Example 19
For CTLA-4, a commercially available recombinant CTLA-4 (R&D systems) at 100 ng/100 μL/well was immobilized on an ELISA plate (Nunc) at room temperature for 1 hour. After washing, the plate was blocked with Block Ace at room temperature for 1 hour. The periplasm fractions of wild-type CTLA4-3-1/its modified antibody were diluted to 100 μL/well with Block Ace and reacted at 37° C. for 1 hour. Detection was performed with a combination of biotin-labeled anti-Kappa Ab (Southern Biotechnology) and peroxidase (HRP)-labeled streptavidin (Vector Lab.). Absorbance at 650 nm/450 nm was measured with Microplate Reader Vmax (Molecular Devices).