For this, after the elution step, the mAbs were incubated with glycine-HCl (pH 2.7, 15 min at room temperature), after which the supernatants were neutralized and processed for the estimation of any residual phage titers. Finally, although mAb-bound phage clones were eluted by competitive displacement with excess of the homologous Ag, we consistently monitored the efficiency of this procedure at each step. Such experiments yielded K d values ranging from 200 to 400 μM, providing a rough estimate of the low-affinity cutoff of our selection protocol. A subset ( n = 10) of the phage clones selected by the affinity-matured mAbs was also processed further to determine the affinity of their binding to the selecting mAb. Titers that were significantly less than 10 2 PFU/ml were routinely obtained from these latter groups, confirming the germline mAb-dependent specificity of our protocol. For example, each round of selection and elution with a given germline Ab was always accompanied by a parallel group, where the corresponding affinity-matured derivative (e.g., mAb 36-71 for mAb 36-65 or mAb Bg53-5 for BBE6.12H3) was included instead. Several control experiments were performed with the above to ensure the validity and specificity of our screening procedure. Next, individual phage clones were amplified and the insert was identified by nucleotide sequencing.

Furthermore, at the end of each selection cycle, only specifically bound phage was eluted by competitive displacement with a 10-fold molar excess (2 μM based on a valency of 2/Ab molecule) of the homologous Ag (i.e., either NP 23-BSA or Ars-BSA). To minimize nonspecific reactivities, preadsorption against nonspecific mouse IgG followed by protein G-Sepharose (blocked with BSA) first preceded each round of selection. The pellet was washed (10 × 1 ml) with TBS (pH 7.5) containing 0.1% Tween 20. Subsequent to this, bound complexes were then separated by first incubating with protein G-Sepharose (at room temperature for 15 min), followed by centrifugation. Briefly, the library was incubated with mAb in a total volume of 200 μl for 1 h at room temperature. The V κL genes for mAbs 36-65 and PC7bM belonged to subgroups V and III, respectively, whereas mAb BBE6.12H3 uses a λ1 L chain.Īliquots of the above library were first subjected to three rounds of selection, in solution, against 100 nM final concentrations of either mAb BBE6.12H3 or mAb 36-65, strictly following the recommended protocol of the manufacturer. The corresponding sequences for mAb BBE6.12H3 are YDYYGSSYFDY (HCDR3), and ALWYSNHWV (LCDR3) for mAb PC7bM, these are QRTIGTPGAY (HCDR3) and QHIREAYT (LCDR3). For mAb 36-65, the amino acid sequences of the complementarity-determining region 3 of the H chain (HCDR3) and complementarity-determining region 3 of the L chain (LCDR3) are SVYYGGSYYFDY and QQGNTLPRT, respectively. When necessary, the IgM class mAb PC7bM was subsequently converted to its monomeric form by reductive alkylation as described earlier ( 12). All of these mAbs were first obtained as ascites and then purified by affinity chromatography as described earlier ( 5). Manser (Kimmel Cancer Institute, Philadelphia, PA), whereas the anti-peptide hybridoma PC7bM has been described earlier ( 12). The anti-(4-hydroxy-3-nitrophenyl)-acetyl (anti-NP) 3 hybridomas BBE6.12H3and Bg53-5, and the anti- p-azophenylarsonate (anti-Ars) hybridomas 36-65 and 36-71 were a kind gift from Dr. Importantly, it also ensures that the primary response is composed of Abs with a high degree of “evolvability.” In addition to providing for an amplified recognition potential, this networking of individual spectra of Ag specificities simultaneously facilitates the rapid recognition of Ag. Combining site plasticity may facilitate overlaps in such windows, with independent Abs converging onto common determinants with near identical binding affinities. This singular feature confers on each Ab a distinct window of Ag specificities, where the entropic space explored constitutes a thermodynamic signature of that particular Ab. We demonstrate that the pluripotency of germline Abs can subsequently be optimized by binding interactions that correlate with thermodynamic changes indicative of structural adaptations at the interface. This revealed a much greater heterogeneity of binders than could be obtained with mutated daughter Abs that presumably had been selected in vivo by nominal Ag during active immune responses. In this study, germline Abs were used to select clones from a random dodecapeptide phage-display library.

Primary antibody repertoire