Journal: bioRxiv
Article Title: Non-human primate LIBRA-Seq accelerates neutralizing antibody discovery in RM vaccinated against HIV-1
doi: 10.64898/2025.12.19.695353
Figure Lengend Snippet: ( A ) Schematic of the LIBRA-seq approach. Fluorophore and DNA oligo conjugated streptavidin are bound to biotinylated HIV-1 Env to create LIBRA-Seq probes. HIV-1 Env specific memory B cells bound to these probes are enriched via FACS prior to 10x capture. DNA libraries are generated from captured RNA and antigen barcodes. Bioinformatic analysis reveals binding profiles of individual memory B cells based on associated antigen barcodes and VDJ sequences. These profiles are used to prioritize clones for downstream functional characterization. (Created with Biorender.com ) ( B ) StvC-AF-Oligo conjugates stain similarly to control Stv-AF. Streptavidin with N-terminal cysteine (StvC) was conjugated to maleimide modified alexa fluorophore (AF) at 10:1 ratio and excess removed by spin column purification before conjugating to amine modified DNA by hydrazone chemistry. StvC-AF-Oligo conjugates were purified from free StvC-AF and DNA by size exclusion chromatography. ( C ) Gel electrophoresis of StvC-AF-Oligo conjugates stained with Coomassie Blue (left) and SYBR DNA Gold (right). ( D ) Staining pmel splenocytes for Gp100-specific T cells with Db-Gp100-tetramers made from various streptavidin conjugates.
Article Snippet: Streptavidin was reduced with 50 molar excess tris(2-carboxyethyl)phosphine (TCEP) for 30 min at 23° C. Alexa fluorophores (AF) 488, 546, and 647 with maleimide group (Thermo Fisher) were dissolved in dimethyl sulfoxide (DMSO) and added at 10 molar excess to streptavidin for 2 hours at 23° C. Excess AF was removed through size exclusion spin columns (BIORAD 7326227) into spin buffer (150 mM NaCl, 100 mM Sodium Phosphate pH 6.5) according to manufacturer instructions.
Techniques: Generated, Binding Assay, Clone Assay, Functional Assay, Staining, Control, Modification, Purification, Size-exclusion Chromatography, Nucleic Acid Electrophoresis
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