Binding analysis of LPS and β₂GPI. Binding of LPS or lipid A to β₂GPI or domains I to IV and V of β₂GPI was investigated with surface plasmon resonance. β₂GPI or domains I to IV or V was immobilized on a CM5 sensor chip, and increasing concentrations of LPS E coli J5 (A) or LPS S minnesota R595 (B) were injected at time points 0, 350, and 700 seconds (100, 300, and 1000nM, respectively, as indicated by black arrows), and every injection was stopped after 100 seconds. Binding of both LPSs to β₂GPI or domain V could be observed (black line), whereas no binding could be detected to domains I to IV (black dotted line). Also, no binding could be observed between lipid A and β₂GPI or domain V (black dotted line). Fitting of the data to a 1-to-1 model revealed a K_D value of 62nM for LPS E coli J5 and 23nM for LPS S minnesota R595 (visualized by the gray line).