期刊
PHARMACEUTICALS
卷 12, 期 4, 页码 -出版社
MDPI
DOI: 10.3390/ph12040176
关键词
antibody-fluorophore conjugate; fluorescence; labelling; bioconjugation
资金
- French National Research Agency under the program Investissements d'avenir Grant Agreement LabEx SynOrg [ANR-11-LABX-0029]
- French National Research Agency under the program Investissements d'avenir Grant Agreement LabEx MAbImprove [ANR-10-LABX-53-01]
- Universite de Tours and Region Centre Val de Loire
- LabEx SynOrg [ANR-11-LABX-0029]
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.
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