A Novel Enzyme Capture - ELISA for the Direct Determination of Factor XIII Activity

Background: Activated factor XIII (fXIII) mechanically stabilizes fibrin clots by catalyzing the formation of intramolecular crosslinks and retards plasmin-mediated fibrinolysis by incorporating intermolecular α2-antiplasmin (α2AP):fibrin crosslinks. High quality quantitative fXIII activity assays are required for the accurate and timely diagnosis of fXIII deficiency. Currently available high-throughput assays indirectly measure the transglutaminase activity of fXIII via detection of ammonia that is elaborated upon formation of ε-N-(γ-glutamyl)-lysyl crosslinking bonds. Unfortunately, these assays can be confounded by fXIII-independent ammonia producing and consuming reactions present in plasma. We thus set out to develop an assay which directly quantifies the transglutaminase activity. We hypothesized that fXIII could be captured in a microtiter plate with anti-fibrinogen antibodies and, following an activation step, its activity determined in a kinetic fashion via the rate of α2AP incorporation (measured in a subsequent ELISA step).

Methods: Co-immunoprecipitation and western blotting techniques were utilized to confirm fXIII/fibrinogen interactions and to determine the presence of basal interactions between fibrinogen and α2AP. Simultaneously, this approach enabled the determination of suitable antibody pairs for use in the enzyme capture-ELISA (EC-ELISA). In microtiter plates, fibrinogen-captured fXIII was activated with excess thrombin, physiologic calcium, and GPRP peptide (to block fibrin polymerization) in the presence of excess α2AP. FXIII activity was stopped with a potent fXIII-inhibitor (iodoacetamide) at predetermined time points. Subsequently, α2AP incorporation was determined with a primary anti-α2AP antibody and secondary HRP-conjugated antibody.

Results: Co-immunoprecipitation and western blotting experiments confirmed that both fXIII and α2AP circulate in complex with fibrinogen, as previously reported by others. However, α2AP incorporation significantly increased following incubation with activated factor XIII. Using the EC-ELISA method, we were able to demonstrate that α2AP is incorporated in a manner consistent with first order kinetics.

Discussion: These experiments demonstrate the feasibility of an EC-ELISA assay to directly measure fXIII activity. Further work will be required to determine the sensitivity of this assay in test plasmas with varying known quantities of fXIII and how its performance characteristics compare to currently available indirect activity assays. With further optimization, the EC-ELISA method may lead to an improved, high throughput diagnostic assay for clinical use.