Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition

Abstract

Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15) derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F) was equally well tolerated as the wild type glutamine (Q) at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv–peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine) was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology). Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes.

ImmunoPET Imaging of CD146 in Murine Models of Intrapulmonary Metastasis of Non-Small Cell Lung Cancer

“…Saturation binding assay unveiled the high affinity of YY146 towards CD146 expressed in the H460 cell line with a K D value of 2.84 ± 0.53 nM (Figure 2). This value was within the expected range and confirmed the strong binding affinity of the antibody; most antibodies display K D values in the range of 10 −6 to 10 −9 M, with antibodies showing the highest affinity being ~10 −12 M. 28 . Additionally, a receptor density of CD146 on H460 cells was calculated to be ~9.9 × 10 5 receptors per cell, which guarantees a successful implementation of imaging studies, where expression level of more than 10 5 receptors per cell is considered appropriate.…”

Properties of Two Enterovirus Antibodies that are Utilized in Diabetes Research

“…Peptide array synthesis and binding detection were performed by PEPperPRINT GmbH (PEPperPRINT, Heidelberg, Germany) as reported 38 39 . Permutation scans were carried out on the peptide SESIPALTAAETGHT (containing the MAb 5D-8.1 epitope) and on its variants.…”

A sandwich-type optical immunosensor based on the alkaline phosphatase enzyme for Salmonella thypimurium detection

“…A selective binder shows little cross-reactivity [22] means that it recognizes a given partner with much higher affinity than other partners. Selectivity test results showed that the detector can only developing color when Salmonella is present, indicated by RGB value obtained is lower than the control while E.coli testing result showed that there was no significant differences with the control ( Figure 5).…”