(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 16 December 2010 (16.12.2010) WO 2010/144358 Al (51) International Patent Classification: (74) Agent: GATTARI, Patrick, G.; McDonnell Boehnen GOlN 33/68 (2006.01) GOlN 33/52 (2006.01) Hulbert & Berghoff LLP, 300 South Wacker Drive, Suite 3100, Chicago, IL 60606 (US). (21) International Application Number: PCT/US2010/037613 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, 7 June 2010 (07.06.2010) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (25) Filing Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (26) Publication Language: English KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (30) Priority Data: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 61/185,1 94 8 June 2009 (08.06.2009) US NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, (71) Applicant (for all designated States except US): SIN- TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. GULEX, INC. [US/US]; 1650 Harbor Bay Parkway, Suite 200, Alameda, CA 94502-3012 (US). (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (72) Inventors; and GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, (75) Inventors/Applicants (for US only): GOIX, Philippe, J. ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, [US/US]; 210 Sunnyside Avenue, Piedmont, CA 946 11 TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (US). PUSKAS, Robert [US/US]; 752 Sherwick Terrace, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, Manchester, MO 63021 (US). TODD, John [US/US]; LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, 1096 Orchard Road, Lafayette, CA 94549 (US). LIV¬ SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, INGSTON, Richard [US/US]; 468 Florence Avenue, GW, ML, MR, NE, SN, TD, TG). Webster Groves, MO 63 119 (US). HELD, Douglas [US/ US]; 1219 Weatherton Place, Ballwin, MO 63021 (US). Published: AGEE, Sara [US/US]; 15 17 6th Street, Berkeley, CA — with international search report (Art. 21(3)) 94710 (US). (54) Title: HIGHLY SENSITIVE BIOMARKER PANELS (57) Abstract: Cardiovascular disease, e.g., congestive heart failure, is often first diagnosed after the onset of clinical symptoms, eliminating potential for early intervention. The invention provides a multi-marker immunoassay, including cardiac pathology and vascular inflammation biomarkers, yielding a more sensitive assay for early detection of CHF in plasma. A panel consisting of car- diac pathology (cTnl, BNP) and vascular inflammation (IL-6, TNFα, IL- 17a) biomarkers provided a sensitivity of 94% for associ- ation with CHF. HIGHLY SENSITIVE BIOMARKER PANELS BACKGROUND OF THE INVENTION [0001] Cardiovascular disease is an abnormal function of the heart and/or blood vessels. Included under this designation are such diverse medical conditions as coronary artery disease, congestive heart failure, arrhythmia, atherosclerosis, hypertension, stroke, cerebrovascular disease, peripheral vascular disease and myocardial infarction. In the United States, CVD is a major cause of death. About 40 percent of all deaths in 1997, or about one million people, were attributed to cardiovascular disease. There are an estimated 62 million people with cardiovascular disease and 50 million people with hypertension in this country. [0002] Cardiovascular disease is a progressive process with etiologies in both cardiac muscle (cardio- pathology) and vascular inflammation. The disease process follows a continuum from early onset mild vascular inflammation to severe acute events such as acute myocardial infarction or chronic events such as heart failure. Patients with well recognized physical conditions such as hypertension, obesity, diabetes, metabolic syndrome, hypercholesterolemia are at varying degrees of risk for developing CVD. A challenge facing clinicians who have patients presenting with CVD risk factors is understanding their degree of risk, developing the appropriate treatment plan and then monitoring the patient for improvements in disease risk. [0003] Ample studies have provided compelling evidence that CVD is largely preventable. The causes of cardiovascular disease range from structural defects, to infection, inflammation, environment and genetics. While some risk factors cannot be modified (genetics, age, gender), there are a number of risk factors that can be addressed through lifestyle changes or medically. These controllable risk factors include cigarette smoking, high blood pressure, obesity, diabetes, physical inactivity, and high blood cholesterol level. By the time that heart problems are detected, the underlying cause (atherosclerosis) is usually quite advanced, having progressed for decades. There is therefore increased emphasis on preventing atherosclerosis by modifying risk factors, such as healthy eating, exercise and avoidance of smoking. [0004] CVD, e.g., congestive heart failure (CHF), is often first diagnosed after the onset of clinical symptoms, eliminating potential for early intervention. There is a need for highly sensitive detection of CVD. SUMMARY OF THE INVENTION [0005] In one embodiment, the present invention provides a method for detecting or monitoring a condition in a subject, comprising detecting a first marker in a first sample from the subject and detecting a second marker, wherein the first marker comprises Cardiac Troponin-I (cTnl) or Vascular Endothelial Growth Factor (VEGF), and wherein the limit of detection of the first marker is less than about 20 pg/ml. In some embodiments, the detection of at least one marker comprises contacting the sample with a label for the marker and detecting the presence or absence of the label, wherein detection of the presence of the label indicates the presence of the corresponding marker. In some embodiments, the label comprises a fluorescent moiety, and the detection comprises passing the label through a single molecule detector, wherein the single molecule detector comprises: (a) an electromagnetic radiation source for stimulating the fluorescent moiety; (b) an interrogation space for receiving electromagnetic radiation emitted from the electromagnetic source; and (c) an electromagnetic radiation detector operably connected to the interrogation space for determining an electromagnetic characteristic of the stimulated fluorescent moiety. [0006] In some embodiments, the limit of detection of the first marker ranges from about 10 pg/ml to about 0.01 pg/ml. In some embodiments, the limit of detection of the first marker is less than about 10 pg/ml. In some embodiments, the limit of detection of the first marker is less than about 5 pg/ml. In some embodiments, the limit of detection of the first marker is less than about 1 pg/ml. In some embodiments, the limit of detection of the first marker is less than about 0.5 pg/ml. In some embodiments, the limit of detection of the first marker is less than about 0.1 pg/ml. In some embodiments, the limit of detection of the first marker is less than about 0.05 pg/ml. In some embodiments, the limit of detection of the first marker is less than about 0.01 pg/ml. In some embodiments, the limit of detection of the first marker is less than about 0.005 pg/ml. In some embodiments, the limit of detection of the first marker is less than about 0.001 pg/ml. In some embodiments, the coefficient of variation (CV) of the limit of detection ranges from about 20% to about 1%. In some embodiments, the coefficient of variation (CV) of the limit of detection ranges from about 100% to about 1%. In some embodiments, the coefficient of variation (CV) of the limit of detection ranges from about 75% to about 1%. In some embodiments, the coefficient of variation (CV) of the limit of detection ranges from about 50% to about 1%. In some embodiments, the coefficient of variation (CV) of the limit of detection ranges from about 25% to about 1%. In some embodiments, the coefficient of variation (CV) of the limit of detection ranges from about 20% to about 1%. In some embodiments, the coefficient of variation (CV) of the limit of detection ranges from about 15% to about 1%. In some embodiments, the coefficient of variation (CV) of the limit of detection ranges from about 10% to about 1%. In some embodiments, the coefficient of variation (CV) of the limit of detection ranges from about 5% to about 1%. In some embodiments, the sample size ranges from about 10 µl to about 0.1 µl. In some embodiments, the sample size ranges from about 100 µl to about 0.1 µl. In some embodiments, the sample size ranges from about 75 µl to about 0.1 µl. In some embodiments, the sample size ranges from about 50 µl to about 0.1 µl. In some embodiments, the sample size ranges from about 25 µl to about 0.1 µl. In some embodiments, the sample size ranges from about 20 µl to about 0.1 µl. In some embodiments, the sample size ranges from about 5 µl to about 0.1 µl. In some embodiments, the sample size ranges from about 1 µl to about 0.1 µl. In some embodiments, the sample size is less than about 100 µl. In some embodiments, the sample size is less than about 75 µl. In some embodiments, the sample size is less than about 50 µl. In some embodiments, the sample size is less than about 25 µl.