Journal: Antibody therapeutics

Article Title: Characterization of HZ0412a, a novel potent humanized anti-IL-6 receptor antibody that blocks IL-6R binding to gp130.

doi: 10.1093/abt/tbad008

Figure Lengend Snippet: Figure 1. Characterization of humanized antibody HZ0412a and its variants. (A) Concentration-dependent binding of IL-6R antibodies to rhIL-6. Antibodies at different concentrations were incubated with rhIL-6-mFc fusion proteins that had been coated in 96-well plates. After extensive washing, the amount of antibodies retained in the wells was measured by ELISA. (B) Measurement of HZ0412a-sIL-6R interaction by MP. Typical mass distribution plots of 30 nM HZ0412a with sIL-6R-Fc, at different concentrations (0, 7.5, 15, 30 and 60 nM) are shown. KD was calculated according to the mass distribution plot of Ab (30 nM)/Ag (30 nM) reaction. (C) HZ0412a and its variants do not significantly affect the binding of rhIL-6 to sIL-6R. sIL- 6R-mFc fusion proteins were incubated with surface-coated rhIL-6-His fusion proteins in 96-well plates in the presence of different concentrations of antibodies. After extensive washing, the amount of sIL-6R-mFc fusion proteins retained in the wells were measured by ELISA. (D) HZ0412a and its variants block the binding of IL-6R to gp130 in a concentration-dependent manner. 96-well plates were first coated with purified recombinant gp130-His fusion proteins. Then, rhIL-6-hFc, sIL-6R-mFc and antibodies at various concentrations were added to the wells. After incubation and strict wash, the amount of sIL-6R-mFc fusion proteins retained in the wells were measured by ELISA. All ELISA results are expressed as the absorbance at 450 nm. Tocilizumab was used as a positive control and included in all experiments, which were repeated at least three times. Mean absorbance at 450 nm and SD values are presented.

Article Snippet: Antigen-binding by anti-IL-6R antibodies was measured by using an ELISA kit from R&D Systems (Cat# DY227).

Techniques: Concentration Assay, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Purification, Recombinant, Positive Control

Journal: Antibody therapeutics

Article Title: Characterization of HZ0412a, a novel potent humanized anti-IL-6 receptor antibody that blocks IL-6R binding to gp130.

doi: 10.1093/abt/tbad008

Figure Lengend Snippet: Figure 2. IL-6 signaling is blocked by HZ0412a and its variants in vitro. (A) All humanized IL-6R antibodies inhibited IL-6-induced phosphorylation of STAT3 in DLD-1 cells in a concentration-dependent manner. Cells were stimulated with rhIL-6 and the phosphorylation of STAT3 at Tyr705 was detected by a phosphorylation site–specific antibody as described in section Materials and Methods. A representative western blot result is shown. β actin was chosen to monitor equal loading of samples. (B) Summary of phosphorylation inhibition experiments. The specific phosphorylation signals on western blot were quantified and normalized with corresponding β actin signals. The normalized signals in the absence of IL-6R antibodies are used as a reference (100%), and the degrees of signal reduction are plotted against the concentrations of antibodies. (C) The inhibition of IL-6/IL-1β-induced SAA secretion by IL-6R antibodies in Hep G2 cells is concentration-dependent. Hep G2 cells (2 × 105) were stimulated with IL-6 and IL-1β with or without antibodies as described in section Materials and Methods. The supernatant was collected for the measurement of SAA by ELISA. (D) IL-6-induced DS-1 cell growth can be suppressed by IL-6R antibodies in a concentration-dependent manner. To quantify the DS-1 cell proliferation, the Cell Counting Kit-8 (CCK8) was used, and the results are expressed as mean absorbance at 450 nm with SD values. Tocilizumab was included as a positive control. All experiments were repeated at least three times.

Article Snippet: Antigen-binding by anti-IL-6R antibodies was measured by using an ELISA kit from R&D Systems (Cat# DY227).

Techniques: In Vitro, Phospho-proteomics, Concentration Assay, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay, Cell Counting, Positive Control

Journal: Journal of Clinical Medicine

Article Title: Characteristics of Interleukin-6 Signaling in Elective Cardiac Surgery—A Prospective Cohort Study

doi: 10.3390/jcm11030590

Figure Lengend Snippet: Exaggeration of IL-6, sIL-6R and sgp130. The figure displays individual time course ( left ) and corresponding boxplots ( right ) of perioperative levels of IL-6, sIL-6R and sgp130 levels. The lines on the left are displayed on a a logarithmic scale. Asterisks (*) mark significant differences compared to baseline at p less than 0.05. In the boxplots, the lower boundary of the box indicates the 25th percentile, a black line within the box marks the median, and the upper boundary of the box indicates the 75th percentile. Whiskers above and below the box indicate the 10th and 90th percentiles. Points (°) above and below the whiskers indicate outliers outside the 10th and 90th percentiles (IL-6 outliers above 1200 pg/mL are not shown). Red, blue and green lines indicate IL-6, sIL-6R and sgp130 levels, respectively. Abbreviations: CPB, cardiopulmonary bypass; POD, postoperative day; sgp130, soluble glycoprotein 130; sIL-6R, soluble interleukin-6 receptor.

Article Snippet: Quantitative measurements of IL-6, sIL-6R, and sgp130 concentrations were conducted with 96-well plate ELISAs (Human IL-6 Quantikine ELISA Kit, Human IL-6R alpha Quantikine ELISA Kit and Human soluble gp130 Quantikine ELISA Kit, R&D Systems ® Inc., Minneapolis, MN, USA), according to the manufacturer’s protocol.

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Characteristics of Interleukin-6 Signaling in Elective Cardiac Surgery—A Prospective Cohort Study

doi: 10.3390/jcm11030590

Figure Lengend Snippet: sIL-6/IL-6 and sIL-6R/sgp130 ratio. The lines show the perioperative levels of ( a ) sIL-6/IL-6 and ( b ) sIL-6R/sgp130 ratio at different time points. Red lines indicate patients with continuously lowest levels, blue lines indicate patients with continuously highest levels and grey lines indicate patients with intermediate levels of perioperative sIL6-R/IL-6 ratio measured between timepoint 3 and 9. Abbreviations: CPB, cardiopulmonary bypass; POD, postoperative day; sgp130, soluble glycoprotein 130; sIL-6R, soluble interleukin-6 receptor.

Article Snippet: Quantitative measurements of IL-6, sIL-6R, and sgp130 concentrations were conducted with 96-well plate ELISAs (Human IL-6 Quantikine ELISA Kit, Human IL-6R alpha Quantikine ELISA Kit and Human soluble gp130 Quantikine ELISA Kit, R&D Systems ® Inc., Minneapolis, MN, USA), according to the manufacturer’s protocol.

Techniques: