Figure 4.

Specificity and in vivo immunotoxicity. (a) The specificity of r-D3E8-KE against analogues of IL-6. Human leukemia inhibitory factor (LIF), human oncostatin M (OSM), human IL-11, mouse IL-6 (mIL-6), and bovine serum albumin (BSA) were used (Sigma). Each protein (2 μg/ml) was coated on a 96-well plate, and sequentially reacted with a purified phage (10¹¹ pfu/ml) and HRP-conjugated anti-g8p monoclonal antibody, and absorbance was measured at 450 nm. Error bars indicate a deviation in the triplicate experiments. (b) Humoral immune responses in BALB/C mice. The mice were immunized at each route (i.v. or i.p.) on day 0, 14, and 28, and bled on day 38. The level of IgG specific for r-D3E8-KE or ovalbumin (OVA) was measured using ELISA. The values indicate the means of optical density (OD) and standard deviation at 450 nm. Each group contained five mice. (c) Cytokine responses against r-D3E8-KE in tumor-bearing mice. H1650 cells were subcutaneously injected into the flanks of BALB/c mice, as described in the Methods section. Nude mice with H1650 xenografts were administered intraperitoneally. A 10 mg/kg dose of r-D3E8-KE was given to each mouse every third day for 30 days. The serum cytokine levels were determined at 30 days after administration. The control group was treated identically but given vehicle injections only (PBS). Each group contained ten mice.