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Development of sandwich-type ELISAs for the quantification of rat and murine thrombin activatable fibrinolysis inhibitor in plasma

Abstract:

Background: Altered plasma levels of thrombin activatable fibrinolysis inhibitor (TAFI) are associated with a large number of pathologies. Rat and murine models are frequently used to study the pathophysiological role of TAFI in vivo but immunological tools to quantify rat and murine TAFI are lacking. Objective: The production of monoclonal antibodies (mAb) towards rat TAFI and the development of an ELISA for the quantification of rat and murine TAFI in plasma. Methods and results: Monoclonal antibodies were raised in TAFI-deficient mice towards (activated) recombinant rat TAFI. Pair-wise testing of the mAb revealed three suitable ELISA combinations, namely RT36A3F5/ RT30D8-HRP, RT36A3F5/RT82F12-HRP and RT82F12/RT36A3F5-HRP. All three ELISAs are highly specific for rat and murine TAFI. TAFI concentrations in the lower ngmL-1 range can be determined in plasma samples with a high reproducibility. Comparing TAFI antigen levels measured by these ELISAs with TAFIa activity values determined by activity based assays revealed excellent correlations (R2 >0.98). The average antigen levels of 20 individual rat plasma samples were 16 ± 2 μgmL-1 using the RT36A3F5-RT30D8-HRP, 12 ± 2μgmL-1 using the RT36A3F5-RT82F12-HRP and 21 ± 2μgmL-1 using the RT82F12-RT36A3F5-HRP ELISA. The determined antigen levels in rat plasma are similar to the levels reported for human plasma. Conclusions: We developed three highly specific and extremely sensitive sandwich-type ELISAs for the quantification of rat and murine TAFI in plasma. The described ELISAs will facilitate in vivo investigation on the pathophysiological role of TAFI. © 2007 International Society on Thrombosis and Haemostasis.