Figure 3
Immunological and biochemical natures of the mAb. Panel A shows isotyping and subtyping results by immobilizing anti-human Igs as indicated. The binding profile of the mAb was subsequently revealed by biotinylated CD152-muIg and avidin-peroxidase conjugates. Results indicate that the present mAb belongs to a type of IgG4λ. Panel B indicates the isoelectric point of the monoclonal IgG4λ anti-CD152 (lane 4 and 8), resolved by isoelectric focusing. Monoclonal human myeloma IgG1λ(lane 2 and 6) and IgG4λ (lane 3 and 7) were run in parallel for comparison. The electrophoretic patterns were visualized by either Coomassie brilliant blue staining (lane 1–5) or immunoblot (lane 6–8) with anti-human IgG conjugated with peroxidase and FAST™ DAB. The calculated isoelectric points for human IgG1λ, IgG4λ and anti-CD152 mAb to be approximately in the range of 7.92–8.79, 5.76–6.52 and 7.87–8.41, respectively, based on the calibration against the linear regression of standard protein markers. Panel C outlines the affinity determination by IAsys. Surface plasmon resonance obtained at 25°C for increasing concentrations of anti-CD152 mAb on purified, unlabeled CD152-muIg. The straight line in the inset was obtained from the kobs plot versus ligated Ab concentration and yielded a kdiss (the intercept) of 16.81 and a kass value (the gradient) of 4.20 × 109. Therefore produced a Kd (kdiss/kass) of 4 × 10-9 M.