Published online before print May 1, 2003, doi: 10.1161/01.ATV.0000074145.58172.BD
© 2003 American Heart Association, Inc.
Thrombosis |
Development of a Genotype 325–Specific proCPU/TAFI ELISA
From the Laboratory for Pharmaceutical Biology and Phytopharmacology (A.G., E.B., M.P., P.J.D.), Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium; Laboratory of Haematology (M.-C.A., I.J.-V.), CHU Timone, INSERM EPI 9936, Marseillle, France; Department of Haematology (P.M., B.B.), University Medical Center Utrecht, The Netherlands; and Laboratory of Medical Biochemistry (J.L., D.H.), University of Antwerp, Belgium. P.M. is presently at the Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, the Netherlands.
Correspondence to Ann Gils, Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, E. Van Evenstraat 4, B-3000 Leuven, Belgium. E-mail ann.gils{at}pharm.kuleuven.ac.be
Objective— A Thr/Ile polymorphism at position 325 in the coding region of proCPU has been reported. Immunological assays, fully characterized (including genotype dependency), are required for the quantitation of proCPU levels.
Methods and Results— We have generated a panel of monoclonal antibodies against human, plasma-derived proCPU. Two combinations exhibiting distinct reactivities were selected for measurement of proCPU in plasma. T12D11/T28G6-HRP yielded values of 10.1±3.1 µg/mL (mean±SD, n=86; normal donors), and T32F6/T9G12-HRP yielded values of 5.4±3.0 µg/mL. Grouping according to the 325 genotype demonstrated that T12D11/T28G6-HRP was independent to this polymorphism whereas T32F6/T9G12-HRP revealed a complete lack of reactivity with the Ile/Ile genotype (ie, 0.0±0.0, 4.2±1.7, and 7.3±2.9 µg/mL for the Ile/Ile, Ile/Thr, and Thr/Thr isoforms, respectively). Commercially available antigen assays appeared to be partially dependent on the 325 genotype (eg, 44±8.9% and 100±30% for the Ile/Ile and Thr/Thr isoforms, respectively).
Conclusions— Our data demonstrate that great care should be taken when evaluating proCPU antigen values as a putative causative agent or as a diagnostic risk marker for cardiovascular events.
Key Words: TAFI • proCPU • monoclonal antibody • ELISA • genotype dependent
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