Fig. 2.

Analysis of the impact of negatively charged and tyrosine CDR residues on antibody specificity using alanine-scanning mutagenesis. Non-specific binding of the AF1-Fc mutants to milk proteins was evaluated as described in Fig. 1 (100 nM antibody). The impact of replacing aspartic acid with glutamic acid was also evaluated. The theoretical HCDR3 net charges were calculated at pH 7.4. A two-tailed Student’s t-test was used to evaluate statistical significance for each mutant relative to the parental AF1-Fc antibody [P-values <0.01 (*)]. The average values are for three independent experiments, and the error bars are standard errors.