FIG. 1.

Selection and analysis of anti-DKK1 and anti-FRZB VHH, (A) VHH bind to DKK1 and neutralize DKK1 activity. Concentration range of VHH G5 and H7 was incubated in wells coated with DKK1 (60 nM) or PBS (negative control). After several washes, bound VHH were detected with a mouse anti-VHH serum and a donkey anti-rabbit antibody coupled to a peroxidase. The amount of converted HRP (absorbance at 450 nm; A450 nm) is proportional to the amount of bound VHH. Error bars represent SD (n = 3). (B) Addition of BMP6 stimulates ALP expression in KS483-4C3 cells. The coincubation of BMP6 with DKK1 reversed the ALP expression back to basal levels. The addition of DKK1 to the standard cell culture did not show any significant difference in ALP expression compared with control. The ALP activity is normalized by the total DNA content of KS483 cells after 7 days of culture and expressed as fold induction relative to control. KS483-4C3 cells were stimulated with BMP6, DKK1, and VHH G5 (C) or VHH H7 (D) in a concentration range of 0–70 nM. Coincubation of VHH with DKK1 and BMP6 reversed DKK1-mediated inhibition of BMP6-induced ALP activity in a dose-dependent manner demonstrating effective neutralization of DKK1 activity by the VHH G5 and H7. ALP activity was measured and expressed as relative enzyme activity corrected for DNA and expressed as fold induction relative to BMP6/DKK1/VHH (+/+/−). *P < 0.05. (n = 3). (E) The apparent affinities of different FRZB VHH. Plates were coated with FRZB (60 nM) and blocked with 4% skimmed milk in PBS (MPBS), then incubated with a concentration range of different VHH (0–7 μM). Unbound VHH were washed with PBS-Tween and bound VHH were detected by incubation with mAb M1 and a HRP-conjugated anti-mouse. ALP, alkaline phosphatase; DKK1, Dickkopf 1 homolog; FRZB, frizzled-related protein; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; SD, standard deviation. Color images available online at www.liebertpub.com/scd