Fig. 1. In vitro characterization of soluble human VEGFR-3 and mAb hF4-hF4-3C5. (A) Conditioned media from cells expressing human sR3-AP (), human sR2-AP () or human sR1-AP () were normalized for AP activity and added to 96-well plates coated with VEGF-C_NC. Bound receptors were detected with a rabbit antibody to AP and peroxidase-labeled secondary antibodies. sR3-AP binds to VEGF-C_NC with an EC₅₀ about ten times lower than sR2-AP whereas sR1-AP does not show binding activity. (B) Conditioned media were normalized for AP activity and added to 96-well plates coated with either VEGF-C_NC or VEGF-A. The amount of bound receptor was measured using AP activity. R3-AP (clear bars) binds strongly to VEGF-C_NC but not to VEGF-A. Control media from cells expressing untagged AP shows no binding (black bars). (C) Purified sR3-AP (µ), sR2-AP () or sR1-AP () were added to 96-well plates coated with hF4-3C5. The amount of bound receptor was measured using AP activity. sR3-AP binds to the mAb hF4-3C5 whereas sR1-AP or sR2-AP show no detectable binding. (D) Inhibition of sR3-AP binding to VEGF-C_NC by the mAb hF4-3C5. Saturating amounts of sR3-AP were mixed with various amounts of mAbs or Fab fragments and added to 96-well plates coated with VEGF-C_NC. Blocking of the receptor binding is evident with full-length hF4-3C5 (, IC₅₀=1.3 nM), and Fab fragments of hF4-3C5 (, IC₅₀=2 nM) but not with the irrelevant human mAb IMC-2F8 ().