Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins

A methodology to correlate the absolute surface concentration of protein to the surface plasmon resonance (SPR) response is described. The thickness and the optical constants for each layer on the sensor chip used were determined with different optical techniques. In a flow injection system, the steady-state SPR response was correlated to the absolute amount of radiolabeled protein adsorbed by using a surface scintillation counter. The proteins used, 14C-labeled human transferrin and chymotrypsinogen A, as well as in vivo 35S-labeled monoclonal antibodies, were adsorbed via electrostatic interaction to a carboxymethylated dextran hydrogel on the sensor chip. For these proteins, surface concentrations from 2 to 50 ng mm−2 correspond linearly to the SPR response, with specific response in the range 0.10 ± 0.01° (ng mm−2)−1, independent of protein size. The minimum detectable surface concentration of protein is estimated to be 50 pg mm−2 with this SPR instrument. Optical models have been developed to describe how the SPR response depends on the distribution of the adsorbed protein within the hydrogel volume at the surface. With a thin-film optical program, the theoretical SPR responses for the different models were calculated. Comparison with experimental data shows that the protein is distributed within an approximately 100-nm-thick dextran hydrogel layer.