Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: ICAM-1 IgG autoantibody and sICAM-1 levels across healthy individuals and those with severe COVID-19, high BMI, and autoimmune conditions. (A) Dot plot graph of AUC on the log axis of the optical density at 450 nm measurement by ELISA for determining the levels of IgG antibodies targeting ICAM-1 within plasma from healthy individuals (n = 40), individuals with severe COVID-19 (n = 40), individuals with a high BMI over 30 kg/m 2 (n = 50), and individuals with select autoimmune conditions, SLE or RA (n = 39). (B) Dot plot graph of concentration (pg/mL) on the log axis measured by ELISA for determining the levels of sICAM-1 within plasma from healthy individuals (n = 40), individuals with severe COVID-19 (n = 40), individuals with a high BMI over 30, (n = 50), and individuals with autoimmune conditions SLE or RA (n = 39). Statistical significance was determined using the Mann-Whitney test. P value results are shown.

Article Snippet: Secondary antibodies were purchased from the following: Goat anti-human IgG (109-036-098, Lot# 149163; Jackson ImmunoResearch), Goat Anti-Human IgA alpha chain (ab97215, Lot# GR3373878-8; Abcam), Goat Anti-Human IgM mu chain (ab97205, Lot# GR3396429-1; Abcam), Mouse Anti-Human IgG1 (9054-05, Lot#B2023-NM34C; Southern Biotech), Mouse Anti-Human IgG2 (9060-05, Lot#K3220-YF80; Southern Biotech), Mouse Anti-Human IgG3 (9210-05, Lot#DO720-QB4017; Southern Biotech), and Mouse Anti-Human IgG4 (9200-05, Lot#E1723-RB54B; Southern Biotech).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Concentration Assay, MANN-WHITNEY

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: Correlation of anti-ICAM-1 IgG autoantibody levels with age. (A) Dot plot graph of the AUC of the optical density at 450 nm on the log y-axis of IgG antibodies targeting ICAM-1 within plasma compared with age (x-axis) from healthy pediatric individuals ranging in age from 6 mo to 15 yr old (n = 37). (B) Dot plot graph of the AUC of the optical density at 450 nm on the log y-axis of IgG antibodies targeting ICAM-1 within plasma compared with age (x-axis) from healthy adult individuals ranging in age from 22 to 75 yr old (n = 40). Statistical significance was determined using the Pearson correlation test. P value results are shown.

Article Snippet: Secondary antibodies were purchased from the following: Goat anti-human IgG (109-036-098, Lot# 149163; Jackson ImmunoResearch), Goat Anti-Human IgA alpha chain (ab97215, Lot# GR3373878-8; Abcam), Goat Anti-Human IgM mu chain (ab97205, Lot# GR3396429-1; Abcam), Mouse Anti-Human IgG1 (9054-05, Lot#B2023-NM34C; Southern Biotech), Mouse Anti-Human IgG2 (9060-05, Lot#K3220-YF80; Southern Biotech), Mouse Anti-Human IgG3 (9210-05, Lot#DO720-QB4017; Southern Biotech), and Mouse Anti-Human IgG4 (9200-05, Lot#E1723-RB54B; Southern Biotech).

Techniques: Clinical Proteomics

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: High-resolution IgG epitope mapping of ICAM-1 autoantibodies using ICAM-1 peptide microarrays. (A) Heatmap of z scores for ICAM-1 epitope binding for individual samples (n = 20). ICAM-1 domains 1 through 5 are annotated above the heatmap. (B) Line graphs of median group z scores of ICAM-1 autoantibody binding to individual ICAM-1 peptides in the peptide array. Epitopes of recurrent high binding were qualified as median z scores of ≥0.95 to represent 1 SD ±0.05 above the median, points in red. (C) Table of recurrent high binding peptides, with associated z score and sequence. (D) Table of high binding clusters with associated peptide IDs, z score range, and sequence. Overlapping sequence motifs are bolded, and the common leucine threonine epitope is underlined.

Article Snippet: Secondary antibodies were purchased from the following: Goat anti-human IgG (109-036-098, Lot# 149163; Jackson ImmunoResearch), Goat Anti-Human IgA alpha chain (ab97215, Lot# GR3373878-8; Abcam), Goat Anti-Human IgM mu chain (ab97205, Lot# GR3396429-1; Abcam), Mouse Anti-Human IgG1 (9054-05, Lot#B2023-NM34C; Southern Biotech), Mouse Anti-Human IgG2 (9060-05, Lot#K3220-YF80; Southern Biotech), Mouse Anti-Human IgG3 (9210-05, Lot#DO720-QB4017; Southern Biotech), and Mouse Anti-Human IgG4 (9200-05, Lot#E1723-RB54B; Southern Biotech).

Techniques: Binding Assay, Peptide Microarray, Sequencing

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: Immunoglobulin isotype and subclass determination of anti-ICAM-1 antibodies. (A) Dot plot graph of concentration (µg/mL) on the log y-axis obtained by ELISA for determining the levels of immunoglobulin isotype class within high-autoantibody-presenting plasma, defined as the top 16 positive samples by qualitative ELISA regardless of disease group (n = 16). (B) Dot plot graph of concentration (µg/mL) obtained by ELISA for determining the levels of IgG subclass within high-autoantibody-presenting plasma, defined as the top 16 positive samples by qualitative ELISA regardless of disease group (n = 16). Statistical significance was determined using the Mann-Whitney test. P value results are shown.

Article Snippet: Secondary antibodies were purchased from the following: Goat anti-human IgG (109-036-098, Lot# 149163; Jackson ImmunoResearch), Goat Anti-Human IgA alpha chain (ab97215, Lot# GR3373878-8; Abcam), Goat Anti-Human IgM mu chain (ab97205, Lot# GR3396429-1; Abcam), Mouse Anti-Human IgG1 (9054-05, Lot#B2023-NM34C; Southern Biotech), Mouse Anti-Human IgG2 (9060-05, Lot#K3220-YF80; Southern Biotech), Mouse Anti-Human IgG3 (9210-05, Lot#DO720-QB4017; Southern Biotech), and Mouse Anti-Human IgG4 (9200-05, Lot#E1723-RB54B; Southern Biotech).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, MANN-WHITNEY

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: Anti-ICAM-1 IgG autoantibodies interact with protein glycans. (A) SDS-PAGE separation of ICAM-1 under nonreducing conditions. Lane 1 shows ICAM-1 protein, lane 2 shows ICAM-1 in reaction buffer without PNGase F, and lane 3 shows ICAM-1 with PNGase F. Molecular masses are displayed on the left margin in kDa. (B) Dot plot graph of the AUC of the optical density at 450 nm on the log y-axis obtained by ELISA for determining the levels of ICAM-1 IgG autoantibodies targeting ICAM-1 with glycans (control) compared with enzymatically deglycosylated ICAM-1 (PNGASE) within plasma with high levels of detected ICAM-1 autoantibodies, defined as the top 8 positive samples by qualitative ELISA regardless of disease group (n = 8). Statistical significance was determined using the Wilcoxon matched pairs signed rank test. P value results are shown.

Article Snippet: Secondary antibodies were purchased from the following: Goat anti-human IgG (109-036-098, Lot# 149163; Jackson ImmunoResearch), Goat Anti-Human IgA alpha chain (ab97215, Lot# GR3373878-8; Abcam), Goat Anti-Human IgM mu chain (ab97205, Lot# GR3396429-1; Abcam), Mouse Anti-Human IgG1 (9054-05, Lot#B2023-NM34C; Southern Biotech), Mouse Anti-Human IgG2 (9060-05, Lot#K3220-YF80; Southern Biotech), Mouse Anti-Human IgG3 (9210-05, Lot#DO720-QB4017; Southern Biotech), and Mouse Anti-Human IgG4 (9200-05, Lot#E1723-RB54B; Southern Biotech).

Techniques: SDS Page, Enzyme-linked Immunosorbent Assay, Control, Clinical Proteomics

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: Scandinavian Journal of Immunology

Article Title: Multiparametric Optimization of Human Primary B‐Cell Cultures Using Design of Experiments

doi: 10.1111/sji.70043

Figure Lengend Snippet: Optimization of iGB cultures using a Box–Behnken DOE strategy. (A) Timeline and design table for DOE 3 using a three‐level Box–Behnken design. B cells (1 × 10 3 ) were seeded in 96‐well plates on Day 0. Flow cytometry and TRIFMA were performed on Day 11. (B, C) Pareto plots of the effect size of each factor on B‐cell viability (B) and proliferation (C) after 11 days of iGB culturing. IL‐21 2 indicates a non‐linear contribution from IL‐21. (D–G) Proliferation and viability of each sample from DOE 3 where colour and mean indicated by dotted lines are specific for level of expression of N.40 cells (D), IL‐4 (E), change of medium (F) and IL‐21 (G). (H) Contour plot showing effect of IL‐21 and IL‐4 exposure levels on B‐cell proliferation. (I, J) Pareto plots of parameter effect sizes on the IgG (I) and IgE (J) levels measured in culture medium using TRIFMA. (K) Measured IgG and IgE in culture medium, colour‐coded for the use of high (N.40‐high) or low (N.40‐low) CD40L‐expressing feeder cells. C.M. indicates medium change.

Article Snippet: 0.3 μg/mL Donkey anti‐human IgG (Jackson ImmunoResearch, 709‐005‐098), 2.5 μg/mL Mouse anti‐human IgG1 (SouthernBiotech, 9052‐01), 1 μg/mL Mouse anti‐human IgG3 (SouthernBiotech, 9210‐01), 1 μg/mL Goat anti‐Human IgA (SouthernBiotech, 2053‐01), 0.5 μg/mL Goat anti‐human IgM (SouthernBiotech, 2023‐01) or 2.5 μg/mL anti‐human IgE (SouthernBiotech, 9240‐01), all incubated o/n at 4°C.

Techniques: Flow Cytometry, Expressing

Journal: Scandinavian Journal of Immunology

Article Title: Multiparametric Optimization of Human Primary B‐Cell Cultures Using Design of Experiments

doi: 10.1111/sji.70043

Figure Lengend Snippet: Comparing cytokine delivery methods for iGB cultures. (A) Timeline and design table for DOE 4 and 5. B cells (1 × 10 3 ) were seeded in 96‐well plates on day 0. Flow cytometry and TRIFMA were performed on Day 8. (B) Pareto plot of parameter effect sizes on B‐cell viability in DOE 4. (C, D) Proliferation and viability of each sample from DOE 4 where colour and mean indicated by dotted lines are specific for IL‐4 pulse (C) and level of expression of N.40 cells (D). (E) as (B) but for DOE 5. (F) as (C) but for DOE 5. (G) as (D) for DOE 5. (H) Pareto plot of parameter effect sizes on the IgE levels measured in culture medium of DOE 4 using TRIFMA. (I) Measured IgG and IgE in culture DOE 4 medium, colour‐coded for the use of an IL‐4 pulse at B‐cell seeding (Day 0). (J) as (I) but colour‐coded for the use of high (N.40‐high) or low (N.40‐low) CD40L‐expressing feeder cells. (K) as (H) but for DOE 5. (L) as (I) but for DOE 5. (M) as (J) but for DOE 5.

Article Snippet: 0.3 μg/mL Donkey anti‐human IgG (Jackson ImmunoResearch, 709‐005‐098), 2.5 μg/mL Mouse anti‐human IgG1 (SouthernBiotech, 9052‐01), 1 μg/mL Mouse anti‐human IgG3 (SouthernBiotech, 9210‐01), 1 μg/mL Goat anti‐Human IgA (SouthernBiotech, 2053‐01), 0.5 μg/mL Goat anti‐human IgM (SouthernBiotech, 2023‐01) or 2.5 μg/mL anti‐human IgE (SouthernBiotech, 9240‐01), all incubated o/n at 4°C.

Techniques: Flow Cytometry, Expressing

Journal: Scandinavian Journal of Immunology

Article Title: Multiparametric Optimization of Human Primary B‐Cell Cultures Using Design of Experiments

doi: 10.1111/sji.70043

Figure Lengend Snippet: Secreted and displayed marker investigation of naïve B‐cell iGB culturing. (A) Timeline and design table for DOE 6. B cells (2.8 × 10 4 ) were seeded in 6‐well plates on Day 0. Flow cytometry and TRIFMA were performed on Day 0, 3 (TRIFMA only), 5, 7, 10 and 14. (B‐C) Flow plotting of Day 0, 7 and 14 B cells to illustrate their activation and differentiation measured by their expression of IgD and CD27 (B), and CD38 and CD95 (C). (D) Pareto plot of parameter effect sizes on IgE secretion. (E) Measured IgG1 and IgE in culture DOE 6 medium by TRIFMA, colour‐coded as harvest day of the respective B cells. (F) as (E) but colour‐coded for the presence of an IL‐4 pulse at B‐cell seeding (Day 0). (G) as (F) but colour‐coded for the use of high (N.40‐high) or low (N.40‐low) CD40L‐expressing feeder cells. (H) UMAP clustering of pooled flow cytometry data. (I) UMAP clustering as (H) but colour‐coded for days in culture. (J) UMAP clustering as (H) colour‐coding the naïve B cells seeded at Day 0. (K) UMAP clustering as (H) but colour‐coded as the respective sample conditions for the DOE 6 screen. (L) UMAP clustering as (H) but colour‐coded for respective marker densities measured by flow cytometry median fluorescence intensity. (M) Heatmap of FlowSOM clustering analysis.

Article Snippet: 0.3 μg/mL Donkey anti‐human IgG (Jackson ImmunoResearch, 709‐005‐098), 2.5 μg/mL Mouse anti‐human IgG1 (SouthernBiotech, 9052‐01), 1 μg/mL Mouse anti‐human IgG3 (SouthernBiotech, 9210‐01), 1 μg/mL Goat anti‐Human IgA (SouthernBiotech, 2053‐01), 0.5 μg/mL Goat anti‐human IgM (SouthernBiotech, 2023‐01) or 2.5 μg/mL anti‐human IgE (SouthernBiotech, 9240‐01), all incubated o/n at 4°C.

Techniques: Marker, Flow Cytometry, Activation Assay, Expressing, Fluorescence

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: Nature

Article Title: TCF1 and LEF1 promote B-1a cell homeostasis and regulatory function

doi: 10.1038/s41586-025-09421-0

Figure Lengend Snippet: (a) Coverage-based diversity accumulation curve for Hill numbers of order (q = 0, 1 and 2), i.e., species richness, Shannon entropy and Simpson index, respectively in peritoneal B cell (top) or B-1 cells (bottom). (b) Circular plot of cell number of different clonotypes in TCF1 WT LEF1 WT and TCF1 Δ LEF1 Δ peritoneal B-1 cells. (c) Flow cytometry plots and quantification of frequency of PtC + B-1a cells in TCF1 WT LEF1 WT (n = 12) and TCF1 Δ LEF1 Δ (n = 8) mice. (d) Total serum IgM and IgG3 were determined by ELISA between TCF1 WT LEF1 WT (n = 5) and TCF1 Δ LEF1 Δ (n = 7) mice. (e) Flow cytometry plots and quantification of percentage of spleen B-1 cell derived plasma cells (B-1 PC) (Dump – CD19 – CD43 + IgM + IgD –/lo CD138 + ) (TCF1 WT LEF1 WT n = 5; TCF1 Δ LEF1 Δ n = 5) and B-1 plasmablasts (B-1 PB) (Dump – CD19 + CD43 + IgM + IgD –/lo CD138 + ) (TCF1 WT LEF1 WT n = 12; TCF1 Δ LEF1 Δ n = 10) in B-1 cells. (f) Flow cytometry plots and quantification of percentage of IgG3 + from peritoneal B-1a cells in TCF1 WT LEF1 WT (n = 5) and TCF1 Δ LEF1 Δ (n = 5) mice. Each symbol represents an individual mouse and bars represent median values. Data from n = 3 experiments in total (c, e), or representative of n = 3 experiments (d-f). The lines represent the diversity estimates. The shaded area around the curve represents the 95% confidence intervals (95% CI) (a). Statistical analysis was performed using two-tailed Mann-Whitney U test (c,e-f), one way ANOVA with Tukey multiple-comparison test (d). The exact P values are shown.

Article Snippet: Serum was added in serial dilution before the addition of secondary antibody: alkaline phosphatase (AP)-conjugated goat anti-mouse IgM (1020-04, Southern Biotech) and AP-conjugated goat anti-mouse IgG3 (1100-04, Southern Biotech).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Clinical Proteomics, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature

Article Title: TCF1 and LEF1 promote B-1a cell homeostasis and regulatory function

doi: 10.1038/s41586-025-09421-0

Figure Lengend Snippet: Purified peritoneal cavity TCF1 WT LEF1 WT or TCF1 Δ LEF1 Δ B-1 cells were cultured with LPS, IL-10R-Fc antibody or IgG1 isotype for 3 days. a , b , Density plots ( a ) and quantification ( b ) of the mean ratio of plasma cells (Pl.Cs; CD138 + ):non-plasma cells (CD138 − ; b ). c , Percentage of non-plasma cells undergone more than two divisions. d , Density plots (left) and mean ratio of plasma cells:non-plasma cells (right) after 3 days of LPS stimulation with or without IL-10. e , Representative gating strategy of MYC high and MYC low cells at division 3 (D3). f , g , Density plots of MYC expression in proliferating cells ( f ) and quantification of the percentage of MYC high and MYC low in each division measured by CTV dilution ( g ). h , Representative gating strategy of MYC high and MYC low cells at undivided (D0) and D3 (top) and the ratio of MYC low :MYC high (bottom). i , Histograms of CD86, FCRL5, CD11b and CD19 expression among MYC high and MYC low cells. j , Histograms (left) and quantification (right) of CD86, FCRL5 and CD19 expression on TCF1 WT LEF1 WT or TCF1 Δ LEF1 Δ B-1 cells stimulated with LPS for 3 days that are either D0 or have undergone D3 or D5. k , Histogram of PDL1 expression in D0 and D1 of TCF1 WT LEF1 WT or TCF1 Δ LEF1 Δ B-1 cells stimulated with or without LPS for 3 days. l , Chromatin immunoprecipitation followed by sequencing (ChIP–seq) analysis of TCF1 binding in CD4 + CD8 + thymocytes and ATAC-seq analysis at the Pdl1 locus on B-1a cells from Vh12/Vk4 transgenic mice . Panel l was adapted from refs. , , Springer Nature Ltd. Data are presented as mean ± s.d. Data are representative of n = 2 experiments with a total of 9 mice per genotype, with peritoneal cavity cells from 3 mice being pooled together. Statistical analysis was performed using one-way ANOVA with Tukey multiple-comparison test ( b – d , h ) or two-way ANOVA ( j ). The exact P values are shown.

Article Snippet: Serum was added in serial dilution before the addition of secondary antibody: alkaline phosphatase (AP)-conjugated goat anti-mouse IgM (1020-04, Southern Biotech) and AP-conjugated goat anti-mouse IgG3 (1100-04, Southern Biotech).

Techniques: Purification, Cell Culture, Clinical Proteomics, Expressing, Chromatin Immunoprecipitation, Sequencing, ChIP-sequencing, Binding Assay, Transgenic Assay, Comparison