Monoclonal antibodies specific for prothrombin fragment 1.2 and their use in a quantitative enzyme-linked immunosorbent assay.
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.
Hursting
M J
MJ
Organon Teknika Corp., Durham, NC 27704.
Butman
B T
BT
Steiner
J P
JP
Moore
B M
BM
Plank
M C
MC
Szewczyk
K M
KM
Bell
M L
ML
Dombrose
F A
FA
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay / Q000706:statistics & numerical data; D000939:Epitopes / Q000276:immunology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology; D011516:Prothrombin / Q000032:analysis* / Q000276:immunology; D012016:Reference Values
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; C045879:prothrombin fragment 1.2; D011516:Prothrombin