Figure 1
From: Facile Discovery of a Diverse Panel of Anti-Ebola Virus Antibodies by Immune Repertoire Mining
Isolation of antibodies by mining the paired VH:VL repertoire of draining popliteal lymph node (PLN) antibody-secreting B cells.
(a) Footpad immunization leads to a marked increase in cellularity within the ipsilateral popliteal lymph node relative to the contralateral lymph node (red and black arrows respectively). (b) PLN CD138+ cells isolated by magnetic sorting are deposited into 125 pL wells on PDMS slides that also contain poly(dT) beads. Cells are lysed in situ and mRNA is captured on the poly(dT) beads33. (c) The poly(dT) beads are emulsified and VH:VL amplicons are generated following reverse transcription and overlap extension PCR. (d) VH:VL amplicons are sequenced using Illumina 2 × 250 MiSeq and the highest frequency VH:VL pairs are identified via bioinformatics analysis. (e) Highest frequency VH (orange) and VL (green) genes are synthesized and cloned into IgH and IgL expression vectors containing human IgG1 (blue) and human kappa (yellow) constant regions, respectively. (f) Following co-transfection into Expi293 cells, recombinant IgG antibodies are expressed and purified.
