2.4 Inhibition assay
The inhibition assay was designed to evaluate whether the percentage of
antibody released would have a significant effect on inhibiting
recombinant KLK7 in vitro .
The assay was performed using the FRET substrate Abz-KLYSQ-EDDnp (GenOne
Biotechnologies). Recombinant KLK7 (5 ng/uL) diluted in
phosphate-buffered saline (pH = 7.4) was added to Greiner
CELLSTAR® black polystyrene 96 flat wells (Greiner
Bio-One). Each well was filled with 100 µL of assay buffer (50 mM Tris,
pH = 7.5). Recombinant KLK7 and the assay buffer were incubated for 2
minutes at 37 ºC prior to the addition of the substrate. The substrate
(1 mg/mL) was added to the wells. The total read time was 15 minutes at
37 ºC under agitation. The positive control was only KLK7; to determine
the residual activity of KLK7 against the antibodies, 100 µL of the
aliquots taken at 6, 8 and 24 hours was added prior to the addition of
the substrate. All readings were carried out with a Hitachi F 2500
fluorometer (Hitachi Science & Technology); the excitation and emission
slits were adjusted to 5 nm and wavelengths were set to 380 and 460 nm.
All reads were performed in triplicate.