Fraction collector

Twenty milligrams of purified rabbit antibodies in 1 mL of binding buffer (0.2 M NaHCO₃ and 500 mM NaCl, pH = 8.3) were slowly loaded onto a pre-equilibrated HiTrap NHS-activated HP column (1 mL, GE, Uppsala, Sweden) and incubated for 1 h at room temperature. The column was blocked with ethanolamine (0.5 M in 0.5 M NaCl, pH = 8.3) and then washed three times with sodium acetate buffer (0.1 M in 0.5 M NaCl, pH = 4.0). Then, one hundred micrograms of venom in 1 mL of phosphate buffer was injected into the affinity column with immobilized antibodies at a flow rate of 0.3 mL/min, and the column was washed with 10 mL of PBS (pH = 7.5). The retained proteins were eluted by acetic acid (10%, v/v) at a flow rate of 1 mL/min for 10 min. All procedures were performed at 4 °C by using a fast protein liquid chromatography (FPLC) instrument (GE Healthcare, Piscataway, NJ, USA) equipped with a fraction collector (GE Healthcare) and an ultraviolet (UV) detector. The UV adsorption wavelength was set at UV 280 nm during the sample analysis. The flow through and eluted fractions were collected, lyophilized, and stored at −20 °C for further analysis.

The affinity analysis followed the procedure developed by Fahmi et al. [44 (link)] with slight modifications. To avoid sample degradation, the analytical procedures were performed at 4 °C with fast protein liquid chromatography (GE Healthcare, Piscataway, NJ, USA) equipped with fraction collector (GE Healthcare) and optical detector (UV 280 nm). Five hundred micrograms of venom dissolved in 1 mL of phosphate buffer was injected into the antibody-immobilized affinity column at a flow rate of 0.3 mL/min, followed by a washing step to remove the unbound protein using 10 mL of phosphate-buffered saline (PBS) (pH 7.5). Subsequently, the retained components were eluted with acetic acid (10%, v/v) at a flow rate of 1 mL/min for another 10 min. The flow-through and elution fractions were collected for further antivenomic analysis. The extent of protein binding to the antivenom-immobilized column was calculated based on the peak areas of retained (R) and non-retained (NR) fractions in the RP–HPLC chromatography (UV 280 nm), and the percentage of retained protein (%) was quantified using the equation as follows: % retained protein = 100 − [100 × (NR/(R + NR))].