ASSAY DEVELOPMENT FOR PLASMA FACTOR VIIA-ANTITHROMBIN COMPLEX (FVIIA-AT)
Abstract number: P-S-111
Adam1 M., Vauthier1 C., Morrissey2 J., Woodhams1 B.J.
11R and D, Stago, Gennevilliers, France 22Department of Biochemistry, University of Illinois, Urbana, United States
How-to-cite Adam M, Vauthier C, Morrissey J, Woodhams BJ. ASSAY DEVELOPMENT FOR PLASMA FACTOR VIIA-ANTITHROMBIN COMPLEX (FVIIA-AT). J Thromb Haemost 2007; 5 Supplement 2: P-S-111
Introduction: Tissue factor (TF) when exposed in the vasculature binds allosterically and activates FVIIa. Antithrombin (AT) is an important regulator and binds to FVIIa forming a stable covalent complex which then loses its affinity for TF and is released into the circulation. Plasma FVIIa-AT levels could be an important marker for coagulation activation. We have developed a stable reproducible ELISA for FVIIa-AT.
Methods: ELISA plates were coated and then dried with a F(ab')2 from a FVIIa specific antibody selected because it does not block AT binding to FVIIa (Mab1172). 0.2ml of diluted calibration material or plasma samples was added to the plate and incubated for 2 hours, washed and then incubated for a further 2 hours with 0.2ml of a polyclonal AT antibody conjugated with peroxidase. A lyophilised preparation of FVIIa-AT, prepared from reacting recombinant FVIIa with purified AT, was used for calibration. Activity determined using a liquid TMB substrate
Results: A linear calibration was obtained from 0 to 30pM (y=0.05x + 0.21. r2=0.99). No interference from plasma factors was observed so calibration in FVII deficient plasma is not required simplifying the assay. No difference in recovery was observed when samples were run diluted either 1:20 or 1:40. The within and between assay variation was <8% and <13% respectively. The mean normal value is 208pM 89 n=37 (after exclusion of values outside the 2SD range). In a preliminary study DIC patient samples gave a mean value of 458pM (range 275-700pM n=4).
Conclusions: The assay is stable and reproducible and can now be applied to clinical studies. Very preliminary results show interesting differences between normal and pathological samples.
To cite this abstract use the following format:
Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number
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