Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: IgG reactivity to (A) purified protein derivative (PPD) from Mtb , (B) Mtb cytosolic protein, (C) Mtb culture filtrate, (D) Ag85A and Ag85B, (E) Mtb cell wall, (F) ESAT-6 and CFP-10, and (G) control respiratory syncytial virus (RSV) was determined by customized multiplex Luminex for each individual patient sample with TB over three serial dilutions. Each dot represents relative reactivity for one individual patient sample determined by the area under the curve (AUC), summarizing the median fluorescence intensity (MFI) from serial dilutions. Bars represent median and 95% confidence intervals for latent ( n = 18) and active ( n = 19) TB groups. The p values determined by a Mann-Whitney U test are marked by ^ for significance after adjustment for multiple comparisons by Benjamini-Hochberg.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Purification, Control, Virus, Multiplex Assay, Luminex, Fluorescence, MANN-WHITNEY

Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: (A) Representative chromatograms show patterns of individual glycoforms isolated from the Fc domain of antigen-specific and total bulk IgG in an individual patient with TB, determined by capillary electrophoresis. (B) Violin plots show the relative abundance of sialic acid, galactose, fucose, and bisecting N-acetylglucosamine (GlcNAc) across all individual glycoforms isolated from ESAT-6 and CFP-10 (red) and Mtb cell wall (blue) polyclonal IgG. Each dot represents an individual sample with latent ( n = 18) or active ( n = 19) TB. The median and interquartiles are shown. The dashed lines show the median RSV (green) and total bulk (purple) glycans. The p values determined by a Wilcoxon matched-pairs signed rank test are marked by ^ for significance after adjustment for multiple comparisons by Benjamini-Hochberg.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Isolation, Electrophoresis

Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: (A) Luminescence from the virulent Mtb H37Rv luminescent reporter strain relates to colony-forming units (CFUs). The significance was evaluated by Pearson correlation. (B) To test the effect of antibodies on intracellular Mtb , primary human monocyte-derived macrophages were first infected with the virulent Mtb H37Rv luminescent reporter strain, and the extracellular bacteria were washed away. Then, Mtb -infected primary human monocyte-derived macrophages (MOI = 1) were treated with IgG. Finally, the bacterial burden was quantified with >99% of detectable Mtb in the intracellular as compared to the extracellular medium supernatant compartment. The error bars represent the mean ± SEM. Significance was determined by a Wilcoxon matched-pairs signed rank test. (C) Daily Mtb luminescence measurements representing the median of endemic control, active TB, and latent TB samples are shown for one representative healthy donor of human macrophages. (D) Data from n = 3 healthy human macrophage donors in independent experiments are summarized, with each dot representing the Mtb burden for each individual patient with TB relative to control polyclonal IgG. The median and 95% confidence interval (CI) are shown. The dashed line shows the median of endemic IGRA− control individuals. The significance was determined by a Mann-Whitney U test.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Derivative Assay, Infection, Bacteria, Control, MANN-WHITNEY

Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: (A) The relationships between ESAT-6 and CFP-10 IgG levels and subclasses and intracellular Mtb burden within individuals with latent and active TB were evaluated by Spearman correlation. Heatmaps depict the Spearman rank correlation coefficient, ** p ≤ 0.01, and ^ stands for significance after adjustment for multiple comparisons by Benjamini-Hochberg. The scatterplot shows ESAT-6 and CFP-10 IgG1, and the Mtb burden is shown in the scatterplot, with each dot representing each individual with latent TB. (B) As a control, the relationships between Mtb cell-wall IgG levels and subclasses and intracellular Mtb burden are shown.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Control

Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: (A) Each column in the histogram depicts the intracellular Mtb burden of one individual patient with latent (light gray) and active (dark gray) TB as in . The dashed line represents the intracellular Mtb burden with control IgG. (A and B) The anti- Mtb activity of IgG from each individual patient with TB was determined by the difference in Mtb burden between control and patient IgG (red). (C) For the n = 17 latent and n = 14 active TB samples with detectable anti- Mtb activities relative to ESAT-6 and CFP-10 IgG1, the relationships to ESAT-6 and CFP-10 IgG Fc glycans as determined by Spearman correlations are listed with ^ marking the significance after adjustment for multiple comparisons by Benjamini-Hochberg. (D) N-glycans from anti-ESAT-6 and CFP-10 mAb were enzymatically removed with PNGase F and then used to treat Mtb -infected primary human monocyte-derived macrophages. The intracellular Mtb burden is shown relative to a no-antibody (Ab) control. The graph summarizes data for n = 6 healthy macrophage donors, with each line representing a single donor. (E) An L234A and L235A (LALA) variant of anti-ESAT-6 and CFP-10 mAb was used to treat Mtb -infected primary human monocyte-derived macrophages. Each line represents a single healthy macrophage donor ( n = 9). The significance was determined by a Wilcoxon matched-pairs signed rank test.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Control, Activity Assay, Infection, Derivative Assay, Variant Assay

Journal: Cell Reports

Article Title: Selective functional antibody transfer into the breastmilk after SARS-CoV-2 infection

doi: 10.1016/j.celrep.2021.109959

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Human IgG2-Fc PE , Southern Biotech , CAT # 9060-09.

Techniques: Virus, Luciferase, Recombinant, Produced, Generated, Software, Luminex

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: CD33 reporter cell line constructs. ( a ) Schematic drawing of the two CD33-DAP12 constructs. Both, the full CD33 ecto-domain (CD33M) and the ecto-domain lacking the sialic acid binding domain (CD33 ΔE2 ) were fused to TYROBP/DAP12. ( b ) Schematic drawing of the readouts for the CD33 reporter cell line. CD33 can be activated by ligands or antibodies, which results in increased SYK phosphorylation and consequently increased intracellular calcium levels. ( c ) Gel electrophoresis image of CD33-DAP12 constructs cloned into pcDNA5/FRT after digestion by EcoRI. ( d ) Successful exchange of the viral CMV promoter with the human EEF1A1 promoter was indicated by a second band after digestion with XhoI at 1,339 bp (CD33M) or 958 bp (CD33 ΔE2 ). ( e ) Gel electrophoresis image of pcDNA5/FRT-CD33-DAP12-GCaMP6m plasmids after digestion with XhoI. GCaMP6m positive clones exhibited three bands compared to the control (Ctrl) with only two bands. Gel images were cropped for better visualization. Supplementary Fig. shows the uncropped full-length gel.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Construct, Binding Assay, Phospho-proteomics, Nucleic Acid Electrophoresis, Clone Assay, Control

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: CD33-specific and control antibodies.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Control, Imaging

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: Flow cytometric analysis of CD33 surface expression. ( a ) Schematic drawing of the CD33-DAP12 constructs. Both, the full CD33 ecto-domain (CD33M) and the ecto-domain lacking the sialic acid binding domain (CD33 ∆E2 ) were fused to TYROBP/DAP12. CD33 ∆E2 can be identified by binding of the antibody clone 1c7/1 (blue) but not WM53 or P67.6 (red), whereas all three antibody clones can bind CD33M. ( b ) The CD33-DAP12 and CD33-DAP12-GCaMP6m cells were stained for CD33 surface expression with the antibody clones 1c7/1, WM53 and P67.6. A representative flow cytometry histogram plot for the CD33M-DAP12-GCaMP6m cells is shown (left side). All three tested antibodies were able to stain full-length CD33 on the cell surface. Expression of variant 2 CD33 from CD33 ∆E2 -DAP12 and CD33 ∆E2 -DAP12-GCaMP6m cells was only detected by antibody clone 1c7/1. A representative flow cytometry histogram plot for the CD33 ∆E2 -DAP12-GCaMP6m cells is shown (right side). ( c ) Quantification of CD33 staining showed a high percentage of CD33 expressing cells in the CD33M-DAP12-GCaMP6m line for all three tested antibody clones but only the CD33 antibody clone 1c7/1 was able to detect CD33 in CD33 ∆E2 -DAP12-GCaMP6m expressing cells. The antibody clones WM53 and P67.6 did not show any staining of CD33 ∆E2 -DAP12 expressing cells. ( d ) Quantification of CD33 staining revealed a high percentage of cells in the CD33M-DAP12 line expressed CD33, and was detected by all three antibody clones. CD33 in CD33 ΔE2 -DAP12 expressing cells was only detected by antibody clone 1c7/1. Data are shown as mean + SEM of three to five independent experiments; *** p ≤ 0.001 compared to Secondary Control determined by Welch ANOVA followed by Games-Howell post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Expressing, Construct, Binding Assay, Clone Assay, Staining, Flow Cytometry, Variant Assay, Control

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: pSYK detection in CD33M-DAP12 cell lines. ( a ) pSYK detection in CD33M-DAP12 reporter cells treated with CD33-specific antibodies. Addition of CD33 antibodies P67.6 and 1c7/1 resulted in increased pSYK levels, whilst P67.6 F(ab), WM53 as well as the different isotype IgG1 control antibodies did not show any change in endogenous pSYK levels. ( b ) 1c7/1 and P67.6 dose–response curve in CD33M-DAP12 reporter cells. Addition of CD33 antibody clones 1c7/1 and P67.6 resulted in an increase in endogenous pSYK levels measured 30 min after the treatment. Data are presented as mean ± or + SD; ** p ≤ 0.01 compared to untreated determined by one-way ANOVA analysis followed by Dunnett’s post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Control, Clone Assay

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: Calcium imaging in CD33-DAP12-GCaMP6m reporter cell lines. ( a ) Schematic time line of image acquisition and compound handling. ( b , c ) Calcium imaging analyzed as ΔF/F(t) in CD33-DAP12-GCaMP6m lines. Addition 100 µM dATP led to a strong increase in intracellular calcium levels in both cell lines, CD33M- and CD33 ∆E2 -DAP12-GCaMP6m, with a peak at around 20–25 s. The CD33 antibody clones 1c7/1 and P67.6 evoked a selective intracellular calcium response only in CD33M-DAP12-GCaMP6m cells. The CD33 antibody clones WM53 and P67.6 F(ab) as well as the isotype IgG1/F(ab’)2 antibodies did not show a change in intracellular calcium levels. ( d , e ) The area under the curve as well as the maximum ΔF/F(t) signal calculated from independent experiments showed a significant increase in dATP treated samples in both CD33-DAP12-GCaMP6m lines and a selective increase in CD33M-DAP12-GCaMP6m expressing cells if treated with the CD33 antibody clone P67.6 or 1c7/1. Data are presented mean + SEM; n = 3–6; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 compared to 10 µg/ml IgG1 determined by Welch ANOVA followed by Games-Howell post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Imaging, Clone Assay, Expressing

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: Activation of endogenous CD33 in iPSdMiG by CD33 agonistic antibodies. ( a ) pSYK analysis in TREM2 + DAP12 reporter cells. Addition of anti-TREM2 antibody AF1828 resulted in an increase in endogenous pSYK levels measured 30 min after the treatment only in TREM2 + DAP12 but not DAP12 expressing control reporter cells. Data are presented mean ± SD. ( b ) CD33 antibodies P67.6 and 1c7/1 were able to decrease the increased pSYK/tSYK levels triggered by TREM2 activation in WT iPSdMiG (left). In CD33 −/− (middle) and CD33 ΔE2 (right) iPSdMiG none of the tested antibodies was able to modulate pSYK/tSYK levels after TREM2 activation. Data are presented mean + SEM; n = 3–6; ** p ≤ 0.01 compared to IgG1 (anti-CD33 Ctrl) determined by Welch ANOVA followed by Games-Howell post hoc test. ( c ) CD33 antibodies P67.6 and 1c7/1 decreased the phagocytic uptake of pHrodo S. aureus BioParticles in WT iPSdMiG (left). In CD33 −/− (middle) and CD33 ΔE2 (right) iPSdMiG none of the tested antibodies was able to modulate pHrodo S. aureus BioParticle phagocytosis. Data are presented mean + SEM; n = 3; ** p ≤ 0.01 and * p ≤ 0.05 compared to IgG1 determined by ANOVA followed by Dunnett’s post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Activation Assay, Expressing, Control