Epitope analysis of anti-prothrombin antibody.

(A) A 96-well microtiter plate was coated with 5 μg/mL prothrombin (▪), meizothrombin (●), and the prothrombin–antithrombin III complex (▴) at 4°C for 18 hours. Then the plate was blocked and washed, and increasing concentrations of patient IgG (0-120 μg/mL) were added. Bound IgG was detected using antihuman IgG polyclonal antibody conjugated with horseradish peroxidase (HRPO) and HRPO substrate with absorbance read on a model 3550 Microplate Reader (Bio-Rad). (B) Plates were also coated with prothrombin derivatives and recombinant deletion mutants (fragment 1, F1; fragment 2, F2; fragment 1 + 2, F1 + 2; and α-thrombin), and the ability of the patient IgG to bind was determined in a similar fashion.