Generation of anti-RH5 and anti-CyRPA recombinant and chimeric mAbs (Table S1) has been previously described 16, 36, 37, 43, 57 . These mAbs were transiently expressed in Expi293F HEK cells. Cognate heavy and light chain-coding plasmids were co-transfected at a 1:1 ratio. Supernatants were harvested via centrifugation. All mAbs were purified using a 5 mL
Protein G HP column (Cytiva) on an
ÄKTA Pure FPLC system (Cytiva). Equilibration and wash steps were performed with PBS and mAbs were eluted in 0.1 M glycine pH 2.7. The eluates were pH equilibrated to 7.4 using 1.0 M Tris HCl pH 9.0 and immediately buffer exchanged into DPBS and concentrated using an
Amicon ultra centrifugal concentrator (Millipore) with a molecular weight cut-off of 30 kDa.
Total IgG from rat serum was purified on drip columns packed with
Pierce Protein G agarose resin (Thermo Fisher Scientific). Pierce Protein G IgG binding buffer (Thermo Fisher Scientific) was used to dilute the serum 1:1 before loading as well as for equilibration and wash steps. Bound IgG was subsequently eluted, neutralised and concentrated as for mAbs above.
Williams B.G., King L.D.W., Pulido D., Quinkert D., Lias A.M., Silk S.E., Ragotte R.J., Davies H., Barrett J.R., McHugh K., Rigby C.A., Alanine D.G.W., Barfod L., Shea M.W., Cowley L.A., Dabbs R.A., Pattinson D.J., Douglas A.D., Lyth O.R., Illingworth J.J., Jin J., Carnrot C., Kotraiah V., Christen J.M., Noe A.R., MacGill R.S., King C.R., Birkett A.J., Soisson L.A., Skinner K., Miura K., Long C.A., Higgins M.K, & Draper S.J. (2024). Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex. Nature communications, 15(1).
