METHODS AND COMPOSITIONS FOR DIAGNOSIS AND PROGNOSIS OF APPENDICITIS AND DIFFERENTIATION OF CAUSES OF ABDOMINAL PAIN

Abstract

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in appendicitis patients and in patients at risk for appendicitis. In particular, the invention relates to using as-says that detect one or more biomarkers as diagnostic and prognostic biomarker assays in such patients.

Claims

1. A method of diagnosing appendicitis in a subject, or assigning a likelihood of a future outcome to a subject diagnosed with appendicitis, comprising: performing one or more assays configured to detect one or more biomarkers selected from the group consisting of 72 kDa type IV collagenase, Adiponectin, Advanced glycosylation end product-specific receptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5, CC motif chemokine 1, CC motif chemokine 13, CC motif chemokine 15, CC motif chemokine 17, CC motif chemokine 19, CC motif chemokine 20, CC motif chemokine 21, CC motif chemokine 22, CC motif chemokine 23, CC motif chemokine 24, CC motif chemokine 26, CC motif chemokine 27, CC motif chemokine 3, CC motif chemokine 4, CC motif chemokine 7, CC motif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, Creatine Kinase-MB, CXC motif chemokine 10, CXC motif chemokine 11, CXC motif chemokine 13, CXC motif chemokine 16, CXC motif chemokine 5, CXC motif chemokine 6, CXC motif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide, Glucagon, Glucagon-like peptide 1, Granulocyte-macrophage colony-stimulating factor, Growth-regulated alpha, beta, and gamma proteins, Heparin-binding growth factor 2, Hepatocyte growth factor, Immuno globulin A, Immunoglobulin M, Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, Insulin-like growth factor-binding protein 5, Insulin-like growth factor-binding protein 6, Insulin-like growth factor-binding protein 7, Interferon alpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptor antagonist protein, Interleukin-1 receptor type I, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunit beta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9, Interstitial collagenase, Islet amyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitory factor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophage metalloelastase, Macrophage migration inhibitory factor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor, Pro-interleukin-16, Prolactin, Prostate-specific antigen, Protein S100-A12, Protransforming growth factor alpha, Secretory immunoglobulin A, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligand superfamily member 10, Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosis factor receptor superfamily member 1B, Vascular endothelial growth factor receptor 1, Vascular endothelial growth factor receptor 2, Vascular endothelial growth factor receptor 3, von Willebrand Factor, and WAP four-disulfide core domain protein 2 on a body fluid sample obtained from the subject to provide one or more assay result(s); and correlating the assay result(s) to the occurrence or nonoccurrence of appendicitis in the subject or likelihood of the future outcome to the subject.

2. A method according to claim 1, wherein the performing step comprises introducing the body fluid sample obtained from the subject into an assay instrument which (i) contacts the body fluid sample with one or more binding reagents corresponding to the biomarker(s) being assayed, wherein each biomarker which is assayed binds to its respective specific binding reagent in an amount related to its concentration in the body fluid sample, (ii) generates one or more assay results indicative of binding of each biomarker which is assayed to its respective specific binding reagent; and (iii) displays the one or more assay results as a quantitative result in a human-readable form.

3. A method according to claim 1 or 2, wherein the future outcome is mortality.

4. A method according to claim 1 or 2, wherein the subject is being evaluated for abdominal pain.

5. A method according to claim 1 or 2, wherein the correlating step comprises determining the concentration of each biomarker which is assayed, and individually comparing each biomarker concentration to a corresponding threshold level for that biomarker.

6. A method according to claim 5, wherein the assay instrument performs the correlating step, which comprises determining the concentration of each biomarker which is assayed, individually comparing each biomarker concentration to a corresponding threshold level for that biomarker, and displaying an indication of whether each biomarker does or does not exceed its corresponding threshold in a human-readable form.

7. A method according to claim 2, wherein a plurality of the biomarkers are measured, wherein the assay instrument performs the correlating step, which comprises determining the concentration of each of the plurality of biomarkers, calculating a single value based on the concentration of each of the plurality of biomarkers, comparing the single value to a corresponding threshold level and displaying an indication of whether the single value does or does not exceed its corresponding threshold in a human-readable form.

8. A method according to one of claims 1-7, wherein method provides a sensitivity or specificity of at least 0.7 for the identification of appendicitis when compared to normal subjects.

9. A method according to one of claims 1-7, wherein method provides a sensitivity or specificity of at least 0.7 for the identification of appendicitis when compared to subjects exhibiting symptoms that mimic appendicitis symptoms.

10. A method according to one of claims 1-9, wherein the sample is selected from the group consisting of blood, serum, and plasma.

11. A method for evaluating biomarker levels in a body fluid sample, comprising: obtaining a body fluid sample from a subject selected for evaluation based on a determination that the subject is experiencing symptoms indicative of possible acute appendicitis; and performing one or more analyte binding assays configured to detect one or more biomarkers selected from the group consisting of 72 kDa type IV collagenase, Adiponectin, Advanced glycosylation end product-specific receptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5, CC motif chemokine 1, CC motif chemokine 13, CC motif chemokine 15, CC motif chemokine 17, CC motif chemokine 19, CC motif chemokine 20, CC motif chemokine 21, CC motif chemokine 22, CC motif chemokine 23, CC motif chemokine 24, CC motif chemokine 26, CC motif chemokine 27, CC motif chemokine 3, CC motif chemokine 4, CC motif chemokine 7, CC motif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, Creatine Kinase-MB, CXC motif chemokine 10, CXC motif chemokine 11, CXC motif chemokine 13, CXC motif chemokine 16, CXC motif chemokine 5, CXC motif chemokine 6, CXC motif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide, Glucagon, Glucagon-like peptide 1, Granulocyte-macrophage colony-stimulating factor, Growth-regulated alpha, beta, and gamma proteins, Heparin-binding growth factor 2, Hepatocyte growth factor, Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, Insulin-like growth factor-binding protein 5, Insulin-like growth factor-binding protein 6, Insulin-like growth factor-binding protein 7, Interferon alpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptor antagonist protein, Interleukin-1 receptor type I, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunit beta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9, Interstitial collagenase, Islet amyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitory factor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophage metalloelastase, Macrophage migration inhibitory factor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor, Pro-interleukin-16, Prolactin, Prostate-specific antigen, Protein S100-A12, Protransforming growth factor alpha, Secretory immunoglobulin A, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligand superfamily member 10, Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosis factor receptor superfamily member 1B, Vascular endothelial growth factor receptor 1, Vascular endothelial growth factor receptor 2, Vascular endothelial growth factor receptor 3, von Willebrand Factor, and WAP four-disulfide core domain protein 2 by introducing the body fluid sample obtained from the subject into an assay instrument which (i) contacts the body fluid sample with one or more binding reagents corresponding to the biomarker(s) being assayed, wherein each biomarker which is assayed binds to its respective specific binding reagent in an amount related to its concentration in the body fluid sample, (ii) generates one or more assay results indicative of binding of each biomarker which is assayed to its respective specific binding reagent; and (iii) displays the one or more assay results as a quantitative result in a human-readable form.

12. A method according to claim 11, wherein the assay result(s) are displayed as a concentration of each biomarker which is assayed.

13. A method according to claim 12, wherein the assay instrument further individually compares each biomarker concentration to a corresponding threshold level for that biomarker, and displays an indication of whether each biomarker does or does not exceed its corresponding threshold in a human-readable form.

14. A method according to claim 11, wherein a plurality of the biomarkers are measured, and wherein the assay results(s) comprise a single value calculated using a function that converts the concentration of each of the plurality of biomarkers into a single value.

15. A method according to claim 14, wherein the assay instrument further compares the single value to a corresponding threshold level and displays an indication of whether the single value does or does not exceed its corresponding threshold in a human-readable form.

16. A method for evaluating biomarker levels in a body fluid sample, comprising: obtaining a body fluid sample from a subject selected for evaluation based on a determination that the subject has been diagnosed with acute appendicitis; and performing one or more analyte binding assays configured to detect one or more biomarkers selected from the group consisting of 72 kDa type IV collagenase, Adiponectin, Advanced glycosylation end product-specific receptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5, CC motif chemokine 1, CC motif chemokine 13, CC motif chemokine 15, CC motif chemokine 17, CC motif chemokine 19, CC motif chemokine 20, CC motif chemokine 21, CC motif chemokine 22, CC motif chemokine 23, CC motif chemokine 24, CC motif chemokine 26, CC motif chemokine 27, CC motif chemokine 3, CC motif chemokine 4, CC motif chemokine 7, CC motif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, Creatine Kinase-MB, CXC motif chemokine 10, CXC motif chemokine 11, CXC motif chemokine 13, CXC motif chemokine 16, CXC motif chemokine 5, CXC motif chemokine 6, CXC motif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide, Glucagon, Glucagon-like peptide 1, Granulocyte-macrophage colony-stimulating factor, Growth-regulated alpha, beta, and gamma proteins, Heparin-binding growth factor 2, Hepatocyte growth factor, Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, Insulin-like growth factor-binding protein 5, Insulin-like growth factor-binding protein 6, Insulin-like growth factor-binding protein 7, Interferon alpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptor antagonist protein, Interleukin-1 receptor type I, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunit beta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9, Interstitial collagenase, Islet amyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitory factor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophage metalloelastase, Macrophage migration inhibitory factor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor, Pro-interleukin-16, Prolactin, Prostate-specific antigen, Protein S100-A12, Protransforming growth factor alpha, Secretory immunoglobulin A, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligand superfamily member 10, Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosis factor receptor superfamily member 1B, Vascular endothelial growth factor receptor 1, Vascular endothelial growth factor receptor 2, Vascular endothelial growth factor receptor 3, von Willebrand Factor, and WAP four-disulfide core domain protein 2 by introducing the body fluid sample obtained from the subject into an assay instrument which (i) contacts the body fluid sample with one or more binding reagents corresponding to the biomarker(s) being assayed, wherein each biomarker which is assayed binds to its respective specific binding reagent in an amount related to its concentration in the body fluid sample, (ii) generates one or more assay results indicative of binding of each biomarker which is assayed to its respective specific binding reagent; and (iii) displays the one or more assay results as a quantitative result in a human-readable form.

17. A method according to claim 16, wherein the assay result(s) are displayed as a concentration of each biomarker which is assayed.

18. A method according to claim 17, wherein the assay instrument further individually compares each biomarker concentration to a corresponding threshold level for that biomarker, and displays an indication of whether each biomarker does or does not exceed its corresponding threshold in a human-readable form.

19. A method according to claim 16, wherein a plurality of the biomarkers are measured, and wherein the assay results(s) comprise a single value calculated using a function that converts the concentration of each of the plurality of biomarkers into a single value.

20. A method according to claim 19, wherein the assay instrument further compares the single value to a corresponding threshold level and displays an indication of whether the single value does or does not exceed its corresponding threshold in a human-readable form.

21. A method according to one of claims 16-19, wherein the subject is selected for evaluation of a mortality risk within a period selected from the group consisting of 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, and 12 hours.

22. A method according to one of claims 11-21, wherein the plurality of assays are immunoassays performed by (i) introducing the body fluid sample into an assay device comprising a plurality of antibodies, at least one of which binds to each biomarker which is assayed, and (ii) generating an assay result indicative of binding of each biomarker to its respective antibody.

23. A system for evaluating biomarker levels, comprising: a plurality of reagents which specifically bind for detection a plurality of biomarkers selected from the group consisting of 72 kDa type IV collagenase, Adiponectin, Advanced glycosylation end product-specific receptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5, CC motif chemokine 1, CC motif chemokine 13, CC motif chemokine 15, CC motif chemokine 17, CC motif chemokine 19, CC motif chemokine 20, CC motif chemokine 21, CC motif chemokine 22, CC motif chemokine 23, CC motif chemokine 24, CC motif chemokine 26, CC motif chemokine 27, CC motif chemokine 3, CC motif chemokine 4, CC motif chemokine 7, CC motif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, Creatine Kinase-MB, CXC motif chemokine 10, CXC motif chemokine 11, CXC motif chemokine 13, CXC motif chemokine 16, CXC motif chemokine 5, CXC motif chemokine 6, CXC motif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide, Glucagon, Glucagon-like peptide 1, Granulocyte-macrophage colony-stimulating factor, Growth-regulated alpha, beta, and gamma proteins, Heparin-binding growth factor 2, Hepatocyte growth factor, Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, Insulin-like growth factor-binding protein 5, Insulin-like growth factor-binding protein 6, Insulin-like growth factor-binding protein 7, Interferon alpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptor antagonist protein, Interleukin-1 receptor type I, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunit beta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9, Interstitial collagenase, Islet amyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitory factor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophage metalloelastase, Macrophage migration inhibitory factor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor, Pro-interleukin-16, Prolactin, Prostate-specific antigen, Protein S100-A12, Protransforming growth factor alpha, Secretory immunoglobulin A, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligand superfamily member 10, Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosis factor receptor superfamily member 1B, Vascular endothelial growth factor receptor 1, Vascular endothelial growth factor receptor 2, Vascular endothelial growth factor receptor 3, von Willebrand Factor, and WAP four-disulfide core domain protein 2; an assay instrument configured to (i) receive a body fluid sample, (ii) contact the plurality of reagents with the body fluid sample and (iii) generate and quantitatively display in human readable form one or more assay results indicative of binding of each biomarker which is assayed to a respective specific binding reagent in the plurality of reagents.

24. A system according to claim 23 wherein the reagents comprise a plurality of antibodies, at least one of which binds to each of the biomarkers which are assayed.

25. A system according to claim 24 wherein assay instrument comprises an assay device and an assay device reader, wherein the plurality of antibodies are immobilized at a plurality of predetermined locations within the assay device, wherein the assay device is configured to receive the body fluid sample such that the body fluid sample contacts the plurality of predetermined locations, and wherein the assay device reader interrogates the plurality of predetermined locations to generate the assay results.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates to methods and compositions for diagnosis, differential diagnosis, risk stratification, monitoring, classifying and determination of treatment regimens in patients diagnosed with, or at risk of, appendicitis. In various embodiments, a measured concentration of one or more biomarkers selected from the group consisting of 72 kDa type IV collagenase, Adiponectin, Advanced glycosylation end product-specific receptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5, CC motif chemokine 1, CC motif chemokine 13, CC motif chemokine 15, CC motif chemokine 17, CC motif chemokine 19, CC motif chemokine 20, CC motif chemokine 21, CC motif chemokine 22, CC motif chemokine 23, CC motif chemokine 24, CC motif chemokine 26, CC motif chemokine 27, CC motif chemokine 3, CC motif chemokine 4, CC motif chemokine 7, CC motif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, Creatine Kinase-MB, CXC motif chemokine 10, CXC motif chemokine 11, CXC motif chemokine 13, CXC motif chemokine 16, CXC motif chemokine 5, CXC motif chemokine 6, CXC motif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide, Glucagon, Glucagon-like peptide 1, Granulocyte-macrophage colony-stimulating factor, Growth-regulated alpha, beta, and gamma proteins, Heparin-binding growth factor 2, Hepatocyte growth factor, Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, Insulin-like growth factor-binding protein 5, Insulin-like growth factor-binding protein 6, Insulin-like growth factor-binding protein 7, Interferon alpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptor antagonist protein, Interleukin-1 receptor type I, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunit beta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9, Interstitial collagenase, Islet amyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitory factor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophage metalloelastase, Macrophage migration inhibitory factor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor, Pro-interleukin-16, Prolactin, Prostate-specific antigen, Protein S100-A12, Protransforming growth factor alpha, Secretory immunoglobulin A, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligand superfamily member 10, Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosis factor receptor superfamily member 1B, Vascular endothelial growth factor receptor 1, Vascular endothelial growth factor receptor 2, Vascular endothelial growth factor receptor 3, von Willebrand Factor, and WAP four-disulfide core domain protein 2 or one or more markers related thereto, are correlated to the status of the patient. As described herein, measurement of one or more biomarkers of the present invention may be used, individually or in panels comprising a plurality of biomarkers, in methods and compositions for the diagnosis, prognosis, or differentiation of abdominal pain in order to rule in or out appendicitis and/or a particular outcome. Such markers can be used in diagnosing and treating a subject and/or to monitor the course of a treatment regimen; for screening subjects for the occurrence or risk of a particular disease; and for screening compounds and pharmaceutical compositions that might provide a benefit in treating or preventing such conditions.

[0029] For purposes of this document, the following definitions apply:

[0030] The term subject as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. Further, while a subject is preferably a living organism, the invention described herein may be used in post-mortem analysis as well. Preferred subjects are humans, and most preferably patients, which as used herein refers to living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology. An appendicitis patient is a patient exhibiting symptoms consistent with appendicitis and being evaluated for its presence, absence, or outcome

[0031] Conditions within the differential diagnosis include gallbladder attack, kidney infection, pneumonia, rheumatic fever, diabetic ketoacidosis, ectopic pregnancy, twisted ovarian cyst, hemorrhaging ovarian follicle, urinary tract infection, ulcerative colitis, pancreatitis, intestinal obstruction, pelvic inflammatory disease, diverticulitis, carcinoma of the colon, and aortic aneurysm. In preferred embodiments, the biomarkers of the present invention distinguish appendicitis from one or more of these mimicking conditions.

[0032] Preferably, an analyte such as an appendicitis biomarker is measured in a sample. Such a sample may be obtained from a patient, such as an appendicitis patient. Preferred samples are body fluid samples.

[0033] The term body fluid sample as used herein refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, classification or evaluation of an appendicitis patient of interest, such as a patient or transplant donor. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition. Preferred body fluid samples include blood, serum, plasma, urine, saliva, sputum, and pleural effusions. In addition, one of skill in the art would realize that certain body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

[0034] The term diagnosis as used herein refers to methods by which the skilled artisan can estimate and/or determine the probability (a likelihood) of whether or not a patient is suffering from a given disease or condition. In the case of the present invention, diagnosis includes using the results of an assay, most preferably an immunoassay, for an appendicitis biomarker of the present invention, optionally together with other clinical characteristics, to arrive at a diagnosis (that is, the occurrence or nonoccurrence) of a disease or condition. That such a diagnosis is determined is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of multiple conditions. The skilled clinician does not use biomarker results in an informational vacuum, but rather test results are used together with other clinical indicia to arrive at a diagnosis. Thus, a measured biomarker level on one side of a predetermined diagnostic threshold indicates a greater likelihood of the occurrence of disease in the appendicitis patient relative to a measured level on the other side of the predetermined diagnostic threshold.

[0035] Similarly, a prognostic risk signals a probability (a likelihood) that a given course or outcome will occur. A level or a change in level of a prognostic indicator, which in turn is associated with an increased probability of morbidity or mortality is referred to as being indicative of an increased likelihood of an adverse outcome in a patient.

[0036] As used herein, the term relating a signal to the presence or amount of an analyte reflects the following understanding. Assay signals are typically related to the presence or amount of an analyte through the use of a standard curve calculated using known concentrations of the analyte of interest. As the term is used herein, an assay is configured to detect an analyte if an assay can generate a detectable signal indicative of the presence or amount of a physiologically relevant concentration of the analyte. Because an antibody epitope is on the order of 8 amino acids, an immunoassay configured to detect a marker of interest will also detect polypeptides related to the marker sequence, so long as those polypeptides contain the epitope(s) necessary to bind to the antibody or antibodies used in the assay. The term related marker as used herein with regard to a biomarker such as one of the appendicitis biomarkers described herein refers to one or more fragments, variants, etc., of a particular marker or its biosynthetic parent that may be detected as a surrogate for the marker itself or as independent biomarkers. The term also refers to one or more polypeptides present in a biological sample that are derived from the biomarker precursor complexed to additional species, such as binding proteins, receptors, heparin, lipids, sugars, etc.

[0037] In this regard, the skilled artisan will understand that the signals obtained from an immunoassay are a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., the analyte) and polypeptides containing the necessary epitope(s) to which the antibodies bind. While such assays may detect the full length biomarker and the assay result be expressed as a concentration of a biomarker of interest, the signal from the assay is actually a result of all such immunoreactive polypeptides present in the sample. Expression of biomarkers may also be determined by means other than immunoassays, including protein measurements (such as dot blots, western blots, chromatographic methods, mass spectrometry, etc.) and nucleic acid measurements (mRNA quatitation). This list is not meant to be limiting. With regard to biomarkers which exist in one form as type-I, type-II, or GPI-anchored membrane proteins, such membrane proteins typically comprise a substantial extracellular domain, some or all of which can be detected as soluble forms present in aqueous samples such as blood, serum, plasma, urine, etc., either as cleavage products or as splice variants which delete an effective membrane spanning domain. Preferred assays detect soluble forms of these biomarkers.

[0038] The term positive going marker as that term is used herein refer to a marker that is determined to be elevated in patients suffering from a disease or condition, relative to those not suffering from that disease or condition. The term negative going marker as that term is used herein refer to a marker that is determined to be reduced in patients suffering from a disease or condition, relative to patients not suffering from that disease or condition.

Appendicitis Biomarkers

[0039] The following table provides a list of the biomarkers of the present invention, together with the Swiss-Prot entry number for the human precursor. As noted above, these biomarkers are referred to for convenience herein as appendicitis biomarkers.

TABLE-US-00001 SwissProtNum Preferred Name Q15848 Adiponectin Q15109 Advanced glycosylation end product-specific receptor P01023 Alpha-2 macroglobulin P02765 Alpha-2-HS-glycoprotein P02771 Alpha-fetoprotein Q15389 Angiopoietin-1 P03973 Antileukoproteinase P08519 Apolipoprotein(a) P15941 Cancer Antigen 15-3 N/A Cancer Antigen 19-9 Q16790 Carbonic anhydrase 9 P06731 Carcinoembryonic antigen-related cell adhesion molecule 5 P22362 C-C motif chemokine 1 Q99616 C-C motif chemokine 13 Q16663 C-C motif chemokine 15 Q92583 C-C motif chemokine 17 Q99731 C-C motif chemokine 19 P78556 C-C motif chemokine 20 O00585 C-C motif chemokine 21 O00626 C-C motif chemokine 22 P55773 C-C motif chemokine 23 O00175 C-C motif chemokine 24 Q9Y258 C-C motif chemokine 26 Q9Y4X3 C-C motif chemokine 27 P10147 C-C motif chemokine 3 P13236 C-C motif chemokine 4 P80098 C-C motif chemokine 7 P80075 C-C motif chemokine 8 P00450 Ceruloplasmin P01233 Choriogonadotropin subunit beta P45452 Collagenase 3 P01308 C-Peptide (aa57-87) P12277; Creatine Kinase-MB P06732 P02778 C-X-C motif chemokine 10 O14625 C-X-C motif chemokine 11 O43927 C-X-C motif chemokine 13 Q9H2A7 C-X-C motif chemokine 16 P42830 C-X-C motif chemokine 5 P80162 C-X-C motif chemokine 6 Q07325 C-X-C motif chemokine 9 P01034 Cystatin-C Q9UNN8 Endothelial protein C receptor P51671 Eotaxin P00533 Epidermal growth factor receptor P02792; Ferritin P02794 P02671; Fibrinogen P02675; P02679 P09681 Gastric inhibitory polypeptide P01275 Glucagon P01275 Glucagon-like peptide 1 (aa98-127; aa98-128) P04141 Granulocyte-macrophage colony-stimulating factor P09341; Growth-regulated alpha, beta, and gamma proteins P19875; P19876 P09038 Heparin-binding growth factor 2 P14210 Hepatocyte growth factor N/A Immunoglobulin A n/a Immunoglobulin M n/a Immunoglogulin G1 N/A Immunoglogulin G2 N/A Immunoglogulin G3 n/a Immunoglogulin G4 P01308 Insulin P08833 Insulin-like growth factor-binding protein 1 P18065 Insulin-like growth factor-binding protein 2 P17936 Insulin-like growth factor-binding protein 3 P22692 Insulin-like growth factor-binding protein 4 P24593 Insulin-like growth factor-binding protein 5 P24592 Insulin-like growth factor-binding protein 6 Q16270 Insulin-like growth factor-binding protein 7 P01563 Interferon alpha-2 P01579 Interferon gamma P01583 Interleukin-1 alpha P01584 Interleukin-1 beta P18510 Interleukin-1 receptor antagonist protein P14778 Interleukin-1 receptor type I P27930 Interleukin-1 receptor type II P20809 Interleukin-11 P29459; Interleukin-12 P29460 P29460 Interleukin-12 subunit beta P35225 Interleukin-13 P40933 Interleukin-15 P60568 Interleukin-2 Q9NYY1 Interleukin-20 Q9HBE4 Interleukin-21 Q9NPF7; Interleukin-23 P29460 Q8IZJ0 Interleukin-28A Q8IU54 Interleukin-29 P08700 Interleukin-3 O95760 Interleukin-33 P05112 Interleukin-4 P05113 Interleukin-5 P08887 Interleukin-6 receptor subunit alpha P40189 Interleukin-6 receptor subunit beta P13232 Interleukin-7 P15248 Interleukin-9 P03956 Interstitial collagenase P10997 Islet amyloid polypeptide P08727 Keratin, type I cytoskeletal 19 (aa311-367) P21583 Kit ligand P41159 Leptin P15018 Leukemia inhibitory factor P47992 Lymphotactin P01374 Lymphotoxin-alpha P09603 Macrophage colony-stimulating factor 1 P39900 Macrophage metalloelastase P14174 Macrophage migration inhibitory factor P09237 Matrilysin P01033 Metalloproteinase inhibitor 1 P16035 Metalloproteinase inhibitor 2 P35625 Metalloproteinase inhibitor 3 Q99727 Metalloproteinase inhibitor 4 P02144 Myoglobin P01298 Pancreatic prohormone P10082 Peptide YY P01133 Pro-epidermal growth factor Q14005 Pro-interleukin-16 P01236 Prolactin P07288 Prostate-specific antigen P80511 Protein S100-A12 P01135 Protransforming growth factor alpha N/A Secretory immunoglobulin A P02743 Serum amyloid P-component P49771 SL cytokine P48061 Stromal cell-derived factor 1 P08254 Stromelysin-1 P40225 Thrombopoietin Q969D9 Thymic stromal lymphopoietin P01375 Tumor necrosis factor P50591 Tumor necrosis factor ligand superfamily member 10 P48023 Tumor necrosis factor ligand superfamily member 6 P19438 Tumor necrosis factor receptor superfamily member 1A P20333 Tumor necrosis factor receptor superfamily member 1B P17948 Vascular endothelial growth factor receptor 1 P35968 Vascular endothelial growth factor receptor 2 P35916 Vascular endothelial growth factor receptor 3 P04275 von Willebrand Factor Q14508 WAP four-disulfide core domain protein 2 P05231 Interleukin-6 P08253 72 kDa type IV collagenase

Marker Assays

[0040] In general, immunoassays involve contacting a sample containing or suspected of containing a biomarker of interest with at least one antibody that specifically binds to the biomarker. A signal is then generated indicative of the presence or amount of complexes formed by the binding of polypeptides in the sample to the antibody. The signal is then related to the presence or amount of the biomarker in the sample. Numerous methods and devices are well known to the skilled artisan for the detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.

[0041] The assay devices and methods known in the art can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest. Suitable assay formats also include chromatographic, mass spectrographic, and protein blotting methods. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. One skilled in the art also recognizes that robotic instrumentation including but not limited to Beckman ACCESS, Abbott AXSYM, Roche ELECSYS, Dade Behring STRATUS systems are among the immunoassay analyzers that are capable of performing immunoassays. But any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.

[0042] Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in assays. Solid phases that may be used to immobilize specific binding members include include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. Antibodies or other polypeptides may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding. In an example of the later case, antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface.

[0043] Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied. Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

[0044] Preparation of solid phases and detectable label conjugates often comprise the use of chemical cross-linkers. Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non-specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups. Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable. Imidoesters are also very useful for protein-protein cross-links. A variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation, are commercially available.

[0045] In certain aspects, the present invention provides kits for the analysis of the described appendicitis biomarkers. The kit comprises reagents for the analysis of at least one test sample which comprise at least one antibody that binds an appendicitis biomarker. The kit can also include devices and instructions for performing one or more of the diagnostic and/or prognostic correlations described herein. Preferred kits will comprise an antibody pair for performing a sandwich assay, or a labeled species for performing a competitive assay, for the analyte. Preferably, an antibody pair comprises a first antibody conjugated to a solid phase and a second antibody conjugated to a detectable label, wherein each of the first and second antibodies bind an appendicitis biomarker. Most preferably each of the antibodies are monoclonal antibodies. The instructions for use of the kit and performing the correlations can be in the form of labeling, which refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use. For example, the term labeling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits.

Antibodies

[0046] The term antibody as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., antigen binding sites, (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term antibody.

[0047] Antibodies used in the immunoassays described herein preferably specifically bind to an appendicitis biomarker of the present invention. The term specifically binds is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody specifically binds if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s). Preferably the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In preferred embodiments, preferred antibodies bind with affinities of at least about 10.sup.7 M.sup.1, and preferably between about 10.sup.8 M.sup.1 to about 10.sup.9 M.sup.1, about 10.sup.9 M.sup.1 to about 10.sup.10 M.sup.1, or about 10.sup.10 M.sup.1 to about 10.sup.12 M.sup.1.

[0048] Affinity is calculated as K.sub.d=k.sub.off/k.sub.on (k.sub.off is the dissociation rate constant, K.sub.on is the association rate constant and K.sub.d is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labeled ligand at various concentrations (c). The data are graphed using the Scatchard equation: r/c=K(nr): where r=moles of bound ligand/mole of receptor at equilibrium; c=free ligand concentration at equilibrium; K=equilibrium association constant; and n=number of ligand binding sites per receptor molecule. By graphical analysis, r/c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

[0049] The term epitope refers to an antigenic determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0050] Numerous publications discuss the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.

[0051] The antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) are present.

[0052] The antibodies so identified may then be further analyzed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.

[0053] While the present application describes antibody-based binding assays in detail, alternatives to antibodies as binding species in assays are well known in the art. These include receptors for a particular target, aptamers, etc. Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist. High-affinity aptamers containing modified nucleotides can confer improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions, and may include amino acid side chain functionalities.

Assay Correlations

[0054] The term correlating as used herein in reference to the use of biomarkers refers to comparing the presence or amount of the biomarker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known to be free of a given condition. Often, this takes the form of comparing an assay result in the form of a biomarker concentration to a predetermined threshold selected to be indicative of the occurrence or nonoccurrence of a disease or the likelihood of some future outcome.

[0055] Selecting a diagnostic threshold involves, among other things, consideration of the probability of disease, distribution of true and false diagnoses at different test thresholds, and estimates of the consequences of treatment (or a failure to treat) based on the diagnosis. For example, when considering administering a specific therapy which is highly efficacious and has a low level of risk, few tests are needed because clinicians can accept substantial diagnostic uncertainty. On the other hand, in situations where treatment options are less effective and more risky, clinicians often need a higher degree of diagnostic certainty. Thus, cost/benefit analysis is involved in selecting a diagnostic threshold.

[0056] Suitable thresholds may be determined in a variety of ways. For example, one recommended diagnostic threshold for the diagnosis of acute myocardial infarction using cardiac troponin is the 97.5th percentile of the concentration seen in a normal population. Another method may be to look at serial samples from the same patient, where a prior baseline result is used to monitor for temporal changes in a biomarker level.

[0057] Population studies may also be used to select a decision threshold. Reciever Operating Characteristic (ROC) arose from the field of signal dectection therory developed during World War II for the analysis of radar images, and ROC analysis is often used to select a threshold able to best distinguish a diseased subpopulation from a nondiseased subpopulation. A false positive in this case occurs when the person tests positive, but actually does not have the disease. A false negative, on the other hand, occurs when the person tests negative, suggesting they are healthy, when they actually do have the disease. To draw a ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision threshold is varied continuously. Since TPR is equivalent with sensitivity and FPR is equal to 1-specificity, the ROC graph is sometimes called the sensitivity vs (1-specificity) plot. A perfect test will have an area under the ROC curve of 1.0; a random test will have an area of 0.5. A threshold is selected to provide an acceptable level of specificity and sensitivity.

[0058] In this context, diseased is meant to refer to a population having one characteristic (the presence of a disease or condition or the occurrence of some outcome) and nondiseased is meant to refer to a population lacking the characteristic. While a single decision threshold is the simplest application of such a method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may be assigned with relatively high confidence, and above a second threshold the presence of disease may also be assigned with relatively high confidence. Between the two thresholds may be considered indeterminate. This is meant to be exemplary in nature only.

[0059] In addition to threshold comparisons, other methods for correlating assay results to a patient classification (occurrence or nonoccurrence of disease, likelihood of an outcome, etc.) include decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a patient belongs to one classification out of a plurality of classifications.

[0060] Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given biomarker. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. The area under the curve (AUC) of a ROC plot is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. The area under the ROC curve may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

[0061] As discussed above, suitable tests may exhibit one or more of the following results on these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding specificity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; at least 75% sensitivity, combined with at least 75% specificity; a ROC curve area of greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an odds ratio different from 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a positive likelihood ratio (calculated as sensitivity/(1-specificity)) of greater than 1, at least 2, more preferably at least 3, still more preferably at least 5, and most preferably at least 10; and or a negative likelihood ratio (calculated as (1-sensitivity)/specificity) of less than 1, less than or equal to 0.5, more preferably less than or equal to 0.3, and most preferably less than or equal to 0.1

[0062] Additional clinical indicia may be combined with the appendicitis biomarker assay result(s) of the present invention. Other clinical indicia which may be combined with the appendicitis biomarker assay result(s) of the present invention includes demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, or renal insufficiency), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), etc.

[0063] Combining assay results/clinical indicia in this manner can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, etc. This list is not meant to be limiting.

Selecting a Treatment Regimen

[0064] Once a diagnosis is obtained, the clinician can readily select a treatment regimen that is compatible with the diagnosis. The skilled artisan is aware of appropriate treatments for numerous diseases discussed in relation to the methods of diagnosis described herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research Laboratories, Whitehouse Station, N.J., 1999. In addition, since the methods and compositions described herein provide prognostic information, the markers of the present invention may be used to monitor a course of treatment. For example, improved or worsened prognostic state may indicate that a particular treatment is or is not efficacious.

[0065] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

EXAMPLE 1. IMMUNOASSAY FORMAT

[0066] Analytes are measured using standard sandwich enzyme immunoassay techniques. A first antibody which binds the analyte is immobilized in wells of a 96 well polystyrene microplate. Analyte standards and test samples are pipetted into the appropriate wells and any analyte present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase-conjugated second antibody which binds the analyte is added to the wells, thereby forming sandwich complexes with the analyte (if present) and the first antibody. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution comprising tetramethylbenzidine and hydrogen peroxide is added to the wells. Color develops in proportion to the amount of analyte present in the sample. The color development is stopped and the intensity of the color is measured at 540 nm or 570 nm. An analyte concentration is assigned to the test sample by comparison to a standard curve determined from the analyte standards.

EXAMPLE 2. USE OF ANALYTE AS A MARKER FOR ASSESSING PATIENTS FOR APPENDICITIS

[0067] Patients from the emergency department (ED) classified as positive for appendicitis according to clinical diagnosis at the study site were selected as a diseased cohort (Cohort 1). Plasma and urine samples from each patient in Cohort 1 were collected at the time of appendectomy. Cohort 2 was a normal population sampled separately. The concentrations of the analyte in these samples were measured by standard immunoassay methods using commercially available assay reagents. A receiver operating characteristic (ROC) curve was generated using the concentrations, and the performance of the analyte is assessed by the area under the ROC curve (AUC). The two-tailed p-value of the AUC for the analyte was also calculated to determine statistical significance. Inc/Dec indicates if the marker is increasing or decreasing in Cohort 1 relative to Cohort 2.

TABLE-US-00002 TABLE 1 Urine samples Increasing/ Preferred Name Units Decreasing p Adiponectin ng/ml inc 8.68E07 Advanced glycosylation end pg/ml dec 2.82E01 product-specific receptor Alpha-2 macroglobulin ug/ml inc 1.51E04 Alpha-2-HS-glycoprotein ng/ml inc 1.97E02 Angiopoietin-1 ng/ml dec 7.54E01 Antileukoproteinase pg/ml dec 9.51E01 Apolipoprotein(a) ng/ml dec 6.85E05 Carbonic anhydrase 9 ng/ml inc 3.80E01 C-C motif chemokine 1 pg/ml inc 7.88E01 C-C motif chemokine 13 pg/ml dec 3.19E01 C-C motif chemokine 15 pg/ml dec 1.45E01 C-C motif chemokine 17 pg/ml inc 6.55E01 C-C motif chemokine 19 pg/ml dec 5.62E02 C-C motif chemokine 20 pg/ml dec 2.49E01 C-C motif chemokine 21 pg/ml inc 7.47E01 C-C motif chemokine 22 pg/ml inc 7.81E01 C-C motif chemokine 23 ng/ml inc 5.63E01 C-C motif chemokine 24 pg/ml inc 3.00E02 C-C motif chemokine 26 pg/ml dec 6.44E01 C-C motif chemokine 27 pg/ml inc 3.18E01 C-C motif chemokine 3 pg/ml dec 3.36E01 C-C motif chemokine 4 pg/ml inc 6.33E01 C-C motif chemokine 7 pg/ml inc 6.44E01 C-C motif chemokine 8 pg/ml inc 8.29E01 Ceruloplasmin ng/ml dec 8.02E02 C-Peptide pg/ml dec 3.35E04 Creatine Kinase-MB ng/ml inc 7.18E01 C-X-C motif chemokine 10 pg/ml inc 6.59E02 C-X-C motif chemokine 11 pg/ml inc 6.35E01 C-X-C motif chemokine 13 pg/ml dec 6.57E01 C-X-C motif chemokine 16 ng/ml inc 4.61E01 C-X-C motif chemokine 5 pg/ml inc 8.20E01 C-X-C motif chemokine 6 pg/ml inc 8.93E06 C-X-C motif chemokine 9 pg/ml inc 2.25E01 Cystatin-C ng/ml dec 2.01E01 Endothelial protein C receptor ng/ml inc 1.26E02 Eotaxin pg/ml inc 7.99E01 Epidermal growth factor receptor pg/ml inc 7.11E01 Ferritin pg/ml inc 5.59E06 Fibrinogen ng/ml inc 7.56E01 Gastric inhibitory polypeptide pg/ml inc 6.44E01 Glucagon pg/ml dec 8.18E01 Glucagon-like peptide 1 pg/ml inc 8.18E01 Granulocyte-macrophage colony- pg/ml dec 9.87E02 stimulating factor Heparin-binding growth factor 2 pg/ml dec 7.31E01 Hepatocyte growth factor ng/ml dec 6.68E01 Immunoglobulin A ng/ml inc 6.83E01 Immunoglobulin M ng/ml inc 2.64E08 Immunoglogulin G1 ng/ml dec 1.37E02 Immunoglogulin G2 ng/ml dec 2.19E01 Immunoglogulin G3 ng/ml dec 7.25E01 Immunoglogulin G4 ng/ml inc 4.56E03 Insulin pg/ml dec 4.31E02 Insulin-like growth factor- ng/ml dec 2.00E03 binding protein 1 Insulin-like growth factor- ng/ml dec 0.00E+00 binding protein 2 Insulin-like growth factor- ng/ml dec 1.96E01 binding protein 3 Insulin-like growth factor- ng/ml inc 8.52E01 binding protein 4 Insulin-like growth factor- ng/ml dec 6.96E01 binding protein 5 Insulin-like growth factor- ng/ml inc 1.17E01 binding protein 6 Insulin-like growth factor- ng/ml inc 2.36E01 binding protein 7 Interferon alpha-2 pg/ml dec 2.86E01 Interferon gamma pg/ml inc 8.31E01 Interleukin-1 alpha pg/ml dec 5.10E01 Interleukin-1 beta pg/ml dec 9.67E01 Interleukin-1 receptor pg/ml dec 4.62E02 antagonist protein Interleukin-1 receptor type I pg/mL inc 3.16E01 Interleukin-1 receptor type II pg/mL inc 9.27E01 Interleukin-11 pg/ml dec 7.53E03 Interleukin-12 pg/ml inc 8.48E01 Interleukin-12 subunit beta ng/ml dec 5.34E02 Interleukin-13 pg/ml dec 8.78E01 Interleukin-15 pg/ml dec 2.31E01 Interleukin-2 pg/ml dec 5.87E01 Interleukin-20 pg/ml dec 3.22E01 Interleukin-21 pg/ml dec 4.17E01 Interleukin-23 pg/ml inc 6.19E01 Interleukin-28A pg/ml inc 4.52E01 Interleukin-29 pg/ml inc 3.74E02 Interleukin-3 pg/ml inc 3.48E01 Interleukin-33 pg/ml inc 2.14E01 Interleukin-4 pg/ml inc 5.71E01 Interleukin-5 ng/ml inc 8.08E01 Interleukin-6 receptor subunit alpha pg/ml inc 6.70E01 Interleukin-6 receptor subunit beta pg/ml dec 6.63E01 Interleukin-7 pg/ml dec 8.59E01 Interleukin-9 pg/ml inc 4.32E01 Islet amyloid polypeptide pg/ml dec 8.78E01 Kit ligand pg/ml dec 3.64E01 Leptin pg/ml dec 3.21E01 Leukemia inhibitory factor pg/ml dec 8.27E01 Lymphotactin pg/ml inc 4.48E01 Lymphotoxin-alpha pg/ml dec 6.44E01 Macrophage colony-stimulating pg/ml inc 1.01E01 factor 1 Metalloproteinase inhibitor 1 pg/ml inc 7.52E01 Metalloproteinase inhibitor 2 pg/ml inc 1.78E01 Metalloproteinase inhibitor 3 pg/ml inc 8.45E01 Metalloproteinase inhibitor 4 pg/ml dec 1.62E01 Mix of Growth-regulated alpha, pg/ml inc 4.65E01 beta, and gamma proteins Myoglobin ng/ml dec 1.14E02 Pancreatic prohormone pg/ml dec 5.89E01 Peptide YY pg/ml dec 8.18E01 Pro-epidermal growth factor pg/ml inc 5.02E02 Pro-interleukin-16 pg/ml dec 9.39E01 Protein S100-A12 ng/ml dec 2.49E02 Protransforming growth factor alpha pg/ml inc 5.62E02 Secretory immunoglobulin A ng/ml inc 9.18E01 Serum amyloid P-component ng/ml inc 1.02E04 SL cytokine pg/ml dec 7.11E01 Stromal cell-derived factor 1 pg/ml dec 7.03E02 Thrombopoietin pg/ml dec 1.15E02 Thymic stromal lymphopoietin pg/ml inc 1.81E03 Tumor necrosis factor pg/ml dec 8.29E01 Tumor necrosis factor ligand pg/ml inc 1.24E01 superfamily member 10 Tumor necrosis factor receptor pg/ml dec 5.55E03 superfamily member 1A Tumor necrosis factor receptor pg/ml dec 4.11E02 superfamily member 1B Vascular endothelial growth factor pg/ml dec 1.11E01 receptor 1 Vascular endothelial growth factor pg/ml dec 6.61E01 receptor 2 Vascular endothelial growth factor pg/ml dec 9.11E01 receptor 3 von Willebrand Factor ng/ml inc 2.52E04

TABLE-US-00003 TABLE 2 Plasma samples Increasing/ Preferred Name Units Decreasing p 72 kDa type IV collagenase pg/ml inc 1.73E14 Adiponectin ng/ml inc 8.92E01 Advanced glycosylation end product-specific receptor pg/ml inc 3.24E01 Alpha-2 macroglobulin ug/ml dec 1.52E01 Alpha-2-HS-glycoprotein ng/ml inc 4.26E04 Alpha-fetoprotein pg/ml inc 8.85E01 Angiopoietin-1 ng/ml inc 1.75E02 Antileukoproteinase pg/ml dec 3.89E01 Apolipoprotein(a) ng/ml inc 3.51E01 Cancer Antigen 15-3 U/ml inc 9.18E06 Cancer Antigen 19-9 U/ml inc 6.13E01 Carbonic anhydrase 9 ng/ml inc 3.78E02 Carcinoembryonic antigen-related cell adhesion molecule 5 pg/ml dec 9.44E01 C-C motif chemokine 1 pg/ml dec 1.79E01 C-C motif chemokine 13 pg/ml inc 5.89E03 C-C motif chemokine 15 pg/ml dec 1.67E01 C-C motif chemokine 17 pg/ml inc 1.69E01 C-C motif chemokine 19 pg/ml inc 2.24E03 C-C motif chemokine 20 pg/ml dec 4.43E01 C-C motif chemokine 21 pg/ml dec 1.43E02 C-C motif chemokine 22 pg/ml dec 3.28E06 C-C motif chemokine 23 ng/ml inc 1.25E08 C-C motif chemokine 24 pg/ml inc 3.32E01 C-C motif chemokine 26 pg/ml inc 1.54E01 C-C motif chemokine 27 pg/ml dec 1.44E02 C-C motif chemokine 3 pg/ml dec 1.81E01 C-C motif chemokine 4 pg/ml inc 3.44E04 C-C motif chemokine 7 pg/ml inc 8.34E01 C-C motif chemokine 8 pg/ml dec 9.23E01 Ceruloplasmin ng/ml dec 9.62E02 Choriogonadotropin subunit beta mU/ml inc 2.52E02 Collagenase 3 pg/ml dec 4.64E01 C-Peptide pg/ml dec 2.52E05 Creatine Kinase-MB ng/ml inc 3.37E01 C-X-C motif chemokine 10 pg/ml inc 1.08E01 C-X-C motif chemokine 11 pg/ml dec 6.95E02 C-X-C motif chemokine 13 pg/ml dec 5.59E03 C-X-C motif chemokine 16 ng/ml dec 1.47E02 C-X-C motif chemokine 5 pg/ml dec 9.76E01 C-X-C motif chemokine 6 pg/ml inc 1.04E01 C-X-C motif chemokine 9 pg/ml inc 4.93E01 Cystatin-C ng/ml inc 8.02E07 Endothelial protein C receptor ng/ml dec 3.19E01 Eotaxin pg/ml inc 1.20E07 Epidermal growth factor receptor pg/ml dec 6.80E01 Ferritin pg/ml dec 1.92E04 Fibrinogen ug/ml dec 5.85E02 Gastric inhibitory polypeptide pg/ml dec 0.00E+00 Glucagon pg/ml dec 8.82E01 Glucagon-like peptide 1 pg/ml dec 6.75E04 Granulocyte-macrophage colony-stimulating factor pg/ml inc 2.07E02 Heparin-binding growth factor 2 pg/ml inc 7.22E01 Hepatocyte growth factor ng/ml inc 0.00E+00 Immunoglobulin A ng/ml dec 4.10E03 Immunoglobulin M ng/ml dec 5.79E01 Immunoglogulin G1 ng/ml dec 4.93E01 Immunoglogulin G2 ng/ml dec 3.03E01 Immunoglogulin G3 ng/ml dec 2.08E04 Immunoglogulin G4 ng/ml dec 4.64E01 Insulin pg/ml dec 8.32E05 Insulin-like growth factor-binding protein 1 ng/ml dec 2.19E10 Insulin-like growth factor-binding protein 2 ng/ml dec 8.69E01 Insulin-like growth factor-binding protein 3 ng/ml inc 1.75E01 Insulin-like growth factor-binding protein 4 ng/ml dec 5.07E01 Insulin-like growth factor-binding protein 5 ng/ml dec 9.25E04 Insulin-like growth factor-binding protein 6 ng/ml inc 1.68E07 Insulin-like growth factor-binding protein 7 ng/ml dec 7.07E05 Interferon alpha-2 pg/ml inc 1.55E01 Interferon gamma pg/ml inc 1.04E02 Interleukin-1 alpha pg/ml dec 8.53E01 Interleukin-1 beta pg/ml dec 1.45E01 Interleukin-1 receptor type I pg/mL dec 1.60E03 Interleukin-1 receptor type II pg/mL inc 7.29E01 Interleukin-11 pg/ml inc 5.15E01 Interleukin-12 pg/ml inc 5.94E01 Interleukin-12 subunit beta ng/ml dec 9.09E01 Interleukin-13 pg/ml inc 4.61E01 Interleukin-15 pg/ml dec 7.13E02 Interleukin-2 pg/ml dec 1.90E03 Interleukin-20 pg/ml inc 5.51E01 Interleukin-21 pg/ml dec 9.01E01 Interleukin-23 pg/ml dec 5.67E01 Interleukin-28A pg/ml dec 4.44E01 Interleukin-29 pg/ml dec 3.04E01 Interleukin-3 pg/ml inc 6.55E01 Interleukin-33 pg/ml dec 5.24E01 Interleukin-4 pg/ml dec 4.79E01 Interleukin-5 ng/ml inc 2.65E01 Interleukin-6 receptor subunit alpha pg/ml dec 2.88E01 Interleukin-6 receptor subunit beta pg/ml dec 5.47E01 Interleukin-7 pg/ml inc 7.57E01 Interleukin-9 pg/ml dec 5.16E01 Interstitial collagenase pg/ml dec 1.78E05 Islet amyloid polypeptide pg/ml inc 2.96E07 Keratin, type I cytoskeletal 19 (aa311-367) pg/ml dec 9.39E01 Kit ligand pg/ml inc 8.87E01 Leptin pg/ml dec 8.44E01 Leukemia inhibitory factor pg/ml dec 3.49E01 Lymphotactin pg/ml dec 5.21E02 Lymphotoxin-alpha pg/ml inc 6.48E01 Macrophage colony-stimulating factor 1 pg/ml dec 7.41E04 Macrophage metalloelastase pg/ml inc 8.74E01 Macrophage migration inhibitory factor pg/ml inc 5.75E06 Matrilysin pg/ml dec 1.59E01 Metalloproteinase inhibitor 1 pg/ml dec 9.56E01 Metalloproteinase inhibitor 2 pg/ml inc 4.10E09 Metalloproteinase inhibitor 3 pg/ml inc 2.28E01 Metalloproteinase inhibitor 4 pg/ml inc 4.21E01 Mix of Growth-regulated alpha, beta, and gamma proteins pg/ml inc 2.33E01 Myoglobin ng/ml dec 5.72E01 Pancreatic prohormone pg/ml inc 2.44E13 Peptide YY pg/ml inc 1.32E03 Pro-epidermal growth factor pg/ml inc 1.23E02 Pro-interleukin-16 pg/ml inc 3.21E01 Prolactin pg/ml dec 2.55E11 Prostate-specific antigen pg/ml dec 7.49E01 Protein S100-A12 ng/ml dec 2.02E04 Protransforming growth factor alpha pg/ml inc 3.50E02 Serum amyloid P-component ng/ml dec 4.77E01 SL cytokine pg/ml dec 1.27E01 Stromal cell-derived factor 1 pg/ml inc 8.60E01 Stromelysin-1 pg/ml dec 2.76E06 Thrombopoietin pg/ml inc 1.76E04 Thymic stromal lymphopoietin pg/ml dec 5.75E01 Tumor necrosis factor pg/ml dec 1.15E01 Tumor necrosis factor ligand superfamily member 10 pg/ml inc 1.03E06 Tumor necrosis factor ligand superfamily member 6 pg/ml dec 3.96E01 Tumor necrosis factor receptor superfamily member pg/ml dec 1.20E07 1A Tumor necrosis factor receptor superfamily member pg/ml dec 1.03E02 1B Vascular endothelial growth factor receptor 1 pg/ml inc 6.04E07 Vascular endothelial growth factor receptor 2 pg/ml dec 2.63E03 Vascular endothelial growth factor receptor 3 pg/ml inc 8.51E01 von Willebrand Factor ng/ml dec 8.01E01 WAP four-disulfide core domain protein 2 pg/ml dec 4.13E04

[0068] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

[0069] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0070] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0071] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms comprising, consisting essentially of and consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0072] Other embodiments are set forth within the following claims.