Mab2926

The pBMP10 ELISA detects Pro:BMP10 and ProBMP10, but not the BMP10 GFD (Hodgson et al., 2019 (link)). For plasma pBMP10 measurements, plates were coated with 0.5 µg/well of anti‐human BMP10 (MAB2926; R&D Systems) and blocked as above. Plasma samples (30 μL) were premixed with 70μL BioRad diluent, supplemented to give final concentrations of 0.5% Triton X‐100, 0.2% GS, and 4.5 mM EDTA. Dilutions of the furin‐cleaved purified Pro:BMP10 standard (97.65–100000 pg/ml GFD equivalent), produced as described previously (Jiang et al., 2016 (link)), were prepared to give the same final concentrations of additives. After washing, biotinylated anti‐human BMP10 propeptide (0.04 µg/well BAF3956; R&D Systems) in 1% BSA, 0.2% goat serum was added. Assays were then processed as described for BMP9. All data are presented as the concentration of the GFD component. All samples were measured in duplicate. The limit of detection of was 445.2 pg/ml and lower limit of quantification was 1484 pg/ml.

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.

A high binding 96-well plate was coated with 0.25 μg/well of anti-human BMP10 GF-domain antibody (cat. No. MAB2926, R&D Systems) in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 °C overnight. The wells were washed with PBS containing 0.05% Tween 20 (PBST) followed by blocking with 1% BSA in PBS (BSA/PBS) at room temperature for 2 hours. In parallel, 25 ng Pro:BMP10 samples were premixed with BMPRII ECD WT (at 1:0, 1:1, 1:5, 1:25, 1:125 molar ratio) or mutants (at 1:125 molar ratio) in 1% BSA/PBS at room temperature for 30 minutes. The plate was washed with PBST before samples were added. After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody (0.04 μg/well, cat. No. BAF3956, R&D Systems) in 1% BSA/PBS was added. After washing, ExtrAvidin®-Alkaline phosphatase (cat. No. E2636, SIGMA) diluted 1:400 in 1% BSA/PBS was added. The assay was then developed with 0.67 mg/ml 4-Nitrophenyl phosphate disodium salt hexahydrate (cat. No. S0942, SIGMA) in 1 M Diethanolamine, 0.5 mM MgCl₂, pH 9.8 and absorbance was measured at 405 nm. The experiment was repeated three times, with technical duplicates each time. All values are presented as the ratio of OD_405nm of samples to Pro:BMP10 only sample.