Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

• HEPARIN BINDING PROTEINS SENSORS FOR HEPARIN DETECTION
• Heparin is a highly heterogeneous glycosaminoglycan (GAG), a family of polysaccharides with alternating uronic acid and aminoglycoside residues that is extracted from mast cells of porcine intestinal mucosa or bovine lung.
• GAG glycosaminoglycan
• AT HI antithrombin HI
• heparin detection is very important in the treatment of a number of diseases and therapeutic procedures. There is a need for accurate and simple direct means for detecting heparin.
• molecules for detecting heparin and for example, molecules that can quantitate heparin, and methods of using these molecules.
• compositions comprising a heparin binding molecules and nucleic acids thereof, as well as methods for making the protein and the nucleic acid, and methods of using the heparin binding protein and nucleic acid thereof.
• Fig. 1 shows the partial tetrasaccharide structures of HA and heparin.
• Fig. 2 shows the schematic preparation of GST-HBI, GST-HB2 and GST-HB3 constructs.
• Panel A shows RHAMM(518-580).
• Panel B shows the cloning strategy.
• Fig. 3 shows expression and purification of GST-HB proteins.
• Panel A shows SDS/PAGE of post-sonication supernatant protein expression; boxes show the GST alone and GST-HB fusion proteins.
• Panel B shows protein purification on GSH-Sepharose beads, following elution of GST and GST-HB proteins with GSH.
• the lanes are 1,GST; 2, GST- HB1; 3, GST-HB2; 4, GST-HB3.
• Fig. 4 shows protein titration for three GST-HB proteins using ELISA with immobilized heparin. Key: diamonds, GST alone; squares, GST-HBI; triangle, GST-HB2; cross, GST-HB3.
• Fig. 5 shows competition ELISAs for three GST-HB proteins using immobilized heparin.
• Competitors Panel A: HA, CS-A, CS-C, UFH; Panel B, HS, 5 ⁇ g/ml and 200 ⁇ g/ml; KS, 5 ⁇ g/ml and 200 ⁇ g/ml. Control: no competitor added.
• Fig. 6 shows quantitative competitive ELISAs using immobilized heparin and detection with GST-HB3, A: HA (Mw 190 kDa); B: CS-A; C: CS-C; D: UFH.
• Fig. 7 shows measurement of UFH by ELISA with immobilized heparin and GST- HB3 detection.
• Panel A shows Serial 1:2 dilutions;
• Panel B shows log-log plot showing linear range over three decades of UFH concentrations.
• Fig. 8 shows ELISA quantification of heparin standards in human plasma. Key: squares, UFH; triangles, LMWH.
• Fig. 9 shows the plasmid construction for a heparin binding molecule.
• Fig. 10 shows a competitive ELISA performed with multiple glycosaminoglycans using biotinylated heparin on a streptavidin-coated plate. Chondroitin sulfate (CS)-A, CS- C, HA, keratan sulfate (KS), heparan sulfate (HS), and unfractionated heparin (UFH) were selected as competitors in a range of 5 ⁇ g/ml-200 ⁇ g/ml.
• FIG. 11 shows a competitive ELISA a clinical assay using both standard well formats.
• the assay is useful for both the traditional unfractionated heparin (UFH) and the newer low molecular weight heparins (LMWH). Due to the hydrophilic nature of heparin, streptavidin-coated microtiter plates treated with commercially available biotinylated heparin are used.
• Fig. 12 shows a sandwich format ELISA. A "capture protein" is used to coat the wells. HB3-GST is used as the detection probe.
• Fig. 13 shows quality control (QC) of a heparin coated surface.
• Fig. 14 shows the effect of adding human plasma on heparin ELISA.
• Fig. 15 shows the effect of NaCl on heparin ELISA. Key: diamonds, 150 mM, squares, 300 mM, triangles, 500 mM, cross, 750 mM, snowflake, 1000 mM.
• Fig. 16 shows analysis of polyelectrolyte theory data for heparin-HB3 binding using a log K vs. log[NaCl] plot.
• Fig. 17 shows unfractionated heparin was the only glycosaminoglycan that reacted with HBP in specificity studies.
• Fig. 18 shows an example of a heparin ELISA, wherein the heparin is bound to the inside ofthe microplate well.
• Fig. 19 shows an example of an ELISA plate setup.
• Fig. 20 shows a competitive ELISA binding reaction. Unknowns and standards were added to the wells, then HB3-HRP was added. The sample was then incubated.
• Fig. 21 shows how the assay of Fig. 20 appears after a wash step. TMB was added, then the sample was incubated, stop reagent added, and the plates were read at 450nm.
• Fig. 22 shows a low molecular weight heparin (LMWH) ELISA. All major clinical
• LMWHs are bound.
• Fig. 23 shows an unfractionated heparin (UFH) ELISA. All major clinical UFHs are bound.
• Fig. 24 shows an ELISA of enoxaparin in plasma. This assay can be used to detect how much heparin is in the plasma of a subj ect.
• Fig. 25 shows an ELISA of UFH in plasma. This assay can be used to detect how much heparin is in the plasma of a subject.
• Fig. 26 shows synthetic heparin vs. tinzaparin using a LMWH ELISA. The synthetic heparins are easily measurable using this assay.
• Fig. 27 shows an extended range ELISA. Heparin can be detected at less than 0.1
• Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use ofthe antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each ofthe ranges are significant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
• HBM heparin binding molecule
• compositions comprising heparin binding molecules (HBM), wherein the heparin binding molecules are comprised of at least one heparin binding unit. Also disclosed are nucleic acids that encode heparin binding molecules. These compositions aid in the detection of heparin.
• the compositions are typically composed of a number of parts, each of which can be a variety of molecules or compositions. Each part ofthe compositions, how to make them, and how to use them is discussed below. 1.
• Heparin binding molecules can be any molecule that binds heparin.
• the HBM can be comprised of one or more individual units, called heparin binding units (HBUs).
• HBUs heparin binding units
• the molecules bind heparin so that the HBM-heparin complexes can be detected.
• HBMs can be linked or combined with any other molecule that may be useful for detection ofthe HBM, manipulation ofthe HBM, or, for example, purification ofthe HBM.
• the HBM will be a peptide, but as discussed herein the peptides can be modified in many ways to provide a variety of useful characteristics, including increased affinity for heparin, or increased stability, or to, for example, attach the peptide to a solid support.
• any known heparin binding molecule could be used in conjunction with an HBU or HBM disclosed herein.
• a) Peptide HBMs In certain embodiments the HBM is a peptide based molecule, meaning that one or more ofthe HBU is a peptide based molecule.
• the HBU is comprised ofthe sequence found in SEQ ID NO: 1, which is two basic amino acids flanking a seven amino acid stretch (hereinafter called BX B). The BX 7 B molecule is known to be minimally required for binding to hyaluronan 41 ' 60 . This domain has been identified in the N- terminal end of H3P molecules (a precursor to a hyaluronan binding molecule).
• Hyaluronan is an unsulfated and homogenous glycosaminoglycan (GAG), with a regular repeating disaccharide consisting of alternating glucoronic acid and N-acetylglucosamine residues in alternating ⁇ -1,4- and ⁇ -1,3 glycosidic linkages.
• Heparin has 1,4-glycoside linkages and no regular repeat unit; it is heterogenous, having 2 epimeric uronic acids, and both N- and O-sulfation.
• One type of protein that contains a HBU is the RHAMM protein (SEQ ID NO: 7).
• RHAMM belongs to a heterogeneous group of proteins designated hyaladherins, which are linked by their common ability to bind hyaluronan.
• RHAMM mediates cell migration and proliferation 48 , and isoforms can be found in cytoplasm as well as on the surfaces of activated leukocytes, subconfluent fibroblasts 49 ' 50 and endothelial cells 51 .
• RHAMM expression in cell-surface variants promoted tumor progression in selected types of cancer cells 52 .
• Intracellular RHAMM has been shown to bind to cytoskeletal proteins, to associate with erk kinase, and to mediate the cell cycle through its interaction with pp60 v"src' 53 .
• the BX 7 B molecule is found within RHAMM. It is understood that in certain embodiments the HBM is not a RHAMM protein, for example, having SEQ ID NO: 7.
• the HBU can also be a portion ofthe RHAMM molecule.
• RHAMM has been found to contain a 62- amino acid heparin binding domain (HABD) with two base- rich BX B motifs, which possesses an overall helix-turn-helix structure (SEQ ID NO: 6, Example 1). This molecule binds with high affinity to heparin as well as to HA.
• GST fusion proteins containing one, two, or three copies ofthe RHAMM HABD (HBl, HB2, and HB3, respectively) were cloned, expressed, and purified. The affinity of these proteins for HA and heparin was determined by competitive ELISA.
• the ELISA employed an immobilized ligand, i.e., biotinylated hyaluronan or biotinylated heparin (HA), bound to a streptavidin-coated microtiter plate.
• immobilized ligand i.e., biotinylated hyaluronan or biotinylated heparin (HA)
• HA biotinylated heparin
• GST-HB3 detected calibration standards of both UFH and LMWH that had been added to plasma at very low levels.
• an ELISA using biotinylated heparin as the immobilized ligand confirmed that affinity increased with the HABD copy number.
• the three-copy construct, GST-HB3, showed excellent sensitivity; 0.1 U/ml free heparin was readily measured.
• GST- HB3 showed a minimum 100-fold selectivity for heparin over other glycosaminoglycans.
• the plot of log Ka vs. log [Na + ] showed between two and three ionic interactions per heparin-HB3 binding based on polyelectrolyte theory (PET).
• HBU Heparin Binding Unit
• HBM can be anything that binds heparin, but in many embodiments they will be peptide based molecules.
• SEQ TO NO:l, BX B is an example of a HBU.
• a HBM is simply composed of a HBU.
• HBUs are typically linked together to form HBMs, although this is not required for the compositions to display heparin binding activity, as only one HBU is required to form an HBM.
• An HBM can comprise a single HBU, or an HBU linked to a second HBU, or a first, second, and third HBU all linked together, and so on, for example.
• HBUs There can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more HBUs linked together. It is understood that that they can be linked in series, i.e. one HBU linked to no more than two other HBUs, or they can be linked in aggregate, i.e., one HBU can be linked to more than two HBUs, such as 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10, or more HBUs.
• the HBUs can be linked via a cleavable bond.
• Such cleavable linkers allow the individual heparin binding units to be released under reducing conditions, oxidizing conditions, or by hydrolysis of an ester, amide, hydrazide, or similar linkage.
• Such linkers may include succinates, disulfide-containing chains, and diol-containing chains. It is understood that one HBM can contain different HBUs, linked by different linkers, for example, different cleavable linkers, cleavable linkers and non-cleavable linkers, and so forth. They may also include short peptides with specific targeting sequences for lysosomes and for lysosomal degradation, such as Gly-Phe-Leu-Gly. Other examples include a flexible linker, such as (GlySer) Gly. Other linkers can be used as well, including peptide linkers, peptide analog linkers, and so forth.
• the polypeptide linker may be from 1 or 2 amino acids to 100 amino acids in length, or more, with every specific length and combination between 1 and 100 disclosed herein, or between 4 to 50 residues, or optimally between 8 and 30 amino acids in length. Sequences that permit proper folding ofthe recombinant HBUs expressable in heterologous expression systems could also, for example, use Thr, and/or Ala residues in place of some Ser, Gly residues, and other amino acids may be tolerated. Alternatively, the HBUs may be connected with synthetic, flexible non-peptide linkers, such as polyethylene glycol linkers. It is understood that when HBUs comprise a protein they can be a recombinant protein, meaning they are made using molecular biology techniques.
• HBM fusion proteins The HBM can be part of a fusion protein.
• the HBM can be fused to a glutathione S-transferase (GST) gene.
• GST glutathione S-transferase
• Other fustion partners include but are not limited to His tags (polyhistidine fusion system, vector pET-21d), c-myc tags, FLAG tags, thioredoxin fusions, or maltose binding protein (MBP) fusions, for example.
• GST gene fusion system is an integrated system that can be used for the expression, purification and detection of fusion proteins produced in bacterial, yeast, mammalian and insect cells.
• the sequence encoding the GST protein is incorporated into an expression vector, generally upstream of the multi-cloning site.
• the sequence encoding the protein of interest is then cloned into the vector.
• Induction ofthe vector results in expression of a fusion protein- the protein of interest fused to the GST protein.
• the fusion protein can then be released from the cells and purified. Purification ofthe fusion protein is facilitated by the affinity ofthe GST protein for glutathione residues. Glutathione residues are coupled to a resin and the expressed protein product is brought into contact with the resin.
• the fusion protein will bind to the glutathione-resin complex and all other non-specific proteins can be washed off.
• the fusion protein can then be released from the resin using a mild elution buffer which is of low pH.
• the pH can be from about 0.1 to about 7.0, or from about 1.0 to about 6.0, or from about 2.0 to about 5.0. It is possible to remove the GST from the protein of interest by using a number of different enzymes such as, for example, thrombin and factor X, which cleave specific sites between the GST and the protein of interest. Fusion proteins can also be detected easily, with a number of GST antibodies available on the market.
• HBM and reporter molecules The HBM can also comprise reporter molecules.
• the reporter molecules can be any molecule that allows for detection ofthe HBM. It is understood that the reporter molecules, can also be linked to the target, ofthe HBM, such as heparin.
• the reporter molecules can be anything that allows for detection ofthe HBM or a molecule bound to the HBM.
• the reporter molecules can be any chemiluminescent or bioluminescent molecules, but they could also be phosphorescent or radioactive, for example.
• chemiluminescent or bioluminescent molecules but they could also be phosphorescent or radioactive, for example.
• reporter molecules include, but are not limited to bacterial alkaline phosphatase (BAP) green fluorescent protein (GFP), beta-glucuronidase (GUS), secreted alkaline phosphatase (SEAP), red fluorescent protein (RFP), horseradish peroxidase conjugation (HRP) and luciferase.
• HBM fusion proteins can be comprised of capture tags or capture tag receptors. The capture tags can be used to separate molecules which have a capture tag away from molecules which do not.
• a capture tag is any compound that can be associated with a HBM or HBU, or any other composition discussed herein, and which can be used to separate compounds or complexes having the capture tag from those that do not.
• a capture tag is a compound, such as a ligand or hapten, that binds to or interacts with another compound called a capture tag receptor, such as a ligand-binding molecule or an antibody. It is also preferred that such interaction between the capture tag and the capturing component, capture tag receptor, be a specific interaction, such as between a hapten and an antibody or a ligand and a ligand-binding molecule.
• a capture tag and capture tag receptor combination can be referred to as a capture tag system.
• Suitable capture tags include hapten or ligand molecules that can be coupled to the disclosed compositions such as an HBM or HBU.
• Preferred capture tags described in the context of nucleic acid probes, have been described by Syvanen et al., Nucleic Acids Res., 14:5037 (1986)), which can be adapted for protein use.
• Preferred capture tags include biotin, which can be incorporated into nucleic acids or proteins (Langer et ah, Proc. Natl. Acad. Sci. USA 78:6633 (1981)) and captured using the capture tag receptors, streptavadin or biotin-specific antibodies.
• a preferred hapten for use as a capture tag is digoxygenin (Kerkhof, Anal. Biochem.
• capture tags can be captured by antibodies which recognize the compound.
• Antibodies useful as capture tags can be obtained commercially or produced using well established methods. For example, Johnstone and Thorpe, bnmunochemistry In Practice
• any antigen: antibody combination can be used as a capture tagxapture tag receptor, forming a capture tag system.
• One type of capture tag is the anti-antibody method.
• anti-antibody antibodies and their use are well known.
• anti-antibody antibodies that are specific for antibodies of a certain class for example, IgG, IgM
• antibodies of a certain species for example, anti-rabbit antibodies
• Another type of capture tag is one which can form selectable cleavable covalent bonds with other molecules of choice.
• a preferred capture tag of this type is one which contains a sulfer atom.
• An HBU or HBM or any other molecule which is associated with this capture tag can be purified by retention on a thiolpropyl sepharose column.
• Capture tags can be associated with the disclosed compositions, such as HBM or HBU, and then the [capure tag:HBM], for example, complex is selectively isolated from the molecules which are not associated with the capture tag. There is then a capture tag receptor (CTR) that can interact with the capture tag complex.
• CTR capture tag receptor
• the capture tags or CTRs can be associated with any type of support, such as a solid support.
• CTRs or capture tags When a CTR is bound to a solid support, capture tag complexes are bound to CTRs of this type they can be effectively purified from the unwanted molecules because the solid support allows for successive washing to remove unbound molecules.
• Supports that the CTRs or capture tags can be coupled to can be any solid material to which the CTRs or capture tags can be adhered or coupled.
• Supports can have any useful form including thin films or membranes, beads, bottles, dishes, fibers, woven fibers, shaped polymers, particles and microparticles. Certain forms of supports are plates and beads, and another form are magnetic beads.
• Adhering or coupling assay components to a substrate is preferrably accomplished by adhering or coupling CTRs or capture tags to the substrate.
• the CTRs or capture tags can then mediate adherance of an assay component such as a primer or protein, or for example, an HBM or HBU, by binding to, or interacting with, a capture tag on the component.
• CTRs or CTs immobilized on a substrate allow capture ofthe associated molecules, such as an HBM or HBU, on the substrate. Such capture provides a convenient means of washing away reaction components that might interfere with subsequent detection steps.
• CTRs or CTs By attaching different CTRs or CTs to different regions of a solid-state detector, different molecules, such as HBMs or HMUs can be captured at different, and therefore diagnostic, locations on the solid-state detector.
• CTRs or CTs specific for up to 96 different molecules can be immobilized on a microtiter plate, each in a different well. Capture and detection will occur only in those wells corresponding to the specific capture tag system for winch the corresponding sample molecules are made.
• Methods for immobilization of oligonucleotides to substrates are well established. Oligonucleotides, including oligonucleotide capture docks, can be coupled to substrates using established coupling methods. For example, suitable attachment methods are described by Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994), and
• Some substrates useful in the disclosed assays have detection antibodies attached to one or more molecules in the assay, such as the capture tag or the molecule attached to the capture tag, or the target sample, the substrate for the molecule attached to the capture tag.
• molecules can be specific for a molecule of interest.
• Captured molecules of interest can then be detected by binding of a second, reporter molecule, such as an antibody.
• a second, reporter molecule such as an antibody.
• Such a use of antibodies in a solid-state detector allows assays to be developed for the detection of any molecule for which antibodies can be generated.
• Methods for immobilizing antibodies to solid-state substrates are well established. Immobilization can be accomplished by attachment, for example, to aminated surfaces, carboxylated surfaces or hydroxylated surfaces using standard immobilization chemistries.
• attachment agents are cyanogen bromide, succinimide, aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable agents, epoxides and maleimides.
• a preferred attachment agent is glutaraldehyde.
• Antibodies can be attached to a support by chemically cross-linking a free amino group on the antibody to reactive side groups present within the solid-state support.
• antibodies may be chemically cross-linked to a support that contains free amino or carboxyl groups using glutaraldehyde or carbodiimides as cross-linker agents.
• aqueous solutions containing free antibodies are incubated with the solid-state substrate in the presence of glutaraldehyde or carbodiimide.
• the reactants can be incubated with 2% glutaraldehyde by volume in a buffered solution such as 0.1 M sodium cacodylate at pH 7.4.
• Solid-state samples are solid-state substrates or supports to which target molecules or target sequences have been coupled or adhered, for example, through capture tag technology. Target molecules or target sequences are preferably delivered in a target sample or assay sample.
• One form of solid-state sample is an array sample.
• An array sample is a solid-state sample to which multiple different target samples or assay samples have been coupled or adhered in an array, grid, or other organized pattern.
• Solid-state substrates for use in solid-state samples can include any solid material to which target molecules or target sequences can be coupled or adhered. This includes materials such as acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumarate, collagen, glycosaminoglycans, and polyamino acids.
• materials such as acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid
• Solid-state substrates can have any useful form including thin films or membranes, beads, bottles, dishes, slides, fibers, woven fibers, shaped polymers, particles and microparticles.
• Preferred forms for a solid-state substrate are microtiter dishes and glass slides.
• One form of microtiter dish is the standard 96-well type.
• Target molecules and target sequences immobilized on a solid-state substrate allow formation of target-specific molecule combinations localized on the solid-state substrate. Such localization provides a convenient means of washing away reaction components that might interfere with subsequent detection steps, and a convenient way of assaying multiple different samples simultaneously. Diagnostic combinations can be independently formed at each site where a different sample is adhered.
• the methods described above for can be used.
• the target molecule is a protein or a polysaccharide
• the protein or polysaccharide can be immobilized on a solid-state substrate generally as described above for the immobilization of antibodies.
• solid-state substrate is a glass slide to which up to 256 separate target or assay samples have been adhered as an array of small dots. Each dot is preferably from 0.1 to 2.5 mm in diameter, and most preferably around 2.5 mm in diameter.
• Such microarrays can be fabricated, for example, using the method described by Schena et ah, Science 270:487-470 (1995).
• microarrays can be fabricated on poly-L-lysine-coated microscope slides (Sigma) with an arraying machine fitted with one printing tip.
• the tip is loaded with l ⁇ l of a DNA sample (0.5 mg/ml) from, for example, 96-well microtiter plates and deposited -0.005 ⁇ l per slide on multiple slides at the desired spacing.
• the printed slides can then be rehydrated for 2 hours in a humid chamber, snap-dried at 100°C for 1 minute, rinsed in 0.1% SDS, and treated with 0.05% succinic anhydride prepared in buffer consisting of 50% l-methyl-2-pyrrolidinone and 50% boric acid.
• the DNA on the slides can then be denatured in, for example, distilled water for 2 minutes at 90°C immediately before use.
• Microarray solid-state samples can be scanned with, for example, a laser fluorescent scanner with a computer-controlled XY stage and a microscope objective.
• a mixed gas, multiline laser allows sequential excitation of multiple fluorophores.
• CTs and CTRs and solid supports and solid state components can be used in any combination.
• a given assay system may have more than one capture tag system employed, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more systems employed.
• different combinations or solid supports and solid states can be used in any given system.
• HBM heparin binding activity Disclosed are HBMs and variants that bind heparin with a Kd of less than or equal to
• HBMs and variants that bind heparin with an affinity that is at least 2, 4, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 300, or 500 fold greater than the affinity with which it binds another aminoglycosan, such as HA.
• the HBM can bind molecules other than heparin.
• HBMs can also bind dextran sulfate, dermatan sulfate, and heparan sulfate.
• heparin can be used interchangeably with these molecules, and they can be detected and quantified using the same methods disclosed to detect and quantify heparin. Also disclosed are HBMs and variants that have residual heparin binding activity of at least between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,.
• the various binding affinities for heparin can be determined as disclosed herein or using any assay for determining binding constants, such as equilibrium dialysis or column chromatography. It is also understood that each individual HBM variant also has a base heparin binding rate which can be detennined from the disclosed residual heparin amounts. It is understood that these percentages of base heparin binding rates can be calculated from a base residual heparin amount obtained at any time, which provides data in the analytical range ofthe assay unless otherwise indicated. Disclosed are variants of HBMs that have the property of being able to bind heparin.
• HBMs that bind heparin with at least 5%, 10%, 15%, 20%, 25%, 30% 35%,
• each individual HBM variant discussed also has a base heparin binding activity which can be determined from the amount of residual heparin, as disclosed below. It is understood that these percentages of activity can be calculated from a base residual heparin binding activity obtained at any time which provides data in the analytical range ofthe assay, unless otherwise indicated.
• the residual heparin represents the amount of heparin that remains, typically after a
• the residual heparin is quantified by taking the ratio ofthe residual heparin after incubation with an HBM to the residual heparin after incubation with buffer.
• the residual heparin can be calculated by subtracting the residual heparin from 100 (100 represents a state of effectively no inhibition). It is understood that if variants of HBMs obtain better binding activity, the timing ofthe reaction can be decreased, to for example, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute.
• the incubation can be increased to, for example, 12, 14, 16, 18, 20, 25, 30, 45, or 60 minutes.
• One or more assays can be performed with different incubation times to obtain residual heparin amounts that fall between 1 and 100, and, for example, at least two times can be performed for a given HBM so that it can be verified that the assay is being performed in the analytical range.
• variants refers to variations in the sequence of either a nucleic acid or a peptide molecule.
• variants designate specific properties dependent on the specific substitutions denoted, however, other substitutions, deletions, and/or insertions, for example, conservative substitutions, insertions, and/or deletions at positions other than the specifically denoted positions are also contemplated provided the variants retain the disclosed activities.
• variants that produce HBMs that have the properties disclosed herein.
• substitutions wherein the substitutions are made at positions B ls B 2 , X 1? X 2 , X 3 , X 4 , X 5 , X 6 , or X 7 ofthe BiX 7 B 2 molecule, either alone or in combination.
• variants which have 8 amino acids or 6 amino acids between B ⁇ and B 2 .
• the ⁇ and B 2 represent basic amino acids and the X 1-7 or X 1-6 or X 1-8 represent any amino acid other than an acidic amino acid as long as one X is a basic amino acid.
• X 1-7 or X 1-6 or X ⁇ -8 can be Gly, Ala, Val, Leu, He, Ser, Thr, Tyr, Cys, Met, Asn, Gin, Arg, Lys, His, Phe, Trp, Pro, but not Asp or Glu, and within the string there must be at least one Arg, Lys, or His. It is understood that every embodiment ofthe B 1 X 1 .
• B 1 X 1-8 B 2 is specifically disclosed. Applicants have not written each specific species within these sets out, but it is understood that each and every species is specifically disclosed and can be either considered a part of certain embodiments or not a part of certain embodiments. Examples of different B 1 X 1-6 B ) B K ⁇ . 7 B 2 , or B ! X 1-8 B 2 molecules can be found, for example, in Table 1. Other examples can be found by for example performing different Blast analysis relating to the varying HBUs disclosed herein. Also disclosed are variants with substitutions to the RHAMM (518-580) molecule.
• HBM proteins and RHAMM proteins As discussed herein there are numerous variants ofthe HBM proteins and RHAMM proteins that are known and herein contemplated.
• derivatives ofthe HBM and RHAMM proteins which also function in the disclosed methods and compositions.
• Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications.
• amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
• Immunogenic fusion protein derivatives are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross- linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion.
• Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule.
• These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
• substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis.
• Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
• Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
• the mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
• substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 2 and 3 and are referred to as conservative substitutions. TABLE 2: Amino Acid Abbreviations
• substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area ofthe substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site or (c) the bulk ofthe side chain.
• the substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
• an electropositive side chain e.g., lysyl, arginyl, or histidyl
• an electronegative residue e.g., glutamyl or aspartyl
• substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).
• Deletions of cysteine or other labile residues also may be desirable.
• Deletions or substitutions of potential proteolysis sites e.g. Arg
• Arg is accomplished for example by deleting one ofthe basic residues or substituting one by glutaminyl or histidyl residues.
• Certain post-translational derivatizations are the result ofthe action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions.
• nucleic acid sequences related to a specific protein sequence i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives ofthe protein sequences.
• each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular species from which that protein arises is also known and herein disclosed and described.
• amino acid and peptide analogs which can be incorporated into the disclosed compositions.
• D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 2 and Table 3.
• the opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs.
• These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al., Methods in Molec. Biol.
• Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage.
• peptide analogs can have more than one atom between the bond atoms, such as ⁇ -alanine, ⁇ -aminobutyric acid, and the like.
• Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activities), reduced antigenicity, and others.
• D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such.
• SEQ ID NO:l sets forth a particular sequence of an HBU and SEQ ID NO:7 sets forth a particular sequence of a RHAMM protein.
• variants of these and other proteins herein disclosed which have at least, 60% or 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence.
• the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
• conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 60%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% homology to a particular sequence wherein the variants are conservative mutations.
• one way to define any known variants and derivatives or those that might arise, ofthe disclosed genes and proteins herein is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein.
• variants of genes and proteins herein disclosed typically have at least, about 40, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence.
• the homology can be calculated after aligning the two sequences so that the homology is at its highest level. Another way of calculating homology can be performed by published algorithms.
• Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.
• the same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M.
• a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more ofthe calculation methods described above.
• a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any ofthe other calculation methods.
• a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any ofthe other calculation methods.
• a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).
• hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
• Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide.
• the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature ofthe reaction all affect whether two nucleic acid molecules will hybridize.
• selective hybridization conditions can be defined as stringent hybridization conditions.
• stringency of hybridization is controlled by both temperature and salt concentration of either or both ofthe hybridization and washing steps.
• the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm.
• the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids).
• a preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C.
• Stringency of hybridization and washing if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
• stringency of hybridization and washing if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.
• selective hybridization is by looking at the amount (percentage) of one ofthe nucleic acids bound to the other nucleic acid.
• selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent ofthe limiting nucleic acid is bound to the non-limiting nucleic acid.
• the non-limiting primer is in for example, 10 or 100 or 1000 fold excess.
• This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k , or where only one ofthe nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k ⁇ .
• Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation.
• selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent ofthe primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent ofthe primer molecules are extended.
• Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.
• homology it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any ofthe methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein. It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein. 2.
• Nucleic Acids There are a variety of molecules disclosed herein, such as various variant HBMs. It is understood that these peptide based molecules can be encoded by a number of nucleic acids, including for example the nucleic acids that encode, for example, SEQ ID NO.T. It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U. a) Sequences There are a variety of sequences related to BX 7 B, RHAMM, and subsections of
• RHAMM such as HABD
• HABD HABD
• Genbank database which can be accessed at www.pubmed.gov.
• SEQ ID NO: 1 One particular sequence set forth in SEQ ID NO: 1 is used herein, as an example, to exemplify the disclosed compositions and methods.
• Nucleic acids comprising a sequence, wherein the sequence encodes a heparin binding peptide are disclosed.
• SEQ ID NO: 8 is the nucleic acid molecule corresponding to the peptide sequence of SEQ JO NO: 7.
• SEQ JD NO: 10 is the nucleic acid molecule corresponding to the peptide sequence of SEQ ID NO: 9.
• SEQ ID NO: 12 is the nucleic acid molecule corresponding to the peptide sequence of SEQ ID NO: 11. It is understood that the description related to this sequence is applicable to any sequence related to HBMs unless specifically indicated otherwise.
• the HBMs can be fused to various molecules such as fluorescent, chromogenic, or GST molecules. Nucleic acids corresponding to those molecules are also disclosed.
• the HBM nucleic acid can further comprise a BAP nucleic acid, for instance.
• the HBM nucleic acid can also further comprise and EGFP nucleic acid.
• the HBM nucleic acid can also further comprise a bacterial GST nucleic acid.
• the nucleic acid can be contained in a vector, such as a plasmid, for example.
• a vector such as a plasmid
• examples of such vectors are well known in the art.
• Primers and/or probes can be designed for any HBM related nucleic acid sequence given the information disclosed herein and known in the art.
• Primers and probes Disclosed are compositions including primers and probes, which are capable of interacting with nucleic acids related to HBMs as disclosed herein. In certain embodiments the primers are used to support DNA amplification reactions.
• the primers will be capable of being extended in a sequence specific manner.
• Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition ofthe nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence ofthe product produced by the extension ofthe primer.
• Extension ofthe primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred.
• the primers are used for the DNA amplification reactions, such as PCR or direct sequencing.
• the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner.
• the disclosed primers hybridize with the nucleic acids related to HBMs or regions ofthe nucleic acids related to the HBMs or they hybridize with the complement ofthe nucleic acids related to the HBMs or complement of a region ofthe nucleic acids related to the HBM gene.
• the primers and probes can be any size that meets the requirements of being a primer or probe including, but not limited to 3, 4, or 5 nucleotides long
• the size ofthe primers or probes for interaction with the nucleic acids related to the HBMs in certain embodiments can be any size that supports the desired enzymatic manipulation ofthe primer, such as DNA amplification or the simple hybridization ofthe probe or primer.
• a typical primer or probe for nucleic acids related to the HBMs would be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475,
• the primers for the nucleic acids related to HBMs typically will be used to produce an amplified DNA product that contains an HBM.
• the size ofthe product will be such that the size can be accurately determined to within 3, or 2 or 1 nucleotides.
• this product is at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,
• primers which are useful with the present invention for amplifying the HABD molecule include the following: SEQ ID NO: 2 5'-CGGGATCCGGTGCTAGCCGTGACTCCTATGCACAGCTCCTTGG-3* SEQ ID NO: 3 5'-GGAGCGGTCGACACGGATGCCCAGAGCTTTATCTAATTC-3' SEQ ID NO: 4 5'-GATCCGGTCTCGAGGGAAGTGGTTCTGGAAGTGGTTCAGGTTCGGGTA GCGGATCTGGTTCAGGAAGTGGTT-3* SEQ IDNO: 5 5'-CTAGAACCACTTCCTGAACCAGATCCGCTACCCGAACCTGAACCACTT CCAGAACCACTTCCCTCGAGACCG-3' B.
• compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted. It is understood that general molecular biology techniques, such as those disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) are available for making the disclosed molecules and practicing the disclosed methods unless otherwise noted. 1. Nucleic acid synthesis
• the nucleic acids such as the oligonucleotides to be used as primers, can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method.
• Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laborato ⁇ y Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen- Biosearch, Burlington, MA or ABI Model 380B). Synthetic methods useful for making oligonucleotides are also described by Ikuta et al., Ai ⁇ n. Rev. Biochem.
• amino acids or peptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA).
• Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry Applied Biosystems, Inc., Foster City, CA.
• a peptide or polypeptide corresponding to the disclosed proteins for example, can be synthesized by standard chemical reactions.
• a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
• the peptide or polypeptide is independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions. For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abralnnsen L et al., Biochemistry, 30:4151 (1991)).
• native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments.
• This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
• the first step is the chemoselective reaction of an unprotected synthetic peptide—thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product.
• this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et al, LBiol.Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
• unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result ofthe chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)).
• This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry TV. Academic Press, New York, pp. 257-267 (1992)).
• the HBU can be used in a vector for plasmid construction.
• Basic recombinant DNA methods like plasmid preparation, restriction enzyme digestion, polymerase chain reaction, ligation, transformation and protein synthesis were performed according to well-established protocols familiar to those skilled in the art, 61 or as recommended by the manufacturer ofthe enzymes or kit.
• a method for making a fusion protein construct comprising amplifying a reporter nucleic acid, restricting the amplified reporter nucleic acid, and ligating the restricted reporter nucleic acid to an HBM nucleic acid, thereby creating a fusion protein construct.
• an additional step of transforming a bacterial host with the fusion protein construct can then be carried out.
• the HBM nucleic acid can be fused to a GST nucleic acid prior to ligating the HBM nucleic acid to the restricted reporter nucleic acid.
• a method for making a fusion protein nucleic acid comprising amplifying a reporter nucleic acid, restricting the amplified reporter nucleic acid, and ligating the restricted reporter nucleic acid to an HBM nucleic acid, thereby creating a fusion protein nucleic acid.
• an additional step of transforming a bacterial host with the fusion protein nucleic acid can then be carried out.
• the HBM nucleic acid can be fused to a GST nucleic acid prior to ligating the HBM nucleic acid to the restricted reporter nucleic acid.
• the fusion protein can then be expressed and purified.
• One method of making an HBM construct comprises amplifying RHAMM cDNA, for example (SEQ ID NO: 7), digesting the amplified RHAMM, ligating the amplified RHAMM into a vector, and obtaining a product from the vector.
• the method can further comprise introducing a linker into the product, linearizing the vector, and ligating the product into the vector then obtaining a second product from the vector. These steps can be repeated to obtain a third product from the vector as well.
• a 62-amino acid heparin binding domain with two base-rich BX 7 B motifs can be used as an individual HBU, and the units can be linked together to form an HBM (this is the HABD molecule referred to above).
• RHAMM(518-580) cDNA (the 62-amino acid heparin binding domain) can be inserted in a vector such as pGEX-ERL.
• Primers with cleavage sites can then be used to amplify RHAMM(518-580), and the PCR product can then be digested with and ligated into the modified pGEX vector that had been also digested to obtain a construct.
• This construct is referred to as HBl.
• a linker, such as (GlySer)9Gly can then be introduced into the vector and then ligated with another cDNA that had been digested to give an HB2 recombinant construct.
• This construct is considered a heparin binding molecule (HBM).
• HBM heparin binding molecule
• an HB3 construct can be synthesized by repeating the steps above with another linker and amplified cDNA. This construct is also considered an HBM.
• Each ofthe plasmids, as well as the empty vector, can then be transformed into a bacterial host. The desired peptide can then be purified. Fusion proteins can be created in order to facilitate detection or purification.
• One method of making a fusion protein nucleic acid comprises ligating an HBM nucleic acid into a reporter plasmid, thereby creating a fusion protein nucleic acid. The fusion protein can then be expressed and purified.
• a fusion protein can be made using the GST molecule, as disclosed above.
• the fusion protein can be created by using a plasmid inserted into a host.
• the host can be any cell capable of producing a fusion protein.
• One of ordinary skill in the art would be able to use a host to form such a fusion protein.
• the host can be bacterial, such as E. coli, for example.
• E. coli expression plasmids can be generated that carry fusions ofthe appropriate gene fragments.
• EGFP, BAP, and GST-HBM are readily expressed in soluble form in E. coli, for example. Once expressed, all three proteins are relatively stable in a variety of salt, detergent, pH, mildly oxidizing, and denaturing buffers. This allows flexibility to modify purification or assay methods.
• the HBM gene can also be placed in EGFP and pFLAG- BAP, for example, utilizing restriction sites.
• pFLAG-BAP carries an OMP-A leader peptide, which results in the secretion ofthe fusion protein into culture media.
• Growth of E. coli in defined media will allow direct purification by ion-exchange chromatography. Isolation of EGFP-HBM can be achieved using an anti-GFP affinity column.
• C. Methods of Using Heparin is a highly heterogeneous glycosaminoglycan (GAG), a family of polysaccharides with alternating uronic acid and aminoglycoside residues that is extracted from mast cells of porcine intestinal mucosa or bovine lung.
• GAG glycosaminoglycan
• heparin In blood, heparin interacts with AT-HI, which blocks activation of factor Xa and thereby prevents blood coagulation. 3 The anticoagulant effect of heparin is mediated through this interaction, which markedly accelerates the rate of AT HI inhibition of thrombin (factor IJa) and factor Xa.
• UHF unfractionated free heparin
• LMWH low molecular weight heparin
• Unfractionated heparin (UFH) polysaccharides are heterogeneous in length and anticoagulation activity and range in mass from 5000 to 30,000 Da.
• Low-molecular- weight heparins (LMWH) are produced from unfractionated heparin to yield smaller polysaccharides with average molecular masses of 4000-5000 kDa. These shorter molecules lose the ability to accelerate AT IJJ inhibition of thrombin but retain the ability to catalyze factor Xa inhibition. Decreased in vivo protein binding improves LMWH bioavailability and leads to more predictable anticoagulant response.
• Another important aspect of LMWH treatment is that it may be administered as a subcutaneous injection as opposed to an intravenous admimstration of UFH.
• Plasma heparin levels can be detected by several clinically-approved methods: (i) determination of activated coagulation time (ACT), (ii) activated partial thromboplastin time (APTT) 12 , (iii) the heparin management test (HMT) 13 ' 14 or (iv) the anti-factor Xa assay. 15 Another chemical method measured heparin by monitoring inhibition of thrombin activity on a fluorogenic substrate ; however, this method lacked the sensitivity required for clinical use. For over 30 years, the measurement of APTT has remained the most widely used tool for prescribing and monitoring the use of anticoagulants in patients. The APTT is a global screening test of coagulation used to evaluate the intrinsic coagulation pathway.
• heparin It is affected by many variables in addition to heparin, including coagulopathies, inhibitors, and increases of factor Vm and fibrinogen. Secondly, there is no agreement on what value shouldbe used for the denominator of APTT ratios: mean or upper limit APTT of a reference range for normal, or a patient's pretreatment APTT. Most importantly, commercial APTT reagent sensitivities to heparin vary widely. In addition, there are potential surface-to-volume effects when small samples are employed, and the effects that sample processing can have on both the coagulation and thrombotic pathways. Collectively, these factors can introduce significant analytical error when performing an APTT.
• the anti-factor Xa assay is a chromogenic assay that is based on heparin' s ability to inactivate factor Xa in the clotting cascade. In this method, both factor Xa and antithrombin HI are present in excess and the residual factor Xa activity is inversely proportional to the heparin concentration. The assumption is made that the patient has a normal concentration of antithrombin DI. It is recommended to also measure the antithrombin HI levels for all patients when using the anti factor Xa assay. During LMWH therapy there are highly significant differences between anti factor Xa activity results obtained with different assays. The mean of results by one technique have been more than twice those by another.
• the HRD requires 0.5-2 hr for 90% reduction of heparin in blood, and employs an exchange cell in which the heparin diffuses out ofthe plasma and is trapped on the bead-immobilized affinity ligand.
• a combination approach i.e., adding a polyethylene glycol (PEG) 3400 linker, and using 100-kDa PLL pre-coating ofthe fiber membranes, substantially amplifies the protamine removal properties.
• a small cartridge can adsorb 60 mg/g fiber, an 8-fold enhancement over immobilized protamine alone. Immobilized heparinase has also been evaluated for extracorporeal heparin removal.
• heparin can be neutralized by binding to an HBM.
• HBM protamine sulfate
• protamine sulfates and HBMs can be used in conjunction, or the HBM can be used in place of protamine for neutralization ofthe anticoagulant effects of heparin.
• HBM can be used in smaller quantity, thereby alleviating the negative effects of protamine.
• HBM can bind to heparin molecules that protamine does not bind, including synthetic heparins. HBM does not have the allergic effects that protamine can have in some subjects.
• Heparin is the anticoagulant most used for this purpose and is typically immobilized onto the surface of these medical devices. Heparin immobilization can be accomplished by microwave-plasma activation of polypropylene fabrics, followed by grafting of acrylic acid and covalent heparin binding through amide linkages.
• a non-cytotoxic crosslinked collagen suitable for endothelial cell seeding was modified with N-hydroxysuccinimide and carbodiimide chemistry, coupling collagen lysine residues to heparin carboxylates.
• Another alternative is to modify hydrophobic device surfaces by ionic complexation using a polymerizable cationic hpid to form a 60 nm thin layer. All surfaces are subject to patchiness or modification and crazing/cracking as a result of flexing ofthe surface. Determining the uniformity of heparin coating is an important area of quality control (QC).
• QC to show the success of heparin immobilization on devices often consists of testing for adsorbed proteins and soluble activation markers such as antithrombin, thrombin, high-molecular-weight-kininogen (HMWK), and fibrinogen binding capacity.
• soluble activation markers such as antithrombin, thrombin, high-molecular-weight-kininogen (HMWK), and fibrinogen binding capacity.
• HMWK high-molecular-weight-kininogen
• fibrinogen binding capacity 34 ' 35 Others have used clinical methods such as APTT or anti-factor Xa methods to determine the anticoagulant activity of a heparin coating 36 or the relative surface content of sulfur to demonstrate immobilization of heparin on a blood pump.
• Platelet activation and flow cytometry in a whole blood assay has been employed to test heparin-coated tantalum stents and gold-coated stainless steel stents.
• Furthemore the HBMs and HBUs and HABDs can be used to detect to bind any heparin molecule, including those recited herein.
• Various assays can be used in to detect heparin, including ELISAs, fluorescent based assays, APTT (Activated Partial Thrombin Time) assays, and others disclosed herein.
• assays can be used in order to quantify the amount of heparin in a sample.
• One example of a method for determining the amount of heparin in a sample comprises incubating the sample with an HBM in a first incubation, thereby forming a HBM mixture, wherein the HBM mixture allows for the formation of an HBM-heparin complex
• Heparin can be detected in blood, plasma, serum, urine, sputum, peritoneal fluid, or any other bodily fluid for which analytical data are desired. Heparin can also be visualized on a coated surface. Both low molecular weight heparins (LMWH) and unfractionated heparin (UFH) can be detected by the methods described herein.
• LMWH low molecular weight heparins
• UH unfractionated heparin
• the HBMs disclosed herein bind all major unfractionated heparins, such as bovine, porcine, Sigma, high antithrombin affinity fraction, high affinity fraction, and inactive portions of heparin (figures 23 and 25). High affinity heparin fractions are able to bind to AT m, while inactive fragments are defined as those fragments not capable of binding to AT HI.
• the detection assays described herein can detect inactive fragments.
• Inactive fragments can be quantitated, for example, by using an assay that detects only heparin that binds AT m in conjunction with the assays described herein that bind either inactive or active portions of heparin, and conducting a a subtraction assay to determine the amount of bound inactive heparin.
• These inactive fragments are useful to control inflammation, for example, and can be used in a manner similar to chondroitin sulfate.
• Unfractionated heparin can also be detected over an extended range, for example, Figure 27 shows the detection of heparin at less than 0.1 U/ml concentrations.
• LMWHs include those found in Table 4, for example, all of which are detectable by the methods described herein.For example, in Figures 22 and 24, dalteparin, enoxaparin, tinzaparin, ardeparin, and parnaparin are all able to be bound by the HBMs described herein. Synthetic heparin can also be readily detected ( Figure 26).
• these and other LMWH and heparin molecules are and can be used for administration to a subject in need of anticoagulation properties for a period of time. As these molecules are metabolized at different rates and different amounts, for example, can be given, it is advantageous to be able to monitor the amount ofthe administered heparin or LMWH, for example, in real time. It is understood that the disclosed HBMs and HABD and methods of using them are capable of performing this monitoring. Also disclosed are methods of restoring blood coagulation paramaters in a subject in need thereof.
• One method of detecting heparin comprises obtaining a sample, applying the sample to an assay, wherein the assay utilizes an HBM, and detecting the heparin. Also contemplated is a method comprising obtaining a sample, contacting the sample with an HBM, and assaying for HBM-heparin complexes. Also contemplated is a method comprising mixing an HBM and heparin sample together, forming an HBM mixture, and determining if an HBM-heparin complex is present. Specific embodiments are disclosed below. As described above, all types of heparin molecules can be detected, both long and short chain, as well as synthetic heparin.
• heparin detection assays can be used in quality control, pharmacokinetics, protamine sulfate optimization, and correlation assays to determine heparin antiproliferative and anti- inflammatory effects.
• Heparin detection assays can also be used to measure oral, inhalation, and depo-administered heparin in a subject, and to measure leaching of heparin speharose or heparin-coated medical devices such as stents.
• ELISAs ELISAs are widely used in clinical research and diagnostics. Any standard ELISA plate can be used with the disclosed embodiments, including but not limited to 96 and 384 well formats. Both the traditional unfractionated heparin (UFH) as well as low molecular weight heparins (LMWH) can be used.
• Heparin can also be directly conjugated to plastic using NHS-heparin (N-hydroxysuccinimide heparin) or other activators.
• NHS-heparin N-hydroxysuccinimide heparin
• 50 to 50,000 ng/ml biotinylated heparin on streptavidin plates can be used.
• 100 to 10,000 ng/ml can also be used.
• More heparin on the plate gives the ability to detect high ranges of heparin in samples, while low levels of heparin on the plate gives a more sensitive test, allowing assay of lower levels of heparin.
• the heparin from the sample and the immobilized heparin then compete for heparin binding sites on the HBM.
• Binding ofthe HBM to the immobilized heparin can be detected using a secondary reagent such as HRP conjugated antibody that recognizes the HBM via a tag, such as GST. This is followed by detection of secondary reagent activity using a detection agent such as TMB. Color development can then be stopped and absorbance can be measured. The signal produced is inversely proportional to the amount of heparin present in the analyte, as the heparin ofthe analyte competes for the HBM binding to the heparin coated plate. A series of increasing concentrations of heparin can be performed in conjunction with the assay to allow for determination ofthe amount of heparin present by comparison to the standard curve.
• a secondary reagent such as HRP conjugated antibody that recognizes the HBM via a tag, such as GST.
• TMB detection agent
• Color development can then be stopped and absorbance can be measured.
• the signal produced is inversely proportional to the amount of heparin present in the analyt
• the capture protein is GST-HB3 fusion protein in which the GST has been cleaved, and the remaining HB3 protein is utilized as the capture protein.
• Fluorescent-based methods can also be used to visualize HBMs bound to heparin.
• the HBM can be fused with a fluorescent molecule such as BAP or GFP, for example.
• Alkaline phosphatase fusion constructs are routinely used in subcellular protein localization.
• fluorescent dyes can be chemically conjugated to the HBM. Plasma, serum, or blood can be used as the analyte.
• a serum based heparin assay eliminates the need for drawing a separate citrated tube of blood, thus decreasing the total volume of blood needed to be drawn from a patient.
• a serum based heparin assay allows the sample to come from the same tube of blood as for other assays. In subjects having only a heparin level drawn, there is a need to draw an additional tube of blood prior to drawing a citrated tube, as a means of clearing the activated tissue factor proteins that would affect a clotting cascade based assay. The elimination of this extra tube provides both time and cost savings.
• the assay can be optimized using different amounts of HBM or other reagents. A multivariate experimental design program can be used to optimize the results.
• Variables can include pH, constitution of buffers, timing for incubations, and concentrations of biotinylated heparin, HBM, and conjugated antibody.
• the heparin can be UFH or LMWH.
• Sandwich format ELISAs In a sandwich assay format, the detection signal increases with increasing heparin concentrations in the analyte rather than decreasing, as is the case with the competitive assay format described above. First a "capture protein" is selected to coat the wells. In one example, HB3-GST is used as the HBM molecule, however, any ofthe coating methods described herein can be used in the sandwich format ELISA.
• the GST tag ofthe HB3 protein is cleaved and then the cleaved HB3 is immobilized in the wells of a microtiter plate as the capture molecule.
• An alternative approach is to utilize a completely different polycationic species as the capture ligand. This has the advantages of avoiding aggregation, being more economical and easy to prepare in advance, and provide two different affinity ligands for maximal differentiation.
• capture ligands are employed. Examples of such capture ligands include protamine and poly-L-lysine (PLL). Synthetic polycationic polymers can also be used. The polycationic polypeptide is adsorbed and coated to the wells.
• the analyte is then be added to the wells and allowed to equilibrate.
• HBM is added to the wells. Binding ofthe HBM to the heparin can be detected using the HRP conjugated anti-GST antibody as in the competitive assay, for example (Example 8).
• This step can be followed by colorimetric detection ofthe HRP activity with TMB. Color development is stopped by acidification, and absorbance read. Signal increases as increased amounts of heparin in the analyte are captured by the capture protein.
• a series of heparin standards can be used as controls in this assay format.
• the sandwich format provides increased signal with increasing heparin in the sample being analyzed, hi contrast to APTT or anti-Xa assays, direct heparin detection can be performed in serum, rather than plasma, as it does not rely on the clotting cascade.
• a multivariate experimental design can be used to optimize this assay.
• the assay can be performed in blood, plasma, or serum, for example.
• Fluorescent Based Assays A fluorescent-based assay can be used for both UFH and LMWH.
• streptavidin-coated microtiter plates can be used which have been treated with biotinylated heparin.
• the wells can be blocked and stabilized with a protein free coating solution.
• BAP is used as the fluorescent molecule
• the BAP -HBM reagent can be added to the analyte for which heparin levels are being determined and allowed to equilibrate.
• This BAP-HBM-analyte mixture is then added to the wells ofthe heparin coated microtiter plate.
• the unknown heparin and the immobilized heparin will compete for heparin binding sites on the BAP -HBM. Binding of the BAP-HBM to the plate can then be detected colorimetrically using a substrate that will react with the BAP tag present on the HBM.
• the level of heparin can be quantified utilizing an HBM.
• the amount of heparin in plasma can be determined by spiking the plasma with heparin calibration standards.
• Figure 24 shows the measurement of enoxaparin in plasma plotted in log.
• Figure 25 shows that unfractionated heparin can also be measured quantitatively in plasma.
• Competitive and sandwich assay formats can be compared with identical samples. Aliquots of plasma can be mixed with equal volumes of serial dilutions prepared from heparin. Relative absorbance vs.
• heparin concentration (log/log) can then be plotted to obtain calibration curves.
• the optimal range for heparin measurements is from 100 ng/ml to 2000 ng/ml for UFH and from 400 ng/ml to 2000 ng/ml for LMWH. With parallel Anti-Xa assay experiment, this corresponds to 0.1-5 U/ml for UFH and 0.3-2 U/ml for LMWH, suitable for therapeutic levels in plasma, which are generally between 0.1-1.0 U/ml.
• heparin was bound to the inside of a a microplate well ( Figure 18). Various concentrations of standards were then placed in the wells, which amounts can vary according to the need thereof ( Figure 19).
• heparin concentrations were then placed in the remaining wells, and a competitive binding reaction using HRP-HB3 was carried out. The sample was then incubated, and a reaction took place between the HB3 and the heparin ( Figure 20).
• HBM-HRP is capable of detecting levels of heparin between lng/ml to 100,000 ng/ml.
• the HBM is capable of detecting levels of heparin between 10 ng/ml and 10,000 ng/ml.
• the HBM is capable of detecting levels of heparin between 100 ng/ml to 2000 ng/ml.
• b) Method of Detecting Heparin on a Coated Surface During surgical procedures when a patient's blood contacts uncoated medical devices, the device surfaces modify plasmatic proteins, promote platelet aggregation, and activate the complement system, unleashing thrombus formation. Thus, it is necessary to use an anticoagulant to keep this process from starting. Heparin is an anticoagulant most used for this purpose and is typically immobilized on to the surface of many surgical instruments and instruments for use in hospitals. Because ofthe tremendous importance of these instruments having an appropriate, evenly-applied layer of heparin, quality control of these instruments is vital.
• heparin application to instruments in solution tends to degrade over time, due to cations in solution that attach to the anions on the chain, removing the bond to the cation on the surface and allowing that part ofthe chain to enter the solution.
• heparin coated stents which are used to combat the issue of restenosis following angioplasty. Quality control of these stents using the methods disclosed below allows for the visualization ofthe uniformity of heparin coating on a stent, saving time and money compared to the standard quality control methods now employed.
• One method of detecting heparin on a coated surface comprises conjugating the HBM to HRP, then detecting the HRP by fluorescence, colorimetry, or chemiluminescence.
• Another method of detecting heparin on a coated surface comprises exposing the surfaces to an HBM fused to a reporter molecule, washing the coated surface to remove excess HBM fused to the reporter molecule, and assaying for the reporter molecule.
• the reporter molecule can be visualized and the uniformity of heparin on the coated surface determined.
• HBMs fused to fluorescent reporter molecules can be used, by way of example.
• the device surface is exposed to the HBM fusion protein, and then fluorescent microscopy can be utilized to detect the level of fluorescence given off by the surface. Flexing and recollapsing ofthe instrument or stent cracks and grazes the coating so discontinuities can be visualized.
• Fluorescence can be detected by, for example, using microscopy, or other detectors.
• an apparatus comprising an implantable medical device, such as a stent, which can be coated with HBMs during manufacture. These devices have an advantage over other medical devices as they can alleviate the need to coat these devices directly with heparin.
• heparin can be secondarily coated onto the device by presenting it to the HBM during manufacture.
• the device can be implanted into a subject and the attached HBM allowed to bind to endogenous glycosaminoglycans, including heparin.
• Neutralizing heparin can be done in vivo in order to stop the effects of heparin in a subject. Removing heparin can be done ex vivo in order to clear it from the subject. Removing or neutralizing heparin from blood, plasma, or serum is often needed in a clinical setting. Heparin must be removed or neutralized from the blood for surgical or other reasons. For example, when patients undergo cardiac surgical procedures, such as angioplasty or coronary artery bypass graft surgery, blood thinners such as heparin are commonly administered prior to the procedure to prevent blood clots.
• Heparin Blood tends to clot when subjected to foreign instruments, such as a bypass machine or balloons used in angioplasty.
• the heparin can be removed by immobilizing an HBM, exposing the HBM to a sample, and removing the heparin from the sample of fluid.
• Affinity chromatography can be used, for example, to remove heparin from a sample.
• HBMs can also be used to neutralize heparin by administering it to a subject in need thereof. Heparin can be removed from the sample at the rate of 1 to 10%, 10 to 20%, 20 to
• heparin is adsorbed to glutathione-Sepharose, in the identical mamier employed for purification of GST-HBM. This anchors the HBM by the high affinity, but non-covalent, GSH-GST interaction.
• the HBM can also be biotinylated for use with companion streptavidin beads.
• the sample containing heparin to be removed is then contacted with the beads, thereby causing an HBM-heparin interaction which removes the heparin from the sample.
• HBM an also be activated and applied to a bead.
• HBM can be applied to activated beads.
• HBM binds more strongly to longer chained heparins allowing an affinity purification of heparin based on molecular size. Varying the number of HBU in the HBM used for purification can allow preferential binding of various sizes of heparin molecules.
• the HBM is covalently attached to beads, hi one example, a GST- HBM construct is covalently attached to AffiGel-10 NHS-activated beads by formation of an amide linkage between lysine residues of GST and the activated ester ofthe agarose beads. This has the possibility of modifying an HBM lysine residue, but a significant number of linkages will still occur to GST, and only those linkages that preserve HBM- heparin binding are important.
• Reductive Amination An HBM can also be linked by reductive amination to a bead.
• a GST-HBM can be linked by reductive amination with NaBH 3 CN at pH ranging from 4.0 to 6.0, more specifically in the range of pH 4.5 to 5.5, more specifically at pH 5.0, to a periodate-activated-epoxy-activated agarose bead.
• the resulting secondary amine linkage to protein lysine residues also covalently immobilizes the heparin-binding domain.
• the beads are then exposed to a heparin-containing sample, and the heparin is immobilized on the beads.
• Kits Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein.
• kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods.
• the kits could include primers to perform the amplification reactions discussed in certain embodiments ofthe methods, as well as the buffers and enzymes required to use the primers as intended.
• An example of a kit for a heparin ELISA comprises a microplate, an HBM, and a color-developing reagent, control standards, a wash buffer, and instructions such as the Accucolor Heparin Kit from Sigma, control standards, such as, heparin salt products, and wash buffers such as, PBS or TBS with detergent Tween-20 added.
• the microplate can be, for example, a heparin coated or HBM-coated microplate.
• the HBM can optionally be linked to an enzyme for detection.
• the kit can optionally include an HBM-GST and anti-GST-HRP. Examples of kits and instructions for their use can be found in Examples 9-12, for example.
• Another example of a kit comprises a bedside heparin quick test.
• This kit comprises an immunochemical test, and instructions.
• the immunological test can be similar to a one step pregnancy test.
• the test can comprise a strip that containing an HBM and a molecule that changes color when heparin is detected.
• a sample of urine or blood can be placed in an application window.
• the fluid fraction along with its dissolved components including the heparin, move along with the liquid front.
• the HBM which can be in great excess, the heparin can react with the HBM.
• the HBM triggers an enzyme to start making an insoluble dye, which upon accumulating causes the vertical bar on the "plus sign" to become visible.
• the test can optionally include a control window.
• the control window shows a plus to indicate that the HBM in the paper had not become damaged.
• the test can use urine, blood, sputum, serum, or plasma, for example, to detect heparin.
• Another example of a kit includes an HBM fused to a fluorescent molecule.
• the HBM can be a fusion protein, for example.
• the fluorescent molecule can be any fluorescent molecule capable of allowing for the detection ofthe HBM.
• fluorescent molecules can be used. Examples include GFP and BAP.
• This kit can also comprise any ofthe various HBM molecules and their variants disclosed above.
• Another example of a kit includes an extracorporeal heparin removal device (HRD) kit.
• HRD extracorporeal heparin removal device
• This kit comprises an HBM molecule as an affinity capture ligand Basically, in one example, sterilized beads containing immobilized HBM would be contained in a sterile tube through which a bodily fluid such as blood would be passed. The heparin would be captured on the beads while the remaining fluid constituents would pass through un-

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