Figure 3. Characterization of anti-TNF-alpha antibody.
(A) Binding affinity of anti-mouse-TNF-alpha antibody. The equilibrium constant of anti-mTNF-alpha antibody 9C6 was measured by Surface Plasmon Resonance (SPR) using a CM5 sensor chip in a BIACORE3000 at 25°C. Recombinant mouse-TNF-alpha was captured in the flow cell. Running buffer and different concentrations of anti-mTNFa mAb 9C6 (0.64, 1.28, 2.56, 5.12, 10.24, and 20.48 µM diluted in running buffer) were applied to the antigen-containing flow cell for 3 min at a flow rate of 30 µl/min. The Kd of 9C6 was calculated as 1.4 nM. (B) Anti-mTNF-alpha mAb 9C6 neutralizes TNF-alpha-induced cytotoxicity in L929 cells. 3.5×105 L929 cells were treated with 1 µg/ml actinomycin D and 3 ng/ml mouse TNF-alpha for 15 hours. Different dilutions of anti-mTNFa mAb 9C6 (with a starting concentration of 18 µg/ml) were added to the assay. Cell death was measured by the MTT assay (Thiazolyl Blue Tetrazolium Bromide staining, read at 570 nm) [20]. (C) Anti-TNF-α mAb 9C6 protects mice from LPS-induced sepsis. 8 week-old C57BL/6 female mice, 8 mice/group, were injected intraperitoneally with 25 mg/kg LPS, along with 2.5 mg/kg anti-TNF-α mAb 9C6 or isotype control antibody. The number of surviving mice was recorded every six hours.