Human Anti-C5 Recombinant Antibody (clone SKY59/RO7112689) (CAT#: PABC-462)

Figure 1 Western blot (WB) of human C5.

C5 (1µg) was resolved on 7.5% PAGE under NR and R conditions and processed for WB with RO7112689 or goat anti-human C5. C5; NR; 190 kDa (intact C5), R; 75 kDa (C5β chain). Goat anti-human C5 detected the native C5 in NR conditions and both C5α (115 kDa) and C5β chains in R conditions, whereas RO7112689 detected C5 in NR conditions and only the C5 β chain in R conditions.

Zelek, W. M., Stott, M., Walters, D., Harris, C. L., & Morgan, B. P. (2018). Characterizing a pH‐switch anti‐C5 antibody as a tool for human and mouse complement C5 purification and cross‐species inhibition of classical and reactive lysis. Immunology, 155(3), 396-403.

Figure 2 Haemolysis assays.

A) Haemolysis assay on run-through fractions (each 4ml) collected from pooled NHS passed over the RO7112689 column; less than 10% lysis seen on each fraction; complete depletion of human C5. NHS (1:10) was used as the positive control (100% lysis). B) Repletion with C5; C5 sourced either from RO7112689 purification or a commercial source was added back to neat C5 -depleted serum (C5D) at 75 µg/ml; both C5 sources fully restored haemolytic activity but commercial C5 restored serum was less lytic; calculated CH50 values were: RO7112689 C5, 63.4 Units; commercial C5, 40.2 Units.

Zelek, W. M., Stott, M., Walters, D., Harris, C. L., & Morgan, B. P. (2018). Characterizing a pH‐switch anti‐C5 antibody as a tool for human and mouse complement C5 purification and cross‐species inhibition of classical and reactive lysis. Immunology, 155(3), 396-403.

Figure 3 ELISA for human and mouse C5.

A C5 sandwich ELISA was generated using RO7112689 as capture antibody and polyclonal anti -human C5 as detection. Standard curves were generated using either human C5 (A) or mouse C5 (B). NHS samples from 25 healthy donors and NMS samples from 11 male C57/Bl mice were measured (C).

Zelek, W. M., Stott, M., Walters, D., Harris, C. L., & Morgan, B. P. (2018). Characterizing a pH‐switch anti‐C5 antibody as a tool for human and mouse complement C5 purification and cross‐species inhibition of classical and reactive lysis. Immunology, 155(3), 396-403.

Figure 1 Anti-C5 Recombinant Antibody (PABC-462) in WB

Western blot analysis of PABC-462 was performed with Recombinant Human C5 Protein (His tag) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with PABC-462 (2 μg/mL) and HRP Goat Anti-human IgG (H&L) as a secondary antibody. Chemiluminescent detection was performed.
Lane 1: Reducing Antigen (0.1 μg)
Lane 2: Reducing Antigen (0.3 μg)
Lane 3: Reducing Antigen (0.6 μg)

Figure 2 Anti-C5 Recombinant Antibody (PABC-462) in WB

Western blot analysis of PABC-462 was performed with Recombinant Human C5 Protein (His tag) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with PABC-462 (2 μg/mL) and HRP Goat Anti-human IgG (H&L) as a secondary antibody. Chemiluminescent detection was performed.
Lane 1: Non-Reducing Antigen (0.1 μg)
Lane 2: Non-Reducing Antigen (0.3 μg)
Lane 3: Non-Reducing Antigen (0.6 μg)

Figure 3 Anti-C5 Recombinant Antibody (PABC-462) in DB

Dot blot analysis of PABC-462 was performed by coating with Recombinant Human C5 Protein (His tag). PABC-462 (2 μg/mL) were probed and HRP Goat Anti-human IgG (H&L) as a secondary antibody.