Victor2 multilabel counter

Serum sVAP-1 was measured by time-resolved immunofluorometric assay at BioTie Therapies Corp., Finland. A biotin-conjugated monoclonal anti-human VAP-1 antibody was adsorbed onto a microtiter plate. Detection of bound serum VAP-1 was performed using a different europium-conjugated anti-human VAP-1 antibody. Time-resolved fluorescence was measured using a fluorometer (Victor²Multilabel Counter, PerkinElmer) at 615 nm. Serum VAP-1 concentration was quantified on the basis of a reference sample of highly purified human serum VAP-1 (Biovian Ltd).

Two microliters of a serial dilution of Damna in DMSO (1 ×10⁻² – 2.5 ×10⁻⁸ M), Damna-NS in DI water (2 ×10⁻² – 5 ×10⁻⁸ M), and Blank-NS in DMSO (1 ×10 ⁰ – 2.5 ×10⁻⁶ M) were mixed with 12 µL of 20 mM NADPH-regenerating system in PBS solution pH 7.4 and 5 µL of 0.5 mg/mL of liver S9 fraction and were then added to 100 mM PBS solution pH 7.4 to make the final volume up to 200 µL. The incubation was carried out at 37 °C for 60 min with gentle shaking. After 60 min of reaction, the reaction was terminated by adding 200 µL DMSO.
For in vitro drug metabolism assay, after incubation of test samples with liver S9 fractions, 2 µL of a serial dilution of incubated Damna in DMSO (1 ×10⁻⁴ – 2.5 ×10⁻¹⁰ M), incubated Damna-NS in DI water (1.4 ×10⁻⁵ – 3.5 ×10⁻¹¹ M) and incubated Blank-NS in DMSO (2 ×10⁻⁸ – 5 ×10⁻¹⁴ M) were added into a black 96-well plate. Two hundred microliters of yeast strains (BLYES and BLYR) were added into each well of the black 96-well plate. All samples were performed in triplicate. Bioluminescence was measured every 60 min for 5 h in a Perkin-Elmer Victor² Multilabel Counter with an integration time of 1 s per well. Incubated 17β-estradiol (1 ×10⁻⁴ – 2.5 ×10⁻¹⁰ M) was used as a positive control for the estrogenic activity assay. Vehicle (DMSO or DI water) was used as a negative control.