Vmax microplate reader

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).

Example 10

The expressed and purified recombinant SEB at 200 ng/100 μL/well was immobilized on an ELISA plate (Nunc) at 4° C. overnight and, after washing, blocked with 1% BSA-PBS at 4° C. overnight. The periplasm fractions or the culture supernatant fractions of wild-type SEB3-2-7/its modified antibody were diluted to 100 μL/well with 1% BSA-PBS 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).

Example 23

In order to investigate whether Fd chain and L chain are assembled together to form a correct molecular form of Fab, the following sandwich ELISA system was utilized. Anti-c-myc tag Ab 9E10 at 200 ng/100 μL/well was immobilized on an ELISA plate (Nunc) at room temperature for 1 hour. After washing, the plate was blocked with 1% BSA-PBS at room temperature for 1 hour. Each of the fractions were diluted to 100 μL/well with 1% BSA-PBS and reacted at 37° C. for 1 hour. Detection was performed with HRP-labeled anti-E tag Ab. Absorbance at 650 nm/450 nm was measured with Microplate Reader Vmax (Molecular Devices).

As a result, it was found that, while no detection was observed for wild-type CTLA4-3-1 Fab-E, a concentration-dependent reaction was observed for P15R modified Fab-E with several ten times higher reaction for the periplasm fractions thereof (FIG. 12). These results confirmed that the P15R modification also highly increased an expression level of a complex of Fd chain and L chain.

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).

Example 10

The expressed and purified recombinant SEB at 200 ng/100 μL/well was immobilized on an ELISA plate (Nunc) at 4′C. overnight and, after washing, blocked with 1% BSA-PBS at 4° C. overnight. The periplasm fractions or the culture supernatant fractions of wild-type SEB3-2-7/its modified antibody were diluted to 100 μL/well with 1% BSA-PBS 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).

Example 14

For FasL, a commercially available recombinant Fas Ligand (R&D systems) with His-tag at 50 ng/100 μL/well was immobilized on an NI chelate plate (Nunc) at room temperature for 2 hours. The culture supernatant fractions of wild-type RNOK203/its modified antibody were diluted to 100 μL/well with Block Ace (Dainippon Pharma Co., Ltd.) 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).