Deliverables and timelines
- Polyclonal antibody serum: 100 ml of frozen serum of an immunised animal including ELISA results determining alpaca serum response titres. For immunisation 400 µg to 800 µg antigen is required and additional 40 µg for evaluation of the immune response. Time required: 8 weeks.
- Isolated lymphocytes: typically 1x108 cells, stored frozen in aliquots of 2x107 in 1 ml Trizol reagent including ELISA results determining alpaca serum immune titres. Time required: 8 weeks.
- Phagemid library in E. coli: typically >1x108 CFUs 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: 10.5 weeks.
- Per target 2 x 96-well masterplates with target-enriched and ELISA-screened individual clones, each clone frozen in 160 µl 2xYT/25% glycerol; including ELISA results. For selection, additional 50 µg of target antigen is needed and for ELISA screening of colonies another 100 µg. Time required: 15 weeks.
- Amino acid sequence of the identified binder(s). A selection of ELISA-positive clones are sent for sequencing (usually one 96-well microtitre plate per target antigen). The nucleic acid sequences are translated, aligned and individual VHH families are determined. Depending on target complexity and immunogenicity, anywhere between 1 to more than 30 different target-specific binders are identified per target antigen (most often 10-15 unique sequences). Time required: approximately 16 weeks / 4 mths.