U.S. patent application number 13/475479 was filed with the patent office on 2012-09-13 for blood biomarkers for bone fracture and cartilage injury.
This patent application is currently assigned to GENERA INSTRAZIVANJA d.o.o.. Invention is credited to Lovorka Grgurevic, Boris Macek, Slobodan Vukicevic.
Application Number | 20120231477 13/475479 |
Document ID | / |
Family ID | 39926006 |
Filed Date | 2012-09-13 |
United States Patent
Application |
20120231477 |
Kind Code |
A1 |
Grgurevic; Lovorka ; et
al. |
September 13, 2012 |
Blood Biomarkers for Bone Fracture and Cartilage Injury
Abstract
Blood biomarkers are described for use in methods and
compositions to determine whether an individual has sustained a
bone fracture or a cartilage injury.
Inventors: |
Grgurevic; Lovorka; (Zagreb,
HR) ; Macek; Boris; (Zagreb, HR) ; Vukicevic;
Slobodan; (Zagreb, HR) |
Assignee: |
GENERA INSTRAZIVANJA d.o.o.
Kalinovica
HR
|
Family ID: |
39926006 |
Appl. No.: |
13/475479 |
Filed: |
May 18, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12451100 |
Oct 23, 2009 |
8182998 |
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PCT/US2008/005463 |
Apr 28, 2008 |
|
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13475479 |
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60926316 |
Apr 26, 2007 |
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Current U.S.
Class: |
435/7.21 ;
435/7.92 |
Current CPC
Class: |
G01N 2333/495 20130101;
G01N 33/6887 20130101; G01N 2333/78 20130101; G01N 2800/10
20130101; G01N 2800/56 20130101; G01N 33/6872 20130101 |
Class at
Publication: |
435/7.21 ;
435/7.92 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Claims
1. A method of detecting an injury to cartilage in a human
individual comprising measuring the level of cartilage acidic
protein 1 (CRTAC-1) in a first blood sample obtained from a first
individual and comparing the level of CRTAC-1 in the first blood
sample to the level of CRTAC-1 determined in a second blood sample
from the first individual or a second individual when the first or
second individual did not have a cartilage injury, wherein a level
of CRTAC-1 in the first blood sample that is at least about 20%
higher than the level of CRTAC-1 in the second blood sample
indicates that the first individual has sustained a cartilage
injury.
2. The method according to claim 1, wherein the step of measuring
the level of CRTAC-1 in the first blood sample comprises contacting
the first blood sample with a binding partner for CRTAC-1 to form a
binding complex between the binding partner and CRTAC-1 present in
the first blood sample.
3. The method according to claim 2, wherein the binding complex
formed between the binding partner and CRTAC-1 present in the first
blood sample is detected by a detectable label present on the
binding partner.
4. The method according to claim 2, wherein the binding complex
formed between the binding partner and CRTAC-1 present in the first
blood sample is detected by adding an antibody that binds to said
binding partner or that binds to said CRTAC-1 present in said
binding complex and detecting said antibody by a detectable label
present on said antibody.
5. The method according to any one of claims 2-4, wherein the
binding partner for CRTAC-1 is a binding protein or an aptamer that
binds CRTAC-1.
6. The method according to claim 5, wherein said binding protein
for CRTAC-1 is an antibody to CRTAC-1.
7. A method of monitoring the state of a cartilage injury in an
individual comprising measuring the level of cartilage acidic
protein 1 (CRTAC-1) in a first blood sample previously obtained
from the individual and comparing the level of CRTAC-1 in the first
blood sample with the level of CRTAC-1 measured in a second blood
sample, wherein the second blood sample was obtained within two
weeks, inclusive, after the first blood sample from the individual,
and determining whether the level of CRTAC-1 in the second blood
sample has increased, decreased, or remained the same relative to
the level in the first blood sample.
8. A method for determining the time proximity of an individual
from a cartilage injury comprising: (a) obtaining a blood sample
from said individual at a first time point, (b) obtaining at least
one additional blood sample from said individual at a later time
point, (c) determining the concentration of cartilage acidic
protein 1 (CRTAC-1) in at least said first blood sample and said at
least one additional blood sample, wherein said first time point
and said later time point are within two weeks of each other,
wherein a determination that an increase in CRTAC-1 concentration
of at least about 20% has occurred between said first time point
and said later time point indicates that the individual has
suffered a cartilage injury within two weeks prior to said later
time point, wherein a determination that a decrease in CRTAC-1
concentration of at least about 4.5% has occurred between said
first time point and said later time point indicates that the
individual has suffered a cartilage injury more than six weeks but
less than ten weeks prior to said later time point, wherein a
determination that the concentration of CRTAC-1 has neither
increased or decreased more than about 2% between said first time
point and said later time point indicates that the individual has
not suffered a cartilage injury for at least ten weeks prior to
said later time point.
9. The method according to claim 8, wherein said first time point
and said at later time point are within one week of each other.
10. A kit for use in measuring the level of cartilage acidic
protein 1 (CRTAC-1) in a blood sample previously obtained from an
individual to determine whether said individual has sustained a
cartilage injury comprising: a binding partner that binds to
cartilage acidic protein 1 (CRTAC-1); an antibody that binds the
binding partner when the binding partner is bound to CRTAC-1;
instructions for using the kit to measure the level of CRTAC-1 in a
blood sample previously obtained from an individual to determine if
said individual has sustained a cartilage injury.
11. A kit for use in detecting the presence of transforming growth
factor beta receptor III (TGF.beta.rIII) in a blood sample
previously obtained from an individual to determine whether said
individual has sustained a bone fracture comprising: a binding
partner that binds to transforming growth factor beta receptor III
(TGF.beta.rIII); an antibody that binds the binding partner when
the binding partner is bound to TGF.beta.rIII; instructions for
using the kit to detect TGF.beta.rIII in a blood sample previously
obtained from an individual to determine if said individual has
sustained a bone fracture.
12. The kit according to claim 10 or claim 11, further comprising a
device to obtain a sample of blood from an individual.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/451,100 filed Oct. 23, 2009, which is a United States
national stage filing under 35 USC .sctn.371 of international
application No. PCT/US2008/005463, filed Apr. 28, 2008, designating
the US, which claims priority to U.S. Provisional Application No.
60/926,316, filed Apr. 26, 2007.
FIELD OF THE INVENTION
[0002] This invention is in the field of diagnosis of injuries.
More specifically, the invention discloses means and methods for a
rapid and accurate detection of bone and cartilage injury in a
human patient and other mammals based on the release into the
peripheral circulation of proteins involved with bone and cartilage
repair.
BACKGROUND OF THE INVENTION
[0003] The blood of humans and other mammals is now known to be
rich with a large amount of previously unstudied molecules that
could reflect the ongoing physiologic state of various tissues. As
blood flows through most of the tissues of the human body, the
origins of plasma proteins may be diverse. The complex mixture of a
blood plasma proteome from a healthy human individual is expected
to comprise well known blood component proteins such as albumin and
other known proteins in a relatively high abundance and various
other proteins that originate from circulating blood cells.
[0004] Bone normally undergoes continuous turnover and remodeling
comprising bone formation and bone resorption; two opposite and
well balanced processes. A number of proteins related to this
normal regenerative process may be found in the plasma and/or urine
of healthy individuals in amounts that are generally correlated
with a relative decrease or increase in bone turnover activity.
See, e.g., Anderson et al., Mol. Cell. Proteomics, 1: 845-867
(2002). Following fracture, a large number of growth factors,
cytokines, and their cognate receptors involved in bone repair are
highly expressed at the fracture site in the first hours following
injury. Skeletal tissues are the main source of such proteins,
while some are released from associated inflammatory cells at the
site of injury. See, e.g., Barnes et al., J. Bone Miner. Res., 14:
1805-1815 (1999).
[0005] Injuries to bones and cartilage are routinely assessed and
monitored using such well known standard methods such as X-rays,
bone scans, and magnetic resonance imaging (MRI). However, such
methods typically require transporting a patient to a location that
contains the machinery necessary to carry out such analyses. Yet
there are many situations in which it would be advantageous to be
able to determine whether a patient has sustained a bone fracture
and/or cartilage injury without the need or benefit of X-ray, bone
scan, or MRI studies. In addition, injuries to bone and cartilage
are not always evident by such methods. Accordingly, needs remain
for additional means and methods to detect and assess bone and
cartilage injuries in patients.
SUMMARY OF THE INVENTION
[0006] The invention described herein solves the above problems by
providing a rapid and accurate method for detecting a bone fracture
and cartilage injuries in a human individual using a sample of the
individual's blood. The invention is based on the discoveries that
transforming growth factor beta receptor III (TGF.beta.rIII), which
normally is not present in the blood of a healthy human individual,
appears in the peripheral blood when a bone is fractured and that
the level of cartilage acidic protein 1 (CRTAC-1) is significantly
elevated in the peripheral blood of an individual that has an
injury to cartilage. TGF.beta.rIII and elevated levels of CRTAC-1
continue to be detected in the blood for a period of time for at
least 24 weeks after time of injury. Moreover, the detection of
TGF.beta.rIII or of elevated levels of CRTAC-1 in the blood
provides a diagnostic test for the presence of injury to bone or
cartilage, respectively, that may be more sensitive than commonly
employed methods, such as X-ray, bone scan, and magnetic resonance
imaging (MRI), which require specialized instruments. Furthermore,
methods described herein can be routinely carried out to monitor
bone fracture or cartilage injury in a patient. The methods
described herein may be readily carried out using any of a variety
of formats.
[0007] A method described herein may be carried out on whole blood
or a fraction thereof, such as plasma or serum. Preferably, the
plasma portion of blood is used in the methods described
herein.
[0008] In one embodiment, the invention provides a method of
detecting a fracture in a bone of a human individual in which a
sample of blood is obtained from the human individual and assayed
for the presence of transforming growth factor beta receptor III
(TGF.beta.rIII), wherein the detection of TGF.beta.rIII in the
blood sample indicates that the individual has sustained a bone
fracture.
[0009] Methods of detecting TGF.beta.rIII and/or CRTAC-1 in a
sample of blood obtained from an individual according to the
invention may also be used to measure (quantitate) the level of
TGF.beta.rIII and/or CRTAC-1 present in the sample of blood and
thereby in the blood of the individual.
[0010] Since TGF.beta.rIII is known to play a role in enhancing
TGF-.beta. signal transduction in the process of bone formation
(osteogenesis), the presence of TGF.beta.rIII in a sample of blood
obtained from an individual may also be used as an indication that
the process of osteogenesis is stimulated in the individual.
Likewise, because CRTAC-1 is a component in the process of
cartilage formation (chondrogenesis), the determination that there
is an elevated level or an increasing level of CRTAC-1 over time in
one blood sample relative to another obtained from an individual
may be used as an indication that the process of chondrogenesis is
stimulated in the individual.
[0011] In addition to methods for detecting whether an individual
has sustained an injury to bone or cartilage, the invention also
provides methods that may be used to routinely monitor the time
course of injury to bone or cartilage in an individual without the
use of X-rays, bone scans, or MRI studies. Such methods are
especially useful in monitoring individuals that may be at
increased risk of bone fracture or cartilage injury, such as an
individual afflicted with osteoarthritis, osteoporosis, or a
genetic disease that impairs osteogenesis or chondrogenesis, such
as osteogenesis imperfecta (OI, "brittle bone disease") in which
the bones of an individual are unusually brittle and susceptible to
fracture. Such methods may also be used in monitoring an individual
who may be suspected of having a bone or cartilage injury but is
incapable of effective communication, such as infants; speaking
impaired individuals; stroke patients; and patients in an altered
consciousness state (ACS), such as coma, near coma, persistent
vegetative state, vegetative state, or minimally conscious state.
Such methods may also find use in the forensic analysis of injuries
to an individual.
[0012] In another embodiment, the invention provides a method for
monitoring the state of a bone fracture in an individual comprising
assaying for the level of transforming growth factor beta receptor
III (TGF.beta.rIII) in a first blood sample and in a second blood
sample, wherein the second blood sample was obtained within two
weeks, preferably one week, inclusive, after the first blood sample
from the individual, and comparing the level of TGF.beta.rIII in
the first blood sample with that of the second blood sample. In a
further embodiment, a significant increase in the level of
TGF.beta.rIII of greater than about 20% or more between the first
(earlier obtained) blood sample and the second (later obtained)
blood sample indicates that the individual has sustained a bone
fracture that occurred within about 1 to about 2 weeks prior to the
time of the second blood sample. In another embodiment, a decrease
in the level of TGF.beta.rIII of greater than about 10% or more
between the first blood sample and the second blood sample
indicates that the individual has sustained a bone fracture that
occurred within about 2 to about 6 weeks prior to the time of the
second blood sample. In yet another embodiment, where the level of
TGF.beta.rIII remains essentially steady, i.e., where the change in
the level of TGF.beta.rIII between the first and second blood
sample is of about 6% or less, including no increase or decrease,
this indicates that the individual has sustained a bone fracture
but has sustained no new bone fracture for at least about 6 weeks
(more preferably, within about 6 to about 24 weeks) prior to the
time of the second blood sample.
[0013] In another embodiment, the invention provides a method of
detecting a cartilage injury in a human individual in which a
sample of blood is obtained from the individual and assayed to
determine the level of cartilage acidic protein 1 (CRTAC-1) in the
blood sample, wherein a level of CRTAC-1 in the blood sample that
is significantly higher, that is, at least about 20% or more higher
than the level previously determined in a sample of blood from the
individual indicates that the individual has sustained a cartilage
injury. Following a cartilage injury in an individual, the level of
CRTAC-1 in a blood sample of the individual increases dramatically
in the first weeks following the injury, rising by at least about
40% (including as much as about 50%, about 60%, about 70%, about
80%, about 90%, and about 100%) higher than the level of CRTAC-1 in
a blood sample from the individual prior to or at the time of the
cartilage injury, or in comparison to a level previously determined
in a blood sample from the individual or to a reference level or
range of concentration for CRTAC-1 as determined from a healthy
population of individuals not suffering from a cartilage
injury.
[0014] In another embodiment, the invention provides a method for
monitoring the state of a cartilage injury in an individual
comprising assaying for the level of cartilage acidic protein 1
(CRTAC-1) in a first blood sample and in a second blood sample,
wherein the second blood sample was obtained from the individual
within two weeks, preferably within a week, inclusive, of the first
blood sample from the individual, and comparing the level of
CRTAC-1 in the first blood sample with that in the second blood
sample. In a further embodiment, an increase in the level of
CRTAC-1 of about 20% or more, more preferably about 24% or more,
between the first (earlier obtained) blood sample and the second
(later obtained) blood sample indicates that the individual has
sustained a cartilage injury that occurred within about 1 to about
2 weeks prior to the time of the second blood sample. In still a
further embodiment, a decrease in the level of CRTAC-1 of about
4.5% or more between the first blood sample and the second blood
sample indicates that the individual has sustained a cartilage
injury that occurred within about 6 to about 10 weeks prior to the
time of the second blood sample. In another embodiment, where the
level of CRTAC-1 measured in the first and second blood sample
remains essentially steady, i.e., where the change in the level of
CRTAC-1 is less than 2% between the first blood sample and the
second blood sample, including no increase or decrease, indicates
that the individual is in a steady state and has sustained no new
cartilage injury for at least about 10 weeks prior to the time of
the second blood sample.
[0015] Preferably, in a method described herein, a blood sample is
assayed using a binding partner that specifically binds
TGR.beta.rIII or CRTAC-1 as its cognate binding partner (cognate
ligand). Binding partners include binding proteins and aptamers.
Binding proteins useful in the methods and compositions described
herein include, but are not limited to, full-length immunoglobulin
antibody molecules comprising four polypeptide chains, i.e., two
heavy (H) chains and two light (L) chains, wherein each pair of
heavy and light chains forms an antigen binding site. Other binding
proteins useful in the methods and compositions described herein
include any of a variety of recombinant antibody constructs that
possess an antigen binding site, including without limitation, a
functional antibody fragment, such a Fab, F(ab').sub.2, and Fv; a
hybrid antibody, such as a chimeric or humanized antibody; a single
chain antibody (scFv); a diabody; a dual-variable domain
immunoglobulin molecule; and the like. Such antibody binding
proteins are especially advantageous as they may be employed in any
of a variety of immunoassay formats in which a blood sample of an
individual is brought into contact with an antibody binding protein
for TGF.beta.rIII and/or an antibody binding protein for CRTAC-1
under conditions suitable for the formation of a binding complex
formed between the binding protein and the binding partner, which
complex can then be detected using any of a variety methods
available in the art for detecting antibody/antigen
immunocomplexes.
[0016] A binding partner that binds TGF.beta.rIII or CRTAC-1 may
also have a detectable label (tag) or other molecule that permits
detection of a binding complex formed between the binding partner
and TGF.beta.rIII or CRTAC-1 in a method described herein. Such
detectable labels and other molecules are well known in the art and
include, without limitation, fluorescent labels, radiolabels,
colorimetric molecules, affinity beads, and the like.
[0017] Formats used for immunoassays to detect antibody/antigen
immunocomplexes may also be employed in the methods and
compositions described herein. Such formats for detecting or
measuring the level of TGF.beta.rIII or CRTAC-1 in a sample of
blood according to the invention include, but are not limited to,
enzyme linked immunoadsorbent assay (ELISA), immunoprecipitations,
immunoblotting, affinity chromatography, assay strips, dip sticks,
and the like, wherein the blood sample is brought into contact with
a binding protein for TGF.beta.rIII or CRTAC-1 and the resulting
binding complex detected.
[0018] In yet another embodiment, the invention provides a kit for
detecting or measuring the level of TGF.beta.rIII and/or CRTAC-1 in
a sample of blood from an individual to determine if the individual
has sustained a bone fracture or cartilage injury. Such kits may
comprise a binding partner for TGF.beta.rIII and/or CRTAC-1, one or
more buffers or solutions for carrying out the assay, and
instructions that indicate how to use the kit to detect the
presence of or measure the level of TGF.beta.rIII and/or CRTAC-1 in
a blood sample and to determine whether the individual has
sustained a bone fracture or cartilage injury.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows graphs of the level (ng/ml) of transforming
growth factor beta receptor III (TGF.beta.rIII) in blood samples
(plasma) from patients that sustained a single traumatic fracture
in the tibia as determined with 24 hours of injury and continuing
for 24 weeks after injury as described in Example 2, below.
Diamonds indicate the average level of TGF.beta.rIII in blood
samples from patients (n=26) in which the bone fracture healed over
the course of 24 weeks following injury ("union"). Squares indicate
the average level of TGF.beta.rIII in blood samples from patients
(n=4) in which the fracture did not heal over the course of 24
weeks following injury ("delayed union"). See description, infra,
for details.
[0020] FIG. 2 shows graphs of the level (ng/ml) of cartilage acidic
protein 1 (CRTAC-1) in blood samples (plasma) from patients that
sustained a single fracture in the middle shaft or in the distal
portion of the tibia without cartilage injury ("union", "delayed
union") and in blood samples from patients that sustained a
fracture in the middle shaft of the tibia and also a fracture in
the distal portion extending into the ankle joint with visible
dislocation of the joint and damage to the cartilage layer
("cartilage damage") as described in Example 2, below. Triangles
indicate the average level of CRTAC-1 in blood samples from
patients (n=8) that sustained bone fractures and cartilage damage
as determined over the course of 24 weeks following injury
("cartilage damage"). Diamonds indicate the average level of
CRTAC-1 in blood samples from patients (n=26) that sustained a bone
fracture that healed over the course of the 24 weeks following
injury ("union"). Squares indicate the average level of CRTAC-1 in
blood samples from patients (n=4) that sustained a bone fracture
that did not heal over the course of 24 weeks following injury
("delayed union"). See description, infra, for details.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The invention is based on the discoveries that transforming
growth factor beta receptor III (TGF.beta.rIII) appears in the
peripheral blood of human individuals that sustain a bone fracture
and that elevated levels of cartilage acidic protein 1 (CRTAC-1)
appear in the peripheral blood of human individuals that sustain
cartilage injury. Neither TGF.beta.rIII nor CRTAC-1 was previously
known to be released to the peripheral circulation as the result of
injury to bone or cartilage tissue. The detection of TGF.beta.rIII
and elevated levels of CRTAC-1 in the blood of an individual may
provide an even more sensitive indication of bone fracture and
cartilage injury, respectively, than traditional diagnostic methods
such as X-rays and bone scans for bone fractures and magnetic
resonance imaging (MRI) for cartilage injury. Accordingly,
TGF.beta.rIII and CRTAC-1 are useful as blood biomarkers for bone
fracture and cartilage injury, respectively.
[0022] Methods described herein may also be used in assessing or
monitoring injury to bone and cartilage in individuals who are
incapable of effective communication to a healthcare professional.
Such individual may include, but are not limited to, infants;
speaking impaired individuals; stroke patients; and patients in an
altered consciousness state (ACS), such as coma, near coma,
persistent vegetative state, vegetative state, or minimally
conscious state. Methods described herein may also find use in the
forensic analysis of injuries.
[0023] The methods and compositions of the present invention are
contemplated to be applied to the detection or measurement of
either or both TGF.beta.rIII or CRTAC-1 in a sample of blood and
are understood to encompass the detection in whole blood or
fractions thereof, such as plasma or serum. Particularly preferred
is the use of blood plasma in the methods and compositions
described herein.
[0024] The methods and compositions of the present invention are
especially contemplated to benefit human subjects, but they are
suitable for any mammalian subject that expresses a TGF.beta.rIII
or a CRTAC-1.
[0025] In order that the invention may be more fully understood,
the follow terms are defined.
[0026] Unless indicated otherwise, when the terms "about" and
"approximately" are used in combination with an amount, number, or
value, then that combination describes the recited amount, number,
or value alone as well as the amount, number, or value plus or
minus 10% of that amount, number, or value. By way of example, the
phrases "about 40%" and "approximately 40%" disclose both "40%" and
"from 36% to 44%, inclusive".
[0027] A "binding partner" is any molecule, including any
polypeptide, immunoglobulin, nucleic acid, or fragment thereof,
which specifically binds a cognate binding partner (cognate ligand)
at one or more sites. Examples of binding partner/cognate ligand
pairs include antibody/antigen, receptor/ligand,
biotin/streptavidin, and enzyme/substrate. A binding partner that
is a polypeptide may also be referred to as a "binding protein".
Binding partners useful in the methods and compositions described
herein include antibody molecules specific for TGF.beta.rIII or
CRTAC-1. A binding partner that is a nucleic acid is referred to as
an aptamer.
[0028] A "TGF.beta.rIII binding partner" is any binding partner
molecule, including any polypeptide, immunoglobulin, or fragment
thereof, which specifically binds transforming growth factor beta
receptor III (TGF.beta.rIII) or an epitope thereof at one or more
sites in the molecule.
[0029] A "CRTAC-1 binding partner" is any binding partner molecule,
including any polypeptide, immunoglobulin, or fragment thereof,
which specifically binds cartilage acidic protein 1 (CRTAC-1) or an
epitope thereof at one or more sites in the molecule.
[0030] A "TGF.beta.rIII antibody" refers to a binding protein that
contains at least one antigen binding site that binds TGF.beta.rIII
or an epitope thereof. Similarly, a "CRTAC-1 antibody" refers to a
binding protein that contains at least one antigen binding site
that binds CRTAC-1 or an epitope thereof.
[0031] An "antibody" includes any of the classes of full-length
mammalian immunoglobulin classes (such as IgG, IgM, IgA, IgE, IgD)
and subclasses thereof. An "antibody" may also be any fragment of a
full-length immunoglobulin that binds the same antigen, such a Fab,
F(ab').sub.2, and Fv fragments, as well as binding molecules that
may be produced by protein engineering or recombinant DNA
technology, including but not limited to, a chimeric antibody,
which comprises a binding domain or complementarity determining
regions (CDRs) of an immunoglobulin fused or inserted into another
immunoglobulin; a humanized antibody, which comprises the CDRs from
a non-human antibody inserted into the framework of a human
antibody molecule; a single chain antibody (scFv); and a diabody
(see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90:
6444-6448 (1993)).
[0032] An antibody useful in the methods and compositions described
herein may be monovalent, i.e., having a single binding site for
binding a single antigen (or epitope) molecule, or multivalent,
i.e., having more than one binding sites for binding more than one
antigen (or epitope). A classic IgG antibody molecule has two
antigen binding sites and, thus, is bivalent.
[0033] An antibody useful in the methods and compositions described
herein may be monospecific, i.e., binding a single type of antigen
(or epitope), or multispecific, i.e., binding two or more different
antigens (or epitopes). A classic IgG antibody molecule that has
two identical antigen binding sites is thus monospecific with
respect to the type of antigen (or epitope) that it can bind. A
bispecific antibody binding partner useful in the invention can
bind at least one molecule of TGF.beta.rIII and at least one
molecule of CRTAC-1. Bispecific antibody molecules may be
heterodimers of two halves of two different full-length
immunoglobulin molecules. For example, bispecific antibodies have
been described using "quadroma" technology that fuses two different
hybridoma cell lines, each capable of expressing a monoclonal
antibody that binds a different antigen. Random pairing of light
and heavy chains of the two monoclonal antibodies include
heterodimers comprising a pair of heavy and light chains of one
monoclonal antibody associated with a pair of heavy and light
chains of the other monoclonal antibody (see, e.g., Milstein et
al., Nature, 305: 537-540 (1983)). A variety of other bispecific
antibody molecules have been described using protein engineering
and recombinant DNA technology (see, e.g., Kriangkum et al.,
Biomol. Eng., 18(2): 31-40 (2001)). Bispecific antibodies useful in
the invention may include, but are not limited to, bispecific
diabodies (e.g., Holliger et al. (1993); Holliger et al., Cancer
Immunol. Immunother., 45: 128-130 (1997); Wu et al., Immunotech.,
2(1): 21-36 (1996)), bispecific tandem scFv molecules, Fab
mulitmers (see, e.g., Miller et al, J. Immunol., 170: 4854-4861
(2003)), and dual variable domain immunoglobulins (see, e.g., Wu et
al., Nature Biotechnology, (Oct. 14, 2007)).
[0034] A composition or method described herein as "comprising" one
or more named elements or steps is open-ended, meaning that the
named elements or steps are essential, but other elements or steps
may be added within the scope of the composition or method. To
avoid prolixity, it is also understood that any composition or
method described as "comprising" (or which "comprises") one or more
named elements or steps also describes the corresponding, more
limited composition or method "consisting essentially of" (or which
"consists essentially of") the same named elements or steps,
meaning that the composition or method includes the named essential
elements or steps and may also include additional elements or steps
that do not materially affect the basic and novel characteristic(s)
of the composition or method. It is also understood that any
composition or method described herein as "comprising" or
"consisting essentially of" one or more named elements or steps
also describes the corresponding, more limited, and closed-ended
composition or method "consisting of" (or "consists of") the named
elements or steps to the exclusion of any other unnamed element or
step. In any composition or method disclosed herein, known or
disclosed equivalents of any named essential element or step may be
substituted for that element or step.
[0035] It is also understood that an element or step "selected from
the group consisting of" refers to one or more of the elements or
steps in the list that follows, including combinations of any two
or more of the listed elements or steps.
[0036] The meanings of other terms will be evident to those skilled
in the art including the meanings known in fields of orthopedic
medicine, molecular biology, immunology, and diagnostic
methodologies.
[0037] Cartilage acidic protein 1 (CRTAC-1) is a glycosylated
extracellular matrix protein that has been isolated from human
articular cartilage secreted by chondrocytes. In cell culture,
CRTAC-1 has been described as a candidate marker to distinguish the
chondrocyte-like phenotype and activity from osteoblast-like and
mesenchymal stem cells (Steck et al., Matrix Biol., 26: 30-41
(2007)).
[0038] Transforming growth factor beta receptor III (TGF.beta.rIII)
is known to be involved in developmental and regenerative
processes. For example, TGF.beta.rIII plays an essential role in
murine and chick development, and TGF.beta.rIII knockout mice have
an embryonic lethal phenotype. TGF.beta.rIII functions as a
co-receptor for TGF-.beta. signal transduction by enhancing
TGF-.beta. binding to its receptor TGF.beta.rII and thereby
increasing TGF-.beta. signaling (see, e.g., Massague, J., Ann. Rev.
Biochem., 67: 753-791 (1998)). Enhancement of TGF-.beta. signaling
is important in bone fracture healing wherein TGF-.beta.1 and its
receptor TGF.beta.rII, together with extracellular matrix proteins
osteocalcin and collagen type I, are involved in a coordinated
manner to promote proper healing of bone fractures.
[0039] Transforming growth factor beta receptor III (TGF.beta.rIII)
and cartilage acidic protein 1 (CRTAC-1) have not previously been
reported to be a component in the blood of humans. Both proteins
were detected in plasma from human individuals that sustained long
bone fractures (see, Example 1, below). Further analysis revealed
that TGF.beta.rIII is normally not found in the blood of healthy
human individuals but is present in the blood of individuals who
have sustained a bone fracture. In contrast, CRTAC-1 may be found
in the blood of healthy individuals but will be present at elevated
levels in the peripheral blood in an individual that has sustained
a cartilage injury. Moreover, the level of both proteins in
circulating blood show a similar pattern of change in the weeks
following the injuries with which we have found them to be
associated: the levels fluctuate between wide limits within the
first ten weeks following injury, first increasing significantly
and precipitously in the first 2 or 4 weeks (for TGF.beta.rIII and
CRTAC-1, respectively), then falling significantly over the next 6
weeks or so, then finally leveling off to essentially a steady
state, neither increasing nor decreasing significantly for 14 weeks
or longer. See FIGS. 1 and 2.
[0040] According to the invention, a level of CRTAC-1 in a blood
sample from of an individual that is at least about 20% higher than
a previously determined level of CRTAC-1 in a sample of blood from
the individual (i.e., a baseline level) or at least about 20%
higher than an estimated baseline level from healthy (non-injured)
individuals or historical controls indicates that individual has
sustained a cartilage injury.
[0041] According to the invention, a preferred method for detecting
a bone fracture in a human individual comprises the steps of
obtaining a sample of blood from the human individual and assaying
the sample of blood for the presence of transforming growth factor
beta receptor III (TGF.beta.rIII), wherein the detection of
TGF.beta.rIII in the blood sample indicates that the individual has
sustained a fracture in a bone.
[0042] Any fracture of bone stimulates the process of bone
formation, i.e., osteogenesis, to regenerate bone and heal the
fracture. Since TGF.beta.rIII is known to play a role in enhancing
TGF-.beta. signal transduction in the process of bone formation
(osteogenesis), the presence of TGF.beta.rIII in a sample of blood
obtained from an individual may also be used as an indication that
osteogenesis has been stimulated in response to a fracture.
Likewise, because CRTAC-1 is a component in the process of
cartilage formation (chondrogenesis), the determination that the
level of CRTAC-1 in the blood of an individual is elevated may be
used as an indication that the process of chondrogenesis has been
stimulated in response to a cartilage injury.
[0043] Fractures in bone as detected by a method described herein
may result from any of a variety of conditions including, trauma
and bone diseases. Metabolic bone diseases include, but are not
limited to, osteoarthritis, osteoporosis, and osteogenesis
imperfecta (OI, "brittle bone disease"). Fractures may occur in
patients with osteoarthritis where a full thickness defect in a
joint so depletes the articular cartilage that normally cushions
two opposing bones that the two bones make contact and grind
against one another and eventually fracture the surface of either
or both of the opposing bones. Osteoporosis and OI are examples of
bone diseases in which the bones of an individual are or can become
unusually brittle and susceptible to fracture. For example,
clinically undetected fractures, such as microfractures, can occur
in trabeculi within vertebrae or in the proximal and distal
metaphyseal areas of bones of individuals with osteoporosis. In the
case of OI, bones can be so brittle that relatively minor trauma
(bumps) cause fractures that would not normally occur in the bones
of healthy individuals. Accordingly, testing a sample of peripheral
blood for the presence of TGF.beta.rIII as described herein may be
advantageously used to routinely monitor for bone fracture in a
variety of patients without the need of equipment and time involved
in subjecting such patients to X-ray or bone scan procedures.
Moreover, methods described herein may indicate fractures that
cannot be detected using conventional X-rays and bone scans, such
as microfractures and occluded fractures.
[0044] As described herein (see, Example 2, infra), a study of
human patients who have sustained a traumatic bone fracture has
revealed that the relative level of transforming growth factor beta
receptor III (TGF.beta.rIII) in the blood of an individual over
time provides useful information regarding not only whether a
fracture has occurred but the state of bone fracture in an
individual. Representative data are shown in the graphs of FIG. 1
in which the level of TGF.beta.rIII as measured in blood samples
from a population of bone fracture patients is followed over time.
One of the graphs in FIG. 1 shows the level of TGF.beta.rIII in
blood samples from a portion of the patient population in which the
bone fractures healed by 24 weeks from injury ("union"). The other
graph in FIG. 1 shows the level of TGF.beta.rIII in blood samples
from the portion of the patient population in which the bone
fractures did not heal by 24 weeks from injury ("delayed union").
The data in FIG. 1 indicate that following fracture, the level of
TGF.beta.rIII in blood follows a relatively steep rise within about
2 weeks following injury, followed by a relatively symmetrical
steep decline within about a further 2 weeks, i.e., between about 2
weeks and about 4 weeks following fracture, followed by a slower
rate of decline at about 4 weeks to about 6 weeks following
fracture, and, thereafter, from about 6 weeks out to about 24 weeks
following injury, there is an extended leveling off in the case of
fracture healing or a gradual rise in the case of delayed
union.
[0045] Thus, another aspect of the invention is a method for
monitoring the state of a bone fracture in an individual comprising
assaying for the level of transforming growth factor beta receptor
III (TGF.beta.rIII) in a first blood sample and in a second blood
sample, wherein the second blood sample was obtained within two
weeks, preferably within a week, inclusive, after the first blood
sample from the individual, and comparing the level of
TGF.beta.rIII in the first blood sample with that of the second
blood sample. Moreover, an analysis of the representative data
provided in FIG. 1, permits a number of correlations to be made
between the change in the level of TGF.beta.rIII present in such a
first (earlier obtained) blood sample and a second (later obtained)
blood sample with respect to the state of bone fracture in the
individual including, but not limited to:
[0046] an increase in the level of TGF.beta.rIII of at least about
20% or more between the first blood sample and the second blood
sample indicates that the individual has sustained a bone fracture
that occurred within about 1 to about 2 weeks prior to the time of
the second blood sample;
[0047] a decrease in the level of TGF.beta.rIII of at least about
10% or more between the first blood sample and the second blood
sample indicates that the individual has sustained a bone fracture
that occurred within about 2 to about 6 weeks prior to the time of
the second blood sample;
[0048] a change in the level of TGF.beta.rIII of about 6% or less,
including no (0%) increase or decrease, indicates that the
individual has sustained a bone fracture and has sustained no new
bone fracture within about 6 weeks to about 24 weeks prior to the
time of the second blood sample.
[0049] Damage to cartilage may occur as the result of trauma or a
progressive disease that affects the cartilage tissue in joints or
other parts of the body. For example, fractures to the distal
portion of the tibia can extend into the ankle joint and damage the
cartilage layer on the surface of the calcanear bone and/or the
distal tibial joint surface (see, Example 2, infra). Injuries to
cartilage are typically detected by magnetic resonance imaging
(MRI). Clearly, an MRI can be useful in both locating a site of
cartilage injury and assessing the particular damage that has
occurred in cartilage tissue. However, methods described herein for
testing a blood sample for elevated levels of CRTAC-1 may be used
to determine if an individual has sustained a cartilage injury
without the use of an MRI. Moreover, methods described herein can
be used to determine whether an MRI study is even necessary. In
particular, testing a sample of blood for an elevated level of
expression of CRTAC-1 as described herein is a convenient means for
determining whether a cartilage injury may even exist in an
individual. Moreover, obtaining a blood sample to determine the
level of CRTAC-1 as described herein may be significantly less
stressful for some individuals than being subjected to the
constraint, noise, time, and expense involved in conducting an MRI
study, especially if the analysis of the blood sample indicates
there is no cartilage injury so that an MRI procedure is
unnecessary.
[0050] Methods described herein to determine whether an individual
has sustained a cartilage injury may comprise the step of comparing
a level of CRTAC-1 in a blood sample from the individual with a
reference level or reference range of concentration of CRTAC-1 that
is indicative of the level of CRTAC-1 present in the blood of
healthy human individuals that do not have a cartilage injury. A
reference level or reference concentration range of CRTAC-1 that is
indicative of normal cartilage health may be obtained from a
population of healthy individuals with normal healthy cartilage
tissue. The use of reference levels or reference ranges of
concentrations for a blood biomarker is the basis for virtually
every biomarker currently used in blood tests to assess the health
of human patients. Accordingly, persons skilled in the art of
optimizing diagnostic blood testing for use with respect to human
individuals are familiar with the procedures for gathering and
qualifying reference levels or concentration ranges of a particular
biomarker in the blood of a population of healthy individuals that
would be indicative of normal health (e.g., cartilage health) and
the levels of the biomarker that would indicate relevant injury,
disease, or condition (e.g., cartilage injury).
[0051] Alternatively, a method described herein may compare a level
of CRTAC-1 measured in a blood sample from an individual with one
or more levels of CRTAC-1 measured in one or more other blood
samples that were obtained from the same individual at a different
point in time (earlier or later). For example, testing the blood of
an individual on a routine basis to monitor the change in the level
of CRTAC-1 present in the blood over time, such as during periodic
check-ups with a healthcare professional, is one way to provide a
baseline CRTAC-1 level for an individual. A pronounced increase in
the level of CRTAC-1 from such a baseline level of CRTAC-1
indicates that the individual has sustained an injury to cartilage
tissue. Preferably, such a database for an individual provides
levels of CRTAC-1 in one or more prior blood samples that were
obtained from the individual when the individual is considered
(e.g., by a healthcare professional) to have healthy cartilage,
i.e., to not have sustained a cartilage injury.
[0052] A comparative study of levels of CRTAC-1 in blood from human
patients is described in Example 2 (infra). All patients in the
study sustained a traumatic fracture in the tibia. A portion of the
population of fracture patients sustained a single fracture in the
middle shaft or in the distal portion of the tibia and no cartilage
injury. Among these patients with a single fracture, a portion
("union") healed over the course of 24 weeks after the time of
injury (traumatic event). The other portion ("delayed union")
failed to heal over the same 24 week post trauma. Another group of
patients ("cartilage damage") sustained a fracture in the distal
portion of the tibia that extended into the ankle joint with a
visible dislocation of the joint and damage to the cartilage layer
on the surface of the calcanear bone and/or distal tibial joint
surface and, thus, sustained a cartilage injury.
[0053] FIG. 2 shows graphs of the levels of CRTAC-1 in blood of
patients in the three groups in the comparative study over the
course of 24 weeks from the time of injury. The levels of CRTAC-1
in blood from the patients with a single fracture and no cartilage
damage ("union" and "delayed union") serve as a control for the
background fracture in the patients with cartilage damage
("cartilage damage"). In the case of the "union" and "delayed
union" groups in FIG. 2, the level of CRTAC-1 initially rose
relatively steeply within about the first 2 weeks from the time of
injury, followed by a period of more gradual rise for about another
two weeks, i.e., at about 2 to about 4 weeks from injury, and
thereafter, leveled off or gradually declined over the course of
about 4 to about 24 weeks. In comparison, the level of CRTAC-1 in
blood from the "cartilage damage" group rose relatively steeply
over the first 2 weeks from time of injury, followed by a period of
considerably slower increase or leveling off at about 2 to about 6
weeks from the time of injury, followed by a period of decline for
about four weeks, i.e., at about 6 to about 10 weeks, and
thereafter, gradually declined and leveled off over the course of
about 10 weeks to about 24 weeks from time of injury until it
reached the approximately same level or lower than first measured
after injury.
[0054] FIG. 2, shows that throughout the 24 week study, the level
of CRTAC-1 in patients with cartilage injury ("cartilage damage")
was always at least about 40% higher than the level of CRTAC-1 in
blood from patients without cartilage injury ("union", "delayed
union"). Accordingly, in one aspect of the invention, a level of
CRTAC-1 in a sample of blood from an individual indicates that the
individual has sustained a cartilage injury when the level of
CRTAC-1 is at least approximately 40% (including, in order of
increasing preference, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, and at
least about 100%) higher than a level of CRTAC-1 previously
determined in a sample of blood from the individual when the
individual was known to have healthy cartilage or known to not have
sustained a cartilage injury.
[0055] Another aspect of the invention is a method of monitoring
the state of a cartilage injury in an individual comprising
assaying for the level of cartilage acidic protein 1 (CRTAC-1) in a
first blood sample and in a second blood sample, wherein the second
blood sample was obtained from the individual within two weeks,
preferably within a week, inclusive, of the first blood sample from
the individual, and comparing the level of CRTAC-1 in the first
blood sample with that in the second blood sample. Moreover, an
analysis of the representative data provided in FIG. 2, permits a
number of correlations to be made between the change in the level
of CRTAC-1 present in such a first (earlier obtained) blood sample
and a second (later obtained) blood sample with respect to the
state of cartilage injury in the individual including, but not
limited to:
[0056] an increase in the level of CRTAC-1 of at least about 20% or
more (more preferably at least about 24% or more), between the
first blood sample and the second blood sample indicates that the
individual has sustained a cartilage injury that occurred within
about 1 to about 2 weeks prior to the time of the second blood
sample;
[0057] a decrease in the level of CRTAC-1 of about 4.5% or more
between the first blood sample and the second blood sample
indicates that the individual has sustained a cartilage injury that
occurred within about 6 to about 10 weeks prior to the time of the
second blood sample;
[0058] a change (increase or decrease) in the level of CRTAC-1 of
less than 2% between the first blood sample and the second blood
sample indicates that the individual is in a steady state and has
sustained no new cartilage injury for at least about 10 weeks prior
to the time of the second blood sample.
[0059] The accuracy of correlating a level or a difference in level
of TGF.beta.rIII or CRTAC-1 biomarkers in one or more blood samples
obtained from an individual with the presence of a bone fracture or
cartilage injury or with the state of bone fracture or cartilage
injury in the individual will improve to the extent that the assays
used to measure levels of TGF.beta.rIII or CRTAC-1 biomarkers in
blood samples are exactly repeated or comparably as accurate as
those used to produce the data in Example 2 (infra) and FIG. 1 (for
TGF.beta.rIII) and FIG. 2 (for CRTAC-1). Furthermore, it will be
obvious to practitioners that when monitoring the level of
TGF.beta.rIII or CRTAC-1 biomarkers for determining the stage of
recovery from a bone or cartilage injury that the frequency of
measurements will have an impact on the accuracy of the method,
i.e., with more frequent blood samples taken closer together
increasing the ability to correctly determine the stage of an
injury in a patient or the proximity to an injury event. Yet
practitioners will also recognize that measurements of
TGF.beta.rIII and CRTAC-1 biomarkers in blood samples that are
taken over an extremely short period of time may undercut the
accuracy or significance of the methods described herein.
Therefore, a sufficient period of time must pass between time
points at which blood samples are taken to permit the detection of
a change in the level of a biomarker to become evident. Preferably,
blood samples employed in methods described herein are not taken
more frequently than one per day.
[0060] Any of a variety of means for the detection of TGF.beta.rIII
and/or CRTAC-1 in a sample of blood may be employed in the methods
and compositions described herein, including detection using liquid
chromatography and mass spectrometry. TGF.beta.rIII or CRTAC-1 may
also be detected in a sample of blood from an individual by
contacting the blood sample with a binding partner for
TGF.beta.rIII or a binding partner for CRTAC-1.
[0061] Most preferably, a binding partner used to detect
TGF.beta.rIII or CRTAC-1 is an antibody molecule. Antibodies may be
obtained commercially or generated by various methods known in the
art. An antibody may be a polyclonal antibody, a monoclonal
antibody, or a recombinant antibody molecule.
[0062] The use of an antibody molecules as the binding partner to
detect TGF.beta.rIII or CRTAC-1 is particularly advantageous as
antibodies may be employed in various formats and protocols known
in the art for the detection (immunodetection) and measurement
(quantitation) of a target antigen (TGF.beta.rIII or CRTAC-1) in a
sample. Such formats and protocols for the immunodetection or
quantitation of TGF.beta.rIII or CRTAC-1 in a sample of blood may
include, without limitation, enzyme linked immunoadsorbent assays
(ELISAs), immunoblots (e.g., Western blots), immunoprecipitations,
immunoaffinity chromatography, and dip sticks. In such formats and
protocols, antibodies may be immobilized on the surface of a solid
substrate, e.g., by adsorbing or linking the antibodies to the
surface of the substrate. Examples of immobilized antibodies on the
surface of a solid substrate may take any of a variety of forms
known in the art including, but not limited to, the surface of a
magnetic or chromatographic matrix particle, the surface of the
wells of a microtiter assay plate, and the surface of pieces or
sheets of a solid substrate material (e.g., plastic, nylon, wood,
cellulose, nitrocellulose, cellulose acetate, glass, cotton,
fiberglass, and the like). Pieces of solid substrate material
containing immobilized antibody adsorbed may be used as assay
strips (or dipsticks) that can be dipped into or otherwise brought
into contact with a blood sample either manually or robotically and
then removed to detect the presence of bound antigen.
[0063] Protocols in which an antibody or other binding partner are
adsorbed or linked to the surface of a substrate preferably include
the pretreatment of the antibody containing substrate with a
protein mixture (e.g., gelatin, bovine serum albumin, and the like)
to block undesired non-specific binding of molecules to the surface
of the substrate.
[0064] An immunocomplex formed by the binding of an antibody to its
cognate antigen may be directly detected by the presence of a
detectable label or tag molecule attached to the antibody or
indirectly detected by the use of another molecule, such as another
antibody, which can in turn be detected. Detectable labels for use
with antibodies are well known in the art and include, but are not
limited to, fluorescent labels, radioactive labels, biotin and
streptavidin (or avidin) based detection systems, bioluminescent
labels, chemiluminescent labels, and enzymes linked to an antibody
that are capable of reacting with colorigenic substrates to produce
a detectable signal. A signal generated by such systems may be
readily detected visually or by an appropriate instrument and in
some cases quantified, e.g., by fluorimetry, epifluorescence
microscopy, confocal scanning laser microscopy, a luminometer, or a
colorimetric assay. Robotic instruments are also available that
permit the reading of multiple samples with minimal human
intervention.
[0065] A sandwich assay is a type of indirect assay for an
immunocomplex. A sandwich assay may use a first antibody (the
capture antibody) that will bind to its cognate target antigen
(e.g., TGF.beta.rIII or CRTAC-1) in a sample to form an
immunocomplex. The sample containing the capture antibody may then
be reacted with a second antibody molecule (the detection antibody)
that can bind to an epitope on the capture antibody or to an
epitope that may be available on the antigen in the immunocomplex.
The detection antibody may carry a detectable label or component of
a signal generation system available in the art. By way of example
and without intending to limit the invention, a capture antibody
may be a murine IgG antibody to TGF.beta.rIII or CRTAC-1, and the
detection antibody may be a goat anti-murine IgG antibody that is
conjugated to a detectable fluorescent tag molecule.
[0066] The invention further contemplates a method for detecting or
measuring the level of TGF.beta.rIII and the level of CRTAC-1 in
peripheral blood of a human individual comprising the steps of
obtaining a sample of peripheral blood from the individual and
assaying the sample of blood for the level of TGF.beta.rIII and
CRTAC-1. Preferably, the level TGF.beta.rIII and CRTAC-1 in the
blood sample is determined by contacting the blood sample with a
binding partner for TGF.beta.rIII and a binding partner for
CRTAC-1. Depending on the format of the assay, the blood sample may
be brought into contact with each binding partner separately (i.e.,
in separate assays), consecutively in the same assay, or
simultaneously in the same assay.
[0067] Materials necessary for detection of TGF.beta.rIII or
CRTAC-1 in a sample of blood (or plasma or serum) are conveniently
assembled into a kit, so that personnel treating or transporting a
trauma victim can determine quickly whether a bone fracture has
been sustained by a patient. A preferred kit of the invention
comprises a first (capture) binding partner(s) for either or each
of TGF.beta.rIII or CRTAC-1 immobilized on a solid substrate
material, such as an anti-TGF.beta.rIII or anti-CRTAC-1 antibody
immobilized on an assay strip, the wells of a microtiter plate, or
on beads or particles; a second (capture) binding partner that will
bind the first binding partner and that contains a detectable label
or component to produce a detectable signal; and instructions that
indicate how to use the kit to carry out the assay to detect either
or both TGF.beta.rIII and CRTAC-1 in a peripheral blood sample.
Beads, assay strips, or microtiter plates containing immobilized
first binding partner molecules in kits of the invention may be
packaged in a variety conditions, including a dry, unhydrated
state; a freeze-dried or dehydrated state; or a hydrated state in a
physiological buffer solution. Kits may also contain a device for
obtaining a sample of blood from an individual (e.g., a syringe or
small pin to obtain a few drops of blood). Kits may also contain
other solutions for washing, for blocking non-specific binding, or
for signal generation may also be included in the kits of the
invention. In a preferred embodiment, a kit of the invention
comprises capture binding partner(s) immobilized on a solid
substrate, such as a bead, an assay strip or a microtiter plate,
which has also been pre-treated to prevent interference by
non-specific binding of molecules to the substrate.
[0068] The methods and compositions described herein may find use
in rapid diagnosis for bone or cartilage injury by emergency and
medical personnel or in the periodic monitoring of the condition of
a bone fracture or cartilage injury. The nature of the methods and
compositions described herein makes it possible to perform
diagnosis and monitoring of bone and cartilage injuries in a
variety of environments, including ambulances or other mobile
medical facilities, laboratories, hospitals, emergency rooms,
sanitoria, homes, and other private facilities.
[0069] Additional embodiments and features of the invention will be
apparent from the following non-limiting examples.
EXAMPLES
Example 1
Identification of Candidate Protein Biomarkers for Bone and
Cartilage Metabolism in Plasma from Human Patients with Long Bone
Fractures
[0070] In this study, samples of blood were drawn from human
patients with an acute bone fracture and analyzed for expression of
candidate biomarkers for bone fracture and fracture healing. The
plasma proteins of patients were characterized by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and affinity
purification, followed by tandem mass spectrometry liquid
chromatography (LC-MS/MS). LC-MS/MS provides picomolar level of
detection of proteins expressed in the plasma samples. Following
identification of proteins expressed in the plasma of the fracture
patients, those species that are associated with bone and cartilage
metabolism were singled out for further analysis.
Materials and Methods
Plasma Collection
[0071] Human blood plasma samples were supplied by the Clinic of
Traumatology in Zagreb, Croatia. The approval for the collecting
samples was obtained from the Ethics Committee of the same
institution. Blood samples from 25 adult humans with a single long
bone fracture (21-60 years of age) were drawn into syringes
containing 3.8% sodium citrate to form an anticoagulant-to-blood
ratio (v/v) 1:9. Plasma was obtained by centrifugation (15 min at
3000.times.g), and aliquots of each adult blood sample were pooled
for the further analysis. Aliquot samples were stored at
-80.degree. C. until used.
Affinity Column Purification
[0072] Pooled plasma of patients with a single-bone fracture (80
ml) was diluted two-fold with 10 mM sodium phosphate buffer (pH 7),
and applied to a column of heparin Sepharose affinity
chromatography matrix (Amersham Pharmacia Biotech), previously
equilibrated with 10 mM sodium phosphate buffer (pH 7). Bound
proteins were eluted from the column with 10 mM sodium phosphate
buffer (pH 7) containing 1 M and 2 M NaCl. Eluted fractions were
precipitated with saturated ammonium sulfate (SAS) to final
concentration of 35%.
SDS-PAGE and In-Gel Digestion
[0073] Samples were run on a NUPAGE.RTM. 10% Bis-Tris SDS-PAGE
system (Invitrogen, Carlsbad, Calif.) using MOPS SDS buffer system,
and subsequently stained with Coomassie staining kit (NuPAGE,
Invitrogen) following manufacturer's instructions. After staining,
each of the seven gel lanes was sliced into 12 pieces, and the
corresponding pieces were combined. The pieces were then subjected
to in-gel reduction, alkylation, and trypsin digestion as described
previously (Grgurevic et al., J. Nephrol., 20: 311-319 (2007)). Gel
pieces were washed two times with acetonitrile/25 mM
NH.sub.4HCO.sub.3, reduced by incubation with 10 mM dithiothreitol
(DTT) for 45 minutes at 56.degree. C., and carboxyamidomethylated
by incubation in 55 mM iodoacetamide for 45 minutes at room
temperature. Trypsin (Promega) was added to dried gel pieces (150
ng per piece, diluted in 25 mM NH.sub.4HCO.sub.3) and incubated
overnight at 37.degree. C. Tryptic peptides were extracted with
formic acid/acetonitrile/H.sub.2O (10:20:70); and 100%
acetonitrile, dried and resuspended in trifluoroacetic
acid/acetonitrile/H.sub.2O (1:2:97) for MS analysis.
Mass Spectrometry
[0074] Tryptic peptides were analyzed by liquid chromatography-mass
spectrometry (LC-MS). An Agilent 1100 nanoflow HPLC system (Agilent
Technologies) was coupled to a LTQ-Orbitrap mass spectrometer
(Thermo Scientific) using a nano-electrospray LC-MS interface
(Proxeon Biosystems). Peptides were loaded on a home-made 75 .mu.m
C.sub.18 HPLC column in solvent "A" (0.5% acetic acid in Milli-Q
water) and eluted with a 70-minute segmented linear gradient of
10%-60% solvent "B" (80% acetonitrile, 0.5% acetic acid in Milli-Q
water) at a flow rate of ca. 250 nL/min. Mass spectrometer was
operated in the positive ion mode. Each measurement cycle consisted
of a full MS scan acquired in the orbitrap analyzer at a resolution
of 60000, and MS/MS fragmentation of the five most-intense ions in
the linear ion trap analyzer. To further improve mass accuracy, the
lock-mass option was used as described previously (Olsen et al.,
Mol. Cell. Proteomics, 4: 2010-2021 (2005)). This resulted in a
typical peptide average absolute mass accuracy of less than 1
ppm.
[0075] Peak lists were generated using in-house developed software
(Raw2 msm) (Olsen et al., 2005), and searched against concatenated
forward and reverse ("decoy") IPI human database (version 3.13)
using Mascot search engine (Matrix Science). Searches were done
with trypsin specificity (2 missed cleavages allowed),
carboxyamidomethylation as fixed modification, and oxidized
methionine as variable modification. Precursor ion and fragment ion
mass tolerances were 10 ppm and 0.5 Da, respectively. Results of
the database search were validated in the MSQuant software
(available from SOURECEFORGE.NET.RTM.). Only peptides with a mass
deviation lower than 5 ppm were accepted; two peptides were
required for protein identification. Gene ontology (GO) analysis
was performed using ProteinCenter software package (Proxeon
Biosystems).
Results
[0076] Pooled plasma samples were subjected to heparin affinity
chromatography to enrich for proteins specific for bone and
cartilage, many of which are known to have heparin binding domains.
This also partially removed highly abundant plasma proteins, such
as albumin, immunoglobulins, transferin, and haptoglobulin.
Fractions of interest were collected, precipitated, with ammonium
sulfate and separated on one dimensional SDS-PAGE gels. Gel bands
were excised, digested with trypsin, and analyzed by LC-MS/MS.
Peptide fragmentation spectra were searched against the human IPI
protein database, and the results of the database search were
validated using MSQuant software. Only peptides with a mass
deviation lower than 5 ppm were accepted; two peptides were
required for protein identification, which led to an overall
false-positive rate of less than 1% at both the peptide and the
protein level.
[0077] In total, two hundred and thirteen nonredundant proteins
were identified in the in-gel analysis of pooled plasma proteins
from 25 patients with a bone fracture. Gene ontology (GO) analysis
of plasma proteins showed that a majority (63.8%) of detected
proteins were of extracellular origin, whereas only a small number
(7.5%) were of intracellular (cytosol and nucleus) origin.
Interestingly, a relatively high number (35.2%) of membrane related
proteins were also detected.
[0078] According to molecular function analysis, 37.6% of detected
proteins had catalytic properties, 18.3% were classified as signal
transducers, and 13.1% as transporters.
[0079] In terms of biological activity, a significant proportion of
detected proteins were involved in cell growth and proliferation
(21.1%), transport (23.9%) and coagulation (13.1%).
Identification of Bone- and Cartilage-Related Proteins
[0080] From the proteins initially identified by the methodology
described above in the pooled plasma samples of individuals with a
long bone fracture, the twelve proteins listed in Table 1, below,
were considered as having possible involvement in bone and
cartilage metabolism.
TABLE-US-00001 TABLE 1 Proteins in Plasma of Bone Fracture Patients
Related to Bone and Cartilage Formation Previously GO console:
Identified in Protein IPI Accession No. Molecular Function Plasma
transforming growth 304865.3 receptor activity No factor beta
receptor III signal transducer (TGF.beta.rIII) splice isoform 1 of
451624.1 metal ion binding No cartilage acidic protein 1 precursor
(CRTAC-1) extracellular matrix 3351.2 signal transducer Yes protein
1 precursor structural molecule transporter activity transforming
growth 18219.1 protein binding No factor beta induced protein IG-H3
precursor (TGF.beta. IG-H3) splice isoform 2 of 220701.3 enzyme
regulator activity Yes collagen alpha 3 (VI) protein binding chain
precursor structural molecule type IV collagenase 27780.1 catalytic
activity Yes precursor enzyme regulator activity metal ion binding
alpha 3 type VI collagen 22200.2 enzyme regulator activity No
isoform 1 precursor protein binding structural molecule procollagen
C proteinase 299738.1 nucleic acid binding No enhancer protein
protein binding precursor isoform long of collagen 22822.4 metal
ion binding Yes alpha-1 (XVIII) chain protein binding precursor
structural molecule hyaluron binding protein 41065.3 catalytic
activity Yes 2 precursor metalloproteinase 32292.1 catalytic
activity Yes inhibitor 1 precursor enzyme regulator metal ion
binding splice isoform A of 24825.2 not known Yes proteoglycan-4
precursor
[0081] As noted in Table 1, above, among the twelve proteins
considered to be involved in bone and cartilage metabolism, five
were not previously identified in plasma.
[0082] Cartilage acidic protein 1 (CRTAC-1) was identified for the
first time in plasma with 28 peptides and an average peptide Mascot
score of 53.
[0083] Transforming growth factor beta receptor III (TGF.beta.rIII)
was identified for the first time in plasma with four specific
peptides and an average Mascot score of 44.
[0084] Transforming growth factor beta induced protein IG-H3
(TGF.beta.-IG-H3) was also identified for the first time in plasma
with 20 peptides and an average peptide Mascot score of 57.
[0085] Among extracellular matrix proteins which were not
previously detected in plasma was the alpha 3 type VI collagen
isoform 1 identified with two peptides and an average peptide
Mascot score of 60.
[0086] Previously identified in plasma, the splice isoform A of the
proteoglycan-4 (or lubricin) was identified with two peptides and
an average peptide Mascot score of 60.
[0087] Extracellular matrix proteins previously identified in
plasma included: isoform long of collagen alpha-1 (XVIII) chain
precursor (or endostatin) identified with five peptides and an
average Mascot score of 36, splice isoform 2 of collagen alpha 3
(VI) chain precursor identified with ten peptides and an average
Mascot score of 62, extracellular matrix protein 1 precursor
identified with 57 peptides and an average Mascot score of 54, and
type IV collagenase precursor (or matrix metalloproteinase-2, MMP2)
identified with three peptides and an average Mascot score of 74.
MMP-2 degrades extra-cellular proteins and disrupts the
subendothelial basement membrane, thus enabling the transmigration
of inflammatory cells. Metalloproteinase inhibitor 1 precursor
(TIMP-1) was identified with five peptides and an average peptide
Mascot score of 49.
Example 2
Monitoring Plasma Levels of Transforming Growth Factor .beta.
Receptor III (TGF.beta.rIII) as Blood Biomarker for Bone Fracture
and Cartilage Acidic Protein 1 (CRTAC-1) as Blood Biomarker for
Cartilage Injury
[0088] In this study, the blood of human patients who sustained an
acute bone fracture was monitored for the presence of transforming
growth factor .beta. receptor III (TGF.beta.rIII) and cartilage
acidic protein 1 (CRTAC-1).
Material and Methods
Patients
[0089] Within 24 hours of injury, thirty (30) patients (24-67 years
of age) who sustained a fracture of the tibia were enlisted in this
study. All patients gave written informed consent, and the study
procedures were in accordance with the Ethics Committee of Clinics
of Traumatology of the Medical School of Zagreb. The criterion for
inclusion in the study was that a patient had a radiologically
confirmed fracture in the middle shaft or in the distal portion of
the tibia. An additional eight (8) patients had a fracture in the
distal portion of the tibia that extended into the ankle joint with
a visible dislocation of the joint and damage to the cartilage
layer on the surface of the calcanear bone and/or distal tibial
joint surface as diagnosed by magnetic resonance imaging (MRI).
Venous Blood Samples
[0090] From all included patients, peripheral venous blood was
drawn at periodic intervals according to a standardized time
pattern at day 1, 3, and 7 following injury, and then at 2, 6, 10,
14, 18, and 24 weeks following fracture. If the fracture healing
was delayed, the blood samples were collected periodically until
bony consolidation was achieved. Blood was drawn into syringes
containing 3.8% sodium citrate to form an anticoagulant-to-blood
ratio (v/v) 1:9. Plasma was obtained by centrifugation (15 min at 3
000.times.g), and aliquots of plasma samples were stored at
-80.degree. C. until used.
Radiological Evaluation
[0091] Physical examinations and radiographs were completed to
assess the evidence of a bone union. At 24 weeks after injury,
fractures were pronounced as healed or as non-union by two
independent radiologists. All patients underwent surgery to insert
an interlocking nail into the fracture. X-rays were taken
pre-operatively, immediately postoperatively, and then at regular
bi-weekly intervals up to 24 weeks following surgery. X-rays were
taken in two positions, i.e., an anterio-posterior view and a
latero-lateral view. A fracture was pronounced as healed when all
four cortices healed. However, partial healing was graded when one,
two, or three cortices rebridged. Additional injury of the
calcanear or distal tibial joint cartilage was confirmed by
MRI.
Measurement of TGF.beta.rIII and CRTAC-1 in Blood Plasma
[0092] An enzyme-linked immunoadsorbent assay (ELISA) was developed
to measure blood concentrations of TGF.beta.rIII and CRTAC-1 in
plasma from patient throughout the follow-up (24 week) period.
Polyclonal antibodies were raised in rabbits immunized with
specific human peptides of TGF.beta.rIII and CRTAC-1 by standard
methods. A monoclonal antibody against TGF.beta.rIII was purchased
from Santa Cruz (A-4: sc-74511-mouse monoclonal antibody). All
samples from 30 patients were measured twice, and a mean value was
then included in the final median range.
[0093] The ELISA for TGF.beta.rIII specifically detects the
biologically active soluble form of the TGF.beta.rIII in human
plasma with a sensitivity of 10 pg/ml. The minimal detectable dose
of TGF.beta.rIII ranged from 5.5 to 35 pg/ml.
[0094] The ELISA for CRTAC-1 in human plasma provided measurable
levels of CRTAC-1 within a range of 10.5 to 55 pg/ml.
Formation of Cross-Linked Antibody-Protein G Complex and
Immunoprecipitation
[0095] Rabbit polycloncal antibody (Genera Research Laboratory)
against the soluble form of TGF.beta.rIII or against CRTAC-1 was
incubated with protein G agarose beads for 15 minutes on a shaker.
The antibody-protein G bead samples were centrifuged for 2 minutes
on 12,000.times.g, and the supernatants removed. Formalin (500
.mu.l of 4% formalin) was then added to the pellet and incubated
for another 30 minutes on the shaker. The samples were centrifuged
for 2 minutes on 12,000.times.g, and the supernatants removed. The
resulting pellets (antibody cross-linked to protein G beads) were
resuspended in a phosphate-buffered saline (PBS) and added to
collected plasma samples for immunoprecipitation of cognate
antigen, i.e., TGF.beta.rIII or CRTAC-1.
[0096] The mixtures of plasma samples and antibody cross-linked
beads were incubated overnight to allow formation of
immunocomplexes between antigen (TGF.beta.rIII or CRTAC-1) in the
plasma samples and the antibody cross-linked beads. The samples
were then centrifuged for 2 minutes on 12,000.times.g to obtain
pellets comprising immunocomplexes formed between TGF.beta.rIII or
CRTAC-1 and antibody cross-linked beads. The supernatants were
removed, and the pellets were washed three times with a
phosphate-buffered saline and prepared for gel electrophoresis.
Gel Electrophoresis and Western Immunoblotting
[0097] Aliquots of samples were analyzed by electrophoresis and
immunoblotting in a Novex mini-gel electrophoresis system. Gel
electrophoresis sample buffer was added to each pellet. The samples
were denatured by heating at 99.degree. C. for 3 minutes followed
by centrifugation for 2 minutes on 12,000.times.g. Supernatants
were then analyzed on a 10% polyacrylamide/SDS gel (Invitrogen).
After electrophoresis, proteins in the gels were transferred by
electroblotting to nitrocellulose membranes and incubated first
with rabbit antibody against TGF.beta.rIII and rabbit antibody
against CRTAC-1. The bound antibodies were detected with alkaline
phosphatase-conjugated anti-rabbit IgG immunoglobulin
(immunodetection kit, Invitrogen).
Results
[0098] Physical examinations and radiographs were completed to
assess the evidence of a bone union in patients. At 24 weeks after
injury, 26 fractures were pronounced as healed by two independent
radiologists, while four (4) patients had non-union fractures. Six
patients had an additional injury of the joint cartilage based on
MRI analysis.
Reference Values
[0099] The postoperative TGF.beta.rIII reference level in plasma of
patients with normal fracture healing was 30.6.+-.7.5 ng/ml (15-47
ng/ml). The level of TGF.beta.rIII in plasma of patients with
delayed union was 32.4.+-.8.2 ng/ml (18-52 ng/ml) without a
significant difference (P=0.861).
[0100] The plasma concentration of CRTAC-1 in patients with normal
fracture healing was in the range of 50.4.+-.9.1 ng/ml. In patients
with a delayed union fracture, the value was 51.2.+-.5.3 ng/ml.
Plasma concentrations of CRTAC-1 in patients with an additional
joint cartilage injury was 83.4.+-.7.8 ng/ml (67-112 ng/ml), which
was significantly higher than in patients without a joint cartilage
injury (P<0.01, Wilcoxon test).
Time Courses
[0101] Levels of transforming growth factor .beta. receptor III
(TGF.beta.rIII) in plasma of patients with a bone fracture that
healed ("union") and in plasma of patients with a bone fracture
that did not heal ("delayed union") over the course of 24 weeks
following injury are shown in Table 2, below.
TABLE-US-00002 TABLE 2 Plasma Levels of TGF.beta.rIII in Patients
with Bone Fracture Week After TGF.beta.rIII TGF.beta.rIII Injury
(union*, ng/ml) (delayed union.sup..dagger-dbl., ng/ml) 1 32.3 31 2
45.4 40 4 28 20 6 26 15 10 32.5 15.5 14 33.4 17 18 32.7 17.8 24
33.1 21 *"union" refers to patients with a bone fracture that
healed within 24 weeks of injury; .sup..dagger-dbl."delayed union"
refers to patients with a bone fracture that did not heal within 24
weeks of injury
[0102] As can be seen from a graph of the data in Table 2 (FIG. 1),
in patients with normal bone fracture healing ("union") as well as
in patients with delayed bone fracture healing ("delayed union"),
TGF.beta.rIII plasma concentrations reached their highest values at
week 2 following injury. After the second week, TGF.beta.rIII
plasma levels declined in patients with delayed union, and at 5
weeks following injury, TGF.beta.rIII concentrations were below the
reference level of this group of patients. In patients with a
normal fracture healing ("union" in FIG. 1), TGF.beta.rIII plasma
concentrations also decreased after the second week following
injury. However, the plasma value did not fall below the reference
level, and slightly increased at 10 weeks following injury.
Thereafter, the level did not change towards the end of the follow
up period. See, FIG. 1. These data indicate that TGF.beta.rIII is
particularly useful as a blood biomarker for detecting bone
fracture.
[0103] With respect to the quantitative levels of TGF.beta.rIII in
the blood samples of the patients, the data in FIG. 1 indicate that
if the level of TGF.beta.rIII falls below 20 ng/ml after week 4,
then there is a possibility that a delayed union or non-union
fracture has developed. The data in FIG. 1 also indicate that the
maintenance of blood levels of TGF.beta.rIII above 25 ng/ml after
week 4 reflects a normal healing process as confirmed by X-rays and
clinical exam and a high probability that the bone will fully
regenerate by week 24.
[0104] Table 3, below, shows the levels of cartilage acidic protein
1 (CRTAC-1) in plasma of patients with a bone fracture that healed
("union"), in plasma of patients with a bone fracture that did not
heal ("delayed union"), and in plasma of patients that sustained a
bone fracture and an articular cartilage injury ("cartilage
damage") over the course of 24 weeks following injury.
TABLE-US-00003 TABLE 3 Plasma Levels of CRTAC-1 in Patients with
Bone Fractures and Cartilage Injuries Week Plasma CRTAC-1 Plasma
CRTAC-1 CRTAC-1 After (union*, (delayed nion.sup..dagger-dbl.,
(cartilage damage.sup..dagger., Injury ng/ml) ng/ml) ng/ml) 1 42 46
78 2 55 53 97 4 58 55 100 6 56 50 98 10 54 48 80 14 55 49 78 18 50
47 76 24 48 46 76 *"union" refers to patients with bone fracture
that healed within 24 weeks of injury; .sup..dagger-dbl."delayed
union" refers to patients with bone fracture did not heal within 24
weeks of injury; .sup..dagger."cartilage damage" refers to patients
that sustained bone fracture and cartilage injury
[0105] As can be seen from a graph of the data in Table 3 (FIG. 2),
in patients that sustained a bone fracture and also damage to
articular cartilage ("cartilage damage"), the concentration of
CRTAC-1 in the blood rose within a week of injury and persisted at
a level that was clearly higher than the level of CRTAC-1 in the
blood of patients that sustained a bone fracture without articular
cartilage damage, whether the fracture healed ("union") or did not
heal ("delayed union") within 24 weeks following injury. These data
indicate that CRTAC-1 is useful as a blood biomarker for cartilage
injury.
[0106] All publications, patent applications, patents, and other
documents cited herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0107] Other variations and embodiments of the invention described
herein will now be apparent to those skilled in the art, and all
such variants and alternative embodiments of the invention are
intended to be encompassed within the foregoing description and the
claims that follow.
* * * * *