U.S. patent application number 13/918465 was filed with the patent office on 2013-10-17 for detection of degradation products of feline nt-probnp.
The applicant listed for this patent is IDEXX Laboratories, Inc.. Invention is credited to Michael Atkinson, Mahalakshmi Yerramilli, Murthy V.S.N. Yerramilli.
Application Number | 20130274453 13/918465 |
Document ID | / |
Family ID | 47259855 |
Filed Date | 2013-10-17 |
United States Patent
Application |
20130274453 |
Kind Code |
A1 |
Yerramilli; Mahalakshmi ; et
al. |
October 17, 2013 |
Detection of Degradation Products of Feline NT-proBNP
Abstract
A method for determining the amount of NT-proBNP in blood
samples from felines. The method includes detecting degradation
products of feline NT-proBNP by various methods, including using
antibodies, kits and devices.
Inventors: |
Yerramilli; Mahalakshmi;
(Falmouth, ME) ; Atkinson; Michael; (Gorham,
ME) ; Yerramilli; Murthy V.S.N.; (Falmouth,
ME) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IDEXX Laboratories, Inc. |
Westbrook |
ME |
US |
|
|
Family ID: |
47259855 |
Appl. No.: |
13/918465 |
Filed: |
June 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13485286 |
May 31, 2012 |
8481333 |
|
|
13918465 |
|
|
|
|
61491580 |
May 31, 2011 |
|
|
|
Current U.S.
Class: |
530/391.3 ;
530/391.1 |
Current CPC
Class: |
G01N 2333/58 20130101;
G01N 33/74 20130101; G01N 2800/32 20130101; G01N 33/54306
20130101 |
Class at
Publication: |
530/391.3 ;
530/391.1 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Claims
1. An immunoassay device comprising a solid support having a first
antibody bound thereto a first antibody specific for a fragment of
feline NT-proBNP selected from the group consisting of:
TABLE-US-00008 (SEQ ID NO: 5) ASEASAIQELLDGLRDTVSEL; (SEQ ID NO: 9)
LGPLQQGHSPAESWEAQEEPPARVLAPHDNVLR; and (SEQ ID NO: 10)
LQQGHSPAESWEAQEEPPARVLAPHDNVLR.
2. The device of claim 1, wherein the first antibody is specific
for SEQ ID NO: 5.
3. The device of claim 1, wherein the first antibody is specific
for SEQ ID NO:9.
4. The device of claim 1, wherein the first antibody is specific
for SEQ ID NO:10.
5. The device of claim 1, wherein the first antibody is a
monoclonal antibody.
6. The device of claim 5, wherein the first antibody is raised
against the fragment of feline NT-proBNP.
7. The device of claim 1, wherein the first antibody is a
polyclonal antibody.
8. The device of claim 7, wherein the first antibody is raised the
fragment of feline NT-proBNP.
9. The device of claim 1, further comprising a second antibody
being specific for the fragment of feline NT-proBNP.
10. The device of claim 9, wherein the second antibody is a
monoclonal antibody.
11. The device of claim 10, wherein the monoclonal antibody is
raised the fragment of feline NT-proBNP.
12. The device of claim 9, wherein the second antibody is a
polyclonal antibody.
13. The device of claim 12, wherein the second antibody is raised
the fragment of feline NT-proBNP.
14. The device of claim 9, wherein the second antibody is
labeled.
15. The device of claim 1, further comprising a labeled detection
reagent that competes with the fragment for binding with the first
antibody.
16. The device of claim 15, wherein the first antibody is specific
for SEQ ID NO: 5.
17. The device of claim 15, wherein the first antibody is specific
for SEQ ID NO:9.
18. The device of claim 15, wherein the first antibody is specific
for SEQ ID NO:10.
19. The device of claim 15, wherein the first antibody is a
monoclonal antibody.
20. The device of claim 15, wherein the first antibody is a
polyclonal antibody.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/485,286, filed May 31, 2012, which claims
benefit of U.S. Provisional Application No. 61/491,580 filed May
31, 2011, both of which are incorporated herein by reference in
their entirety.
SEQUENCE LISTING STATEMENT
[0002] The sequence listing is filed in this application in
electronic format only and is incorporated by reference herein. The
sequence listing text file "10-999-US-CON SeqListing_ST25.txt" was
created on Jun. 12, 2013, and is 7.13 bytes in size.
BACKGROUND
[0003] 1. Field of the Invention
[0004] The invention is related to the detection of NT-proBNP in
biological samples from felines.
[0005] 2. Description of Related Art
[0006] Heart diseases play an important role in animal health,
including non-human animals. Heart diseases concern, for example,
the cardiac valves and the cardiac muscle. Because the heart is
capable of compensating functional impairment by working harder,
such diseases in most cases remain hidden, with the consequence
that the state of the heart will deteriorate due to the increased
load on the heart. The symptoms resulting from heart diseases, such
as fatigue, circulatory insufficiency, and languor are usually
recognized when the animal's heart is no longer able to compensate
for the weakness. In such a case, the heart disease has progressed
to the point that curing the disease is no longer possible.
[0007] While chronic cardiac valve and cardiac muscle changes are
usually not curable, the use of medicaments can slow the further
progress of the disease. Therefore, an early diagnosis for heart
diseases is preferred. Routinely, physical methods are used for
this purpose, such as auscultation of the heart sounds, the
recording of an electrocardiogram, and X-ray and ultrasonic
examinations. These examination methods have the disadvantage that
they are carried out only when already visible or audible defects
of the heart are recognized. Furthermore, physical examination
methods require suitable and generally expensive devices in order
to carry out a respective diagnosis.
[0008] In many heart diseases, such as, e.g., heart decompensation
and dilated cardiomyopathy, a peptide hormone--the so-called BNP
(brain natriuretic peptide)--is secreted by heart muscle tissue.
Since this hormone is produced in the heart and is increasingly
produced in case of an overstress and congestion of the heart,
determining the BNP level in blood is a suitable means for
evaluating cardiac insufficiency.
[0009] BNP as well as other natriuretic peptides play an important
part in regulating water balance and blood pressure. If the cardiac
wall is dilated, it secrets BNP in increasing amounts, which in
turn causes an excretion of sodium and liquid via the kidneys and a
dilation of the blood vessels. These factors can lower the blood
pressure and the filling level of the heart. BNP is synthesized by
the cells of the cardiac muscle as proBNP, which is cleaved into
N-terminal proBNP (NT-proBNP) and BNP. Both parts of the
polypeptide are delivered to the blood and can be determined
therein.
[0010] The utility of both BNP and NT-proBNP as makers for cardiac
disease in veterinary patients (e.g., dogs and cats) has been
demonstrated in numerous studies. For instance, BNP and NT-proBNP
assays have been shown to be effective as a diagnostic test for
dogs, as illustrated by two studies, which report remarkably
similar sensitivity and specificity (85% and 82% respectively) for
differentiating the cause of clinical signs that may be
attributable to cardiac disease in dogs. See Oyama M A, et al.,
"Assessment of serum N-terminal pro-B-type natriuretic peptide
concentration for differentiation of congestive heart failure from
primary respiratory tract disease as the cause of respiratory signs
in dogs," Journal of American Veterinary Medical Association
(December 2009); Boswood et al., "The diagnostic accuracy of
different natriuretic peptides in the investigation of canine
cardiac disease," JSAP 2007 1-7. In cats, the clinical challenge is
different, as the most common cardiac disease in cats is
hypertrophic cardiomyopathy. This disease remains occult or
`silent` with very few clinical signs that are appreciable to the
pet owner until the disease is very advanced.
[0011] A number of immunoassays for the detection of NT-proBNP are
known. These assays use polyclonal or monoclonal antibodies
specific for epitopes within various regions of the peptide. These
methods, however, are subject to variability because NT-proBNP is
further processed ex vivo by various proteases in the blood serum
and plasma. Therefore, immunoassays for NT-proBNP are inherently
vulnerable to inconsistency due to the ex vivo degradation of the
protein over time. Therefore, samples to be tested for NT-proBNP
are typically refrigerated and efforts are made to conduct sample
analysis as quickly as possible following taking of the sample.
[0012] Accordingly, the inventors have identified a need in the art
for a method of determining NT-proBNP that can measure NT-proBNP
without regard to when a sample was taken and without cumbersome
handling requirements for the sample.
SUMMARY
[0013] In one aspect, the invention is directed to a method for
determining the amount of NT-proBNP in a sample from a feline. The
method includes determining the amount of a polypeptide selected
from a sequence consisting essentially of one of
ASEASAIQELLDGLRDTVSEL (SEQ ID NO: 5),
LGPLQQGHSPAESWEAQEEPPARVLAPHDNVLR (SEQ ID NO:9) and
LQQGHSPAESWEAQEEPPARVLAPHDNVLR (SEQ ID NO:10) in the sample, and
correlating the amount of the polypeptide in the sample to the
amount of NT-proBNP in the sample.
[0014] In various aspects, the amount of polypeptide can be
determined with mass spectrometry or an immunoassay. For instance,
the immunoassay can include (a) contacting the sample with a first
antibody that specifically binds a polypeptide selected from a
sequence consisting essentially of one of SEQ ID NO:5, SEQ ID NO:9
and SEQ ID NO: 10, (b) determining the amount of binding of the
NT-proBNP in the sample to the first antibody, and (c) associating
the amount of binding to the amount of NT-proBNP in the sample. In
one example, the determining step includes further includes
contacting a complex formed by the first antibody and the NT-proBNP
in the sample with a second antibody specific for feline NT-proBNP
that is directly or indirectly conjugated to a label and detecting
the amount of the label associated with the complex. In this
aspect, the first antibody and the second antibody do not compete
for binding to the same epitope on NT-proBNP. The second antibody
may be specific for one of SEQ ID NO:5, SEQ ID NO:9 and SEQ ID NO:
10.
[0015] The method may also include the use of a standard, and
includes contacting a standard comprising recombinant NT-proBNP
with the first antibody, detecting the binding of the standard to
the first antibody, and comparing the amount of binding of the
first antibody to the NT-proBNP in the sample to the amount of
binding of the first antibody to the standard. Similarly, the
method may include contacting a standard comprising recombinant
NT-proBNP with the first antibody and the second antibody, and
comparing a signal from the label of the second antibody that is
bound to the NT-proBNP in the sample to the signal from the label
of the second antibody that is bound to the standard.
[0016] In selected aspects of the invention, the antibodies are
monoclonal or polyclonal antibodies raised against a polypeptide
selected from a sequence consisting essentially of one of SEQ ID
NO:5, SEQ ID NO:9 and SEQ ID NO: 10.
[0017] In a further aspect, the invention is directed to a method
for determining the presence or amount of NT-proBNP in a biological
sample. The method includes: (a) forming a mixture of the sample
with a first monoclonal antibody that specifically binds NT-proBNP,
wherein the first monoclonal antibody is conjugated to a label; (b)
allowing the NT-proBNP in the sample and the first monoclonal
antibody to form a complex; (c) contacting the mixture with a
second antibody that binds to NT-proBNP wherein the second antibody
is immobilized on a solid phase; and (d) detecting the presence or
amount of the label on the solid phase.
[0018] In yet another aspect, the invention is directed to a method
for determining the presence or amount of NT-proBNP in a biological
sample, which includes detecting a degradation product of NT-proBNP
in the sample. The method can use a mass spectrometer or at least
one antibody or two antibodies directed towards two different
epitopes on the degradation product.
[0019] Still further, the invention is directed to a kit for
determining the presence, absence or level of feline NT-proBNP in a
sample. The components of the kit include a solid support; a first
antibody, the first antibody being specific for a first epitope on
a fragment of feline NT-proBNP, the fragment being less than the
full length of feline NT-proBNP and being stable in feline serum or
plasma at room temperature after at least 192 hours from taking the
sample; and a second antibody being specific for a second epitope
on the fragment of feline NT-proBNP, the second epitope being
different than the first epitope.
[0020] The invention is also directed to an immunoassay device that
includes: a solid support having a first antibody bound thereto,
the first antibody being specific for a first epitope on a fragment
of feline NT-proBNP; a fragment of feline NT-proBNP being bound to
the first antibody, the fragment being less than the full length of
feline NT-proBNP and being stable in feline serum or plasma at room
temperature after at least 192 hours from taking the sample; and a
second antibody being specific for a second epitope on the fragment
of feline NT-proBNP, the second epitope being different than the
first epitope, the second antibody being bound to the fragment of
feline NT-proBNP.
[0021] In various aspects of the kit and the device, the fragment
of feline NT-proBNP is selected from the group consisting of
ASEASAIQELLDGLRDTVSEL (SEQ ID NO: 5);
LGPLQQGHSPAESWEAQEEPPARVLAPHDNVLR (SEQ ID NO:9) and
LQQGHSPAESWEAQEEPPARVLAPHDNVLR (SEQ ID NO:10).
[0022] In a further aspect, the invention is direct to a method for
determining the presence or amount of feline NT-proBNP in a
biological sample. The method includes: [0023] (a) providing a
first antibody that binds at least one of the following
peptides:
TABLE-US-00001 [0023] (SEQ ID NO: 5) ASEASAIQELLDGLRDTVSEL; (SEQ ID
NO: 6) DGLRDTVSEL; (SEQ ID NO: 7) LDGLRDTVSELQEAQM; (SEQ ID NO: 8)
LGPLQQGHSPAESWEAQEEPPAR; (SEQ ID NO: 9)
LGPLQQGHSPAESWEAQEEPPARVLAPHDNVLR; (SEQ ID NO: 10)
LQQGHSPAESWEAQEEPPARVLAPHDNVLR; and (SEQ ID NO: 11) VLAPHDNVLR;
[0024] (b) providing a sample suspected of containing feline
NT-proBNP or fragments thereof; [0025] (c) contacting the sample
with the first antibody to provide an antibody/peptide complex;
[0026] (d) providing a second antibody that forms a complex with
the antibody/peptide complex; and [0027] (e) detecting the
presence, absence or amount of the antibody/peptide/antibody
complex.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1 provides an alignment of BNP polypeptide sequences
from canine, feline and human (SEQ ID NOS 1, 2 and 3).
[0029] FIG. 2 shows the results of the degradation of feline
synthetic NT-proBNP in plasma.
[0030] FIGS. 3 and 4 show the relative amounts of NT-proBNP
polypeptide fragments in plasma samples after 0, 24, 96 and 192
hours of spiking with feline synthetic NT-proBNP.
DETAILED DESCRIPTION
[0031] Degradation products of NT-proBNP can be determined to
provide an accurate and reliable measurement of the original amount
of NT-proBNP in a fresh blood sample. Because the stable
degradation products, and not the NT-proBNP itself, are determined,
the urgency to test the sample immediately or to refrigerate the
sample prior to testing is eliminated.
[0032] There are several ways to assay a sample (e.g. whole blood,
serum or plasma) for NT-proBNP or its degradation products. Mass
Spectrometry, for example, can be used to look for a particular
peptide sequence (e.g. full length NT-proBNP or a portion thereof).
Immunoassay techniques can also be used to directly or indirectly
detect full length NT-proBNP or a portion thereof. In a direct
assay format, monoclonal antibodies, polyclonal antibodies or
combinations thereof can be used to capture and detect full length
or fragments of NT-proBNP (e.g.,"sandwich" assays where one
antibody captures the target analyte and a second, labeled antibody
detects the captured analyte).
[0033] When assaying for NT-proBNP fragments in a sandwich assay,
it is important to ensure the antibody pair is specific for
epitopes that exist on the target, i.e. the assay will miss the
fragment if one or more of the epitopes are cleaved from the
original peptide or otherwise become compromised or inactivated.
For example, assuming the line below is full length feline
|NT-proBNP|. Over time, the peptide degrades into portions {A}, [B]
and <C>, where [B] and <C> are degradation products of
A:
##STR00001##
[0034] An antibody pair, one directed to an epitope on [B] and the
other to an epitope on <C>, will work in a sandwich assay for
{A} (or the full length peptide) as long as {A} does not degrade
into [B] and <C>. If {A} degrades into [B] and <C>, a
working assay for this section of the full peptide would need to
have an antibody pair directed towards active epitopes on either
[B] or <C>.
[0035] Alternatively, in a competition immunoassay format, only one
epitope of a target fragment need be identified and targeted with
either a monoclonal or polyclonal antibody. In this format, if the
fragment/epitope is present, it will bind with the antibody and
"compete" with a detection reagent that would have otherwise bound
to the antibody.
[0036] Preferred target NT-proBNP fragments (discussed in detail,
below) are those that exist in detectable concentrations over time.
By determining those fragments that are stable over time one can
choose Mass Spectrometry parameters or antibodies to detect and
determine the concentration of the stable fragment. Once one
determines the concentration of a stable fragment, the original
amount of NT-proBNP in the sample can be determined.
[0037] In order to assess the extent of heart damage or disease,
the determination of the quantity of NT-proBNP in a sample is
helpful. Quantitative determination of the degradation products of
NT-proBNP (i.e., a polypeptide fragment of native feline NT-proBNP)
can be accomplished by several methods. Once the amount of one or
more of its degradation products (the analyte) in a sample is
determined, the amount NT-proBNP in the sample, prior to
degradation, can be determined.
[0038] Accordingly, in one aspect the invention is directed to
determining the amount of NT-proBNP in a sample from a feline,
wherein the method includes determining the amount of a degradation
product of NT-proBNP. The amount of NT-proBNP in the sample can be
used as a measurement of cardiac disease in the animal.
[0039] Substantial homology between species exists for BNP and
NT-proBNP. For example, FIG. 1 provides an alignment of BNP
polypeptide sequences from human, canine and feline (SEQ ID NOS
1-3). As described above, BNP is processed prior to secretion from
muscle cells as the N-terminal pro-BNP (NT-proBNP) and BNP.
[0040] Feline Synthetic NT-proBNP (fsNT-proBNP) has the following
polypeptide sequence (SEQ ID NO:4):
TABLE-US-00002 HPLGGPGPAS EASAIQELLD GLRDTVSELQ EAQMALGPLQ
QGHSPAESWE AQEEPPARVL APHDNVLRAL RRLGSSKM
[0041] As shown in FIG. 2, feline NT-proBNP is degraded (cleaved in
at least one location) nearly 80% after 24 hours ex vivo in plasma.
Because the peptide is quickly degraded, epitopes specific for
various antibodies may no longer be available for binding to the
antibodies. In addition, when an assay relies upon antibody pairs
that bind to the same polypeptide, cleavage through degradation of
the polypeptide in the region between the two epitopes recognized
by the antibodies results in the antibodies not longer being able
to associate and provide a signal that is indicative of the
association.
[0042] For nomenclature purposes, the feline NT-proBNP sequence can
be loosely divided into two regions: the N-terminal region, which
roughly includes the N-terminal half of the peptide, and the
C-terminal region, which generally includes the C-terminal half of
the peptide. Preferred degradation products of NT-proBNP are shown
in FIGS. 3 and 4, wherein NT-proBNP was incubated in feline plasma
and sampled after 4, 96 and 192 hours .
[0043] Preferred Feline NT-proBNP N-Terminal Peptide Plasma
Degradents (FIG. 3):
TABLE-US-00003 (SEQ ID NO: 5) ASEASAIQELLDGLRDTVSEL; (SEQ ID NO: 6)
DGLRDTVSEL; and (SEQ ID NO: 7) LDGLRDTVSELQEAQM.
[0044] Preferred Feline NT-proBNP C-Terminal Peptide Plasma
Degradents (FIG. 4):
TABLE-US-00004 (SEQ ID NO: 8) LGPLQQGHSPAESWEAQEEPPAR; (SEQ ID NO:
9) LGPLQQGHSPAESWEAQEEPPARVLAPHDNVLR; (SEQ ID NO: 10)
LQQGHSPAESWEAQEEPPARVLAPHDNVLR; and (SEQ ID NO: 11) VLAPHDNVLR.
[0045] The most prevalent polypeptides after 192 hours were:
ASEASAIQELLDGLRDTVSEL (SEQ ID NO: 5) in the N-terminal region; and
LGPLQQGHSPAESWEAQEEPPARVLAPHDNVLR (SEQ ID NO: 9) and
LQQGHSPAESWEAQEEPPARVLAPHDNVLR (SEQ ID NO:10) in the C-terminal
region.
[0046] The invention also provides antibodies that selectively bind
to one of the NT-proBNP degradation products, as well as variants
and further fragments thereof. As used herein, an antibody
selectively binds a target peptide when it binds the target peptide
and does not significantly bind to unrelated proteins. An antibody
is still considered to selectively bind a peptide even if it also
binds to other proteins that are not substantially homologous with
the target peptide so long as such proteins share homology with a
fragment or domain of the peptide target of the antibody. In this
case, it would be understood that antibody binding to the peptide
is still selective despite some degree of cross-reactivity.
[0047] As used herein, an antibody is defined in terms consistent
with that recognized within the art: they are multi-subunit
proteins produced by a mammalian organism in response to an antigen
challenge. Chimeric antibodies are also within the scope of the
invention. The antibodies of the present invention include
polyclonal antibodies and monoclonal antibodies, as well as
fragments of such antibodies, including, but not limited to, Fab or
F(ab').sub.2, and Fv fragments.
[0048] Many methods are known for generating and/or identifying
antibodies to a given target peptide. Several such methods are
described by Harlow, Antibodies, Cold Spring Harbor Press,
(1989).
[0049] In general, to generate antibodies, an isolated peptide is
used as an immunogen and is administered to a mammalian organism,
such as a rat, rabbit or mouse. In one aspect, the antibodies of
the invention are monoclonal antibodies produced by a mouse myeloma
cell line. This cell line can be made by fusing a mouse myeloma
cell line with the spleen cells from mice that have been injected
with the NT-proBNP fragments described herein and suitable carrier
proteins that are well known to those of skill in the art.
[0050] The antibodies can be used to isolate NT-proBNP or a
fragment thereof by standard techniques, such as affinity
chromatography or immunoprecipitation. In one aspect, the invention
is directed to an immunological method for detecting the presence
of an amount of NT-proBNP in a biological sample. The invention
provides a method, a device and a kit that uses one or more feline
NT-proBNP monoclonal antibodies. In another aspect, the method
includes calibrators and standards comprising one or more NT-proBNP
polypeptides.
[0051] Stable fragments NT-proBNP can also be used to purify
polyclonal antibodies from sera (e.g. from chickens, goats, sheep
and/or donkeys that have been immunized with all or a portion of
the NT-proBNP sequence).
[0052] As used herein, antibodies that have been raised against a
particular polypeptide sequence refer to polyclonal or monoclonal
antibodies that have been obtained after immunizing an animal with
the polypeptide, such as well known in the art, usually conjugated
to a suitable carrier.
[0053] "Binding specificity" or "specific binding" refers to the
substantial recognition of a first molecule for a second molecule,
for example a polypeptide and a polyclonal or monoclonal antibody,
or an antibody fragment (e.g. a Fv, single chain Fv, Fab', or
F(ab').sub.2 fragment) specific for the polypeptide.
[0054] A "specific binding pair" is a set of two different
molecules, where one molecule has an area on its surface or in a
cavity that specifically binds to, and is therefore complementary
to, an area on the other molecule. "Specific binding partner"
refers to one of these two complementarily binding molecules.
"Specific binding pair" may refer to a ligand and a receptor, for
example. In another example, the specific binding pair might refer
to an immunological pair, for example an antigen and antibody.
[0055] "Substantial binding" or "substantially bind" refer to an
amount of specific binding or recognizing between molecules in an
assay mixture under particular assay conditions. In its broadest
aspect, substantial binding relates to the difference between a
first molecule's incapability of binding or recognizing a second
molecule, and the first molecules capability of binding or
recognizing a third molecule, such that the difference is
sufficient to allow a meaningful assay to be conducted
distinguishing specific binding under a particular set of assay
conditions, which includes the relative concentrations of the
molecules, and the time and temperature of an incubation. In
another aspect, one molecule is substantially incapable of binding
or recognizing another molecule in a cross-reactivity sense where
the first molecule exhibits a reactivity for a second molecule that
is less than 25%, preferably less than 10%, more preferably less
than 5% of the reactivity exhibited toward a third molecule under a
particular set of assay conditions, which includes the relative
concentration and incubation of the molecules. Specific binding can
be tested using a number of widely known methods, e.g, an
immunohistochemical assay, an enzyme-linked immunosorbent assay
(ELISA), a radioimmunoassay (RIA), or a western blot assay.
[0056] A "blood sample" refers to a whole blood sample from an
animal or its components (e.g., serum, plasma, etc.).
[0057] A "label" is any molecule that is bound (via covalent or
non-covalent means, alone or encapsulated) to another molecule or
solid support and that is chosen for specific characteristics that
allow detection of the labeled molecule. Generally, labels are
comprised of, but are not limited to, the following types:
particulate metal and metal-derivatives, radioisotopes, catalytic
or enzyme-based reactants, chromogenic substrates and chromophores,
fluorescent and chemiluminescent molecules, and phosphors. The
utilization of a label produces a signal that may be detected by
means such as detection of electromagnetic radiation or direct
visualization, and that can optionally be measured.
[0058] The label employed in the current invention could be, but is
not limited to: alkaline phosphatase; glucose-6-phosphate
dehydrogenase ("G6PDH"); horse radish peroxidase (HRP);
chemiluminescers such as isoluminol, fluorescers such as
fluorescein and rhodamine compounds; ribozymes; and dyes.
[0059] The label can directly produce a signal, and therefore
additional components are not required to produce a signal.
Alternatively, a label may need additional components, such as
substrates or co-enzymes, in order to produce a signal. The
suitability and use of such labels useful for producing a signal
are discussed in U.S. Pat. No. 6,489,309, and U.S. Pat. No.
5,185,243, which are incorporated by reference herein in their
entirety. For example, a label may be conjugated to the specific
binding partner in a non-covalent fashion. Alternatively, the label
may be conjugated to the specific binding partner covalently. U.S.
Pat. No 3,817,837, and U.S. Pat. No. 3,996,345, which are
incorporated by reference herein in their entirety, describe in
detail example of various ways that a label may be non-covalently
or covalently conjugated to the specific binding partner.
[0060] Solid phase means a porous or non-porous water insoluble
material. Such materials include a support or a surface such as the
wall of a reaction vessel. The support can be hydrophilic or
capable of being rendered hydrophilic and includes inorganic
powders such as silica, magnesium sulfate, and alumina; natural
polymeric materials, particularly cellulosic materials and
materials derived from cellulose, such as fiber containing papers,
e.g., filter paper, chromatographic paper, etc.; synthetic or
modified naturally occurring polymers, such as nitrocellulose,
cellulose acetate, poly(vinyl chloride), polyacrylamide, cross
linked dextran, agarose, polyacrylate, polyethylene, polypropylene,
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene
terephthalate), nylon, poly(vinyl butyrate), etc.; either used by
themselves or in conjunction with other materials; glass available
as Bioglass, ceramics, metals, and the like. Natural or synthetic
assemblies such as liposomes, phospholipid vesicles, and cells can
also be employed.
[0061] Binding of specific binding pair members to a support or
surface may be accomplished by well-known techniques, commonly
available in the literature. See, for example, "Immobilized
Enzymes," Ichiro Chibata, Halsted Press, New York (1978) and
Cuatrecasas, J. Biol. Chem., 245:3059 (1970). The surface can have
any one of a number of shapes, such as strip, rod, particle,
including bead, and the like. In one aspect, the polypeptides of
the invention include a N-terminal cysteine residue to assist in
binding the polypeptides to the solid phase.
[0062] The method of the invention can be optimized in many ways
and one of skill in the art could simultaneously adjust the sample
dilutions, reagent concentrations, incubation temperatures and
times used in the method to accomplish detection of NT-proBNP.
[0063] To be useful in the detection methods of the present
invention, the polypeptides are obtained in a substantially pure
form, that is, typically from about 50% w/w or more purity,
substantially free of interfering proteins and contaminants.
Preferably, the polypeptides are isolated or synthesized in a
purity of at least 80% w/w, and more preferably, in at least about
95% w/w purity. Using conventional protein purification techniques,
homogeneous polypeptide compositions of at least about 99% w/w
purity can be obtained. For example, the proteins may be purified
by use of the antibodies described hereinafter using the
immunoabsorbant affinity columns described hereinabove.
[0064] The method of the invention may be accomplished using
immunoassay techniques well known to those of skill in the art,
including, but not limited to, using microplates and lateral flow
devices. In one embodiment, an antibody specific for NT-proBNP
protein is immobilized on a solid support at a distinct location.
Following addition of the sample, detection of protein-antibody
complexes on the solid support can be by any means known in the
art. For example, U.S. Pat. No. 5,726,010, which is incorporated
herein by reference in its entirety, describes an example of a
lateral flow device, the SNAP.RTM. immunoassay device (IDEXX
Laboratories), useful in the present invention. In another aspect,
the solid support is a well of a microtiter plate.
[0065] Immobilization of one or more analyte capture reagents,
e.g., antibodies to NT-proBNP, onto a device or solid support is
performed so that an analyte capture reagent will not be washed
away by the sample, diluent and/or wash procedures. One or more
analyte capture reagents can be attached to a surface by physical
adsorption (i.e., without the use of chemical linkers) or by
chemical binding (i.e., with the use of chemical linkers). Chemical
binding can generate stronger attachment of specific binding
substances on a surface and provide defined orientation and
conformation of the surface-bound molecules.
[0066] In another aspect, the invention includes one or more
labeled specific binding reagents that can be mixed with a test
sample prior to application to a device for of the invention. In
this case it is not necessary to have labeled specific binding
reagents deposited and dried on a specific binding reagent pad in
the device. A labeled specific binding reagent, whether added to a
test sample or pre-deposited on the device, can be for example, a
labeled NT-proBNP monoclonal antibody.
[0067] The detection method may include the use of a standard such
as a recombinant NT-proBNP polypeptide. The standard can be mixed
with the monoclonal antibody or antibodies in the same manner as
the sample. The amount of binding between the monoclonal antibody
or antibodies and the standard can be compared to the amount of
binding of the antibodies to the protein in the sample.
Accordingly, because the amount of NT-proBNP in the standard is
known, the amount of protein in the sample can be determined.
[0068] Any or all of the above embodiments can be provided as a
kit. In one particular example, such a kit can include, for
example, a solid support, a first antibody specific for a first
epitope on a fragment of feline NT-proBNP. The fragment can be less
than the full length of feline NT-proBNP and stable in feline serum
or plasma at room temperature after at least 192 hours from taking
the sample. Such fragments are described above; for example SEQ ID
NO:5, SEQ ID NO:9 and SEQ ID NO:10. The kit can also include a
second antibody being specific for a second epitope on the fragment
of feline NT-proBNP, the second epitope being different than the
first epitope.
[0069] The kit can include a device complete with specific binding
reagents (e.g., a non-immobilized labeled specific binding reagent
and an immobilized analyte capture reagent) and wash reagent, as
well as detector reagent and positive and negative control
reagents, if desired or appropriate. In addition, other additives
can be included, such as stabilizers, buffers, and the like. The
relative amounts of the various reagents can be varied, to provide
for concentrations in solution of the reagents that substantially
optimize the sensitivity of the assay. Particularly, the reagents
can be provided as dry powders, usually lyophilized, which on
dissolution will provide for a reagent solution having the
appropriate concentrations for combining with a sample.
[0070] The device may also include a liquid reagent that transports
unbound material (e.g., unreacted fluid sample and unbound specific
binding reagents) away from the reaction zone (solid phase). A
liquid reagent can be a wash reagent and serve only to remove
unbound material from the reaction zone, or it can include a
detector reagent and serve to both remove unbound material and
facilitate analyte detection. For example, in the case of a
specific binding reagent conjugated to an enzyme, the detector
reagent includes a substrate that produces a detectable signal upon
reaction with the enzyme-antibody conjugate at the reactive zone.
In the case of a labeled specific binding reagent conjugated to a
radioactive, fluorescent, or light-absorbing molecule, the detector
reagent acts merely as a wash solution facilitating detection of
complex formation at the reactive zone by washing away unbound
labeled reagent.
[0071] Two or more liquid reagents can be present in a device, for
example, a device can comprise a liquid reagent that acts as a wash
reagent and a liquid reagent that acts as a detector reagent and
facilitates analyte detection.
[0072] A liquid reagent can further include a limited quantity of
an "inhibitor", i.e., a substance that blocks the development of
the detectable end product. A limited quantity is an amount of
inhibitor sufficient to block end product development until most or
all excess, unbound material is transported away from the second
region, at which time detectable end product is produced.
[0073] In one aspect, the device is provided upon binding of
NT-proBNP from the sample of the antibody immobilized on the
device. Accordingly, the device has a a solid support having a
first antibody bound thereto, the first antibody being specific for
a first epitope on a fragment of feline NT-proBNP. A fragment of
feline NT-proBNP becomes bound to the first antibody, the fragment
being less than the full length of feline NT-proBNP. This fragment
is, for example, one of the fragments describe above and is stable
in feline serum or plasma at room temperature after at least 192
hours from taking the sample. The device also includes a second
antibody being specific for a second epitope on the fragment of
feline NT-proBNP, the second epitope being different than the first
epitope, the second antibody being bound to the fragment of feline
NT-proBNP. Therefore, the device is complete upon sandwiching the
fragment between the first and second antibodies, as would occur
upon application of a sample containing NT-proBNP to the
device.
[0074] Detection of NT-proBNP by Mass Spectrometry
[0075] Peptides and peptide fragments of the disclosure may be
detected using standard mass spectrometric techniques. In mass
spectrometry (MS), the analyte is first vaporized and ionized, then
the ions are directed to a mass analyzer, in which electromagnetic
fields are used to separate species by mass. Lastly, a detector
quantifies the abundance of each species.
[0076] Standard ionization techniques that are well known in the
art include electrospray ionization (ESI) and matrix-assisted laser
desorption ionization (MALDI), which are commonly used with liquid
and solid biological samples. Other ionization technologies known
in the art include thermal ionization (TIMS), spark ionization,
secondary ion mass spectrometry (SIMS), atmospheric pressure
chemical ionization (APCI), ion attachment ionization, direct
analysis in real time (DART), desorption/ionization on silicon
(DIOS), thermospray ionization, fast atom bombardment (FAB), field
desorption, glow discharge, and inductively couple plasma (ICP).
Examples of well-known mass analyzers include sector field,
time-of-flight (TOF), quadrupole, quadrupole ion trap (IT), linear
quadrupole ion trap, Fourier transform ion cyclotron resonance
(FT-ICR), and orbitrap mass analyzers. Examples of well-known
detectors include electron multiplier, Faraday cup, microchannel
plate, and Daly detectors.
[0077] Mass analyzers segregate ions according to their
mass-to-charge ratio (m/z, where m is mass and z is charge). For
example, a peptide of molecular weight P with two positive charges
will have a net mass of (P+2) due to the ionizing presence of two
protons, and will appear on a mass spectrum at m/z=(P+2)/2. Thus,
in general, a molecule of molecular weight M with charge Z will
appear on a mass spectrum at m/z=(M+Z)/Z.
[0078] Mass spectrometry can be combined with liquid chromatography
(LC) or gas chromatography (GC) to facilitate detection and
identification of compounds residing within a complex mixture. In
liquid chromatography-mass spectrometry (LC/MS), the analyte
solution is first passed through a high-performance liquid
chromatography (HPLC) column, which may separate components by a
characteristic such as hydrophobicity, partition coefficient,
polarity, bioaffinity, charge, or size using well-known
chromatographic techniques such as reversed-phase chromatography,
normal phase chromatography, displacement chromatography, partition
chromatography, ion exchange chromatography, size exclusion
chromatography, or bioaffinity chromatography. The mass
spectrometer then generates a mass spectrum for each peak in the
HPLC chromatogram. Using LC/MS, a single species of interest can be
separated and identified from within complex biological mixtures
such as plasma or serum.
[0079] LC can also be combined with a tandem mass spectrometer
(MS/MS), which is capable of multiple rounds of mass spectrometry
for purposes such as protein identification. An MS/MS system can
isolate and stabilize an individual ion species appearing in a mass
spectrum, which is then fragmented using any one of several
well-known techniques, such as collision-induced dissociation
(CID), electron capture dissociation (ECD), electron transfer
dissociation (ETD), infrared multiphoton dissociation (IRMPD), and
blackbody infrared radiative dissociation (BIRD). The fragments are
themselves analyzed by MS to generate a fragment mass spectrum,
also called a fragmentation pattern. In the case of protein
identification, an experimentally derived fragmentation pattern
(generated from a sample with an unknown protein or peptide) can be
compared with fragmentation patterns that have been predicted from
information in protein sequence or nucleotide sequence databases. A
high degree of similarity between experimental and predicted
fragmentation patterns leads to identification of the unknown
protein in the sample.
[0080] Other features and advantages of the invention will be
apparent from the following Examples. The following are provided
for exemplification purposes only and are not intended to limit the
scope of the invention described in broad terms above. All
references cited in this disclosure are incorporated herein by
reference.
EXAMPLES
[0081] LC/MS analyses were obtained using a Thermo-Scientific
ACCELA UHPLC and LTQ ORBITRAP DISCOVERY.TM. mass spectrometry
systems.
[0082] For plasma extraction, 100 uL of plasma was precipitated by
addition of 200 uL methanol in a 1.5 mL protein LoBind eppendorf
tube. After vortexing for 10 seconds, the precipitate was pelleted
by centrifugation at 13,000 rpm for 15 minutes at 10.degree. C.
using a benchtop centrifuge. The supernatant was transferred to a
limited volume glass HPLC vial and analyzed by LC/MS as described
in the following examples.
Example 1
MS Analysis of Degradation Kinetics of NT-proBNP in Feline
Plasma
[0083] Degradation kinetics of whole feline synthetic (fs)
NT-proBNP in feline plasma were investigated by adding 1.0 .mu.g
fsNT-proBNP to 1 mL of feline plasma prepared as described above.
Aliquots of the plasma were analyzed by LC/MS at 0, 4, 24, and 48
hours after addition of the peptide. As shown in FIG. 2,
approximately 80% of the peptide was degraded after 24 hours.
[0084] The LC gradient for this example is shown in Table 1, where
Solvent C is 0.1% formic acid in water and Solvent D is 0.1% formic
acid in acetonitrile.
TABLE-US-00005 TABLE 1 No Time A % B % C % D % .mu.L/min 1 0 0 0
100 0 300 2 5 0 0 100 0 300 3 23 0 0 65 35 300 4 26 0 0 65 35 300 5
44 0 0 5 95 300 6 46 0 0 5 95 300 7 46.5 0 0 100 0 300 8 60 0 0 100
0 300 Column: Acquity UPLC BEH300 C.sub.18 1.7 .mu.M 2.1 id .times.
150 mm length Guard Column: vanguard BEH C.sub.18 1.7 uM Injection
volume: 25 .mu.L Tray temp: 10.degree. C. Column oven temp:
45.degree. C. MS run time: 60.0 minutes
[0085] The mass spec parameters for degradation kinetics are shown
in Table 2:
TABLE-US-00006 TABLE 2 MS run time: 60.0 minutes Divert valve:
Divert Time Valve State 0.00 To waste 4.95 To source 55 To waste
Number of Scan Events: 1 Tune Method: 031710 peptide tune
[0086] Scan Event Details: [0087] 1: FTMS+c norm res=30000
o(375.0-2000.0) [0088] CV=0.0V [0089] Default Charge State: 2
[0090] Activation Q: 0.250 [0091] Activation Time: 30.000 [0092]
CV=0.0V
[0093] MS Tune File Values [0094] Source Type: ESI [0095] Capillary
Temp (.degree. C.): 250.00 [0096] Sheath gas Flow: 24.0 [0097] Aux
Gas Flow: 13.0 [0098] Sweep Gas Flow: 0 [0099] ITMS MSn AGC Target:
10000 [0100] FTMS Injection waveforms: off [0101] FTMS AGC Target:
500000 [0102] Source voltage (kV): 4.50 [0103] Source current
(.mu.A): 100.00 [0104] Capillary Voltage (V): 68.28 [0105] Tube
Lens (V): 130.00 [0106] Skimmer Offset (V): 0.00 [0107] Multipole
RF Amplifier (Vp-p): 550.00 [0108] Multipole 00 offset (V): -1.60
[0109] Lens 0 Voltage (V): -2.70 [0110] Multipole 0 offset (V):
-2.70 [0111] Lens 1 Voltage (V): -11.00 [0112] Gate Lens offset
(V): -60.00 [0113] Multipole 1 offset (V): -10.5 [0114] Front Lens
(V): -5.18 [0115] ITMS MSn microscans: 3 [0116] ITMS MSn Max Ion
Time (ms): 100.00 [0117] FTMS full microscans: 1 [0118] FTMS full
Max Ion Time (ms): 500.00
[0119] The Mass Spec parameters used for determining peptide
degradation products are shown in Table 3.
TABLE-US-00007 TABLE 3 MS run time: 60.0 minutes Divert valve:
Divert Time Valve State 0.00 To waste 4.95 To source 55 To waste
Number of Scan Events: 6 Tune Method: 031710 peptide tune
[0120] Scan Event Details: [0121] 1: FTMS+c norm res=30000
o(375.0-2000.0) [0122] CV=0.0V [0123] 2: ITMS+c norm Dep MS/MS Most
intense ion from (1) [0124] Activation Type: CID [0125] Min. Signal
Required: 500.0 [0126] Isolation Width: 1.50 [0127] Normalized
Coll. Energy: 35.0 [0128] Default Charge State: 2 [0129] Activation
Q: 0.250 [0130] Activation Time: 30.000 [0131] CV=0.0V [0132] 3:
ITMS+c norm Dep MS/MS 2nd most intense ion from (1) [0133]
Activation Type: CID [0134] Min. Signal Required: 500.0 [0135]
Isolation Width: 2.00 [0136] Normalized Coll. Energy: 35.0 [0137]
Default Charge State: 2 [0138] Activation Q: 0.250 [0139]
Activation Time: 30.000 [0140] CV=0.0V [0141] 4: ITMS+c norm Dep
MS/MS 3rd most intense ion from (1) [0142] Activation Type: CID
[0143] Min. Signal Required: 500.0 [0144] Isolation Width: 2.00
[0145] Normalized Coll. Energy: 35.0 [0146] Default Charge State: 2
[0147] Activation Q: 0.250 [0148] Activation Time: 30.000 [0149]
CV=0.0V [0150] 5: ITMS+c norm Dep MS/MS 4th most intense ion from
(1) [0151] Activation Type: CID [0152] Min. Signal Required: 500.0
[0153] Isolation Width: 1.50 [0154] Normalized Coll. Energy: 35.0
[0155] Default Charge State: 2 [0156] Activation Q: 0.250 [0157]
Activation Time: 30.000 [0158] CV=0.0V [0159] 6: ITMS+c norm Dep
MS/MS 5th most intense ion from (1) [0160] Activation Type: CID
[0161] Min. Signal Required: 500.0 [0162] Isolation Width: 1.50
[0163] Normalized Coll. Energy: 35.0 [0164] Default Charge State: 2
[0165] Activation Q: 0.250 [0166] Activation Time: 30.000 [0167]
CV=0.0V
[0168] Data Dependent Settings: [0169] Use separate polarity
settings disabled [0170] Parent Mass List: (none) [0171] Reject
Mass List: (none) [0172] Neutral Loss Mass List: (none) [0173]
Product Mass List: (none) [0174] Neutral loss in top: 3 [0175]
Product in top: 3 [0176] Most intense if no parent masses found not
enabled [0177] Add/subtract mass not enabled [0178] FT master scan
preview mode enabled [0179] Charge state screening enabled [0180]
Monoisotopic precursor selection enabled [0181] Non-peptide
monoisotopic recognition not enabled [0182] Charge state rejection
enabled [0183] Unassigned charge states: rejected [0184] Charge
state 1: not rejected [0185] Charge state 2: not rejected [0186]
Charge state 3: not rejected [0187] Charge states 4+: not
rejected
[0188] Global Data Dependent Settings: [0189] Use global parent and
reject mass lists not enabled [0190] Exclude parent mass from data
dependent selection not enabled [0191] Exclusion mass width
relative to mass [0192] Exclusion mass width relative to low (ppm):
20.000 [0193] Exclusion mass width relative to high (ppm): 20.000
[0194] Parent mass width relative to mass [0195] Parent mass width
relative to low (ppm): 10.000 [0196] Parent mass width relative to
high (ppm): 10.000 [0197] Reject mass width relative to mass [0198]
Reject mass width relative to low (ppm): 20.000 [0199] Reject mass
width relative to high (ppm): 20.000 [0200] Zoom/UltraZoom scan
mass width by mass [0201] Zoom/UltraZoom scan mass width low: 5.00
[0202] Zoom/UltraZoom scan mass width high: 5.00 [0203] FT SIM scan
mass width low: 5.00 [0204] FT SIM scan mass width high: 5.00
[0205] Neutral Loss candidates processed by decreasing intensity
[0206] Neutral Loss mass width by mass [0207] Neutral Loss mass
width low: 0.50000 [0208] Neutral Loss mass width high: 0.50000
[0209] Product candidates processed by decreasing intensity [0210]
Product mass width by mass [0211] Product mass width low: 0.50000
[0212] Product mass width high: 0.50000 [0213] MS mass range:
0.00-1000000.00 [0214] MSn mass range by mass [0215] MSn mass
range: 0.00-1000000.00 [0216] Use m/z values as masses not enabled
[0217] Analog UV data dep. not enabled [0218] Dynamic exclusion
enabled [0219] Repeat Count: 2 [0220] Repeat Duration: 30.00 [0221]
Exclusion List Size: 500 [0222] Exclusion Duration: 60.00 [0223]
Exclusion mass width relative to mass [0224] Exclusion mass width
relative to low (ppm): 20.000 [0225] Exclusion mass width relative
to high (ppm): 20.000 [0226] Expiration: disabled [0227] Isotopic
data dependence not enabled [0228] Mass Tags data dependence not
enabled4
[0229] MS Tune File Values [0230] Source Type: ESI [0231] Capillary
Temp (.degree. C.): 250.00 [0232] Sheath gas Flow: 24.0 [0233] Aux
Gas Flow: 13.0 [0234] Sweep Gas Flow: 0 [0235] ITMS MSn AGC Target:
10000 [0236] FTMS Injection waveforms: off [0237] FTMS AGC Target:
500000 [0238] Source voltage (kV): 4.50 [0239] Source current
(.mu.A): 100.00 [0240] Capillary Voltage (V): 68.28 [0241] Tube
Lens (V): 130.00 [0242] Skimmer Offset (V): 0.00 [0243] Multipole
RF Amplifier (Vp-p): 550.00 [0244] Multipole 00 offset (V): -1.60
[0245] Lens 0 Voltage (V): -2.70 [0246] Multipole 0 offset (V):
-2.70 [0247] Lens 1 Voltage (V): -11.00 [0248] Gate Lens offset
(V): -60.00 [0249] Multipole 1 offset (V): -10.5 [0250] Front Lens
(V): -5.18 [0251] ITMS MSn microscans: 3 [0252] ITMS MSn Max Ion
Time (ms): 100.00 [0253] FTMS full microscans: 1 [0254] FTMS full
Max Ion Time (ms): 500.00
Example 2
Characterization of Stable Degradation Products of Reline
NT-proBNP
[0255] To characterize the fsNT-proBNP stable degradation products
present after incubation in feline plasma, fsNT-proBNP was added to
feline plasma at 1, 5, and 10 .mu.g/mL and incubated at room
temperature. At 0, 24, 48, 96, and 192 hours, 100 .mu.L aliquots of
the plasma were collected and combined with 200 .mu.L of methanol
to form a precipitate. After centrifugation, the supernatant was
analyzed for stable peptides by LC/MS as described in Example
1.
[0256] The relative abundance of several fragments from the
N-terminal, capture, and C-terminal regions were evaluated over
time. These data are shown in FIGS. 3 and 4, and indicate that the
most stable fragment sequences over time were ASEASAIQELLDGLRDTVSEL
(SEQ ID NO: 5) from the N-terminal region and
LGPLQQGHSPAESWEAQEEPPARVLAPHDNVLR (SEQ ID NO: 9) and
LQQGHSPAESWEAQEEPPARVLAPHDNVLR (SEQ ID NO:10); from the C-terminal
region.
Example 3
Identification of Stable Epitopes of Feline NT-proBNP
[0257] To identify stable peptide epitopes, a database containing
the amino acid sequence of fsNT-proBNP (SEQ ID NO:1) in FASTA
format was created. The RAW file of interest acquired from the
LC-MS run was uploaded along with the FASTA database to Proteome
Discoverer software (v 1.0 Thermo Scientific) for identification of
the stable epitopes using SEQUEST.
[0258] The peptides that satisfy the criteria as directed in the
Proteome Discover Software were indentified as stable epitopes for
a particular time series.
Example 4
Immunoassay for Feline NT-proBNP
[0259] Immunoassays can be conducted using monoclonal antibodies
specific for stable feline NT-proBNP degradation fragments (SEQ ID
NOS: 5, 9 and 10) at, for example, 24, 48, 72 and 96 hours. Sheep
polyclonal antibodies raised against various fragment of feline
NT-proBNP can be coated on a solid phase. Monoclonal antibodies can
be raised against raised against any of SEQ ID NOS:5, 9 and 10, and
conjugated at either or both the N and C terminus with a protein
carrier, for example, (PEG)6-Cys-KLH. The antibodies can be labeled
with HRP or another label using standard techniques known in the
art. Feline plasma incubated with the solid phase for 1 hour
followed by a second incubation with the labeled antibodies allow
for the detection of the label on the solid phase when the
polyclonal and monoclonal antibodies bind to the same degradation
fragment of feline NT-proBNP.
[0260] The examples given above are merely illustrative and are not
meant to be an exhaustive list of all possible embodiments,
applications or modifications of the invention. Thus, various
modifications and variations of the described methods and systems
of the invention will be apparent to those skilled in the art
without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific embodiments, it should be understood that the invention as
claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying
out the invention which are obvious to those skilled in molecular
biology, immunology, chemistry, biochemistry or in the relevant
fields are intended to be within the scope of the appended
claims.
[0261] It is understood that the invention is not limited to the
particular methodology, protocols, and reagents, etc., described
herein, as these may vary as the skilled artisan will recognize. It
is also to be understood that the terminology used herein is used
for the purpose of describing particular embodiments only, and is
not intended to limit the scope of the invention.
[0262] The embodiments of the invention and the various features
and advantageous details thereof are explained more fully with
reference to the non-limiting embodiments and/or illustrated in the
accompanying drawings and detailed in the following description. It
should be noted that the features illustrated in the drawings are
not necessarily drawn to scale, and features of one embodiment may
be employed with other embodiments as the skilled artisan would
recognize, even if not explicitly stated herein.
[0263] Any numerical values recited herein include all values from
the lower value to the upper value in increments of one unit
provided that there is a separation of at least two units between
any lower value and any higher value. As an example, if it is
stated that the concentration of a component or value of a process
variable such as, for example, size, angle size, pressure, time and
the like, is, for example, from 1 to 90, specifically from 20 to
80, more specifically from 30 to 70, it is intended that values
such as 15 to 85, 22 to 68, 43 to 51, 30 to 32, etc. are expressly
enumerated in this specification. For values which are less than
one, one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 as
appropriate. These are only examples of what is specifically
intended and all possible combinations of numerical values between
the lowest value and the highest value enumerated are to be
considered to be expressly stated in this application in a similar
manner.
[0264] Particular methods, devices, and materials are described,
although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention. The disclosures of all references and publications cited
herein are expressly incorporated by reference in their entireties
to the same extent as if each were incorporated by reference
individually.
Sequence CWU 1
1
111140PRTCanis familiarismisc_featureCanine preproBNP; SwissProt
P16859 1Met Glu Pro Cys Ala Ala Leu Pro Arg Ala Leu Leu Leu Leu Leu
Phe 1 5 10 15 Leu His Leu Ser Pro Leu Gly Gly Arg Pro His Pro Leu
Gly Gly Arg 20 25 30 Ser Pro Ala Ser Glu Ala Ser Glu Ala Ser Glu
Ala Ser Gly Leu Trp 35 40 45 Ala Val Gln Glu Leu Leu Gly Arg Leu
Lys Asp Ala Val Ser Glu Leu 50 55 60 Gln Ala Glu Gln Leu Ala Leu
Glu Pro Leu His Arg Ser His Ser Pro 65 70 75 80 Ala Glu Ala Pro Glu
Ala Gly Gly Thr Pro Arg Gly Val Leu Ala Pro 85 90 95 His Asp Ser
Val Leu Gln Ala Leu Arg Arg Leu Arg Ser Pro Lys Met 100 105 110 Met
His Lys Ser Gly Cys Phe Gly Arg Arg Leu Asp Arg Ile Gly Ser 115 120
125 Leu Ser Gly Leu Gly Cys Asn Val Leu Arg Lys Tyr 130 135 140
2132PRTFelis catusmisc_featureFeline preproBNP; SwissProt Q9GLK4
2Met Asp Pro Lys Thr Ala Leu Leu Arg Ala Leu Leu Leu Leu Leu Phe 1
5 10 15 Leu His Leu Ser Pro Leu Gly Gly Arg Ser His Pro Leu Gly Gly
Pro 20 25 30 Gly Pro Ala Ser Glu Ala Ser Ala Ile Gln Glu Leu Leu
Asp Gly Leu 35 40 45 Arg Asp Thr Val Ser Glu Leu Gln Glu Ala Gln
Met Ala Leu Gly Pro 50 55 60 Leu Gln Gln Gly His Ser Pro Ala Glu
Ser Trp Glu Ala Gln Glu Glu 65 70 75 80 Pro Pro Ala Arg Val Leu Ala
Pro His Asp Asn Val Leu Arg Ala Leu 85 90 95 Arg Arg Leu Gly Ser
Ser Lys Met Met Arg Asp Ser Arg Cys Phe Gly 100 105 110 Arg Arg Leu
Asp Arg Ile Gly Ser Leu Ser Gly Leu Gly Cys Asn Val 115 120 125 Leu
Arg Arg His 130 3134PRTHomo sapiensmisc_featureHuman preproBNP;
SwissProt P16860 3Met Asp Pro Gln Thr Ala Pro Ser Arg Ala Leu Leu
Leu Leu Leu Phe 1 5 10 15 Leu His Leu Ala Phe Leu Gly Gly Arg Ser
His Pro Leu Gly Ser Pro 20 25 30 Gly Ser Ala Ser Asp Leu Glu Thr
Ser Gly Leu Gln Glu Gln Arg Asn 35 40 45 His Leu Gln Gly Lys Leu
Ser Glu Leu Gln Val Glu Gln Thr Ser Leu 50 55 60 Glu Pro Leu Gln
Glu Ser Pro Arg Pro Thr Gly Val Trp Lys Ser Arg 65 70 75 80 Glu Val
Ala Thr Glu Gly Ile Arg Gly His Arg Lys Met Val Leu Tyr 85 90 95
Thr Leu Arg Ala Pro Arg Ser Pro Lys Met Val Gln Gly Ser Gly Cys 100
105 110 Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
Cys 115 120 125 Lys Val Leu Arg Arg His 130 478PRTArtificial
SequenceFeline Synthetic NT-proBNP (fsNT-proBNP) 4His Pro Leu Gly
Gly Pro Gly Pro Ala Ser Glu Ala Ser Ala Ile Gln 1 5 10 15 Glu Leu
Leu Asp Gly Leu Arg Asp Thr Val Ser Glu Leu Gln Glu Ala 20 25 30
Gln Met Ala Leu Gly Pro Leu Gln Gln Gly His Ser Pro Ala Glu Ser 35
40 45 Trp Glu Ala Gln Glu Glu Pro Pro Ala Arg Val Leu Ala Pro His
Asp 50 55 60 Asn Val Leu Arg Ala Leu Arg Arg Leu Gly Ser Ser Lys
Met 65 70 75 521PRTArtificial SequencePreferred Feline NT-proBNP
N-Terminal Peptide Plasma Degradent 5Ala Ser Glu Ala Ser Ala Ile
Gln Glu Leu Leu Asp Gly Leu Arg Asp 1 5 10 15 Thr Val Ser Glu Leu
20 610PRTArtificial SequencePreferred Feline NT-proBNP N-Terminal
Peptide Plasma Degradent 6Asp Gly Leu Arg Asp Thr Val Ser Glu Leu 1
5 10 716PRTArtificial SequencePreferred Feline NT-proBNP N-Terminal
Peptide Plasma Degradent 7Leu Asp Gly Leu Arg Asp Thr Val Ser Glu
Leu Gln Glu Ala Gln Met 1 5 10 15 823PRTArtificial
SequencePreferred Feline NT-proBNP C-Terminal Peptide Plasma
Degradent 8Leu Gly Pro Leu Gln Gln Gly His Ser Pro Ala Glu Ser Trp
Glu Ala 1 5 10 15 Gln Glu Glu Pro Pro Ala Arg 20 933PRTArtificial
SequencePreferred Feline NT-proBNP C-Terminal Peptide Plasma
Degradent 9Leu Gly Pro Leu Gln Gln Gly His Ser Pro Ala Glu Ser Trp
Glu Ala 1 5 10 15 Gln Glu Glu Pro Pro Ala Arg Val Leu Ala Pro His
Asp Asn Val Leu 20 25 30 Arg 1030PRTArtificial SequencePreferred
Feline NT-proBNP C-Terminal Peptide Plasma Degradent 10Leu Gln Gln
Gly His Ser Pro Ala Glu Ser Trp Glu Ala Gln Glu Glu 1 5 10 15 Pro
Pro Ala Arg Val Leu Ala Pro His Asp Asn Val Leu Arg 20 25 30
1110PRTArtificial SequencePreferred Feline NT-proBNP C-Terminal
Peptide Plasma Degradent 11Val Leu Ala Pro His Asp Asn Val Leu Arg
1 5 10
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