Determination of the Degree of Grafting

The degree of grafting (DG) was evaluated by determining the decrease of free amino groups of the conjugates by the o-phthalaldehyde (OPA) method (17). The OPA reagent was freshly prepared before testing, and 200 μl of the sample and 4 ml of OPA solution were added to the test tube. After mixing well, all tubes were placed in the water bath at 35°C for 2 min, and then, the absorbance of the solution was measured at 340 nm. The above experiment was repeated with OVA and monosaccharide mixture without grafting as a control. The DG was evaluated by the following equation:

where C₀ and C_t represent the free amino group content of native OVA and conjugates, respectively.