Anti m13 hrp

The plates with serial dilutions of the polyclonal phage from the three rounds were used for selecting random monoclonal scFv phage for each of the six anti-C1q fractions. The selection was on the basis of colony morphology. The selected clones were inoculated into 100 µL/well 2xTY-Amp in 96-well plates (Cell Well, Corning, Glendale, AZ, USA) and grown with shaking (190 revs/min) overnight at 37 ℃. The overnight cultures were transferred into a new 96-well plate by inoculation of 2 µL in 200 µL fresh 2xTY-Amp and cultivated for one hour. Then, 50 µL of 2xTY-Amp containing 10¹⁰ PFU of VCS-Ml3 was added to each well and the plate was incubated at 37 °C for 30 min without shaking. The cells were spun down by centrifugation at 3000 rev/min at 4 °C, resuspended in 200 µL/well 2xTY-Amp-Kan and grown overnight at 30 °C, 120 rev/min shaking. The supernatant containing monoclonal phage was tested for binding of antigen by immunoblot. Phage that displayed scFv were incubated with antigen-coated nitrocellulose membranes. Bound phage was detected by anti-M13-HRP (Sigma-Aldrich, Darmstadt, Germany) and DAB (diaminobenzidine, Sigma-Aldrich, St. Louis, MO, USA).

The live attenuated PPRV/N/75/1 vaccine strain was outsourced from Botswana Vaccine Institute, Botswana for immunisation of the alpaca. Whole killed PPRV antigen mixture generated from completely inactivated PPRV was obtained from The Pirbright Institute, United Kingdom for affinity selection of PPRV-reactive nanobodies. For enzyme-linked immunosorbent assay (ELISA) tests, rabbit anti-camel VHH antibody, goat anti-rabbit-horseradish peroxide (HRP, BioRad, Hercules, CA, USA), anti-M13-HRP, mouse anti-His tag antibody, and goat anti-mouse alkaline phosphatases (Sigma-Aldrich, St. Louis, MO, USA) were all provided by Vrije Universiteit Brussel, Brussels, Belgium and all were used according to the manufacturer’s instructions.