 780Leu Tyr Cys His Gln Leu Asn Ile Cys Ser Lys Val Lys
Ala Glu Val785 790 795 800Gln Asn Leu Gly Gly Glu Leu Val Val Ser
Gly Val Asp Ser Ala Met805 810 815Ser Leu Ile Gln Ala Ala Lys Asn
Leu Met Asn Ala Val Val Gln Thr820 825 830Val Lys Ala Ser Tyr Val
Ala Ser Thr Lys Tyr Gln Lys Ser Gln Gly835 840 845Met Ala Ser Leu
Asn Leu Pro Ala Val Ser Trp Lys Met Lys Ala Pro850 855 860Glu Lys
Lys Pro Leu Val Lys Arg Glu Lys Gln Asp Glu Thr Gln Thr865 870 875
880Lys Ile Lys Arg Ala Ser Gln Lys Lys His Val Asn Pro Val Gln
Ala885 890 895Leu Ser Glu Phe Lys Ala Met Asp Ser Ile900
9055953PRTHomo sapiensmisc_feature(1)..(953)Alpha-2 Catenin,
Swissprot Accession P26232 5Met Thr Ser Ala Thr Ser Pro Ile Ile Leu
Lys Trp Asp Pro Lys Ser1 5 10 15Leu Glu Ile Arg Thr Leu Thr Val Glu
Arg Leu Leu Glu Pro Leu Val20 25 30Thr Gln Val Thr Thr Leu Val Asn
Thr Ser Asn Lys Gly Pro Ser Gly35 40 45Lys Lys Lys Gly Arg Ser Lys
Lys Ala His Val Leu Ala Ala Ser Val50 55 60Glu Gln Ala Thr Gln Asn
Phe Leu Glu Lys Gly Glu Gln Ile Ala Lys65 70 75 80Glu Ser Gln Asp
Leu Lys Glu Glu Leu Val Ala Ala Val Glu Asp Val85 90 95Arg Lys Gln
Gly Glu Thr Met Arg Ile Ala Ser Ser Glu Phe Ala Asp100 105 110Asp
Pro Cys Ser Ser Val Lys Arg Gly Thr Met Val Arg Ala Ala Arg115 120
125Ala Leu Leu Ser Ala Val Thr Arg Leu Leu Ile Leu Ala Asp Met
Ala130 135 140Asp Val Met Arg Leu Leu Ser His Leu Lys Ile Val Glu
Glu Ala Leu145 150 155 160Glu Ala Val Lys Asn Ala Thr Asn Glu Gln
Asp Leu Ala Asn Arg Phe165 170 175Lys Glu Phe Gly Lys Lys Met Val
Lys Leu Asn Tyr Val Ala Ala Arg180 185 190Arg Gln Gln Glu Leu Lys
Asp Pro His Cys Arg Asp Glu Met Ala Ala195 200 205Ala Arg Gly Ala
Leu Lys Lys Asn Ala Thr Met Leu Tyr Thr Ala Ser210 215 220Gln Ala
Phe Leu Arg His Pro Asp Val Ala Ala Thr Arg Ala Asn Arg225 230 235
240Asp Tyr Val Phe Lys Gln Val Gln Glu Ala Ile Ala Gly Ile Ser
Asn245 250 255Ala Ala Gln Ala Thr Ser Pro Thr Asp Glu Ala Lys Gly
His Thr Gly260 265 270Ile Gly Glu Leu Ala Ala Ala Leu Asn Glu Phe
Asp Asn Lys Ile Ile275 280 285Leu Asp Pro Met Thr Phe Ser Glu Ala
Arg Phe Arg Pro Ser Leu Glu290 295 300Glu Arg Leu Glu Ser Ile Ile
Ser Gly Ala Ala Leu Met Ala Asp Ser305 310 315 320Ser Cys Thr Arg
Asp Asp Arg Arg Glu Arg Ile Val Ala Glu Cys Asn325 330 335Ala Val
Arg Gln Ala Leu Gln Asp Leu Leu Ser Glu Tyr Met Asn Asn340 345
350Thr Gly Arg Lys Glu Lys Gly Asp Pro Leu Asn Ile Ala Ile Asp
Lys355 360 365Met Thr Lys Lys Thr Arg Asp Leu Arg Arg Gln Leu Arg
Lys Ala Val370 375 380Met Asp His Ile Ser Asp Ser Phe Leu Glu Thr
Asn Val Pro Leu Leu385 390 395 400Val Leu Ile Glu Ala Ala Lys Ser
Gly Asn Glu Lys Glu Val Lys Glu405 410 415Tyr Ala Gln Val Phe Arg
Glu His Ala Asn Lys Leu Val Glu Val Ala420 425 430Asn Leu Ala Cys
Ser Ile Ser Asn Asn Glu Glu Gly Val Lys Leu Val435 440 445Arg Met
Ala Ala Thr Gln Ile Asp Ser Leu Cys Pro Gln Val Ile Asn450 455
460Ala Ala Leu Thr Leu Ala Ala Arg Pro Gln Ser Lys Val Ala Gln
Asp465 470 475 480Asn Met Asp Val Phe Lys Asp Gln Trp Glu Lys Gln
Val Arg Val Leu485 490 495Thr Glu Ala Val Asp Asp Ile Thr Ser Val
Asp Asp Phe Leu Ser Val500 505 510Ser Glu Asn His Ile Leu Glu Asp
Val Asn Lys Cys Val Ile Ala Leu515 520 525Gln Glu Gly Asp Val Asp
Thr Leu Asp Arg Thr Ala Gly Ala Ile Arg530 535 540Gly Arg Ala Ala
Arg Val Ile His Ile Ile Asn Ala Glu Met Glu Asn545 550 555 560Tyr
Glu Ala Gly Val Tyr Thr Glu Lys Val Leu Glu Ala Thr Lys Leu565 570
575Leu Ser Glu Thr Val Met Pro Arg Phe Ala Glu Gln Val Glu Val
Ala580 585 590Ile Glu Ala Leu Ser Ala Asn Val Pro Gln Pro Phe Glu
Glu Asn Glu595 600 605Phe Ile Asp Ala Ser Arg Leu Val Tyr Asp Gly
Val Arg Asp Ile Arg610 615 620Lys Ala Val Leu Met Ile Arg Thr Pro
Glu Glu Leu Glu Asp Asp Ser625 630 635 640Asp Phe Glu Gln Glu Asp
Tyr Asp Val Arg Arg Gly Thr Ser Val Gln645 650 655Thr Glu Asp Asp
Gln Leu Ile Ala Gly Gln Ser Ala Arg Ala Ile Met660 665 670Ala Gln
Leu Pro Gln Glu Glu Lys Ala Lys Ile Ala Glu Gln Val Glu675 680
685Ile Phe His Gln Glu Lys Ser Lys Leu Asp Ala Glu Val Ala Lys
Trp690 695 700Asp Asp Ser Gly Asn Asp Ile Ile Val Leu Ala Lys Gln
Met Cys Met705 710 715 720Ile Met Met Glu Met Thr Asp Phe Thr Arg
Gly Lys Gly Pro Leu Lys725 730 735Asn Thr Ser Asp Val Ile Asn Ala
Ala Lys Lys Ile Ala Glu Ala Gly740 745 750Ser Arg Met Asp Lys Leu
Ala Arg Ala Val Ala Asp Gln Cys Pro Asp755 760 765Ser Ala Cys Lys
Gln Asp Leu Leu Ala Tyr Leu Gln Arg Ile Ala Leu770 775 780Tyr Cys
His Gln Leu Asn Ile Cys Ser Lys Val Lys Ala Glu Val Gln785 790 795
800Asn Leu Gly Gly Glu Leu Ile Val Ser Gly Thr Gly Val Gln Ser
Thr805 810 815Phe Thr Thr Phe Tyr Glu Val Asp Cys Asp Val Ile Asp
Gly Gly Arg820 825 830Ala Ser Gln Leu Ser Thr His Leu Pro Thr Cys
Ala Glu Gly Ala Pro835 840 845Ile Gly Ser Gly Ser Ser Asp Ser Ser
Met Leu Asp Ser Ala Thr Ser850 855 860Leu Ile Gln Ala Ala Lys Asn
Leu Met Asn Ala Val Val Leu Thr Val865 870 875 880Lys Ala Ser Tyr
Val Ala Ser Thr Lys Tyr Gln Lys Val Tyr Gly Thr885 890 895Ala Ala
Val Asn Ser Pro Val Val Ser Trp Lys Met Lys Ala Pro Glu900 905
910Lys Lys Pro Leu Val Lys Arg Glu Lys Pro Glu Glu Phe Gln Thr
Arg915 920 925Val Arg Arg Gly Ser Gln Lys Lys His Ile Ser Pro Val
Gln Ala Leu930 935 940Ser Glu Phe Lys Ala Met Asp Ser Phe945
9506781PRTHomo sapiensmisc_feature(1)..(781)beta-Catenin, Swissprot
Accession P35222 6Met Ala Thr Gln Ala Asp Leu Met Glu Leu Asp Met
Ala Met Glu Pro1 5 10 15Asp Arg Lys Ala Ala Val Ser His Trp Gln Gln
Gln Ser Tyr Leu Asp20 25 30Ser Gly Ile His Ser Gly Ala Thr Thr Thr
Ala Pro Ser Leu Ser Gly35 40 45Lys Gly Asn Pro Glu Glu Glu Asp Val
Asp Thr Ser Gln Val Leu Tyr50 55 60Glu Trp Glu Gln Gly Phe Ser Gln
Ser Phe Thr Gln Glu Gln Val Ala65 70 75 80Asp Ile Asp Gly Gln Tyr
Ala Met Thr Arg Ala Gln Arg Val Arg Ala85 90 95Ala Met Phe Pro Glu
Thr Leu Asp Glu Gly Met Gln Ile Pro Ser Thr100 105 110Gln Phe Asp
Ala Ala His Pro Thr Asn Val Gln Arg Leu Ala Glu Pro115 120 125Ser
Gln Met Leu Lys His Ala Val Val Asn Leu Ile Asn Tyr Gln Asp130 135
140Asp Ala Glu Leu Ala Thr Arg Ala Ile Pro Glu Leu Thr Lys Leu
Leu145 150 155 160Asn Asp Glu Asp Gln Val Val Val Asn Lys Ala Ala
Val Met Val His165 170 175Gln Leu Ser Lys Lys Glu Ala Ser Arg His
Ala Ile Met Arg Ser Pro180 185 190Gln Met Val Ser Ala Ile Val Arg
Thr Met Gln Asn Thr Asn Asp Val195 200 205Glu Thr Ala Arg Cys Thr
Ala Gly Thr Leu His Asn Leu Ser His His210 215 220Arg Glu Gly Leu
Leu Ala Ile Phe Lys Ser Gly Gly Ile Pro Ala Leu225 230 235 240Val
Lys Met Leu Gly Ser Pro Val Asp Ser Val Leu Phe Tyr Ala Ile245 250
255Thr Thr Leu His Asn Leu Leu Leu His Gln Glu Gly Ala Lys Met
Ala260 265 270Val Arg Leu Ala Gly Gly Leu Gln Lys Met Val Ala Leu
Leu Asn Lys275 280 285Thr Asn Val Lys Phe Leu Ala Ile Thr Thr Asp
Cys Leu Gln Ile Leu290 295 300Ala Tyr Gly Asn Gln Glu Ser Lys Leu
Ile Ile Leu Ala Ser Gly Gly305 310 315 320Pro Gln Ala Leu Val Asn
Ile Met Arg Thr Tyr Thr Tyr Glu Lys Leu325 330 335Leu Trp Thr Thr
Ser Arg Val Leu Lys Val Leu Ser Val Cys Ser Ser340 345 350Asn Lys
Pro Ala Ile Val Glu Ala Gly Gly Met Gln Ala Leu Gly Leu355 360
365His Leu Thr Asp Pro Ser Gln Arg Leu Val Gln Asn Cys Leu Trp
Thr370 375 380Leu Arg Asn Leu Ser Asp Ala Ala Thr Lys Gln Glu Gly
Met Glu Gly385 390 395 400Leu Leu Gly Thr Leu Val Gln Leu Leu Gly
Ser Asp Asp Ile Asn Val405 410 415Val Thr Cys Ala Ala Gly Ile Leu
Ser Asn Leu Thr Cys Asn Asn Tyr420 425 430Lys Asn Lys Met Met Val
Cys Gln Val Gly Gly Ile Glu Ala Leu Val435 440 445Arg Thr Val Leu
Arg Ala Gly Asp Arg Glu Asp Ile Thr Glu Pro Ala450 455 460Ile Cys
Ala Leu Arg His Leu Thr Ser Arg His Gln Glu Ala Glu Met465 470 475
480Ala Gln Asn Ala Val Arg Leu His Tyr Gly Leu Pro Val Val Val
Lys485 490 495Leu Leu His Pro Pro Ser His Trp Pro Leu Ile Lys Ala
Thr Val Gly500 505 510Leu Ile Arg Asn Leu Ala Leu Cys Pro Ala Asn
His Ala Pro Leu Arg515 520 525Glu Gln Gly Ala Ile Pro Arg Leu Val
Gln Leu Leu Val Arg Ala His530 535 540Gln Asp Thr Gln Arg Arg Thr
Ser Met Gly Gly Thr Gln Gln Gln Phe545 550 555 560Val Glu Gly Val
Arg Met Glu Glu Ile Val Glu Gly Cys Thr Gly Ala565 570 575Leu His
Ile Leu Ala Arg Asp Val His Asn Arg Ile Val Ile Arg Gly580 585
590Leu Asn Thr Ile Pro Leu Phe Val Gln Leu Leu Tyr Ser Pro Ile
Glu595 600 605Asn Ile Gln Arg Val Ala Ala Gly Val Leu Cys Glu Leu
Ala Gln Asp610 615 620Lys Glu Ala Ala Glu Ala Ile Glu Ala Glu Gly
Ala Thr Ala Pro Leu625 630 635 640Thr Glu Leu Leu His Ser Arg Asn
Glu Gly Val Ala Thr Tyr Ala Ala645 650 655Ala Val Leu Phe Arg Met
Ser Glu Asp Lys Pro Gln Asp Tyr Lys Lys660 665 670Arg Leu Ser Val
Glu Leu Thr Ser Ser Leu Phe Arg Thr Glu Pro Met675 680 685Ala Trp
Asn Glu Thr Ala Asp Leu Gly Leu Asp Ile Gly Ala Gln Gly690 695
700Glu Pro Leu Gly Tyr Arg Gln Asp Asp Pro Ser Tyr Arg Ser Phe
His705 710 715 720Ser Gly Gly Tyr Gly Gln Asp Ala Leu Gly Met Asp
Pro Met Met Glu725 730 735His Glu Met Gly Gly His His Pro Gly Ala
Asp Tyr Pro Val Asp Gly740 745 750Leu Pro Asp Leu Gly His Ala Gln
Asp Leu Met Asp Gly Leu Pro Pro755 760 765Gly Asp Ser Asn Gln Leu
Ala Trp Phe Asp Thr Asp Leu770 775 7807585PRTHomo
sapiensmisc_feature(1)..(585)Involucrin, Swissprot Accession P07476
7Met Ser Gln Gln His Thr Leu Pro Val Thr Leu Ser Pro Ala Leu Ser1 5
10 15Gln Glu Leu Leu Lys Thr Val Pro Pro Pro Val Asn Thr His Gln
Glu20 25 30Gln Met Lys Gln Pro Thr Pro Leu Pro Pro Pro Cys Gln Lys
Val Pro35 40 45Val Glu Leu Pro Val Glu Val Pro Ser Lys Gln Glu Glu
Lys His Met50 55 60Thr Ala Val Lys Gly Leu Pro Glu Gln Glu Cys Glu
Gln Gln Gln Lys65 70 75 80Glu Pro Gln Glu Gln Glu Leu Gln Gln Gln
His Trp Glu Gln His Glu85 90 95Glu Tyr Gln Lys Ala Glu Asn Pro Glu
Gln Gln Leu Lys Gln Glu Lys100 105 110Thr Gln Arg Asp Gln Gln Leu
Asn Lys Gln Leu Glu Glu Glu Lys Lys115 120 125Leu Leu Asp Gln Gln
Leu Asp Gln Glu Leu Val Lys Arg Asp Glu Gln130 135 140Leu Gly Met
Lys Lys Glu Gln Leu Leu Glu Leu Pro Glu Gln Gln Glu145 150 155
160Gly His Leu Lys His Leu Glu Gln Gln Glu Gly Gln Leu Lys His
Pro165 170 175Glu Gln Gln Glu Gly Gln Leu Glu Leu Pro Glu Gln Gln
Glu Gly Gln180 185 190Leu Glu Leu Pro Glu Gln Gln Glu Gly Gln Leu
Glu Leu Pro Glu Gln195 200 205Gln Glu Gly Gln Leu Glu Leu Pro Glu
Gln Gln Glu Gly Gln Leu Glu210 215 220Leu Pro Gln Gln Gln Glu Gly
Gln Leu Glu Leu Ser Glu Gln Gln Glu225 230 235 240Gly Gln Leu Glu
Leu Ser Glu Gln Gln Glu Gly Gln Leu Glu Leu Ser245 250 255Glu Gln
Gln Glu Gly Gln Leu Lys His Leu Glu His Gln Glu Gly Gln260 265
270Leu Glu Val Pro Glu Glu Gln Met Gly Gln Leu Lys Tyr Leu Glu
Gln275 280 285Gln Glu Gly Gln Leu Lys His Leu Asp Gln Gln Glu Lys
Gln Pro Glu290 295 300Leu Pro Glu Gln Gln Met Gly Gln Leu Lys His
Leu Glu Gln Gln Glu305 310 315 320Gly Gln Pro Lys His Leu Glu Gln
Gln Glu Gly Gln Leu Glu Gln Leu325 330 335Glu Glu Gln Glu Gly Gln
Leu Lys His Leu Glu Gln Gln Glu Gly Gln340 345 350Leu Glu His Leu
Glu His Gln Glu Gly Gln Leu Gly Leu Pro Glu Gln355 360 365Gln Val
Leu Gln Leu Lys Gln Leu Glu Lys Gln Gln Gly Gln Pro Lys370 375
380His Leu Glu Glu Glu Glu Gly Gln Leu Lys His Leu Val Gln Gln
Glu385 390 395 400Gly Gln Leu Lys His Leu Val Gln Gln Glu Gly Gln
Leu Glu Gln Gln405 410 415Glu Arg Gln Val Glu His Leu Glu Gln Gln
Val Gly Gln Leu Lys His420 425 430Leu Glu Glu Gln Glu Gly Gln Leu
Lys His Leu Glu Gln Gln Gln Gly435 440 445Gln Leu Glu Val Pro Glu
Gln Gln Val Gly Gln Pro Lys Asn Leu Glu450 455 460Gln Glu Glu Lys
Gln Leu Glu Leu Pro Glu Gln Gln Glu Gly Gln Val465 470 475 480Lys
His Leu Glu Lys Gln Glu Ala Gln Leu Glu Leu Pro Glu Gln Gln485 490
495Val Gly Gln Pro Lys His Leu Glu Gln Gln Glu Lys His Leu Glu
His500 505 510Pro Glu Gln Gln Asp Gly Gln Leu Lys His Leu Glu Gln
Gln Glu Gly515 520 525Gln Leu Lys Asp Leu Glu Gln Gln Lys Gly Gln
Leu Glu Gln Pro Val530 535 540Phe Ala Pro Ala Pro Gly Gln Val Gln
Asp Ile Gln Pro Ala Leu Pro545 550 555 560Thr Lys Gly Glu Val Leu
Leu Pro Val Glu His Gln Gln Gln Lys Gln565 570 575Glu Val Gln Trp
Pro Pro Lys His Lys580 5858968PRTHomo
sapiensmisc_feature(1)..(968)p120, Swissprot Accession O60716 8Met
Asp Asp Ser Glu Val Glu Ser Thr Ala Ser Ile Leu Ala Ser Val1 5 10
15Lys Glu Gln Glu Ala Gln Phe Glu Lys Leu Thr Arg Ala Leu Glu Glu20
25 30Glu Arg Arg His Val Ser Ala Gln Leu Glu Arg Val Arg Val Ser
Pro35 40 45Gln Asp Ala Asn
Pro Leu Met Ala Asn Gly Thr Leu Thr Arg Arg His50 55 60Gln Asn Gly
Arg Phe Val Gly Asp Ala Asp Leu Glu Arg Gln Lys Phe65 70 75 80Ser
Asp Leu Lys Leu Asn Gly Pro Gln Asp His Ser His Leu Leu Tyr85 90
95Ser Thr Ile Pro Arg Met Gln Glu Pro Gly Gln Ile Val Glu Thr
Tyr100 105 110Thr Glu Glu Asp Pro Glu Gly Ala Met Ser Val Val Ser
Val Glu Thr115 120 125Ser Asp Asp Gly Thr Thr Arg Arg Thr Glu Thr
Thr Val Lys Lys Val130 135 140Val Lys Thr Val Thr Thr Arg Thr Val
Gln Pro Val Ala Met Gly Pro145 150 155 160Asp Gly Leu Pro Val Asp
Ala Ser Ser Val Ser Asn Asn Tyr Ile Gln165 170 175Thr Leu Gly Arg
Asp Phe Arg Lys Asn Gly Asn Gly Gly Pro Gly Pro180 185 190Tyr Val
Gly Gln Ala Gly Thr Ala Thr Leu Pro Arg Asn Phe His Tyr195 200
205Pro Pro Asp Gly Tyr Ser Arg His Tyr Glu Asp Gly Tyr Pro Gly
Gly210 215 220Ser Asp Asn Tyr Gly Ser Leu Ser Arg Val Thr Arg Ile
Glu Glu Arg225 230 235 240Tyr Arg Pro Ser Met Glu Gly Tyr Arg Ala
Pro Ser Arg Gln Asp Val245 250 255Tyr Gly Pro Gln Pro Gln Val Arg
Val Gly Gly Ser Ser Val Asp Leu260 265 270His Arg Phe His Pro Glu
Pro Tyr Gly Leu Glu Asp Asp Gln Arg Ser275 280 285Met Gly Tyr Asp
Asp Leu Asp Tyr Gly Met Met Ser Asp Tyr Gly Thr290 295 300Ala Arg
Arg Thr Gly Thr Pro Ser Asp Pro Arg Arg Arg Leu Arg Ser305 310 315
320Tyr Glu Asp Met Ile Gly Glu Glu Val Pro Ser Asp Gln Tyr Tyr
Trp325 330 335Ala Pro Leu Ala Gln His Glu Arg Gly Ser Leu Ala Ser
Leu Asp Ser340 345 350Leu Arg Lys Gly Gly Pro Pro Pro Pro Asn Trp
Arg Gln Pro Glu Leu355 360 365Pro Glu Val Ile Ala Met Leu Gly Phe
Arg Leu Asp Ala Val Lys Ser370 375 380Asn Ala Ala Ala Tyr Leu Gln
His Leu Cys Tyr Arg Asn Asp Lys Val385 390 395 400Lys Thr Asp Val
Arg Lys Leu Lys Gly Ile Pro Val Leu Val Gly Leu405 410 415Leu Asp
His Pro Lys Lys Glu Val His Leu Gly Ala Cys Gly Ala Leu420 425
430Lys Asn Ile Ser Phe Gly Arg Asp Gln Asp Asn Lys Ile Ala Ile
Lys435 440 445Asn Cys Asp Gly Val Pro Ala Leu Val Arg Leu Leu Arg
Lys Ala Arg450 455 460Asp Met Asp Leu Thr Glu Val Ile Thr Gly Thr
Leu Trp Asn Leu Ser465 470 475 480Ser His Asp Ser Ile Lys Met Glu
Ile Val Asp His Ala Leu His Ala485 490 495Leu Thr Asp Glu Val Ile
Ile Pro His Ser Gly Trp Glu Arg Glu Pro500 505 510Asn Glu Asp Cys
Lys Pro Arg His Ile Glu Trp Glu Ser Val Leu Thr515 520 525Asn Thr
Ala Gly Cys Leu Arg Asn Val Ser Ser Glu Arg Ser Glu Ala530 535
540Arg Arg Lys Leu Arg Glu Cys Asp Gly Leu Val Asp Ala Leu Ile
Phe545 550 555 560Ile Val Gln Ala Glu Ile Gly Gln Lys Asp Ser Asp
Ser Lys Leu Val565 570 575Glu Asn Cys Val Cys Leu Leu Arg Asn Leu
Ser Tyr Gln Val His Arg580 585 590Glu Ile Pro Gln Ala Glu Arg Tyr
Gln Glu Ala Ala Pro Asn Val Ala595 600 605Asn Asn Thr Gly Pro His
Ala Ala Ser Cys Phe Gly Ala Lys Lys Gly610 615 620Lys Asp Glu Trp
Phe Ser Arg Gly Lys Lys Pro Ile Glu Asp Pro Ala625 630 635 640Asn
Asp Thr Val Asp Phe Pro Lys Arg Thr Ser Pro Ala Arg Gly Tyr645 650
655Glu Leu Leu Phe Gln Pro Glu Val Val Arg Ile Tyr Ile Ser Leu
Leu660 665 670Lys Glu Ser Lys Thr Pro Ala Ile Leu Glu Ala Ser Ala
Gly Ala Ile675 680 685Gln Asn Leu Cys Ala Gly Arg Trp Thr Tyr Gly
Arg Tyr Ile Arg Ser690 695 700Ala Leu Arg Gln Glu Lys Ala Leu Ser
Ala Ile Ala Asp Leu Leu Thr705 710 715 720Asn Glu His Glu Arg Val
Val Lys Ala Ala Ser Gly Ala Leu Arg Asn725 730 735Leu Ala Val Asp
Ala Arg Asn Lys Glu Leu Ile Gly Lys His Ala Ile740 745 750Pro Asn
Leu Val Lys Asn Leu Pro Gly Gly Gln Gln Asn Ser Ser Trp755 760
765Asn Phe Ser Glu Asp Thr Val Ile Ser Ile Leu Asn Thr Ile Asn
Glu770 775 780Val Ile Ala Glu Asn Leu Glu Ala Ala Lys Lys Leu Arg
Glu Thr Gln785 790 795 800Gly Ile Glu Lys Leu Val Leu Ile Asn Lys
Ser Gly Asn Arg Ser Glu805 810 815Lys Glu Val Arg Ala Ala Ala Leu
Val Leu Gln Thr Ile Trp Gly Tyr820 825 830Lys Glu Leu Arg Lys Pro
Leu Glu Lys Glu Gly Trp Lys Lys Ser Asp835 840 845Phe Gln Val Asn
Leu Asn Asn Ala Ser Arg Ser Gln Ser Ser His Ser850 855 860Tyr Asp
Asp Ser Thr Leu Pro Leu Ile Asp Arg Asn Gln Lys Ser Asp865 870 875
880Lys Lys Pro Asp Arg Glu Glu Ile Gln Met Ser Asn Met Gly Ser
Asn885 890 895Thr Lys Ser Leu Asp Asn Asn Tyr Ser Thr Pro Asn Glu
Arg Gly Asp900 905 910His Asn Arg Thr Leu Asp Arg Ser Gly Asp Leu
Gly Asp Met Glu Pro915 920 925Leu Lys Gly Thr Thr Pro Leu Met Gln
Asp Glu Gly Gln Glu Ser Leu930 935 940Glu Glu Glu Leu Asp Val Leu
Val Leu Asp Asp Glu Gly Gly Gln Val945 950 955 960Ser Tyr Pro Ser
Met Gln Lys Ile965
* * * * *