WO2007070021A1 - Detection de la calcification de nanoparticules, et proteines y associees - Google Patents

Detection de la calcification de nanoparticules, et proteines y associees Download PDF

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Publication number
WO2007070021A1
WO2007070021A1 PCT/US2005/044589 US2005044589W WO2007070021A1 WO 2007070021 A1 WO2007070021 A1 WO 2007070021A1 US 2005044589 W US2005044589 W US 2005044589W WO 2007070021 A1 WO2007070021 A1 WO 2007070021A1
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Prior art keywords
proteins
particle
calcifying nano
factor
disease
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PCT/US2005/044589
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English (en)
Inventor
E. Olavia Kajander
Katja Aho
Neve Ciftioglu
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Nanobac Pharmaceuticals Incorporated
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Priority to PCT/US2005/044589 priority Critical patent/WO2007070021A1/fr
Publication of WO2007070021A1 publication Critical patent/WO2007070021A1/fr

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the disclosed invention is generally in the field of calcification and calcifying bodies and specifically in the area of calcifying nano-particles.
  • the present invention discloses methods and compositions for the identication of calcifying nano- particles and protein/calcifying nanoparticles complexes and the correlation of said particles to various diseases. BACKGROUND OF THE INVENTION
  • Calcifying nano-particles are approximately 200 nm in size and appear to multiply in the biological mode, meaning their growth curve has the same characteristics as that of a life form, i.e., certain doubling time (typically around 3 days), plus a lag, a logarithmic, a stationary and even a death phase.
  • the particles are passageable apparently indefinitely in cell culture media (Kajander and Ciftcioglu, Proc. Natl. Acad. Sci. USA 95, 8274 (1998)).
  • the main structural component identified, without question, is bonelike apatite (Kajander and Ciftcioglu, Proc. Natl. Acad. Sci.
  • CNPs have been isolated from kidney stones (Ciftcioglu et al., Kidney Int. 56, 1893 (1999); Khullar et al., Urol. Res. 32, 190 (2004)), gall stones (Wen et al., Chin Med.
  • CNPs have been clearly differentiated from known biological entities: eubacteria, archaea, virus, prions and eukaryotes (Aho and Kajander, J. Clin. Microbiol. 41, 3460 (2003)).
  • CNPs have been shown to form mineral calcium or hydroxy apatite coatings on their surfaces.
  • the hydroxy apatite surface acts an a mineral calcium substrate for the binding of calcium binding proteins (CaBP). Proteins that associate with the CNP
  • CNP/HA complex Hydroxy apatite complex
  • CNP/HA CaBP complex may undergo a conformational change. Subsequently, the CNP/HA CaBP complex may attract or bind proteins that have an affinity to the aforementioned bound CaBPs.
  • Neoeopitope formation is causal for multiple binding by host proteins.
  • Crosslink formation is causal for multiple binding of host protein and stabilizes the structure so that it is stable and can withstand washing steps, for example, detergents, freeze thawing, etc., step involved in assays and storage functions.
  • Copending applications 11/102,798 , 11/180,921, and 11/182,076 disclose methods and compositions for the treatment of CNPs and are incorporated by reference hererin.
  • Commonly assigned patents 6,706,290 (Eradication of Nanobacteria) and 5,135,851 (Culture and Detection Methods for the Sterile Filterable autonomously replicating biological particles) are incorporated by reference herein.
  • the disclosed methods and compositions generally involve detecting one or more proteins present on a calcifying nano-particle. It has been discovered that particular proteins become associated with calcifying nano- particles. This association provides a means for detecting, classifying, analyzing, categorizing, and assessing calcifying nano-particles. Detecting particular proteins while associated with a calcifying nano-particle can be used to indicate the presence and type of calcifying nano-particle, which can be used to indicate the presence of, or disposition to, diseases or conditions. Multiple proteins on a calcifying particle can be detected. The presence or absence of particular proteins and the pattern of the presence and absence of particular proteins can be used to indicate the presence and type of calcifying nano- particle.
  • the disclosed method can involve detecting calcifying particles by detecting one or more proteins on the calcifying particle.
  • the method generally can involve detecting at least one protein on the calcifying particle by binding at least one compound to the protein and detecting the bound compound.
  • Binding a compound to the protein can involve, for example, an antibody.
  • the antibody can be the compound and also can be the means of specific binding of the compound to the protein.
  • a compound can be associated with an antibody with the antibody mediating binding of the compound to the protein. Detecting the bound compound can be accomplished by, for example, detecting the compound directly or indirectly.
  • the compound can be detected using, for example, a microarray, coded beads, flow cytometry, ELISA, mass spectrometry, fluorescence, chemiluminescence, spectrophotometry, chromatography, electrophoresis, or a combination.
  • a microarray coded beads
  • flow cytometry cytometry
  • ELISA electrophoresis
  • fluorescence chemiluminescence
  • spectrophotometry chromatography
  • electrophoresis electrophoresis
  • particular proteins and other components are found on calcifying nano-particles and that detection of such proteins and components can serve to detect, classify, analyze, categorize, and assess calcifying nano-particles.
  • detection of two or more particular proteins in association is indicative and/or characteristic of calcifying nano- particles.
  • detection of a particular protein on a calcifying nano- particle is indicative and/or characteristic of calcifying nano-particles.
  • the presence of the protein on the calcifying nano-particle and/or the identity of combinations of particular proteins serve as identifying characteristics of calcifying nano-particles.
  • Said proteins can undergo a conformational change as result of being associated with calcifying nano-particles.
  • calcium binding proteins will bind to the mineral calcium or hydroxy apaptite coating that surrounds calcifying nano-particles in the circulatory sytem of a mammal.
  • This speficity of conformational changed proteins on the surface of the calcifying nano-particles provides for the specific discovery, detection, classification, analysis, categorization, and assessment of calcifying nano-particles as described herein is useful for diagnosing, assessing, and/or monitoring diseases associated with calcification and calcifying nano- particles, the progress of such diseases, and the progress of treatment of such diseases.
  • Calcifying nano-particles are implicated in and represent a risk factor for disease. For example, as described in Example 1, calcifying nano-particles can stimulate a novel blood coagulation mechanism. This mechanism can explain why thrombosis occurs in diseases associated with calcification and calcifying nano-particles. Because of this discovery, detection, classification, analysis, categorization, and assessment of calcifying nano-particles as described herein is useful for diagnosing, assessing, and/or monitoring diseases associated with calcification and calcifying nano-particles, the progress of such diseases, and the progress of treatment of such diseases.
  • Disclosed is a method for detecting calcifying nano-particles where the method comprises detecting calcifying nano-particles by detecting one or more proteins on the calcifying nano-particles.
  • Also disclosed is a method for detecting one or more proteins where the method comprises detecting one or more proteins on a calcifying nano-particle.
  • the identified proteins identify a disease or condition with which calcifying nano-particles having the identified proteins are related or associated.
  • Also disclosed is a method of assessing the prognosis of a disease or condition where the method comprises identifying one or more proteins on a calcifying nano-particle from a subject.
  • the identified proteins identify calcifying nano-particles that are related to or associated with the prognosis of the disease or condition.
  • Also disclosed is a method of identifying a subject at risk of a disease or condition where the method comprises identifying one or more proteins on a calcifying nano-particle from a subject.
  • the identified proteins identify calcifying nano-particles that are related to or associated with a risk of developing a disease or condition.
  • calcifying nano-particle comprises one or more of the proteins selected from the group consisting of proteins with a Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kappa B, Osteopontin, Factor XfXa, CDl 4, Prothrombin, Factor DC, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin-1, B2 microglobulin, CD 18, Larninin, Antitrypsin, Notch-1, BSA, LBP, PTX3, Complement C5, Fibrinogen, D-Dimer, Factor V, gamm
  • proteins that bind to calcium binding proteins may bind to said calcium binding protein/calcifying nano-particles complex including but not limited to Fetuin binding proteins, Thrombin binding proteins, Troponin binding proteins, Tropomyosin binding proteins, GLA Matric binding proteins, Fibrin binding proteins, Kallikrein binding proteins, Factor binding proteins, Matrix metalloprotinease binding proteins, Platelet glycol binding proteins, NF Kappa B binding protein, Factor X binding protein.
  • Table 9 shows representative proteins.
  • compositions comprising a calcifying nano-particle and one or more compounds bound to one or more proteins on the calcifying nano-particle. Also disclosed is a composition of a calcifying nano-particle comprising a hydroxy apatite (mineral calcium phosphate) coating.
  • composition of a calcifying nano-particle comprising said calcifying nanoparticle and a mineral calcium hydroxy apatite coating containing bound proteins that may be conformationally changed. Also disclosed is a method of determining the progress of treatment of a subj ect having calcifying nano-particles, where the method comprises detecting one or more proteins on calcifying nano-particles in a sample from the subject, and repeating the detection in another sample from the subject following treatment. A change in the level, amount, concentration, or a combination of calcifying nano-particles in the subject indicates the progress of the treatment of the subject.
  • compositions comprising apatite and a coating material, where, for example, the coating material limits exposure of the blood of a subject when the composition is in a subject.
  • the present applications may provide for testing of implants of other devices for the detection of CNPs, for example, stents, prosthetics, articificial valves, etc. Artificial devices are commonly covered with calcific biofilms.
  • Also disclosed is a method of testing biocompatibility comprising testing blood coagulation in the absence of anticoagulants. Also disclosed is a method of testing materials that will be exposed to circulating blood for formation of calcific biofilm formation.
  • the term "protein” is meant to include both proteins in there natural state or proteins that have undergone a conformational change, be it primary or primary and secondary hereafter.
  • Calcifying nano-particles can be detected by detecting one or more of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A 5 Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kap ⁇ a B, Osteopontin, Factor X/Xa, CD 14, Prothrombin, Factor IX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin- 1, B2 microglobulin, CD 18, Laminin, Antitrypsin, Notch- 1, B
  • proteins that bind to calcium binding proteins may bind to said calcium binding protein/calcifying nano-particles complex including but not limited to Fetuin binding proteins, Thrombin binding proteins, Troponin binding proteins, Tropomyosin binding proteins, GLA Matric binding proteins, Fibrin binding proteins, Kallikrein binding proteins, Factor binding proteins, Matrix metalloprotinease binding proteins, Platelet glycol binding proteins, NF Kappa B binding protein, Factor X binding protein.
  • Table 9 shows representative proteins.
  • Calcifying nano-particles can be detected by detecting two or more proteins on the calcifying nano-particles.
  • Calcifying nano-particles can be detected by detecting one or more proteins with a GLA-containing domain.
  • Calcifying nano-particles can be detected by detecting one or more proteins with a calcium binding domain. Calcifying nano-particles can be captured, identified, or both prior to, simultaneous with, or following detection of one or more of the proteins. Capture or identification of the calcifying nano- particle can indicate that the detected proteins are on the calcifying nano-particles. Calcifying nano-particles can be captured by binding at least one compound to one or more of the proteins, wherein the compound is or becomes immobilized. Calcifying nano- particles can be identified by binding at least one compound to one or more of the proteins, wherein the calcifying nano-particles are separated based on the compound. Calcifying nano-particles can be separated by fluorescence activated sorting.
  • One or more of the proteins can be detected by binding at least one compound to the protein and detecting the bound compound. Detection of two or more bound compounds can indicate that the proteins to which the compounds are bound are on the calcifying nano-particle. The two or more compounds can be detected in the same location or at the same time.
  • the compounds can be an antibody, where the antibody is specific for the protein.
  • the calcifying nano-particles can comprise calcium phosphate and one or more of the proteins.
  • the proteins can be detected by detecting any combination of 100 or fewer of the proteins selected from the group consisting of proteins with a Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kappa B, Osteopontin, Factor X/Xa, CD14, Prothrombin, Factor IX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin-1, B2 microglobulin, CD 18, Laminin, Antitrypsin, Notch- 1, BSA, LBP, PTX3, Complement C5, Fibrinogen, D-Dimer, Factor V, gamma-Gla residues, TF
  • the proteins can be detected by detecting any combination of 75 or fewer of the proteins.
  • the proteins can be detected by detecting any combination of 50 or fewer of the proteins.
  • the proteins can be detected by detecting any combination of 25 or fewer of the proteins.
  • the proteins can be detected by detecting any combination of 10 or fewer of the proteins.
  • the proteins can be detected by detecting any combination of 7 or fewer of the proteins.
  • the proteins can be detected by detecting any combination of 3 or fewer of the proteins.
  • the combination of proteins can be detected in the same assay.
  • the combination of proteins can be detected simultaneously.
  • the combination of proteins can be detected on the same calcifying nano-particle.
  • the combination of proteins can be detected on or within the same device.
  • the combination of proteins detected can constitute a pattern of proteins.
  • the pattern can indicate or identify a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the pattern can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the pattern can identify the type of calcifying nano-particles detected.
  • the proteins can be detected by detecting the presence or absence of any combination of 100 or fewer of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-ka ⁇ a B, Osteopontin, Factor XZXa, CD 14, Prothrombin, Factor IX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain,
  • the pattern of the presence or absence of the proteins can indicate or identify a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the pattern of the presence or absence of the proteins can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the pattern of the presence or absence of the proteins can identify the type of calcifying nano- particles detected.
  • the presence of one or more of the proteins can indicate or identify a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the presence of one or more of the proteins can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the presence of one or more of the proteins can identify the type of calcifying nano-particles detected.
  • the absence of one or more of the proteins indicates or identifies a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the absence of one or more of the proteins can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the absence of one or more of the proteins can identify the type of calcifying nano-particles detected.
  • the proteins can be detected using any suitable composition, apparatus, or technique, for example, a microarray, coded beads, flow cytometry, ELISA, mass spectrometry, fluorescence, chemiluminescence, spectrophotometry, chromatography, electrophoresis, or a combination.
  • the proteins on the calcifying nano-particle can be detected by (a) capturing the calcifying nano-particle, (b) binding a detection compound to one or more of the proteins, and (c) detecting the detection compound.
  • the proteins on the calcifying nano-particle can be detected by (a) binding a detection compound to one or more of the proteins, (b) capturing the calcifying nano-particle, and (c) detecting the detection compound.
  • the calcifying nano-particle can be captured by binding a capture compound to one or more of the proteins, where the capture compound is or becomes immobilized.
  • the proteins to which capture compounds bind can mediate capture, where the detection compound can be bound to one of the proteins, where the calcifying nano-particle can be characterized by determining which proteins mediate capture of the calcifying nano-particle to which the detected detection compound is bound.
  • the capture compound can be bound to one of the proteins, where the detection compounds detected can indicate which of the proteins is present on the calcifying nano-particle, where the calcifying nano-particle can be characterized by which proteins are present on the calcifying nano-particle.
  • the identified proteins can identify the type of calcifying nano-particle.
  • the identified type of calcifying nano-particle can be related to or associated with a disease or condition.
  • the identified proteins can identify a disease or condition with which calcifying nano-particles having the identified proteins are related or associated.
  • the identified proteins can identify a disease or condition that is caused by calcifying nano- particles having the identified proteins.
  • the identified proteins can identify a disease or condition in which calcifying nano-particles having the identified proteins are produced.
  • Subjects in which pathological thrombosis can occur via apatite-mediated clotting are useful targets for the disclosed methods.
  • Such subjects can include (1) Patients with vulnerable plaque rupture exposing atheroma calcification; (2) Patients undergoing angioplasty or heart-lung machine perfusion; (3) Patients with massive bone fractures or dislocated implants releasing potentially apatite particles; (4) Patients with implants, catheters, wires or stents subject to calcium encrustation; (5) Cancer patients with soft tissue calcification; and (6) Healthy or sick people with CNPs in their blood or positive calcification scores in arteries.
  • the composition can comprise a calcifying nano-particle and one or more compounds bound to two or more proteins on the calcifying nano-particle.
  • the compound can comprise an antibody, where the antibody is specific for the protein.
  • the compound can block the calcifying nano-particle.
  • FIGS. IA- IE are diagrams showing an example of Surface Antigen Pattern Immunoassay (SAPIA).
  • SAPIA Surface Antigen Pattern Immunoassay
  • Figures 2A and 2B are graphs of levels of signal generated for various proteins in SAPIA performed on positive ( Figure 2A) and negative ( Figure 2B) serum and plasma samples showing same levels in serum and plasma.
  • Figure 3 is a scatterplot of SAPIA results for clotting matrix GLA proteins, fibrinogen and tissue factor, and CNP capture ELISA results.
  • Figure 4 is a graph of levels of signal generated for various proteins in SAPIA showing the presence of pro-thrombin fragments and oesteocalcin in CNPsas measured by sepia.
  • Figures 5 A and 5B are graphs of prothrombin activation on apatite using bovine ( Figure 5A) and human ( Figure 5B) prothrombin.
  • Figure 6 is a graph of whole blood clotting times for various materials using glass slide test.
  • Figure 7 is a diagram of apatite-mediated clotting pathway.
  • Figure 8 is a diagram of a model for conformational changes caused by apatite/blood calcium binding as exemplified by prothrombin.
  • Figure 9 is a diagram of formation of fibrin in response to thrombotic event due to CNPs how thrombin bound to apatite surface activates formation of fibrin.
  • Figures 1OA shows boxplots of individual disease states.
  • Figures 1OB shows boxplots of individual proteins correlating with disease.
  • Figure 1OC shows protein stip plots.
  • Figure 11 is a graph of clinomics samples for 15 diseases associated with CNPs. Marker values can be obtained from the disease.
  • Figure 12 is a graph depicting urine expression showing physiological differentiations of various CNP isolates 99m Tc.
  • Figure 13 is a graph of CNP antigen (U/niL) for Pacreatitis, Rheumatoid Arthritis and Cholecystitis.
  • Figure 14 is a boxplot of biomarkers for negative endometrioid adenocarcinoma.
  • Figure 15 is a boxplot for biomarkers for positive endometrioid adenocarcinoma.
  • Figure 16 is scatterplot of markers for aortic data.
  • Figure 17 is a scatterplot of markers for arthritis data.
  • Figure 18 is a scatterplot of markers for cholecystitis data.
  • Figure 19 is a scatterplot of markers for endometrioid data.
  • Figure 20 is a- scatterplot of markers for kidney stones data.
  • Figure 21 is a scatterplot of markers for Parkinson's data.
  • Figure 22 is a scatterplot of markers for prostate data.
  • Figure 23 is a scatterplot of markers for prostatitis data.
  • the disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.
  • the disclosed methods and compositions generally involve detecting one or more proteins present on a calcifying nano-particle. It has been discovered that particular proteins become associated with calcifying nano- particles. This association provides a means for detecting, classifying, analyzing, categorizing, and assessing calcifying nano-particles.
  • Detecting particular proteins while associated with a calcifying nano-particle can be used to indicate the presence and type of calcifying nano-particle, which can be used to indicate the presence of, or disposition to, diseases or conditions.
  • Multiple proteins on a calcifying particle can be detected. Proteins may experience a conformational change resultant from association and/or binding to the califying nano-particle. Proteins associated with calcifying nano-particles may undergo secondary conformational changes. Proteins may bind to proteins associated to calcifying nanoparticles.
  • the presence or absence of particular proteins and the pattern of the presence and absence of particular proteins can be used to indicate the presence and type of calcifying nano-particle.
  • the disclosed method can involve detecting calcifying particles by detecting one or more proteins on the calcifying particle.
  • the method generally can involve detecting at least one protein on the calcifying particle by binding at least one compound to the protein and detecting the bound compound.
  • Binding a compound to the protein can involve, for example, an antibody.
  • the antibody can be the compound and also can be the means of specific binding of the compound to the protein.
  • a compound can be associated with an antibody with the antibody mediating binding of the compound to the protein.
  • Detecting the bound compound can be accomplished by, for example, detecting the compound directly or indirectly.
  • the compound can be detected using, for example, a microarray, coded beads, flow cytometry, ELISA, mass spectrometry, fluorescence, chemiluminescence, spectrophotometry, chromatography, electrophoresis, or a combination.
  • compositions and methods are known for the detection of analytes and such can be used in and with the disclosed compositions and methods for the detection of calcifying nano-particles and proteins on calcifying nano- particles. Some such compositions and methods are described herein and others are known to those of skill in the art.
  • Detection of two or more proteins associated with calcifying nanoparticles enables the generation of a patterns that are useful for diagnosing, assessing, and/or monitoring diseases.
  • the origin and activity of said detected proteins is usefull in the determination of a potential or active disease state in the host.
  • Calcifying nano-particles are implicated in and represent a risk factor for disease. For example, as described in the Example, calcifying nano-particles can stimulate a novel blood coagulation mechanism. This mechanism can explain why thrombosis occurs in diseases associated with calcification and calcifying nano-particles. Because of this discovery, detection, classification, analysis, categorization, and assessment of calcifying nano-particles as described herein is useful for diagnosing, assessing, and/or monitoring diseases associated with calcification and calcifying nano-particles, the progress of such diseases, and the progress of treatment of such diseases.
  • calcifying nano-particle comprises one or more of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kap ⁇ a B, Osteopontin, Factor XIXa, CDU, Prothrombin, Factor JX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin- 1 5 B2 microglobulin, CD 18, Laminin, Antitrypsin, Notch-1, BSA, LBP, PTX3, Complement C5, Fibrinogen, D-Dimer Factor V, gamma
  • composition comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that collects said calcium binding proteins.
  • a compositon comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that has calcium binding proteins associated thereon and proteins that bind to said calcium binding proteins.
  • compositions comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that has calcium binding proteins associated thereon wherein said calcium binding proteins undergo a primary conformation change as a result of said association
  • composition comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating containing bound calcium binding binding proteins that may experience conformational changes and wherein secondary bound proteins thereon experience conformational changes.
  • composition comprising a calcifying nano-particle and one or more compounds bound to one or more proteins on the calcifying nano-particle.
  • compositions comprising apatite and a coating material, where, for example, the coating material limits exposure of the blood of a subject when the composition is in a subj ect.
  • the composition can comprise a calcifying nano-particle and one or more compounds bound to two or more proteins on the calcifying nano-particle.
  • the compound can comprise an antibody, where the antibody is specific for the protein.
  • the compound can block the calcifying nano-particle.
  • the disclosed method can make use of compounds that can bind to calcifying nano-particles, such as compounds that can bind proteins on calcifying nano-particles.
  • Detection compounds and capture compounds are examples of such compounds.
  • Compounds for use in the disclosed methods can be any compound, molecule, material or substance that can bind to a calcifying nano-particle and/or a protein on a calcifying nano- particle. It is preferred that the compound bind specifically to the calcifying nano-particle or protein. Such specificity allows detection and identification of calcifying nano-particles and proteins.
  • Useful compounds include antibodies and molecules that can bind to proteins on calcifying nano-particles such as ligands, substrates, proteins, cofactors, coenzymes.
  • Useful compounds include compounds, such as antibodies, that can bind to proteins with a Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kappa B, Osteopontin, Factor XJXa, CD 14, Prothrombin, Factor IX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin-1, B2 microglobulin, CD 18, Laminin, Antitrypsin, Notch-1, BSA, LBP, PTX3, Complement C5, Fibrinogen, D-Dimer, Factor V, gamma-Gla residues, TF-VIIa, Complement 3c3, Comp
  • the disclosed compounds can be used for detection and capture of calcifying nano-particles and/or proteins on calcifying nano-particles.
  • detecting compounds can be used for detection and capture compounds can be used for capture of calcifying nano-particles and/or proteins on calcifying nano-particles.
  • Detection and identification of calcifying nano-particles and proteins on calcifying nano-particles can be facilitated by including labels on the disclosed compounds. Useful labels and their use are described elsewhere herein. Detection of compounds bound to calcifying nano-particles and/or proteins on calcifying nano-particles indicates the presence of the bound calcifying nano-particles and/or proteins on calcifying nano-particles.
  • the disclosed compounds can be detected, for example, via labels on the compounds, by direct detection of the compounds (via an intrinsic feature of the compounds, for example), or by binding a secondary compound to the primary compound and detecting the secondary compound.
  • the secondary compound can include a label.
  • labels can be used.
  • labels can be incorporated into, coupled to, or associated with, compounds, detection compound, capture compound (such as compounds to be bound to proteins).
  • a label can include, for example, a fluorescent dye, a member of binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence.
  • labels can be detected using nuclear magnetic resonance, electron paramagnetic resonance, surface enhanced raman scattering, surface plasmon resonance, fluorescence, phosphorescence, chemiluminescence, resonance raman, microwave, photometry, mass spectrometry, or a combination.
  • Substances suitable for detectably labeling proteins include, for example, fluorescent dyes (also known herein as fluorochromes and fluorophores), chromophores, and enzymes that react with colorometric substrates (e.g., horseradish peroxidase).
  • fluorescent dyes also known herein as fluorochromes and fluorophores
  • chromophores and enzymes that react with colorometric substrates (e.g., horseradish peroxidase).
  • colorometric substrates e.g., horseradish peroxidase
  • each protein can be associated with a distinct label compound for simultaneous and/or multiplex detection.
  • Labels can be detected using a detection device or apparatus suitable for the label to be detected, such as a fluorimeter, spectrophotomer, or mass spectrometer, the presence of a signal indicating the presence of the corresponding protein.
  • Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5- Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5- Carboxytetramethylrhodamine (5-TAMRA); 5 -Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4- methylcoumarin; 7-Aminoactmomycm D (7 -AAD); 7-Hydroxy-4- 1 methylcoumariii; 9- Amino-6-chloro-2-methoxyacridine (ACMA
  • Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro- Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43TM; FM 4-46; Fura RedTM (high pH); Fura RedTM/Fluo-3 ; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type' non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGF
  • labels include molecular or metal barcodes, mass labels, and labels detectable by nuclear magnetic resonance, electron paramagnetic resonance, surface enhanced raman scattering, surface plasmon resonance, fluorescence, phosphorescence, chemiluminescence, resonance raman, microwave, photometry, mass spectrometry, or a combination.
  • Mass labels are compounds or moieties that have, or which give the labeled component, a distinctive mass signature in mass spectroscopy. Mass labels are useful when mass spectroscopy is used for detection.
  • Preferred mass labels are peptide nucleic acids and carbohydrates.
  • Combinations of labels can also be useful. For example, color- encoded microbeads having, for example, 256 unique combinations of labels, are useful for distinguishing numerous components. For example, 256 different ligator-detectors can be uniquely labeled and detected allowing multiplexing and automation of the disclosed method.
  • Metal barcodes a form of molecular barcode, can be, for example, 30-300 nm diameter by 400-4000 nm multilayer multi metal rods. These rods can be constructed by electrodeposition into an alumina mold, then the alumina is removed leaving these small multilayer objects behind.
  • the system can have multiple zones encoded using multiple different metals where the metals have different reflectivity and thus appear lighter or darker in an optical microscope depending on the metal. For example, up to 12 zones can be encoded in up to 7 different metals. This allows practically unlimited identification codes.
  • the metal bars can be coated with glass or other material, which can facilitate attachment of the bars to compounds to be labeled. The bars can be identified from the light dark pattern of the barcode.
  • Epitopes can be used as labels.
  • Epitopes (that is, a portion of a molecule to which an antibody binds) can be composed of sugars, lipids or amino acids.
  • Epitope tags are useful for the labeling and detection of proteins when an antibody to the protein is not available. Due to their small size, they are unlikely to affect the tagged protein's biochemical properties.
  • Epitope tags generally range from 10 to 15 amino acids long and are designed to create a molecular handle for the protein.
  • An epitope tag can be placed anywhere within the protein, but typically they are placed on either the amino or carboxyl terminus to minimize any potential disruption in tertiary structure and thus function of the protein. Any short stretch of amino acids known to bind an antibody could become an epitope tag.
  • Useful epitope tags include c-myc (a 10 amino acid segment of the human protooncogene myc), haemoglutinin (HA) protein, His ⁇ , Green flourescent protein (GFP), digoxigenin (DIG), and biotin. Flourescent dyes, such as those described herein, can also be used as epitope tags.
  • Calcifying nano-particles and proteins on calcifying nano-particles can be any from any source, such as an animal.
  • the disclosed method is performed using a sample that contains (or is suspected of containing) calcifying nano-particles.
  • a sample can be any sample of interest.
  • the source, identity, and preparation of many such samples are known.
  • the sample can be, for example, a sample from one or more cells, tissue, or bodily fluids such as blood, urine, semen, lymphatic fluid, cerebrospinal fluid, or amniotic fluid, or other biological samples, such as tissue culture cells, buccal swabs, mouthwash, stool, tissues slices, and biopsy aspiration.
  • Types of useful samples include blood samples, urine samples, semen samples, lymphatic fluid samples, cerebrospinal fluid samples, amniotic fluid samples, biopsy samples, needle aspiration biopsy samples, cancer samples, tumor samples, tissue samples, cell samples, cell lysate samples, and/or crude cell lysate samples.
  • the sample can be from any organism of interest that contains or is suspected of containing calcifying nano-particles.
  • the sample can be animal, non-human animals, vertebrate, non-human vertebrate, invertebrate, insect, amphibian, avian, reptilian, fish, mammalian, non-human mammalian, rodent, farm animal, domesticated animal, bovine, porcine, murine, feline, canine, or human.
  • the term subject can refer to any animal or any member of any subgroup or classification of animal, including those listed above and elsewhere herein.
  • patient can refer to any animal under care or treatment, such as a veterinary patient or human patient. D. Solid Supports
  • Solid supports are solid-state substrates or supports with which molecules, such as analytes and analyte binding molecules, can be associated.
  • Analytes such as calcifying nano-particles and proteins, can be associated with solid supports directly or indirectly.
  • analytes can be directly immobilized on solid supports.
  • Analyte capture agents such a capture compounds, can also be immobilized on solid supports.
  • a preferred form of solid support is an array.
  • Another form of solid support is an array detector.
  • An array detector is a solid support to which multiple different capture compounds or detection compounds have been coupled in an array, grid, or other organized pattern.
  • Solid-state substrates for use in solid supports can include any solid material to which molecules can be coupled.
  • Solid-state substrates can have any useful form including thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers, particles, beads, microparticles, or a combination.
  • Solid-state substrates and solid supports can be porous or non-porous.
  • a preferred form for a solid-state substrate is a microtiter dish, such as a standard 96-well type.
  • a multiwell glass slide can be employed that normally contain one array per well. This feature allows for greater control of assay reproducibility, increased throughput and sample handling, and ease of automation.
  • Different compounds can be used together as a set.
  • the set can be used as a mixture of all or subsets of the compounds used separately in separate reactions, or immobilized in an array.
  • Compounds used separately or as mixtures can be physically separable through, for example, association with or immobilization on a solid support.
  • An array can include a plurality of compounds immobilized at identified or predefined locations on the array. Each predefined location on the array generally can have one type of component (that is, all the components at that location are the same). Each location will have multiple copies of the component.
  • the spatial separation of different components in the array allows separate detection and identification of calcifying nano-particles and proteins. Although preferred, it is not required that a given array be a single unit or structure.
  • the set of compounds may be distributed over any number of solid supports.
  • each compound may be immobilized in a separate reaction tube or container, or on separate beads or microparticles.
  • Different modes of the disclosed method can be performed with different components (for example, different compounds specific for different proteins) immobilized on a solid support.
  • Some solid supports can have capture compounds, such as antibodies, attached to a solid-state substrate.
  • capture compounds can be specific for calcifying nano- particles or a protein on calcifying nano-particles. Captured calcifying nano-particles or proteins can then be detected by binding of a second, detection compound, such as an antibody.
  • the detection compound can be specific for the same or a different protein on the calcifying nano-particle.
  • 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 the heterobifimctional cross-linker N-[ ⁇ - Maleimidobutyryloxy] succinimide ester (GMBS).
  • Antibodies can be attached to a substrate by chemically cross-linking a free amino group on the antibody to reactive side groups present within the solid-state substrate.
  • antibodies may be chemically cross-linked to a substrate that contains free amino, carboxyl, or sulfur groups using glutaraldehyde, carbodiimides, or GMBS, respectively, as cross-linker agents.
  • aqueous solutions containing free antibodies are incubated with the solid-state substrate in the presence of glutaraldehyde or carbodiimide.
  • a preferred method for attaching antibodies or other proteins to a solid-state substrate is to functionalize the substrate with an amino- or thiol-silane, and then to activate the functionalized substrate with a homobifunctional cross-linker agent such as (Bis-sulfo-succinimidyl suberate (BS 3 ) or a heterobifunctional cross-linker agent such as GMBS.
  • a homobifunctional cross-linker agent such as (Bis-sulfo-succinimidyl suberate (BS 3 ) or a heterobifunctional cross-linker agent such as GMBS.
  • Thiol- derivatized slides are activated by immersing in a 0.5 mg/ml solution of GMBS in 1% dimethylformamide, 99% ethanol for 1 hour at room temperature. Antibodies or proteins are added directly to the activated substrate, which are then blocked with solutions containing agents such as 2% bovine serum albumin, and air-dried. Other standard immobilization chemistries are known by those of skill in the art.
  • Each of the components (compounds, for example) immobilized on the solid support preferably is located in a different predefined region of the solid support.
  • Each of the different predefined regions can be physically separated from each other of the different regions.
  • the distance between the different predefined regions of the solid support can be either fixed or variable. For example, in an array, each of the components can be arranged at fixed distances from each other, while components associated with beads will not be in a fixed spatial relationship. In particular, the use of multiple solid support units (for example, multiple beads) will result in variable distances.
  • Components can be associated or immobilized on a solid support at any density. Components preferably are immobilized to the solid support at a density exceeding 400 different components per cubic centimeter.
  • Arrays of components can have any number of components. For example, an array can have at least 1,000 different components immobilized on the solid support, at least 10,000 different components immobilized on the solid support, at least 100,000 different components immobilized on the solid support, or at least 1,000,000 different components immobilized on the solid support.
  • kits for detecting calcifying nano-particles the kit comprising one or more detection compounds, one or more capture compounds, and one or more solid supports.
  • the kits also can contain one or more buffers.
  • mixtures formed by performing or preparing to perform the disclosed method For example, disclosed are mixtures comprising a calcifying nano-particle, a detection compound, and a capture compound.
  • performing the method creates a number of different mixtures. For example, if the method includes 3 mixing steps, after each one of these steps a unique mixture is formed if the steps are performed separately.
  • a mixture is formed at the completion of all of the steps regardless of how the steps were performed.
  • the present disclosure contemplates these mixtures, obtained by the performance of the disclosed methods as well as mixtures containing any disclosed reagent, composition, or component, for example, disclosed herein.
  • Systems useful for performing, or aiding in the performance of, the disclosed method.
  • Systems generally comprise combinations of articles of manufacture such as structures, machines, devices, and the like, and compositions, compounds, materials, and the like. Such combinations that are disclosed or that are apparent from the disclosure are contemplated.
  • systems comprising a calcifying nano-particle, a detection compound, and a solid support.
  • Data structures used in, generated by, or generated from, the disclosed method.
  • Data structures generally are any form of data, information, and/or objects collected, organized, stored, and/or embodied in a composition or medium.
  • the disclosed method, or any part thereof or preparation therefor, can be controlled, managed, or otherwise assisted by computer control.
  • Such computer control can be accomplished by a computer controlled process or method, can use and/or generate data structures, and can use a computer program. These include such techniques as neural network that may quickly analyze and interpret data for clinical diagnosis and interpreations to indicated a disease state.
  • Such computer control, computer controlled processes, data structures, and computer programs are contemplated and should be understood to be disclosed herein. Uses
  • the disclosed methods and compositions are applicable to numerous areas including, but not limited to, detecting, analyzing and assessing the significance of calcifying nano-particles.
  • Other uses include, for example, detecting one or more proteins on a calcifying nano-particle, characterizing a calcifying nano-particle, diagnosing a disease or condition, assessing the prognosis of a disease or condition, identifying a subject at risk of a disease or condition, determining the progress of treatment of a subject having calcifying nano-particles, testing biocompatibility comprising testing blood coagulation in the absence of anticoagulants, and testing materials that will be exposed to circulating blood for formation of calcific biofilm formation.
  • Other uses are disclosed, apparent from the disclosure, and/or will be understood by those in the art.
  • the disclosed methods generally involve detecting one or more proteins present on a calcifying nano-particle. It has been discovered that particular proteins become associated with calcifying nano-particles. This association provides a means for detecting, classifying, analyzing, categorizing, and assessing calcifying nano- particles. Detecting particular proteins while associated with a calcifying nano-particle can be used to indicate the presence and type of calcifying nano-particle, which can be used to indicate the presence of, or disposition to, diseases or conditions. Multiple proteins on a calcifying particle can be detected. The presence or absence of particular proteins and the pattern of the presence and absence of particular proteins can be used to indicate the presence and type of calcifying nano-particle.
  • the disclosed method can involve detecting calcifying particles by detecting one or more proteins on the calcifying particle.
  • the method generally can involve detecting at least one protein on the calcifying particle by binding at least one compound to the protein and detecting the bound compound.
  • Binding a compound to the protein can involve, for example, an antibody.
  • the antibody can be the compound and also can be the means of specific binding of the compound to the protein.
  • a compound can be associated with an antibody with the antibody mediating binding of the compound to the protein. Detecting the bound compound can be accomplished by, for example, detecting the compound directly or indirectly.
  • the compound can be detected using, for example, a microarray, coded beads, coated beads, flow cytometry, ELISA, mass spectrometry, fluorescence, chemiluminescence, spectrophotometry, chromatography, electrophoresis, or a combination.
  • Detection and identification of calcifying nano-particles and proteins on calcifying nano-particles can be facilitated by including labels on the disclosed compounds. Useful labels and their use are described elsewhere herein. Detection of compounds bound to calcifying nano-particles and/or proteins on calcifying nano-particles indicates the presence of the bound calcifying nano-particles and/or proteins on calcifying nano- particles.
  • the disclosed compounds can be detected, for example, via labels on the compounds, by direct detection of the compounds (via an intrinsic feature of the compounds, for example), or by binding a secondary compound to the primary compound and detecting the secondary compound.
  • the secondary compound can include a label.
  • Disclosed is a method for detecting calcifying nano-particles where the method comprises detecting calcifying nano-particles by detecting one or more proteins on the calcifying nano-particles.
  • composition comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that collects said calcium binding proteins.
  • a compositon comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that has calcium binding proteins associated thereon and proteins that bind to said calcium binding proteins.
  • a hydroxy apatite calcium phosphate mineral
  • composition comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that has calcium binding proteins associated thereon wherein said calcium binding proteins undergo a primary conformation change as a result of said association
  • a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that has calcium binding proteins associated thereon wherein said calcium binding proteins undergo a primary conformation change as a result of said association
  • composition comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating containing bound calcium binding binding proteins that may experience conformational changes and s secondary bound proteins thereon that experience conformational changes.
  • a hydroxy apatite calcium phosphate mineral
  • Also disclosed is a method for detecting one or more proteins comprising detecting one or more proteins on a calcifying nano-particle. Also disclosed is a method of characterizing a calcifying nano-particle, where the method comprises identifying one or more proteins on a calcifying nano-particle.
  • Also disclosed is a method of diagnosing a disease or condition where the method comprises identifying one or more proteins on a calcifying nano-particle from a subject.
  • the identified proteins identify a disease or condition with which calcifying nano-particles having the identified proteins are related or associated.
  • Also disclosed is a method of assessing the prognosis of a disease or condition where the method comprises identifying one or more proteins on a calcifying nano-particle from a subject.
  • the identified proteins identify calcifying nano-particles that are related to or associated with the prognosis of the disease or condition.
  • Also disclosed is a method of identifying a subject at risk of a disease or condition where the method comprises identifying one or more proteins on a calcifying nano-particle from a subject.
  • the identified proteins identify calcifying nano-particles that are related to or associated with a risk of developing a disease or condition.
  • a method of determining the progress of treatment of a subj ect having calcifying nano-particles where the method comprises detecting one or more proteins on calcifying nano-particles in a sample from the subject, and repeating the detection in another sample from the subject following treatment.
  • a change in the level, amount, concentration, or a combination of calcifying nano-particles in the subject indicates the progress of the treatment of the subj ect.
  • Also disclosed is a method of testing biocompatibility comprising testing blood coagulation in the absence of anticoagulants.
  • Calcifying nano-particles can be detected by detecting one or more of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kap ⁇ a B, Osteopontin, Factor X/Xa, CD 14, Prothrombin, Factor IX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin- 1, B2 microglobulin, CD 18, Laminin, Antitrypsin, Notch- 1, BSA, LBP, PTX3, Complement C5, Fibrin
  • Calcifying nano-particles can be detected by detecting two or more proteins on the calcifying nano-particles. Calcifying nano-particles can be detected by detecting one or more proteins with a GLA-containing domain. Calcifying nano-particles can be detected by detecting one or more proteins with a calcium binding domain. Calcifying nano- particles can be captured, identified, or both prior to, simultaneous with, or following detection of one or more of the proteins. Capture or identification of the calcifying nano- particle can indicate that the detected proteins are on the calcifying nano-particles. Calcifying nano-particles can be captured by binding at least one compound to one or more of the proteins, wherein the compound is or becomes immobilized. Calcifying nano- particles can be identified by binding at least one compound to one or more of the proteins, wherein the calcifying nano-particles are separated based on the compound. Calcifying nano-particles can be separated by fluorescence activated sorting.
  • One or more of the proteins can be detected by binding at least one compound to the protein and detecting the bound compound. Detection of two or more bound compounds can indicate that the proteins to which the compounds are bound are on the calcifying nano-particle. The two or more compounds can be detected in the same location or at the same time.
  • the compounds can be an antibody, where the antibody is specific for the protein.
  • the calcifying nano-particles can comprise calcium phosphate and one or more of the proteins.
  • the proteins can be detected by detecting any combination of 10 or fewer of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kappaB, Osteopontin, Factor X/Xa, CD 14, Prothrombin, Factor FX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin-1, B2 microglobulin, CD 18, Laminin, Antitrypsin, Notch- 1 , BSA, LBP, PTX3, Complement C5, Fibrinogen, D-Dimer, Factor V, gamma-Gla residues, TF-VI
  • Said proteins may or may not undergo conformational changes.
  • the proteins can be detected by detecting any combination of 7 or fewer of the proteins.
  • the proteins can be detected by detecting any combination of 5 or fewer of the proteins.
  • the proteins can be detected by detecting any combination of 3 or fewer of the proteins.
  • the combination of proteins can be detected in the same assay.
  • the combination of proteins can be detected simultaneously.
  • the combination of proteins can be detected on the same calcifying nano-particle.
  • the combination of proteins can be detected on or within the same device.
  • the combination of proteins detected can constitute a pattern of proteins.
  • the pattern can indicate or identify a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the pattern can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the pattern can identify the type of calcifying nano-particles detected.
  • Disease associated with patholical clarification include, but are not limited to for example, heart or circulatory diseases such as Arteriosclerosis, Atherosclerosis, Coronary Heart Disease, Chronic Heart Failure, Valve Calcifications, Arterial Aneurysms, Calcific Aortic Stenosis, Transient Cerebral Ischemia, Stroke, Peripheral Vascular Disease, Monckeberg's Disease, Vascular Thrombosis; Dental Diseases such as Dental Plaque, Gum Disease (dental pulp stones), calcification of the dentinal papilla, and Salivary Gland Stones; Chronic Infection Syndromes such as Chronic Fatigue Syndrome; Kidney and Bladder Stones, Gall Stones, Pancreas and Bowel Diseases such as Pancreatic Duct Stones, Crohn's Disease, Colitis Ulcerosa; Blood disorders
  • Ear Diseases such as Otosclerosis, Degeneration of Otoliths and Symptoms from the Vestibular Organ and Inner Ear (Vertigo and Tinnitus); Thyroglossal cysts, Thyroid Cysts, Ovarian Cysts; Cancer such as Meningiomas, Breast Cancer, Prostate Cancer, Thyroid Cancer, Serous Ovarian Adenocarcinoma; Skin diseases such as Calcinosis Cutis, Skin Stones, Calciphylaxis, Psoriasis, Eczema, Lichen Ruber Planus or Lichen Simple Cysts;, Choroid Plexus Calcification, Neuronal Calcification, Calcification of the FaIx Cerebri, Calcification of the Intervertebral Cartilage or Disc, Intercranial or Cerebral Calcification, Rheumatoid Arthritis, Calcific Tenditis, Oseoarthritis, Fibromyalg
  • the proteins can be detected by detecting the presence or absence of any combination of 10 or fewer of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kappa B, Osteopontin, Factor X/Xa, CDU, Prothrombin, Factor FX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin-1, B2 microglobulin, CD 18, Laminin, Antitrypsin, Notch- 1, BSA, LBP, PTX3, Complement C5, Fibrinogen, D-Dimer, Factor V, gamma-Gla residues,
  • proteins that bind to calcium binding proteins may bind to said calcium binding protein/calcifying nano-particles complex including but not limited to Fetuin binding proteins, Thrombin binding proteins, Troponin binding proteins, Tropomyosin binding proteins, GLA Matric binding proteins, Fibrin binding proteins, Kallikrein binding proteins, Factor binding proteins, Matrix metalloprotinease binding proteins, Platelet glycol binding proteins, NF Kappa B binding protein, Factor X binding protein.
  • Table 9 shows representative proteins. Said proteins may or may not undergo conformational changes.
  • the pattern of the presence or absence of the proteins can indicate or identify a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the pattern of the presence or absence of the proteins can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the pattern of the presence or absence of the proteins can identify the type of calcifying nano- particles detected.
  • the presence of one or more of the proteins can indicate or identify a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the presence of one or more of the proteins can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the presence of one or more of the proteins can identify the type of calcifying nano-particles detected.
  • the absence of one or more of the proteins indicates or identifies a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the absence of one or more of the proteins can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the absence of one or more of the proteins can identify the type of calcifying nano-particles detected.
  • Diseases associated with CNPs and pathological calcification include, but are not limimted to, for example, heart or circulatory diseases such as Arteriosclerosis, Atherosclerosis, Coronary Heart Disease, Chronic Heart Failure, Valve Calcifications, Arterial Aneurysms, Calcific Aortic Stenosis, Transient Cerebral Ischemia, Stroke, Peripheral Vascular Disease, Monckeberg's Disease, Vascular Thrombosis; Dental
  • Ear Diseases such as Otosclerosis, Degeneration of Otoliths and Symptoms from the Vestibular Organ and Inner Ear (Vertigo and Tinnitus); Thyroglossal cysts, Thyroid Cysts, Ovarian Cysts; Cancer such as Meningiomas, Breast Cancer, Prostate Cancer, Thyroid Cancer, Serous Ovarian Adenocarcinoma; Skin diseases such as Calcinosis Cutis, Skin Stones, Calciphylaxis, Psoriasis, Eczema, Lichen Ruber Planus or Lichen Simple Cysts;, Choroid Plexus Calcification, Neuronal Calcification, Calcification of the FaIx Cerebri, Calcification of the Intervertebral Cartilage or Disc, Intercranial or Cerebral Calcification, Rheumatoid Arthritis, Calcific Tenditis, Oseoarthritis, Fibromyalgia, Bone Spurs, Diff
  • the proteins can be detected using any suitable composition, apparatus, or technique, for example, a microarray, coded beads, flow cytometry, ELISA, mass spectrometry, fluorescence, chemiluminescence, spectrophotometry, chromatography, electrophoresis, or a combination.
  • the disclosed method can use an immunassy detecting approximagtely 100 or fewer difrrerent antigens on the same particles using only one tracer antibody for all of said detected target antigens or epitopes. Thereby utilitizing only one standard curve to provide quantitation of the target antigens and/or epitopes (focused on the use of the antibody).
  • the disclosed method is especially suitable for biogenic particles, such as CNPs, due to stable surface structure due to crosslinking of proteins and binding to the HA.
  • the disclosed method is not limited to CNPs.
  • the disclosed method can be utilitzed with viruses, spores, bacteria with stable capsules or similar stable substrate, microparticles in blood, plasma, and the like.
  • the particles may be captured using antibodies from any source or based on chemical regions from any source.
  • the proteins on the calcifying nano-particle can be detected by (a) capturing the calcifying nano-particle, (b) binding a detection compound to one or more of the proteins, and (c) detecting the detection compound.
  • the proteins on the calcifying nano-particle can be detected by (a) binding a detection compound to one or more of the proteins, (b) capturing the calcifying nano-particle, and (c) detecting the detection compound.
  • the calcifying nano-particle can be captured by binding a capture compound to one or more of the proteins, where the capture compound is or becomes immobilized.
  • the proteins to which capture compounds bind can mediate capture, where the detection compound can be bound to one of the proteins, where the calcifying nano-particle can be characterized by determining which proteins mediate capture of the calcifying nano-particle to which the detected detection compound is bound.
  • the capture compound can be bound to one of the proteins, where the detection compounds detected can indicate which of the proteins is present on the calcifying nano-particle, where the calcifying nano-particle can be characterized by which proteins are present on the calcifying nano-particle.
  • the identified proteins can identify the type of calcifying nano-particle.
  • the identified type of calcifying nano-particle can be related to or associated with a disease or condition.
  • the identified proteins can identify a disease or condition with which calcifying nano-particles having the identified proteins are related or associated.
  • the identified proteins can identify a disease or condition that is caused by calcifying nano- particles having the identified proteins.
  • the identified proteins can identify a disease or condition in which calcifying nano-particles having the identified proteins are produced.
  • Subjects in which pathological thrombosis can occur via apatite-mediated clotting are useful targets for the disclosed methods.
  • Such subjects can include (1) Patients with vulnerable plaque rupture exposing atheroma calcification; (2) Patients undergoing angioplasty or heart-lung machine perfusion; (3) Patients with massive bone fractures or dislocated implants releasing potentially apatite particles; (4) Patients with implants, catheters, wires or stents subject to calcium encrustation; (5) Cancer patients with soft tissue calcification; and (6) Healthy or sick people with CNPs in their blood or positive calcification scores in arteries.
  • Some forms of the disclosed methods involve detection and/or identification of calcifying nano-particles and/or proteins on calcifying nano-particles.
  • Molecules of interest including calcifying nano-particles, proteins, and/or proteins in or on a calcifying nano-particle— can be detected using any suitable technique.
  • Molecules of interest to be detected can be in any sample, any composition or any other context.
  • Detection and identification of calcifying nano-particles and proteins on calcifying nano-particles can be facilitated by including labels on the disclosed compounds. Useful labels and their use are described elsewhere herein.
  • Detection of compounds bound to calcifying nano-particles and/or proteins on calcifying nano-particles indicates the presence of the bound calcifying nano-particles and/or proteins on calcifying nano-particles.
  • the disclosed compounds can be detected, for example, via labels on the compounds, by direct detection of the compounds (via an intrinsic feature of the compounds, for example), or by binding a secondary compound to the primary compound and detecting the secondary compound.
  • the secondary compound can include a label.
  • Molecules that interact with or bind to the disclosed calcifying nano-particles and proteins, such as antibodies to a protein can be detected using known techniques.
  • the detecting molecule (the compound that binds the protein of interest such as a detecting compound or capture compound) can include a label. Calcifying nano- particles and/or proteins can be contacted with the labeled molecules (such as detection compounds and capture compounds) under conditions effective and for a period of time sufficient to allow the formation of complexes. The complexes can then be generally washed to remove any non-specifically bound labeled molecules, and the remaining label in the complexes can then be detected. Detection of the label indicates the presence of the detecting molecule which in turn indicates the presence of the protein of interest or other analyte.
  • the labeled molecules such as detection compounds and capture compounds
  • an additional molecule or moiety is brought into contact with, or generated at the site of, the complex of the protein of interest and the detecting molecule.
  • a signal-generating molecule or moiety such as an enzyme can be attached to or associated with the detecting molecule.
  • the signal-generating molecule can then generate a detectable signal at the site of the immunocomplex.
  • an enzyme when supplied with suitable substrate, can produce a visible or detectable product at the site of the immunocomplex.
  • ELISAs use this type of indirect labeling.
  • an additional molecule (which can be referred to as a binding agent) that can bind to the protein of interest can be contacted with the protein complex.
  • the additional molecule can have a label or signal-generating molecule or moiety.
  • the additional molecule can be termed a secondary molecule or compound. If the secondary molecule is an antibody it can be termed a secondary antibody.
  • the complexes can be contacted with the labeled molecules under conditions effective and for a period of time sufficient to allow the formation of secondary complexes. The secondary complexes can then be generally washed to remove any non- specifically bound labeled secondary molecules, and the remaining label in the secondary complexes can then be detected.
  • the additional molecule can also be or include one of a pair of molecules or moieties that can bind to each other, such as the biotin/avidin pair.
  • the detecting molecule can include the other member of the pair.
  • Other modes of indirect labeling include the detection of primary complexes by a two step approach.
  • a molecule which can be referred to as a first binding agent, such as an antibody, that has binding affinity for the protein of interest can be used to form secondary complexes, as described above.
  • the secondary complexes can be contacted with another molecule (which can be referred to as a second binding agent) that has binding affinity for the first binding agent, again under conditions effective and for a period of time sufficient to allow the formation of complexes (thus forming tertiary complexes).
  • the second binding agent can be linked to a detectable label or signal-generating molecule or moiety, allowing detection of the tertiary complexes thus formed.
  • This system can provide for signal amplification. Methods for detecting and measuring signals generated by labels are known.
  • radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by measurement or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary detection label coupled to the antibody.
  • detection molecules are molecules which interact with a molecule of interest (such as a calcifying nano-particle and/or proteins) and to which one or more detection labels are coupled.
  • labels can be distinguished temporally via different fluorescent, phosphorescent, or chemiluminescent emission lifetimes. Multiplexed time-dependent detection is described in Squire et al., J. Microscopy 197(2):136-149 (2000), and WO 00/08443.
  • Quantitative measurement of the amount or intensity of a label can be used. For example, quantitation can be used to determine if a given label, and thus the labeled component, is present at a threshold level or amount.
  • a threshold level or amount is any desired level or amount of signal and can be chosen to suit the needs of the particular form of the method being performed.
  • Methods that involve the detection of a substance, such as a protein or an antibody to a specific protein include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection.
  • Label-free assays are generally diagnostic means of determining the presence or absence of a specific protein, or an antibody to a specific protein, in a sample.
  • Protein separation methods are additionally useful for evaluating physical properties of the protein, such as size or net charge.
  • Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein, or antibody to a specific protein, in a sample.
  • in vivo detection is useful for evaluating the spatial expression patterns of the substance, i.e., where the substance can be found in a subject, tissue or cell.
  • Assay and detection techniques described herein use various terms, such as antigen, substance, molecule, analyte, etc., to refer molecules of interest that are to be bound or detected. Use of particular terms is not intended to be limiting. Unless the context clearly indicates otherwise, the assays and detection techniques described herein can be used to assay and detect calcifying nano-particles and proteins, such as proteins on calcifying nano-particles and/or proteins on or associated with the proteins on the calcifying nano-particles. As such, the calcifying nano-particles and proteins can be considered the antigen, substance, molecule, analyte, etc. that is bound and/or detected in the assay or detection technique.
  • Assay and detection techniques described herein refer, at various times, to the use of antibodies, such as antibodies that bind to or are specific for antigens, proteins, molecules, etc. Although many forms of the described assays and detection techniques are typically performed using antibodies, the assays and techniques for use in the disclosed methods is not intended to be limiting. Unless the context clearly indicates otherwise, the assays and detection techniques described herein that are described as using (or that commonly used) antibodies can be used with any suitable compound that can bind to the disclosed calcifying nano-particles and proteins. 1. SAPIA
  • SAPIA Surface Antigen Pattern Immunoassay
  • Said components can include, but are not limited to, proteins, peptides, isopeptide bonds, carbohydrates, lipids (fatty acids, phospholipids), endotoxin, heparin sulfate, calcium phosphate, and or nucleic acids (nucleic acid binding proteins associated with HA, amyloid protein P, etc. as associated on the particles.).
  • stable particles include spores, virus, certain bateria, any colloidal size mineral, metal biological or synthetic material particles (capable of binding antigens to its surface) and calcifying nanoparticles.
  • SAPIA calcifying nano-particles and/or components on calcifying nano-particles.
  • SAPIA allows detection of the presence of multiple proteins on CNPs ( Figures 1 A-IE).
  • An example and demonstration of SAPIA is described in the Example 1.
  • capture compounds such as antibodies specific for one or more proteins on calcifying nano-particles, are immobilized on a solid support.
  • capture compounds specific for multiple proteins on calcifying nano-particles can be situated on a single solid support and/or in an array.
  • SAPIA generally involves capture of calcifying nano-particles on a solid support via binding of one or more proteins on the calcifying nano-particles to capture compounds on the solid support.
  • the captured calcifying nano-particles can then be detected and/or identified by binding a detection compound to the calcifying nano-particles and/or one or more proteins on the calcifying nano-particles and/or one or more of proteins bound to said proteins.
  • a detection compound to the calcifying nano-particles and/or one or more proteins on the calcifying nano-particles and/or one or more of proteins bound to said proteins.
  • an array of capture compounds specific for different proteins on calcifying nano-particles is used, thus capturing calcifying nano- particles at each array location where a capture compound is present that can bind a protein on the calcifying nano-particles.
  • each type of calcifying nano-particle can bind to multiple locations where multiple different capture compounds are present in the array, hi this way detection of the presence of calcifying nano-particles at a given array location can identify a protein on the calcifying nano-particle. Such detection can be accomplished with detection compounds that bind to a single type of protein on calcifying nano-particles because only the presence of calcifying nano-particles needs to be detected.
  • capture of calcifying nano-particles can be via a single type of protein on calcifying nano- particles and detection can be via multiple types of proteins on calcifying nano-particles or capture and detection can each be via multiple types of proteins on calcifying nano- particles.
  • Immunodetection methods can be used for detecting, binding, purifying, removing and quantifying various molecules including the disclosed proteins. Further, antibodies and ligands to the disclosed calcifying nano-particles and proteins can be detected. For example, the disclosed proteins can be employed to detect antibodies having reactivity therewith. This is useful, for example, to detect whether a subject has been exposed to or has developed antibodies against a protein. Standard immunological techniques are described, e.g., in Hertzenberg, et al., Weir's Handbook of Experimental Immunology, vols. 1-4 (1996); Coligan, Current Protocols in Immunology (1991); Methods in Enzymology, vols.
  • immunoassays are enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmune precipitation assays (RIPA), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAP/ FLAP).
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • RIPA radioimmune precipitation assays
  • immunobead capture assays Western blotting
  • dot blotting dot blotting
  • gel-shift assays Flow cytometry
  • protein arrays multiplexed bead arrays
  • magnetic capture in vivo imaging
  • FRET fluorescence resonance energy transfer
  • FRAP/ FLAP fluorescence recovery/
  • immunoassays involve contacting a sample suspected of containing a molecule of interest (such as the disclosed calcifying nano-particles and proteins) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest (such as antibodies to the disclosed proteins) with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • a molecule of interest such as the disclosed calcifying nano-particles and proteins
  • an antibody to a molecule of interest such as antibodies to the disclosed proteins
  • the sample-antibody composition such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • the sample used can be any sample that is suspected of containing a molecule of interest (or an antibody to a molecule of interest).
  • the sample can be, for example, one or more cells, tissue, or bodily fluids such as blood, urine, semen, lymphatic fluid, cerebrospinal fluid, or amniotic fluid, or other biological samples, such as tissue culture cells, buccal swabs, mouthwash, stool, tissue slices, tissue sections, homogenized tissue extract, cell membrane preparation, biopsy aspiration, archeological samples such as bone or mummified tissue, infection samples, nosocomial infection samples, production samples, drug preparation samples, biological molecule production samples, protein preparation samples, lipid preparation samples, and/or carbohydrate preparation samples, and separated or purified forms of any of the above.
  • tissue or bodily fluids
  • tissue culture cells such as blood, urine, semen, lymphatic fluid, cerebrospinal fluid, or amniotic fluid
  • other biological samples such as tissue culture cells, buccal swabs, mouthwash, stool, tissue slices, tissue sections, homogenized tissue extract, cell membrane preparation, biopsy aspiration, archeological samples such as bone or mummified tissue,
  • Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as the disclosed proteins or their antibodies) in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process.
  • a molecule of interest such as the disclosed proteins or their antibodies
  • the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label. See, for example, U.S.
  • Immunoassays that involve the detection of a substance, such as a protein or an antibody to a specific protein, include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection.
  • Label-free assays are generally diagnostic means of determining the presence or absence of a specific protein, or an antibody to a specific protein, in a sample.
  • Protein separation methods are additionally useful for evaluating physical properties of the protein, such as size or net charge.
  • Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein, or antibody to a specific protein, in a sample.
  • in vivo detection is useful for evaluating the spatial expression patterns of the substance, i.e., where the substance can be found in a subject, tissue or cell.
  • the molecular complexes can be visible to the naked eye, but smaller amounts may also be detected and measured due to their ability to scatter a beam of light.
  • the formation of complexes indicates that both reactants are present, and in immunoprecipitation assays a constant concentration of a reagent antibody can be used to measure specific antigen and reagent antigens can be used to detect specific antibody. If the reagent species is previously coated onto cells (as in hemagglutination assay) or very small particles (as in latex agglutination assay),
  • clumping of the coated particles is visible at much lower concentrations.
  • assays based on these elementary principles are in common use, including Ouchterlony immunodiffusion assay, rocket Immunoelectrophoresis, and immunoturbidometric and nephelometric assays.
  • the main limitations of such assays are restricted sensitivity (lower detection limits) in comparison to assays employing labels and, in some cases, the fact that very high concentrations of analyte can actually inhibit complex formation, necessitating safeguards that make the procedures more complex.
  • a variety of instruments can directly detect molecular interactions (binding, for example). Many are based on an evanescent wave on a sensor surface with immobilized ligand, which allows continuous monitoring of binding.
  • Detection of calcifying nano-particles and/or proteins can involve the separation of the calcifying nano-particles and/or proteins by electophoresis.
  • proteins are fractionated first on the basis of one physical property, and, in a second step, on the basis of another.
  • isoelectric focusing can be used for the first dimension, conveniently carried out in a tube gel, and SDS electrophoresis in a slab gel can be used for the second dimension.
  • One example of a procedure is that of O'Farrell, P.H., High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem.
  • Western Blot analysis allows the determination of the molecular mass of a protein and the measurement of relative amounts of the protein present in different samples. Detection methods include chemiluminescence and chromagenic detection. Standard methods for Western Blot analysis can be found in, for example, D.M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent 4,452,901, each herein incorporated by reference in their entirety for their teaching regarding Western Blot methods. Generally, proteins are separated by gel electrophoresis, usually SDS-PAGE.
  • the proteins are transferred to a sheet of special blotting paper, e.g., nitrocellulose, though other types of paper, or membranes, can be used.
  • the proteins retain the same pattern of separation they had on the gel.
  • the blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose.
  • An antibody is then added to the solution which is able to bind to its specific protein
  • chromogem ' c substrate e.g. alkaline phosphatase or horseradish peroxidase
  • chemiluminescent substrates e.g. alkaline phosphatase or horseradish peroxidase
  • Other possibilities for probing include the use of fluorescent or radioisotope labels (e.g., fluorescein, T).
  • Probes for the detection of antibody binding can be conjugated anti-immunoglobulins, conjugated staphylococcal Protein A (binds IgG), or probes to biotinylated primary antibodies (e.g., conjugated avidin/ streptavidin).
  • the power of the technique lies in the simultaneous detection of a specific protein by means of its antigenicity, and its molecular mass: proteins are first separated by mass in the SDS-PAGE, then specifically detected in the immunoassay step.
  • protein standards (ladders) can be run simultaneously in order to approximate molecular mass of the protein of interest in a heterogeneous sample.
  • Calcifying nano-particles and proteins can be detecting when captured or bound to a solid support (e.g., tube, well, bead, or cell).
  • a solid support e.g., tube, well, bead, or cell.
  • capture assays include Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), Flow cytometry, protein array, multiplexed bead assay, and magnetic capture.
  • RIA Radioimmunoassay
  • Radioimmunoassay is a quantitative assay for detection of binding complexes using a radioactively labeled substance (radioligand), either directly or indirectly, to measure the binding of the unlabeled substance to a specific antibody or other compound that can bind to the substance.
  • RIA involves mixing a radioactive substance (because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes 125 I or 131 I are often used) with antibody or other compound that can bind to the substance.
  • the antibody or other compound is generally linked to a solid support, such as the tube or beads.
  • Unlabeled or "cold" substance is then adding in known quantities and the amount of labeled substance displaced is measured. Initially, the radioactive substance is bound. When cold substance is added, the two compete for binding sites - and at higher concentrations of cold substance, more binds to the antibody or compound, displacing the radioactive variant. The bound substance is separated from the unbound in solution and the radioactivity of each used to plot a binding curve. The technique is both extremely sensitive, and specific. ii. ELISAs Enzyme-Linked Immunosorbent Assay (ELISA), or more generically termed EIA (Enzyme ImmunoAssay), is an immunoassay that can detect an antibody specific for a protein.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • EIA Enzyme ImmunoAssay
  • a detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme reacts in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • Enzymes which can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, /3-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha.-glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • ELISA procedures see Voller, A. et al.. J.
  • ELISA techniques are know to those of skill in the art.
  • antibodies that can bind to proteins can be immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing a marker antigen can be added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen can be detected. Detection can be achieved by the addition of a second antibody specific for the target protein, which is linked to a detectable label.
  • ELISA is a simple "sandwich ELISA.” Detection also can be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • competition ELISA Another variation is a competition ELISA.
  • test samples compete for binding with known amounts of labeled antigens or antibodies.
  • the amount of reactive species in the sample can be determined by mixing the sample with the known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunecomplexes.
  • Antigen or antibodies can be linked to a solid support, such as in the form of plate, beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody.
  • a solid support such as in the form of plate, beads, dipstick, membrane or column matrix
  • any remaining available surfaces of the wells can then be "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera.
  • a nonspecific protein that is antigenically neutral with regard to the test antisera.
  • These include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • Such coating and blocking can be used with other capture assays and with other forms of the disclosed methods involving capture and/or solid supports.
  • a secondary or tertiary detection means rather than a direct procedure can also be used.
  • the immobilizing surface is contacted with the control sample to be tested under conditions effective to allow immunecomplex (antigen/antibody) formation. Detection of the immunecomplex then requires a labeled secondary binding agent, or a secondary binding agent in conjunction with a labeled third binding agent.
  • Under conditions effective to allow immunecomplex (antigen/antibody) formation means that the conditions include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents can also assist in the reduction of nonspecific background.
  • solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween.
  • suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps can typically be from about 1 minute to twelve hours, at temperatures of about 20° to 30° C, or can be incubated overnight at about 0° C to about 10° C.
  • the contacted surface can be washed so as to remove non-complexed material.
  • a washing procedure can include washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immunecomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immunecomplexes can be determined.
  • the second or third antibody can have an associated label to allow detection, as described elsewhere herein.
  • This can be an enzyme that can generate color development upon incubating with an appropriate chromogenic substrate.
  • one can contact and incubate the first or second immunecomplex with a labeled antibody for a period of time and under conditions that favor the development of further immunecomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
  • the amount of label can be quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-(3-ethyl- benzthiazoline-6-sulfonic acid [ABTS] and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation can then be achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. iii.
  • Flow Cytometry Flow Cytometry, fluorescent activated cell sorting (FACS), fluorescence activated sorting, and flow microfluorometry provide a means of scanning individual cells or particles for the presence of a molecule of interest. Although commonly used for analysis of cells, these techniques can be used in the disclosed method to detect, analyze and identify calcifying nano-particles and/or proteins on calcifying nano-particles.
  • Flow Cytometry is the characterization of single cells or particles as they pass at high speed through a laser beam. While a hematologist can count 200 cells in less than a minute by hand (hemocytometer) on a stage microscope, a flow cytometer can discriminate cells at speeds up to 50,000 cells/second.
  • the Flow component is a fluidics system that precisely delivers the cells at the intersection of the laser beam and light gathering lens by hydrodynamic focusing (a single stream of cells is injected and confined within an outer stream at greater pressure).
  • the laser acting as a light source develops parameters of light scatter as well as exciting the fluorescent molecules used to label the cell.
  • Cells are characterized individually by their physical and/or chemical properties (Kohler, G. and Milstein, C. (1975) Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity. Nature 256: p. 495-49) which provide analytical parameters capable of accurate quantitation of the number of molecules/cell through Quantitative Flow Cytometry (QFCM).
  • QFCM Quantitative Flow Cytometry
  • the physical (morphological) profile of a cell or particle can be observed by combining forward light scatter (FS) and orthogonal or side light scatter (SSC).
  • FS forward light scatter
  • SSC side light scatter
  • FS forward light scatter
  • SSC side light scatter
  • This measurement is an indication of the cell's or particle's unique refractive index.
  • Side scatter is the light that is reflected 90° to the laser beam (all fluorescence is emitted and therefore collected at this angle) and is an indication of density or surface granularity.
  • a short list of some of the information that can be discerned by multiparameter (multi-color) Flow Cytometry includes; Apoptosis (programmed cell death), Cell Type, DNA Content, Enzyme Activity, Intracellular Proteins, Cell Surface Antigens,
  • Cytoplasmic Granularity Surface Membrane Integrity
  • Intracellular [Ca++]-Signal Transduction DNA Synthesis-Proliferation
  • Cell Surface Receptors Intracellular Cytokines
  • Oxidative Metabolism Intracellular pH, RNA Content, and Cell Size.
  • Antibodies can provide a useful tool for the analysis and quantitation of markers of individual cells.
  • flow cytometric analyses are described in Melamed, et al., Flow Cytometry and Sorting (1990); Shapiro, Practical Flow Cytometry (1988); and Robinson, et al., Handbook of Flow Cytometry Methods (1993), each herein incorporated by reference in its entirety for their teaching regarding FACS.
  • proteins are detected with antibodies that have been conjugated to fluorescent molecules such as FITC, PE, Texas Red, APC, etc. Molecules on the cell or particle surface can be detected.
  • the width of the laser beam maximum peak fluorescence is achieved within approximately 10 nsec as the excited outer orbital electrons return to their more stable ground state and emit a photon of light at a longer wavelength (e.g., 520 nm for FITC) than that at which they were excited.
  • Photomultiplier tubes PMT's detect these faint fluorescent signals and their sole role is to change discrete packets of light called photons (hv) into electrons and amplify them by producing as much as 10 million electrons for every photon captured.
  • Fluorescence-activated cell sorting and fluorescence-activated sorting are types of flow cytometry.
  • FACS is a method for sorting a suspension of biologic cells into two or more containers, one cell at a time.
  • FACS can also be performed on particles such as calcifying nano-particles (in which case it can be referred to as fluorescence-activated sorting).
  • Fluorescence-activated cell sorting is based upon specific light scattering and fluorescence characteristics of each cell or particle.
  • the cell or particle suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells and particles relative to their diameter.
  • a vibrating mechanism causes the stream of cells and particles to break into individual droplets.
  • the system is adjusted so that there is a low probability of more than one cell or particle being in a droplet.
  • the flow passes through a fluorescence measuring station where the fluorescence character of interest of each cell or particle is measured.
  • An electrical charging ring is placed just at the point where the stream breaks into droplets.
  • a charge is placed on the ring based on the immediately prior fluorescence intensity measurement and the opposite charge is trapped on the droplet as it breaks from the stream.
  • the charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge.
  • Protein arrays are solid-phase ligand binding assay systems using immobilised proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles.
  • the assays are highly parallel (multiplexed) and often miniaturised (microarrays, protein chips). Their advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and giving an abundance of data for a single experiment. Bioinformatics support is important; the data handling demands sophisticated software and data comparison analysis. However, the software can be adapted from that used for DNA arrays, as can much of the hardware and detection systems.
  • Such systems and techniques of protein arrays can be used to detect calcifying nano-particles and/or proteins on calcifying nano-particles.
  • capture array in which ligand-binding reagents, which are usually antibodies but can also be alternative protein scaffolds, peptides or nucleic acid aptamers, are used to detect target molecules in mixtures such as plasma or tissue extracts.
  • ligand-binding reagents which are usually antibodies but can also be alternative protein scaffolds, peptides or nucleic acid aptamers, are used to detect target molecules in mixtures such as plasma or tissue extracts.
  • capture arrays can be used to carry out multiple immunoassays in parallel, both testing for several analytes in individual sera for example and testing many serum samples simultaneously.
  • proteomics capture arrays are used to quantitate and compare the levels of proteins in different samples in health and disease, i.e. protein expression profiling.
  • Proteins other than specific ligand binders are used in the array format for in vitro functional interaction screens such as protein-protein, protein- DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. They may also be used to correlate the polymorphic changes resulting from SNPs with protein function.
  • the capture reagents themselves are selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets. Analysis of multiple proteins on calcifying nano-particles can be performed using such techniques.
  • sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for peptides. Many of these methods can be automated for high throughput production.
  • Protein arrays have been designed as a miniaturisation of familiar immunoassay methods such as ELISA and dot blotting, often utilizing fluorescent readout, and facilitated by robotics and high throughput detection systems to enable multiple assays to be carried out in parallel.
  • Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.
  • microdrops of protein delivered onto planar surfaces are the most familiar format
  • alternative architectures include CD centrifugation devices based on developments in microfluidics [Gyros] and specialised chip designs, such as engineered microchannels in a plate [The Living ChipTM, Biotrove] and tiny 3D posts on a silicon surface [Zyomyx].
  • Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads [Luminex, Bio-Rad] and semiconductor nanocrystals [QDotsTM, Quantum Dots], and barcoding for beads [UltraPlexTM, Smartbeads] and multimetal microrods [NanobarcodesTM particles, Nanoplex Technologies]. Beads can also be assembled into planar arrays on semiconductor chips [LEAPS technology, Bio Array Solutions].
  • Immobilization of proteins involves both the coupling reagent and the nature of the surface being coupled to.
  • a good protein array support surface is chemically stable before and after the coupling procedures, allows good spot morphology, displays minimal nonspecific binding, does not contribute a background in detection systems, and is compatible with different detection systems.
  • the immobilization method used are reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation, and compatible with retention of fully functional protein activity.
  • Orientation of the surface-bound protein is recognized as an important factor in presenting it to ligand or substrate in an active state; for capture arrays the most efficient binding results are obtained with orientated capture reagents, which generally require site-specific labeling of the protein.
  • Both covalent and noncovalent methods of protein immobilization are used and have various pros and cons. Passive adsorption to surfaces is methodologically simple, but allows little quantitative or orientational control; it may or may not alter the functional properties of the protein, and reproducibility and efficiency are variable.
  • Covalent coupling methods provide a stable linkage, can be applied to a range of proteins and have good reproducibility; however, orientation may be variable, chemical derivatization may alter the function of the protein and requires a stable interactive surface.
  • Biological capture methods utilizing a tag on the protein provide a stable linkage and bind the protein specifically and in reproducible orientation, but the biological reagent must first be immobilized adequately and the array may require special handling and have variable stability.
  • Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents.
  • VersalinxTM system [Prolinx]
  • reversible covalent coupling is achieved by interaction between the protein derivatised with phenyldiboronic acid, and salicylhydroxamic acid immobilized on the support surface. This also has low background binding and low intrinsic fluorescence and allows the immobilized proteins to retain function.
  • Noncovalent binding of unmodified protein occurs within porous structures such as HydroGelTM [PerkinElmer], based on a 3- dimensional polyacrylamide gel; this substrate is reported to give a particularly low background on glass microarrays, with a high capacity and retention of protein function.
  • Widely used biological coupling methods are through biotin/streptavidin or hexahistidine/Ni interactions, having modified the protein appropriately.
  • Biotin may be conjugated to a poly-lysine backbone immobilised on a surface such as titanium dioxide [Zyomyx] or tantalum pentoxide [Zeptosens].
  • Array fabrication methods include robotic contact printing, ink-jetting, piezoelectric spotting and photolithography.
  • a number of commercial arrayers are available [e.g. Packard Biosience] as well as manual equipment [V & P Scientific].
  • Bacterial colonies can be robotically gridded onto PVDF membranes for induction of protein expression in situ.
  • Fluorescence labeling and detection methods are widely used. The same instrumentation as used for reading DNA microarrays is applicable to protein arrays.
  • capture (e.g. antibody) arrays can be probed with fluorescently labeled proteins from two different cell states, in which cell lysates are directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and mixed, such that the color acts as a readout for changes in target abundance.
  • Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) [PerkinElmer Lifesciences].
  • TSA tyramide signal amplification
  • Planar waveguide technology [Zeptosens] enables ultrasensitive fluorescence detection, with the additional advantage of no intervening washing procedures.
  • High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label [Luminex] or the properties of semiconductor nanocrystals [Quantum Dot].
  • Luminex phycoerythrin
  • Quantum Dot the properties of semiconductor nanocrystals
  • Capture arrays form the basis of diagnostic chips and arrays for expression profiling. They employ high affinity capture reagents, such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers, to bind and detect specific target ligands in high throughput manner.
  • Antibody arrays have the required properties of specificity and acceptable background, and some are available commercially [BD Biosciences Clontech, BioRad, Sigma]. Antibodies for capture arrays are made either by conventional immunisation (polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage or ribosome display libraries [Cambridge Antibody Technology, Biolnvent, Aff ⁇ tech, Biosite]. In addition to the conventional antibodies, Fab and scFv fragments, single V-domains from camelids or engineered human equivalents [Domantis] may also be useful in arrays.
  • 'scaffold' refers to ligand-binding domains of proteins, which are engineered into multiple variants capable of binding diverse target molecules with antibody-like properties of specificity and affinity.
  • the variants can be produced in a genetic library format and selected against individual targets by phage, bacterial or ribosome display.
  • Such ligand-binding scaffolds or frameworks include 'Affibodies' based on Staph, aureus protein A [Affibody], 'Trinectins' based on fibronectins [Phylos] and 'Anticalins' based on the lipocalin structure [Pieris]. These can be used on capture arrays in a similar fashion to antibodies and may have advantages of robustness and ease of production.
  • Non-protein capture molecules notably the single-stranded nucleic acid aptamers which bind protein ligands with high specificity and affinity, are also used in arrays [SomaLogic].
  • Aptamers are selected from libraries of oligonucleotides by the SelexTM procedure and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Photocrosslinking to ligand reduces the crossreactivity of aptamers due to the specific steric requirements.
  • Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, universal fluorescent protein stains can be used to detect binding.
  • Protein analytes binding to antibody arrays may be detected directly or via a secondary antibody in a sandwich assay. Direct labeling is used for comparison of different samples with different colors. Where pairs of antibodies directed at the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; they also give the possibility of detection of protein modifications. Label- free detection methods, including mass spectrometry, surface plasmon resonance and atomic force microscopy, avoid alteration of ligand.
  • An alternative to an array of capture molecules is one made through 'molecular imprinting' technology, in which peptides (e.g.
  • ProteinChip® array [Ciphergen]
  • Solid phase chromatographic surfaces bind proteins with similar characteristics of charge or hydrophobicity from mixtures such as plasma or tumour extracts
  • SELDI-TOF mass spectrometry is used to detection the retained proteins.
  • This technology differs from the protein arrays under discussion here since, in general, it does not involve immobilization of individual proteins for detection of specific ligand interactions.
  • protein arrays can be in vitro alternatives to the cell-based yeast two-hybrid system and may be useful where the latter is deficient, such as interactions involving secreted proteins or proteins with disulphide bridges.
  • a multiplexed bead assay such as for example the BDTM Cytometric Bead Array, is a series of spectrally discrete particles that can be used to capture and quantitate soluble analytes. The analyte is then measured by detection of a fluorescence-based emission and flow cytometric analysis. Multiplexed bead assay generates data that is comparable to ELISA based assays, but in a "multiplexed" or simultaneous fashion. Concentration of unknowns is calculated for the cytometric bead array as with any sandwich format assay, i.e. through the use of known standards and plotting unknowns against a standard curve.
  • Magnetic Capture Antibody-coated magnetic particles can be used to capture and selectively separate analytes, such as calcifying nano-particles, from solution.
  • target-specific antibody is bound to a magnetic particle (often termed an immunobead). After reaction time to allow binding of immunobead and target, a strong magnetic field is applied to selectively separate the captured target-particle complexes from the milieu. 7.
  • Imunocytochemistry and immunohistochemistry are techniques for identifying cellular or tissue constituents, respectively, by means of antigen-antibody interactions.
  • the methods generally involve administering to an animal or subject an imaging-effective amount of a detectably-labeled protein-specific antibody or fragment thereof, and then detecting the location of the labeled antibody in the sample cell or tissue.
  • An "imaging effective amount” is an amount of a detectably-labeled antibody, or fragment thereof, that when administered is sufficient to enable later detection of binding of the antibody or fragment in the specific cell or tissue.
  • the effective amount of the antibody-marker conjugate is allowed sufficient time to come into contact with reactive antigens that are present within the tissues of the subject, and the subject is then exposed to a detection device to identify the detectable marker.
  • Antibody conjugates or constructs for imaging thus have the ability to provide an image of the tissue, for example, through fluorescence microscopy, laser scanning confocal microscopy (LSCM), magnetic resonance imaging (MRT), SEM, TEM, x-ray imaging, computerized emission tomography and the like.
  • Fluorescence microscopy and laser scanning confocal microscopy (LSCM) involve the detection of fluorochrome labels, such as those provided herein.
  • Wide-field fluorescence microscopy is a very widely used technique to obtain both topographical and dynamic information. It relies on the simultaneous illumination of the whole sample.
  • the source of light is usually a mercury lamp, giving out pure white light.
  • Optical filters are then used in order to select the wavelength of excitation light (the excitation filter).
  • Excitation light is directed to the sample via a dichroic mirror (i.e., a mirror that reflects some wavelengths but is transparent to others) and fluorescent light detected by a camera (usually a CCD camera).
  • a dichroic mirror i.e., a mirror that reflects some wavelengths but is transparent to others
  • fluorescent light detected by a camera usually a CCD camera.
  • LSCM differs from wide-field fluorescence microscopy in a number of ways.
  • the light source in LSCM is one or more laser(s). This has two consequences. Firstly, the excitation light bandwidth is determined by the source, not the excitation filter and thus is much narrower than in fluorescence microscopy (2-3 nm rather than 20 - 30 nm).
  • the laser beam has to be rapidly scanned across the area in a series of lines, much like a TV image is generated.
  • the fluorescence detected at each point is measured in a photomultiplier tube (PMT) and an image built up.
  • PMT photomultiplier tube
  • the major difference between fluorescence microscopy and LSCM, however, is the pinhole, which is a device that removes unwanted, out-of-focus fluorescence, giving an optical slice of a 3-dimensional image. This "optical slicing" allows the observer to see inside the object of interest and gives clearer images, with more fine detail observable.
  • This method of illumination also has advantages in that it is possible to illuminate selected regions of the visual field allowing complex photobleaching protocols to be carried out to investigate the rates of lateral travel of fluorophores, etc. and for the excitation of different fluorophores in different regions of the same cell.
  • images can be obtained at different depths. Each image is called a z-section, and can be used to reconstruct an image of the 3- dimensional object.
  • Multi-Photon LSCM is a variation of LSCM that involves the generation of high energy fluorescence using low energy incident light. This is achieved by delivering multiple photons of excitation light to the same point in space in a sufficiently short time that the energy effectively is summed and so acts as a higher energy single photon.
  • multiphoton LSCM is innately confocal, i.e., no pinhole is required. Excitation of the fluorophore can only occur where the two photons can interact. Given the quadratic nature of the probability of two photons interacting with the fluorophore in the necessary timescale, excitation only occurs in the focal plane of the objective lens, which provides cleaner images.
  • Elements particularly useful in MPJ include the nuclear magnetic spin-resonance isotopes 157 Gd, 55 Mn, 162 Dy, 52 Cr, and 56 Fe, with gadolinium often being preferred. Radioactive substances, such as technicium 99 " 1 or indium ⁇ , that can be detected using a gamma scintillation camera or detector, also can be used. Further examples of metallic ions suitable for use in the current methods are 123 1, 131 I, 97 Ru, 67 Cu, 67 Ga, 1251, 68 Ga, 72 As, 89 Zr, and 201 Tl. A radionuclide can be bound to an antibody either directly or indirectly by using an intermediary functional group.
  • Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetracetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylene diaminetetracetic acid
  • Administration of the antibodies can be done as disclosed herein.
  • Nucleic acid approaches for antibody delivery also exist.
  • Antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment.
  • the delivery of the nucleic acid can be by any means, as disclosed herein, for example.
  • Administration of the antibody can be local or systemic and accomplished intravenously, intra-arterially, via the spinal fluid or the like. Administration also can be intradermal or intracavitary, depending upon the body site under examination. After a sufficient time has lapsed for the labeled antibody or fragment to bind to the diseased tissue, for example 30 minutes to 48 hours, the area of the subject under investigation can then be examined by an imaging technique, such as those described herein.
  • the distribution of the bound radioactive isotope and its increase or decrease with time can be monitored and recorded. By comparing the results with data obtained from studies of clinically normal individuals, the presence and extent of the diseased tissue can be determined.
  • the exact imaging protocol will necessarily vary depending upon factors specific to the subject, and depending upon the body site under examination, method of administration, type of label used and the like. One of ordinary skill in the art will be able to determine which imaging protocol to use based on these factors.
  • Effective dosages and schedules for administering the compositions can be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are affected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • a typical daily dosage of the antibody used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • FRET Fluorescence resonance energy transfer
  • a prerequisite for this phenomenon is the very close proximity of both chromophores.
  • a result of FRET is the decrease/loss of emission by the donor chromophore while at the same time emission by the acceptor chromophore is observed.
  • a further result of FRET is shortening of the duration of the donor excited state as detected by a reduction in the fluorescence lifetime.
  • a pair of 2 chromophores which can interact in the above described manner is called a "donor-acceptor-pair" for FRET.
  • FRET fluorophore
  • This transfer is due to dipole-dipole interactions between the emission dipole of the donor and the absorption dipole of the acceptor and depends on the separation distance, the orientation between the dipoles, and the extent of overlapping energy levels (the overlap integral).
  • the inverse sixth order dependence of FRET on separation distance produces an extremely steep decline of the FRET efficiency over a couple of nanometers.
  • the typical distance for most pairs at which 50% of the molecules engage in FRET lies in the order of magnitude of average protein diameter (4-6 nm), giving rise to detectable FRET at a maximum distance of about 10 nm.
  • FRET is a very popular method to assess (fluorescently labeled) protein-protein interactions and protein conformational changes.
  • FRET can be used to detect calcifying nano-particles and/or proteins on calcifying nano-particles.
  • FRET fluorescence emission intensity-based methods that are based on the loss of donor emission and concomitant gain of acceptor emission. These are: sensitized acceptor emission, ratio imaging, acceptor photobleaching-induced donor unquenching, and anisotropy microscopy. 2) Fluorescence decay kinetics-based methods that are based on the reduced donor photobleaching phenomenon and reduced donor fluorescence lifetime (or duration of the excited state) in the presence of FRET. These are: donor photobleaching kinetics and fluorescence lifetime imaging microscopy (FLEVl).
  • FLEVl fluorescence lifetime imaging microscopy
  • FRAP (Reits andNeefjes (2001) Nat Cell Biol,Jwi;3(6) :E145-7) is a technique that reports on diffusion of fluorescently labeled biomolecules in living cells.
  • a high-power laser beam is used to photodestruct labeled biomolecules in a defined area of the cell. Diffusion (and transport) of molecules from neighboring non- illuminated areas can then repopulate the illuminated area, leading to a time-dependent recovery of fluorescence in this area. For the recovery kinetics, the diffusional recovery can be determined.
  • FLIP fluorescence loss in photobleaching
  • the high-power laser illuminates the same area in the cell for a longer period. Diffusionally connected areas in the cell, outside of the illuminated area will loose total fluorescence intensity due to continuous delivery and photodestruction in the illuminated area.
  • FRAP and FLIP can be used to detect and follow the movements of calcifying nano- particles.
  • FLAP fluorescence localization after photobleaching
  • Disclosed is a method for detecting calcifying nano-particles comprising detecting calcifying nano-particles by detecting one or more proteins on the calcifying nano-particles.
  • Also disclosed is a method for detecting one or more proteins the method comprising detecting one or more proteins on a calcifying nano-particle.
  • Also disclosed is a method of diagnosing a disease or condition comprising identifying one or more proteins on a calcifying nano-particle from a subject, wherein the identified proteins identify a disease or condition with which calcifying nano- particles having the identified proteins are related or associated.
  • Also disclosed is a method of assessing the prognosis of a disease or condition comprising identifying one or more proteins on a calcifying nano-particle from a subject, wherein the identified proteins identify calcifying nano-particles that are related to or associated with the prognosis of the disease or condition.
  • Also disclosed is a method of identifying a subject at risk of a disease or condition comprising identifying one or more proteins on a calcifying nano-particle from a subject, wherein the identified proteins identify calcifying nano-particles that are related to or associated with a risk of developing a disease or condition.
  • the calcifying nano- particle comprises one or more of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kappa B, Osteopontin, Factor X/Xa, CD14, Prothrombin, Factor IX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CPvP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin-1, B2 microglobulin, CD 18, Laminin, Antitrypsin, Notch-1, BSA, LBP, PTX3, Complement C5, Fibrinogen, D-Dimer, Factor V, gamm
  • composition comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that collects said calcium binding proteins.
  • a compositon comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that has calcium binding proteins associated thereon and proteins that bind to said calcium binding proteins .
  • a hydroxy apatite calcium phosphate mineral
  • composition comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that has calcium binding proteins associated thereon wherein said calcium binding proteins undergo a primary conformation change as a result of said association
  • a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating that has calcium binding proteins associated thereon wherein said calcium binding proteins undergo a primary conformation change as a result of said association
  • composition comprising a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating containing bound calcium binding binding proteins that may experience conformational changes and wherein secondary bound proteins thereon experience conformational changes.
  • a calcifying nano-particle where the calcifying nano-particle is covered in a hydroxy apatite (calcium phosphate mineral) coating containing bound calcium binding binding proteins that may experience conformational changes and wherein secondary bound proteins thereon experience conformational changes.
  • composition comprising a calcifying nano-particle and one or more compounds bound to one or more proteins on the calcifying nano-particle.
  • a method of determining the progress of treatment of a subj ect having calcifying nano-particles comprising detecting one or more proteins on calcifying nano-particles in a sample from the subject, and repeating the detection in another sample from the subject following treatment, wherein a change in the level, amount, concentration, or a combination of calcifying nano-particles in the subject indicates the progress of the treatment of the subject.
  • compositions comprising apatite and a coating material, where, for example, the coating material limits exposure of the blood of a subject when the composition is in a subject.
  • Also disclosed herein is a method of testing biocompatibility comprising testing blood coagulation in the absence of anticoagulants and a method of testing materials that will be exposed to circulating blood for formation of calcific biofilm formation.
  • the calcifying nano-particles can be detected by detecting one or more of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kappa B, Osteopontin, Factor X/Xa, CD 14, Prothrombin, Factor IX, Fetuin B, CD40,
  • the calcifying nano-particles can be detected by detecting two or more proteins on the calcifying nano-particles.
  • the calcifying nano-particles can be detected by detecting one or more proteins with a GLA-containing domain.
  • the calcifying nano-particles can be detected by detecting one or more proteins with a calcium binding domain.
  • the calcifying nano-particles can be captured, identified, or both prior to, simultaneous with, or following detection of one or more of the proteins. Capture or identification of the calcifying nano-particle can indicate that the detected proteins are on the calcifying nano- particles.
  • the calcifying nano-particles can be captured by binding at least one compound to one or more of the proteins, wherein the compound is or becomes immobilized.
  • the calcifying nano-particles can be identified by binding at least one compound to one or more of the proteins, wherein the calcifying nano-particles are separated based on the compound.
  • the calcifying nano-particles can be separated by fluorescence activated sorting.
  • One or more of the proteins can be detected by binding at least one compound to the protein and detecting the bound compound. Detection of two or more bound compounds can indicate that the proteins to which the compounds are bound are on the calcifying nano-particle.
  • the two or more compounds can be detected in the same location or at the same time.
  • At least one of the compounds can be an antibody, wherein the antibody is specific for the protein.
  • the calcifying nano-particles can comprise calcium phosphate and one or more of the proteins.
  • the proteins can be detected by detecting any combination of 10 or fewer of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kappa B, Osteopontin, Factor X/Xa, CD14, Prothrombin, Factor IX, Fetuin B, CD40,
  • the proteins can be detected by detecting any combination of 100 or fewer of the proteins.
  • the proteins can be detected by detecting any combination of 5 or fewer of the proteins.
  • the proteins can be detected by detecting any combination of 3 or fewer of the proteins.
  • the combination of proteins can be detected in the same assay.
  • the combination of proteins can be detected simultaneously.
  • the combination of proteins can be detected on the same calcifying nano-particle.
  • the combination of proteins can be detected on or within the same device.
  • the combination of proteins detected can constitute a pattern of proteins.
  • the pattern can indicate or identify a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination including but not limited to for example, heart or circulatory diseases such as Arteriosclerosis, Atherosclerosis, Coronary Heart Disease, Chronic Heart Failure, Valve Calcifications, Arterial Aneurysms, Calcific Aortic Stenosis, Transient Cerebral Ischemia, Stroke, Peripheral Vascular Disease, Monckeberg's Disease, Vascular Thrombosis; Dental Diseases such as Dental Plaque,
  • Gum Disease (dental pulp stones), calcification of the dentinal papilla, and Salivary Gland Stones; Chronic Infection Syndromes such as Chronic Fatigue Syndrome; Kidney and Bladder Stones, Gall Stones, Pancreas and Bowel Diseases such as Pancreatic Duct Stones, Crohn's Disease, Colitis Ulcerosa; Blood disorders; Adrenal Calcification; Liver Diseases such as Liver Cirrhosis and Liver Cysts; Testicular Microliths, Chronic
  • Adenocarcinoma Skin diseases such as Calcinosis Cutis, Skin Stones, Calciphylaxis, Psoriasis, Eczema, Lichen Ruber Planus or Lichen Simple Cysts;, Choroid Plexus Calcification, Neuronal Calcification, Calcification of the FaIx Cerebri, Calcification of the Intervertebral Cartilage or Disc, Mercranial or Cerebral Calcification, Rheumatoid Arthritis, Calcific Tenditis, Oseoarthritis, Fibromyalgia, Bone Spurs, Diffuse Interstitial Skeletal Hyperostosis, Intracranial Calcifications such as Degenerative Disease Processes and Dementia; Erythrocyte-Related Diseases involving Anemia, Intraerythrocytic Nanobacterial Infection and Splenci Calcifications; Chronic Obstructive Pulmonary Disease, Broncholiths, Bronchial Stones, Neuro
  • the proteins can be detected by detecting the presence or absence of any combination of 10 or fewer of the proteins selected from the group consisting of proteins Bovine CaBP-HA complex, Fetuin A, Calmodulin, Tissue Transglutaminase II, MMP-9, MMP-3, CD 42b, NF-kappa B, Osteopontin, Factor X/Xa, CD14, Prothrombin, Factor IX, Fetuin B, CD40, Myeloperoxidase, Fibronectin, Factor VII, Tissue factor, Human complement 5b-9, Human CRP, Matrix GLA protein, CD61, Kappa Light Chain, Macrophage Ll Protein, Factor XIIIA, hsp 60, Fibrillin-1, B2 microglobulin, CD 18,
  • the pattern of the presence or absence of the proteins can indicate or identify a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the pattern of the presence or absence of the proteins can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the pattern of the presence or absence of the proteins can identify the type of calcifying nano- particles detected.
  • the presence of one or more of the proteins can indicate or identify a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the presence of one or more of the proteins can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the presence of one or more of the proteins can identify the type of calcifying nano-particles detected.
  • the absence of one or more of the proteins indicates or identifies a disease or condition, a risk of a disease or condition, the severity of a disease or condition, or a combination.
  • the absence of one or more of the proteins can indicate or identify a treatment to inhibit, remove or prevent the calcifying nano-particles.
  • the absence of one or more of the proteins can identify the type of calcifying nano-particles detected.
  • the proteins can be detected using a microarray, coded beads, coated beads, flow cytometry, ELISA, mass spectrometry, fluorescence, chemiluminescence, spectrophotometry, chromatography, electrophoresis, or a combination.
  • the proteins on the calcifying nano-particle can be detected by (a) capturing the calcifying nano-particle, (b) binding a detection compound to one or more of the proteins, and (c) detecting the detection compound.
  • the proteins on the calcifying nano-particle can be detected by (a) binding a detection compound to one or more of the proteins, (b) capturing the calcifying nano-particle, and (c) detecting the detection compound.
  • the calcifying nano-particle can be captured by binding a capture compound to one or more of the proteins, where the capture compound is or becomes immobilized.
  • the proteins to which capture compounds bind can mediate capture, where the detection compound can be bound to one of the proteins, where the calcifying nano-particle can be characterized by determining which proteins mediate capture of the calcifying nano-particle to which the detected detection compound is bound.
  • the capture compound can be bound to one of the proteins, where the detection compounds detected can indicate which of the proteins is present on the calcifying nano-particle, where the calcifying nano-particle can be characterized by which proteins are present on the calcifying nano-particle.
  • the identified proteins can identify the type of calcifying nano-particle.
  • the identified type of calcifying nano-particle can be related to or associated with a disease or condition.
  • the identified proteins can identify a disease or condition with which calcifying nano-particles having the identified proteins are related or associated.
  • the identified proteins can identify a disease or condition that is caused by calcifying nano- particles having the identified proteins.
  • the identified proteins can identify a disease or condition in which calcifying nano-particles having the identified proteins are produced.
  • Subjects in which pathological thrombosis can occur via apatite-mediated clotting are useful targets for the disclosed methods.
  • Such subjects can include (1) Patients with vulnerable plaque rupture exposing atheroma calcification; (2) Patients undergoing angioplasty or heart-lung machine perfusion; (3) Patients with massive bone fractures or dislocated implants releasing potentially apatite particles; (4) Patients with implants, catheters, wires or stents subject to calcium encrustation; (5) Cancer patients with soft tissue calcification; and (6) Healthy or sick people with CNPs in their blood or positive calcification scores in arteries.
  • Such people in the last category can be identified using the disclosed compositions and methods.
  • the composition can comprise a calcifying nano-particle and one or more compounds bound to two or more proteins on the calcifying nano-particle.
  • the compound can comprise an antibody, wherein the antibody is specific for the protein.
  • the compound can block the calcifying nano-particle.
  • Example 1 In this example evidence is presented of host molecules involving two families of calcium binding Gla-proteins, calcification-defense system and clotting Gla-proteins, simultaneously binding to apatite surfaces and calcifying nano-particles. Thus, it was discovered that both Gla-systems participate in the body's calcification-defense by spatially blocking apatite surfaces. It was also realized that this creates a novel clotting mechanism. Thrombosis (the clotting of blood within an artery or vein) is a major cause of death and serious illness. Patients with circulatory, autoimmune and renal diseases, diabetes and cancers have abnormal ongoing coagulation often leading to thrombosis.
  • a clotting test was developed to measure effects of various surfaces, including apatite and calcifying nano-particles (CNPs), on blood clotting in vitro.
  • CNPs calcifying nano-particles
  • a multiplex surface antigen pattern test was also developed to demonstrate the pattern of clotting factors and their activators on the surface of CNPs isolated from human plasma and serum. This multiplex surface antigen pattern test is an example of the disclosed method for detecting calcifying nano- particles. The significance of this novel calcium mediated clotting mechanism is far- reaching since many diseases have a thrombotic component which may cause death.
  • Clinical experience in cardiovascular medicine suggested that contact of blood with exposed calcified surface leads to thrombi (Halloran and Bekavac, Neuroimaging. 2004 Oct;14(4):385-7; Demer, Int. J.
  • CAC coronary artery calcification
  • Vitamin- K-dependent, gamma-carboxyglutamic acid (Gla)-containing domains of clotting proteins in this family are homologous and are responsible for phospholipid membrane association considered to be the substratum for clotting activation cascades (Nelsestuen, Trends Cardiovasc. Med. 9, 162 (1999)). Normal hemostasis results in platelet activation, aggregation and more thrombin generation (Dumas et al., Science 301, 222 (2003)) leading to a clot covering the damaged area. Clot growth is stopped by anticoagulation cascades activating inhibitors of clotting.
  • Ga gamma-carboxyglutamic acid
  • factor Xa and thrombin are assumed to diffuse through the developing clot, filled with their specific inhibitors, to the surface of the growing clot. Formation of a large thrombus blocking a blood vessel is difficult to explain with this hypothesis, and has been experimentally shown to be insufficient (Hathcock and Nemerson, Blood 104, 123
  • clotting factors acting as proteolytic executors of the clotting cascade are calcium-binding proteins also known to bind to apatite/calcium phosphate via their calcium binding GIa domains.
  • the classical models imply that the Gla-domains undergo calcium dependent conformation changes before or concomitant with binding to phospholipid membrane. It was discovered that calcium phosphate surfaces serve the dual function as a suitable substratum (replacing phospholipid membrane) and as activators in normal and pathological blood clotting.
  • CNPs are controversial in their content and genetic characterization, critics and proponents alike agree that CNPs have a calcium phosphate mineral surface (Kajander and Ciftcioglu, Proc. Natl. Acad. Sci. U S A. 95, 8274 (1998); Cisar et al., Proc. Natl. Acad. Sci. U S A. 97, 11511 (2000); VaIi et al, Geochim. Cosmochim. Acta 65, 63 (2001); Miller et al., Am. J. Physiol. Heart Circ. Physiol. 287, Hl 115 (2004); Ciftcioglu et al., Kidney Int. 67, 483 (2005)). 1. Materials and Methods
  • CNPs Calcifying nano-particles
  • Tissues were processed to paraffin blocks, sectioned, deparaffmized and stained with H&E and with TUNEL assay for apoptotic changes with In situ Cell Death Detection Kit, AP (Roche) according to the manufacturer's instructions. Tissues were pretreated for TUNEL staining with 20 ⁇ g/ml proteinase K (Sigma, molecular biology grade) in 10 mM Tris/HCl, pH 7.4 for 15 min at room temperature. Apoptotic changes were evaluated with light microscopy.
  • the method used detected apoptosis based on labeling of DNA strand-breaks using modified nucleotide labeling by terminal deoxynucleotidyl transferase visualized with enzymic reaction using Fast Red substrate (Roche). No changes were observed in control rats exposed to sterile PBS. The study was approved by the Ethics Committee of the University of Kuopio. iii. Thrombosis detection after i.v. injection of 99m Tc-labeled apatite or CNPs in rabbits
  • Clotting induced by apatite was detected initially by using standard whole blood clotting time tube tests, with added glass beads, incubated at +37°C water bath with or without apatite.
  • the clotting times were of the order of 2 minutes and did not allow precise evaluation of subtle changes by extraneous materials on clotting time due to need of sample preparation time, such as mixing the extraneous minerals.
  • This could not be amended by using anti-coagulated blood samples (citrate or EDTA), reconstituted with 25 to 50 mM CaCl 2 at start of the test, because such samples clotted poorly indicating irreversible interference by the anticoagulant to some important player(s) in clotting.
  • a novel test platform was developed using glass slides (Menzel-Glaser, Braunschweig, Germany) incubated at room temperature. The method allows for measuring changes in the clotting time induced by contact with foreign surfaces, i.e. plain glass or coated glass, and for studying the effects of drugs on the clotting induced by foreign surface. Glass slides were coated with synthetic apatite (Poser and Price, J. Biol. Chem. 254, 43 (1979)) and controlled by TEM and EDX analysis (Kajander and giftcioglu, Proc. Natl. Acad. Sci. USA 95, 8274 (1998)).
  • apatite colloidal suspension (10% pellet containing suspension) was pipetted to each slide, and slides were dried +37°C overnight.
  • Commercial heat-fixed CNP-coated slides were obtained from Nanobac Oy, Kuopio, Finland. Plain glass slides without further processing were used as a foreign surface.
  • Effect of Calcium EDTA, disodium EDTA, and clodronate on blood clotting time was investigated by adding 10 ⁇ l solution to a plain glass slide immediately before addition of blood. Calcium EDTA and disodium EDTA were from Fluka. Clodronate was a gift from Professor Jouko Vepsalainen (University of Kuopio).
  • Venous blood was collected with venipuncture from 19 random volunteers participating in CNP epidemiological study (Ethical Committee Approval, Kuopio University). Volunteers signed an informed consent. Blood was collected with venipuncture in siliconized glass serum tubes, EDTA plasma tubes or citrate plasma tubes (Terumo), and was tested immediately after collection for whole blood clotting time on different test platforms.
  • Proteomics on proteins bound to apatite particles Protein-free apatite particles in DMEM (Gibco) without any additives were suspended into 10% FBS-DMEM and were immediately centrifuged at 14 000 rpm, 30 rain at +4°C. The pellet was washed two times by suspending with sterile PBS followed by centrifugation at 13,200 rpm, 20 min at room temperature. Pellet was frozen prior to analysis. Proteomics analysis was provided by Protana, Montreal, Canada. The SDS- boiled samples were subjected to ID SDS-PAGE under reducing conditions. Protein bands were detected by Coomassie staining, excised and processed following standard procedures including: 1. The proteins in the gel plug were reduced with DTT.
  • the peptides produced were extracted in neutral, acidic and basic conditions. vi. Mass Spectrometry Analysis The peptide mixtures were separated by C 18 reverse phase chromatography into a
  • Thermo-Finnigan LTQ-FT ion trap/FTICR hybrid mass spectrometer coupled with a nano- spray interface.
  • the mass spectrometer was operated in data-dependent mode to obtain tandem (ms/ms) spectra of each peptide above an intensity threshold as it emerged from the chromatography column.
  • the raw data files were processed using LCQ-DTA to generate peak lists of the tandem spectra.
  • the processed data was searched with Mascot (Matrix Sciences, London UK) using the NCBI non-redundant database.
  • the Mascot results were curated by mass spectrometry scientists to correlate the results with the raw data (Table 2). vii. Nanocapture and SAPIA ELISA Methods
  • Nanocapture ELISA kit Nabac Oy
  • the test measures presence of CNPs in human serum or plasma, with a measurement range from 0 to 640 units (Pretorius et al., HIV Med. 5, 391 (2004)).
  • the capture kit uses separate step-wise capture and detection reactions involving two monoclonal antibodies targeted on different surface epitopes on the CNPs.
  • SAPIA Surface Antigen Pattern Immunoassay
  • SAPIA plates were made by coating high binding polystyrene ELISA plates (Coming, USA) with antibodies against anti-calcification proteins and GIa clotting factors and control antibodies. SAPIA was controlled by using antibodies against human serum albumin, D-Dimer, NF- ⁇ B and fibronectin as these proteins were not expected to be specifically bound on particle surface ( Figure IA). Monoclonal antibodies were diluted at a final concentration of 1 ⁇ g/ml with IX PBS, pH 7.4, 100 ⁇ l/well to ELISA plates and incubated at +4°C overnight. Polyclonal antibodies were diluted to a concentration of 10 ⁇ g/ml and plates were coated as above.
  • prothrombin Activation of prothrombin by apatite in vitro Human prothrombin >95 % pure (Calbiochem) and two samples of bovine prothrombin >98 % pure (ICN, Aurora, OH and American Diagnostica, Stamford, CT) were diluted to a concentration of 10 ⁇ g/ml, 1 ⁇ g/ml and 0.1 ⁇ g/ml in 25 niM Tris, 150 mM NaCl and 5 mM CaCl 2 , pH 7.4 (which is the substrate buffer for thrombin). 20 ⁇ l of prothrombin solution was mixed with 20 ⁇ l apatite (Poser and Price, J. Biol. Chem.
  • Serum and plasma samples from 6 healthy volunteers were used for measurement of thrombin and FXa activity in particles captured with SAPIA using plates coated with antibodies against CNPs, thrombin and Factor XJXa.
  • 50 ⁇ l of serum or plasma samples were pipetted onto plates and 50 ⁇ l of Assay Buffer (0.05 M Tris, 0.15 M NaCl, 0,05% Proclin 300, pH 7.5 with 1% mouse serum) was added. Plates were incubated 1 hour at room temperature with moderate shaking. Plates were washed 4 times, before 100 ⁇ l specific substrate was added.
  • Assay Buffer 0.05 M Tris, 0.15 M NaCl, 0,05% Proclin 300, pH 7.5 with 1% mouse serum
  • Three substrates were used for thrombin: Bx-Phe-Val-Arg- pNA HCl (Bachem), Sar-Pro-Arg-pNA (Bachem) and /33-Ala-Gly-Arg-pNA-acetate (Sigma, St. Louis, MI).
  • One substrate was used for Factor Xa, CH 3 -D-CHA-GIy- Arg- pNA-AcOH (Sigma).
  • Thrombin substrates Bx-Phe-Val-Arg-pNA HCl (0.136 mg/ml) and Sar-Pro-Arg-pNA (0.25 mg/ml) were in 25 mM Tris, 150 mM NaCl, 5 mM CaCI2, pH 7.4; and /3-Ala-Gly-Arg-pNA-acetate (1 mM) in 50 mM Tris, 100 mM NaCl, 5 mM CaCl 2 , pH 7.4.
  • Factor Xa substrate was CH 3 -D-CHA-Gly-Arg-pNA-AcOH (0.5 mM) in 50 mM Tris, 100 mM NaCl, 5 mM CaCl 2 , pH 7.4.
  • Thrombin substrates Bx-Phe-Val-Arg-pNA HCl and /3-Ala-Gly-Arg-pNA-acetate failed to give positive signals.
  • Factor Xa substrate CH 3 -D-CHA-GIy- Arg-pNA- AcOH gave weak positive results for serum samples after 18 hours incubation. Results did not correlate with the presence of CNPs. Thus, the results indicate only non-specific binding of thrombin and Factor Xa activity to ELISA plate which was present only in serum samples.
  • the CNP-bound antigens must have been in an inactive form, as is expected in blood samples of healthy people.
  • Immunohistochemical staining for antigen pattern analysis Paraffin-embedded arterial tissue blocks representing various forms of severe atherosclerotic lesions were obtained from commercial sources (Clinomics BioSciences, Inc., Pittsfield MA 01201. Tissue samples were collected from New York area and processed under Institutional Review Board permit). Thin sections were cut using standard techniques. Sections were deparaffinized without decalcification and stained with monoclonal antibodies for antigen pattern analysis mapping calcification defense proteins, clotting factors and CNPs. The staining protocol was tailored for each antibody,
  • apatite formation includes several metastable calcium phosphate intermediate phases (NancoUas, Pure & Appl. Chem,l 1, 1673 (1992)).
  • BCP Basic calcium phosphate
  • Synthetic colloidal apatite was used as a control while performing acute toxicity studies for calcifying nano-particles (CNPs).
  • CNPs nano-particles
  • both iv injected apatite and CNPs caused ischemia-type tissue damage in the kidneys of rats.
  • the pathognomic feature in ischemia-reperfusion kidney damage is that glomeruli are saved whereas tubuli die (Park et al., Am. J. Physiol. Renal Physiol. 282, F352 (2002)).
  • the kidney damage was dose-dependent, and did not occur when two microliter or less apatite was injected.
  • Control animals receiving only phosphate buffered saline (PBS) did not show histological changes in kidneys. There were also signs of thrombotic events in large blood vessels and cardiac chamber walls.
  • PBS phosphate buffered saline
  • Standard blood coagulation tests e.g., activated partial thromboplastin time, prothrombin time
  • thromboplastin time e.g., activated partial thromboplastin time, prothrombin time
  • apatite surface Counteracting the anticoagulants with high calcium chloride concentrations, as is required in the tests, creates non-physiological competition for binding between free calcium (tens of times higher than the physiological) and calcium phosphate surface.
  • the apatite surface would be modified by a solution high in calcium, forming other forms of calcium minerals on the surface (e.g., octacalcium phosphate) (Boskey, J. Phys. Chem. 93, 1628 (1989)).
  • Apatite is stable under physiological calcium and phosphate concentrations. Therefore, to study the effects of apatite on clotting, a whole blood clotting slide test was developed.
  • plain objective glass, or objective glass coated with various forms of apatite, or test drugs were used as test platforms. 200 ⁇ L of freshly collected human blood was applied on the slides, which were tilted ⁇ 30°, 15 tilts per minute, at room temperature. Clotting time was established at the time when droplet contents stopped moving. The test indicated that clotting was two times faster on apatite coating compared to the control slide. CNP coating also decreased clotting time significantly (Figure 6; Table 1).
  • the method was controlled by using EDTA or citrate plasma samples, which never clotted, even when exposed to apatite coated test platforms.
  • Calcium EDTA and a small concentration of the calcium binding drug etidronate did not affect the clotting time. Therefore the test appropriately measured clotting triggered by a foreign surface, glass. It was surprising that the apatite surface was superior at inducing clotting over the untreated glass, the traditionally used foreign surface in clotting tests. ii. How does apatite cause clotting?
  • tissue factor which is a 40 kD membrane-spanning protein expressed normally by almost all cells, except the endothelium. Endothelial damage exposes tissue factor, which binds and allosterically activates a serine protease, factor Vila (FVIIa), in the presence of calcium.
  • the intrinsic pathway commences upon exposure to a foreign negatively charged surface, activating calcium-dependent conformational changes of clotting factors resulting in binding to a platelet or other phospholipid membrane, and leading to an activation-amplification cascade which eventually activates FX resulting in thrombin release.
  • Proteomics analysis revealed prothrombinase complex on apatite surface together with players of complement, antibodies and protease inhibitors. Although the use of serum to test clotting factors is not preferred, this proved the ability of apatite surface to bind clotting factors and provided information about what proteins can bind in biological situations, for instance on CNPs.
  • SAPIA profiles of CNPs using plasma and serum samples were practically identical ( Figure 3). These results indicated that serum samples can be used for the test. The results also indicated that particles with this specific antigen surface pattern can be isolated from human blood without any culturing steps. SAPIA results were stable after freeze-thawing, detergent (Tween20), EDTA or citrate application. Evidence was found that the detected proteins are cross- linked (very little protein released by SDS boiling). The stability of CNPs makes them amenable to surface antigen mapping with SAPIA technique which involves extended step-wise incubations separated by numerous washings before the detection. This feature of CNPs also allows the use of harsh treatments, when useful or desired, in other assays and detection methods.
  • SAPIA indicated that clotting factors V, VII, IX, X, tissue factor-FVIIa complex, fibrin, fibrinogen, FXIIIa, fragments of factor II, thrombin and prothrombin Fragment 1, but not prothrombin Fragment 2 are on CNPs (Tables 2 and 3; Figure 2). Both matrix
  • thrombin is retained in the particle. There may be mechanism(s) to retain it, such as crosslinking, or complex formation.
  • thrombin is known to make a complex with FXIII and fibrin (Aliens et al., Blood 100, 743 (2002)), which were also found on the particle.
  • FXIII and fibrin Aliens et al., Blood 100, 743 (2002)
  • apatite binds clotting factors and their activators, concentrating them in close proximity, thus providing the necessary players for clotting on a suitable substratum ( Figures 7-9).
  • GIa residues in the GIa domain are known to bind to apatite. Free blood calcium completes the activation by binding to the rest of Gla-residues ( Figure 8).
  • Calcium phosphate the key element in apatite, is a normal body constituent, therefore cannot be regarded as foreign surface that activates the intrinsic pathway of clotting.
  • tissue factor the key player in the extrinsic pathway
  • FVIIa FVIIa
  • the diagram depicts a novel platform, formation of complexes and activation of a clotting cascade on apatite surfaces. It was also shown that apatite itself can contribute to conformational changes leading to activation of prothrombin on apatite surfaces to release active thrombin. This non-enzymatic activation was much less rapid without the added clotting cascade players, yet proves the essential role that apatite plays. Prothrombin activation involves initial reactions with calcium, followed by a membrane prothrombinase complex formation leading to a thrombin release (Borowski et al, J. Biol. Chem. 261, 14969 (1986)).
  • apatite-mediated clotting cascade as with other clotting cascades, would have to be meticulously controlled by anti-thrombin, Protein S and C, heparin and other anti- clotting mechanisms and fibrinolytic systems to maintain a fine balance between activation and inhibition.
  • Tissue factor found on CNPs shows that apatite particles can activate clotting using extrinsic pathway players. This process could be controlled by inhibitors, for example, by tissue factor inhibitor pathway (TFIP), which is likely since CNPs were not more active than apatite, which lacked the presence of the tissue factor.
  • TFIP tissue factor inhibitor pathway
  • This example shows for two forms of apatite that sudden circulatory exposure leads to thrombotic events, indicating that exposure of blood to apatite can have catastrophic results.
  • Thrombosis was found when blood in a vessel was suddenly exposed to apatite pellet (colloidal) volume in excess of two microliters.
  • Apatite exposure of this magnitude could take place as a consequence of, for example, bone fracture, rapture of vulnerable plaque revealing pathological vascular calcification, or in any situation where circulatory apatite particle counts would become locally high, for example, after rapture of a cyst filled with them.
  • Apatite-mediated clotting can have an important physiological function in bone physiology.
  • Large bones have cancellous surface compartments with a diameter larger than largest blood vessels.
  • bone fracture often leads to clots up to 10 centimeters in diameter that must be made relatively rapidly to prevent the victim from bleeding to death.
  • Exposed apatite could serve as the platform, providing booster power for clotting, since the hollow bone cannot reduce its diameter as damaged blood vessels do via vasoconstriction, and the bone has few tissue factor sources.
  • Bone trabeculae are covered with only a monocellular layer, endostium, and the cortical bone has very low cell density (no subendothelial cells available with cell tissue factor carrying membranes as present in other tissues).
  • tissue factor-mediated clotting could take place in bone, but based on the results in this example it can be seen that the exposed bone could allow apatite-mediated clotting.
  • bone contains significant amounts of clotting GIa proteins. Those proteins are present at 1 - 2 % level of the non-collagen proteins in bone They could act with the bone Gla-protein osteocalcin, which was also found on CNPs, to control bone mineralization and/or provide protection against bleeding after bone fracture, where large areas of calcified surface are exposed. Gla-proteins are also found in kidney stones, suggesting a role in stone formation via both mineralization and thrombin production via thrombotic events or other mechanisms.
  • Prothrombin Fl is the most common protein associated with kidney stones, and thrombin has been detected in urine in kidney diseases. Thrombogenic mechanisms have been proposed for kidney stone formation (Stoller et al., J. Urol. 171, 1920 (2004)). There is a very high incidence of calcifying nano-particles in disease processes known to be associated with calcification/thrombosis, for example, 97.5% associated with carotid stenosis, whereas only 10% association in Crohn's disease.
  • CNPs are detectable just below the endothelium, they can contribute to thrombotic clotting together with the circulating CNPs when the endothelium lining is damaged.
  • the results in this example indicate a role for an apatite-mediated clotting system in thrombotic events.
  • Studies on thrombogenicity of biomaterials have examined heparin stabilized apatite, or heparinized animals. Since heparin is an anticoagulant, such studies do not reveal thrombotic potential adequately. Thus, biocompatible materials may not be hemocompatible. Apatite coated implants are widely used due to their bone biocompatibility.
  • compositions comprising apatite and a coating material, where, for example, the coating material limits exposure of the blood of a subject when the composition is in a subject.
  • results in this example may be due to the ISO 10993-4. It requires the use of citrate or hirudin blood, or plasma and allows their application on implant materials while performing hemocompatibility testing (Seyfert et al., Biomolecular Engineering 19, 91 (2002)).
  • the results in this example indicate that ISO 10993-4 required conditions cannot be used to detect blood clotting on apatite.
  • Such subject include (1) Patients with vulnerable plaque rupture exposing atheroma calcification; (2) Patients undergoing angioplasty or heart-lung machine perfusion; (3) Patients with massive bone fractures or dislocated implants releasing potentially apatite particles; (4) Patients with implants, catheters, wires or stents subject to calcium encrustation; (5) Cancer patients with soft tissue calcification; and (6) Healthy or sick people with CNPs in their blood or positive calcification scores in arteries.
  • Such people in the last category can be identified using the disclosed compositions and methods.
  • This example describes a newly discovered pathophysiological mechanism linking pathological calcification to thrombosis.
  • Blood anti-calcification Gla-proteins and GIa- clotting factor proteins were shown to bind to calcium phosphate surfaces creating a novel clotting mechanism capable of causing thrombosis where blood is in contact with apatite or CNPs. This was shown by detecting thrombosis after IV injections of apatite and CNPs in vivo in rats and rabbits, leading to thrombotic events, including ischemia- reperfusion damage.
  • a whole blood coagulation slide test was developed to measure effects of various surfaces, including apatite and CNP, on blood clotting in vitro.
  • Tables 11 and 12 illustrate the results of SAPIA testing.
  • Table 11 shows raw absorbance data in the upper half of the table for 97 proteins and components measured in 16 human serum pools. The lower half illustrates units per ml. The pools were obtained by mixing the serum from 1-5 donors for each pool, pooled according to capture ELISA results that showed similar antigen levels.
  • Table 12 shows the statistical analysis as generated from the raw data of Table 11.
  • the table shows correlation between the markers (100x100). Correlation coefficients greater than 0.5 indicate positive correlation (with low p values) and those values approaching 0.0 indicate a negative correlation. Therefore, statistical review via the generation of, for example, of box plots or scatter plots enables one skilled in the art to visualize data patterns that may be useful in the assessment, diagnosis, and therapeutic selection for certain diseases and/or conditions.
  • Various algorithmic methods may be applied, for example, by multiplying, dividing, addition, or subtraction for various antigen values. These algorithms may be used in the diagnosis of diseases and/or conditions. Data may be further analyzed via more sophisticated techniques, for instance, cluster analysis, neural network, or multivariate loigistic regression techniques.
  • Neural networks are a well-established technology for solving prediction and classification problems, using training and testing data to build a model.
  • the data involves historical data sets containing input variables, or data fields, which correspond to an output.
  • the network uses the training data to "learn" the solution to the problem by example. Since the network learns in this way, no complex models need to be created. Also, it is not necessary for your data to be complete or show a clear trend - neural networks can still converge to a solution under these conditions.
  • Logistic regression is part of a category of statistical models called generalized linear models. This broad class of models includes ordinary regression and ANOVA, as well as multivariate statistics such as ANCOVA and loglinear regression. An excellent treatment of generalized linear models is presented in Agresti (1996).
  • Logistic regression allows one to predict a discrete outcome, such as group membership, from a set of variables that maybe continuous, discrete, dichotomous, or a mix of any of these.
  • the dependent or response variable is dichotomous, such as presence/absence or success/failure.
  • Discriminant analysis is also used to predict group membership with only two groups. However, discriminant analysis can only be used with continuous independent variables. Thus, in instances where the independent variables are a categorical, or a mix of continuous and categorical, logistic regression is preferred.
  • the most important biomarkers are the presence or absence or MHC-I, Macrophage Scavenger Receptor, Osteocalcin, PGRP-I, PSA 5 Aquaporin-4.
  • the results may be better analyzed by comparison of specific marker values to the capture results.
  • the ratio of marker Macrophage 1:1.5 to (or and approximate 30 fold difference) capture whereas in prostatitis the ratio is 1:0.5.
  • the ratio result (prostate cancer) MHC-I to capture is about 5% whereas the ratio in Prostatitis is almost 1.0 or a 20 fold difference.
  • Osteocalcin shows importance as either a presence and absence value as it is not present in Cancer.
  • PSA shows a value of approximately 0.076 in Prostatitis and 0.03 in prostate cancer, or approximately 2 fold differences. This is a very small factor in favor of prostate cancer. In TG2 (labvision) the difference is .0009 in Prostatitis and approximately 0.15 in prostate cancer, a difference of approximately 166 fold.
  • Psammoma Endometrioid adenocarecenoma the most important biomarkers that are present or absent are MHC-I, Cystatin A, osteocalcin, PGRP-I Beeta, PSA, Labvisoin TG-2, Aquaporin-4.
  • Psammoma Endometrioid adenocarecenoma had 8 groups with extremely high calcification that may be, for instance, easily separated by the correlation of the presence or absence MSR and PGRP-I Beeta and Aquaporin-4. Notable is that some normal positive high value had high PSA.
  • disease specific marker tests results indicate that since the measurements were made using human blood samples different patterns of antigens on CNP may be explained only by assuming that those markers were bound on the surface of the CNP at the specific location of the pathological process. Therefore, these markers as associated with the CNP may be used to diagnose pathological processes, diseases, and ongoing processes leading to pathological problems (risk analysis and therapy follow up). This is due to the fact that different tissue and cells contain different (and the same) types of specific markers. It is well known that markers for diseases can be present YEARS before the onset of disease.
  • biomarkers can detectable prior to clinical diagnosis of disease and may be used as risk factor analysis or early detection of diseases including, but are not limited to, for example, heart or circulatory diseases such as Arteriosclerosis, Atherosclerosis, Coronary Heart Disease, Chronic Heart Failure, Valve Calcifications, Arterial Aneurysms, Calcific Aortic Stenosis, Transient Cerebral Ischemia, Stroke, Peripheral Vascular Disease, Monckeberg's Disease, Vascular Thrombosis; Dental Diseases such as Dental Plaque, Gum Disease (dental pulp stones), calcification of the dentinal papilla, and Salivary Gland Stones; Chronic Infection Syndromes such as Chronic Fatigue Syndrome; Kidney and Bladder Stones, Gall Stones, Pancreas and Bowel Diseases such as Pancreatic Duct Stones, Crohn's Disease, Colitis Ulcerosa; Blood disorders; Adrenal Calcification; Liver Diseases such as Liver Cirrhosis and Liver Cyst
  • Figure 12 shows the excretion in urine from a RAT.
  • the excretion kinetics in the urine were very different. The most pronounced differentiation was shown with the Kindey stone isolate.
  • Table 8 is a list of some proteins that can be on CNPs.
  • Table 9 is a list of proteins and compounds that can be associated with CNPs and proteins on CNPs.
  • Table 10 shows calculated unit per ml data from 8 diseases using 14 markers and 10 patient samples for each disease.
  • Table 11 shows the use of SAPIA technique to map Proteins associated with CNPS (Raw Data plus units per ml data).
  • Table 12 shows a table on correlation on SAPIA results for various proteins and antigens on CNPs (coefficients and significances).
  • Nontreat - Clodrona -38.68 -158.69 81.34
  • Nontreat - CaIEDTA -35.68 -155.69 84.34
  • Nontreat - Nanobact 133.00 33.33 232.67 ***
  • Apolipoprotein A-II (antimicrobial peptide 21 21.00 1
  • Alpha-2-antiplasmin precursor 2 13.28 1 Table 3. List of antibodies used in the SAPIA test and immunohistochemical staining (IHS).
  • Testican-3 precursor SPARC/osteonectin, CWCV, and Kazal-like domains
  • TM Thrombomodulin precursor (Fetomodulin) (CD141 antigen).
  • TM Thrombomodulin precursor (Fetomodulin) (CD141 antigen).
  • TRBM HUMAN P07204
  • Name THBD
  • Synonyms THRM ⁇ - Homo sapiens (Human)
  • Uromodulin precursor (Tamm-Horsfall urinary glycoprotein) (THP).
  • THP Tamm-Horsfall urinary glycoprotein
  • VILIP Visinin-like protein 1
  • HLP3 Hippocalcin-like protein 3
  • VISL1 HUMAN P62760
  • Name VSNL1
  • Synonyms VISL1 ⁇ - Homo sapiens (Human)
  • Nan-04-294 343.062 640.000 40.458 25.798 1.954 43.247 1.087 3.206 1.118 1.118
  • Nan-04-315 14.989 0.200 11.784 5.908 0.653 14.396 0.435 0.191 0.093 0.093
  • Nan-04-315 14.989 1.950 0.036 4.712 0.000
  • RhO O Number of Observations var8 var9 varlO varll
  • RhO O Number of Observations varl2 varl3 varl4 varl5
  • RhO O Number of Observations var24 var25 var26 var27
  • RhO O Number of Observations var32 var33 var34 var35
  • Rho 0 Number of Observations var69 var70 var71 var72

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Abstract

La présente invention concerne des procédés et des compositions pour la détection l'analyse et l'évaluation de l'importance de la calcification de nanoparticules. D'une manière générale, les procédés et compositions de l'invention comprennent la détection d'une ou de plusieurs protéines présentes sur la nanoparticule en cours de calcification. On a découvert que des protéines particulières s'associent à des nanoparticules en cours de calcification. Cette association fournit un moyen pour la détection, la classification, l'analyse, la catégorisation, et l'évaluation de nanoparticules en cours de calcification. La détection de protéines particulières pendant leur association avec une nanoparticule en cours de calcification peut être utilisée pour indiquer la présence ou le type de nanoparticule en cours de calcification, qui peut servir d'indication de la présence de, ou la disposition à, des maladies ou conditions. Une pluralité de protéines sur une particule en cours de calcification peuvent être détectées. La présente ou l'absence de protéines particulières et la configuration de la présence ou de l'absence de protéines particulières peuvent être utilisées pour indiquer la présence et le type de nanoparticule en cours de calcification.
PCT/US2005/044589 2005-12-09 2005-12-09 Detection de la calcification de nanoparticules, et proteines y associees WO2007070021A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2468368C1 (ru) * 2011-06-17 2012-11-27 Федеральное государственное автономное образовательное учреждение высшего профессионального образования "Казанский (Приволжский) Федеральный Университет" (ФГАОУ ВПО КФУ) Способ детектирования парамагнитных комплексов марганца как маркеров атеросклероза
WO2013104104A1 (fr) * 2012-01-09 2013-07-18 苏州工业园区为真生物医药科技有限公司 Marqueur de diagnostic et d'indication pour le cancer du sein
CN103901207A (zh) * 2013-05-07 2014-07-02 上海良润生物医药科技有限公司 Cystatin S和CA15-3在制备诊断和预示乳腺癌标志物中的应用
US10670611B2 (en) 2014-09-26 2020-06-02 Somalogic, Inc. Cardiovascular risk event prediction and uses thereof
US11143659B2 (en) 2015-01-27 2021-10-12 Arterez, Inc. Biomarkers of vascular disease
WO2022182832A1 (fr) * 2021-02-24 2022-09-01 Board Of Regents, The University Of Texas System Procédés de traitement et d'analyse de polypeptides
US11435365B2 (en) 2010-06-03 2022-09-06 Idexx Laboratories, Inc. Markers for renal disease
KR102543112B1 (ko) * 2023-04-12 2023-06-13 주식회사 브레디스헬스케어 유체 시료 내에 극미량으로 존재하는 분석 대상 물질의 입자를 검출하기 위한 방법

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006052924A2 (fr) * 2004-11-08 2006-05-18 Nanobac Life Sciences Procedes et compositions de complexes proteines-hydroxy-apatites et leur application pour tester et moduler un systeme immunologique contenant un nouveau test in vitro pour la detection d'anticorps contre les complexes proteines-hydroxy-apatites liant le calcium

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006052924A2 (fr) * 2004-11-08 2006-05-18 Nanobac Life Sciences Procedes et compositions de complexes proteines-hydroxy-apatites et leur application pour tester et moduler un systeme immunologique contenant un nouveau test in vitro pour la detection d'anticorps contre les complexes proteines-hydroxy-apatites liant le calcium

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "NANOCAPTURE ELISA KIT", NANOBAC OY, 7 July 2003 (2003-07-07), Science Park BioTeknia, Kuopio, Finland, XP002430847 *
G. HUDELIST ET AL: "Presence of nanobacteria in psammoma bodies of ovarian cancer: evidence for pathogenetic role in intratumoral biomineralization.", HISTOPATHOLOGY DEC 2004, vol. 45, no. 6, December 2004 (2004-12-01), United Kingdom, pages 633 - 637, XP002430846, ISSN: 0309-0167 *

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US11933792B2 (en) 2010-06-03 2024-03-19 Idexx Laboratories, Inc. Markers for renal disease
US11435365B2 (en) 2010-06-03 2022-09-06 Idexx Laboratories, Inc. Markers for renal disease
RU2468368C1 (ru) * 2011-06-17 2012-11-27 Федеральное государственное автономное образовательное учреждение высшего профессионального образования "Казанский (Приволжский) Федеральный Университет" (ФГАОУ ВПО КФУ) Способ детектирования парамагнитных комплексов марганца как маркеров атеросклероза
GB2513771A (en) * 2012-01-09 2014-11-05 Suzhou Microdiag Biomedicine Co Ltd Breast cancer diagnosis and indicaton marker
CN104136630A (zh) * 2012-01-09 2014-11-05 苏州工业园区为真生物医药科技有限公司 诊断和预示乳腺癌的标志物
GB2513771B (en) * 2012-01-09 2020-05-27 Suzhou Microdiag Biomedicine Co Ltd Biomarkers for breast cancer predictions and diagnoses
WO2013104104A1 (fr) * 2012-01-09 2013-07-18 苏州工业园区为真生物医药科技有限公司 Marqueur de diagnostic et d'indication pour le cancer du sein
CN103901207A (zh) * 2013-05-07 2014-07-02 上海良润生物医药科技有限公司 Cystatin S和CA15-3在制备诊断和预示乳腺癌标志物中的应用
US10670611B2 (en) 2014-09-26 2020-06-02 Somalogic, Inc. Cardiovascular risk event prediction and uses thereof
US11143659B2 (en) 2015-01-27 2021-10-12 Arterez, Inc. Biomarkers of vascular disease
US11821905B2 (en) 2015-01-27 2023-11-21 Arterez, Inc. Biomarkers of vascular disease
WO2022182832A1 (fr) * 2021-02-24 2022-09-01 Board Of Regents, The University Of Texas System Procédés de traitement et d'analyse de polypeptides
KR102543112B1 (ko) * 2023-04-12 2023-06-13 주식회사 브레디스헬스케어 유체 시료 내에 극미량으로 존재하는 분석 대상 물질의 입자를 검출하기 위한 방법

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