Monoclonal antibodies specific for prothrombin fragment 1.2 and their use in a quantitative enzyme-linked immunosorbent assay

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 39; no. 4; p. 583
• Main Authors: Hursting, M J, Butman, B T, Steiner, J P, Moore, B M, Plank, M C, Szewczyk, K M, Bell, M L, Dombrose, F A
• Format: Journal Article
• Language: English
• Published: England 01-04-1993
• Subjects: Adult; Animals; Antibodies, Monoclonal - immunology; Antibody Specificity; Enzyme-Linked Immunosorbent Assay - statistics & numerical data; Epitopes - immunology; Humans; Mice; Mice, Inbred BALB C; Peptide Fragments - analysis; Peptide Fragments - immunology; Prothrombin - analysis; Prothrombin - immunology; Reference Values
• ISSN: 0009-9147
• DOI: 10.1093/clinchem/39.4.583

Prothrombin fragment 1.2 (F1.2) is an activation peptide generated during a critical event of blood coagulation, the conversion of prothrombin to thrombin. As a marker of thrombin generation, F1.2 has clinical potential in assessing thrombotic risk and monitoring anticoagulant therapy. In developing a highly specific, monoclonal antibody-based immunoassay of human plasma F1.2, we generated six murine anti-F1.2 monoclonal antibodies, using as immunogen a synthetic peptide (sequence: CGSD-RAIEGR) similar to the unique carboxyl terminus of F1.2. Each antibody bound F1.2 but not prothrombin. Epitope mapping studies with one antibody (5-3B) showed that optimum binding required six to eight amino acids plus a terminal arginine to emulate the F1.2 carboxyl terminus. A quantitative sandwich ELISA for human plasma F1.2 was configured with monoclonal antibody 5-3B as the capture antibody and peroxidase-labeled polyclonal antibodies to the F1.2 amino-terminal region as detector antibodies. Calibrators were prepared by adding purified F1.2, 0-10 nmol/L, to F1.2-depleted plasma. Assay characteristics included the following: mean (+/- SD) analytical recovery of 98% +/- 13%; no interference from lipemia, hemolysis, icterus, or thrombolytic agents; 0.08 nmol/L sensitivity; and mean intra- and interassay imprecision (three lots) < 12% at both low and high concentrations of F1.2.