SPR analysis of C3 and C5 binding to C3b and C4b. (A-D) Estimation of equilibrium dissociation constants for C3 and C5 binding to C3b and C4b. After biotinylation of C3b and C4b molecules at their thioester moiety, 1500 and 2100 response units (RUs), respectively, were immobilized onto a streptavidin SAP sensor chip. C3 was flowed over in concentrations ranging from 0.023 to 6 µM, C5 in concentrations from 0.011 to 3 µM. The equilibrium dissociation constant K_D was estimated by plotting the steady-state response over the corresponding concentration and using a 1:1 steady-state affinity model. (E) C3 binding to C3b in presence of Cp40. A total of 1800 RUs of biotinylated C3b was immobilized on a SAP sensor chip. C3, at a concentration of 0.6 µM, was applied to the chip (orange) before or (brown) after the immobilized C3b had been exposed to Cp40. The binding of Cp40 (6 µM) to C3b was also assayed (yellow). Finally, a C3 (0.6 µM) together with a 10-fold excess of Cp40 was flowed over the Cp40-saturated C3b surface (blue). (F) C3 binding to C4b despite Cp40 excess. A total of 1700 RUs of biotinylated C4b was immobilized on a SAP sensor chip. C3, at a concentration of 0.35 µM, was flowed over the chip alone (orange) or in mixture with Cp40 at a concentration 10-fold over C3 (gray). Cp40 alone did not bind (yellow). The number of independent SPR assays performed were: 1 typical assay of 2 independent assays (A-B,F); 1 assay was performed (C-D,E).