Donkey anti sheep hrp conjugated antibody

To measure FH present in mouse plasma, ELISA plates (Nunc) were coated with monoclonal anti-FH (2A5) at 1 μg/ml. After overnight incubation at 4°C, plates were blocked with 1% w/v BSA at room temperature for 1 hour. After washing, plates were incubated with diluted mouse plasma (1:1000) for 1 hour at room temperature. Plates were washed and then incubated with sheep anti–mouse FH (1:5000, Abcam Ab8842) for 1 hour at room temperature and then detected by adding donkey anti-sheep HRP-conjugated antibody (1:5000, 713-035-147, lot 125113, Jackson ImmunoResearch Laboratories Inc. via Stratech Scientific) for 1 hour at room temperature. A standard curve was compiled using the known concentration of purified mouse CFH.

Mouse C3 was detected using goat anti-mC3 antibody (MP Biomedicals) in combination with an HRP-conjugated goat anti-mC3 polyclonal antibody (MP Biomedicals). Results were quantified using a standard curve generated from pooled mouse sera containing a known quantity of mC3 [in house, Imperial College London and as previously described (63 (link))]. For hFH levels, ELISA plates were coated with 2µg/ml of an anti-FH monoclonal antibody (OX24, Thermo scientific MA1-70057) in 0.2M carbonate buffer. After washing and blocking with PBS-1% BSA, mouse plasma (1:166,600) was added in PBS-0.1% Tween-20-1% BSA. After incubation and washing, plates were incubated with a polyclonal sheep anti-hFH antibody (1:20,000, Abcam) for 1 h. After washing, plates were incubated with a donkey anti-sheep HRP conjugated antibody (1:20,000, Jackson ImmunoResearch via Stratech,UK) for 1 h. After washing, enzymatic activity was developed using TMB substrate. Results were quantified using a standard curve generated using purified hFH (Complement Technologies) and interpolated from a four-parameter linear regression curve generated in GraphPad Prism9.