Cytotox one substrate reagent

Example 10

In order to demonstrate whether bispecific HBV×CD3 IgM binding molecules can kill target cells in the presence of CD8+ T-cell acute lymphoblastic leukemia (TALL) cells, co-culture experiments can be performed. HBsAg-expressing cells (about 6×10³ cells), e.g., hepatocellular carcinoma cells such as Alexander hepatoma cells (PLC/PRF/5; ATCC CRL-8024) or recombinant cells expressing HBsAg-L, are co-cultured with 3×10⁴ TALL cells (ATCC CRL-11386) in the presence of different concentrations of test compounds in 45 μL total volume of RPMI 1640 media supplemented with 10% heat-inactivated FBS per well on a 384-well black tissue culture plate. After 24 hours of incubation at 37° C. in a 5% CO₂ incubator, 15 μL of CytoTox-ONE substrate reagent (Promega, G7891) is added to each well to measure the level of LDH released from dead cells. The plates are shaken briefly to mix the reagents, and then incubated at room temperature for 90 min before measuring fluorescence signal (485 nm for excitation and 615 nm for emission) on an EnVision plate reader (Perkin-Elmer). The data is then analyzed with GraphPad Prism to determine the EC₅₀.

The breadth and scope of the disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined in accordance with the following claims and their equivalents.

Example 10

In order to demonstrate whether bispecific HBV×CD3 IgM binding molecules can kill target cells in the presence of CD8+ T-cell acute lymphoblastic leukemia (TALL) cells, co-culture experiments can be performed. HBsAg-expressing cells (about 6×10³ cells), e.g., hepatocellular carcinoma cells such as Alexander hepatoma cells (PLC/PRF/5; ATCC CRL-8024) or recombinant cells expressing HBsAg-L, are co-cultured with 3×10⁴ TALL cells (ATCC CRL-11386) in the presence of different concentrations of test compounds in 45 μL total volume of RPMI 1640 media supplemented with 10% heat-inactivated FBS per well on a 384-well black tissue culture plate. After 24 hours of incubation at 37° C. in a 5% CO₂ incubator, 15 μL of CytoTox-ONE substrate reagent (Promega, G7891) is added to each well to measure the level of LDH released from dead cells. The plates are shaken briefly to mix the reagents, and then incubated at room temperature for 90 min before measuring fluorescence signal (485 nm for excitation and 615 nm for emission) on an EnVision plate reader (Perkin-Elmer). The data is then analyzed with GraphPad Prism to determine the EC₅₀.

The breadth and scope of the disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined in accordance with the following claims and their equivalents.