(A) K_D of mAbs generated from WT and TACI-deficient mice. K_D (y axis) in nM (the reciprocal of avidity is depicted in blue) of monoclonal anti-NP antibodies (mAbs) encoded by the canonical VH1-72 (VH186.2) was determined by ascertaining the concentration at equilibrium at which one-half was bound and one-half unbound (25, 45). mAbs from WT mice had an average K_D of 4.6 × 10^–10, while mAbs from TACI-deficient mice had an average K_D of 2.4 × 10^–10 (P = 0.0079, unpaired t test). (B) Comparison of mAb K_D determined by real-time measurement of association and dissociation rates and BLI, with K_D determined at equilibrium by ELISA. (C) Impact of TACI on the mutation frequency of VH1-72 encoded by anti-NP antibodies. Frequency of sequences with a given number of mutations is plotted. TACI deficiency increased the frequency of independent mutations by more than 2-fold (P < 0.001, contingency analysis). (D) Relationship between frequency of mutations and antibody avidity in response to NP. Frequency of mutations in VH1-72 sequences is depicted in relation to avidity at equilibrium of the corresponding antibodies. Avidity correlated with the number of mutations (P < 0.0001, Wilcoxon matched-pairs, signed-rank test). (E) Localization of aa substitutions in distinct clones encoding VH1-72 heavy chains of anti-NP antibodies obtained from WT and TACI-deficient mice. K_D for the corresponding sequences was determined by BLI (values are shown on the right). V(D)J, variable(V), diversity (D) and joining (J) gene segments junction region of the Ig heavy chain exon.