Journal: Frontiers in Immunology

Article Title: Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species

doi: 10.3389/fimmu.2020.612402

Figure Lengend Snippet: Direct and sandwich ELISA to determine mAb binding to human, monkey, rat and mouse C7. (A) Direct ELISA: plates were coated with human, monkey (cyno), rat or mouse C7; mAbs were tested in a dilution series (0–10 µg/ml). The graph shows representative binding with the 10 µg/ml capture. (B) Sandwich ELISA: the new mAbs were paired as capture and detect with human C7 used in a dilution series (0–5 µg/ml). The graph shows representative binding with 5 µg/ml C7. As a positive control, polyclonal anti-C7 (goat anti-C7) was used as capture with each of the mAb as detect; rat C7 captured on goat anti-C7 (goat anti-C7; rat C7) was also tested with the mAb as detect. (C) Sandwich ELISA to detect pre-formed TCC in activated NHS; TCC in activated NHS (and NRS for 2H2) was captured on aE11 anti-C9 and the novel mAbs and the positive control E2 anti-C8 mAb used to detect the TCC complex captured. (D) TCC complexes generated in NHS (or NRS for 2H2) in the presence of each of the new mAb (biotinylated) were captured on avidin-coated plates, then polyclonal antibodies against each of the terminal complement proteins used to test the presence of the respective components in the complex. The error bars are standard errors of triplicates. All experiments were repeated three times with the same results.

Article Snippet: Binding of the mAbs to pre-formed TCC was tested in ELISA; serum activated as above in the absence of mAbs (1 in 50 dilution in 0.2% BSA-PBS-T) was transferred to plates coated with aE11 anti-C9 neo-specific antibody (5 µg/ml, Hycult Biotech, # HM2167), incubated 1 h at 37°C and blocked with 2% BSA (1 h at 37°C); after washing, the new biotinylated mAb or as positive control, an in house mAb known to bind aE11-captured TCC (E2 anti-C8; 1 in 1000 dilution in 0.2% BSA-PBS-T) were added and incubated for 1 h at 37°C, after washing, Streptavidin-HRP (1 in 5000 dilution in 0.2% BSA-PBS-T, Fisher Scientific, # 21130) was added, plates incubated 1 h at 37°C and the assay developed as described above.

Techniques: Sandwich ELISA, Binding Assay, Direct ELISA, Positive Control, Generated, Avidin-Biotin Assay