Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies to Enable Combination Multiplexing

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 ∼6-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 ∼3-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.

Figure 1

Oligo-based ext. MuSIC probe labeling of antibodies. (A,B) Graphic depicting MuSIC probe labeling. The NHS ester of the linker reacts with free amines on the antibody. Fluorophore-end-labeled (external-extrinsic) donor and acceptor strands are annealed onto the docking strand to form the oligo complex. The azide on the docking strand in the oligo complex is reacted with the free DBCO group on the linker to covalently bind the oligo complex to the antibody. There are multiple free amines on each antibody, allowing for the linker to attach at multiple sites increasing the degree of labeling. (C) Comparison of fluorescence intensity of PBMCs stained with CF488A conventional labeling kit vs Atto488 ext. MuSIC probes. Error bars are standard errors from triplicate measurements.

Figure 2

Fluorescence signal change from docking strand. (A) Comparison of fluorescence emission spectra, excited at 470 nm, of the Atto488 5′ donor and 3′ acceptor strands hybridized to the docking strand and when alone in solution with and without the docking strand. Data are representative from triplicate. (B) Change in fluorescence intensity of 15 fluorescent oligos when hybridized to the docking strand. Error bars are standard error from triplicate.

Figure 3

Int. labeling method increases the intensity relative to the ext. labeling method. (A) Int. oligo complex containing the Int. donor and acceptor strands and the azide strand. (B) Comparison of relative fluorescence intensity of the Atto488 probes using the int. and ext. oligo complexes (470 nm excitation). Data are representative from triplicate.

Figure 4

Differentiating static and dynamic quenching by time-resolved fluorescence and fluorescence correlation spectroscopy. Atto488 int. oligo complex fitting is shown in blue, with the raw data in light blue, and Atto488 ext. oligo complex fitting is shown in orange, with the raw data in light orange. (A) Normalized fluorescence decays between the Atto488 int. oligo complex and the Atto488 ext. oligo complex. The difference in fluorescence lifetimes is visible by the difference in the slope of the decays. Residuals for the fitting model are shown on top. (B) Normalized fluorescence correlation between the Atto488 int. oligo complex and the Atto488 ext. oligo complex. The difference in dark triplet states is visible in the offset between the start of the curves and correlation times (t_c) ∼ 10^–3 ms. The curve overlaps between t, being 10^–1 and 10, indicating similar diffusion coefficients between samples. Residuals for the fitting model are shown on top. (C) Anisotropy rotational correlation between the Atto488 int. oligo complex and the Atto488 ext. oligo complex. The slight offset in rotational times could be due to differences in the flexibility of the int. and ext. oligo complexes.

Figure 5

Comparing int. oligo complex and conventionally labeled antibodies in cell-based experiments. (A) Signal increase of PBMCs stained with Atto488 int.-labeled CD8 antibodies versus CF488A conventionally labeled CD8 antibodies. (B) Percentage of CD8+ lymphocytes in PBMC for int.-labeled CD8 antibodies compared to CF488A conventional-labeled CD8 antibodies. Measurements are in triplicate, and error bars are standard error.

Figure 6

Unmixing experiments for int. oligo-labeled antibodies. (A) Three different antibodies were labeled with three different oligo complexes: Atto488, Atto647, and the Atto488/647 combination. (B) The median fluorescence intensity (MFI) of positive cells in each group was used to generate the spectrum and identify the peak channel. Each cell’s fluorescence value at this peak channel was normalized by dividing it by the group’s MFI at the same channel. Similarly, the unmixed value of each cell was normalized by dividing it by its corresponding median value to generate the normalized unmixed relative abundance. The normalized fluorescence intensity in each cell is plotted versus its normalized unmixed relative abundance from PBMC singly stained with each antibody, demonstrating a good correlation between intensity and unmixed values. (C) Data exported from singly stained PBMC were subjected to unmixing and histograms observed, showing good specificity. (D) PBMC were gated for positivity for the three different antibodies from histograms from either standard intensity-based metrics or unmixed quantification, in either singly stained or triple-stained cell samples. There is overall good agreement between all methods demonstrating confidence in the approach. In all panels, measurements are based on triplicate, and error bars are standard error. *: p < 0.05 (t test).