Fig. 3

Optimization of conditions for conjugation of Ab to ∆F-BMP-FA. a Optimization of wash cycle number. Amount of linked Ab was maximal for ∆F-BMP-FA from seven wash cycles. b Optimization of ∆F-BMP-FA weight used for linkage to 300 µg Ab. Optimal weight ratio was 1:3. c Optimization of linkage temperature and time. Maximal linkage rate was observed for linking for 2 h at 30 °C. d Optimization of coupling buffer used for linkage of BMP to Ab. 1: 10 mmol/L PBS, pH 7.4. 2: 10 mmol/L Tris–HCl, pH 7.4. 3: 10 mmol/L HEPES, pH 7.4. 4: 10 mmol/L Na₂HPO₃-C₆H₈O₇, pH 7.4. 5: 10 mmol/L KH₂PO₃-NaOH, pH 7.4. Buffer #2 (10 mmol/L Tris–HCl, pH 7.4) was selected for use in subsequent experiments