In Vitro Characterization and Stability Profiles of Antibody–Fluorophore Conjugates Derived from Interchain Cysteine Cross-Linking or Lysine Bioconjugation - Université de Tours Accéder directement au contenu
Article Dans Une Revue Pharmaceuticals Année : 2019

In Vitro Characterization and Stability Profiles of Antibody–Fluorophore Conjugates Derived from Interchain Cysteine Cross-Linking or Lysine Bioconjugation

Emilie Allard-Vannier
Igor Chourpa

Résumé

Fluorescent labelling of monoclonal antibodies (mAbs) is classically performed by chemical bioconjugation methods. The most frequent labelling technique to generate antibody-fluorophore conjugates (AFCs) involves the bioconjugation onto the mAb lysines of a dye bearing an N-hydroxysuccinimide ester or an isothiocyanate group. However, discrepancies between labelling experiments or kits can be observed, related to reproducibility issues, alteration of antigen binding, or mAb properties. The lack of information on labelling kits and the incomplete characterization of the obtained labelled mAbs largely contribute to these issues. In this work, we generated eight AFCs through either lysine or interchain cysteine cross-linking bioconjugation of green-emitting fluorophores (fluorescein or BODIPY) onto either trastuzumab or rituximab. This strategy allowed us to study the influence of fluorophore solubility, bioconjugation technology, and antibody nature on two known labelling procedures. The structures of these AFCs were thoroughly analyzed by mass spectroscopy, and their antigen binding properties were studied. We then compared these AFCs in vitro by studying their respective spectral properties and stabilities. The shelf stability profiles and sensibility to pH variation of these AFCs prove to be dye-, antibody- and labelling-technology-dependent. Fluorescence emission in AFCs was higher when lysine labelling was used, but cross-linked AFCs were revealed to be more stable. This must be taken into account for the design of any biological study involving antibody labelling.
Fichier principal
Vignette du fichier
pharmaceuticals-12-00176.pdf (1.17 Mo) Télécharger le fichier
pharmaceuticals-12-00176-s001.pdf (1.98 Mo) Télécharger le fichier
Origine : Fichiers éditeurs autorisés sur une archive ouverte
Licence : CC BY - Paternité
Licence : CC BY - Paternité

Dates et versions

hal-02425275 , version 1 (03-11-2023)

Identifiants

Citer

Camille Martin, Guillaume Brachet, Cyril Colas, Emilie Allard-Vannier, Claire Kizlik-Masson, et al.. In Vitro Characterization and Stability Profiles of Antibody–Fluorophore Conjugates Derived from Interchain Cysteine Cross-Linking or Lysine Bioconjugation. Pharmaceuticals, 2019, 12 (4), pp.176. ⟨10.3390/ph12040176⟩. ⟨hal-02425275⟩
122 Consultations
2 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More