Journal: Journal of proteomics

Article Title: Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease.

doi: 10.1016/j.jprot.2014.12.020

Figure Lengend Snippet: Fig. 2 – Changes in IgG subclass-specific glycoforms in liver disease. Average intensity (mean ± SEM) of indicated glycoforms in three patient groups: A. IgG1 (n = 5 per group), B. IgG2, CTRL (n = 5), CIR (n = 5), HCC (n = 4); C. IgG3, CTRL (n = 4), CIR (n = 3), HCC (n = 4); D. IgG4, CTRL (n = 4), HCC (n = 4).

Article Snippet: Purified full length native human IgG1, 2, and 4 (Abcam, Cambridge, MA), IgG3 (Athens Research & Technology, Athens, GA), IgA and IgM (Lee Biosolutions, St. Louis, MO) proteins were used to generate isotope dilution calibration curves.

Techniques:

Journal: Journal of proteomics

Article Title: Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease.

doi: 10.1016/j.jprot.2014.12.020

Figure Lengend Snippet: Fig. 3 – Contribution of specific glycoforms to variability between IgG subclasses. Indicated glycoforms of IgG1, 2 and 3 were compared in intensity in a healthy control participant. Glycoform intensities for each subclass were normalized to intensity of glycoform FA2G0 in the same IgG subclass and analyzed by principal component analysis. *: glycoforms that significantly contribute to differences among IgG subclasses.

Article Snippet: Purified full length native human IgG1, 2, and 4 (Abcam, Cambridge, MA), IgG3 (Athens Research & Technology, Athens, GA), IgA and IgM (Lee Biosolutions, St. Louis, MO) proteins were used to generate isotope dilution calibration curves.

Techniques: Control

Journal: Journal of the American Society of Nephrology

Article Title: Inhibition of Toll-Like Receptor-7 (TLR-7) or TLR-7 plus TLR-9 Attenuates Glomerulonephritis and Lung Injury in Experimental Lupus

doi: 10.1681/asn.2006101162

Figure Lengend Snippet: Figure 5. Glomerular immune complex and C3c deposits in MRLlpr/lpr mice. Paraffin-embedded renal sections were stained for IgG1 and IgG2a as indicated. In saline-treated mice, IgG1 and IgG2a localize to the mesangium and the glomeru- lar capillary wall. Note that IRS 661– and IRS 954–treated MRLlpr/lpr mice show less IgG2a deposits. Frozen renal sec- tions were stained for complement factor C3c. Note the robust glomerular complement activation in saline-treated MRLlpr/lpr mice, which is hardly detectable in IRS 661– and IRS 954–treated MRLlpr/lpr mice. Images are representative of 10 mice in each group. Magnification, 400.

Article Snippet: After incubation with mouse serum, dsDNA-specific IgG, IgG1, IgG2a, IgG2b, and IgG3 were detected by ELISA (Bethyl Labs, Montgomery, TX).

Techniques: Staining, Saline, Activation Assay

Journal: Journal of the American Society of Nephrology

Article Title: Inhibition of Toll-Like Receptor-7 (TLR-7) or TLR-7 plus TLR-9 Attenuates Glomerulonephritis and Lung Injury in Experimental Lupus

doi: 10.1681/asn.2006101162

Figure Lengend Snippet: Figure 6. Localization of IRS 661 and IRS 954 in kidneys of MRLlpr/lpr mice after intraperitoneal injection. Rhodamine-la- beled IRS 661 and IRS 954 were intraperitoneally injected into 16-wk-old MRLlpr/lpr mice, and renal tissue was harvested 2 h later. (A) Fluorescence imaging of frozen sections showed up- take of labeled IRS 661 in glomeruli (encircled) in a mesangial and capillary staining pattern as well as in tubular epithelial cells. Co-staining with IgG showed partial co-localization of IRS 661 with glomerular IgG deposits. (B) At higher magnification, granular deposits of IRS 661 in tubular epithelial cells (left) and glomerular cells (right) can be seen. (C) Co-staining with a FITC-labeled Mac-2 antibody identified IRS 661 to co-localize with renal macrophages (arrow), whereas other renal macro- phages remain IRS 661 negative. Magnification, 400 in A and C.

Article Snippet: After incubation with mouse serum, dsDNA-specific IgG, IgG1, IgG2a, IgG2b, and IgG3 were detected by ELISA (Bethyl Labs, Montgomery, TX).

Techniques: Injection, Fluorescence, Imaging, Labeling, Staining

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: EBioMedicine

Article Title: Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

doi: 10.1016/j.ebiom.2015.11.041

Figure Lengend Snippet: Adjuvants impact the durability of protection conferred by eVLP vaccination. (a) C57BL/6 mice were vaccinated IM two times with 10 μg of eVLP and challenged four (short-term) weeks or twenty-two (long-term) weeks after the vaccine boost. Data in A are pooled from 8 individual studies with 6–10 animals/group. Fisher's exact test: survival in the short-term group was significantly higher than in the long-term group (p < 0.0001). (b) C57BL/6 mice were vaccinated IM two times with 10 μg of eVLP and challenged at the indicated days after the second vaccination. n = 9 or 10/group. Cochran-Armitage test: percentage surviving declined as time to challenge increased (p = 0.0046). There was a significant difference (p = 0.03) between survival on Day 77 and Day 175. (c) Serum samples collected from animals in (B) one week prior to challenge were subjected to an ELISA for the evaluation of anti-GP IgG, IgG1, and IgG2c antibody titers. Red symbols indicate titers of animals that succumbed to challenge while black indicate titers of survivors; red symbols with black outlines indicate that one of these two animals succumbed to challenge, but animal tags were indeterminate after challenge. Median and IQR shown. (d) C57BL/6 mice were vaccinated two times (IM) with VLP, with or without the indicated adjuvants. Animals were challenged twenty-two weeks after the vaccine boost. Data in D are pooled from at least 4 separate studies with a total of at least 35 animals per group. P-values comparing VLP alone to vaccination with VLP and adjuvant are shown, calculated using Fisher's exact tests with stepdown Bonferroni correction. V = VLP, VP = VLP + PolyICLC, VC = VLP + CpG, VM = VLP + MPLA, and VA = VLP + alhydrogel.

Article Snippet: Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05).

Techniques: Enzyme-linked Immunosorbent Assay, Adjuvant

Journal: EBioMedicine

Article Title: Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

doi: 10.1016/j.ebiom.2015.11.041

Figure Lengend Snippet: Adjuvants have variable impact on IgG subclasses and antibody neutralization. (a) Serum was collected 14 days and 147 days after the vaccine boost (days 35 and 168, respectively) and evaluated for anti-GP IgG, IgG1, IgG2c, and IgG3 levels using an ELISA. Data shown are pooled from at least two separate experiments per group. (b) Pairwise comparison using DSCF multiple pairwise comparison was used and p values greater than 0.05 are shown as “ns”. (c) Summary of results shown in A, B and D. (d) Neutralizing antibody titers were evaluated using the PsVNA, with titers giving 80% neutralization shown; median and IQR shown. Samples within each group were selected randomly from three separate studies for evaluation in the assay. Pairwise comparison using post-hoc Tukey's studentized range test procedure was used to evaluate differences between groups at both days 35 and day 168, where “*” indicates 0.01 < p < 0.05, “**” indicates 0.001 < p < 0.01, “***” indicates 0.0001 < p < 0.001, and “****” indicates p < 0.0001.

Article Snippet: Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05).

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Comparison

Journal: EBioMedicine

Article Title: Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

doi: 10.1016/j.ebiom.2015.11.041

Figure Lengend Snippet: CD8 T cell deficiency does not impact short term or long term survival of vaccinated C57BL/6J mice. (a) Mice were treated IM twice with saline, VLP, VLP and polyICLC, or VLP and CpG. Four weeks after the second vaccination, mice were challenged. Closed symbols represent wild type C57BL/6J mice and open symbols indicate CD8-deficient mice. (b) Mice were vaccinated on the same schedule as in A, but challenge occurred 22 weeks after the second vaccination. (c) Two weeks after the second vaccination and 1 week prior to challenge, blood was collected from vaccinated animals and evaluated for anti-GP IgG antibody titers. (D) A subset of vaccinated animals was euthanized 4 days after the vaccine boost to evaluate CD4 + T cell responses. Median response of C57BL/6J mice and CD8-deficient mice is shown. N = 8–10 per treated group for A–C and D presents data pooled from two separate evaluations of 4–8 mice per group each. “*” indicates 0.005 < p < 0.05.

Article Snippet: Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05).

Techniques: Saline