The design schema is shown on the left. A 2 mg/ml sample of mouse FLT-1-Fc protein (471-F1; R&D Systems, Minneapolis, MN) was immobilized on an ELISA plate. After blocking with 1% BSA, the plate was incubated in 2 ng/ml mouse VEGF-A (493-MV-005; R&D) or mouse PlGF-2 (465-PL-010; R&D) at room temperature (RT) for 2 hr. Then, each well was incubated in (A) 2 ng/ml mouse VEGF-A, (B) 2 ng/ml mouse VEGF-A + 5 mg/ml isotype IgG, (C) 2 ng/ml mouse VEGF-A + 5 mg/ml anti-FLT-1 IgG (AF471; R&D), (D) 2 ng/ml mouse PlGF-2, (E) 2 ng/ml mouse PlGF-2 + 5 mg/ml isotype IgG, or (F) 2 ng/ml mouse PlGF-2 + 5 mg/ml anti-FLT-1 IgG at RT for 6 hr. Using biotinylated anti-VEGF-A (BAF493; R&D) or biotinylated anti-PlGF-2 (BAF465; R&D) and streptavidin-HRP (DY998; R&D), VEGF-A or PlGF-2 was detected. The results (row C vs row F) show that FLT-1 neutralizing antibody desequestered VEGF-A but not PlGF-2 from FLT-1. *p<0.0001 by two-tailed Student t test.