Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcoreTM binding data. Analysis of the interaction of NC10 Fab with an anti-idiotype Fab′

Kortt, Alexander A.; Gruen, L.C.; Oddie, Geoffrey W.

doi:10.1002/(sici)1099-1352(199705/06)10:3<148::aid-jmr360>3.0.co;2-f

Cited by 16 publications

(6 citation statements)

References 14 publications

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“…1D, inset). Thus, the K d values obtained using the kinetic or the affinity measurements were similar, indicating that the potential mass transport complications and the analyte (dEGR–FIXa) rebinding during the dissociation phase are minimal under our experimental conditions [12]. Moreover, the binding of dEGR–FIXa was ∼ 3.7‐fold weaker in 1.1 m m Ca 2+ than in 5 m m Ca 2+ or 1.1 m m Ca 2+ /0.6 mm Mg 2+ .…”

mentioning

confidence: 54%

Contribution of magnesium in binding of factor IXa to the phospholipid surface: implications for vitamin K‐dependent coagulation proteins

Messer

Velander

Bajaj

2009

Journal of Thrombosis and Haemostasis

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mentioning

confidence: 54%

Contribution of magnesium in binding of factor IXa to the phospholipid surface: implications for vitamin K‐dependent coagulation proteins

Messer

Velander

Bajaj

2009

Journal of Thrombosis and Haemostasis

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“…Data were corrected for instrument and bulk artifacts by double referencing (15) a surface-immobilized with capture antibody without scFvFc-scFv using Scrubber version 2.0c software (BioLogic Software). The transformed data were fit to a 1:1 binding model in Biacore T200 evaluation software v1.0 (GE Healthcare), which includes a parameter for mass transfer (16).…”

Section: Methodsmentioning

confidence: 85%

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

Terraube

Tam

et al. 2016

Journal of Biological Chemistry

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Background: Antibody v-domains in scFv format often suffer from aggregation and stability issues that restrict formulation. Results: Structural and empirical analyses of an optimized scFv revealed that three V L -CDR3 mutations were sufficient to mediate significant stability and affinity improvements. Conclusion: scFv issues were resolved via removal of side-chain clashes at the V L /V H interface. Significance: CDR-restricted mutagenesis delivers stability-optimized molecules for high concentration dosing.

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“…Affinity measurement is thus a critical step in the evaluation of mAbs for diagnostic or prophylactic applications, and may also be key to select mAbs for use in the identification of microbial antigens suitable for vaccine development. Considering the caveats of currently available technologies to quantify affinity [8] , [12] , [14] , [15] , [20] , there is a need for a rapid high-throughput method using low quantities of antibody, to allow efficient characterization of large numbers of mAbs. We developed a computational approach based on data generated by a microfluidic system.…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, these assays rely on endpoint analysis, as is the case for enzyme-linked immunosorbent assay (ELISA) titration [7] . Drawbacks of the first category are the complicated measurement of the long dissociation times imposed by very high-affinity (K D ≤ 10 −12 M) binding, and the potential of artifacts due to mass-transport effects and steric hindrance of reagents [8] . ELISA-based methods can handle wider K D ranges (10 −13 to 10 −07 M [9] ), but are based on in-solution measurements performed after the equilibrium between antigen-bound and free antibodies has been reached [10] .…”

Section: Introductionmentioning

confidence: 99%

Computational modeling of microfluidic data provides high-throughput affinity estimates for monoclonal antibodies

Budroni¹,

Buricchi²,

Cavallone³

et al. 2021

Computational and Structural Biotechnology Journal

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Affinity measurement is a fundamental step in the discovery of monoclonal antibodies (mAbs) and of antigens suitable for vaccine development. Innovative affinity assays are needed due to the low throughput and/or limited dynamic range of available technologies. We combined microfluidic technology with quantum-mechanical scattering theory, in order to develop a high-throughput, broad-range methodology to measure affinity. Fluorescence intensity profiles were generated for out-of-equilibrium solutions of labelled mAbs and their antigen-binding fragments migrating along micro-columns with immobilized cognate antigen. Affinity quantification was performed by computational data analysis based on the Landau probability distribution. Experiments using a wide array of human or murine antibodies against bacterial or viral, protein or polysaccharide antigens, showed that all the antibody-antigen capture profiles (n = 841) generated at different concentrations were accurately described by the Landau distribution. A scale parameter W , proportional to the full-width-at-half-maximum of the capture profile, was shown to be independent of the antibody concentration. The W parameter correlated significantly (Pearson’s r [ p– value]: 0.89 [3 × 10 −8 ]) with the equilibrium dissociation constant K D , a gold-standard affinity measure. Our method showed good intermediate precision (median coefficient of variation: 5%) and a dynamic range corresponding to K D values spanning from ~10 −7 to ~10 −11 Molar. Relative to assays relying on antibody-antigen equilibrium in solution, even when they are microfluidic-based, the method’s turnaround times were decreased from 2 days to 2 h. The described computational modelling of antibody capture profiles represents a fast, reproducible, high-throughput methodology to accurately measure a broad range of antibody affinities in very low volumes of solution.

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Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcoreTM binding data. Analysis of the interaction of NC10 Fab with an anti-idiotype Fab′

Cited by 16 publications

References 14 publications

Contribution of magnesium in binding of factor IXa to the phospholipid surface: implications for vitamin K‐dependent coagulation proteins

Contribution of magnesium in binding of factor IXa to the phospholipid surface: implications for vitamin K‐dependent coagulation proteins

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

Computational modeling of microfluidic data provides high-throughput affinity estimates for monoclonal antibodies

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