Methods for diagnosis and prognosis of renal injury and renal failure using insulin-like growth factor-binding protein 7 and metalloproteinase inhibitor 2

Abstract

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in sepsis patients. In particular, the invention relates to using assays that detect one or more biomarkers selected from the group consisting of Insulin-like growth factor-binding protein 7, Beta-2-glycoprotein 1, Metalloproteinase inhibitor 2, Alpha-1 Antitrypsin, Leukocyte elastase, Serum Amyloid P Component, C-X-C motif chemokine 6, Immunoglobulin A, Immunoglobulin G subclass I, C-C motif chemokine 24, Neutrophil collagenase, Cathepsin D, C-X-C motif chemokine 13, Involucrin, Interleukin-6 receptor subunit beta, Hepatocyte Growth Factor, CXCL-1, -2, -3, Immunoglobulin G subclass II, Metalloproteinase inhibitor 4, C-C motif chemokine 18, Matrilysin, C-X-C motif chemokine 11, and Antileukoproteinase as diagnostic and prognostic biomarker assays of renal injury in the sepsis patient.

Claims

1. A method for treating acute kidney injury comprising: (a) obtaining a urine sample from a subject determined to have sepsis; (b) performing a first assay to measure a level or concentration of insulin-like growth factor-binding protein 7 in the urine sample and performing a second assay to measure a level or concentration of metalloproteinase inhibitor 2 in the urine sample; (c) calculating a composite value comprising the level or concentration of insulin-like growth factor-binding protein 7 and the level or concentration of metalloproteinase inhibitor 2; (d) comparing the composite value of (c) to a first predetermined threshold, wherein the first predetermined threshold comprises a predetermined level or concentration of urinary insulin-like growth factor-binding protein 7 and a predetermined level or concentration of urinary metalloproteinase inhibitor 2; (e) identifying a subject at risk for occurrence of RIFLE I or F within 24 hours of the time at which the urine sample is obtained from the subject when the composite value is higher as compared to the first predetermined threshold; and (f) treating the identified subject of (e), wherein the treating comprises initiating kidney replacement therapy, withdrawing delivery of compounds known to be damaging to the kidney, kidney transplant, or modifying diuretic administration, wherein the insulin-like growth factor-binding protein 7 comprises amino acid residues 27-282 of SEQ ID NO: 1.

2. The method of claim 1, wherein the level or concentration of urinary insulin-like growth factor-binding protein 7 obtained from the first assay is elevated as compared to a second predetermined threshold, wherein the second predetermined threshold comprises the predetermined level or concentration of urinary insulin-like growth factor-binding protein 7.

3. The method of claim 1, wherein the level or concentration of urinary metalloproteinase inhibitor 2 obtained from the second assay is elevated as compared to a third predetermined threshold, wherein the third predetermined threshold comprises the predetermined level or concentration of urinary metalloproteinase inhibitor 2.

4. The method of claim 1, comprising measuring the concentration of the insulin-like growth factor-binding protein 7 and the metalloproteinase inhibitor 2 in the sample.

5. The method of claim 1, wherein the sepsis is severe sepsis or septic shock.

6. The method of claim 1, wherein performing a first assay comprises contacting all or a portion of the urine sample with a first binding reagent which specifically binds to the insulin-like growth factor-binding protein 7, and generating a first assay result indicative of the level or concentration of the insulin-like growth factor-binding protein 7, and wherein performing a second assay comprises contacting all or a portion of the urine sample with a second binding reagent which specifically binds to metalloproteinase inhibitor 2, and generating a second assay result indicative of the level or concentration of the metalloproteinase inhibitor 2 to the second binding reagent.

7. The method of claim 1, wherein the composite value is obtained using a function that combines the level or concentration of insulin-like growth factor-binding protein 7 and the level or concentration of metalloproteinase inhibitor 2 into a single composite value.

8. The method of claim 1, wherein risk for occurrence of RIFLE I or F acute kidney injury is within 12 hours of the time at which urine sample is obtained.

9. The method of claim 1, wherein the subject is identified at risk for occurrence of RIFLE I within 24 hours of the time at which the urine sample is obtained from the subject.

10. The method of claim 1, wherein the subject is identified at risk for occurrence of RIFLE F within 24 hours of the time at which the urine sample is obtained from the subject.

11. The method of claim 1, wherein the subject has suffered from an injury to kidney function, reduced kidney function, or acute kidney failure.

12. The method of claim 1, wherein the subject is in RIFLE stage R.

13. A method for treating acute kidney injury comprising: (a) obtaining a urine sample from a subject determined to have sepsis; (b) measuring a level or concentration of insulin-like growth factor-binding protein 7 in the sample as compared to a first predetermined threshold, wherein the insulin-like growth factor-binding protein 7 comprises amino acid residues 27-282 of SEQ ID NO: 1, wherein the first predetermined threshold comprises a predetermined level of concentration of urinary insulin-like growth factor-binding protein 7; (c) measuring a level or concentration of metalloproteinase inhibitor 2 in the sample as compared to a second predetermined threshold, wherein the metalloproteinase inhibitor 2 comprises amino acid residues 27-220 of SEQ ID NO: 3, wherein the second predetermined threshold comprises a predetermined level or concentration of the metalloproteinase inhibitor 2; (d) identifying the subject at risk for occurrence of RIFLE I or F within 24 hours of the time at which the urine sample is obtained from the subject when the level of insulin-like growth factor-binding protein 7 in the sample is elevated as compared to the first predetermined threshold and the level of metalloproteinase inhibitor 2 in the sample is elevated as compared to the second predetermined threshold; and (e) treating the identified subject of (e), wherein the treating comprises initiating kidney replacement therapy, withdrawing delivery of compounds known to be damaging to the kidney, kidney transplant, or modifying diuretic administration.

14. The method of claim 13, comprising measuring the concentration of insulin-like growth factor-binding protein 7 and metalloproteinase inhibitor 2 in the sample.

15. The method of claim 13, wherein sepsis is severe sepsis or septic shock.

16. The method of claim 13, wherein measuring the level or concentration of insulin-like growth factor-binding protein 7 comprises contacting all or a portion of the urine sample with a first binding reagent which specifically binds to the insulin-like growth factor-binding protein 7, and generating a first assay result indicative of the level or concentration the insulin-like growth factor-binding protein 7, and wherein measuring the level or concentration of metalloproteinase inhibitor 2 comprises contacting all or a portion of the urine sample with a second binding reagent which specifically binds to metalloproteinase inhibitor 2, and generating a second assay result indicative of the level or concentration of the metalloproteinase inhibitor 2 to the second binding reagent.

17. The method of claim 16, further comprising combining the first and second assay result using a function that converts the first and second assay result into a single combined assay result.

18. The method of claim 13, wherein the risk for occurrence of RIFLE I or F acute kidney injury is within 12 hours of the time at which urine sample is obtained.

19. The method of claim 1, wherein the metalloproteinase inhibitor 2 comprises amino acid residues 27-220 of SEQ ID NO: 3.

20. The method of claim 1, wherein the insulin-like growth factor-binding protein 7 consists of amino acid residues 27-282 of SEQ ID NO: 1.

21. The method of claim 20, wherein the metalloproteinase inhibitor 2 consists of amino acid residues 27-220 of SEQ ID NO: 3.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention relates to methods and compositions for diagnosis, differential diagnosis, risk stratification, monitoring, classifying and determination of treatment regimens in sepsis patients diagnosed with sepsis. In various embodiments, a measured concentration of one or more biomarkers selected from the group consisting of Insulin-like growth factor-binding protein 7, Beta-2-glycoprotein 1, Metalloproteinase inhibitor 2, Alpha-1 Antitrypsin, Leukocyte elastase, Serum Amyloid P Component, C-X-C motif chemokine 6, Immunoglobulin A, Immunoglobulin G subclass I, C-C motif chemokine 24, Neutrophil collagenase, Cathepsin D, C-X-C motif chemokine 13, Involucrin, Interleukin-6 receptor subunit beta, Hepatocyte Growth Factor, CXCL-1, -2, -3, Immunoglobulin G subclass II, Metalloproteinase inhibitor 4, C-C motif chemokine 18, Matrilysin, C-X-C motif chemokine 11, and Antileukoproteinase or one or more markers related thereto, are correlated to the renal status of the sepsis patient.

(2) The kidney is responsible for water and solute excretion from the body. Its functions include maintenance of acid-base balance, regulation of electrolyte concentrations, control of blood volume, and regulation of blood pressure. As such, loss of kidney function through injury and/or disease results in substantial morbidity and mortality. A detailed discussion of renal injuries is provided in Harrison's Principles of Internal Medicine, 17.sup.th Ed., McGraw Hill, New York, pages 1741-1830, which are hereby incorporated by reference in their entirety. Renal disease and/or injury may be acute or chronic. Acute and chronic kidney disease are described as follows (from Current Medical Diagnosis & Treatment 2008, 47.sup.th Ed, McGraw Hill, New York, pages 785-815, which are hereby incorporated by reference in their entirety): Acute renal failure is worsening of renal function over hours to days, resulting in the retention of nitrogenous wastes (such as urea nitrogen) and creatinine in the blood. Retention of these substances is called azotemia. Chronic renal failure (chronic kidney disease) results from an abnormal loss of renal function over months to years.

(3) Acute renal failure (ARF, also known as acute kidney injury, or AKI) is an abrupt (typically detected within about 48 hours to 1 week) reduction in glomerular filtration. This loss of filtration capacity results in retention of nitrogenous (urea and creatinine) and non-nitrogenous waste products that are normally excreted by the kidney, a reduction in urine output, or both. It is reported that ARF complicates about 5% of hospital admissions, 4-15% of cardiopulmonary bypass surgeries, and up to 30% of intensive care admissions. ARF may be categorized as prerenal, intrinsic renal, or postrenal in causation. Intrinsic renal disease can be further divided into glomerular, tubular, interstitial, and vascular abnormalities. Major causes of ARF are described in the following table, which is adapted from the Merck Manual, 17.sup.th ed., Chapter 222, and which is hereby incorporated by reference in their entirety:

(4) TABLE-US-00001 Type Risk Factors Prerenal ECF volume depletion Excessive diuresis, hemorrhage, GI losses, loss of intravascular fluid into the extravascular space (due to ascites, peritonitis, pancreatitis, or burns), loss of skin and mucus membranes, renal salt- and water-wasting states Low cardiac output Cardiomyopathy, MI, cardiac tamponade, pulmonary embolism, pulmonary hypertension, positive-pressure mechanical ventilation Low systemic vascular Septic shock, liver failure, antihypertensive drugs resistance Increased renal vascular NSAIDs, cyclosporines, tacrolimus, hypercalcemia, resistance anaphylaxis, anesthetics, renal artery obstruction, renal vein thrombosis, sepsis, hepatorenal syndrome Decreased efferent ACE inhibitors or angiotensin II receptor blockers arteriolar tone (leading to decreased GFR from reduced glomerular transcapillary pressure, especially in patients with bilateral renal artery stenosis) Intrinsic Renal Acute tubular injury Ischemia (prolonged or severe prerenal state): surgery, hemorrhage, arterial or venous obstruction; Toxins: NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, streptozotocin Acute glomerulonephritis ANCA-associated: Crescentic glomerulonephritis, polyarteritis nodosa, Wegener's granulomatosis; Anti- GBM glomerulonephritis: Goodpasture's syndrome; Immune-complex: Lupus glomerulonephritis, postinfectious glomerulonephritis, cryoglobulinemic glomerulonephritis Acute tubulointerstitial Drug reaction (eg, -lactams, NSAIDs, sulfonamides, nephritis ciprofloxacin, thiazide diuretics, furosemide, phenytoin, allopurinol, pyelonephritis, papillary necrosis Acute vascular Vasculitis, malignant hypertension, thrombotic nephropathy microangiopathies, scleroderma, atheroembolism Infiltrative diseases Lymphoma, sarcoidosis, leukemia Postrenal Tubular precipitation Uric acid (tumor lysis), sulfonamides, triamterene, acyclovir, indinavir, methotrexate, ethylene glycol ingestion, myeloma protein, myoglobin Ureteral obstruction Intrinsic: Calculi, clots, sloughed renal tissue, fungus ball, edema, malignancy, congenital defects; Extrinsic: Malignancy, retroperitoneal fibrosis, ureteral trauma during surgery or high impact injury Bladder obstruction Mechanical: Benign prostatic hyperplasia, prostate cancer, bladder cancer, urethral strictures, phimosis, paraphimosis, urethral valves, obstructed indwelling urinary catheter; Neurogenic: Anticholinergic drugs, upper or lower motor neuron lesion

(5) In the case of ischemic ARF, the course of the disease may be divided into four phases. During an initiation phase, which lasts hours to days, reduced perfusion of the kidney is evolving into injury. Glomerular ultrafiltration reduces, the flow of filtrate is reduced due to debris within the tubules, and back leakage of filtrate through injured epithelium occurs. Renal injury can be mediated during this phase by reperfusion of the kidney. Initiation is followed by an extension phase which is characterized by continued ischemic injury and inflammation and may involve endothelial damage and vascular congestion. During the maintenance phase, lasting from 1 to 2 weeks, renal cell injury occurs, and glomerular filtration and urine output reaches a minimum. A recovery phase can follow in which the renal epithelium is repaired and GFR gradually recovers. Despite this, the survival rate of sepsis patients with ARF may be as low as about 60%.

(6) A commonly reported criteria for defining and detecting AKI is an abrupt (typically within about 2-7 days or within a period of hospitalization) elevation of serum creatinine. Although the use of serum creatinine elevation to define and detect AKI is well established, the magnitude of the serum creatinine elevation and the time over which it is measured to define AKI varies considerably among publications. Traditionally, relatively large increases in serum creatinine such as 100%, 200%, an increase of at least 100% to a value over 2 mg/dL and other definitions were used to define AKI. However, the recent trend has been towards using smaller serum creatinine rises to define AKI. The relationship between serum creatinine rise, AKI and the associated health risks are reviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14:265-270, 2005 and Chertow et al, J Am Soc Nephrol 16: 3365-3370, 2005, which, with the references listed therein, are hereby incorporated by reference in their entirety. As described in these publications, acute worsening renal function (AKI) and increased risk of death and other detrimental outcomes are now known to be associated with very small increases in serum creatinine. These increases may be determined as a relative (percent) value or a nominal value. Relative increases in serum creatinine as small as 20% from the pre-injury value have been reported to indicate acutely worsening renal function (AKI) and increased health risk, but the more commonly reported value to define AKI and increased health risk is a relative increase of at least 25%. Nominal increases as small as 0.3 mg/dL, 0.2 mg/dL or even 0.1 mg/dL have been reported to indicate worsening renal function and increased risk of death. Various time periods for the serum creatinine to rise to these threshold values have been used to define AKI, for example, ranging from 2 days, 3 days, 7 days, or a variable period defined as the time the patient is in the hospital or intensive care unit. These studies indicate there is not a particular threshold serum creatinine rise (or time period for the rise) for worsening renal function or AKI, but rather a continuous increase in risk with increasing magnitude of serum creatinine rise.

(7) One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605, 2004, hereby incorporated by reference in its entirety) investigated both increases and decreases in serum creatinine. Patients with a mild fall in serum creatinine of 0.1 to 0.3 mg/dL following heart surgery had the lowest mortality rate. Patients with a larger fall in serum creatinine (more than or equal to 0.4 mg/dL) or any increase in serum creatinine had a larger mortality rate. These findings caused the authors to conclude that even very subtle changes in renal function (as detected by small creatinine changes within 48 hours of surgery) seriously effect patient's outcomes. In an effort to reach consensus on a unified classification system for using serum creatinine to define AKI in clinical trials and in clinical practice, Bellomo et al., Crit Care. 8(4):R204-12, 2004, which is hereby incorporated by reference in its entirety, proposes the following classifications for stratifying AKI patients:

(8) Risk: serum creatinine increased 1.5 fold from baseline OR urine production of <0.5 ml/kg body weight/hr for 6 hours;

(9) Injury: serum creatinine increased 2.0 fold from baseline OR urine production<0.5 ml/kg/hr for 12 h;

(10) Failure: serum creatinine increased 3.0 fold from baseline OR creatinine >355 mol/l (with a rise of >44) or urine output below 0.3 ml/kg/hr for 24 h or anuria for at least 12 hours;

(11) And included two clinical outcomes:

(12) Loss: persistent need for renal replacement therapy for more than four weeks.

(13) ESRD: end stage renal diseasethe need for dialysis for more than 3 months.

(14) These criteria are called the RIFLE criteria, which provide a useful clinical tool to classify renal status. As discussed in Kellum, Crit. Care Med. 36: S141-45, 2008 and Ricci et al., Kidney Int. 73, 538-546, 2008, each hereby incorporated by reference in its entirety, the RIFLE criteria provide a uniform definition of AKI which has been validated in numerous studies.

(15) More recently, Mehta et al., Crit. Care 11:R31 (doi:10.1186.cc5713), 2007, hereby incorporated by reference in its entirety, proposes the following similar classifications for stratifying AKI patients, which have been modified from RIFLE:

(16) Stage I: increase in serum creatinine of more than or equal to 0.3 mg/dL (26.4 mol/L) or increase to more than or equal to 150% (1.5-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 6 hours;

(17) Stage II: increase in serum creatinine to more than 200% (>2-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 12 hours;

(18) Stage III: increase in serum creatinine to more than 300% (>3-fold) from baseline OR serum creatinine354 mol/L accompanied by an acute increase of at least 44 mol/L OR urine output less than 0.3 mL/kg per hour for 24 hours or anuria for 12 hours.

(19) The CIN Consensus Working Panel (McCollough et al, Rev Cardiovasc Med. 2006; 7(4):177-197, hereby incorporated by reference in its entirety) uses a serum creatinine rise of 25% to define Contrast induced nephropathy (which is a type of AKI). Although various groups propose slightly different criteria for using serum creatinine to detect AKI, the consensus is that small changes in serum creatinine, such as 0.3 mg/dL or 25%, are sufficient to detect AKI (worsening renal function) and that the magnitude of the serum creatinine change is an indicator of the severity of the AKI and mortality risk.

(20) Although serial measurement of serum creatinine over a period of days is an accepted method of detecting and diagnosing AKI and is considered one of the most important tools to evaluate AKI patients, serum creatinine is generally regarded to have several limitations in the diagnosis, assessment and monitoring of AKI patients. The time period for serum creatinine to rise to values (e.g., a 0.3 mg/dL or 25% rise) considered diagnostic for AKI can be 48 hours or longer depending on the definition used. Since cellular injury in AKI can occur over a period of hours, serum creatinine elevations detected at 48 hours or longer can be a late indicator of injury, and relying on serum creatinine can thus delay diagnosis of AKI. Furthermore, serum creatinine is not a good indicator of the exact kidney status and treatment needs during the most acute phases of AKI when kidney function is changing rapidly. Some patients with AKI will recover fully, some will need dialysis (either short term or long term) and some will have other detrimental outcomes including death, major adverse cardiac events and chronic kidney disease. Because serum creatinine is a marker of filtration rate, it does not differentiate between the causes of AKI (pre-renal, intrinsic renal, post-renal obstruction, atheroembolic, etc) or the category or location of injury in intrinsic renal disease (for example, tubular, glomerular or interstitial in origin). Urine output is similarly limited, Knowing these things can be of vital importance in managing and treating patients with AKI.

(21) For purposes of this document, the following definitions apply:

(22) As used herein, an injury to renal function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable reduction in a measure of renal function. Such an injury may be identified, for example, by a decrease in glomerular filtration rate or estimated GFR, a reduction in urine output, an increase in serum creatinine, an increase in serum cystatin C, a requirement for renal replacement therapy, etc Improvement in Renal Function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable increase in a measure of renal function. Preferred methods for measuring and/or estimating GFR are described hereinafter.

(23) As used herein, reduced renal function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.1 mg/dL (8.8 mol/L), a percentage increase in serum creatinine of greater than or equal to 20% (1.2-fold from baseline), or a reduction in urine output (documented oliguria of less than 0.5 ml/kg per hour).

(24) As used herein, acute renal failure or ARF is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.3 mg/dl (26.4 mol/1), a percentage increase in serum creatinine of greater than or equal to 50% (1.5-fold from baseline), or a reduction in urine output (documented oliguria of less than 0.5 ml/kg per hour for at least 6 hours). This term is synonymous with acute kidney injury or AKI.

(25) As used herein, the term Insulin-like growth factor-binding protein 7 or IGFBP7 refers to one or more polypeptides present in a biological sample that are derived from the Insulin-like growth factor-binding protein 7 precursor (human precursor: Swiss-Prot Q16270 (SEQ ID NO: 1))

(26) TABLE-US-00002 10203040 MERPSLRALLLGAAGLLLLLLPLSSSSSSDTCGPCEPASC 50607080 PPLPPLGCLLGETRDACGCCPMCARGEGEPCGGGGAGRGY 90100110120 CAPGMECVKSRKRRKGKAGAAAGGPGVSGVCVCKSRYPVC 130140150160 GSDGTTYPSGCQLRAASQRAESRGEKAITQVSKGTCEQGP 170180190200 SIVTPPKDIWNVTGAQVYLSCEVIGIPTPVLIWNKVKRGH 210220230240 YGVQRTELLPGDRDNLAIQTRGGPEKHEVTGWVLVSPLSK 250260270280 EDAGEYECHASNSQGQASASAKITVVDALHEIPVKKGEGAEL

(27) The following domains have been identified in Insulin-like growth factor-binding protein 7:

(28) TABLE-US-00003 Residues Length Domain ID 1-26 26 Signal peptide 27-282 256 Insulin-like growth factor-binding protein 7

(29) As used herein, the term Beta-2-glycoprotein 1 refers to one or polypeptides present in a biological sample that are derived from the Beta-2-glycoprotein 1 precursor (human precursor: Swiss-Prot P02749 (SEQ ID NO: 2)).

(30) TABLE-US-00004 10203040 MISPVLILFSSFLCHVAIAGRTCPKPDDLPFSTVVPLKTF 50607080 YEPGEEITYSCKPGYVSRGGMRKFICPLTGLWPINTLKCT 90100110120 PRVCPFAGILENGAVRYTTFEYPNTISFSCNTGFYLNGAD 130140150160 SAKCTEEGKWSPELPVCAPIICPPPSIPTFATLRVYKPSA 170180190200 GNNSLYRDTAVFECLPQHAMFGNDTITCTTHGNWTKLPEC 210220230240 REVKCPFPSRPDNGFVNYPAKPTLYYKDKATFGCHDGYSL 250260270280 DGPEEIECTKLGNWSAMPSCKASCKVPVKKATVVYQGERV 290300310320 KIQEKFKNGMLHGDKVSFFCKNKEKKCSYTEDAQCIDGTI 330340 EVPKCFKEHSSLAFWKTDASDVKPC

(31) The following domains have been identified in Beta-2-glycoprotein 1:

(32) TABLE-US-00005 Residues Length Domain ID 1-19 19 Signal sequence 20-345 326 Beta-2-glycoprotein 1

(33) In addition, several naturally occurring variants have been identified:

(34) TABLE-US-00006 Residue Change 5 V to A 107 S to N 154 R to H 266 V to L 325 C to G 335 W to S

(35) As used herein, the term Metalloproteinase inhibitor 2 refers to one or more polypeptides present in a biological sample that are derived from the Metalloproteinase inhibitor 2 precursor (human precursor: Swiss-Prot P16035 (SEQ ID NO: 3)).

(36) TABLE-US-00007 10203040 MGAAARTLRLALGLLLLATLLRPADACSCSPVHPQQAFCN 50607080 ADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFK 90100110120 GPEKDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDG 130140150160 KMHITLCDFIVPWDTLSTTQKKSLNHRYQMGCECKITRCP 170180190200 MIPCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGS 210220 CAWYRGAAPPKQEFLDIEDP

(37) The following domains have been identified in Metalloproteinase inhibitor 2:

(38) TABLE-US-00008 Residues Length Domain ID 1-26 26 Signal peptide 27-220 194 Metalloproteinase inhibitor 2

(39) As used herein, the term alpha-1-antitrypsin refers to one or more polypeptides present in a biological sample that are derived from the alpha-1-antitrypsin precursor (human precursor: Swiss-Prot P01009 (SEQ ID NO: 4)).

(40) TABLE-US-00009 10203040 MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHH 50607080 DQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVS 90100110120 IATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGF 130140150160 QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKK 170180190200 LYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKEL 210220230240 DRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTV 250260270280 KVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPD 290300310320 EGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGT 330340350360 YDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKA 370380390400 VLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIE 410 QNTKSPLFMGKVVNPTQK

(41) The following domains have been identified in alpha-1-antitrypsin:

(42) TABLE-US-00010 Residues Length Domain ID 1-24 24 signal sequence 25-418 394 alpha-1-antitrypsin

(43) As used herein, the term leukocyte elastase refers to one or more polypeptides present in a biological sample that are derived from the leukocyte elastase precursor (human precursor: Swiss-Prot P08246 (SEQ ID NO: 5)).

(44) TABLE-US-00011 10203040 MTLGRRLACLFLACVLPALLLGGTALASEIVGGRRARPHA 50607080 WPFMVSLQLRGGHFCGATLIAPNFVMSAAHCVANVNVRAV 90100110120 RVVLGAHNLSRREPTRQVFAVQRIFENGYDPVNLLNDIVI 130140150160 LQLNGSATINANVQVAQLPAQGRRLGNGVQCLAMGWGLLG 170180190200 RNRGIASVLQELNVTVVTSLCRRSNVCTLVRGRQAGVCFG 210220230240 DSGSPLVCNGLIHGIASFVRGGCASGLYPDAFAPVAQFVN 250260 WIDSIIQRSEDNPCPHPRDPDPASRTH

(45) The following domains have been identified in leukocyte elastase:

(46) TABLE-US-00012 Residues Length Domain ID 1-27 315 signal sequence 28-29 2 pro-peptide 30-267 238 leukocyte elastase

(47) As used herein, the term Serum amyloid P-component refers to one or more polypeptides present in a biological sample that are derived from the Serum amyloid P-component precursor (human precursor: Swiss-Prot P02743 (SEQ ID NO: 6)).

(48) TABLE-US-00013 10203040 MNKPLLWISVLTSLLEAFAHTDLSGKVFVFPRESVTDHVN 50607080 LITPLEKPLQNFTLCFRAYSDLSRAYSLFSYNTQGRDNEL 90100110120 LVYKERVGEYSLYIGRHKVTSKVIEKFPAPVHICVSWESS 130140150160 SGIAEFWINGTPLVKKGLRQGYFVEAQPKIVLGQEQDSYG 170180190200 GKFDRSQSFVGEIGDLYMWDSVLPPENILSAYQGTPLPAN 210220 ILDWQALNYEIRGYVIIKPLVWV

(49) The following domains have been identified in Serum amyloid P-component:

(50) TABLE-US-00014 Residues Length Domain ID 1-19 19 Signal peptide 20-223 204 Serum amyloid P-component 20-222 203 Serum amyloid P-component (1-203)

(51) As used herein, the term C-X-C motif chemokine 6 refers to one or more polypeptides present in a biological sample that are derived from the C-X-C motif chemokine 6 precursor (human precursor: Swiss-Prot P80162 (SEQ ID NO: 7))

(52) TABLE-US-00015 102030405060 MSLPSSRAARVPGPSGSLCALLALLLLLTPPGPLASAGPVSAVLTELRCTCLRVTLRVNP 708090100110 KTIGKLQVFPAGPQCSKVEVVASLKNGKQVCLDPEAPFLKKVIQKILDSGNKKN

(53) The following domains have been identified in C-X-C motif chemokine 6:

(54) TABLE-US-00016 Residues Length Domain ID 1-37 37 Signal peptide 38-114 77 CXC motif chemokine 6 40-114 75 CXC motif chemokine 6 (N-processed variant 1) 43-114 72 CXC motif chemokine 6 (N-processed variant 2) 46-114 69 CXC motif chemokine 6 (N-processed variant 3)

(55) As used herein, the term C-C motif chemokine 24 refers to one or more polypeptides present in a biological sample that are derived from the C-C motif chemokine 24 precursor (human precursor: Swiss-Prot 000175 (SEQ ID NO: 8)).

(56) TABLE-US-00017 10203040 MAGLMTIVTSLLFLGVCAHHIIPTGSVVIPSPCCMFFVSK 50607080 RIPENRVVSYQLSSRSTCLKAGVIFTTKKGQQFCGDPKQE 90100110 WVQRYMKNLDAKQKKASPRARAVAVKGPVQRYPGNQTTC

(57) The following domains have been identified in C-C motif chemokine 24:

(58) TABLE-US-00018 Residues Length Domain ID 1-26 26 Signal peptide 27-119 93 C-C motif chemokine 24

(59) As used herein, the term Neutrophil collagenase (also known as MMP-8 and matrix metalloproteinase 8) refers to one or more polypeptides present in a biological sample that are derived from the Neutrophil collagenase precursor (human precursor: Swiss-Prot P22894 (SEQ ID NO: 9)).

(60) TABLE-US-00019 102030405060 MFSLKTLPFLLLLHVQISKAFPVSSKEKNTKTVQDYLEKFYQLPSNQYQSTRKNGTNVIV 708090100110120 EKLKEMQRFFGLNVTGKPNEETLDMMKKPRCGVPDSGGFMLTPGNPKWERTNLTYRIRNY 130140150160170180 TPQLSEAEVERAIKDAFELWSVASPLIFTRISQGEADINIAFYQRDHGDNSPFDGPNGIL 190200210220230240 AHAFQPGQGIGGDAHFDAEETWTNTSANYNLFLVAAHEFGHSLGLAHSSDPGALMYPNYA 250260270280290300 FRETSNYSLPQDDIDGIQAIYGLSSNPIQPTGPSTPKPCDPSLTFDAITTLRGEILFFKD 310320330340350360 RYFWRRHPQLQRVEMNFISLFWPSLPTGIQAAYEDFDRDLIFLFKGNQYWALSGYDILQG 370380390400410420 YPKDISNYGFPSSVQAIDAAVFYRSKTYFFVNDQFWRYDNQRQFMEPGYPKSISGAFPGI 430440450460 ESKVDAVFQQEHFFHVFSGPRYYAFDLIAQRVTRVARGNKWLNCRYG

(61) The following domains have been identified in Neutrophil collagenase:

(62) TABLE-US-00020 Residues Length Domain ID 1-20 20 Signal peptide 21-100 80 Activation peptide 101-467 367 Neutrophil collagenase

(63) As used herein, the term Cathepsin D refers to one or more polypeptides present in a biological sample that are derived from the Cathepsin D precursor (human precursor: Swiss-Prot P07339 (SEQ ID NO: 10)).

(64) TABLE-US-00021 102030405060 MQPSSLLPLALCLLAAPASALVRIPLHKFTSIRRTMSEVGGSVEDLIAKGPVSKYSQAVP 708090100110120 AVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNLWVPSIHCKLLDIACWIH 130140150160170180 HKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQVFG 190200210220230240 EATKQPGITFIAAKFDGILGMAYPRISVNNVLPVFDNLMQQKLVDQNIFSFYLSRDPDAQ 250260270280290300 PGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQVEVASGLTLCKEGCEAIVDTGTSL 310320330340350360 MVGPVDEVRELQKAIGAVPLIQGEYMIPCEKVSTLPAITLKLGGKGYKLSPEDYTLKVSQ 370380390400410 AGKTLCLSGFMGMDIPPPSGPLWILGDVFIGRYYTVFDRDNNRVGFAEAARL

(65) The following domains have been identified in Capthesin D:

(66) TABLE-US-00022 Residues Length Domain ID 1-18 18 Signal peptide 19-64 46 Activation peptide 65-412 348 Cathepsin D 65-161 348 Cathepsin D light chain 169-412 348 Cathepsin D heavy chain

(67) As used herein, the term C-X-C Motif chemokine 13 refers to one or more polypeptides present in a biological sample that are derived from the C-X-C Motif chemokine 13 precursor (human precursor: Swiss-Prot 043927 (SEQ ID NO: 11)).

(68) TABLE-US-00023 102030405060 MKFISTSLLLMLLVSSLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGC 708090100 PRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTLPVPVFKRKIP

(69) The following domains have been identified in C-X-C Motif chemokine 13:

(70) TABLE-US-00024 Residues Length Domain ID 1-22 22 Signal peptide 23-109 87 CXC Motif chemokine 13

(71) As used herein, the term Involucrin refers to one or more polypeptides present in a biological sample that are derived from the Involucrin precursor (human precursor: Swiss-Prot P07476 (human precursor: SEQ ID NO: 12)).

(72) TABLE-US-00025 102030405060 MSQQHTLPVTLSPALSQELLKTVPPPVNTHQEQMKQPTPLPPPCQKVPVELPVEVPSKQE 708090100110120 EKHMTAVKGLPEQECEQQQKEPQEQELQQQHWEQHEEYQKAENPEQQLKQEKTQRDQQLN 130140150160170180 KQLEEEKKLLDQQLDQELVKRDEQLGMKKEQLLELPEQQEGHLKHLEQQEGQLKHPEQQE 190200210220230240 GQLELPEQQEGQLELPEQQEGQLELPEQQEGQLELPEQQEGQLELPEQQEGQLELPQQQE 250260270280290300 GQLELSEQQEGQLELSEQQEGQLKHLEHQEGQLEVPEEQMGQLKYLEQQEGQLKHLDQQE 310320330340350360 KQPELPEQQMGQLKHLEQQEGQPKHLEQQEGQLEQLEEQEGQLKHLEQQEGQLEHLEHQE 370380390400410420 GQLGLPEQQVLQLKQLEKQQGQPKHLEEEEGQLKHLVQQEGQLKHLVQQEGQLEQQERQV 430440450460470480 EHLEQQVGQLKHLEEQEGQLKHLEQQQGQLEVPEQQVGQPKNLEQEEKQLELPEQQEGQV 490500510520530540 KHLEKQEAQLELPEQQVGQPKHLEQQEKHLEHPEQQDGQLKHLEQQEGQLKDLEQQKGQL 550560570580 EQPVFAPAPGQVQDIQPALPTKGEVLLPVEHQQQKQEVQWPPKHK

(73) As used herein, the term Interleukin-6 receptor subunit beta refers to one or more polypeptides present in a biological sample that are derived from the Interleukin-6 receptor subunit beta precursor (human precursor: Swiss-Prot P40189 (SEQ ID NO: 13))

(74) TABLE-US-00026 102030405060 MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHV 708090100110120 NANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITI 130140150160170180 ISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPT 190200210220230240 SCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSIL 250260270280290300 KLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIR 310320330340350360 CMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEAN 370380390400410420 GKILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACD 430440450460470480 FQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTVHRT 490500510520530540 YLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWD 550560570580590600 QLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGG 610620630640650660 KDGPEFTFTTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSK 670680690700710720 SHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN 730740750760770780 TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRS 790800810820830840 ESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSV 850860870880890900 NEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEG 910 MPKSYLPQTVRQGGYMPQ

(75) Most preferably, the Interleukin-6 receptor subunit beta assay detects one or more soluble forms of Interleukin-6 receptor subunit beta. Interleukin-6 receptor subunit beta is a type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of Interleukin-6 receptor subunit beta generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form. In the case of an immunoassay, one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in Interleukin-6 receptor subunit beta:

(76) TABLE-US-00027 Residues Length Domain ID 1-22 22 Signal peptide 23-918 896 Interleukin-6 receptor subunit beta 642-918 277 Cytoplasmic domain 620-641 21 transmembrane domain 23-619 597 Extracellular domain 330-918 589 Missing in isoform 2 325-329 5 RPSKA (SEQ ID NO: 14) .fwdarw. NIASF (SEQ ID NO: 15) in isoform 2

(77) As used herein, the term Hepatocyte growth factor refers to one or more polypeptides present in a biological sample that are derived from the Hepatocyte growth factor precursor (human precursor: Swiss-Prot P14210 (SEQ ID NO: 16)).

(78) TABLE-US-00028 102030405060 MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK 708090100110120 TKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYE 130140150160170180 NKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNP 190200210220230240 RGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP 250260270280290300 HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL 310320330340350360 ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS 370380390400410420 ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME 430440450460470480 DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL 490500510520530540 DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD 550560570580590600 LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP 610620630640650660 NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG 670680690700710720 AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

(79) The following domains have been identified in Hepatocyte growth factor:

(80) TABLE-US-00029 Residues Length Domain ID 1-31 31 signal sequence 32-494 463 Hepatocyte growth factor alpha chain 495-728 234 Hepatocyte growth factor beta chain

(81) As used herein, the term Metalloproteinase inhibitor 4 refers to one or polypeptides present in a biological sample that are derived from the Metalloproteinase inhibitor 4 precursor (human precursor: Swiss-Prot Q99727 (SEQ ID NO: 17)).

(82) TABLE-US-00030 102030405060 MPGSPRPAPSWVLLLRLLALLRPPGLGEACSCAPAHPQQHICHSALVIRAKISSEKVVPA 708090100110120 SADPADTEKMLRYEIKQIKMFKGFEKVKDVQYIYTPFDSSLCGVKLEANSQKQYLLTGQV 130140150160170180 LSDGKVFIHLCNYIEPWEDLSLVQRESLNHHYHLNCGCQITTCYTVPCTISAPNECLWTD 190200210220 WLLERKLYGYQAQHYVCMKHVDGTCSWYRGHLPLRKEFVDIVQP

(83) The following domains have been identified in Metalloproteinase inhibitor 4:

(84) TABLE-US-00031 Residues Length Domain ID 1-27 27 Signal sequence 28-224 197 Metalloproteinase inhibitor 4

(85) As used herein, the term C-C motif chemokine 18 refers to one or more polypeptides present in a biological sample that are derived from the C-C motif chemokine 18 precursor (human precursor: Swiss-Prot P55774 (SEQ ID NO: 18)).

(86) TABLE-US-00032 102030405060 MKGLAAALLVLVCTMALCSCAQVGTNKELCCLVYTSWQIPQKFIVDYSETSPQCPKPGVI 7080 LLTKRGRQICADPNKKWVQKYISDLKLNA

(87) The following domains have been identified in C-C motif chemokine 18:

(88) TABLE-US-00033 Residues Length Domain ID 1-20 20 Signal peptide 21-89 69 C-C motif chemokine 18 21-88 68 CCL 18 (1-68) 23-89 67 CCL 18 (3-69) 24-89 66 CCL 18 (4-69)

(89) As used herein, the term Matrilysin refers to one or more polypeptides present in a biological sample that are derived from the Matrilysin precursor (Swiss-Prot P09237 (human precursor: SEQ ID NO: 19))

(90) TABLE-US-00034 102030405060 MRLTVLCAVCLLPGSLALPLPQEAGGMSELQWEQAQDYLKRFYLYDSETKNANSLEAKLK 708090100110120 EMQKFFGLPITGMLNSRVIEIMQKPRCGVPDVAEYSLFPNSPKWTSKVVTYRIVSYTRDL 130140150160170180 PHITVDRLVSKALNMWGKEIPLHFRKVVWGTADIMIGFARGAHGDSYPFDGPGNTLAHAF 190200210220230240 APGTGLGGDAHFDEDERWTDGSSLGINFLYAATHELGHSLGMGHSSDPNAVMYPTYGNGD 250260 PQNFKLSQDDIKGIQKLYGKRSNSRKK

(91) The following domains have been identified in Matrilysin:

(92) TABLE-US-00035 Residues Length Domain ID 1-17 17 signal peptide 18-94 77 activation peptide 95-267 173 Matrilysin

(93) As used herein, the term C-X-C motif chemokine 11 refers to one or more polypeptides present in a biological sample that are derived from the C-X-C motif chemokine 11 precursor (human precursor: Swiss-Prot 014625 (SEQ ID NO: 20))

(94) TABLE-US-00036 102030405060 MSVKGMAIALAVILCATVVQGFPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKI 708090 EVIITLKENKGQRCLNPKSKQARLIIKKVERKNF

(95) The following domains have been identified in C-X-C motif chemokine 11:

(96) TABLE-US-00037 Residues Length Domain ID 1-21 21 signal peptide 22-94 73 CXC motif chemokine 11

(97) As used herein, the term C-X-C motif chemokines-1, -2, and -3 refers to one or more polypeptides present in a biological sample that are common to the C-X-C motif chemokines-1, -2, and -3 precursors (Swiss-Prot accession numbers of the human precursors: C-X-C motif chemokine-1 (P09341), -2 (P19875), and -3 (P19876)).

(98) CXC motif chemokine-1 is also known as Growth-regulated alpha protein (human precursor Swiss-Prot P09341 (SEQ ID NO: 21)).

(99) TABLE-US-00038 102030405060 MARAALSAAPSNPRLLRVALLLLLLVAAGRRAAGASVATELRCQCLQTLQGIHPKNIQSV 708090100 NVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSN

(100) The following domains have been identified in Growth-regulated alpha protein:

(101) TABLE-US-00039 Residues Length Domain ID 1-34 34 Signal peptide 35-107 73 Growth-regulated alpha protein 38-107 70 GRO-alpha (4-73) 39-107 69 GRO-alpha (5-73) 40-107 68 GRO-alpha (6-73)

(102) CXC motif chemokine-2 is also known as Macrophage inflammatory protein 2-alpha (human precursor Swiss-Prot P19875 (SEQ ID NO: 22)).

(103) TABLE-US-00040 102030405060 MARATLSAAPSNPRLLRVALLLLLLVAASRRAAGAPLATELRCQCLQTLQGIHLKNIQSV 708090100 KVKSPGPHCAQTEVIATLKNGQKACLNPASPMVKKIIEKMLKNGKSN

(104) The following domains have been identified in Macrophage inflammatory protein 2-alpha:

(105) TABLE-US-00041 Residues Length Domain ID 1-34 34 Signal peptide 35-107 73 CXC motif chemokine 2 39-107 69 GRO-beta (5-73)

(106) CXC motif chemokine-2 is also known as Growth-regulated protein gamma (human precursor Swiss-Prot P19876 (SEQ ID NO: 23)).

(107) TABLE-US-00042 102030405060 MAHATLSAAPSNPRLLRVALLLLLLVAASRRAAGASVVTELRCQCLQTLQGIHLKNIQSV 708090100 NVRSPGPHCAQTEVIATLKNGKKACLNPASPMVQKIIEKILNKGSTN

(108) The following domains have been identified in C-X-C motif chemokine 3:

(109) TABLE-US-00043 Residues Length Domain ID 1-34 34 Signal peptide 35-107 73 CXC motif chemokine 3 39-107 73 GRO-gamma (5-73)

(110) As used herein, the term Antileukoproteinase refers to one or more polypeptides present in a biological sample that are derived from the Antileukoproteinase precursor (Swiss-Prot P03973 (SEQ ID NO: 24)).

(111) TABLE-US-00044 102030405060 MKSSGLFPFLVLLALGTLAPWAVEGSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGK 708090100110120 KRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMG 130 MCGKSCVSPVKA

(112) The following domains have been identified in Antileukoproteinase:

(113) TABLE-US-00045 Residues Length Domain ID 1-25 25 signal sequence 26-132 107 Antileukoproteinase

(114) As used herein, the term IgA refers to an antibody having two subclasses (IgA1 and IgA2) and which can exist in a dimeric form linked by a J chain (called secretory IgA, or sIgA). In its secretory form, IgA is the main immunoglobulin found in mucous secretions, including tears, saliva, colostrum and secretions from the genito-urinary tract, gastrointestinal tractprostate and respiratory epithelium. It is also found in small amounts in blood. IgA may be measured separately from other immunoglobulins such as IgG or IgM, for example, using antibodies which bind to the IgA -chain.

(115) As used herein, the terms IgG1 and IgG subclass I refer to subclass 1 of the glycoprotein immunoglobulin G (IgG), a major effector molecule of the humoral immune response in man. Antibodies of the IgG class express their predominant activity during a secondary antibody response. The basic immunoglobulin G molecule has a four-chain structure, comprising two identical heavy (H) chains and two identical light (L) chains, linked together by inter-chain disulfide bonds. Each heavy chain is encoded by 4 distinct types of gene segments, designated V.sub.H (variable), D (diversity), J.sub.H (joining) and C.sub.H(constant). The variable region of the heavy chain is encoded by the V.sub.H, D and J.sub.H segments. The light chains are encoded by the 3 gene segments, V.sub.L, J.sub.L and C.sub.L. The variable region of the light chains is encoded by the V.sub.L and J.sub.L segments.

(116) As used herein, the terms IgG2 and IgG subclass II refer to subclass 2 of the glycoprotein immunoglobulin G (IgG), a major effector molecule of the humoral immune response in man. Antibodies of the IgG class express their predominant activity during a secondary antibody response. The basic immunoglobulin G molecule has a four-chain structure, comprising two identical heavy (H) chains and two identical light (L) chains, linked together by inter-chain disulfide bonds. Each heavy chain is encoded by 4 distinct types of gene segments, designated V.sub.H (variable), D (diversity), J.sub.H (joining) and C.sub.H(constant). The variable region of the heavy chain is encoded by the V.sub.H, D and J.sub.H segments. The light chains are encoded by the 3 gene segments, V.sub.L, J.sub.L and C.sub.L. The variable region of the light chains is encoded by the V.sub.L and J.sub.L segments.

(117) The length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231 and since it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges (23). IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks a glycine residue, it is relatively short and contains a rigid poly-proline double helix, stabilised by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule (24). IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix (25,26). In IgG3 the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and IgG2.

(118) The four IgG subclasses also differ with respect to the number of inter-heavy chain disulfide bonds in the hinge region (26). The structural differences between the IgG subclasses are also reflected in their susceptibility to proteolytic enzymes. IgG3 is very susceptible to cleavage by these enzymes, whereas IgG2 is relatively resistant. IgG1 and IgG4 exhibit an intermediary sensitivity, depending upon the enzyme used. Since these proteolytic enzymes all cleave IgG molecules near or within the hinge region, it is likely that the high sensitivity of IgG3 to enzyme digestion is related to its accessible hinge. Another structural difference between the human IgG subclasses is the linkage of the heavy and light chain by a disulfide bond. This bond links the carboxy-terminal of the light chain with the cysteine residue at position 220 (in IgG) or at position 131 (in IgG2, IgG3 and IgG4) of the CH1 sequence of the heavy chain.

(119) As a consequence of the structural differences, the four IgG subclasses may be distinguished from one another, for example using antibodies that are specific for differences between the isoforms. In the present application, a level of IgG1 is determined using an assay which distinguishes this subclass, relative to the other subclasses.

(120) 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 kidney injury markers 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.

(121) 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.

(122) The term positive going marker as that term is used herein refer to a marker that is determined to be elevated in sepsis patients suffering from a disease or condition, relative to sepsis patients 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 sepsis patients suffering from a disease or condition, relative to sepsis patients not suffering from that disease or condition.

(123) The term sepsis patient 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 sepsis patient is preferably a living organism, the invention described herein may be used in post-mortem analysis as well. Preferred sepsis patients 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.

(124) Preferably, an analyte is measured in a sample. Such a sample may be obtained from a sepsis patient, or may be obtained from biological materials intended to be provided to the sepsis patient. For example, a sample may be obtained from a kidney being evaluated for possible transplantation into a sepsis patient, and an analyte measurement used to evaluate the kidney for preexisting damage. Preferred samples are body fluid samples.

(125) 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 a sepsis 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, cerebrospinal fluid, 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.

(126) 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 a kidney injury marker of the present invention, optionally together with other clinical characteristics, to arrive at a diagnosis (that is, the occurrence or nonoccurrence) of an acute renal injury or ARF for the sepsis patient from which a sample was obtained and assayed. 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 sepsis patient relative to a measured level on the other side of the predetermined diagnostic threshold.

(127) 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 (e.g., worsening renal function, future ARF, or death) is referred to as being indicative of an increased likelihood of an adverse outcome in a patient.

(128) Marker Assays

(129) 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.

(130) 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.

(131) 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, TentaGel resins (Rapp Polymere GmbH), AgroGel resins (I.L.S.A. Industria Lavorazione Sottoprodotti Animali S.P.A.), polyethylene glycol and acrylamide (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.

(132) 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.).

(133) 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.

(134) In certain aspects, the present invention provides kits for the analysis of the described kidney injury markers. The kit comprises reagents for the analysis of at least one test sample which comprise at least one antibody that a kidney injury marker. 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 that bind a kidney injury marker. 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.

(135) Antibodies

(136) 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.

(137) Antibodies used in the immunoassays described herein preferably specifically bind to a kidney injury marker 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.

(138) 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.

(139) 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.

(140) 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.

(141) 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.

(142) 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.

(143) 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 conferring 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.

(144) Assay Correlations

(145) 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.

(146) 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.

(147) 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.

(148) Population studies may also be used to select a decision threshold. Reciever Operating Characteristic (ROC) arose from the field of signal dectection theory 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 1specificity, the ROC graph is sometimes called the sensitivity vs (1specificity) 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.

(149) 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.

(150) 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 sepsis patient belongs to one classification out of a plurality of classifications. 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.

(151) 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

(152) Additional clinical indicia may be combined with the kidney injury marker assay result(s) of the present invention. These include other biomarkers related to renal status. Other clinical indicia which may be combined with the kidney injury marker 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, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urine total protein measurement, a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a renal papillary antigen 1 (RPA1) measurement; a renal papillary antigen 2 (RPA2) measurement; a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, and/or a renal failure index calculated as urine sodium/(urine creatinine/plasma creatinine). Other measures of renal function which may be combined with the kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17.sup.th Ed., McGraw Hill, New York, pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47.sup.th Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by reference in their entirety.

(153) 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.

(154) Diagnosis of Acute Renal Failure

(155) As noted above, the terms acute renal (or kidney) injury and acute renal (or kidney) failure as used herein are defined in part in terms of changes in serum creatinine from a baseline value. Most definitions of ARF have common elements, including the use of serum creatinine and, often, urine output. Patients may present with renal dysfunction without an available baseline measure of renal function for use in this comparison. In such an event, one may estimate a baseline serum creatinine value by assuming the patient initially had a normal GFR. Glomerular filtration rate (GFR) is the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman's capsule per unit time. Glomerular filtration rate (GFR) can be calculated by measuring any chemical that has a steady level in the blood, and is freely filtered but neither reabsorbed nor secreted by the kidneys. GFR is typically expressed in units of ml/min:

(156) $\mathrm{GFR}=\frac{\mathrm{Urine}\mathrm{Concentration}\mathrm{Urine}\mathrm{Flow}}{\mathrm{Plasma}\mathrm{Concentration}}$

(157) By normalizing the GFR to the body surface area, a GFR of approximately 75-100 ml/min per 1.73 m.sup.2 can be assumed. The rate therefore measured is the quantity of the substance in the urine that originated from a calculable volume of blood.

(158) There are several different techniques used to calculate or estimate the glomerular filtration rate (GFR or eGFR). In clinical practice, however, creatinine clearance is used to measure GFR. Creatinine is produced naturally by the body (creatinine is a metabolite of creatine, which is found in muscle). It is freely filtered by the glomerulus, but also actively secreted by the renal tubules in very small amounts such that creatinine clearance overestimates actual GFR by 10-20%. This margin of error is acceptable considering the ease with which creatinine clearance is measured.

(159) Creatinine clearance (CCr) can be calculated if values for creatinine's urine concentration (U.sub.Cr), urine flow rate (V), and creatinine's plasma concentration (P.sub.Cr) are known. Since the product of urine concentration and urine flow rate yields creatinine's excretion rate, creatinine clearance is also said to be its excretion rate (U.sub.CrV) divided by its plasma concentration. This is commonly represented mathematically as:

(160) $C_{\mathrm{Cr}}=\frac{U_{\mathrm{Cr}}V}{P_{\mathrm{Cr}}}$

(161) Commonly a 24 hour urine collection is undertaken, from empty-bladder one morning to the contents of the bladder the following morning, with a comparative blood test then taken:

(162) $C_{\mathrm{Cr}}=\frac{U_{\mathrm{Cr}}24\text{-}\mathrm{hour}\mathrm{volume}}{P_{\mathrm{Cr}}2460\mathrm{mins}}$

(163) To allow comparison of results between people of different sizes, the CCr is often corrected for the body surface area (BSA) and expressed compared to the average sized man as ml/min/1.73 m2. While most adults have a BSA that approaches 1.7 (1.6-1.9), extremely obese or slim patients should have their CCr corrected for their actual BSA:

(164) $C_{\mathrm{Cr}\text{-}\mathrm{corrected}}=\frac{C_{\mathrm{Cr}}1.73}{\mathrm{BSA}}$

(165) The accuracy of a creatinine clearance measurement (even when collection is complete) is limited because as glomerular filtration rate (GFR) falls creatinine secretion is increased, and thus the rise in serum creatinine is less. Thus, creatinine excretion is much greater than the filtered load, resulting in a potentially large overestimation of the GFR (as much as a twofold difference). However, for clinical purposes it is important to determine whether renal function is stable or getting worse or better. This is often determined by monitoring serum creatinine alone. Like creatinine clearance, the serum creatinine will not be an accurate reflection of GFR in the non-steady-state condition of ARF. Nonetheless, the degree to which serum creatinine changes from baseline will reflect the change in GFR. Serum creatinine is readily and easily measured and it is specific for renal function.

(166) For purposes of determining urine output on a Urine output on a mL/kg/hr basis, hourly urine collection and measurement is adequate. In the case where, for example, only a cumulative 24-h output was available and no patient weights are provided, minor modifications of the RIFLE urine output criteria have been described. For example, Bagshaw et al., Nephrol. Dial. Transplant. 23: 1203-1210, 2008, assumes an average patient weight of 70 kg, and patients are assigned a RIFLE classification based on the following: <35 mL/h (Risk), <21 mL/h (Injury) or <4 mL/h (Failure).

(167) Selecting a Treatment Regimen

(168) Once a diagnosis is obtained, the clinician can readily select a treatment regimen that is compatible with the diagnosis, such as initiating renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, kidney transplantation, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, initiating goal directed therapy, etc. 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.

(169) 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: Septic Sepsis Patient Sample Collection

(170) The objective of this study was to collect samples from patients expected to be in the ICU for at least 48 hours were enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

(171) Inclusion Criteria: males and females 18 years of age or older and which either acquire sepsis or have sepsis on admission.

(172) Exclusion Criteria

(173) known pregnancy;

(174) institutionalized individuals;

(175) previous renal transplantation;

(176) known acutely worsening renal function prior to enrollment (e.g., any category of RIFLE criteria);

(177) received dialysis (either acute or chronic) within 5 days prior to enrollment or in imminent need of dialysis at the time of enrollment;

(178) known infection with human immunodeficiency virus (HIV) or a hepatitis virus;

(179) meets only the SBP<90 mmHg inclusion criterion set forth above, and does not have shock in the attending physician's or principal investigator's opinion.

(180) After providing informed consent, an EDTA anti-coagulated blood sample (10 mL) and a urine sample (25-30 mL) are collected from each patient. Blood and urine samples are then collected at 4 (0.5) and 8 (1) hours after contrast administration (if applicable); at 12 (1), 24 (2), and 48 (2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the sepsis patient is hospitalized. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, Calif. The study urine samples are frozen and shipped to Astute Medical, Inc.

Example 2: Immunoassay Format

(181) 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.

(182) Concentrations for the various markers are reported as follows:

(183) TABLE-US-00046 Insulin-like growth factor-binding protein 7 ng/ml Beta-2-glycoprotein 1 ng/ml Metalloproteinase inhibitor 2 pg/ml Alpha-1 Antitrypsin ng/ml Neutrophil Elastase ng/ml Serum Amyloid P Component ng/ml CXC motif chemokine 6 pg/ml Immunoglobulin A ng/ml Immunoglobulin G, subclass I ng/ml C-C motif chemokine 24 pg/ml Neutrophil collagenase pg/ml Cathepsin D pg/ml CXC motif chemokine 13 pg/ml Involucrin ng/ml Interleukin-6 receptor subunit beta pg/ml Hepatocyte Growth Factor pg/ml CXCL-1, -2, -3 mix pg/ml Immunoglobulin G, subclass II ng/ml Metalloproteinase inhibitor 4 pg/ml C-C motif chemokine 18 ng/ml Matrilysin pg/ml CXC motif chemokine 11 pg/ml Antileukoproteinase (WAP4) pg/ml

Example 3: Use of Kidney Injury Markers for Evaluating Sepsis Patients

(184) Patients from the sepsis study (Example 1) were classified by kidney status as non-injury (0), risk of injury (R), injury (I), and failure (F) according to the maximum stage reached within 7 days of enrollment as determined by the RIFLE criteria. EDTA anti-coagulated blood samples (10 mL) and a urine samples (25-30 mL) were collected from each patient at enrollment, 4 (0.5) and 8 (1) hours after contrast administration (if applicable); at 12 (1), 24 (2), and 48 (2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the sepsis patient is hospitalized. Markers were each measured by standard immunoassay methods using commercially available assay reagents in the urine samples and the plasma component of the blood samples collected.

(185) Two cohorts were defined to represent a diseased and a normal population. While these terms are used for convenience, diseased and normal simply represent two cohorts for comparison (say RIFLE 0 vs RIFLE R, I and F; RIFLE 0 vs RIFLE R; RIFLE 0 and R vs RIFLE I and F; etc.). The time prior max stage represents the time at which a sample is collected, relative to the time a particular patient reaches the lowest disease stage as defined for that cohort, binned into three groups which are +/12 hours. For example, 24 hr prior which uses 0 vs R, I, F as the two cohorts would mean 24 hr (+/12 hours) prior to reaching stage R (or I if no sample at R, or F if no sample at R or I).

(186) A receiver operating characteristic (ROC) curve was generated for each biomarker measured and the area under each ROC curve (AUC) is determined Patients in Cohort 2 were also separated according to the reason for adjudication to cohort 2 as being based on serum creatinine measurements (sCr), being based on urine output (UO), or being based on either serum creatinine measurements or urine output. Using the same example discussed above (0 vs R, I, F), for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements alone, the stage 0 cohort may include patients adjudicated to stage R, I, or F on the basis of urine output; for those patients adjudicated to stage R, I, or F on the basis of urine output alone, the stage 0 cohort may include patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements; and for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the stage 0 cohort contains only patients in stage 0 for both serum creatinine measurements and urine output. Also, in the data for patients adjudicated on the basis of serum creatinine measurements or urine output, the adjudication method which yielded the most severe RIFLE stage is used.

(187) The ability to distinguish cohort 1 from Cohort 2 was determined using ROC analysis. SE is the standard error of the AUC, n is the number of sample or individual patients (pts, as indicated). Standard errors are calculated as described in Hanley, J. A., and McNeil, B. J., The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology (1982) 143: 29-36; p values are calculated with a two-tailed Z-test. An AUC<0.5 is indicative of a negative going marker for the comparison, and an AUC>0.5 is indicative of a positive going marker for the comparison.

(188) Various threshold (or cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 are determined OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

(189) In the following tables 1-12, a population which either acquire sepsis days 1-7 or have sepsis on admission are used as the disease cohort; in tables 13-24, only those patients with sepsis on admission were included.

(190) TABLE-US-00047 Lengthy table referenced here US10935548-20210302-T00001 Please refer to the end of the specification for access instructions.

(191) TABLE-US-00048 Lengthy table referenced here US10935548-20210302-T00002 Please refer to the end of the specification for access instructions.

(192) TABLE-US-00049 Lengthy table referenced here US10935548-20210302-T00003 Please refer to the end of the specification for access instructions.

(193) TABLE-US-00050 Lengthy table referenced here US10935548-20210302-T00004 Please refer to the end of the specification for access instructions.

(194) TABLE-US-00051 Lengthy table referenced here US10935548-20210302-T00005 Please refer to the end of the specification for access instructions.

(195) TABLE-US-00052 Lengthy table referenced here US10935548-20210302-T00006 Please refer to the end of the specification for access instructions.

(196) TABLE-US-00053 Lengthy table referenced here US10935548-20210302-T00007 Please refer to the end of the specification for access instructions.

(197) TABLE-US-00054 Lengthy table referenced here US10935548-20210302-T00008 Please refer to the end of the specification for access instructions.

(198) TABLE-US-00055 Lengthy table referenced here US10935548-20210302-T00009 Please refer to the end of the specification for access instructions.

(199) TABLE-US-00056 Lengthy table referenced here US10935548-20210302-T00010 Please refer to the end of the specification for access instructions.

(200) TABLE-US-00057 Lengthy table referenced here US10935548-20210302-T00011 Please refer to the end of the specification for access instructions.

(201) TABLE-US-00058 Lengthy table referenced here US10935548-20210302-T00012 Please refer to the end of the specification for access instructions.

(202) TABLE-US-00059 Lengthy table referenced here US10935548-20210302-T00013 Please refer to the end of the specification for access instructions.

(203) TABLE-US-00060 Lengthy table referenced here US10935548-20210302-T00014 Please refer to the end of the specification for access instructions.

(204) TABLE-US-00061 Lengthy table referenced here US10935548-20210302-T00015 Please refer to the end of the specification for access instructions.

(205) TABLE-US-00062 Lengthy table referenced here US10935548-20210302-T00016 Please refer to the end of the specification for access instructions.

(206) TABLE-US-00063 Lengthy table referenced here US10935548-20210302-T00017 Please refer to the end of the specification for access instructions.

(207) TABLE-US-00064 Lengthy table referenced here US10935548-20210302-T00018 Please refer to the end of the specification for access instructions.

(208) TABLE-US-00065 Lengthy table referenced here US10935548-20210302-T00019 Please refer to the end of the specification for access instructions.

(209) TABLE-US-00066 Lengthy table referenced here US10935548-20210302-T00020 Please refer to the end of the specification for access instructions.

(210) TABLE-US-00067 Lengthy table referenced here US10935548-20210302-T00021 Please refer to the end of the specification for access instructions.

(211) TABLE-US-00068 Lengthy table referenced here US10935548-20210302-T00022 Please refer to the end of the specification for access instructions.

(212) TABLE-US-00069 Lengthy table referenced here US10935548-20210302-T00023 Please refer to the end of the specification for access instructions.

(213) TABLE-US-00070 Lengthy table referenced here US10935548-20210302-T00024 Please refer to the end of the specification for access instructions.

(214) 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.

(215) 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.

(216) 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.

(217) 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.

(218) Other embodiments are set forth within the following claims.

(219) TABLE-US-LTS-00001 LENGTHY TABLES The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).