Antibody active site contributions to fluorescein ligand binding

A panel of idiotypically cross-reactive murine monoclonal antibodies (Mabs) isolated in response to fluorescein (Fl) was generated and segregated based on Fl affinity. Intermediate affinity prototype Mab 9-40 (IgG1, $\kappa$; K$\sb{\rm a}$ = 3.3 $\times$ 10$\sp7$M$\sp{-1}$) possessed $>$90% active site quaternary structural homology to the intermediate affinity 9-40 idiotype family (comprised of 12-40, 3-24, 10-25, 5-14 and 5-27). The 9-40 family was 40-100% and 10-40% idiotypically related to high affinity prototype Mab 4-4-20 (IgG2a, $\kappa$; K$\sb{\rm a}$ = 1.8 $\times$ 10$\sp{10}$M$\sp{-1}$) and low affinity prototype Mabs 3-13 and 3-17 (K$\sb{\rm a}$ $\sim$ 5.0 $\times$ 10$\sp4$M$\sp{-1}$), respectively. Mabs 4-4-20 and 3-13/3-17 were idiotypically distinct. This anti-Fl panel spanned a greater affinity range than any previously reported idiotype family and was exploited to define specific active site residues (and their interactions with antigen) responsible for the observed Fl binding characteristics. To begin active site structure-function assessments, V$\sb{\rm H}$ and V$\sb\kappa$ primary structures were obtained. Except for 5-27 and 3-13, highly homologous V$\sb{\rm H}$III(C) genes were utilized. D region length and sequence variabilities were seen; however, compensatory J$\sb{\rm H}$4 (4-4-20 and 3-24) or J$\sb{\rm H}$3 sequence lengths resulted in HCDR3 + FR4 to be a constant 18 amino acids. Each Mab rearranged $>$95% homologous V$\sb\kappa$II genes to J$\sb\kappa$1, except 10-25 (J$\sb\kappa$5) and 3-13 (J$\sb\kappa$4). In addition, 4-4-20 Fl contact residues were determined by x-ray crystallographic studies. Mabs 5-14, 9-40, 12-40 and 3-24 possessed identical Fl contact residues as 4-4-20 except for L34 His substitution for Arg. Primary structure comparisons, therefore, implicated L34 Arg responsible for increased 4-4-20 affinity. To verify this, Fl binding assays, following in vitro H and L chain reassociations, were performed. Results indicated: (1) L34 Arg was responsible for increased 4-4-20 Fl affinity; (2) L96 Trp was critical for at least an affinity of $\sim$10$\sp7$M$\sp{-1}$; and (3) Fl contact residue orientations were potentially affected by HCDR3 and/or C$\sb{\rm H}$1 residue differences. Recombinant 4-4-20 molecules (generated by an in vivo H chain expression system) that expressed IgG1 and IgG2b H chains possessed identical Fl binding patterns as 4-4-20 (IgG2a). This implicated HCDR3 residues for subtle binding differences observed between the various idiotype family members.U of I OnlyETDs are only available to UIUC Users without author permissio