| Autor: |
Sonia Budroni, Francesca Buricchi, Andrea Cavallone, Gianfranco Volpini, Alessandra Mariani, Paola Lo Surdo, Christoph J. Blohmke, Giuseppe Del Giudice, Duccio Medini, Oretta Finco |
| Jazyk: |
angličtina |
| Rok vydání: |
2021 |
| Předmět: |
|
| Zdroj: |
Computational and Structural Biotechnology Journal, Vol 19, Iss , Pp 3664-3672 (2021) |
| Druh dokumentu: |
article |
| ISSN: |
2001-0370 |
| DOI: |
10.1016/j.csbj.2021.06.024 |
| Popis: |
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 KD, a gold-standard affinity measure.Our method showed good intermediate precision (median coefficient of variation: 5%) and a dynamic range corresponding to KD 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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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