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: medRxiv

Article Title: Serological Analysis Reveals an Imbalanced IgG Subclass Composition Associated with COVID-19 Disease Severity

doi: 10.1101/2020.10.07.20208603

Figure Lengend Snippet: Reactivity of IgM, IgA, and IgG specific to the SARS-CoV-2 nucleocapsid, RBD, S1 subunit, or S2 subunit of the full patient cohort (a ) or grouped by disease severity (b) . Index value represents the raw MFI divided by the background cutoff value determined by a panel of 93 normal human serum specimens. Statistical significance was determined by the non-parametric Kruskall-Wallace test where * p ≥ 0.05 ** p ≥ 0.001 *** p ≥ 0.0001, and **** p ≥ 0.00001.

Article Snippet: R-phycoerythrin (PE)-conjugated goat anti-human Ig, goat anti-human IgG, anti-human IgM, anti-human IgA, mouse anti-human IgG1, mouse anti-human IgG2, mouse anti-human IgG3, and mouse anti-human IgG4 were purchased (Southern Biotech).

Techniques:

Journal: medRxiv

Article Title: Serological Analysis Reveals an Imbalanced IgG Subclass Composition Associated with COVID-19 Disease Severity

doi: 10.1101/2020.10.07.20208603

Figure Lengend Snippet: (a) Reactivity of IgG1, IgG2, and IgG3 to SARS-CoV-2 nucleocapsid, RBD, S1 subunit, or S2 subunit of the full patient cohort (b) or grouped by disease severity. Index value represents the raw MFI divided by the background cutoff value determined by a panel of 93 normal human serum specimens. Statistical significance was determined by the non-parametric Kruskall-Wallace test where * p ≤ 0.05 ** p ≤ 0.001 *** p ≤ 0.0001, and **** p ≤ 0.00001.

Article Snippet: R-phycoerythrin (PE)-conjugated goat anti-human Ig, goat anti-human IgG, anti-human IgM, anti-human IgA, mouse anti-human IgG1, mouse anti-human IgG2, mouse anti-human IgG3, and mouse anti-human IgG4 were purchased (Southern Biotech).

Techniques:

Journal: medRxiv

Article Title: Serological Analysis Reveals an Imbalanced IgG Subclass Composition Associated with COVID-19 Disease Severity

doi: 10.1101/2020.10.07.20208603

Figure Lengend Snippet: (a) Three-dimensional scatter plot depicting the optimal feature set of disease severity-associated features (age, log10-transformed index values (MFI/cutoff) for S1-specific IgG3, and RBD-specific IgG1) as determined by ordered probit regression and backwards stepwise selection by Akaike information criterion. Data is displayed as the distribution of mild (yellow), moderate (orange), and severe (red) disease severities across variables in 478 patients.

Article Snippet: R-phycoerythrin (PE)-conjugated goat anti-human Ig, goat anti-human IgG, anti-human IgM, anti-human IgA, mouse anti-human IgG1, mouse anti-human IgG2, mouse anti-human IgG3, and mouse anti-human IgG4 were purchased (Southern Biotech).

Techniques: Transformation Assay, Selection

Journal: medRxiv

Article Title: Serological Analysis Reveals an Imbalanced IgG Subclass Composition Associated with COVID-19 Disease Severity

doi: 10.1101/2020.10.07.20208603

Figure Lengend Snippet: (a) IgG/Subclass index ratios for IgG1, 2, and 3 specific to the nucleocapsid, RBD, S1, and S2 subunits in mild, moderate, and severe patients. (b) Heat map represents the average MFI of each severity group divided by the average MFI of the entire data set. * denotes statistical significance. (c) Spearman’s correlation coefficient comparing the IgG response to the PRNT90 neutralizing titer.

Article Snippet: R-phycoerythrin (PE)-conjugated goat anti-human Ig, goat anti-human IgG, anti-human IgM, anti-human IgA, mouse anti-human IgG1, mouse anti-human IgG2, mouse anti-human IgG3, and mouse anti-human IgG4 were purchased (Southern Biotech).

Techniques:

Journal: Cell reports. Medicine

Article Title: Systems serology in cystic fibrosis: Anti-Pseudomonas IgG1 responses and reduced lung function.

doi: 10.1016/j.xcrm.2023.101210

Figure Lengend Snippet: Figure 2. Characterization of anti-Pseudomonas responses by surface binding and membrane protein immunoassays (A) Summary of the positive percentage of antibody surface binding against PA46 at the indicated dilution. Representative flow results and comparisons of CF versus non-CF are in Figures S1A and S1B. (B) Overview of anti-OMP46 level in patients with CF with different lung function at the indicated dilutions. (C) Workflow of screening specific antigens from OMP46 interacting with CF sera using LC-MS. Two samples each from ppFEV1 >90% CF and ppFEV1 <70% CF groups were used. IgG antibodies in CF sera were bound with protein A beads via its Fc region and specific antigens from OMP46 via its Fab region. The im- munobinding proteins were purified and analyzed through liquid chromatography (LC)-MS, in which OprI was identified as the top candidate; see STAR Methods for detailed information. (D) Summary of all ant-OprI IgG isotype levels in patients with CF and non-CF control subjects. (E) Serial dilution in 2-fold of determining anti-OprI IgG titer shown in diluted curve (left) and area under curve (right) using ELISA. (F) The anti-OprI IgG1 (left) and IgG4 (right) titers in three groups. All summaries were visualized as polar plots, n = 10 in each group; the asterisk represents significant differences in the isotype between CF with reduced and healthy lung function cohorts using Mann-Whitney test, *p < 0.05. Area under curve (AUC) comparison was using Kruskal-Wallis test, ****p < 0.0001.

Article Snippet: All used antibodies were listed: Goat antihuman IgG BIOT (1:50 dilution, SouthernBiotech#2040-08); Mouse anti-human IgG1 Hinge BIOT (1:50 dilution, SouthernBiotech#9052-08); Mouse anti-human IgG2 Fc BIOT (1:50 dilution, SouthernBiotech#9070-08); Mouse anti-human IgG3 Hinge BIOT (1:50 dilution, SouthernBiotech#9210-08); Mouse anti-human IgG4 Fc BIOT (1:50 dilution, SouthernBiotech#9052-08); Streptavidin-PE (1:250 dilution, eBioscience #12-4317-87)

Techniques: Binding Assay, Membrane, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Control, Serial Dilution, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Comparison

Journal: Cell reports. Medicine

Article Title: Systems serology in cystic fibrosis: Anti-Pseudomonas IgG1 responses and reduced lung function.

doi: 10.1016/j.xcrm.2023.101210

Figure Lengend Snippet: Figure 4. Key antibody biophysical and func- tional profiling replication using an indepen- dent validation cohort (A) Serial dilution in 2-fold of determining anti-OprI IgG titer shown in diluted curve (left) and AUC (right) using ELISA. (B) Anti-OprI IgG1 (left) and IgG4 (right) in the sera of CF validation cohorts. (C) ADCD analysis mediated by CF sera from vali- dation cohorts (n = 10 each). All statistical compar- isons were conducted using Mann-Whitney test, *p < 0.05, ****p < 0.0001.

Article Snippet: All used antibodies were listed: Goat antihuman IgG BIOT (1:50 dilution, SouthernBiotech#2040-08); Mouse anti-human IgG1 Hinge BIOT (1:50 dilution, SouthernBiotech#9052-08); Mouse anti-human IgG2 Fc BIOT (1:50 dilution, SouthernBiotech#9070-08); Mouse anti-human IgG3 Hinge BIOT (1:50 dilution, SouthernBiotech#9210-08); Mouse anti-human IgG4 Fc BIOT (1:50 dilution, SouthernBiotech#9052-08); Streptavidin-PE (1:250 dilution, eBioscience #12-4317-87)

Techniques: Biomarker Discovery, Serial Dilution, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Cell reports. Medicine

Article Title: Systems serology in cystic fibrosis: Anti-Pseudomonas IgG1 responses and reduced lung function.

doi: 10.1016/j.xcrm.2023.101210

Figure Lengend Snippet: Figure 6. Change in anti-Oprl serologies following modulator treatment (A) Paired comparison of anti-Oprl IgG1 in plasma acquired from a cohort of adult subjects with CF immediately prior to and on average 5 months post-ETI treatment initiation (n = 20). Plasma dilution 1:256. Comparison was performed using paired t test, **p < 0.01. (B) Plasma serial dilution in 2-fold of anti-OprI IgG1 titer shown in diluted curve (left) and AUC (right). (C) Change in Pseudomonas abundance (log10 CFU/mL) in the same cohort as measured by quantitative cultures prior to and >1 month after ETI treatment initiation. Comparisons were made using unpaired t test, ***p < 0.001. (D) Regression analysis of the anti-OprI IgG1 predictor of clinical outcomes after ETI. Statistical comparisons were analyzed by a linear mixed model (via the lmerTest R package) using study visit as the only fixed effect and a random intercept for each subject.

Article Snippet: All used antibodies were listed: Goat antihuman IgG BIOT (1:50 dilution, SouthernBiotech#2040-08); Mouse anti-human IgG1 Hinge BIOT (1:50 dilution, SouthernBiotech#9052-08); Mouse anti-human IgG2 Fc BIOT (1:50 dilution, SouthernBiotech#9070-08); Mouse anti-human IgG3 Hinge BIOT (1:50 dilution, SouthernBiotech#9210-08); Mouse anti-human IgG4 Fc BIOT (1:50 dilution, SouthernBiotech#9052-08); Streptavidin-PE (1:250 dilution, eBioscience #12-4317-87)

Techniques: Comparison, Clinical Proteomics, Serial Dilution

Journal: Translational Psychiatry

Article Title: Heterogeneity of anti-Caspr2 antibodies: specificity and pathogenicity

doi: 10.1038/s41398-025-03677-w

Figure Lengend Snippet: A Diagram of the structure of P13F4 and P6B2 with their unique sequences for the heavy and light chains. B Isotype determination with a sandwich ELISA done by coating a plate with various anti-IgG isotype subtypes, adding purified antibody to each well, and measuring their optical density (OD). P13F4 and P6B2 are IgG2 isotype. C Anti-Casp2 binding on HEK293 cells (top) and GnTI-HEK293 cells (bottom). P13F4 and P6B2 bind preferentially to human Casp2 and less to mouse Caspr2 compared to the null binding of an IgG2-matched isotype control (CON2). D Binding of P13F4 (10 μg/mL) and P6B2 (10 μg/mL) to adult mouse brain sections, PFA-fixed (first column), snap-frozen (second column) and E15.5 whole embryo sections (third column). In brain tissue, P6B2 (second row) binds only to PFA-fixed adult sections, whereas P13F4 (first row) binds to both PFA-fixed adult and E15.5 embryonic sections. IgG2b isotype control third row. Representative images of the CA1 region of the hippocampus (adult, first and second column) and cortical plate (E15.5, third column) at 20x. E Binding of P13F4 (10 μg/mL) and P6B2 (10 μg/mL) to primary neuronal cultures (fixed at DIV14) stained with primary antibodies. Hybridoma anti-Caspr2 antibodies are labeled in green, a commercial anti-Caspr2 antibody (ABN1380) in red, and their co-localization in yellow. Images (P13F4 top, P6B2 middle, IgG2b isotype control bottom) taken at 40x. F Assessment of male mice exposed to anti-Caspr2 antibodies in utero reveals that mice in the P13F4 group have normal behavior in the social interaction task but less time in the center of the chamber during the open field task and more distance covered in the light-dark box task. Mice in the P6B2 group show less body contact with an unfamiliar mouse in the social interaction task. Statistical testing done with ANOVA followed by Tukey post-hoc test. G Sholl analysis of pyramidal neurons from the CA1 and CA2 regions of the hippocampus reveals that P6B2-exposed males have significantly shorter dendrite length when compared to male mice exposed to isotype control in both CA1( P < 0.007, ICC=0.174) and CA2 neurons ( P = 0.0084, ICC = 0.214). Insets (at right in each panel) show representative tracing of CA1 and CA 2 neurons.

Article Snippet: Anti-Caspr2 antibodies were tested against each isotype by adding supernatant to the ELISA plate followed by an AP conjugated antibody to the same isotype Goat anti-mouse IgG1 (Southern biotech # 1070-04), Goat anti-mouse IgG2a, (Southern biotech # 1081-04), Goat anti-mouse IgG2b (Southern biotech # 1090-04), and Goat anti-mouse IgG3 (Southern biotech # 1100-04).

Techniques: Sandwich ELISA, Purification, Binding Assay, Control, Staining, Labeling, In Utero

Journal: Translational Psychiatry

Article Title: Heterogeneity of anti-Caspr2 antibodies: specificity and pathogenicity

doi: 10.1038/s41398-025-03677-w

Figure Lengend Snippet: A Diagram of the structure of P11G7 and P9C7 with their unique sequences for the heavy and light chains. B Isotype determination with a sandwich ELISA done by coating a plate with various anti-IgG isotype subtypes, adding purified antibody to each well, and measuring their optical density (OD). P11G7 and P9C7 are clearly IgG1 isotype. C Anti-Casp2 binding on HEK293 cells (top) and GnTI-HEK293 cells (bottom). P11G7 and P9C7 bind to both human Casp2 and mouse Caspr2 compared to an IgG1-matched isotype control (CON1). D Binding of P11G7 (10 μg/mL) and P9C7 (10 μg/mL) to adult mouse brain sections, PFA-fixed (first column), snap-frozen (second column) and E15.5 whole embryo sections (third column). P11G7 (top row) binds to all sections, whereas P9C7 (second row) binds to snap-frozen and embryonic sections but not PFA-fixed sections. IgG1 isotype control third row. Representative images of the CA1 region of the hippocampus (adult, first and second column) and cortical plate (E15.5, third column) at 20x. E Binding of P11G7 (10 μg/mL) and P9C7 (10 μg/mL) to primary neuronal cultures (fixed at DIV14) stained with primary antibodies. Hybridoma anti-Caspr2 antibodies are labeled in green, a commercial anti-Caspr2 antibody (ABN1380) in red, and their co-localization in yellow. Images (P11G7 top, P9C7 middle, IgG1 isotype control bottom) taken at 40x. F Assessment of male mice exposed to anti-Caspr2 antibodies in utero reveals that mice in the P11G7 group have no differences in any of the tasks compared to the CON1 group, while mice in the P9C7 group has less body contact with an unfamiliar mouse in the social interaction task, and less time in the center of the chamber in the open field task. Statistical testing done with ANOVA followed by Tukey post-hoc test. ( G ) Sholl analysis of pyramidal neurons from the CA1 and CA2 regions of the hippocampus reveals no significant differences in dendritic length between the groups. Insets (at right in each panel) show representative tracing of CA1 and CA2 neurons.

Article Snippet: Anti-Caspr2 antibodies were tested against each isotype by adding supernatant to the ELISA plate followed by an AP conjugated antibody to the same isotype Goat anti-mouse IgG1 (Southern biotech # 1070-04), Goat anti-mouse IgG2a, (Southern biotech # 1081-04), Goat anti-mouse IgG2b (Southern biotech # 1090-04), and Goat anti-mouse IgG3 (Southern biotech # 1100-04).

Techniques: Sandwich ELISA, Purification, Binding Assay, Control, Staining, Labeling, In Utero