Human Anti-IL15 Recombinant Antibody (clone 4H10) (CAT#: HPAB-S0072-YC)

Figure 1 Monoclonal ELISA to identify E. coli clones producing Fab-pIII fusion molecules specific for IL-15 after L chain shuffling.

A. After two rounds of solution-phase sorting, 96 E. coli clones were randomly selected, and monoclonal ELISA was performed to identify specific binders. More than 60% of the Fab clones tested showed discrete binding to MBP-IL-15 but not to MBP only. B. After the second round of biopanning 48 E. coli colonies were randomly selected, and a monoclonal ELISA was performed. The ELISA results revealed that 21 clones showed discrete binding to MBP-IL-15 but not to MBP.

Hur, B. U., Choi, H. J., Song, S. Y., Yoon, J. B., Liu, L. K., & Cha, S. H. (2010). Development of the dual-vector system-III (DVS-III), which facilitates affinity maturation of a Fab antibody via light chain shuffling. Immunology letters, 132(1-2), 24-30.

Figure 2 Effects on initial absorbance by adding increasing concentrations of NH4SCN into the anti-IL-15 ELISA.

The affinity index of the parental 4H10 turned out to be 0.96 M, and all of the 4H10 variants showed higher affinity indices than that of 4H10. The affinity index of the 4H10-LS was 1.96 M, and 5 Fab clones isolated by biopanning seemed to have higher affinities than that of the 4H10-LS. Notably, the affinity index of the 4H10-LP4 was higher than 4 M, implying that it had the highest affinity of all of the Fab clones.

Hur, B. U., Choi, H. J., Song, S. Y., Yoon, J. B., Liu, L. K., & Cha, S. H. (2010). Development of the dual-vector system-III (DVS-III), which facilitates affinity maturation of a Fab antibody via light chain shuffling. Immunology letters, 132(1-2), 24-30.

Figure 3 Competitive inhibitory ELISA for estimating affinities of anti-IL-15 Fabs.

Culture supernatants containing soluble Fab molecules were harvested from E. coli clones expressing 4H10 or 4H10-LP4, respectively, and pre-incubated with 10⁻⁶ to 10⁻¹⁰ M concentration of MBP-IL-15. Standard ELISA was then performed using microtiter plates coated with MBP-IL-15 antigen. Goat anti-human kappa Ab-conjugated with HRPO was used as a secondary antibody.

Hur, B. U., Choi, H. J., Song, S. Y., Yoon, J. B., Liu, L. K., & Cha, S. H. (2010). Development of the dual-vector system-III (DVS-III), which facilitates affinity maturation of a Fab antibody via light chain shuffling. Immunology letters, 132(1-2), 24-30.