Fig. 1.

Hypoxic HeLa cells can be used for generation of a hypoxia-specific phage library. a Indicated cells were grown under normoxic (21 % O₂) (red) and hypoxic (1 % O₂) (blue) conditions for 24 h, and CAIX levels were determined by a cell-based ELISA as described in Materials and Methods in the ESM. b Workflow of phage display selections: 1. Llamas are immunized with hypoxic HeLa cells; 2. During the immune response peripheral B-lymphocytes generate CAIX-specific heavy chain-only antibodies; 3. peripheral B-lymphocytes are isolated and RNA is extracted. After reverse transcriptase PCR, antibody specific DNA is ligated into a phagemid vector; 4. Phages expressing nanobodies at their surface are produced in E. coli bacteria; 5. Two rounds of phage display selections are performed in a 96-wells format coated with recombinant CAIX.