BACKGROUND: Studies of disease associations with γ′ fibrinogen, a newly emerging risk factor for cardiovascular disease, have been hampered by the lack of a standardized and well-characterized assay. METHODS: We developed an immunometric technique to measure γ′ fibrinogen concentrations in plasma and studied the clinical utility of this test in samples from healthy individuals enrolled in the Framingham Offspring Study and in a separate case/control study of coronary artery disease (CAD). Monoclonal antibody 2.G2.H9, specific for the unique carboxyl terminal peptide of the fibrinogen γ′ chain, was used as capture antibody. Sheep antihuman fibrinogen/horseradish peroxidase conjugate was used for detection, with 3,3′,5,5′-tetramethylbenzidine as substrate. We evaluated the linearity, imprecision, analytical specificity, and lower limit of quantification of the assay. We determined the reference interval for γ′ fibrinogen in healthy individuals from the Framingham Offspring Study (n = 2879) and quantified associations between γ′ fibrinogen and cardiovascular disease risk factors. The sensitivity and specificity of γ′ fibrinogen in evaluating CAD patients (n = 133) was determined with ROC curve analysis. RESULTS: The γ′ fibrinogen ELISA had within-run CVs of 13.4% at 0.127 g/L and 4.8% at 0.416 g/L. The limit of quantification at an imprecision of 20% was 0.10 g/L. The reference interval for healthy individuals was 0.088-0.551 g/L. ROC curve analysis of results from patients with CAD yielded an area under the curve of 0.76, with a diagnostic accuracy of 0.78 at a decision threshold of 0.30 g/L. CONCLUSIONS: γ′ Fibrinogen shows excellent utility for cardiovascular risk analysis.