High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

Figure 5 Dissociation constant (K_D) of the chimeric Fab. The K_D was determined using an ELISA based method. Different amounts of HBsAg were incubated with concentrated periplasmic extract of the clone expressing the anti-HBs chimeric Fab for 16 h and unbound antibody was detected by ELISA using rabbit anti-human antibody-HRP (Dako). The data was fitted to the equation A_O/(A_O–A) = K_D*1/[Ag]+1, where Ao = absorbance when the antibody was incubated without any antigen, A = absorbance corresponding to free antibody after incubation with the antigen. Dissociation constant of the chimeric Fab was determined from the slope of the straight line (K_D = 8.166±0.14 nmol/L).