Mab3209

Hyaluronan levels from mouse plasma (40-weeks-old females) were quantified according to the manufacturer’s recommendation (DHYALO Biotechne, Minneapolis, MN, USA).
BMP9 ELISA measurement has been previously described [4 (link)]. MAB3209 (R&D Systems) antibody for BMP9 was used as capture antibody and detection was performed using biotinylated antibody (anti mature BMP9: BAF3209, R&D Systems). Plasma of 12 (129/Ola WT) and 12 (C57BL/6) mice were quantified.

Lactating dams were injected i.p. once on P3 with PBS, mouse monoclonal isotype control Abs (15 mg/kg, IgG2b, MAB004; 15 mg/kg, IgG2a, MAB003; R&D Systems), or mouse monoclonal anti-BMP9 and anti-BMP10 Abs (15 mg/kg, IgG2b, MAB3209; 15 mg/kg, IgG2a, MAB2926; R&D Systems, respectively). As controls, groups of neonates were directly injected i.p. on P3 with PBS or the same mouse monoclonal isotype control or anti-BMP9 and anti-BMP10 Abs (15 mg/kg). Neonates were euthanized by CO₂ asphyxiation on P6 and non-heparinized blood was collected. Neonates were enucleated and eyes fixed in 4% paraformaldehyde for 20 min on ice and retinas were isolated.

ELISAs were used to measure IgG2a, IgG2b, and anti-BMP9 Ab levels in mouse serum. IgG2a and IgG2b ELISAs were performed as per the manufacturer’s instructions (eBioscience). For the anti-BMP9 Ab ELISA, 96-well ELISA plates (Maxisorp, Nunc) were coated with 100 μL of 1 μg/mL recombinant BMP9 (R&D Systems) in coating buffer (15 mM K₂HPO₄, 25 mM KH₂PO₄, 0.1 M NaCl₂, 0.1 mM EDTA, 7.5 mM NaN₃) and incubated overnight at 4 °C. Plates were then washed 3 times with 0.05% Tween PBS (PBST) and blocked for 1 h at room temperature (RT) with 1% BSA in PBS. After washing 3 times with PBST, serial dilutions of individual mouse serum samples and reference mouse anti-BMP9 Ab (MAB3209, R&D Systems) (diluted in 1% BSA PBS) were prepared and 100 μL/well were incubated for 2 h at RT. After 3 more washes, 100 μL/well horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobins (Igs) secondary Ab (Southern Biotech, diluted 1:500 in 1% BSA PBS) was incubated for 1 h at RT. TMB substrate was added after 5 washes and the reaction was allowed to develop for 30 min at RT. The optical density was measured at 450 nm using a TECAN GENios Pro plate reader.