Figure 6. VL chain K53 is responsible for the temperature-dependent binding of 2B5 to PEG.(A) Recombinant 2B5 or 2B5 in which VH V23 (2B5ΔV) or VL K53 (2B5ΔK) were replaced by the corresponding amino acid of the parental 3.3 antibody were purified from mammalian cell culture medium. (B) Graded concentrations of recombinant 2B5 (■), 2B5ΔK (○), or 2B5ΔV (●) antibodies were added to microplate wells coated with linear amino-PEG at the indicated temperatures. After 1 h, the wells were washed and antibody binding was determined by adding HRP-conjugated donkey anti-mouse IgG Fc antibodies, followed by ABTS substrate. The mean absorbance values (405 nm) of triplicate determinations are shown. Bars, SD.