JP2008538610A - Method for measuring glycoprotein IBα (GPIBα) protein - Google Patents

Abstract

  The present invention provides a novel assay system for detecting the presence, concentration and binding activity of GPIbα in a biological sample. A method for measuring 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 that binds to GPIbα; (C) adding a detection compound specific for GPIbα; (d) adding a complexing compound that binds to the binding protein from step (b); and (e) a detection compound from step (c). Wherein a positive detection signal indicates the presence of GPIbα in the biological sample.

Description

  This application claims priority from US Provisional Application No. 60/673926, filed Apr. 22, 2005, which is hereby incorporated by reference in its entirety. .

  The present invention belongs to the field of biochemical assay systems, particularly for measuring proteins. More specifically, the present invention relates to the detection and quantification of glycoprotein Ibα (GPIbα) protein.

  In vascular biology, platelet function is the cornerstone of proper homeostasis and hemostasis. Platelets contribute to the maintenance of normal blood circulation by maintaining vascular integrity and controlling bleeding after injury (Ruggeri, J. Clin. Invest. 99: 559-564 (1997)). Platelet thrombus formation is a defense mechanism necessary for survival, but may also contribute to diseases such as myocardial infarction, particularly in the microenvironment of atherosclerosis (Fuster, N. Engl. J. Med. 326: 242-250 (1992)). Furthermore, one of the major causes of morbidity and mortality in developed countries is acute thrombotic arterial occlusion (Ruggeri, J. Clin. Invest. 99: 4559-564 (1997)). This underscores the relevance of research focused on elucidating the mechanism of platelet response to vascular injury and on commercial means to detect components of this complex pathway.

  Of importance to the mechanism for the platelet response is its receptor complex, glycoprotein (GP) Ib / IX / V. This platelet receptor binds directly to von Willebrand factor (vWF), which forms a bridge to the damaged vessel wall (Miura, J. Biol. Chem. 275: 7539-7546 (2000)). This effect is induced by vWF associated with the subendothelial matrix and is regulated by shear stress caused by blood flow in microvessels (Turitto, Blood 65: 823-829 (1985)). This event results in further interaction between the damaged blood vessel wall-derived collagen receptor and platelets, forming a hemostatic thrombus that blocks platelet activation, platelet coagulation, and endothelial injury and prevents blood leakage. (Girma, Blood 70: 605-615 (1987)).

  The platelet receptor GPIb / IX / V that binds to the vWF-collagen matrix is composed of four subunits, GPIbα, GPIbβ, GPIX and GPV (Modderman, J. Biol. Chem. 267: 364-369. (1992)). The most important of these subunits, based on functionality and size, is the 150 kDa GPIbα chain (Uff, J. Biol. Chem. 277: 35657-35663 (2002)). GPIbα is involved in initial adhesion to vWF by binding to various sites on the A1 domain of vWF. GPIbα mutations can lead to bleeding disorders, Bernard-Soulier syndrome (BSS) and vWF Wilbrand disease (Pt-vWD). vWF is the main ligand of GPIbα, but other proteins have been identified that bind to this glycoprotein, including thrombin, kininogen, factor XI, factor XII, P-selectin and Mac-1 (Uff, J Biol.Chem.277: 35657-35663 (2002)).

  Since the biological significance of GPIbα is clear, the need for a simple and definitive bioassay to identify and quantify GPIbα has become apparent. Currently available bioassays for quantifying GPIbα are time consuming, labor intensive, limited to certain isoforms, and expensive to operate. In vivo methods include the rat tail vein hemorrhage model and the Folts canine animal model, but the ristocetin-induced platelet coagulation assay (RIPA) constitutes an in vitro assay currently available (Folts, Circulation 83: IV3 IV 14 (1991), Dejana, Thromb. Haemost. 48: 108-111 (1982), Weiss J. Clin. Invest. 52: 2708-16 (1973)). These assays involve significant background-to-assay variation with high background noise, limited sensitivity, high cost, and the need for intensive work. Therefore, there is a need for an assay system that can accurately and rapidly detect and quantify a wide range of GPIbα levels in several types of biological samples. More specifically, there is a need for a method that can accurately and efficiently measure GPIbα in a laboratory environment and in the clinical and diagnostic areas. A new method for detecting the biological activity of GPIbα that is simple, accurate and highly reproducible is highly desirable.

  The present invention fulfills these needs and also provides related advantages. Additional objects and advantages of the present invention may be set forth in part in the description which follows, and in part will be obvious from the description, and may be confirmed 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.

  It should 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 Although the importance of GPIbα in vascular biology is well recognized, there is no simple and efficient test or diagnostic assay to detect it or measure its biological activity . The invention described herein relates to the development of an efficient, reproducible and inexpensive assay for detecting and quantifying the biological activity of GPIbα. The present invention applies to both clinical and laboratory environments. Since the present invention also allows sensitive and specific identification of different isoforms of GPIbα, it can be very useful in performing quality control and monitoring GPIbα production levels. The sensitivity and specificity of the assay includes, but is not limited to, the determination of isoforms that may be contaminating the GPIbα production and purification process, and the active GPIbα-Fc fusion protein and non-active protein Allows identification.

  The present invention is directed to an assay for measuring the presence of GPIbα in a biological sample, the method comprising: (a) providing a substance comprising GPIbα; (b) a substance from step (a), Contacting with a binding protein that binds to GPIbα; (c) adding a detection compound specific for GPIbα; (d) adding a complexing compound that binds to the binding protein from step (b); and (E) detecting a detection compound from step (c), wherein a positive detection signal indicates the presence of GPIbα in the biological sample.

  In another embodiment, the invention is directed to an assay for detecting the protein concentration of GPIbα in a biological sample, the method comprising: (a) providing a substance comprising GPIbα; (b) step ( a) contacting the derived material with a binding protein that binds to GPIbα; (c) adding a detection compound specific for GPIbα; (d) complexing that binds to the binding protein from step (b). Adding a compound; and (e) detecting the detection compound from step (c), wherein the detection signal compared to a known standard curve is the protein concentration of GPIbα in the biological sample. Show.

  In yet another embodiment, the present invention is directed to an assay for detecting the biological activity of GPIbα in a biological sample, the method comprising: (a) providing a substance comprising GPIbα; (b ) Contacting the substance from step (a) with a binding protein that binds to GPIbα; (c) adding a detection compound specific for GPIbα; (d) binding to the binding protein from step (b). Adding a complexing compound; (e) detecting the detection compound from step (c), wherein the detection signal compared to a known standard curve is the protein concentration of GPIbα in the biological sample. And (f) calculating the binding activity of GPIbα.

  In yet another embodiment, the present invention is directed to an assay for detecting a GPIbα isoform by measuring the binding activity of the GPIbα isoform in a biological sample, the method comprising (a) GPIbα (B) contacting the substance from step (a) with a binding protein that binds to GPIbα; (c) adding a detection compound specific for GPIbα; (D) adding a complexed compound that binds to the binding protein from step (b); (e) detecting the detection compound from step (c), where detection relative to a known standard curve The signal indicates the protein concentration of GPIbα in the biological sample; (f) calculating the binding activity of the isoform of GPIbα; and (g) the isoform Comparing the binding activity of the foam to that of a known GPIbα control.

  The accompanying drawings, which are incorporated in and constitute a part of this specification, together with the description of the specification, serve to explain the principles of the invention.

  Unless defined otherwise, 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 hereby incorporated by reference in their entirety. Moreover, 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 claims.

Abbreviations and Definitions The following abbreviations are used herein. “ATCC” means American Type Culture Collection, “vWF” means von Willebrand factor, “GPIbα” means glycoprotein 1b-α, “CHO” means Chinese hamster ovary, “NIH” “3T3” means National Institute of Health 3T3.

  As used herein, the term “antibody” includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, genetically engineered antibodies, bispecific antibodies, antibody fragments, and reactive portions of antibodies. Means a single strand to represent. The preparation of each of the antibody forms referred to above is well known in the art.

  As used herein, the term “biological sample” means, without limitation, any cell, prokaryotic or eukaryotic organism, any tissue or organ, or any product of its recombinant technique or genetic manipulation. To do. A “biological sample” may be a plasma sample, a cell culture supernatant, or a buffer from a purification process. In the case of plasma, its origin can be any mammal, including but not limited to monkeys, mice, rats, rabbits, guinea pigs, gerbils, pigs, dogs, horses and humans. Plasma is the part of whole blood that contains soluble proteins. Alternatively, the assay can be performed on whole blood without separating the plasma. The cell culture supernatant can be isolated from any cell culture system that expresses GPIbα. The cell line can be selected from any CHO cell line, NIH-3T3 cell line, or any cell line obtained from ATCC, all engineered to express GPIbα.

  The term “isoform” as used herein includes, but is not limited to, low molecular weight (LMW) GPIbα, high molecular weight (HMW) GPIbα (see FIG. 11), other mutant forms (V1, V2, V3), and By means of a small molecule GPIbα including but not limited to fully sulfated or partially sulfated GPIbα protein (see FIG. 10).

DESCRIPTION The present invention is a method for specifically and sensitively detecting and quantifying the presence of GPIbα or GPIbα-like contaminants 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 that binds to GPIbα; (c) specific for GPIbα. (D) adding a complexing compound that binds to the binding protein from step (b); and (e) detecting the detection compound from step (c). Where a positive detection signal indicates the presence of GPIbα in the biological sample.

  In one embodiment, the biological material comprising GPIbα can be plasma, cell line supernatant, or buffer. In another embodiment, the plasma can be from a mammal selected from monkeys, mice, rats, rabbits, guinea pigs, dogs, horses or humans. The supernatant can be derived from CHO cell lines, NIH 3T3 cell lines, or cells obtained from ATCC.

  In yet another embodiment, the detection compound specific for GPIbα is an antibody, or more preferably a Fab fragment of an antibody. In another embodiment, the binding protein specific for GPIbα is a protein having an active binding site different from that of the antibody. In another embodiment, the binding protein specific for GPIbα is a fragment of vWF, such as the A1 domain of vWF. This fragment may be a recombinant form of the AWF domain of vWF. Alternatively, the binding protein specific for GPIbα is a complete vWF protein. In another embodiment, the binding protein specific for GPIbα is biotinylated or His-tagged.

  In yet another embodiment, the complexing compound that binds to the binding protein of step (d) is streptavidin-coated magnetic beads or anti-His-coated magnetic beads.

  Another embodiment is directed to a detection compound specific for GPIbα that is labeled with a chemiluminescent material. More specifically, the Fab fragment of an antibody specific for GPIbα is labeled with a chemiluminescent substance.

  In another aspect, detecting the detection compound that binds to the binding protein of step (e) comprises exposing the chemiluminescent material and measuring the excitation of the chemiluminescent material that correlates with the presence of GPIbα.

  A further embodiment of the present invention, streptavidin-coated magnetic beads, is placed in contact with biotinylated vWF that can bind to GPIbα in an analyte or biological sample (FIG. 1). An antibody tagged with a chemiluminescent material and specific for another antigenic site on GPIbα is contacted with this biotinylated vWF for 2 hours (FIG. 1). After this step, the beads are contacted with biotinylated vWF for 30 minutes. This procedure is distinguished from ELISA because it only has a maximum of 2 steps and no washing step is required. The contact site is between GPIbα and the A1 domain of vWF (FIG. 2). Detection of bound GPIbα involves exposing the entire complex (consisting of streptavidin-coated beads, biotinylated vWF, GPIbα and tagged GPIbα-specific chemiluminescent antibodies) and measuring the light signal emitted on the detector (FIG. 1).

  In another embodiment of the invention, the streptavidin-coated magnetic beads are replaced with anti-His-coated magnetic beads. These beads are placed in contact with a His-labeled A1 domain that can bind to GPIbα in a biological sample. An antibody that is tagged with a chemiluminescent material and specific for another antigenic site on GPIbα. Detection of bound GPIbα measures the light signal emitted to the detector as well as exposing the entire complex (consisting of streptavidin-coated magnetic beads, biotinylated vWF, GPIbα and tagged GPIbα specific chemiluminescent antibodies) (FIG. 1).

  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 of plasma, its origin can be from any mammal, including but not limited to monkeys, mice, rats, rabbits, guinea pigs, dogs, horses and humans. Plasma is the part of whole blood that contains soluble proteins. Alternatively, the assay can be performed on whole blood from which plasma has not been separated. The cell culture supernatant can be isolated from any cell culture system that expresses GPIbα. The cell line can be selected from the group consisting of a CHO cell line, NIH-3T3 cell line, or any cell line obtained from ATCC, all engineered to express GPIbα. The purification buffer containing 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 also as analytes Can be used.

  Another aspect of the present invention is a method for measuring the protein concentration of GPIbα in a biological sample, the method comprising: (a) providing a substance comprising GPIbα; (b) from step (a) Contacting the substance with a binding protein that binds to GPIbα; (c) adding a detection compound specific for GPIbα; (d) adding a complexing compound that binds to the binding protein from step (b). And (e) detecting the detection compound from step (c), wherein the detection signal compared to a known standard curve is indicative of the protein concentration of GPIbα in the biological sample.

  In one embodiment, the biological material comprising GPIbα may be selected from plasma, cell line-derived supernatant, or buffer. In another aspect, the plasma can be from a mammal, including but not limited to monkeys, mice, rats, rabbits, guinea pigs, dogs, horses or humans. The supernatant can be derived from a CHO cell line, a NIH 3T3 cell line, or a cell line obtained from ATCC.

  In another embodiment, the detection compound specific for GPIbα is an antibody or a Fab fragment of an antibody. In another embodiment, the binding protein specific for GPIbα is a protein having an active binding site different from the active binding site of the antibody. In another embodiment, the binding protein specific for GPIbα is a fragment of vWF, such as the A1 domain of vWF. This fragment may be a recombinant form of the AWF domain of vWF. Alternatively, the binding protein specific for GPIbα is a complete vWF protein. In another embodiment, the binding protein specific for GPIbα is biotinylated or His-tagged.

  In another embodiment, the complexing compound that binds to the binding protein of step (d) is streptavidin-coated magnetic beads or anti-His-coated magnetic beads.

  In another embodiment, the antibody specific for GPIbα is labeled with a chemiluminescent material. More specifically, the Fab fragment specific for GPIbα is labeled with a chemiluminescent substance.

  In another embodiment of the invention, the protein concentration of GPIbα is generated using a known amount of GPIbα bound to either vWF (FIG. 3) or the recombinant A1 domain of vWF (FIG. 4). , And comparing optical density readings from unknown biological samples with known standard readings. Streptavidin-coated magnetic beads are placed in contact with biotinylated vWF that can bind to GPIbα in the analyte. The antibody is tagged with a chemiluminescent material and is specific for another antigenic site on GPIbα. Detection and quantification of bound GPIbα is performed by exposing the entire complex and measuring the light signal emitted to the detector. The measured value (optical density) can then be compared with a value generated from a known standard, and the concentration of unknown GPIbα can be extrapolated (FIGS. 3 and 4).

  In another aspect of the invention, the vWF that is biotinylated and used to bind GPIbα in a biological sample can be one of three variant forms of V1, V2, and V3. All three of these mutant forms have additional binding activity over the wild-type form of vWF, the binding activity being V3> V2> V1 (FIG. 5). A similar trend is observed when testing the ability to bind the mutant form of A1, but the difference between the mutant and total binding activity is less pronounced than when using the entire vWF (FIG. 5). .

  In another aspect of the invention, the low molecular weight (LMW) and high molecular weight binding activity of the control GPIbα (FIG. 6). This aspect of the invention allows for the differentiation of GPIbα from other closely related isoforms, a process of particular interest when producing and purifying GPIbα under quality control. FIG. 6 shows the difference in percent binding (expressed as Clip product) of four different cleaved LMW isoforms of GPIbα compared to the wild type control GPIbα. The binding activity in this assay is reduced to 42.5% and almost no signal is generated for other LMW isoforms that do not have an Fc moiety or binding protein, which is a molecule that is intact with the cleaved species. It is demonstrated that can be identified.

  In FIGS. 7A and 7B, the drug substance (BDS) of GPIbα was stored at 4 ° C. for different times. As time increased, the percentage of high molecular weight (HMW) samples increased. In the binding assay, samples stored for longer times at 4 ° C. showed reduced binding activity. This inverse correlation allows the present invention to be used to monitor the level of aggregation of GPIbα% HMW in a sample (ie, a sample with a high percentage of HMW showed lower binding activity).

  In yet another aspect of the invention, the sulfated form of GPIbα has a differential binding activity, and fully sulfated GPIbα has a higher binding activity than non-sulfated GPIbα (FIGS. 8 and 9). .

  Another aspect of the present invention is a method for calculating the binding activity of GPIbα in a biological sample, the method comprising: (a) providing a substance comprising GPIbα; (b) from step (a) Contacting the substance with a binding protein that binds to GPIbα; (c) adding a detection compound specific for GPIbα; (d) adding a complexing compound that binds to the binding protein from step (b). (E) detecting the detection compound from step (c), wherein the detection signal compared to a known standard curve indicates the protein concentration of GPIbα in the biological sample; and (f) GPIbα Calculating the binding activity of

  In one aspect, the biological material comprising GPIbα may be selected from plasma, cell line-derived supernatant, or buffer. In another embodiment, the plasma can be from a mammal, including but not limited to monkeys, mice, rats, rabbits, guinea pigs, dogs, horses or humans. The supernatant can be derived from a CHO cell line, a NIH 3T3 cell line, or a cell line obtained from ATCC.

  In another embodiment, the detection compound specific for GPIbα is an antibody or a Fab fragment of an antibody. In another embodiment, the binding protein specific for GPIbα is a protein having an active binding site different from that of the antibody. In another embodiment, the binding protein specific for GPIbα is a fragment of vWF, such as the A1 domain of vWF. This fragment may be a recombinant form of the AWF domain of vWF. More preferably, the binding protein specific for GPIbα is a complete vWF protein. In another embodiment, the binding protein specific for GPIbα is biotinylated or His-tagged.

  In another embodiment, the complexing compound that binds to the binding protein in step (d) is streptavidin-coated magnetic beads or anti-His-coated magnetic beads.

  In some aspects of the invention, the detection compound specific for GPIbα is labeled with a chemiluminescent material. More specifically, the Fab fragment specific for GPIbα is labeled with a chemiluminescent substance. In another embodiment, detecting the detection compound that binds to the binding protein in step (e) further comprises exposing the chemiluminescent material and measuring excitation of the chemiluminescent material, the measurement result comprising: The protein concentration of GPIbα is shown when compared to a known standard curve. In another embodiment, the binding activity X of step (f) is (A / B) × 100%, wherein A is the protein concentration measured for GPIbα by vWF interaction, and B is not limited Is the protein concentration measured by protein A HPLC or Fc capture immunoassay].

  In yet another aspect of the invention, the binding activity of GPIbα is calculated by first calculating the concentration of GPIbα, then the formula A / B × 100%, where A is measured for GPIbα by vWF interaction. And B is the protein concentration measured for the test reference by, but not limited to, a protein A HPLC or Fc capture immunoassay].

  These benefits over the current state of the art are significant. Available GPIbα detection assays include both in vivo and in vitro protocols. One previously reported method for measuring GPIbα-Fc protein activity is the rat tail vein bleeding model described in Example 4. Because GPIbα is essential for homeostasis, specifically the coagulation / bleeding cascade, measuring in vivo bleeding time is an indirect method of measuring GPIbα activity. The disadvantages of using this animal model are its indirectness, time, labor, cost, and large% deviation (at least 30% range).

  Another method previously reported is the Folts canine animal model. This method described in Example 5 is specific for studying unstable angina where GPIbα function is considered critical. Although this method can directly measure the function of GPIbα, similar disadvantages involving time, effort, cost and accuracy issues are also associated with this method.

  Another method used to detect GPIbα binding activity is the surface plasma resonance (SPR) assay. This method can measure binding events between two proteins. However, this method is very time consuming and involves low sensitivity. SPR assays typically consider a logarithmic difference of 1 as background noise.

  A conventional ELISA was also tested as a potential assay for detecting GPIbα. ELISA minimized work, but the results were very non-specific and varied, especially when the capture antigen was vWF.

  An important aspect of the present invention overcomes the challenge of using multimeric vWF as a physiologically relevant reagent in an in vitro situation because the “adhesion” of multimeric vWF is recognized. Was to do. The process of the invention was to develop a homogenous binding reaction that binds vWF to GPIbα in a natural conformation rather than mobilized conditions. Capture beads were added only during the last 30 minutes of the reaction, and then the entire mixture was read on the reader immediately after the final incubation. With this scheme, vWF had little opportunity to bind to other non-specific molecules or surfaces. This is the only in vitro binding activity assay that can specifically and efficiently measure GPIbα activity. Furthermore, instead of the need to initiate aggregation, the sulfated assay, this assay can be used to monitor the expressed GPIbα-Fc fusion protein quickly and precisely. A further advantage is the use of the present invention to screen for small molecules targeted to GPIbα since this assay is very sensitive to the effects from inhibitors.

  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-step or two-step direct assay to measure GPIbα. However, only one step is required to determine GPIbα activity. (2) This assay utilizes chemical bond formation specific and defined for the capture process instead of non-specific bond formation. (3) The detection site is a stable site using electrochemiluminescence (ECL) technique. (4) The detection substrate is recycled and significantly amplifies the read signal. (5) Since this assay lacks a washing step, it has a high assay throughput. (6) This assay has minimal matrix interference since it utilizes highly specific reagents. (7) This assay is highly sensitive because it utilizes reagents with increased specificity. (8) The novel assay system of the present invention results in a signal to background ratio that is 2-5 times greater than other existing detection assays for GPIbα. (9) The novel assay system of the present invention provides a significant linearity measure over two logarithms. Overall, the proposed invention is faster, cheaper, reproducible (see FIG. 12) and more specific than any detection method available in the art (FIGS. 13A and 13B). Sensitive, active GPIbα-Fc fusion protein can be distinguished from the inactive form (FIGS. 5 and 6).

  The following examples are presented to aid the understanding of the present invention and, of course, are not to be construed as specifically limiting the invention described and claimed herein. Such variations of the present invention, including alterations of all equivalents now known or later developed and within the purview of those skilled in the art, and changes in formulation or experimental design are described herein. Should be considered within the scope of the invention as contained in the document.

Example 1: Generation of a standard curve A standard curve buffer is prepared by adding 15 μl of R5CD1 medium to 30 ml of buffer (PBS w / 0.05% Tween 20 and 0.5% BSA). This buffer is sufficient for 90 assay plates (based on 96 well plates). To prepare the standard solution, add 4 μl of the standard stock solution to 20 ml of the standard curve buffer. This results in a standard solution with a concentration of 0.2 μg / ml. Collect 2 ml of the prepared standard solution and prepare a serial dilution by adding 1 ml of standard curve buffer. This is sufficient for 8 assay plates. Dispense 50 μl / well into a 96 well plate. Each plate is processed in a plate reader and a standard curve is generated by plotting optical density (transmission) against the concentration of a known standard solution.

Example 2: Control preparation A control sample with a concentration of 0.08 μg / ml is obtained by adding 20 μl GPIbα reference standard solution to 105 μl medium. Next, 5 μl of the above control is collected and added to 10 ml of buffer to make a 1: 2000 dilution. This is sufficient for 18 assay plates (based on 96 well plates). Dispense 50 μl / well into a 96 well plate.

Example 3: GPIbα-vWF binding assay protocol The following protocol outlines a method for detecting and quantifying GPIbα levels from biological samples. This protocol includes reagents, methods, instruments and software used in the process of detecting the presence of GPIbα and measuring GPIbα protein concentration and binding activity.

  Dispense the GPIbα standard solution (concentration 1 mg / ml; recombinant protein made in-house) in a volume of 50 μl / well to a 96-well microtiter plate (Corning / Costar, Wilkes-Barre, Pa.). Controls are then prepared as above and dispensed at 50 μl / well. For sample preparation, take at least 5 μl from each sample, dilute the sample to 0.08 and 0.04 μg / ml in a final volume of approximately 1 ml (make at least 500 μl) buffer, 50 μl / well Distribute with. To prepare biotin-vWF, biotin-vWF stock solution (concentration 1.31 mg / ml, in-house conjugate per 6 ml of buffer per assay plate. VWF: American Diagnostica, Grenich, CT. Biotin: Pierce, Take 2 μl (Rockford, Ill.). To prepare streptavidin beads, 192 μl of bead stock solution (concentration 10 mg / ml, obtained from IGEN (Bioveris, Gaithersburg, MD) or Dynal Biotech (Lake Success, NY)) in 6 ml per assay plate And dispense at 50 μl / well.

  All standard solutions and controls shall be diluted in R5CD1 medium. Distribute reagents and samples to the plate by the following method. Add 50 μl of standard solution (0.2 μg / ml to 0.005 μg / ml), control or sample, and add 50 μl of anti-Fc ORI-TAG Ab at a final concentration of 0.4 μg / ml (anti-Fc ORI-TAG antibody, Stock solution concentration 1.07 mg / ml, original material: “AffiniPure F (ab ′) 2 fragment goat anti-human IgG, Fcγ fragment specific” from Jackson ImmunoResearch (West Grove, Pa.)). After the tagged antibody, 50 μl of b-vWF (final concentration 0.1 μg / ml) and 50 μl of b-vWF (final concentration 0.1 μg / ml) are added. Incubate for 2 hours at room temperature and mix. After incubation, 50 μl of streptavidin-conjugated beads (final concentration 16 μg beads / well) are added and incubated for 30 minutes at room temperature with mixing. Read the microtiter plate with an M8 or M384 analyzer.

  In order to use an M8 or M384 analyzer, the analyzer must first be calibrated. To accomplish this, it is performed in a “water control” 96-well plate. Prepare a 96-well microplate by adding 250 μL of RODi water to all wells. Place the plate on the analyzer and start reading the plate. The results for all wells should be similar. Next, a 96-well microplate is prepared by adding 250 uL of positive standard to the wells of columns 1, 2, 3, 7, 8, and 12. Add 250 uL of negative standard to the wells of columns 4, 5, 6, 9, 10 and 11. Place the plate on the analyzer and start reading the plate. Continue reading the plate if the information is accurate. Quality control (QC) is acceptable if the total CV of the positive reference material is less than 7% and the value is 80000 count ± 10%. There should be no warning in the right window. If the QC deviates from these guidelines, review the user manual for troubleshooting suggestions. Once the machine is properly calibrated, the plate with the sample to be processed is placed on the stacker, the protocol to be performed is selected, and the plate reader is started. Save all data.

  In order to analyze the collected data, the Microsoft Excel file is opened and the data is downloaded to Softmax-Pro. Analyze data using template settings according to the plate map below. The following represents a grid map for the assay. Standard (std), control and sample (S) are included.

  The standard acceptance guidelines are as follows: The CV of the readings between standard points repeated in at least 6 out of 8 standards must be ≦ 20%. Control acceptance guidelines require that control recovery should be in the range of 80-120%. Sample acceptance guidelines require that the CV of readings between repeated samples should be ≦ 20%. Only readings within the range will be considered.

Example 4: Rat Tail Vein Hemorrhage The following example represents an in vivo assay to determine the efficacy of GPIbα and whether the observed in vitro data correlates with in vivo testing. Thirty-five male Wistar rats (1-2 months old, 200-230 grams; Charles River Laboratories International, Inc., Wilmington, Mass.) Are divided into 5 groups of 7 animals in each group. The five groups of rats are subjected to different dosing schedules described in Table 1 below.

  Each rat is fixed in a rat holder and a subcutaneous incision is made from the rear end base of the rat to the 1.5 inch tail using a sterile razor blade. At the same time, an equal dose (change volume based on weight) is injected intravenously near the incision site. Record bleeding time and percent binding over the next 15 minutes. Data for each group is averaged and plotted as mean ± standard deviation (SD). As can be seen in FIG. 8, the bleeding time of the in vivo animal model correlates with the results of the in vitro binding assay. In FIG. 8, untreated, LVW, loading, complete sulfation, and zero sulfation occupy the low molecular weight isoform of GPIbα, the starting material used for the AEX (anion exchange) column, and six sulfation sites, respectively. And those in which no sulfation site is occupied. FIG. 8 demonstrates the correlation between the in vitro binding assay of the present invention and the rat tail bleeding animal model.

Example 5: Folts Canine Animal Model The following example represents a Folts canine model that is widely accepted as a model for studying the appearance of unstable angina. This considerably predicts the success of antiplatelet and antithrombotic agents in the clinical setting of unstable angina. This model is prepared for thoracotomy and involves the isolation of the left coronary circumflex. An ultrasonic flow probe is placed in the artery to monitor coronary blood flow in real time. Serious wound constriction and vascular injury double invasion initiate platelet accumulation and thrombus formation. When the artery is completely occluded, as shown by zero blood flow, the platelet thrombus is mechanically destroyed by vibrating the occluded thrombus. Repeated thrombus formation is monitored by periodic flow reduction (CFR) in coronary blood flow. Once consistent CFR is observed 5 times, GPIbα and other drugs for comparison are administered therapeutically. The response is scored on a scale of 0 to 4 (Figure 9). In FIG. 9, untreated, monomeric, LMW, loaded, fully sulphated and zero sulphated are untreated, non-cleavable GPIbα, low molecular weight isoforms of GPIbα, starting with AEX (anion exchange) column respectively. Represents substances, those in which 6 sulfation sites are occupied, and those in which no sulfation sites are occupied. Similar to FIG. 8, FIG. 9 demonstrates a significant correlation between the present invention and available in vivo assay systems.

  All references, including publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. To the extent that publications and patents or patent applications that are part of this specification by reference are inconsistent with the disclosure herein, the specification will supersede any such conflicting material, and / or Or intended to precede it.

  It should be understood that all numbers indicating the amounts of ingredients, reaction conditions, etc. used in the specification and claims are modified in all cases by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth herein and in the appended claims are approximations that may vary depending upon the desired properties sought to be acquired by the present invention. At a minimum and without limiting the application of the principle of equivalents to the claims, each numerical parameter should be interpreted in the light of the number of significant digits and the usual rounding approach.

  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 provided by way of example only and are in no way meant to be limiting. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the appended claims.

1 is a schematic representation of a GPIbα binding assay format. Biotin vWF, GPIbα-Fc fusion protein, and BV-tagged anti-human Fc antibody were first mixed and incubated for 2 hours at room temperature. After incubation, streptavidin (SA) beads were added to the mixture and incubated for an additional 30 minutes. Figure 2 is a schematic representation of the GPIbα binding region on vWF. FIG. 5 is a graph showing a standard curve for GPIbα-vWF binding assay. The ratio of signal to background (S / B) for this assay is close to 10, but the S / B of the ELISA GPIbα binding assay is a maximum of 2 with 1 log linearity. FIG. 5 is a graph showing a standard curve for GPIbα-A1 binding assay. It is a graph which shows the binding activity of a product variant. WT corresponds to wild type GPIbα. V1, V2 and V3 are gain-of-function mutants of GPIbα. According to the results of in vivo experiments, the gain-of-function mutant had an increased ability to bind GPIb. In the vWF binding assay, differences between V3, V2, V1 and WT can be easily observed, whereas in the A1 binding assay, the differences between the variants were very limited. Figure 2 is a graph showing the binding activity of four different low molecular weight (LMW) isoforms for the vWF binding assay. The control is an uncleaved GPIbα molecule. Clip, Clip1-276, Clip1-282, and Clip Fc represent different cleavage isoforms of GPIbα. It is a graph which shows the stability test of a GPIb (alpha) sample. GPIbα drug substance (BDS) was stored at 4 ° C. and the percent high molecular weight (HMW) was monitored as a function of time. It is a graph which shows the stability test | inspection of a GPIb (alpha) sample. GPIbα drug substance (BDS) was stored at 4 ° C. and the percent binding activity was monitored as a function of time. 2 is a graph showing a comparison of in vitro and in vivo data from a rat tail vein reaction time assay. Test samples include untreated animal controls for in vivo testing; LMW, GPIbα low molecular weight (typical clip); control, preload, anion exchange (AEX) column separation prior to separation; complete sulfation, all sulfates GPIbα in which the sulfation site is sulfated; and 0 sulfation, GPIbα in which no sulfation site is sulfated. 2 is a graph showing comparison of in vitro and in vivo data from a cants animal model of folts. Test samples included untreated animal controls for in vivo testing; monomer, intact GPIbα intact; LMW1 and LMW2, low molecular weight fraction of GPIbα from AEX column separation; complete sulfation, all sulfates GPIbα in which the sulfation site is sulfated; and 0 sulfation, GPIbα in which no sulfation site is sulfated. It is a graph which shows the sulfated isoform isolate | separated by AEX-HPLC. It is a graph which shows the isoform of GPIb (alpha) isolate | separated by the size in SEC-HPLC. FIG. 6 is a graph showing the reproducibility of the GPIbα assay and the calculated percent variance. Figure 2 is a graph showing the binding specificity of the GPIbα assay relative to a control (Fc part 1). FIG. 2 is a graph showing the binding specificity of the GPIbα assay relative to a control (Fc part 2 and Fc part 3).

Claims (26)

  A method for 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 that binds to GPIbα. (C) adding a detection compound specific for GPIbα; (d) adding a complexing compound that binds to the binding protein from step (b); and (e) from step (c). Detecting a detection compound, wherein the positive detection signal is indicative of the presence of GPIbα in the biological sample.   The method of claim 1, wherein the biological sample comprising GPIbα is plasma.   The method of claim 2, wherein the plasma is derived from a mammal.   4. The method of claim 3, wherein the mammal is a human.   The method of claim 1, wherein the biological sample containing GPIbα is a cell line-derived supernatant.   6. The method of claim 5, wherein the cell line is a CHO cell line.   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.   The method of claim 1, wherein the binding protein comprises a protein having a binding site different from that of the antibody of claim 7.   10. The method of claim 9, wherein the binding protein is a fragment of von Willebrand factor (vWF).   12. The method of claim 10, wherein the fragment is an A1 domain of vWF.   10. The method of claim 9, wherein the binding protein is a complete vWF protein.   The method of claim 1, wherein the binding protein is biotinylated.   12. The method of claim 11, wherein the A1 domain of vWF is His-tagged.   14. The method of claim 13, wherein vWF is biotinylated.   The method of claim 1, wherein the complexing compound that binds to the binding protein is streptavidin-coated magnetic beads.   The method of claim 1, wherein the complexing compound that binds to the binding protein is an anti-His coated magnetic bead.   The method according to claim 7, wherein the detection compound specific for GPIbα is labeled with a chemiluminescent substance.   9. The method of claim 8, wherein the Fab fragment specific for GPIbα is labeled with a chemiluminescent substance.   The method of claim 1, wherein detecting the detection compound in step (e) further comprises exposing the chemiluminescent material and measuring the excitation of the chemiluminescent material.   A method for measuring the protein concentration of GPIbα in a biological sample, comprising: (a) preparing a substance containing GPIbα; (b) contacting the substance from step (a) with a binding protein that binds to GPIbα. (C) adding a detection compound specific for GPIbα; (d) adding a complexing compound that binds to the binding protein from step (b); and (e) from step (c). Detecting a detection compound, wherein the detection signal compared to a known standard curve is indicative of the protein concentration of GPIbα in the biological sample.   22. The method of claim 21, wherein detecting the detection compound further comprises exposing the chemiluminescent material and measuring the excitation of the chemiluminescent material that exhibits a protein concentration of GPIbα when compared to a known standard curve.   A method for detecting the binding activity of GPIbα in a biological sample, comprising: (a) preparing a substance containing GPIbα; (b) contacting the substance derived from step (a) with a binding protein that binds to GPIbα. (C) adding a detection compound specific for GPIbα; (d) adding a complexing compound that binds to the binding protein from step (b); (e) detection from step (c). Detecting the compound, wherein the 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.   (F) Binding activity X is (A / B) × 100% [where A is the protein concentration measured for GPIbα by vWF interaction and B is measured by protein A HPLC or Fc capture immunoassay. 24. The method of claim 23, wherein   A method for detecting a GPIbα isoform by measuring a binding activity of a GPIbα isoform in a biological sample, comprising: (a) preparing a GPIbα-containing substance and a GPIbα-like substance; a) contacting the derived material with a binding protein that binds to GPIbα; (c) adding a detection compound specific for GPIbα; (d) complexing that binds to the binding protein from step (b). (E) detecting the detection compound from step (c), wherein the detection signal compared to a known standard curve indicates the protein concentration of GPIbα in the biological sample; ) Calculating the binding activity of the isoform of GPIbα; and (g) comparing the binding activity of the isoform to the binding activity of a known GPIbα control. , The method comprising.   (F) Binding activity X is (A / B) × 100% [where A is the protein concentration measured for GPIbα by vWF interaction and B is measured by protein A HPLC or Fc capture immunoassay. 26. The method of claim 25, wherein:

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