Anti-human IL20 Recombinant Antibody (7D1) (TAB-563LC) (CAT#: TAB-563LC)

Figure 1 ELISA determination of binding of exemplary anti-IL-20 antibodies to IL-20 constructs fused either N-terminally (gl IL-20) or C-terminally (g2 IL-20) to Gaussia luciferase.

A: 2A11; B: 6E11; C: 6H2; D: 7D1; E: 20A10; F: A non-IL20 specific monoclonal antibody is used as control for non-specific binding. Antibodies 2A11, 6E11 and 20A10 bind with a high affinity to both IL-20 constructs, in contrast remaining antibodies 6H2 and 7D1 bind with a lower affinity to g2 IL-20.

Figure 2 Human-derived anti-IL-20 monoclonal antibodies neutralize recombinant IL-20-mediated STAT3 activation in HEK 293T MSR cells transiently expressing Type I and Type II IL-20 receptors.

Either cells were left untreated or stimulated with recombinant human or murine IL-20 in the absence of antibodies or in the presence of human-derived IL-20 monoclonal antibodies or a human control IgG as indicated (hulgG). Cell lysates were subjected to SDS-PAGE and pSTAT3 levels were visualized in Western blots. Total STAT3, alpha-tubulin and IL-20RB levels serve as loading control. Antibody concentration: 5 μg/ml. A: Control: rhIL-20 and rmlL-20 induce the dose-dependent phosphorylation of STAT3 in HEK 293T MSR cells transiently expressing Type I or Type II IL-20 receptors. Detection of total and phosphorylated STAT3 and IL-20RB-Myc-DDK levels. B: Stimulation with rhIL-20 (10 ng/ml) or rmIL-20 (25 ng/ml). Exemplary antibodies 2A11, 20A10 and 7D1 neutralize rhIL-20, whereas only 20A10 also potently neutralizes rmIL-20.

Figure 3 Human-derived anti-IL-20 monoclonal antibodies neutralize rhIL-20-mediated induction of KZ136-NLuc reporter constructs in HEK 293T MSR cells transiently transfected with IL-20 receptors and KZ136-NLuc.

Cells were stimulated with 120 ng/ml rhIL-20 or 200 ng/ml rmIL-20 in the presence of human-derived IL-20 monoclonal antibodies or of a control human IgG (hulgG) as indicated. rhlL-20 IC50analysis of exemplary antibodies 20A10, 7D1 and 2A11.

Figure 4 IC50analysis of human-derived anti-IL-20 monoclonal antibodies in the chemiluminescent cellular binding assay.

HEK 293T MSR cells transiently expressing Type I or Type II IL-20 receptors were incubated with gl IL-20 supernatants in the presence of human-derived IL-20 monoclonal antibodies, A: 20A10, B: 7D1 and C: 2A11. D: IC50 values of exemplary antibodies 20A10, 7D1 and 2Al l.

Figure 5 Cross-competition

Experimental setup: 96 well microplates (Costar, USA) were coated with Rabbit-anti-GLuc specific antibody diluted 1/250 in PBS O/N at 4 °C. Plates were washed with PBS-T and blocked lh at room temperature with PBS containing 2 % BSA (Sigma, Buchs, Switzerland). 30 μL GLuc-IL-20 was added at a final concentration of 2x106 LU/well and incubated for 2h at room temperature. The chimeric antibodies are premixed with the human competitor antibodies at a final concentration of 0.5 μg/ml vs. 3 μg/ml in 30 μL PBS and added to the plates. Upon incubation for 2 h at room temperature the plates were washed with PBS-T and the binding of the human-mouse chimeric antibodies was determined using a horseradish peroxidase conjugated goat anti-mouse IgG Fc-gamma specific antibody (Jackson Immuno Research, 1 :500 in 0.5 % BSA-PBS) followed by measurement of the HRP activity using a TMB substrate solution (Sigma, Buchs, Switzerland). The differential binding of exemplary human-derived anti-IL-20 monoclonal antibodies with human-mouse (hm) chimeric constructs to distinct binding sites was investigated in cross-competition experiments with 7D1 hm.

Figure 6 SPR analysis

Detailed analysis of the sensograms concerning the binding of human and mouse IL-20 to exemplary antibodies. A 1 : 1 binding kinetic was observed. The antigens were injected in concentrations of 1.5 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. Calculated Affinities (KD values [M]) are indicated in the diagrams.

Figure 7 Mapping of the epitopes of the human anti-IL-20 antibodies.

Binding of antibodies to full length antigens coupled to the microarray. The Y-axis indicated the fluorescence intensity (RFU) upon detection with a Cy5-conjugated secondary antibody.

Figure 8 Mapping of the epitopes of the human anti-IL-20 antibodies.

Primary peptide array of 18mer peptides of human IL-20 against all antibodies

Figure 9 Mapping of the epitopes of the human anti-IL-20 antibodies.

Primary peptide array of 18mer peptides of murine IL-20 against all antibodies.