ELISA for determination of total coagulation factor XII concentration in human plasma

• Published in: Journal of immunological methods Vol. 394; no. 1-2; pp. 32 - 39
• Main Authors: Madsen, Daniel Elenius, Sidelmann, Johannes Jakobsen, Overgaard, Kathrine, Koch, Claus, Gram, Jørgen Brodersen
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 30.08.2013
• Subjects: blood coagulation; blood sampling; Blotting, Western; C1-inhibitor; coagulation; Coagulation factor XII; detection limit; disulfide bonds; ELISA; enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Factor XII - analysis; Fibrinolysis; Humans; Limit of Detection; monoclonal antibodies; serine proteinases; Thrombosis; Validation; volunteers; C1-inhibitor; B; RT; TW; HRP; kDa; MAb; O/N; ANCOVA; SD; FXII; OD; APlC; Strp; FPLC; LOD; PBS; Validation; 4-PL; IV; Fibrinolysis; Thrombosis; LOQ; RD; CV; C1-inh; PK; ELISA; Sr; Coagulation factor XII; within-laboratory standard deviation; horseradish peroxidase; complement C1 esterase inhibitor; streptavidin; estimate of repeatability standard deviation; limit of detection; fast protein liquid chromatography; kilo Dalton; analysis of covariance; standard deviation; room temperature; intravenously; 4-parameter nonlinear logistic curve fitting; S(r); limit of quantitation; enzyme linked immunosorbent assay; over night; anti phospholipid syndrome; tween; phosphate buffered saline; relative dilution; plasma kallikrein; monoclonal antibody; optical density; coefficient of variation
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2013.04.012

Human blood coagulation factor XII (FXII) is the one chain 80kDa zymogen form of the active serine protease α-FXIIa, which consists of a heavy and light chain linked by a disulfide bond, the light chain being responsible for the proteolytical activity. FXII is the first component of the contact dependent pathway of coagulation, but its physiological role is still subject to debate. In the present study we utilized two monoclonal antibodies against the heavy chain of FXII to establish a sandwich enzyme linked immunosorbent assay (ELISA) for quantification of total FXII concentration in human plasma samples. A unique characteristic of this assay is its equal recognition of FXII and inhibitor bound FXII. This is important, as inhibitor complexes of α-FXIIa are formed in vivo as well as during blood sampling and handling. Validation of the assay demonstrated a high sensitivity, with a limit of detection and quantification of 1.2ng/mL and 2.6ng/mL respectively. The coefficients of variation for the repeatability and within-laboratory standard deviations were 2.6% and 5.2% respectively. The reference interval determined from healthy volunteers (n=240) was 10.6–43mg/L. •We established a total human factor XII (FXII) antigen ELISA using two monoclonal antibodies.•The ELISA is suitable for stable and reliable measurement of FXII in human plasma.•Our assay recognizes α-FXIIa C1-inhibitor complexes to the same degree as α-FXIIa alone.•Limit of detection and quantitation was 1.2ng/mL and 2.6ng/mL respectively.•The coefficients of variation for the repeatability and within-laboratory standard deviations were 2.6% and 5.2% respectively.