Treffer: Workflow for the production of mABs from patient-derived individual B-cells.

Wet lab work steps (ellipse-icons with headers) and data processing steps (yellow icons). Sample flow is depicted by blue arrows and information flow is depicted by yellow arrows. The experimental procedure starts with FACS sorting of individual B-cells. cDNA is generated from individual B-cells, and PCR1 and PCR2 serve to amplify antibody chains. Successfully amplified chains, evaluated by capillary electrophoresis ( cELE1 ), are sequenced ( SEQ1 ). Upon positive sequence evaluation with the aBASE software [ 23 ], specific primers are used for the final amplification of the specificity-determining chain parts ( PCR3 ). Amplification is quality-controlled by electrophoresis ( cELE2 ). The specificity-determining regions are cloned by Gibson Assembly ( GiAs ) into plasmids encoding the constant parts of the chains. Plasmids are amplified by transformed ( TRFO ) bacteria after plating ( PLA ) and picking ( PICK ) individual bacterial clones. Prior to quality control by sequencing ( SEQ2 ), amplified plasmids are isolated ( MINI ), and plasmid concentration is measured and adjusted ( ConcAdj ). Based on sequencing results, functional plasmids ( PLA ) are sorted. Matching plasmid pairs ( mAB Pla ) are prepared for transfection ( Traf ) into HEK cells. mABs produced and secreted by HEK cells are harvested ( mABs ), and mAB concentration is quantified ( quant ). Glycerol stocks ( Glyc ) of transformed bacteria and plasmid aliquots ( Pla safe ) serve as safe stocks of plasmids. The colors of the wet lab work step captions indicate different workflow sections: blue – molecular biology; red – microbiology; green – cell biology. The colors of the data processing circle contours and font indicate the utilized technology: red – Python; yellow – Knime; blue – third-party software.