The Blood Coagulation Factor XIII-Microassay Kit uses biotinylated F11 peptide (BiotinpepF11) as the first substrate (amine-acceptor/acyl-donor) and spermine as second substrate (amine-donor/acyl-acceptor).
Usually samples contain the pro-enzyme (FXIII) and a pre-treatment with thrombin (
T056) to obtain activated FXIII (FXIIIa) could be necessary to measure the activity using Blood Coagulation Factor XIII-Microassay Kit. After this activation step, samples suspected of containing activated FXIIIa are incubated with calcium, dithiothreitol (DTT), and Biotin-pepF11 in the wells of microtiter plates to which spermine had been covalently coupled, previously. In the presence of FXIII spermine is incorporated into the gamma-carboxamide of the glutaminyl residue of the biotin-pepF11 to form a biotin-pepF11-gamma-glutamyl spermine.
The system is coupled to Streptavidin labelled peroxidase (SAv-HRP). SAv-HRP is revealed using H
2O
2 as HRP substrate and tetramethyl benzidine as electron acceptor (chromogen).