Figure 4. Isolation of temperature-dependent anti-PEG antibodies by mimicking the germinal center reaction. (A) 3.3/loxP-AID hybridoma cells were enriched for cells binding Alexa Fluor 647-PEG probes (y axis). Surface immunoglobulin was also detected using Alexa Fluor 405-conjugated goat anti-mouse IgG Fc antibody (x axis). The cells were cultured for 2 wk before approximately 3 × 10⁷ cells were sorted each round (S0-S5). Results show binding of fluorescent-labeled PEG vs. surface Ig levels. (B) Binding of PEG-Alexa Fluor 647 to three hybridoma clones (1E3, 1E10 and 2B5) that were isolated from the hybridoma population after round 5 of selection (S5). (C) Amino acid sequences and alignment of immunoglobulin V_H (upper panel) and V_L (lower panel) gene sequences from 1E3, 1E10 and 2B5 antibodies in comparison to the parental 3.3 antibody. Antibody framework regions and CDRs were assigned according to the Kabat numbering system⁶¹ using the website http://www.bioinf.org.uk/abysis.