Bioassay of 1-11E/vIL-10. (A) ELISA showing increased binding of 1-11E/vIL-10 to ROS-CII (CII modified by glycation (Gly) or HOCl) compared with native CII (NT-CII). No binding to hen-egg lysozyme (HEL) was observed. In contrast, C7/vIL-10 bound only to HEL. (B) Western blot analysis showed that 1-11E/vIL10 bound to native CII (Lane 1), ROS modified CII (lane 2, glycated; lane 3, HOCl; lane 4, OH-; lane 5, peroxynitrate) but not to HEL (lane 6). (C). IL-10 bioassay of 1-11E/vIL10 and C7/vIL10. We added 10, 100, or 1,000 ng/ml of fusion protein (white, pattern, and black boxes, respectively) to IL-10-responsive MC-9 cells with (+) or without (-) previous MMP-1 digestion. As a positive control, 10, 100, or 1,000 ng/ml commercial recombinant vIL-10 (vIL-10) was used, whereas MMP-1 alone-treated cells were used as a negative control. After 3 days, cell growth was measured with the Cell Titer Glo assay. Significant enhanced cell growth of the fusion proteins was observed after MMP-1 digestion (P < 0.01 for 1,000 ng/ml). No significant difference in growth was seen between 1-11E/vIL-10, C7/vIL-10, and vIL-10 (P > 0.05).