Gamma' fibrinogen: evaluation of a new assay for study of associations with cardiovascular disease

Abstract

Background: Studies of disease associations with gamma' 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 gamma' 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 gamma' 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 gamma' fibrinogen in healthy individuals from the Framingham Offspring Study (n = 2879) and quantified associations between gamma' fibrinogen and cardiovascular disease risk factors. The sensitivity and specificity of gamma' fibrinogen in evaluating CAD patients (n = 133) was determined with ROC curve analysis.

Results: The gamma' 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: gamma' Fibrinogen shows excellent utility for cardiovascular risk analysis.

Fig. 1. Analytical specificity of 2.G2.H9 towards γ' fibrinogen

The analytical specificity of anti-γ' fibrinogen monoclonal antibody 2.G2.H9 and its ability to differentiate γ' fibrinogen from fibrinogen lacking γ' chains was determined by incubating the indicated dilutions of the antibody with γA/γA or γA/γ' fibrinogen in microtiter wells. Goat anti-mouse IgG/HRP conjugate was added for detection, followed by O-phenylenediamine, and the absorbance was quantitated at 450 nm.

Fig. 2. Curve fitting for the γ' fibrinogen ELISA

A γ' fibrinogen standard curve was generated using the indicated concentrations of purified γ' fibrinogen reconstituted in heat-defibrinated plasma. Capture antibody was anti-γ' fibrinogen monoclonal antibody 2.G2.H9, and detection antibody was a commercial sheep anti-human fibrinogen/HRP conjugate. TMB substrate absorbance was quantitated at 450 nm. The resulting points were fit either to a linear, logarithmic, or second-degree polynomial curve fit.

Fig. 3. Limit of quantification for the γ' fibrinogen ELISA

The limit of quantification of the assay for measurement of γ' fibrinogen concentrations in plasma was determined using eight separate pools of patient plasma. The limit of quantification for the assay was 0.10 g/L, defined as that concentration of γ' fibrinogen giving a within-run CV of 20% or greater.

Fig. 4. γ' fibrinogen concentrations in healthy individuals from the Framingham Offspring Study

γ' fibrinogen was measured in 2,879 participants from the Framingham Offspring Study with no previous history of cardiovascular disease. The reference interval of γ' fibrinogen varied nearly 40-fold, from a low of 0.037 g/L to a high of 1.443 g/L. The 2.5^th and 97.5^th percentile limits for γ' fibrinogen were 0.088 to 0.551 g/L. The median concentration was 0.234 g/L and the mean concentration was 0.255 γ 0.119 g/L (±SD).

Fig. 5. Receiver-operating characteristic curve for γ' fibrinogen in CAD patients

γ' fibrinogen concentrations were measured in a prior study (7) in 133 patients referred for elective diagnostic cardiac catheterization. The receiver-operating characteristic curve of γ' fibrinogen concentrations in CAD cases and controls showed an area under the curve of 0.76. A maximum diagnostic accuracy of 0.78 was found at a decision threshold of 0.30 g/L.