The elucidation of key influence factors for electrostatic adsorption is very important to control protein nonspecific adsorption on modified surfaces. In this study, real time surface plasmon resonance technique is used to characterize the electrostatic adsorption of two proteins (mouse IgG and protein A) on carboxymethyldextran modified surface. The results show that protein solution pH and ionic strength are key influence factors for efficient electrostatic adsorption. The influence of protein solution pH on the amount of electrostatic adsorption depends on the type of the charge and the charge density of both protein and modified matrix on the surface. The electrostatic adsorption process involves a competition between the positively charged protein and other positively charged species in the buffer solution. A decrease of ionic strength leads to an increasing electrostatic adsorption. The kinetic adsorption constants of protein A at different pH values were also calculated and compared.
The kinetic analysis of the interaction between tumor necrosis factor(TNF) and its monoclonal antibody was performed by surface plasmon resonance(SPR) technique. The monoclonal antibody was immobilized to the surface of CM5 sensor chip by amine coupling. TNF at different concentrations was injected across the mAb immobilized surface. The interaction was recorded in real time and could be seen on the sensorgram. One cycle, including association, dissociation and regeneration, lasted no more than 15 min . The interaction results was evaluated using 1∶1 Langmuir binding model. The kinetic rate constants were calculated to be: k a=1.68×10 3 L·mol -1 ·s -1 , k d=1.73×10 -4 s -1 , and the affinity constants K A=9.7×10 6 L·mol -1 , K D=1.03×10 -7 mol·L -1 . The χ \+2 was 3.47, which showed that the interaction is consistent with the 1∶1 model. We can see from the results that although there are two binding sites in one mAb molecule, TNF reacts with each site in an independent and noncooperative manner.
