Sandwich ELISA assays were carried out similarly as described previously8. The plate was coated with 1 μg/mL (50 μL/well) mAb9 in 200 mM Na2CO3 buffer pH 9.6 overnight at 4 °C. The wells were blocked with 1 mg/mL bovine serum albumin in phosphate-buffered saline (PBS) for 1 h at room temperature. Next, the plate was incubated with serial dilutions of 5 μM TPPP/p25 for 1 h at room temperature in PBS. Where indicated, TPPP/p25 was pre-incubated with 100 μM DTE for 30 min at room temperature. In another experimental setup, different TPPP forms at 1 μM concentration were added to the plate coated with mAb in the absence and presence of DTE. Alternatively, 1 μM FL TPPP/p25 or CORE TPPP/p25 was preincubated with 100 μM DTE for 30 min at room temperature, then added to the plate coated with mAb; after removing the excess DTE, the different TPPP/p25 forms were added, and dimers were detected by biotinylated mAb. Then the plate was sequentially incubated with biotinylated mAb (1 μg/mL) and peroxidase conjugated avidin (Calbiochem) (2.5 μg/mL). Both antibodies were in PBS buffer containing 1 mg/mL bovine serum albumin, and incubated for 1 h at room temperature. Between each incubation step the wells were washed thrice with PBS containing 0.05% Tween 20 for 10 min. The presence of antibodies was detected using o-phenylenediamine as substrate. The reaction was stopped after 10 min with 1 M H2SO4, and the absorbance was read at 490 nm with an EnSpire Multimode Reader (Perkin Elmer). The binding constants (Kd) were evaluated from the saturation curves by non-linear curve fitting assuming single binding site hyperbola model using the Origin 8.0 software.