title: A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma.
abstract: Based on the iso-peptidase activity of human plasma FXIII, a novel fluorometric assay that determines FXIII concentrations in human plasma below 0.05 IU/ml is introduced. We considered a peptide sequence derived from alpha(2)-antiplasmin (n =12) to yield high sensitivity. Peptide Abz-NE(Cad-Dnp)EQVSPLTLLK exhibits a K(m) value of 19.8+/-2.8 microM and is used in a concentration of 50 microM. The assay design is suitable for measurements in cuvettes (1 ml volume) as well as for the microtiter plate (MTP) format (0.2 ml volume). It provides linear dose-response curves over a wide range of FXIII concentrations (0.05-8.8 IU/ml). The assay was validated with respect to precision, detection and quantitation limits, accuracy/specificity, linearity, and range. A comparison of the fluorometric assay with the photometric assay for FXIII determinations in plasma pools as well as single donor plasma revealed suitability of the fluorometric assay for FXIII determination in plasma of healthy individuals. FXIII concentrations in plasma samples of patients with severe FXIII deficiency are discussed in the context of FXIII antigen levels. These assays correlate well in the critical range below 0.1 IU/ml, whereas the photometric assay may overestimate residual FXIII activity in severe FXIII-deficient patients.
authors.affiliation: N-Zyme BioTec, D-64295 Darmstadt, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Oertel
authors.forename: Andreas
authors.initials: A
authors.lastname: Hunfeld
authors.forename: Edgar
authors.initials: E
authors.lastname: Specker
authors.forename: Christine
authors.initials: C
authors.lastname: Reiff
authors.forename: Rainer
authors.initials: R
authors.lastname: Seitz
authors.forename: Ralf
authors.initials: R
authors.lastname: Pasternack
authors.forename: Johannes
authors.initials: J
authors.lastname: Dodt
mesh_terms: D019759:Carbon-Nitrogen Lyases / Q000097:blood; D005176:Factor XIII / Q000032:analysis*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D010783:Photometry; D011503:Transglutaminases / Q000097:blood
chemical_list: D005176:Factor XIII; D011503:Transglutaminases; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase