Protocol for Creating Antibodies with Complex Fluorescence Spectra

• Published in: Bioconjugate chemistry 2021-06, Vol.32 (6), p.1156-1166
• Main Authors: McCarthy, Madeline E, Anglin, Caitlin M, Peer, Heather A, Boleman, Sevanna A, Klaubert, Stephanie R, Birtwistle, Marc R
• Format: Article
• Language: English
• Subjects: Antibodies; Antibodies - chemistry; Antibodies - metabolism; Beads; Chemical compounds; Combinations (mathematics); Combinatorial analysis; DNA probes; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; Docking; Flow cytometry; Fluorescence; Fluorophores; Heterogeneity; Labeling; Molecular Docking Simulation; Multiplexing; Music; Nucleic Acid Conformation; Probes; Protein A; Protein Conformation; Single-stranded DNA; Spectra; Spectral signatures; Spectrometry, Fluorescence
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.1c00220

Fluorescent antibodies are a workhorse of biomedical science, but fluorescence multiplexing has been notoriously difficult due to spectral overlap between fluorophores. We recently established proof-of-principal for fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC), which uses combinations of existing fluorophores to create unique spectral signatures for increased multiplexing. However, a method for labeling antibodies with MuSIC probes has not yet been developed. Here, we present a method for labeling antibodies with MuSIC probes. We conjugate a DBCO-Peg5-NHS ester linker to antibodies and a single-stranded DNA “docking strand” to the linker and, finally, hybridize two MuSIC-compatible, fluorescently labeled oligos to the docking strand. We validate the labeling protocol with spin-column purification and absorbance measurements. We demonstrate the approach using (i) Cy3, (ii) Tex615, and (iii) a Cy3-Tex615 combination as three different MuSIC probes attached to three separate batches of antibodies. We created single-, double-, and triple-positive beads that are analogous to single cells by incubating MuSIC probe-labeled antibodies with protein A beads. Spectral flow cytometry experiments demonstrate that each MuSIC probe can be uniquely distinguished, and the fraction of beads in a mixture with different staining patterns are accurately inferred. The approach is general and might be more broadly applied to cell-type profiling or tissue heterogeneity studies in clinical, biomedical, and drug discovery research.