Background: B-Type natriuretic peptide (BNP 1-32 ) as well as the N-terminal fragment of the prohormone containing residues 1-76 (NT-proBNP 1-76 ), both cleavage products of the precursor proBNP 1-108 , are reported to be powerful markers for prognosis and risk stratification of heart failure. However, the intact precursor also circulates in the bloodstream. Assays for the detection of these cleavage products have been developed, but most of these assays may overestimate the concentrations of the cleavage products because they also measure the precursor form. It is therefore important to develop an immunoassay that specifically measures solely proBNP 1-108 in plasma. Methods: After carefully designing the peptide used to immunize mice, we selected a specific monoclonal antibody (mAb Hinge76) that recognizes the cleavage site of proBNP 1-108 , an epitope present only in the precursor form. mAb Hinge76 recognizes recombinant proBNP 1-108 in a dose-dependent manner, without any significant cross-reactivity with either recombinant NT-proBNP 1-76 or synthetic BNP 1-32 . By combining mAb Hinge76 with a polyclonal antibody directed against BNP 1-32 , we were able to set up a proBNP 1-108 -specific sandwich immunoassay able to confirm the presence of proBNP 1-108 in blood samples. Results: From a cohort of 50 healthy persons and 170 patients with congestive heart failure (CHF), our assay was able to differentiate healthy individuals from CHF patients (P <0.005). Interestingly, plasma proBNP 1-108

Objective-Previous proteomics experiments have demonstrated that several proteins are differentially expressed in vulnerable human carotid plaques compared with stable plaques. This study aims to investigate the prognostic value of 13 such circulating biomarkers in patients with coronary artery disease. Approach and Results-Between 2008 and 2011, 768 patients who underwent coronary angiography for acute coronary syndrome or stable angina pectoris were included in a prospective biomarker study. Plasma concentrations of 13 biomarkers were measured in 88 patients who experienced a major adverse cardiovascular event (MACE) within 1 year and 176 control patients without MACE who were matched on age, sex, and number of diseased coronary vessels. MACE comprised all-cause mortality, acute coronary syndrome, unplanned coronary revascularization, and stroke. After adjustment for established cardiovascular risk factors, osteoglycin (OGN; odds ratio per SD increase in ln-transformed OGN, 1.53; 95% confidence interval, 1.11-2.11; P=0.010) and neutrophil gelatinase-associated lipocalin/matrix metalloproteinase 9 (NGAL/MMP9; odds ratio per SD increase in ln-transformed NGAL/MMP9, 1.37; 95% confidence interval, 1.01-1.85; P=0.042) complex were independently associated with MACE during follow-up. These associations were independent of C-reactive protein levels. Cheng et al OGN and NGAL/MMP9 Predict Cardiovascular Outcome 1079human carotid plaques when compared with stable fibrotic plaques. 8 The majority of these proteins, including aciculin, oncogene DJ1 (DJ1), microfibril-associated glycoprotein 4, osteoglycin (OGN), procollagen C proteinase enhancer 1, phosphatidylethanol-amine-binding protein 1, and peroxiredoxin 2, have not yet been investigated as prognostic biomarkers in CAD patients. In addition, evidence exists that neutrophil gelatinase-associated lipocalin (NGAL) and its NGAL/matrix metalloproteinase 9 (NGAL/MMP9) complex display increased expression in (unstable) atherosclerotic plaques.9,10 These proteins have also not yet been investigated as prognostic biomarkers in CAD patients.We have performed a prospective, nested case-control study in a cohort of 768 patients undergoing coronary angiography to investigate whether plasma levels of the above-described novel protein biomarkers are associated with adverse cardiovascular events. We have also evaluated whether these biomarkers improve discrimination and risk reclassification. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results Baseline CharacteristicsMean age of the patients was 64.9 (SD, 10.6) years, 77% were men, and 52% had acute coronary syndrome (Table 1). Percutaneous coronary intervention was performed in 82% of the patients. The group of patients who experienced major adverse cardiovascular event (MACE) during follow-up displayed a higher prevalence of diabetes mellitus (27.3% versus 14.9%; P=0.016) and a tendency toward a higher prevalence of renal insufficiency (11.4% versus 4.6%; P=0.053) compared with the gro...