Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: (a) Schematic of laser-cut PDMS microwell array reversibly assembled on a polystyrene petri dish and workflow of enzymatically amplified silver metallization using Biotin-BSA and poly-HRP-SA. (b) Cell-phone image of silver metallized wells after assay. (c) Biotin-BSA dilution curve obtained from cell-phone image silver darkness quantification in ImageJ. (d) Assay stack for initial biotinyl tyramide signal amplification testing using biotinylated anti-IFN-γ as capture. (e) Dilution curve of silver darkness of increasing biotinylated anti-IFN-γ as target and 10 µg/mL, 1 µg/mL, and 0 µg/mL biotinyl tyramide. All assays were repeated three (n=3) times and fit lines are four-parameter logistic sigmoidal curves.

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques: Amplification

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: (a) Schematic of immunoassay for cytokine detection. (b) Initial LOD optimization for the IFN-γ assay. (c-e) IFN-γ (n=2), IL-2 (n=5), TNF-α (n=2) dilution curves using recombinant cytokines (f-h) Correlation plots of ELISA absorbance values at 450 nm versus silver darkness values for IFN-γ (f), TNF-α (g), and IL-2 (h). All fit lines are four-parameter logistic sigmoidal curves.

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: (a) Schematic demonstrating the stimulation groups and controls. Dilution curves for IFN-γ (b-c), IL-2 (d-e), and TNF-α (f-g) in pooled LTB+ and LTB-clinical supernatant samples. Clinical samples consisted of NIL (negative control), Ag1, Ag2, and Mitogen (positive control) groups (n=2).

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques: Negative Control, Positive Control

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: Individual participant silver darkness values for IFN-γ (a), IL-2 (b), and TNF-α (c) detection from NIL, Ag1, Ag2, and Mitogen samples of IGRA+ and IGRA-patients using a sample dilution of 1:10. Average of n=2 repeats was plotted for each sample.

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: The NewroBus platform: engineered humanized anti-TfR1 nanobodies for efficient brain delivery

doi: 10.1186/s12964-025-02605-1

Figure Lengend Snippet: Fusion to TfR1b-Nb enables CNS delivery of TNFI-Nb1 via human TfR1-mediated transcytosis. a TNFα-based ELISA quantification of TNFI-Nb1 in serum and brain homogenates from Tfr1 h/w and Tfr1 w/w rats, 6 h after intravenous injection. TNFI-Nb1 was detectable in serum but absent in brain tissue, indicating lack of BBB permeability. b SDS-PAGE and SEC-HPLC analysis of the heterodimer composed of TNFI-Nb1 fused to TfR1b-Nb 04B05R3 (Family A), produced in CHO-S cells. C ) TNFα-based ELISA quantification of TNFI-Nb1 ~ 04B05R3 in serum, CSF, and brain homogenates of Tfr1 h/w and Tfr1 w/w rats, 48 h post-injection. The heterodimer was present in serum of both genotypes but detected in CSF and brain only in Tfr1 h/w rats, demonstrating human TfR1-dependent BBB transcytosis. The observed CSF/serum ratio (TNFα-based ELISA) of ~ 1 indicates efficient CNS delivery. Statistical analyses were conducted using unpaired two-tailed t-tests, with p-values reported in the figure panels. Data are presented as mean ± SEM

Article Snippet: The same procedure was used with the following modifications: plates were coated with 0.2 μg/mL biotinylated human TNFα (Acro Biosystems, TNA-H82E3-25ug) instead of TfR1.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Permeability, SDS Page, Produced, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: The NewroBus platform: engineered humanized anti-TfR1 nanobodies for efficient brain delivery

doi: 10.1186/s12964-025-02605-1

Figure Lengend Snippet: TfR1 binding and TNFα inhibitory activity of optimized and humanized heterodimers. a Schematic representation of the 16 humanized and optimized TNFI–TfR1b heterodimers. b Flow cytometry analysis in HEK293 cells stably expressing human TfR1, comparing the binding of heterodimers to that of their parental humanized TfR1b-Nbs. c ) Dose–response apoptosis inhibition curves in WEHI-13VAR cells treated with human TNFα and twofold serial dilutions of TNFI–TfR1b heterodimers (100,000 to 12.21 pM). Apoptosis was measured using a fluorogenic caspase-3/7 activation assay. Caspase-3/7 activity in the TNFα-only samples was defined as 100%. The IC₅₀ values of the heterodimers were comparable to those of the parental humanized nanobodies TNFI-α (IC₅₀ = 48.84 pM, range: 42.11–56.56) and TNFI-β (IC₅₀ = 64.31 pM, range: 57.17–72.31).). Data are presented as mean ± SEM from triplicate measurements and were analyzed using the "Inhibitor vs. Normalized Response" model in GraphPad Prism 10 software

Article Snippet: The same procedure was used with the following modifications: plates were coated with 0.2 μg/mL biotinylated human TNFα (Acro Biosystems, TNA-H82E3-25ug) instead of TfR1.

Techniques: Binding Assay, Activity Assay, Flow Cytometry, Stable Transfection, Expressing, Inhibition, Activation Assay, Software

Journal: Cell Communication and Signaling : CCS

Article Title: The NewroBus platform: engineered humanized anti-TfR1 nanobodies for efficient brain delivery

doi: 10.1186/s12964-025-02605-1

Figure Lengend Snippet: Fusion to TfR1b-Nb enables CNS delivery of TNFI-Nb1 via human TfR1-mediated transcytosis. a TNFα-based ELISA quantification of TNFI-Nb1 in serum and brain homogenates from Tfr1 h/w and Tfr1 w/w rats, 6 h after intravenous injection. TNFI-Nb1 was detectable in serum but absent in brain tissue, indicating lack of BBB permeability. b SDS-PAGE and SEC-HPLC analysis of the heterodimer composed of TNFI-Nb1 fused to TfR1b-Nb 04B05R3 (Family A), produced in CHO-S cells. C ) TNFα-based ELISA quantification of TNFI-Nb1 ~ 04B05R3 in serum, CSF, and brain homogenates of Tfr1 h/w and Tfr1 w/w rats, 48 h post-injection. The heterodimer was present in serum of both genotypes but detected in CSF and brain only in Tfr1 h/w rats, demonstrating human TfR1-dependent BBB transcytosis. The observed CSF/serum ratio (TNFα-based ELISA) of ~ 1 indicates efficient CNS delivery. Statistical analyses were conducted using unpaired two-tailed t-tests, with p-values reported in the figure panels. Data are presented as mean ± SEM

Article Snippet: Biotinylated TNFα , ELISA capture antigen , Acro Biosystems , TNA-H82E3-25ug.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Permeability, SDS Page, Produced, Two Tailed Test