EXPERIMENTAL-DESIGN FOR KINETIC-ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS WITH SURFACE-PLASMON RESONANCE BIOSENSORS

The reaction between antibody immobilized to surfaces, with and withou t a dextran matrix, and antigen in solution was studied using surface plasmon resonance detection. The use of a reference surface made it po ssible to identify conditions where a response related to changes in m atrix conformation could be ignored. It was possible therefore to impr ove data quality by separating signals related to binding events from signals due to differences in refractive index between sample and runn ing buffer. When antigen was injected over antibody immobilized to sur faces with and without dextran matrix the binding curves were virtuall y superimposable. Consequently, no binding artifacts associated with t he dextran matrix were observed. Sets of binding curves obtained with different antigen concentrations were analyzed using numerical integra tion of differential rate equations and global fitting. When data was inconsistent with a one to one reaction it was possible to obtain good fits to an entire data set assuming several other reaction schemes in cluding parallel, competitive and two-state reactions. Thus data analy sis alone was not sufficient to discriminate between different reactio n schemes. In contrast several reaction schemes could be ruled out wit h simple experiments; the duration of antigen injection, and reanalysi s of antigen recovered in fractions from the antibody surface. In view of these findings experimental design appears to be the key to succes sful interaction analysis.