Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies.
bioRxiv
; 2023 Jul 06.
Article
em En
| MEDLINE
| ID: mdl-37461453
ABSTRACT
While 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 intensity 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 ~6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence spectroscopy and fluorescence correlation spectroscopy showed that ~3-fold brightness difference is due to static quenching. Spectral flow cytometry experiments using peripheral blood mononuclear cells stained with anti-CD8 antibodies showed that int. MuSIC probe-labeled antibodies have signal intensity equal to or greater than conventionally-labeled antibodies with similar estimated proportion of CD8+ lymphocytes. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications.
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MEDLINE
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En
Ano de publicação:
2023
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Article