Next Article in Journal
A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems
Previous Article in Journal
Libidibia ferrea (jucá), a Traditional Anti-Inflammatory: A Study of Acute Toxicity in Adult and Embryos Zebrafish (Danio rerio)
Previous Article in Special Issue
Study of Iron Piperazine-Based Chelators as Potential Siderophore Mimetics
Open AccessCommunication

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

1
GICC EA7501, Equipe IMT, Université de Tours, UFR des Sciences Pharmaceutiques, 31 Avenue Monge, 37200 Tours, France
2
Laboratoire d’immunologie, CHRU Tours, 10 Bd Tonnelé, 37000 Tours, France
3
GICC EA7501, Equipe FRAME, Université de Tours, 10 Bd Tonnelé, 37000 Tours, France
4
ICOA UMR 7311 CNRS, Université d'Orléans, Pôle de Chimie, Rue de Chartres, BP 6759, 45067 Orléans CEDEX 2, France
5
CBM, UPR4301 CNRS, Rue Charles Sadron, 45071 Orléans, France
6
NMNS EA 6295, 31 Avenue Monge, 37200 Tours, France
7
INSERM U1100, Université de Tours, 10 Bd Tonnelé, 37000 Tours, France
*
Author to whom correspondence should be addressed.
Pharmaceuticals 2019, 12(4), 176; https://doi.org/10.3390/ph12040176
Received: 22 October 2019 / Revised: 22 November 2019 / Accepted: 25 November 2019 / Published: 2 December 2019
(This article belongs to the Special Issue New Tools for Medicinal Chemists)
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.
Keywords: antibody–fluorophore conjugate; fluorescence; labelling; bioconjugation antibody–fluorophore conjugate; fluorescence; labelling; bioconjugation
Show Figures

Graphical abstract

MDPI and ACS Style

Martin, C.; Brachet, G.; Colas, C.; Allard-Vannier, E.; Kizlik-Masson, C.; Esnault, C.; Respaud, R.; Denevault-Sabourin, C.; Chourpa, I.; Gouilleux-Gruart, V.; Viaud-Massuard, M.-C.; Joubert, N. In Vitro Characterization and Stability Profiles of Antibody–Fluorophore Conjugates Derived from Interchain Cysteine Cross-Linking or Lysine Bioconjugation. Pharmaceuticals 2019, 12, 176.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop