1. Volume 114, Issue 3, Pages 688-700 (February 2018)
The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes 
Ágnes Szabó, Tímea Szendi-Szatmári, László Ujlaky-Nagy, Ildikó Rádi, György Vereb, János Szöllősi, Peter Nagy 
Biophysical Journal 
Volume 114, Issue 3, Pages (February 2018)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
The fluorescence intensity of free and bound antibodies as a function of the degree of labeling. The fluorescence intensity of 20 nM solutions of antibodies with different DOLs was measured using fluorometry. Cells (SKBR-3 for trastuzumab; JY for L368) were labeled with saturating concentrations of the antibodies followed by flow cytometric measurement of the fluorescence intensity of the cell-bound antibody fraction. The fluorescence intensities of the antibody stock and those of the bound fraction were normalized to the intensity measured for the lowest DOL and the normalized intensities are plotted as a function of the DOL. The error bars represent the SE calculated from three measurements. The measured data were fitted according to Eqs. 9 and 10 assuming the quantum yield and the Kd can be expressed as power functions of the number of fluorophores in an antibody species (Qk = kx, Kd,k = Kd,0 (k+1)y, where Qk and Kd,k are the quantum yield and dissociation constant, respectively, of the antibody species having k number of fluorophores, and Kd,0 is the Kd of the unlabeled antibody). The dashed lines show the expected intensity assuming linear dependence of intensity on the DOL. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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3. Figure 2
Model calculation of the fluorescence intensity of antibody stocks and that of the bound fraction of antibodies. The fluorescence quantum yield and the dissociation constant of single antibody molecules were assumed to depend on the single molecule degree of labeling. In model 1 (A, graphs on the left) and model 2 (B, graphs on the right) antibody affinity and dye quantum yield, respectively, are affected more significantly by labeling. The dependence of the quantum yield and the dissociation constant on the single molecule DOL is shown in the bottom. Antibody stock solutions with a certain mean DOL were assumed to contain a Poissonian mixture of antibody species with different single molecule DOLs. The intensity of these antibody stocks and the fluorescence intensity of the cell-bound fraction were calculated as described in the Theory according to Eqs. 9 and 10 (graphs on the top). For calculating the cell-bound curves a total antibody concentration of 20 μg/mL was assumed. So that these graphs are comparable to those measured experimentally (Fig. 1; Fig. S3) the intensities are normalized to those calculated for the lowest DOL. The dashed line shows how the fluorescence intensity would depend on the mean DOL if neither the dissociation constant, nor the quantum yield were influenced by labeling. The mean degree of labeling of the cell-bound fraction is shown in the graphs in the middle. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
Anisotropy of antibody stocks and cell-bound antibodies as a function of the degree of labeling. The fluorescence anisotropy of 20 nM stock solutions of antibodies with different DOLs was measured by fluorometry (●) and the mean anisotropies were fitted according to Runnels and Scarlata (Eq. 15). Results of the fitting are shown in the bottom left corner of the graphs (d = R0/R, and r1 is the estimated anisotropy for a single fluorophore on the antibody, with the 95% confidence intervals displayed in brackets). Antibody stock solutions (○) and cells labeled with antibodies (▲) were immunoprecipitated with protein G and the anisotropy of the isolated antibodies was measured by fluorometry. Error bars represent the SE (n = 3–5).
Biophysical Journal  , DOI: ( /j.bpj )
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5. Figure 4
Fluorescence lifetimes and intensities of antibody stock solutions with different degrees of labeling. The fluorescence lifetimes of antibodies were determined in the frequency domain and were normalized to the lifetime of the antibody with the lowest DOL. The unnormalized lifetimes of the antibody with the lowest DOL are shown in every plot. Fluorescence intensities of the same antibody stock solutions were also determined by fluorometry and the intensity values normalized both by the DOL and by the intensity of the antibody with the lowest DOL are also shown for comparison. Error bars represent the SD (n = 3). The lifetimes of the other two antibodies (trastuzumab, L243) are shown in Fig. S7.
Biophysical Journal  , DOI: ( /j.bpj )
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6. Figure 5
Absorption and excitation spectra of AlexaFluor546 and AlexaFluor647 alone and bound to antibodies. The absorption spectra of antibody-conjugated fluorophores at a concentration of 3 μM were recorded by photometry. Excitation spectra of 20 nM solutions of antibodies were recorded at an emission wavelength of 610 and 690 nm for AlexaFluor546 and AlexaFluor647, respectively. Spectra were normalized to the peak value. The absorption and excitation spectra of unconjugated, free dye molecules were recorded at dye concentrations of 3 μM and 20 nM, respectively. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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7. Figure 6
Single molecule fluorescence measurements of free and cell-bound antibodies. Cells were labeled with the indicated antibodies mixed with a 500-fold or 1000-fold molar excess of unlabeled antibodies to decrease the surface density of labeled, cell membrane-bound antibodies to such an extent that diffraction limited fluorescence spots correspond to single antibodies. Antibody stock solutions of the same kind of antibodies at two different concentrations (0.1 and 0.05 μg/mL) were immobilized on the surface of epoxy-functionalized coverslips. Both the cell-bound antibodies and those covalently cross linked to the surface of epoxy-coated coverslips were imaged under the same conditions. Images were recorded from a layer adjacent to the coverslip in photon counting mode. The intensity of single, diffraction limited fluorescence spots, shown in Fig. S10, was determined from the integral of 2D Gaussian curves fitted to the intensity distribution. The histograms typically represent data from 1000 to 1500 diffraction limited spots. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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