Journal: bioRxiv

Article Title: Affinity maturation of a single-domain antibody via rational mutagenesis informed by artificial intelligence

doi: 10.64898/2026.10.13.758789

Figure Lengend Snippet: A) IL-12 binding of CDR3-sequence clustered clones in ELISA with crude extracts shows four CDR3 clusters with IL-12 binding >2 times background (OD 480 nm of 0.05), selected clones are indicated in orange. Data shown is from a single experiment. B) ELISA dose-response curve of the wildtype QSY-2 clones binding to immobilised IL-12 Bound sdAbs were detected using rabbit-anti-VHH and HRP-conjugated donkey-anti-rabbit antibodies and OPD as substrate. Data represent mean values from a single experiment conducted in technical triplicates.

Article Snippet: Phage display selections were performed on recombinant IL-12 (Sino Biological, CT011-H08H) in three panning rounds.

Techniques: Binding Assay, Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Affinity maturation of a single-domain antibody via rational mutagenesis informed by artificial intelligence

doi: 10.64898/2026.10.13.758789

Figure Lengend Snippet: A) Structure prediction of the IL-12 heterodimer in complex with the lead sdAb 2F3 (left panel) and coloured by plDDT score (right panel). Disulfides and N-linked glycans are shown as sticks. B) predicted Aligned Error (pAE): Expected Position Error versus Aligned Residue C) ELISA dose-response curve of the wildtype QSY-2F3 (2F3_wt) and single amino acid substitution mutants binding to immobilised IL-12. Bound sdAbs were detected using rabbit-anti-VHH and HRP-conjugated donkey-anti-rabbit antibodies and OPD as substrate. Data represent values from a singlet measurement from a single experiment.

Article Snippet: Phage display selections were performed on recombinant IL-12 (Sino Biological, CT011-H08H) in three panning rounds.

Techniques: Residue, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: bioRxiv

Article Title: Affinity maturation of a single-domain antibody via rational mutagenesis informed by artificial intelligence

doi: 10.64898/2026.10.13.758789

Figure Lengend Snippet: A) ELISA dose-response curves of the wildtype 2F3_wt clone, single amino acid substitution and combination mutants binding to immobilised IL-12. Bound sdAbs were detected using rabbit-anti-VHH and HRP-conjugated donkey-anti-rabbit antibodies and OPD as substrate. Data represent mean values from a single experiment conducted in technical triplicates. B) The amino acid substitutions in the G31D (left), S54K (middle) and the A75R (right) mutant introduced extra salt bridges between the sdAb and IL-12, as determined through modeling of the sdAb/IL12 complexes.

Article Snippet: Phage display selections were performed on recombinant IL-12 (Sino Biological, CT011-H08H) in three panning rounds.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Affinity maturation of a single-domain antibody via rational mutagenesis informed by artificial intelligence

doi: 10.64898/2026.10.13.758789

Figure Lengend Snippet: Binding affinities of the 2F3_wt and triple mutant for IL-12 by SPR. Association and dissociation rates from surface plasmon resonance, and binding affinity determined from these rates as well as from an equilibrium fit, in good agreement with each other.

Article Snippet: Phage display selections were performed on recombinant IL-12 (Sino Biological, CT011-H08H) in three panning rounds.

Techniques: Binding Assay, Mutagenesis, SPR Assay

Journal: bioRxiv

Article Title: Powassan Virus NS5 Antagonizes TYK2-Mediated Immune Signaling Pathways

doi: 10.64898/2026.09.07.749923

Figure Lengend Snippet: ( A ) Schematic depicting TYK2 involvement in IFN-λ and IL-12 signaling. ( B ) HepG2 hepatocyte cells were stimulated with IFN-λ 1 (50ng/mL) for 24hr, infected with POWV SP (MOI 1 for 48hr), then subjected to RNA extraction and qRT-PCR for POWV SP NS5 RNA normalized to 18S RNA ( n =3 independent experiments). ( C, D ) HepG2 hepatoma cells ( C ) or HaCaT keratinocyte cells ( D ) stably expressing POWV NS5 or empty vector controls left unstimulated or stimulated with IFN-λ 1 (50ng/mL) for 24hr, then subjected to RNA extraction and qRT-PCR. Ifit1 expression was normalized to 18S RNA ( n =3 independent experiments; ordinary one-way ANOVA with Šidák’s multiple comparisons test). ( E ) HMC3 cells were infected with POWV SP (MOI 0.1 for 48h), left unstimulated or stimulated with 50ng/mL IFN-λ 1 or 5ng/ml IFN-β for 20min, then lysates were subjected to western blotting. ( F ) HEK-Blue IL-12 cells were infected with POWV SP (MOI 1 for 48h), left unstimulated or stimulated with IL-12 (100ng/mL) for 24hr, and SEAP reporter enzyme activity measured in the supernatant (n=3 independent experiments; Mann-Whitney test). ( G ) HEK-Blue IL-12 cells stably expressing an empty vector control were stimulated with increasing concentrations of IL-12 for 24h and the reporter enzyme activity was measured in the supernatant (n=3 independent experiments). ( H ) HEK-Blue IL-12 cells stably expressing WNV NS5, POWV LB NS5, POWV SP NS5 or an empty vector control were left unstimulated or stimulated with IL-12 (100ng/mL) for 24hr, and the reporter enzyme activity was measured in the supernatant (n=3 independent experiments; Brown-Forsythe ANOVA with Dunnett’s T3 multiple comparisons test). For all data, the mean ± SD is shown. Significance is indicated by ∗∗ p < 0.005.

Article Snippet: HEK-Blue IL-12 cells (Invivogen hkb-il12) were cultured according to manufacturer’s instructions.

Techniques: Infection, RNA Extraction, Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Activity Assay, MANN-WHITNEY, Control

Journal: bioRxiv

Article Title: Powassan Virus NS5 Antagonizes TYK2-Mediated Immune Signaling Pathways

doi: 10.64898/2026.09.07.749923

Figure Lengend Snippet: Lysates from HepG2 ( A ) and HaCaT ( B ) cells transduced with lentiviruses to stably express Strep-tagged POWV LB NS5, POWV SP NS5, or empty vector were analyzed via western blot with anti-Strep antibody. ( C ) HEK-Blue IL-12 cells were infected with POWV SP (MOI 1 for 48hr) and viral RNA measured by qRT-PCR. The mean ± SD is shown. ( D ) HEK-Blue IL-12 cells transduced with lentiviruses to stably express Strep-tagged POWV LB NS5, POWV SP NS5, WNV NS5, or empty vector were analyzed via western blot with anti-Strep antibody. Representative images shown.

Article Snippet: HEK-Blue IL-12 cells (Invivogen hkb-il12) were cultured according to manufacturer’s instructions.

Techniques: Transduction, Stable Transfection, Plasmid Preparation, Western Blot, Infection, Quantitative RT-PCR

Journal: Journal of Microbiology and Biotechnology

Article Title: Anti-PD-1 Therapy Alleviates Klebsiella pneumoniae -Induced Liver Abscess by Effectively Activating Kupffer Cells

doi: 10.4014/jmb.2602.02040

Figure Lengend Snippet: ( A ) Western blot detection of PD-1 protein expression; ( B ) In vitro bactericidal activity of Kupffer cells against K. pneumoniae evaluated by enumerating viable bacteria; ( C–E ) IL-6 ( C ), IL-12 ( D ), and TNF-α ( E ) levels measured by ELISA; (F-H) Flow-cytometric detection of IL-6( F ), IL-12( G ), and TNF-α(H) proportions; ( I ) Flow-cytometric analysis of Kupffer-cell phagocytic capacity; ( J ) Western blot detection of SHP2, p-SHP2, STAT1, p-STAT1, NF-κB p65, and p-NF-κB p65. * P < 0.05.

Article Snippet: The concentrations of TNF-α (E-EL-M3063, Elabscience, China), IL-6 (E-EL-M0044, Elabscience, China), and IL-12 (E-EL-M3062, Elabscience, China) in the supernatant were determined with ELISA kits.

Techniques: Western Blot, Expressing, In Vitro, Activity Assay, Bacteria, Enzyme-linked Immunosorbent Assay, Analysis

Journal: Journal of Microbiology and Biotechnology

Article Title: Anti-PD-1 Therapy Alleviates Klebsiella pneumoniae -Induced Liver Abscess by Effectively Activating Kupffer Cells

doi: 10.4014/jmb.2602.02040

Figure Lengend Snippet: ( A ) In vitro bactericidal activity of Kupffer cells against K. pneumoniae evaluated by enumerating viable bacteria; ( B–D ) IL-6 ( B ), IL-12 ( C ), and TNF-α ( D ) levels measured by ELISA; ( E-G ) Flow cytometry detection of IL-6 ( E ), IL-12 ( F ), and TNF-α ( G ) proportions; ( H ) Flow-cytometric analysis of Kupffer-cell phagocytic capacity; ( I ) Western blot detection of SHP2, p-SHP2, STAT1, p-STAT1, NF-κB p65, and p-NF-κB p65. * P < 0.05.

Article Snippet: The concentrations of TNF-α (E-EL-M3063, Elabscience, China), IL-6 (E-EL-M0044, Elabscience, China), and IL-12 (E-EL-M3062, Elabscience, China) in the supernatant were determined with ELISA kits.

Techniques: In Vitro, Activity Assay, Bacteria, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Analysis, Western Blot

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: PBMCs are cultured with or without L . bulgaricus OLL1073R-1 or S. thermophilus OLS3059 for 24 h. The expression of CD86, CD40, and HLA-DR in (A) cDCs and (B) pDCs was analyzed by flow cytometry. Representative histograms (black, control; red, OLL1073R-1; blue, OLS3059) and the corresponding geometric mean fluorescent intensity (gMFI) are shown (n = 6). (C) Representative plots of IL-12 expression in cDCs were shown, and the ratio of IL-12 expressing cells in cDCs were analyzed (n = 5). *: p < 0.05, **: p < 0.01, NS: not significant by Dunnett’s test.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Cell Culture, Expressing, Flow Cytometry, Control

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: (A) Isolated cDCs and pDCs are cultured with or without L . bulgaricus OLL1073R-1 for 24 h. IL-12p40, IFN-α2a, IFN-β levels in supernatants were analyzed (n = 3). (B) PBMCs (n = 4), cDCs-depleted PBMCs (n = 3) and pDCs-depleted PBMCs (n = 3) were cultured with or without L . bulgaricus OLL1073R-1 for 24 h. IFN-α2a, IFN-β levels in supernatants were analyzed. (C) PBMCs, cDCs-depleted PBMCs and pDCs-depleted PBMCs were cultured with or without L . bulgaricus OLL1073R-1 under the stimulation of influenza antigen. The ratio of IFN-γ expressing cells in CD4(+) T EM were analyzed 24 h after incubation (n = 5, *: p < 0.05, NS: not significant by Student’s t-test). IL-12 levels in supernatants were analyzed 20 h after incubation (n = 3). *: p < 0.05, NS: not significant by Mann-Whitney U test unless otherwise noted. Data below detection limit were calculated as zero.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Isolation, Cell Culture, Expressing, Incubation, MANN-WHITNEY

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: PBMCs are cultured with multiple L. bulgaricus strains for 24 h. (A) The ratio of IL-12 expressing cells in cDCs and (B) Geometric mean fluorescent intensity (gMFI) of CD86 expression were analyzed (n = 5). **: p < 0.01, NS: not significant by Dunnett’s test.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Cell Culture, Expressing

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: PBMCs are cultured with or without L . bulgaricus OLL1073R-1 or S. thermophilus OLS3059 for 24 h. The expression of CD86, CD40, and HLA-DR in (A) cDCs and (B) pDCs was analyzed by flow cytometry. Representative histograms (black, control; red, OLL1073R-1; blue, OLS3059) and the corresponding geometric mean fluorescent intensity (gMFI) are shown (n = 6). (C) Representative plots of IL-12 expression in cDCs were shown, and the ratio of IL-12 expressing cells in cDCs were analyzed (n = 5). *: p < 0.05, **: p < 0.01, NS: not significant by Dunnett’s test.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Cell Culture, Expressing, Flow Cytometry, Control

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: (A) Isolated cDCs and pDCs are cultured with or without L . bulgaricus OLL1073R-1 for 24 h. IL-12p40, IFN-α2a, IFN-β levels in supernatants were analyzed (n = 3). (B) PBMCs (n = 4), cDCs-depleted PBMCs (n = 3) and pDCs-depleted PBMCs (n = 3) were cultured with or without L . bulgaricus OLL1073R-1 for 24 h. IFN-α2a, IFN-β levels in supernatants were analyzed. (C) PBMCs, cDCs-depleted PBMCs and pDCs-depleted PBMCs were cultured with or without L . bulgaricus OLL1073R-1 under the stimulation of influenza antigen. The ratio of IFN-γ expressing cells in CD4(+) T EM were analyzed 24 h after incubation (n = 5, *: p < 0.05, NS: not significant by Student’s t-test). IL-12 levels in supernatants were analyzed 20 h after incubation (n = 3). *: p < 0.05, NS: not significant by Mann-Whitney U test unless otherwise noted. Data below detection limit were calculated as zero.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Isolation, Cell Culture, Expressing, Incubation, MANN-WHITNEY

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: PBMCs are cultured with multiple L. bulgaricus strains for 24 h. (A) The ratio of IL-12 expressing cells in cDCs and (B) Geometric mean fluorescent intensity (gMFI) of CD86 expression were analyzed (n = 5). **: p < 0.01, NS: not significant by Dunnett’s test.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Cell Culture, Expressing