A method to identify antibodies against peptide–MHC complexes could enable patient-specific cancer immunotherapeutics
Before each round of selection, a small sample of yeast was stained with tetramer pMHC and an anti-human influenza hemagglutinin tag 6E2 antibody AlexaFluor 488 conjugate for 1 h on ice to confirm the surface expression of the scFv-Aga2 library. For tetramer pMHC staining, biotinylated target pMHCs were incubated with SA coupled to AlexaFluor 647 . After washing of cells twice with PBS-M , they were analyzed using an Accuri C6 flow cytometer .
High Five cells were cultured in Insect X-press medium with gentamicin sulfate . SF9 cells were cultured in SF900-II serum-free medium containing 10% fetal bovine serum , gentamicin sulfate and 2 mM GlutaMAX at 27 °C. Each expression vector for mouse Fab, BiTE, human scFv and human Fab was cotransfected into SF9 cells with BestBac 2.0 and FuGene to create P0 viruses in 2-ml cultures. P1 viruses were used to infect 1–2-l volumes of High Five cells at 2 × 10.
Fluorescence was detected using a spectral image splitter with a dichroic beam splitter combined with bandpass filter 600/37 for detection of Rho11, and filter 690/70 for detection of ATTO643, dividing each emission channel into 256 × 512 pixels. Image stacks of 150 frames were recorded for each cell at a time resolution of 32 ms per frame.
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