Nano-differential scanning fluorimetry as a tool for the assessment of refolded antibody fragments: a case study for anti-Pfs25 single-chain antibodies

Differential Scanning Fluorimetry (DSF) is a valuable and versatile tool in the field of protein and antibody stability studies, providing valuable insights into their thermal stability, and aiding in the optimization of experimental conditions for various applications. Antibody fragments provide armamentarium for researchers and practitioners across various fields, enabling advancements in medicine, diagnostics, research, and industrial applications. In the present study, single-chain antibodies derived from mAb 4B7 and mAb 1245 were expressed in E. coli, refolded, and checked for their binding with the antigen - Pfs25 (transmission-blocking malaria vaccine candidate). We used nano-DSF as a valuable tool to assess thermal stability, consequently aiding in predicting the correct folding of single-chain antibodies.. Employing nano-DSF as a checkpoint enables the determination of whether to proceed with functional and binding studies on the refolded single-chain antibodies

Refolding and characterization of a diabody against Pfs25, a vaccine candidate of Plasmodium falciparum

Pfs25, a vaccine candidate, expressed on the surface of the malarial parasite, plays an important role in the development of Plasmodium falciparum. 1269, a monoclonal antibody targeting the epidermal growth factor-like domain 1 and epidermal growth factor-like domain 3 of Pfs25, blocks the transmission of parasites in mosquitoes. In this study, we refolded 1269-Db, a dimeric antibody fragment referred as diabody, designed from 1269, with a yield of 3 mg/litre of bacterial culture. Structural integrity of the protein was validated with thermal stability, disulphide bond analysis and glutaraldehyde crosslinking experiments. To evaluate the functionality of 1269-Db, recombinant monomeric MBP-Pfs25 was produced from bacteria. Qualitative binding assays demonstrated that 1269-Db recognized the epitopes on Pfs25 in its native, but not the denatured state. An apparent KD of 2.6 nM was determined for 1269-Db with monomeric MBP-Pfs25, using isothermal titration calorimetry. 1269-Db recognized the periphery of zygotes/ookinetes, demonstrating recognition of Pfs25, expressed on the surface of the parasite. As the established refolding method resulted in a functional diabody, the optimized method pipeline for 1269-Db can potentially facilitate engineering of antibody fragments with desired properties