Biophysical methods for the determination of antibody-antigen affinities

Neri, Dario; Montigiani, Silvia; Kirkham, Perry M.

doi:10.1016/s0167-7799(96)10067-6

Cited by 76 publications

(51 citation statements)

References 24 publications

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“…lateral diffusion/clustering) of ␣ V ␤ 3 . In contrast and as shown experimentally for PAC1 Fab and ␣ IIb ␤ 3 , a monovalent ligand is likely to be more sensitive to affinity than avidity modulation (23,30). We found that both penton base and WOW-1 Fab bound specifically and saturably to ␣ V ␤ 3 in an activation-dependent manner.…”

Section: Discussionsupporting

confidence: 61%

“…To accomplish this task, we characterized the binding of soluble monovalent and multivalent ligands to ␣ V ␤ 3 in several cell types, reasoning that a monovalent ligand would be sensitive to affinity modulation and a multivalent ligand would be sensitive to both affinity and avidity modulation (18,23,30). Penton base, a coat protein from adenovirus type 2, was selected as a multivalent ligand because each of its five subunits contains a 50-amino acid RGD tract that mediates virus internalization through ␣ V integrins (8).…”

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confidence: 99%

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Mechanisms and Consequences of Affinity Modulation of Integrin αVβ3 Detected with a Novel Patch-engineered Monovalent Ligand

Pampori¹,

Hato²,

Stupack³

et al. 1999

Journal of Biological Chemistry

156

153

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Integrin ␣ V ␤ 3 mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which ␣ V ␤ 3 is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, ␣ IIb ␤ 3 , has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of ␣ V ␤ 3 , a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single ␣ V integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated ␣ V ␤ 3 , but not to ␣ IIb ␤ 3 , in receptor and cell binding assays. ␣ V ␤ 3 affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K d ‫؍‬ 2.4 M), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K d ‫؍‬ 80 nM), with no change in receptor number. In contrast, ␣ V ␤ 3 in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of ␤ 3 cytoplasmic tails. Up-regulation of ␣ V ␤ 3 affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that ␣ V ␤ 3 is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.Integrins mediate cell adhesion and signaling during many developmental, physiological, and pathological processes (1-4). The ␤ 3 integrin family includes ␣ IIb ␤ 3 , often referred to as the fibrinogen receptor, and ␣ V ␤ 3 , the vitronectin receptor. ␣ IIb ␤ 3 is confined to megakaryocytes and platelets and is required for platelet aggregation through interactions with Arg-Gly-Asp (RGD)-containing adhesive ligands, including fibrinogen and von Willebrand factor (5). ␣ V ␤ 3 is more widely expressed in proliferating endothelial cells, arterial smooth muscle cells, osteoclasts, platelets, and certain subpopulations of leukocytes and tumor cells (6, 7). The list of cognate ligands for ␣ V ␤ 3 overlaps that for ␣ IIb ␤ 3 , but includes others, such as osteopontin, matrix metalloproteinase-2, and adenovirus penton base, which do not interact with ␣ IIb ␤ 3 (6, 8 -10). In the adult organism, ␣ V ␤ 3 has been implicated in processes ranging from wound healing to tumor angiogenesis (11), arterial restenosis (12), osteoporosis (13), tumor progression (14), and adenovirus internalization (8).One fundamental function of integrins is ligand binding, which in many cases is rapidly regulated by a process variously referred to as "integrin activation," "inside-out signaling," or "affinity/avidity modulation" (15-19). Integrin activation encompasses at least two events: 1) modulation of receptor affinity through conformational changes in the ␣␤ heterodimer and 2) modulation...

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Section: Discussionsupporting

confidence: 61%

mentioning

confidence: 99%

Mechanisms and Consequences of Affinity Modulation of Integrin αVβ3 Detected with a Novel Patch-engineered Monovalent Ligand

Pampori¹,

Hato²,

Stupack³

et al. 1999

Journal of Biological Chemistry

156

153

View full text Add to dashboard Cite

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“…This allows for a more accurate comparison of the relative binding affinities of antibodies. Furthermore, this process is free of the mass transport limitations present in direct ELISAs (32,34). Taken together, both the direct and competitive ELISA results form supporting evi- dence that the 2F5 CDR H3 apex is important for mediating access to a possibly recessed epitope, although any underlying specificity of the 2F5 CDR H3 apex residues is not located within the 2F5 core epitope itself.…”

Section: Discussionmentioning

confidence: 53%

“…Indeed, 2F5 CDR H3 mutants were observed to bind differently to gp41 constructs in direct ELISAs, whereas they showed similar binding profiles in competitive ELISAs. Recognition of antibodies to the solid-phase adsorbed antigen may be limited by multiple factors, including the conformation of the antigens on the plate surface and steric repulsive and/or attractive interactions between the antibody molecules themselves (32,35,54). It is therefore not unlikely that removing a flexible hydrophobic moiety at the apex of the 2F5 antigen binding site would cause artifacts in apparent binding affinity for the gp41 epitope bound to the solid phase.…”

Section: Discussionmentioning

confidence: 99%

Ablation of the Complementarity-Determining Region H3 Apex of the Anti-HIV-1 Broadly Neutralizing Antibody 2F5 Abrogates Neutralizing Capacity without Affecting Core Epitope Binding

Julien¹,

Huarte

Maeso

et al. 2010

J Virol

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The identification and characterization of broadly neutralizing antibodies (bnAbs) against HIV-1 has formed a major research focus, with the ultimate goal to help in the design of an effective AIDS vaccine. One of these bnAbs, 2F5, has been extensively characterized, and residues at the apex of its unusually long complementaritydetermining region (CDR) H3 loop have been shown to be crucial for neutralization. Structural studies, however, have revealed that the 100 TLFGVPI 100F apex residues of the CDR H3 loop do not interact directly with residues of its core gp41 epitope. In an attempt to gain better insight into the functional role of this element, we have recombinantly expressed native 2F5 Fab and two mutants in which either the apical Phe100B(H) residue was changed to an alanine or the CDR H3 residues 100 TLFGVPI 100F were replaced by a Ser-Gly dipeptide linker. Isothermal titration calorimetry (ITC) and competitive-binding enzyme-linked immunosorbent assays (ELISAs) rendered strikingly similar affinity constants (K d [dissociation constant] of ϳ20 nM) for linear peptide epitope binding by 2F5 Fabs, independent of the presence or absence of the apex residues. Ablation of the CDR H3 apex residues, however, abolished the cell-cell fusion inhibition and pseudovirus neutralization capacities of 2F5 Fab. We report competitive ELISA data that suggest a role of 2F5 CDR H3 apex residues in mediating weak hydrophobic interactions with residues located at the C terminus of the gp41 membrane proximal external region and/or membrane components in the context of core epitope binding. The present data therefore imply an extended 2F5 paratope that includes weak secondary interactions that are crucial for neutralization of Env-mediated fusion.

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“…Half-lives of the GutR ⅐ DNA complexes were estimated based on the equation (t 1/2 ϭ 0.69/k d ) described by Neri et al (33). GutR is required to induce GutR to have a conformational change so that critical residues involved in ATP binding can be positioned in proper locations.…”

Section: Table I Kinetic Association and Dissociation Rate Constants mentioning

confidence: 99%

Roles of Glucitol in the GutR-mediated Transcription Activation Process in Bacillus subtilis

Poon

Chu

Wong

2001

Journal of Biological Chemistry

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GutR is a 95-kDa glucitol-dependent transcription activator that mediates the expression of the Bacillus subtilis glucitol operon. Glucitol allows GutR to bind tightly to its binding site located upstream of the gut promoter. In this study, a second functional role of glucitol is identified. Glucitol induces GutR to change its conformation and triggers GutR to bind ATP efficiently. After sequential binding of glucitol and ATP to GutR, GutR adopts a new conformation by forming a compact structure that is resistant to trypsin digestion. Under this condition, the ATP⅐glucitiol⅐GutR complex can dissociate slowly from the gutR-binding site (t1 ⁄2 ‫؍‬ 274 min). Interestingly, if ATP in the ATP⅐glucitiol⅐GutR complex is replaced by ADP, GutR adopts another conformation and can dissociate from the gutR-binding site even faster (t1 ⁄2 ‫؍‬ 82 min). In all these GutR-DNA binding studies in the presence of different ligands (glucitol, ATP, or ADP), only the off-rate is affected. The vital role of ATP in the GutRmediated transcription activation process is reflected by the poor transcription from the gut promoter with GutR(D285A) which has a mutation in the motif B of the putative ATP-binding site. A working model for this transcription activation process is presented.

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Biophysical methods for the determination of antibody-antigen affinities

Cited by 76 publications

References 24 publications

Mechanisms and Consequences of Affinity Modulation of Integrin αVβ3 Detected with a Novel Patch-engineered Monovalent Ligand

Mechanisms and Consequences of Affinity Modulation of Integrin αVβ3 Detected with a Novel Patch-engineered Monovalent Ligand

Ablation of the Complementarity-Determining Region H3 Apex of the Anti-HIV-1 Broadly Neutralizing Antibody 2F5 Abrogates Neutralizing Capacity without Affecting Core Epitope Binding

Roles of Glucitol in the GutR-mediated Transcription Activation Process in Bacillus subtilis

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