WO 2006/116167 PCT/US2006/015211
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METHOD FOR DETERMINATION OF GLYCOPROTEIN IBALPHA (GPIBALPHA) PROTEIN
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to provisional U.S. Application Serial No.
60/673,926 filed on April 22,2005, which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention is in the field of biochemical assay systems, particularly for the
measuring of proteins. More specifically, the invention relates to the detection and
quantification of glycoprotein Iba (GPIb) protein.
BACKGROUND OF THE INVENTION
[0003] In vascular biology, platelet function is the corner stone for proper hemostasis and
thrombosis. Platelets contribute to maintaining the normal circulation of blood through the
preservation of vascular integrity and the control of hemorrhage following injury (Ruggeri, J.
Clin. Invest. 99:559-564(1997)). Although the formation of the platelet plug is a defense
mechanism required for survival, it may also contribute to diseases such as myocardial
infarction, especially in an atherosclerotic microenvironment (Fuster, N. Engl. J. Med. 326:242-
250(1992)). Moreover, one of the leading causes of morbidity and mortality in developed
nations is acute thrombotic arterial occlusion (Ruggeri, J. Clin. Invest. 99:4559-564(1997)). This
underscores the relevance of studies focused on unraveling the mechanism of platelet response to
vascular injury, as well as on commercial means to detect components of this complicated
pathway.
[0004] Important in the mechanism for platelet response is its receptor complex, glycoprotein
(GP)Ib/IX/V. This platelet receptor binds directly to von Willerbrand Factor (vWF) which forms
the bridge to the damaged blood vessel wall (Miura, J. Biol. Chem. 275:7539-7546(2000)). This
effect is triggered by vWF in association with the subendothelial matrix and is modulated by
shear stress provided by blood flow in the microvasculature (Turitto, Blood 65:823-829(1985)).

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This event results in further interaction between the collagen receptors from the damaged vessel
wall and the platelets, resulting in platelet activation, platelet aggregation and formation of the
hemostatic plug which seals the endothelial lesion and prevents blood leakage (Girma, Blood
70:605-615(1987)).
[0005] The platelet receptor, GPIb/IX/V, which binds to the vWF-collagen matrix, is
composed of four subunits, GPIb, GPIb GPIX and GPV (Modderman, J. Biol. Chem.
267:364-369(1992)). The most important of these subunits, based on its functionality and size, is
the 150-kDa GPIb chain (Uff, J. Biol. Chem. 277:35657-35663(2002)). GPIb is responsible
for the initial adhesion to vWF by binding to various sites on the Al-domain of vWF. Mutations
in GPIba can result in bleeding disorders, Bernard Soulier syndrome (BSS) and vWF
Willerbrand disease (Pt-vWD). While vWF is the main ligand for GPIba, other proteins have
been identified which bind to this glycoprotein, including thrombin, kininogens, Factor XI,
Factor XII, P-selectin and Mac-1 (Uff, J. Biol. Chem. 277:35657-35663(2002)).
[0006] The biological importance of GPIba is evident, and therefore the need for simple,
deterministic bioassays to identify and quantify GPIba becomes apparent. Currently available
bioassays to quantify GPIba are time consuming, laborious, limited to certain isoforms, and
costly to operate. In vivo methods include the Rat Tail Vein Bleeding Model and the Canine
Folts' Animal Model, while the Ristocetin-induced platelet aggregation assay (RIPA) makes up
the current available in vitro assay (Folts, Circulation 83:IV3-IV14 (1991), Dejana, Thromb.
Haemost. 48:108-111(1982), Weiss J. Clin. Invest 52:2708-16(1973)). These assays are
associated with high background noise, limited sensitivity, high cost, intensive labor
requirements, and provide for significant inter-assay variability. Therefore, a need exists for an
assay system that can accurately and rapidly detect and quantify a wide range of levels of GPIba
in several biological sample types. More specifically, methods that could accurately and
efficiently measure GPIba in research laboratory settings, as well as in the clinical and
diagnostic arenas. A novel method to detect the biological activity of GPIba with simplicity,
accuracy and with a high degree of reproducibility is highly desirable.
[0007] The present invention satisfies these needs and provides related advantages as well.
Additional objects and advantages of the invention will be set forth in part in the description

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which follows, and in part will be obvious from the description, or may be learned by practice of
the invention. The objects and advantages of the invention will be realized and attained by
means of the elements and combinations particularly pointed out in the appended claims.
[0008] It is to be understood that both the foregoing general description and the following
detailed description are exemplary and explanatory only and are not restrictive of the invention,
as claimed.
BRIEF DESCRIPTION OF THE INVENTION
[0009] The importance of GPIb in vascular biology is well recognized and yet there exists
no simple, efficient test or diagnostic assay to detect it or measure its bioactivity. The invention
described herein relates to the development of an efficient, reproducible, and inexpensive assay
method for detecting and quantifying the bioactivity of GPIb. The invention has applications in
both the clinical and research settings. The invention also allows for the sensitive and specific
discrimination of different isoforms of GPIb and, therefore, may be extremely useful in the
performance of quality control and the monitoring of GPIb production levels. The sensitivity
and specificity of the instant assay includes, but is not limited to, the determination of possible
contaminating isoforms in a GPIb production and purification process, allowing for the ability
to distinguish between the active GPIb-Fc fusion protein and the non-active one.
[0010] The present invention is directed to an assay method of determining the presence of
GPIb in a biological sample comprising: (a) providing a substance comprising GPIb;
(b) contacting the substance from step (a) with a binding protein which binds to GPIb; (c)
adding a detection compound specific to GPIb; (d) adding a complexing compound that binds
the binding protein from step (b); and (e) detecting the detection compound from step (c)
wherein a positive detection signal indicates the presence of GPIb in the biological sample.
[0011] In another embodiment, the present invention is directed to an assay method of
detecting the protein concentration of GPIb in a biological sample comprising: (a) providing a
substance comprising GPIb; (b) contacting the substance from step (a) with a binding protein
which binds to GPIb; (c) adding a detection compound specific to GPIb; (d) adding a
complexing compound that binds the binding protein from step (b); and (e) detecting the

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detection compound from step (c) wherein a detection signal compared to a known standard
curve indicates the protein concentration of GPIbα in the biological sample.
[0012] In yet another embodiment, the present invention is directed to an assay method of
detecting the binding activity of GPIbα in a biological sample comprising: (a) providing a
substance comprising GPIbα; (b) contacting the substance from step (a) with a binding protein
which binds to GPIbα; (c) adding a detection compound specific to GPIbα; (d) adding a
complexing compound that binds the binding protein from step (b); (e) detecting the detection
compound from step (c) wherein a detection signal compared to a known standard curve
indicates the protein concentration of GPIbα in the biological sample; and (f) calculating the
binding activity of GPIbα.
[0013] In still another embodiment, the present invention is directed to an assay method of
detecting an isoform of GPIbα by measuring the binding activity of the isoform of GPIbα in a
biological sample comprising: (a) providing a substance comprising GPIbαand GPIbα-like
substances; (b) contacting the substance from step (a) with a binding protein which binds to
GPIbα; (c) adding a detection compound specific to GPIbα, (d) adding a complexing compound
that binds the binding protein from step (b); (e) detecting the detection compound from step (c)
wherein a detection signal compared to a known standard curve indicates the protein
concentration of GPIbα in the biological sample; (f) calculating the binding activity of an
isoform of GPIbα; and (g) comparing the binding activity of the isoform to the binding activity
of a known GPIbα control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The accompanying drawings, which are incorporated in and constitute part of this
specification, and together with the description, serve to explain the principles of the invention.
[0015] Figure 1 is a schematic representation of the GPIbα binding assay format. Biotin
vWF, GPIbα - Fc fusion protein and BV-tagged anti-human Fc antibodies were first mixed and
incubated for 2 hours at room temperature. Following the incubation, streptavidin (SA) beads
were added into the mixture and were incubated for an additional 30 minutes.

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[0016] Figure 2 is a schematic representation of the GPIbα binding region on vWF.
[0017] Figure 3 is a graphic representation of a standard curve for the GPIbα-vWF binding
assay. The signal to background (S/B) ratio for the assay is close to 10, while the S/B of the
ELISA GPIbα binding assay is at its maximum, 2, with 1 log of linearity.
[0018] Figure 4 is a graphic representation of a standard curve for the GPIbα-Al binding assay un
[0019] Figure 5 is a graphic representation of the binding activities of the product variants.
WT corresponds to wild-type GPIbα. VI, V2, and V3 are the gain-of-function variants of
GPIbα. According to in vivo experimental results, the gain-of-function variants had increasing
ability to bind GPlb. In vWF binding assay, the differences between V3, V2, VI, and WT are
readily observable, while in the Al binding assay, the differences between the variants were very
limited.
[0020] Figure 6 is a graphic representation of the binding activity of four different low
molecular weight (LMW) isoforms on the vWF binding assay. The control is an uncleaved
molecule of GPIbα. Clip, Clip 1-276, Clip 1-282 and Clip Fc represent different cleaved
isoforms of GPIbα.
[0021] Figure 7A is a graphic representation of the stability test of GPIbα samples. Bulk
drug substance (BDS) of GPIbα was stored at 4°C and the percentage of high molecular weight
(HMW) was monitored as a function of time.
[0022] Figure 7B is a graphic representation of the stability test of GPIbα samples. Bulk
drug substance (BDS) of GPIbα was stored at 4°C and the percentage of binding activity was
monitored as a function of time.
[0023] Figure 8 is a graphic representation of a comparison of in vitro and in vivo data from
the rat rail vein responding time assay. Test samples include untreated, animal control for in vivo
test; LMW, low molecular weight of GPIbα (typical clip); loading, the control sample prior to
anionic exchange (AEX) column separation; full sulfation, GPIbα with all sulfation sites
sulfated; and 0 sulfation, GPIbα with no sulfation sites sulfated.

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[0024] Figure 9 is a graphic representation of a comparison of in vitro and in vivo data from
the canine folts' animal model. Test samples include untreated, animal control for in vivo test;
Monomer, intact GPIbα without cleavage; LMW1 and LMW2, low molecular weight fractions
of GPIbα from AEX column separation; full sulfation, GPIbα with all sulfation sites sulfated;
and 0 sulfation, GPIbα with no sulfation sites sulfated.
[0025] Figure 10 is a graphic representation of sulfation isoforms separated on AEX-HPLC.
[0026] Figure 11 is a graphic representation of the isoforms of GPIbα separated by size on
SEC-HPLC.
[0027] Figure 12 is a graphic representation of the reproducibility and the percent calculated
variance of the GPIbα assay.
[0028] Figure 13A is a graphic representation of the binding specificity of the GPIbα assay
versus a control (Fc prtn 1).
[0029] Figure 13B is a graphic representation of the binding specificity of the GPIbα assay
versus controls (Fc prtn 2 and Fc prtn 3).
DETAILED DESCRIPTION OF THE INVENTION
[0030] Unless otherwise defined, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
belongs. Although methods and materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, suitable methods and materials are
described below. All publications, patent applications, patents, and other references mentioned
herein are incorporated by reference in their entirety. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting. Other features and advantages of
the invention will be apparent from the detailed description, drawings, and from the claims.

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Abbreviations and Definitions
[0031] The following abbreviations are used herein: "ATCC" means American Type Culture
Collection, "vWF" means von Willerbrand Factor, "GPIbα" means glycoprotein 1 b-alpha,
"CHO" means Chinese Hamster Ovary, "NIH 3T3" means National Institute of Health 3T3.
[0032] As used herein, the term "antibody" means, without limitation, a polyclonal antibody,
a monoclonal antibody, a chimeric antibody, a humanized antibody, a genetically engineered
antibody, a bispecific antibody, antibody fragments and single chains representing the reactive
portions of the antibody. Methods of production of each of the above mentioned antibody forms
are well known in the art.
[0033] As used herein, the term "biological sample" means, without limitation, any cell,
prokaryote or eukaryote, any tissue or organ, or any product of recombinant technology or
genetic engineering thereof. The "biological sample" may also be a plasma sample, a cell-
culture supernatant or a buffer from a purification process. In the case for plasma, the source can
be from any mammal, including but not limited to monkey, mouse, rat, rabbit, guinea pig, gerbil,
pig, dog, horse and human. The plasma is the portion of the whole blood which comprises the
soluble proteins. Alternatively, the assay can be conducted on whole blood without having
separated out the plasma. Cell-culture supernatants can be isolated from any cell culture line
which expresses GPIbα. Cell lines can be selected from CHO cell lines, NIH-3T3 cell lines or
any cell line obtained from the ATCC, any of which has been manipulated to express GPIbα.
[0034] As used herein, the term "isoform" means, without limitation, low molecular weight
(LMW) GPIbα, high molecular weight (HMW) GPIbα (see Figure 11), other variants forms (VI,
V2, V3) and small molecules of GPIbα including, but not limited to, fully-sulfated or partially-
sulfated GPIbα proteins (see Figure 10).

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Description
[0035] The present invention is a method to specifically and sensitively detect and quantify
the presence of GPIbα or a GPIbα-like contaminant in a biological sample. In one embodiment,
the assay comprises: (a) providing a substance comprising GPIbα; (b) contacting the substance
from step (a) with a binding protein which binds to GPIbα; (c) adding a detection compound
specific to GPIbα; (d) adding a complexing compound that binds the binding protein from step
(b); and (e) detecting the detection compound from step (c) wherein a positive detection signal
indicates the presence of GPIbα in the biological sample.
[0036] In one embodiment, the biological substance comprising GPIbα can be plasma,
supernatant from a cell line or a buffer. In another embodiment, the plasma can originate from a
mammal, selected from a monkey, mouse, rat, rabbit, guinea pig, dog, horse or human. The
supernatant can be from a CHO cell line, an NTH 3T3 cell line or a cell line obtained from the
ATCC.
[0037] In yet another embodiment, the detection compound specific to GPIbα is an antibody
or more preferably, a Fab fragment of the antibody. In another embodiment, the binding protein
specific to GPIbα is a protein with an active binding site different from that of the antibody
above. In another embodiment, the binding protein specific to GPIbα is a fragment of vWF,
such as the Al Domain of vWF. This fragment can be a recombinant form of the Al Domain of
vWF. Alternatively, the binding protein specific to GPIbα is the complete vWF protein. In
another aspect, the binding protein specific to GPIbα is biotinylated or His-tagged.
[0038] In still another embodiment, the complexing compound that binds the binding protein
in step (d) is streptavidin-coated magnetic beads or anti-His coated magnetic beads.
[0039] Another embodiment is directed at the detection compound specific to GPIbα that is
labeled with a chemiluminescent substance. More specifically, the Fab fragment of the antibody
specific to GPIbα is labeled with a chemiluminescent substance.
[0040] In another aspect, the detection of the detection compound that binds to the binding
protein in step (e) further comprises exposing a chemiluminescent substance to light and

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measuring the excitation of the chemiluminescent substance which correlates with the presence
ofGPIbα.
[0041] Streptavidin-coated magnetic beads, a further embodiment of the present invention,
are placed in contact with biotinylated vWF, which will bind to the GPIbα in the analyte or
biological sample (Figure 1). An antibody, tagged with a chemiluminescent substance, and
specific for a separate antigenic site on GPIbα is contacted to the biotinylated vWF for a period
of 2 hours (Figure 1). Following this step, the beads are contacted with the biotinylated vWF for
30 minutes. This procedure distinguishes from the ELISA because there is only a maximum of 2
steps and no washing steps are required. The site of contact is between the GPIbα and the Al
domain of the vWF (Figure 2). Detection of the bound GPIbα is carried out by subjecting the
whole complex, (consisting of streptavidin-coated beads, biotinylated vWF, GPIbα and tagged
GPIbα -specific chemiluminescent-antibody), to light and measuring the light signal emitted
onto a detector (Figure 1).
[0042] In another embodiment of the present invention, the streptavidin-coated magnetic
beads are replaced with anti-His-coated magnetic beads. These beads are placed in contact with
His-labeled Al domain, which will bind to GPIbα in the biological sample. An antibody, tagged
with a chemiluminescent substance, and specific for a separate antigenic site on GPIbα.
Detection of the bound GPIbα is carried out by subjecting the whole complex (consisting of
streptavidin-coated beads, biotinylated vWF, GPIbα and tagged GPIbα-specific
chemiluminescent-antibody), to light and measuring the light signal emitted onto a detector
(Figure 1).
[0043] In the present invention, the biological sample or analyte can be selected from, but
not limited to, plasma, cell-culture supernatant or a buffer from a purification process. In the
case for plasma, the source can be from any mammal, including but not limited to monkey,
mouse, rat, rabbit, guinea pig, dog, horse and human. The plasma is the portion of the whole
blood which comprises the soluble proteins. Alternatively, the assay can be conducted on whole
blood without having separated out the plasma. Cell-culture supernatants can be isolated from
any cell culture line which expresses GPIbα. Cell lines can be selected from the group
consisting of CHO cell lines, NIH-3T3 cell lines or any cell line obtained from the ATCC, any of

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which has been manipulated to express GPIbα. Purification buffers comprising GPIbα can be
selected from TRIS, TRIS sodium chloride, glycine, glycine-sodium chloride, sodium acetate,
histidine buffer, and histidine buffer with sodium chloride, sucrose and Tween EDTA can also be
used as analytes.
[0044] Another aspect of the invention is a method of measuring the protein concentration of
GPIbα in a biological sample comprising: (a) providing a substance comprising GPIbα;
(b) contacting the substance from step (a) with a binding protein which binds to GPIαb; (c)
adding a detection compound specific to GPIbα; (d) adding a complexing compound that binds
the binding protein from step (b); and (e) detecting the detection compound from step (c)
wherein a detection signal compared to a known standard curve indicates the protein
concentration of GPIbα in the biological sample.
[0045] In one embodiment, the biological substance comprising GPIbα can be selected from
plasma, supernatant from a cell line or a buffer. In another aspect, the plasma can originate from
a mammal, including but not limited to, a monkey, mouse, rat, rabbit, guinea pig, dog, horse or
human. The supernatant can originate from CHO cell line, an NIH 3T3 cell line or a cell line
obtained from the ATCC.
[0046] In another embodiment, the detection compound specific to GPIbα is an antibody or
alternatively, a Fab fragment of the antibody. In another embodiment, the binding protein
specific to GPIbα is a protein with an active binding site different from that of the antibody
above. In another embodiment, the binding protein specific to GPIbα is a fragment of vWF,
such as the Al Domain of vWF. This fragment can be a recombinant form of the Al Domain of
vWF. Alternatively, the binding protein specific to GPIbα is the complete vWF protein. In
another embodiment, the binding protein specific to GPIbα is biotinylated or His-tagged.
[0047] In another aspect, the complexing compound that binds the binding protein in step (d)
is streptavidin-coated magnetic beads or anti-His-coated magnetic beads.
[0048] In another embodiment, the antibody specific to GPIbα is labeled with a
chemiluminescent substance. More specifically, the Fab fragment specific to GPIbα is labeled
with a chemiluminescent substance.

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[0049] In another embodiment of the present invention, the protein concentration of GPIbα
is determined by generating a standard curve with a known quantity of GPIbα bound to either
vWF (Figure 3) or recombinant Al domain of vWF (Figure 4), and comparing the optical density
readout from an unknown biological sample with that of the known standard. Streptavidin
coated magnetic beads are placed in contact with biotinylated vWF which will bind to the GPIbα
in the analyte. An antibody, tagged with a chemiluminescent substance, and specific for a
separate antigenic site on GPIbα. Detection and quantification of the bound GPIbα is carried out
by subjecting the whole complex to light and measuring the light emitted onto a detector. The
measured values (optical densities) are then compared to the values generated from the known
standard and the concentrations of the unknown GPIbα can be extrapolated (Figure 3 and Figure
4).
[0050] In another aspect of the invention, the vWF, which is biotinylated and used to bind
the GPIbα in the biological sample, can be one of three variant forms, VI, V2 and V3. All three
variant forms have increasing binding activity over the wild-type form of vWF, with binding
activities V3 > V2 > VI (Figure 5). A similar trend is observed when testing the variant forms'
ability to bind Al, but the difference between variant and the total binding activity is less
pronounced than with whole vWF (Figure 5).
[0051] In another aspect of the invention, low molecular weight (LMW) and high molecular weigl
binding activity of a control GPIbα (Figure 6). This aspect of the invention allows for the
discrimination of GPIbα from other close isoforms, a process particularly of interest when
producing and purifying GPIbα in a quality controlled setting. Figure 6 demonstrates the
differential percent binding of 4 different cleaved LMW isoforms of GPIbα (represented as Clip
products) compared to wild-type control GPIbα. The binding activity on the assay is reduced to
42.5%, and for the other LMW isoforms, without the Fc portion or the binding protein, almost no
signal was generated, demonstrating the ability to distinguish the cleaved species from the intact
molecule.
[0052] In Figures 7A and 7B, bulk drug substance (BDS) of GPIbα were stored at 4°C for
different periods of time. With increasing time, the high molecular weight (HMW) percentage of
the sample increased. On the binding assay, the samples with longer storage time at 4°C showed

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decreased binding activity. The inverse correlation allows the invention to be used to monitor
aggregate levels of %HMW of GPIbα in samples (i.e. the samples with high percentage of
HMW showed lower binding activity).
[00S3] In still another aspect of the invention, sulfated forms of GPIbα have differential
binding activities; fully-sulfated GPIbα having a higher binding activity than non-sulfated
GPIbα (Figure 8 and Figure 9).
[0054] Another aspect of the invention is a method of calculating the binding activity of
GPIbα in a biological sample comprising: (a) providing a substance comprising GPIbα; (b)
contacting the substance from step (a) with a binding protein which binds to GPIbα; (c) adding a
detection compound specific to GPIbα; (d) adding a complexing compound that binds the
binding protein from step (b); (e) detecting the detection compound from step (c) wherein a
detection signal compared to a known standard curve indicates the protein concentration of
GPIbα in the biological sample; and (f) calculating the binding activity of GPIbα.
[0055] In one aspect, the biological substance comprising GPIbα can be selected from
plasma, supernatant from a cell line or a buffer. In another embodiment, the plasma can
originate from a mammal, including, but not limited to, a monkey, mouse, rat, rabbit, guinea pig,
dog, horse or human. The supernatant can originate from a CHO cell line, an NIH 3T3 cell line
or a cell line obtained from the ATCC.
[0056] In another embodiment, the detection compound specific to GPIbα is an antibody or
alternatively, a Fab fragment of the antibody. In another embodiment, the binding protein
specific to GPIbα is a protein with an active binding site different from that of the antibody
above. In another embodiment, the binding protein specific to GPIbα is a fragment of vWF,
such as the Al Domain of vWF. This fragment can be a recombinant form of the Al Domain of
vWF. More preferably, the binding protein specific to GPIbα is the complete vWF protein. In
another embodiment, the binding protein specific to GPIbα is biotinylated or His-tagged.
[0057] In another embodiment, the complexing compound that binds the binding protein in
step (d) is streptavidin-coated magnetic beads or anti-His-coated magnetic beads.

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[0058] The detection compound specific to GPIbα is labeled with a chemiluminescent
substance in some aspects of the invention. More specifically, the Fab fragment specific to
GPIbα is labeled with a chemiluminescent substance. In another embodiment, the detection of
the detection compound that binds to the binding protein in step (e) further comprises exposing a
chemiluminescent substance to light and measuring the excitation of the chemiluminescent
substance which, when compared to a known standard curve, indicates the protein concentration
of GPIbα. In another embodiment, the binding activity X of step (f) is calculated by (A/B) x
100% where A is the protein concentration determined for GPIbα by vWF interaction, and B is
the protein concentration determined by, but not limited to, Protein A HPLC or Fc capture
immunoassay.
[0059] In yet another aspect of the present invention, the binding activity of GPIbα is
determined by first calculating the concentration of GPIbα and then applying the formula A/B x
100% where A is the protein concentration determined for GPIbα by vWF interaction, and B is
the protein concentration determined for a test reference by, but not limited to, Protein A HPLC
or Fc capture immunoassay.
[0060] These benefits are significant over the current state of the art. Available GPIbα
detection assays include both in vivo and in vitro protocols. One method reported previously for
measuring GPIbα-Fc protein activities is the Rat Tail Vein Bleeding Model described in
Example 4. As GPIbα is essential in homeostasis, specifically in the clotting/bleeding cascades,
measuring in vivo bleeding time is an indirect way to measure GPIbα activity. The
disadvantages to using this animal model are its indirectness, time consumption, labor
requirements, cost, and the large % deviation (at least at 30% range).
[0061] Another method reported previously is Canine Folts' Animal Model. This method,
described in Example 5, is specific for studying unstable angina in which the function of GPIbα
is considered to be vital. Though this method can directly measure the function of GPIbαFc, the
same disadvantages, including the concerns of time, labor, cost, and precision, are also
associated with this method.

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[0062] Another method used to detect GPIbα binding activity is surface plasma resonance
(SPR) assay. This method is capable of measuring binding events between two proteins.
However, the method is extremely time-consuming and is associated with low sensitivity is low.
SPR assays typically consider a logarithmic difference of 1 to be background noise.
[0063] Conventional ELISA was also tested as a possible assay to detect GPIbα. Though the
ELISA minimized the labor, the results were very non-specific and variable, particularly when
the capture antigen is vWF.
[0064] An important aspect of this invention was to overcome the challenge of using vWF
in a multimer format as a physiologically relevant reagent in an in vitro setting due to its
recognized "stickyness". An inventive step was to develop a homogeneous binding reaction to
allow vWF to bind to GPIbα in a more native conformation rather than a mobilized condition.
The capture beads were added only at the last 30 min. of the reaction, then the whole mixture
was read in the reader right after the last incubation. With this design, vWF had very little
opportunity to bind to other non-specific molecules or surfaces. This is the only in vitro binding
activity assay which is able to specifically and efficiently determine the activity of GPIbα.
Furthermore, instead of the requirement to set-up aggregation, sulfation assays, this assay can be
used to monitor the expressed GPIboc-Fc fusion protein very quickly and precisely. A further
advantage is the use of this invention to screen for small molecules targeted to GPIbα as the
assay is very sensitive to the impact from the inhibitors.
[0065] The novel assay method of the present invention has several advantages over existing
GPIbα detection systems: (1) The present invention described herein utilizes a one- or two-step
direct assay that measures GPIbα and requires only one step to determine GPIbα activity. (2)
The assay employs specific, defined chemical bonding for its capture process, instead of non-
specific bonding. (3) The detection site is a stable site using electrochemiluminescence (ECL)
technology. (4) The detection substrate is recycled allowing for the read-out signal to become
significantly amplified. (5) The assay is devoid of washing steps and therefore has a high assay
throughput. (6) The assay utilizes reagents of increased specificity and therefore has minimum
matrix interference. (7) The assay utilizes reagents of increased specificity and therefore has a
high sensitivity. (8) The novel assay system of the present invention provides a signal-to-

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background ratio 2-5 fold greater than other existing detection assays for GPIbα. (9) The novel
assay system of the present invention provides a significant linearity scale spanning 2 logarithms.
Overall, the proposed invention is faster, cheaper, reproducible (see Figure 12), more specific
(Figures 13 A and 13B), more sensitive and capable of distinguishing the active GPIbα-Fc fusion
protein from the non-active forms (Figures 5 and 6), compared to any method of detection
available in the art.
Examples
[0066] The following examples are set forth to assist in understanding the invention and
should not, of course, be construed as specifically limiting the invention described and claimed
herein. Such variations of the invention, including the substitution of all equivalents now known
or later developed, which would be within the purview of those skilled in the art, and changes in
formulation or minor changes in experimental design, are to be considered to fall within the
scope of the invention incorporated herein.
EXAMPLE 1; Preparation of Standard Curves
[0067] A standard curve buffer is produced by adding 15 (l of R5CD1 media into 30 ml of
Buffer (PBS w/ 0.05 % Tween 20 and 0.5 % BSA). This buffer is sufficient for 90 assay plates
(based on a 96-well plate). To prepare the standard, add 4 l of standard stock to 20 ml of
standard curve buffer. This provides a concentration of 0.2 g/ml of standard. Prepare serial
dilutions by taking 2 ml of prepared standard and adding 1 ml of standard curve buffer. This is
sufficient for 8 assay plates. In a 96-well plate, distribute 50 l/ well. Running each plate
through the plate reader and plotting the optical density (transmission) versus concentration of
known standard generates the standard curve.

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EXAMPLE 2: Preparation of Controls
[0068] A control sample with a concentration 0.08 g/ml is achieved by adding 20 l of
GPIbα reference standard to 105 l media. A 1:2000 dilution is then made by taking 5 l of the
above control and adding it to 10 ml of buffer. This is good for 18 assay plates (based on a 96-
well plate). In a 96-well plate, distribute 50 l / well.
EXAMPLE 3: GPIb-vWF Binding Assay Protocol
[0069] The following protocol outlines the method to detect and quantify levels of GPIbα
from biological samples. This protocol includes reagents, methods, instruments and software
used in the process of detecting the presence of and measuring the protein concentration and
binding activity of GPIbα.
[0070] In a 96-well microtiter plate (Corning/Costar, Wilkes-Barre, PA) distribute GPIbα
standard (cone. 1 mg/ml; recombinant protein made in-house) at a volume of 50 l/well. Then,
prepare controls as described above, and distribute 50 l/well. For sample preparation, take at
least 5 l from each sample and dilute the samples to 0.08 and 0.04 |Llg/ml in buffer, final volume
around lml (make at least 500 l) and distribute 50 l/ well. To prepare biotin-vWF, take 2 l
of biotin-vWF stock (cone. 1.31 mg/ml, conjugate in-house; vWF: American Diagnostica,
Greenwich, CT; Biotin: Pierce, Rockford, IL) per 6 ml of buffer per assay plate. To prepare the
streptavidin beads add 192 |il of beads stock (cone. l0mg/ml; from IGEN, Bioveris,
Gaithersburg, MD, or from Dynal Biotech, Lake Success, NY) to 6 ml per assay plate and
distribute 50 l/ well.
[0071] All standards and controls are to be diluted in R5CD1 media. Distribute the reagents
and samples onto the plates according to the following method: Add 50 l of standard (0.2 g/ml
to 0.005 g/ml), control, or sample; add 50 l of anti-Fc ORI-TAG Ab at a final 0.4 g/ml (anti-
Fc ORI-TAG Antibody, stock concentration. 1.07 mg/ml, original material: "AffiniPure F(ab')2
Fragment Goat Anti-Human IgG, Fe Fragment Specific" from Jackson ImmunoResearch, West
Grove, PA). Following the tagged-antibody, add 50 l of b-vWF (final 0.1 M.g/ml) and 50 I of
b-vWF (final 0.1 g/ml). Incubate for 2 hours at room temperature and mix. Following

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incubation, add 50 l streptavidin-conjugated beads (final 16 g beads/well) and incubate for 30
minutes at room temperature while mixing. Read the microtiter plate on the M8 or M384
Analyzer.
[0072] To use the M8 or M384 analyzer, it must first be calibrated. To achieve this, run a
"water control" 96-well plate. Prepare a 96-well microplate by adding 250 |xL RODi water to all
wells. Place plate in analyzer and begin the plate reader. Results in all the wells should be
similar. Next, prepare a 96-well microplate by adding 250uL of Positive Calibrator to wells in
columns 1,2, 3, 7, 8, and 12. Add 250uL of Negative Calibrator to wells in columns 4, 5, 6, 9,
10, and 11. Place plate in analyzer and begin the plate reader. If the information is accurate,
continue to read plate. Quality control (QC) is acceptable if the overall Positive Calibrator CV is
less than 7% and the values are 80,000 counts + 10%. There should be no warning messages
shown in the right-hand window. If the QC is outside these guidelines, review the user manual
for trouble shooting suggestions. Once the machine is properly calibrated, place the plate with
samples to be run in stacker, select protocol to be run and begin the plate reader. Save all data.

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[0073] To analyze the collected data, open the file in Microsoft Excel and download data
into Softmax-Pro. Analyze data using template set up according to the plate map below. The
following represents a grid map for the assay. Included are the standards (std), controls and
samples (S).

1 2 3 4 5 6 7 8 9 10 11 12
A std std std std std std std std std std control control
B
0.2 0.13 0.089 0.059 0.04 0.026 0.017 0.012 0.008 0.005 0.08 0.04
C 0.08 SI S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12
D

E 0.04 SI S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12
F

G 0.08 S13 S14 S15 S16 S17 S18 S13 S14 S15 S16 S17 S18
H 0.04

[0074] The Standard Acceptance Guidelines are as follows: At least 6 out of 8 standards the
CV of the readings between standard point replicates should be ≤ 20%. The Control Acceptance
Guidelines require that the control recovery needs to be within 80 to 120 %. The Sample
Acceptance Guidelines require that the CV of the readings between sample replicates should be
≤ 20%. Only the reading within range will be taken into consideration.
EXAMPLE 4: Rat Tail Vein Bleeding
[0075] The following example represents an in vivo assay to determine the efficacy of GPIb
and if the observed in vitro data correlates with the in vivo test. Thirty-five (35) male Wister rats
(1-2 months of age, 200-230 grams; Charles River Laboratories International, Inc.,

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Wilmington, MA) are divided into 5 groups of 7 animals. The 5 groups of rats receive different
regimens according to Table 1 below:
Table 1

Group 1 Group 2 Group 3 Group 4 Group 5
Placebo 200 g/ml
Intact GPIb 200 g/ml
LMW GPIb Fully Sulfated
GPIbα
200 g/ml 0 Sulfated
GPIbα
200 g/ml
[0076] Each rat is immobilized in a rat holder and using a sterile razor blade, a subcutaneous
incision is made in the tail, 1.5 inches from the base of the posterior end of the rat.
Simultaneously, equal doses (varying volumes per weight basis) are injected intravenously by
the site of the incision. Over the next 15 minutes, bleeding times and percentage binding are
recorded. Data for each group are averaged, and plotted as mean ± standard deviation (SD). As
can be observed in Figure 8, the in vivo animal model bleeding time data correlate with the in
vitro binding assay results. In figure 8, untreated, LMW, loading, full sulfation, and 0 sulfation
refer to the low molecular weight isoform of GPIbα, starting material used in an AEX (anionic
exchange) column, six sulfation sites occupied and no sulfation occupied respectively. Figure 8
demonstrates the correlation between the in vitro binding assay of the invention, and the Rat Tail
Bleeding Animal model.
EXAMPLE 5: Canine Folts' Animal Model
[0077] The following example represents the Canine Folts' model which is widely accepted
as a model to study aspects of unstable angina. It is quite predictive of the success of anti-
platelet agents and anti-thrombotic agents in the clinical setting of unstable angina. The model is
an open chest preparation and involves isolation of the left circumflex coronary artery. An
ultrasonic flow probe is positioned on the artery to monitor coronary blood flow in real time. A
dual insult of severe stenosis and vessel injury sets up the accumulation of platelets and
thrombus formation. When the artery is completely occluded, as indicated by zero blood flow,

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the platelet plug is mechanically disrupted by shaking the occluding thrombus. Repetitive
thrombus formation is monitored by cyclic flow reductions in coronary blood flow (CFR).
GPIb and other agents for comparison are therapeutically administered upon observing 5
consistent CFRs. The response is scored on a scale 0-4 (Figure 9). In Figure 9, untreated,
monomer, LMW, loading, full sulfation, and 0 sulfation refer to no treatment, uncleaved GPIbα,
the low molecular weight isoform of GPIbα, starting material used in an AEX (anionic
exchange) column, six sulfation sites occupied and no sulfation occupied respectively. Similar
to Figure 8, Figure 9 demonstrates the significant correlation between the invention and the
available in vivo assay systems.
[0078] All references cited herein are incorporated herein by reference in their entirety and
for all purposes to the same extent as if each individual publication or patent or patent
application was specifically and individually indicated to be incorporated by reference in its
entirety for all purposes. To the extent publications and patents or patent applications
incorporated by reference contradict the disclosure contained in the specification, the
specification is intended to supercede and/or take precedence over any such contradictory
material.
[0079] All numbers expressing quantities of ingredients, reaction conditions, and so forth
used in the specification and claims are to be understood as being modified in all instances by the
term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth
in the specification and attached claims are approximations that may vary depending upon the
desired properties sought to be obtained by the present invention. At the very least, and not as an
attempt to limit the application of the doctrine of equivalents to the scope of the claims, each
numerical parameter should be construed in light of the number of significant digits and ordinary
rounding approaches.
[0080] Many modifications and variations of this invention can be made without departing
from its spirit and scope, as will be apparent to those skilled in the art. The specific
embodiments described herein are offered by way of example only and are not meant to be
limiting in any way. It is intended that the specification and examples be considered as

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exemplary only, with a true scope and spirit of the invention being indicated by the following
claims.

WO 2006/116167 PCT/US2006/015211
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Claims
1. A method of detecting the presence of GPIbα in a biological sample comprising:
(a) providing a substance comprising GPIbα; (b) contacting the substance from step (a) with a
binding protein which binds to GPIbα; (c) adding a detection compound specific to GPIbα;
(d) adding a complexing compound that binds the binding protein from step (b); and (e)
detecting the detection compound from step (c) wherein a positive detection signal indicates the
presence of GPIbα in the biological sample.
2. The method of claim 1, wherein the biological sample comprising GPIbα is
plasma.
3. The method of claim 2, wherein the plasma is from a mammal.
4. The method of claim 3, wherein the mammal is a human.
5. The method of claim 1, wherein the biological sample comprising GPIbα is
supernatant from a cell line.
6. The method of claim 5, wherein the cell line is a CHO cell line.
7. The method of claim 1, wherein the detection compound comprises an antibody.
8. The method of claim 7, wherein the detection compound comprises a Fab
fragment.
9. The method of claim 1, wherein the binding protein comprises a protein with a
binding site different from that of the antibody in claim 7.

10. The method of claim 9, wherein the binding protein is a fragment of von
Willerbrand Factor (vWF).
11. The method of claim 10, wherein the fragment is the Al Domain of vWF.
12. The method of claim 9, wherein the binding protein is the complete vWF protein.

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13. The method of claim 1, wherein the binding protein is biotinylated.
14. The method of claim 11, wherein the Al Domain of vWF is His-Tagged.
15. The method of claim 13, wherein the complete vWF is biotinylated.
16. The method of claim 1, wherein the complexing compound that binds the binding
protein is streptavidin-coated magnetic beads.
17. The method of claim 1, wherein the complexing compound that binds the binding
protein is anti-His-coated magnetic beads.
18. The method of claim 7, wherein the detection compound specific to GPIb is
. labeled with a chemiluminescent substance.
19. The method of claim 8, wherein the Fab fragment specific to GPIb is labeled
with a chemiluminescent substance.
20. The method of claim 1, wherein the detection of the detection compound in step
(e) further comprises exposing a chemiluminescent substance to light and measuring the
excitation of the chemiluminescent substance.
21. A method of measuring the protein concentration of GPIb in a biological
sample comprising: (a) providing a substance comprising GPIb; (b) contacting the substance
from step (a) with a binding protein which binds to GPIbα; (c) adding a detection compound
specific to GPIbα; (d) adding a complexing compound that binds the binding protein from step
(b); and (e) detecting the detection compound from step (c) wherein a detection signal compared
to a known standard curve indicates the protein concentration of GPIbα in the biological sample.
22. The method of claim 21, wherein the detection of the detection compound further
comprises exposing a chemiluminescent substance to light and measuring the excitation of the
chemiluminescent substance which, when compared to a known standard curve, indicates the
protein concentration of GPIbα.

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23. A method of detecting the binding activity of GPIbα in a biological sample
comprising: (a) providing a substance comprising GPIbα; (b) contacting the substance from step
(a) with a binding protein which binds to GPIbα; (c) adding a detection compound specific to
GPIbα; (d) adding a complexing compound that binds the binding protein from step (b); (e)
detecting the detection compound from step (c) wherein a detection signal compared to a known
standard curve indicates the protein concentration of GPIbα in the biological sample; and
(f) calculating the binding activity of GPIbα.
24. The method of claim 23, wherein the binding activity X of (f) is calculated by
(A/B) x 100% where A is the protein concentration determined for GPIbα by vWF interaction,
and B is the protein concentration determined by Protein A HPLC or Fc capture immunoassay.
25. A method of detecting an isoform of GPIbα by measuring the binding activity of
the isoform of GPIbα in a biological sample comprising: (a) providing a substance comprising
GPIbα and GPIbα-like substance; (b) contacting the substance from step (a) with a binding
protein which binds to GPIbα; (c) adding a detection compound specific to GPIbα; (d) adding a
complexing compound that binds the binding protein from step (b); (e) detecting the detection
compound from step (c) wherein a detection signal compared to a known standard curve
indicates the protein concentration of GPIbα in the biological sample; (f) calculating the binding
activity of an isoform of GPIbα; and (g) comparing the binding activity of the isoform to the
binding activity of a known GPIbα control.
26. The method of claim 25, wherein the binding activity X of (f) is calculated by
(A/B) x 100% where A is the protein concentration determined for GPIbα by vWF interaction,
and B is the protein concentration determined by Protein A HPLC or Fc capture immunoassay.

The invention provides a novel assay system for detecting the presence, concentration and binding activity of GPIbα in a biological sample. The method for determining the presence of GPIbα in a biological sample comprises: (a) providing a substance comprising GPIbα; (b) contacting the substance from step (a) with a binding protein which binds to GPIbα; (c) adding a detection compound specific to GPIbα; (d) adding a complexing compound that binds the binding protein from step (b); and (e)
detecting the detection compound from step (c) wherein a positive detection signal indicates the presence of GPIbα in the biological
sample.