Journal: mAbs

Article Title: Generation of robust bispecific antibodies through fusion of single-domain antibodies on IgG scaffolds: a comprehensive comparison of formats

doi: 10.1080/19420862.2023.2189432

Figure Lengend Snippet: Antibody binding to PD-L1 and HER2 using FIDA. (a) Assessment of bispecific binding functionality through changes in apparent Rh in response to addition of fluorescent PD-L1-DY490 ± unlabeled HER2 antigens in solution (b) Assessment of bispecific binding functionality through changes in apparent Rh in response to addition of fluorescent HER2-DY490 ± unlabeled PD-L1 antigens in solution. (c) Antibody titration curves investigating binding in mono- and bispecific binding environments. The binding curves were generated by titrating antibody against the fluorescently labeled primary antigen ± an unlabeled secondary antigen. Since the secondary antigen does not carry any fluorescence, it can only affect the signal indirectly through complexation with the bsAb, which is in itself binding the primary antigen. This is to test if the titration curves change in response to addition of the unlabeled secondary antigen. The coloring scheme is the same as in (a) and (b).

Article Snippet: Rh values were obtained by Taylorgrams to FIDA Software v2.3 (Fida Biosystems) with a Taylorgram fraction of 75%.

Techniques: Binding Assay, Titration, Generated, Labeling, Fluorescence

Journal: mAbs

Article Title: Generation of robust bispecific antibodies through fusion of single-domain antibodies on IgG scaffolds: a comprehensive comparison of formats

doi: 10.1080/19420862.2023.2189432

Figure Lengend Snippet: Affinity binding constants and the related goodness-of-fit from antibody titration curves using FIDA. The titration curves were generated by titrating antibodies against a fluorescently labeled antigen ± the unlabeled antigen. The goodness-of-fit is evaluated using R 2 and root mean squared error (RMSE). Affinity values in brackets marked with an asterisk indicate affinity values previously reported elsewhere. 20 , 21 , 33 , 34 .

Article Snippet: Rh values were obtained by Taylorgrams to FIDA Software v2.3 (Fida Biosystems) with a Taylorgram fraction of 75%.

Techniques: Binding Assay, Titration, Generated, Labeling

Journal: bioRxiv

Article Title: Selective Targeting of Pathogenic Tau Seeds via a Novel VHH

doi: 10.1101/2025.01.13.632833

Figure Lengend Snippet: ( A - C ) Immunoprecipitation was performed with purified wild-type VHHs (anti-tau VHH(50) and VHH(510)) and their stabilized counterparts (anti-tau VHH(50M) and VHH(510M)) using Amsphere™ A3 resin across different concentrations of lysate total protein. Bar graphs show the resulting FRET% from v2L tau biosensor cells following IP and elution from ( A ) AD brain lysate, ( B ) CBD brain lysate, and ( C ) P301S mouse brain lysate. A non-specific (NS) VHH was included as a negative control.

Article Snippet: Flow Induced Dispersion Analysis (FIDA) measurement for anti-tau VHH(510M) and VHH(50M) were performed on Fida 1 (Fida Biosystems, Denmark) using a dynamic coated (Fida Biosystems, Denmark) 100cm capillary with 75cm length (Fida Biosystems, Denmark).

Techniques: Immunoprecipitation, Purification, Negative Control

Journal: bioRxiv

Article Title: Selective Targeting of Pathogenic Tau Seeds via a Novel VHH

doi: 10.1101/2025.01.13.632833

Figure Lengend Snippet: ( A - C ) Seed capture from two AD brain samples (AD1, AD2), two CBD brain samples (CBD1, CBD2), and a P301S mouse brain, respectively, using anti-tau VHH(510M) or VHH(50M). ( D ) Immunoprecipitation from a non-tauopathy control brain. Bars indicate mean ± S.D. (n=3). Significance was evaluated against a non-specific VHH (NS VHH) and bead-only controls using two-way ANOVA with Tukey’s multiple comparison test (AD, CBD) or one-way ANOVA with Šídák’s multiple comparisons test (P301S). ***p ≤ 0.0002; **p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Flow Induced Dispersion Analysis (FIDA) measurement for anti-tau VHH(510M) and VHH(50M) were performed on Fida 1 (Fida Biosystems, Denmark) using a dynamic coated (Fida Biosystems, Denmark) 100cm capillary with 75cm length (Fida Biosystems, Denmark).

Techniques: Immunoprecipitation, Control, Comparison

Journal: bioRxiv

Article Title: Selective Targeting of Pathogenic Tau Seeds via a Novel VHH

doi: 10.1101/2025.01.13.632833

Figure Lengend Snippet: ( A ) Dot blots using anti-tau VHH(510M) (left) and anti-tau VHH50M (right) as detection molecules against serial dilutions of lysates from two brains of AD cases (AD1, AD2), a P301S mouse model, and control. ( B ) Quantification of signal intensity at 1.25 µg of total protein, based on integrated optical densities measured with Fiji (version 1.54f). Anti-tau VHH(510M) (left panel) preferentially binds to AD and P301S lysates while showing minimal reactivity to control tissue. By contrast, VHH50M (right panel) shows broadly similar signal across all tested samples. Error bars represent S.D. (n = 4). Statistical significance was assessed with one-way ANOVA followed by Šídák’s multiple comparisons test (p ≤ 0.0029, **p ≤ 0.0001).

Article Snippet: Flow Induced Dispersion Analysis (FIDA) measurement for anti-tau VHH(510M) and VHH(50M) were performed on Fida 1 (Fida Biosystems, Denmark) using a dynamic coated (Fida Biosystems, Denmark) 100cm capillary with 75cm length (Fida Biosystems, Denmark).

Techniques: Control

Journal: bioRxiv

Article Title: Selective Targeting of Pathogenic Tau Seeds via a Novel VHH

doi: 10.1101/2025.01.13.632833

Figure Lengend Snippet: ( A ) Representative thermal denaturation profile of VHH(510M) monitored by circular dichroism (CD) at 215 nm (θ 215 ), measured from 4 °C to 95 °C. Data were fitted to a sigmoidal model to estimate the midpoint of thermal unfolding (T m ). See Figure S7 for additional details. ( B ) In-solution affinity measurement of VHH(510M) using Flow-Induced Dispersion Analysis (FIDA). The hydrodynamic radius (R h ) of fluorescently labeled VHH(510M) was plotted against increasing concentrations of tau 2N4R monomer. Fitting to a 1:1 binding model yielded a K_D of ∼4.2 µM. See Figure S8 for more information. ( C ) Overlaid 2D 1 H, 15 N-HSQC spectrum of 15 N-labeled tau 2N4R alone (black) and in the presence of equimolar VHH(510M) (blue). Several peaks—particularly in the C-terminal region—shift or disappear upon binding, indicating that (VHH510M) recognizes residues at the carboxy-terminus of tau. Insets show enlarged views of selected regions highlighting peak perturbations.

Article Snippet: Flow Induced Dispersion Analysis (FIDA) measurement for anti-tau VHH(510M) and VHH(50M) were performed on Fida 1 (Fida Biosystems, Denmark) using a dynamic coated (Fida Biosystems, Denmark) 100cm capillary with 75cm length (Fida Biosystems, Denmark).

Techniques: Circular Dichroism, Dispersion, Labeling, Binding Assay

Journal: bioRxiv

Article Title: Selective Targeting of Pathogenic Tau Seeds via a Novel VHH

doi: 10.1101/2025.01.13.632833

Figure Lengend Snippet: Shown are representative images from 3-, 6-, 9-, and 12-month-old P301S tauopathy mice stained with the phospho-tau antibody AT8 (green) and anti-tau VHH510M (red). Anti-tau VHH510M labels pathological inclusions that overlap with AT8-positive aggregates but also highlights additional inclusions not recognized by AT8 (white arrows). These observations suggest that VHH(510M) detects both phospho-tau and non-phosphorylated forms of aggregated tau. See Figure S9 for additional images and details.

Article Snippet: Flow Induced Dispersion Analysis (FIDA) measurement for anti-tau VHH(510M) and VHH(50M) were performed on Fida 1 (Fida Biosystems, Denmark) using a dynamic coated (Fida Biosystems, Denmark) 100cm capillary with 75cm length (Fida Biosystems, Denmark).

Techniques: Staining

Journal: bioRxiv

Article Title: Selective Targeting of Pathogenic Tau Seeds via a Novel VHH

doi: 10.1101/2025.01.13.632833

Figure Lengend Snippet: Representative images from tissue microarray sections of fixed frontal cortex of an AD case. Staining with the phospho-tau antibody AT8 (green) and anti-tau VHH(510M) (red) reveals co-localization of tau inclusions in overlapping regions (yellow in overlay). These findings support the selectivity of VHH(510M) for disease-relevant tau aggregates in human AD brains.

Article Snippet: Flow Induced Dispersion Analysis (FIDA) measurement for anti-tau VHH(510M) and VHH(50M) were performed on Fida 1 (Fida Biosystems, Denmark) using a dynamic coated (Fida Biosystems, Denmark) 100cm capillary with 75cm length (Fida Biosystems, Denmark).

Techniques: Microarray, Staining

Journal: The Journal of Physical Chemistry. B

Article Title: Two Receptor Binding Strategy of SARS-CoV-2 Is Mediated by Both the N-Terminal and Receptor-Binding Spike Domain

doi: 10.1021/acs.jpcb.3c06258

Figure Lengend Snippet: FIDA of SARS-CoV-2 spike S1 segment binding to SA containing glycolipids. (a) Schematic of how complex formation by fluorescently labeled S1 will affect the Taylorgrams of FIDA measurements. A single species that includes a fluorescent label will give rise to a single Gaussian line shape, with a Gaussian width σ that increases with an increasing hydrodynamic radius. (b) Taylorgrams recorded for pure 100 nM spike S1 protein, with 200 μM DPPC vesicles, and with 1:9 GM1:DPPC vesicles, partly composed of the SA containing GM1 glycolipid. In the case of the glycolipid-containing vesicles, a double Gaussian line shape is observed, indicating the presence of both free S1 species and S1-vesicle complexes, as opposed to the single Gaussian lineshapes observed for the pure S1 for in the presence of the DPPC vesicles that do not contain glycolipids, indicating the absence of S1-binding in that case. (c) The relative number of bound species for the S1 segments of SARS-CoV, SARS-CoV-2, and MERS-CoV bound to (1:9)-GM1:DPPC and -GM3:DPPC lipid vesicles (lipid concentration in monomer units). Lines show best fit to eq . (d) Estimated dissociation constants (K D s).

Article Snippet: Finally, the injected indicator sample is then flowed toward the detection point with the vesicle sample at 50 mbar for 20 min. All samples were performed in duplicate, and the Taylorgrams were processed using the FIDA data analysis software (Fida Biosystems ApS, Copenhagen, Denmark).

Techniques: Binding Assay, Labeling, Concentration Assay