Polyclonal antibody serum: 30-50 ml of frozen serum of an immunised animal including ELISA results determining alpaca serum response titres. Time required: 8 weeks. For immunisation 400 µg to 800 µg antigen is required and additional 35-50 µg for evaluation of the immune response.
Isolated lymphocytes: typically 1x108 cells, stored frozen in aliquots of 2x108 in 1 ml Trizol reagent including ELISA results determining alpaca serum immune titres. Time required: 8 weeks.
Phagemid library in E. coli: typically 1x107 - 1x108 single colonies in 12 ml total volume, stored frozen in 2xYT/25% glycerol. Time required: 10 weeks.
Phage library consisting of 1x1012-13 precipitated phages, stored frozen in 1 ml PBS/50% glycerol. Time required: 10 weeks.
2x96-well masterplates with target-enriched and ELISA-screened individual clones, each clone frozen in 150 µl 2xYT/25% glycerol; including ELISA results. Time required: 13-14 weeks. For selection, additional 50 µg of target antigen is needed and for ELISA screening of colonies another 100 µg.
Amino acid sequence of the identified binder(s). A selection of ELISA-positive clones are sent for sequencing. The nucleic acid sequences are translated, aligned and individual nanobody families are determined. Usually, 1 to 20 different nanobodies are identified per target. Time required: approximately 15-16 weeks.
