NeuroMab™ Anti-CD282 Antibody(NRP-0422-P735)

Figure 1 Inhibition of host activation by microbial challenge in vivo by mAb T2.5.

Mice were pretreated intraperitoneally with 1 mg mAb T2.5 (solid bars) or not (open bars). Mice were challenged with P3CSK4 and D-galactosamine (ip) 1 h later and sacrificed after 2 or 4 h (n = 4 per group per time point). Serum concentrations of TNFα (a), GROα/KC (human IL-8 homologue) (b), IL-6 (c) and IL-12p40 (d) were analyzed by ELISA (*p<0.05, **p<0.005 , ***p<0.001, Student's t-test for unconnected samples).

Figure 2 Effect of mAb T2.5 administration on viability after TLR2-specific systemic challenge.

Mice were pretreated intraperitoneally with 1 mg mAb T2.5 (solid bars) or not (open bars). Mice were challenged with P3CSK4 and D-galactosamine (ip) 1 h later and sacrificed after 2 or 4 h (n = 4 per group per time point). Serum concentrations of TNFα (a), GROα/KC (human IL-8 homologue) (b), IL-6 (c) and IL-12p40 (d) were analyzed by ELISA (*p<0.05, **p<0.005 , ***p<0.001, Student's t-test for unconnected samples).

Figure 3 Dose-dependent binding of T2.5 to mTLR2ECD and failed binding of TL2.1 to the same antigen in ELISA.

Mice were pretreated intraperitoneally with 1 mg mAb T2.5 (solid bars) or not (open bars). Mice were challenged with P3CSK4 and D-galactosamine (ip) 1 h later and sacrificed after 2 or 4 h (n = 4 per group per time point). Serum concentrations of TNFα (a), GROα/KC (human IL-8 homologue) (b), IL-6 (c) and IL-12p40 (d) were analyzed by ELISA (*p<0.05, **p<0.005 , ***p<0.001, Student's t-test for unconnected samples).

Figure 4 Lack of interaction between TL2.1 and mouse TLR2 in immunoprecipitation and subsequent western blot analysis.

Lysates of 1 × 106 mouse RAW264.7 macrophages or HEK293 cells, 1 μg of each antibody indicated and 20 μl of protein G beads (Santa Cruz, CA, USA) were mixed for pelleting. Immune complexes were analyzed by immunoblot analysis using Flag or murine TLR2-specific antisera.

Figure 5 Inhibition of lipopeptide-induced cell activation by recombinant single-chain and partially humanized hT2.5 antibodies. HT2.5 was overexpressed in HEK293 cells and the supernatant was collected.

Lysates of 1 × 106 mouse RAW264.7 macrophages or HEK293 cells, 1 μg of each antibody indicated and 20 μl of protein G beads (Santa Cruz, CA, USA) were mixed for pelleting. Immune complexes were analyzed by immunoblot analysis using Flag or murine TLR2-specific antisera.

Figure 6 Application of mAb T2.5 in TLR2 specific detection.

Lysates of 1 × 106 mouse RAW264.7 macrophages or HEK293 cells, 1 μg of each antibody indicated and 20 μl of protein G beads (Santa Cruz, CA, USA) were mixed for pelleting. Immune complexes were analyzed by immunoblot analysis using Flag or murine TLR2-specific antisera.