Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) ICN1-transduced human ETPs were treated with either blocking anti-CD44 mAb (515; IgG1) or control IgG1 before transplantation into RAG-2–/– × γc–/– mice. Absolute ICN1+ cell numbers infiltrating the BM of 5 mice/group were analyzed at 3 weeks after transplant. (B) Schematic representation of experiment design (left): RAG-2–/– × γc–/– mice transplanted with ICN1-transduced ETPs received 3 weekly i.p. injections of either blocking anti-CD44 (HP2/9; IgG1) mAb or control IgG1, starting at day 5 after transplant. (Right) Absolute numbers of human ICN1+ cells infiltrating the BM of 4 mice/group analyzed at day 15 after transplant. (C) Human cell numbers infiltrating the BM of RAG-2–/– × γc–/– mice (5/group) after 3 weeks of transplant with ETPs transduced with either ICN1 or CD44. Data in A–C were normalized to 105 transduced input cells (n = 3). (D) Mean percentages ± SEM of human cells infiltrating the BM of RAG-2–/– × γc–/– mice (4/group) transplanted with ETPs cotransduced with ICN1 along with either a CD44-encoding or a control retrovirus (n = 4). (E) Cells obtained from the BM of a T-ALL patient (T-ALL1) were pretreated with either blocking anti-CD44 mAb 515 or control IgG1 and then transplanted into RAG-2–/– × γc–/– mice. Relative T-ALL1 cell numbers infiltrating the BM, PB, spleen, and thymus of 4 mice/group were analyzed at the indicated times after transplant (n = 3). *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: In vivo persistence of HP2/9 mAb on the surface of T-ALL cells was analyzed by FACS using a PE-labeled goat anti-mouse IgG1 Ab (Southern Biotech).

Techniques: Blocking Assay, Transplantation Assay, Transduction

Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) RAG-2–/– × γc–/– mice transplanted with primary human T-ALL1 or T-ALL2 cells (Supplemental Figure 4) received 3 weekly i.p. injections of either blocking anti-CD44 mAb (HP2/9) or control IgG1 during 4 weeks, starting at 1 week (B/C) or 5 weeks (D/E/F) after transplant. When indicated, T-ALL cells recovered from the BM of transplanted mice at the end of treatment were transplanted into secondary hosts. (B) Percentages of T-ALL1 and T-ALL2 cells infiltrating the BM, PB, and spleen of mice treated from weeks 1 to 5 after transplant as shown in A. Mean values from 3 independent experiments with a total of 13 to 18 mice for T-ALL and 3 to 8 mice for T-ALL2 are shown. (C) Image of representative spleens obtained at the end of treatment from mice shown in B. (D) Thorough analysis of anti-CD44 in vivo treatment showing percentages of human T-ALL2 cells infiltrating the BM (left) and PB (right) of 5 mice/group treated from weeks 5 to 9 after transplant as shown in A. Empty symbols represent cell percentages before the onset of Ab treatment. Boxes identify individual donors for secondary transplantations shown in F. (E) Kaplan-Meier survival curve of mice treated with anti-CD44 mAb (HP2/9) or control IgG1 in D. (F) Kaplan-Meier survival curve of secondary recipients transplanted with T-ALL2 cells (33 cells/mouse) obtained from the BM of individual donors represented as boxed in D. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: In vivo persistence of HP2/9 mAb on the surface of T-ALL cells was analyzed by FACS using a PE-labeled goat anti-mouse IgG1 Ab (Southern Biotech).

Techniques: Blocking Assay, In Vivo

Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) NSG mice transplanted with primary T-ALL2 cells received 3 weekly i.p. injections of either blocking anti-CD44 mAb (HP2/9) or control IgG1 during 7 weeks, starting at 1 week after transplant. (B) Image of representative spleens obtained at the end of treatment from mice shown in A. (C) Percentages of T-ALL2 cells infiltrating the BM, PB, and spleen of 5 to 10 mice treated as shown in A. (D) Representative FACS analysis showing persistence of anti-CD44 HP2/9 mAb on the surface of T-ALL cells recovered from the BM of NSG mice represented in A at the end of treatment with either anti-CD44 mAb or IgG1. Bound HP2/9 mAb was detected by reactivity with PE-labeled anti-mouse IgG1 (n = 3). (E) FACS analysis showing levels of HP2/9 anti-CD44 mAb persisting on the surface of T-ALL2 cells that infiltrate the BM of anti-CD44–treated mice shown in A at the indicated weeks after transplant. Shown are MFI values from a total of 7 mice, determined as in D. (F) BM and PB infiltration of T-ALL1 cells transduced with a lentiviral vector encoding GFP and either CD44-specific shRNA (28% transduction efficiency) or a scramble control shRNA (36%). Data show percentages of GFP+ cells within infiltrating CD45+ T-ALL1 cells of 5 NSG mice/group at 11 weeks after transplant. *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: In vivo persistence of HP2/9 mAb on the surface of T-ALL cells was analyzed by FACS using a PE-labeled goat anti-mouse IgG1 Ab (Southern Biotech).

Techniques: Blocking Assay, Labeling, Transduction, Plasmid Preparation, shRNA

Journal: Scientific Reports

Article Title: In vitro complement activation via nucleocapsid and spike proteins of SARS-CoV-2 in COVID-19 patients

doi: 10.1038/s41598-025-20926-6

Figure Lengend Snippet: Comparison of the in vitro C3b and C4b complement depositions via spike (C3b(S) and C4b(S)) or nucleocapsid (C3b(N) and C4b(N)) recombinant proteins between patients with (Ab(+)) or without (Ab(-)) SARS-CoV-2-specific IgM and IgG antibodies from four different COVID-19 groups (CONV, HOSP, HOSP + O 2 and ICU). The in vitro C3b and C4b complement depositions were measured by our in-house complement deposition assays, where we used carrier free recombinant nucleocapsid or spike proteins and non-heat-inactivated patient sera. The p values for the pair-wise group comparisons on the violin plots were calculated by the Mann-Whitney tests. P values of significant differences are marked as p MW between without and with specific antibodies. Non-significant differences are marked as n.s.

Article Snippet: Mouse anti-human IgG1 Fc-HRP (HP6001) , SouthernBiotech , 9054-05.

Techniques: Comparison, In Vitro, Recombinant, MANN-WHITNEY

Journal: Scientific Reports

Article Title: In vitro complement activation via nucleocapsid and spike proteins of SARS-CoV-2 in COVID-19 patients

doi: 10.1038/s41598-025-20926-6

Figure Lengend Snippet: Comparison of the levels of SARS-CoV-2-specific IgM and IgG among four COVID-19 groups (CONV, HOSP, HOSP + O2, ICU) measured by Generic Assays CoV-2 IgM or IgG ( a ). The percentages of SARS-CoV-2-specific IgG or IgM positivity were shown also for four different COVID-19 groups. The numbers represent the mean percentages of SARS-CoV-2-specific IgM in blue or specific IgG in green circle sectors ( b ). The levels of total S- or N-specific IgG were quantified by in-house ELISA, normalized to positive and negative serum samples ( c ). The levels of S-specific and N-specific IgG1 ( d ) and IgG3 ( e ) subclasses were quantified by in-house ELISA calibrated with purified human IgG1 and IgG3. The p -values ( p KW ) were determined by Kruskal-Wallis test.

Article Snippet: Mouse anti-human IgG1 Fc-HRP (HP6001) , SouthernBiotech , 9054-05.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Purification

Journal: Scientific Reports

Article Title: In vitro complement activation via nucleocapsid and spike proteins of SARS-CoV-2 in COVID-19 patients

doi: 10.1038/s41598-025-20926-6

Figure Lengend Snippet: The in vitro C3b and C4b complement depositions via spike (C3b(S) and C4b(S)) recombinant protein are plotted by the levels of S-specific IgG1 and IgG3 in CONV individuals and in the other three hospitalized COVID-19 groups (HOSP, HOSP + O 2 and ICU) ( a ). The in vitro C3b and C4b complement depositions via nucleocapsid (C3b(N) and C4b(N)) recombinant protein are plotted by the levels of N-specific IgG1 and IgG3 in CONV individuals and in the other three hospitalized COVID-19 groups (HOSP, HOSP + O 2 and ICU) ( b ).

Article Snippet: Mouse anti-human IgG1 Fc-HRP (HP6001) , SouthernBiotech , 9054-05.

Techniques: In Vitro, Recombinant

Journal: Scientific Reports

Article Title: In vitro complement activation via nucleocapsid and spike proteins of SARS-CoV-2 in COVID-19 patients

doi: 10.1038/s41598-025-20926-6

Figure Lengend Snippet: Comparison of the levels of SARS-CoV-2-specific IgM and IgG antibodies, and in vivo complement profiles in all COVID-19 patients between low and high in vitro C3b(N) complement depositions. The p values for the pair-wise group comparisons on the violin plots were calculated by the Mann-Whitney tests. Asterisks indicate p value < 0.0143 , considered significant results after 5% FDR correction using the Benjamini-Hochberg method.

Article Snippet: Mouse anti-human IgG1 Fc-HRP (HP6001) , SouthernBiotech , 9054-05.

Techniques: Comparison, In Vivo, In Vitro, MANN-WHITNEY

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: sWTA probe generation and validation. A) schematic representation of sWTA probe generation for all three glycoforms, i.e. RboP +β-1,4-GlcNAc, +α-1,4-GlcNAc, or + β-1,3-GlcNAc. All sWTA probes were made for detection in two different fluorescence channels using streptavidin conjugated to AF647 or BB515. B) dual sWTA probe labeling of protein A-coated beads coated with anti-β-GlcNAc RboP (clone 4497), anti-α-GlcNAc RboP (clone 4461), and isotype IgG1. Data in dot plots represent geometric mean fluorescence intensity (gMFI) signals (fluorophores: AF647 and BB515) on the beads. Q2 and Q4 comprise, respectively, double positive (dual sWTA probe labeling) and double negative (no sWTA probe binding) beads. Signals within Q1 and Q3 represent aspecific binding of, respectively, AF647 and BB515 streptavidin to beads. Histograms are included on the sides of the dot plots to visualize relative amounts of different bead populations within a fluorescent channel.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Biomarker Discovery, Fluorescence, Labeling, Binding Assay

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: sWTA specificity screening of pilot-scale produced mAbs. Binding profiles of 15 B cell-derived mAbs, expressed by HEK293T cells (production levels in ) as human IgG1, to streptavidin-coated beads immobilized with biotinylated sWTA to determine clone reactivity as measured by flow cytometry. Fluorescent signals are depicted in this figure as gMFI fold changes (mean + s.d. of three independent experiments) relative to the condition without antibodies to compensate for technical variation. One-way ANOVA was performed to determine significant binding to glycan-coated beads compared to empty beads. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Produced, Binding Assay, Derivative Assay, Flow Cytometry, Glycoproteomics

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Specificity verification of sWTA-reactive mAbs at equimolar level. Clones that displayed sWTA reactivity in the pilot screening (main ) were selected for large-scale production in HEK293 freestyle cells and purified through protein G agarose. Selected clones were categorized based on their α-GlcNAc A) or β-GlcNAc B) reactivity and relative binding capacities to sWTA beads were determined at a concentration of 3 μg/ml. Beads coated with polyrhamnose (PR) + β-1,3-GlcNAc and empty beads were used as controls for cross-reactivity and background, respectively. IgG1 binding to sWTA beads was measured by flow cytometry and data represent the gMFI mean + s.d. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine significant binding of sWTA-reactive clones to glycan-coated beads compared to empty beads. ns not significant ** P < 0.01, **** P < 0.0001. Index sort data of all sorted clones can be found in .

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Clone Assay, Purification, Binding Assay, Concentration Assay, Flow Cytometry, Glycoproteomics

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Binding of sWTA-reactive mAbs to WTA on S. aureus surface. For each glycoform specificity, one mAb clone was selected to assess bacterial opsonization. A-D) binding of W1C11 (anti-α-GlcNAc), W1F10 (anti-β-GlcNAc, with preference for β-1,3-GlcNAc), W1G7 (anti-β-GlcNAc), and B12 (isotype control) to S. aureus strains N315 Δ spa A), N315 Δ spa Δ tarSP B), Newman Δ spa Δ sbi C), and Streptococcus pyogenes strain 5448 Δ gacH D). IgG1 binding to bacteria was measured using flow cytometry and data represent normalized mean gMFI + s.d. (isotype signals set to 1) of three independent experiments. N315 Δ spa Δ tarSP and S. pyogenes 5448 Δ gacH were included as controls for WTA GlcNAc (species) specificity.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Binding Assay, Control, Bacteria, Flow Cytometry

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Effector functions of anti-WTA clones towards S. aureus . A-C) C3b deposition by sWTA-reactive mAbs onto S. aureus strains N315 Δ spa A), N315 Δ spa Δ tarSP B), and Newman Δ spa Δ sbi C). Data represent C3b binding (normalized gMFI + s.d.) of three independent experiments as measured by flow cytometry. Fluorescent signals are depicted as a fold change relative to the condition without antibodies to compensate for variation in background signals between biological replicates. D) neutrophil-mediated phagocytosis of GFP-expressing S. aureus Newman Δspa Δsbi by anti-WTA mAbs. Displayed data represent percentages of GFP-positive neutrophils and are representative of three biological replicates (individual replicates can be found in ). Curves were generated using nonlinear dose–response fitting model. E) relative phagocytic capacities of the anti-WTA mAbs. Absolute IC 50 values were determined for each replicate individually using nonlinear dose–response fitting model. Black lines represent means of the IC 50 values which are depicted as data points. Assays (A-D) were performed in the presence of 1% IgG-/IgM-depleted human serum as complement source. Statistical differences compared to isotype were determined by one-way ANOVA. ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Clone Assay, Binding Assay, Flow Cytometry, Expressing, Generated

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Discovery and characterization of GAC-specific mAbs. A) schematic representation of sGAC probe generation for two glycoforms, i.e. PR and PR + β-1,3-GlcNAc. All sGAC probes were made for detection in two different fluorescence channels using streptavidin conjugated to Pe-Cy7 or BV421. B) sGAC probe binding to protein beads immobilized with goat polyclonal anti-GAC GlcNAc (Ab9191). N.B. no bead coat option was available to test PR specificity. Data in dot plots represent fluorescence signals (fluorophores: PE-Cy7 and BV421) on the beads. Q2 and Q4 comprise, respectively, double positive (dual sGAC probe labeling) and double negative (no sGAC probe binding) beads. Signals within Q1 and Q3 represent aspecific binding of, respectively, Pe-Cy7 and BV421 streptavidin to beads. C) specificity verification of sGAC-reactive mAbs at equimolar level. Clones were produced in HEK293 freestyle cells and purified through protein G agarose. Relative binding capacities to sGAC beads were determined at a concentration of 3 μg/mL. Beads coated with RboP +β-1,3-GlcNAc and empty beads were used as controls for cross-reactivity and background, respectively. IgG1 binding to sGAC beads was measured by flow cytometry and data represent the mean gMFI ± s.d. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine significant binding of sGAC-reactive clones to glycan-coated beads compared to empty beads. **** P < 0.0001. Index sort data of all sorted clones can be found in . D) binding of sGAC-reactive mAbs to natural GAC on Streptococcus pyogenes . For each glycoform specificity, one mAb clone was selected that showed evident binding to sGAC beads (panel C) and tested for bacterial opsonization. Titration of G1E8 (anti-PR), G1C4 (anti-β-1,3-GlcNAc PR), and B12 (isotype control) to S. pyogenes 5448 Δ emm1 . Bacterial opsonization was determined by measuring IgG1 binding to bacteria using flow cytometry and data represent normalized mean gMFI + s.d. (isotype signals set to 1) of three independent experiments. E) effector functions of anti-GAC clones towards S. pyogenes. C3b deposition by sGAC-reactive mAbs onto S. pyogenes 5448 Δ emm1 . Data represent C3b binding (normalized gMFI + s.d.) of three independent experiments and was measured by flow cytometry. Fluorescent signals are depicted as a fold change relative to the condition without antibodies to compensate for variation in background signals between biological replicates.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Fluorescence, Binding Assay, Labeling, Clone Assay, Produced, Purification, Concentration Assay, Flow Cytometry, Glycoproteomics, Titration, Control, Bacteria