Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies to Enable Combination Multiplexing.
Bioconjug Chem
; 35(7): 1053-1063, 2024 Jul 17.
Article
em En
| MEDLINE
| ID: mdl-38889324
ABSTRACT
Full-spectrum flow cytometry has increased antibody-based multiplexing, yet further increases remain potentially impactful. We recently proposed how fluorescence multiplexing using spectral imaging and combinatorics (MuSIC) could do so using tandem dyes and an oligo-based antibody labeling method. In this work, we found that such labeled antibodies had significantly lower signal intensities than conventionally labeled antibodies in human cell experiments. To improve signal intensity, we tested moving the fluorophores from the original external (ext.) 5' or 3' end-labeled orientation to internal (int.) fluorophore modifications. Cell-free spectrophotometer measurements showed a â¼6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence and fluorescence correlation spectroscopy showed that the â¼3-fold brightness difference is due to static quenching most likely by the oligo or solution in the ext. configuration. Spectral flow cytometry experiments using peripheral blood mononuclear cells show int. MuSIC probe-labeled antibodies (i) retained increased signal intensity while having no significant difference in the estimated % of CD8+ lymphocytes and (ii) labeled with Atto488, Atto647, and Atto488/647 combinations can be demultiplexed in triple-stained samples. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications where spectral detection is available.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Citometria de Fluxo
/
Corantes Fluorescentes
/
Anticorpos
Limite:
Humans
Idioma:
En
Revista:
Bioconjug Chem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Estados Unidos