Quantifying recombinant human activin A

To evaluate rhAA presence in the culture medium, an indirect ELISA was performed. Microtiter plates (NUNC, Netherlands) were coated with 100 µL per well of 0.2 µg standard recombinant human activin A (Isokine™, ORF Genetics, Kópavogur, Iceland), along with rhAA produced from transgenic rice cells in a coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6), and were placed at 4 °C overnight. On the following day, the wells were washed three times with PBST (PBS buffer with 0.05% Tween 20). The plate was then given 200 µL blocking buffer containing 0.1% bovine serum albumin and left at room temperature for 2 h and then washed 3 times with PBST. Subsequently, 100 µL per well of a 1:1000 dilution of inhibin β-A monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was added. After incubating at room temperature for 2 h, the plate was washed with PBST, and 100 µL of a 1:7000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (W4021, Promega, Madison, WI, USA) was added to wells. The microplate was incubated at room temperature for 2 h and washed three times with PBST. The color was developed by the addition of 100 µL per well of phosphatase substrates (S0942, Sigma-Aldrich, St. Louis, MO, USA). Optical density was measured at a wavelength of 405 nm using an ELISA reader (Sunrise, Tecan, Männedorf, Switzerland).