Differential scanning fluorimetry as a measure of functionality of refolded anti-malarial antibody fragments

Abstract

AbstractTransmission blocking monoclonal antibodies, 4B7 and 1245, targeting Pfs25, have been demonstrated to block the developmental stages of the malarial parasite inside the mosquitoes. In this work, anti-Plasmodium antibody fragments of 4B7 and 1245, designated as diabodies, were expressed in bacteria and refolded using a standardized method, with a yield of 0.1 and 0.4 mg per litre of culture, respectively. Dimeric species was detected for 4B7-Db, but not for 1245-Db, using glutaraldehyde cross-linking experiment. The quality of the purified refolded fraction was assessed with differential scanning fluorimetry (DSF). Refolded 4B7-Db, with a melting temperature of 59°C, recognized Pfs25 expressed on the surface of ookinete and zygote stages of Plasmodium in an immunofluorescence-based assay, whereas, 1245-Db, exhibiting a skewed melt profile, showed weak recognition. Further, refolded 4B7-Db recognized the linear epitope, on purified Pfs25 protein, both in the denatured and native state. Differential scanning fluorimetry can be potentially employed as a qualitative measure of functionality, to evaluate refolded proteins, with applicability for antibody engineering in passive immunization.