Journal: Scientific Reports
Article Title: Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients
doi: 10.1038/s41598-024-66558-0
Figure Lengend Snippet: Schematic representation of scFvs library construction and IACT screening ( A ). Co-immunoprecipitation of Nucleocapsid full length protein with scFv anti Nucleocapsid in yeast ( B ) and in HEK 293 T cells ( C ). ( A ) Blood from patients recovered from Covid-19 was collected and tested for the presence of neutralizing antibodies. scFvs libraries were constructed from the RNA isolated from their peripheral blood mononuclear cells and sequenced. On the left panel, the schematic representation of the Single Pot Library of Intracellular Antibodies (SPLINT) library construction is shown. SPLINT libraries are then used in the Intracellular Antibody Capture Technology (IACT), schematically shown on the right panel. The bait and prey plasmids are co-expressed in yeast cells. In those cells in which bait and prey interact, his3 and lacZ genes are activated and these events allow selection. ( B ) Extracts from yeast strains expressing both the LexA-N-FL-HA protein (with LexA at the N-terminus and HA tag at the C-terminus) (69 KDa) and each of the anti N scFvs-VP16 (with VP16 at the C terminus) (#5, 77, 93, 255, 291, 24, 66, 220, 261, 336) or an unrelated scFv (#58F, anti AcK9H358 ) (control) have been immunoprecipitated with anti HA antibody (top panels). Co-immunoprecipitated scFvs (~ 38.5 KDa) were detected with the anti VP16 antibody (bottom panels). ( C ) HEK 293 T cell extracts from cells expressing both the Myc-N-FL protein (with the Myc tag at the N-terminus) (46.8 KDa) and the individual scFvs-HA (with the HA tag at the C-terminus) (# 24, 66, 77, 93, 255, 291, 336 or an unrelated scFv (#58F, anti AcK9H358) used as negative control) have been immunoprecipitated with anti HA antibody (~ 28 KDa) (bottom panel). The co-immunoprecipitated Nucleocapsid protein was detected by the anti Myc antibody (top panel). Uncropped blots are presented in the Supplementary material section “Images of the original blots”.
Article Snippet: PBMC were isolated by density gradient following Lymphoprep protocol (Stemcell Technologies, # 07801) and then 10 7 aliquots were frozen in Trizol (Thermo Fisher Scientific, # 15596026) at − 80 °C until use.
Techniques: Immunoprecipitation, Construct, Isolation, Selection, Expressing, Control, Negative Control
