Selective Binding Assay for VEGFR-1

A binding assay was performed to determine the selectivity of the [¹⁸F]AlF-NODA-scVR1 for the VEGFR-1 receptor based on previously described protocols [16 (link)]. Commercially available HisPur™ Ni-NTA (Nickel-Nitrilotriacetic acid) magnetic beads (Thermo Fisher Scientific, Waltham, MA) were incubated with 1 μg of murine Fc-VEGFR-1 (BPS Biosciences, San Diego, CA) with an N-terminal His-tag at room temperature for 15 minutes in phosphate buffered saline with 1% bovine serum albumin for the tracer only and blocking cohorts. In the control group no VEGFR-1 was used and thus no receptor was bound to the surface of the magnetic beads. The beads were then washed and subsequently incubated for 5 minutes with approximately 1 ng of [¹⁸F]AlF-NODA-scVR1 in the control and tracer cohorts and 1 ng of [¹⁸F]AlF-NODA-scVR1 along with 5.0 μg of scVR1 in the blocking cohort. The beads were then washed by placing vials in a magnetic bead rack and collecting the supernatant. The bound tracer and unbound tracer collected in the washes was measured using a Wizard² automatic γ-counter (PerkinElmer, Waltham, MA).