rabbit anti human igg4 monoclonal antibody  (Cell Marque)

Journal: Frontiers in Cardiovascular Medicine

Article Title: IgG2 rules: N-acetyl-β- D -glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes

doi: 10.3389/fcvm.2022.919700

Figure Lengend Snippet: IgG2 deposits in vivo in RHD valvular tissues. Immunohistochemistry for IgG subclasses reveals strong human IgG2 deposition in RHD heart tissues from four different patients compared to other subclasses as seen by Fast Red stain of IgG subclass deposition. Red staining of cells indicates a positive IgG binding (see arrows). RHD 1 IgG2 staining is 4+, RHD 2 IgG2 staining is 4 +, RHD 3 IgG2 is 3+, RHD 4 IgG2 staining is 4 + . Faint staining IgG3 (RHD 3 0.5+) and IgG4 (RHD 2 1 =, RHD 3 1+) staining was present. No visible IgG1 staining was observed for any of the four RHD samples. IgG subclass deposition is absent from control, non-RHD heart tissue. Magnification 400×.

Article Snippet: Heart tissues from RHD were tested for heart tissue-bound antibody which was detected using biotin-conjugated rabbit anti-human IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies (Sigma Chemical Co.) incubated at 4°C overnight.

Techniques: In Vivo, Immunohistochemistry, Staining, Binding Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: IgG2 rules: N-acetyl-β- D -glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes

doi: 10.3389/fcvm.2022.919700

Figure Lengend Snippet: IgG2 is associated with IFN-γ and IL-17A in RHD hearts. (Top row) hematoxylin and eosin (H&E) staining of four RHD heart tissues showed infiltration of mononuclear cells (arrows in top row) into the heart at the valve surface endothelium (RHD 1) and within the myocardial tissues (RHD 1-4). Focal lesions (large arrow) were observed within the myocardium of RHD4. Non-RHD valvular heart tissue (control) lacked mononuclear cell infiltration and focal lesions. (Middle row) IFN-γ staining of RHD cardiac tissues demonstrates widespread IFN-γ staining of heart tissues was found for RHD1, and RHD3 tissues. Concentrated IFN-γ staining can be seen around invading mononuclear cells (RHD 1-RHD 4). IFN-γ is absent in the control heart tissue. Magnification 400×. (Bottom row) IL-17A staining of RHD cardiac tissues. Four RHD tissues shows IL-17A throughout the heart tissues as well as concentrated around mononuclear cells (arrows) (RHD 1-RHD 4). The control, non-RHD heart tissue had no IL-17A present. Magnification 400×.

Article Snippet: Heart tissues from RHD were tested for heart tissue-bound antibody which was detected using biotin-conjugated rabbit anti-human IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies (Sigma Chemical Co.) incubated at 4°C overnight.

Techniques: Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: IgG2 rules: N-acetyl-β- D -glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes

doi: 10.3389/fcvm.2022.919700

Figure Lengend Snippet: IgG2 in SC CSF targets human neuronal cells. The IgG subclasses from CSF capable of binding to surface antigen on SK-N-SH human neuronal cells were determined by Fast Red staining followed by counterstaining with hematoxylin. Red staining of cells indicates positive IgG binding. IgG2 from SC CSF predominantly targeted the extracellular surface of SK-N-SH cells (SC CSF 1 = 3 + staining and SC CSF 2 = 4 + staining) in comparison to the other subclasses (blue stained cells, 0 staining). Arrows indicate positive reactivity. Very faint IgG2 staining is shown for control CSF (0.5 + staining). In contrast, there is no IgG1, IgG3, or IgG4 binding to human neuronal cells by disease control CSF (blue stained cells, 0 staining). Magnification 400×.

Article Snippet: Heart tissues from RHD were tested for heart tissue-bound antibody which was detected using biotin-conjugated rabbit anti-human IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies (Sigma Chemical Co.) incubated at 4°C overnight.

Techniques: Binding Assay, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: IgG2 rules: N-acetyl-β- D -glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes

doi: 10.3389/fcvm.2022.919700

Figure Lengend Snippet: IgG subclass responses (μg/ml) to GlcNAc in ARF (RHD, SC, Arthritis) sera compared to pharyngitis and healthy control groups. (A) The concentration of GlcNAc-specific IgG1 was significantly elevated in uncomplicated pharyngitis sera in comparison to RHD ( p = 0.001), SC ( p = 0.0052), and healthy control sera ( p = 0.0003), but not to rheumatic arthritis ( p = 0.2667). (B) GlcNAc-specific IgG2 concentrations were significantly elevated in SC sera in comparison to uncomplicated pharyngitis ( p = 0.0052) and healthy control ( p = 0.0016) sera, but not to RHD sera ( p = 0.0653). RHD sera had significant amounts of IgG2 to GlcNAc in comparison to uncomplicated pharyngitis ( p = 0.0431) and healthy control sera ( p = 0.0155). (C) GlcNAc-specific IgG3 in sera from uncomplicated pharyngitis was significantly elevated in comparison to SC ( p = 0.0115) and healthy control ( p = 0.002) sera. (D) GlcNAc-specific IgG4 concentrations were negligible for all groups. P values were calculated by the Mann-Whitney two-tailed t test for comparison of individual sera groups of carditis ( n = 10) SC ( n = 9), arthritis ( n = 3), pharyngitis ( n = 7), and healthy control (n = 8) (Red p value bars are RHD comparisons vs Blue p value bars are SC comparisons vs Black p value bars are pharyngitis comparisons). GlcNAc-specific IgG2 was the significant subclass response to GlcNAc in RHD and SC sera compared to pharyngitis and healthy controls, while GlcNAc-specific IgG1 and IgG3 were significantly elevated in pharyngitis sera. NS, not significant.

Article Snippet: Heart tissues from RHD were tested for heart tissue-bound antibody which was detected using biotin-conjugated rabbit anti-human IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies (Sigma Chemical Co.) incubated at 4°C overnight.

Techniques: Concentration Assay, MANN-WHITNEY, Two Tailed Test

Journal: Frontiers in Cardiovascular Medicine

Article Title: IgG2 rules: N-acetyl-β- D -glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes

doi: 10.3389/fcvm.2022.919700

Figure Lengend Snippet: IgG subclass responses (μg/ml) to the group A streptococcal M protein type 5 (pepM5) in ARF (RHD, SC, Arthritis) sera, pharyngitis and healthy control groups. (A) In rheumatic carditis, IgG1 was the dominant subclass response to pepM5 in comparison to SC ( p = 0.0115), rheumatic arthritis ( p = 0.004), uncomplicated pharyngitis ( p = 0.0330), and healthy control ( p = 0.0002) sera. (B) PepM5-specific IgG2 was significantly elevated in SC sera in comparison to RHD samples ( p = 0.0288). Neither SC nor RHD sera had significant amounts of anti-pepM5 IgG2 in comparison to uncomplicated pharyngitis ( p = 0.3148 and p = 0.0688, respectively). (C) Significantly elevated pepM5-specific IgG3 was found in rheumatic carditis in comparison to SC ( p = 0.0271), pharyngitis ( p = 0.0023), and healthy control ( p = 0.0005) sera. (D) PepM5-specific IgG4 concentrations were negligible in all samples. P values were calculated by the Mann–Whitney two-tailed t test for comparison of individual sera groups of carditis ( n = 10) SC ( n = 9), arthritis ( n = 3), pharyngitis ( n = 7), and healthy control ( n = 8) (Red p value bars are RHD comparisons vs Blue p value bars are SC comparisons vs Black p value bars are pharyngitis comparisons). IgG1 and IgG3 were the significant subclass responses in sera to pepM5 in RHD while IgG2 was significant in SC. NS, not significant.

Article Snippet: Heart tissues from RHD were tested for heart tissue-bound antibody which was detected using biotin-conjugated rabbit anti-human IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies (Sigma Chemical Co.) incubated at 4°C overnight.

Techniques: MANN-WHITNEY, Two Tailed Test

Journal: Frontiers in Cardiovascular Medicine

Article Title: IgG2 rules: N-acetyl-β- D -glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes

doi: 10.3389/fcvm.2022.919700

Figure Lengend Snippet: SC CSF ( n = 3) IgG responses to GlcNAc and PepM5 GAS protein. (A) Total CSF IgG recognition of GlcNAc and PepM5 streptococcal protein. SC CSF demonstrated significantly elevated GlcNAc-specific IgG and pepM5-specific IgG in comparison to disease control CSF ( p = 0.0177 and p = 0.0112, respectively). P values were calculated by the two-tailed Welch’s t test for comparison of SC and control CSF groups. (B) GlcNAc-specific IgG2 dominates SC CSF subclass responses. SC CSF demonstrates significantly higher concentrations of GlcNAc-specific IgG2 ( p = 0.0021) in comparison to disease control CSF. The concentration of SC CSF IgG2 was significantly elevated in comparison to SC CSF IgG1 (0.01) and SC CSF IgG3 (0.0002). P values were calculated by the two-tailed Welch’s t test for comparison of SC and control CSF groups. (C) SC CSF IgG concentrations reactive to pepM5 show slightly elevated levels of IgG1 and IgG3 in comparison to disease control CSF, but were not significantly elevated ( p = 0.1166 and 0.2842, respectively). P values were calculated by the two-tailed Welch’s t test for comparison of SC and control CSF groups. Red dots are SC disease CSF ( n = 3) vs Black dots disease control CSF ( n = 3). NS, not significant.

Article Snippet: Heart tissues from RHD were tested for heart tissue-bound antibody which was detected using biotin-conjugated rabbit anti-human IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies (Sigma Chemical Co.) incubated at 4°C overnight.

Techniques: Two Tailed Test, Concentration Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: IgG2 rules: N-acetyl-β- D -glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes

doi: 10.3389/fcvm.2022.919700

Figure Lengend Snippet: IgG2 is associated with infiltrating mononuclear cells in RHD granuloma. Elevated levels of autoreactive IgG2 were found in association with a mononuclear cell infiltrates in an RHD cardiac valvular tissue granuloma (Aschoff nodule). IgG2 deposition was qualitatively higher in comparison to other IgG subclasses at the valve surface where the granuloma was present (IgG1 0 staining, IgG2 4 + staining, IgG3 2 + staining, IgG4 1 + staining, isotype control 0 staining). Magnification 200×.

Article Snippet: Heart tissues from RHD were tested for heart tissue-bound antibody which was detected using biotin-conjugated rabbit anti-human IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies (Sigma Chemical Co.) incubated at 4°C overnight.

Techniques: Staining

Journal: Journal of Translational Medicine

Article Title: Tumor-B-cell interactions promote isotype switching to an immunosuppressive IgG4 antibody response through upregulation of IL-10 in triple negative breast cancers

doi: 10.1186/s12967-022-03319-5

Figure Lengend Snippet: a , b Tumor cells induce IgG4 class switching in B cells. A representative flow cytometry experiment showing increased IgG4 + cells in a SUM159 and b MDA-MB-231 cells co-cultured with primary peripheral B cells. Activated primary B cells were cultured with or without tumor cells for 5–7 days and analyzed by flow cytometry. Graphs show results of three separate experiments. c Graph represents a relative increase in IgG4 protein expression as detected by ELISA in SUM159 and MDA-MB-231 cells co-cultured with B cells compared to control tumor cells. B cells were cultured with tumor cells for 5 days, and then transferred to a new flask. Cells were cultured for 2 weeks and supernatant was collected an analyzed by ELISA. This experiment was performed in duplicate. Error bars represent standard deviation of technical replicants. ( d , e ) A representative flow cytometry dot- plot showing increased IgG4 positive cells upon co-culture with an EBV-transformed B cell line and ( d ) SUM159 cells or ( e ) MDA-MB-231 cells. Activated B cells were co-cultured with or without tumor cells and a blocking IL-10 antibody for 5–7 days and analyzed by flow cytometry. Increase in IgG4 class switching is prevented by addition of an anti-IL-10 antibody during co-culture. Graphs show the mean of three independent experiments. Error bars represent standard deviations Significance is indicated by asterisk, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.01, ns p > 0.05

Article Snippet: Primary antibodies were incubated overnight at 4 °C with a rabbit monoclonal antibody to IgG4 (Abcam, ab109493, 1:1000), a rabbit polyclonal antibody to IL-4 (Abcam, ab9622, 1:100), and mouse monoclonal antibodies to CD20 (Abcam, ab9475, 1:100) and IL-10 (Santa Cruz, sc-8438, 1:50).

Techniques: Flow Cytometry, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Control, Standard Deviation, Co-Culture Assay, Transformation Assay, Blocking Assay

Journal: Journal of Translational Medicine

Article Title: Tumor-B-cell interactions promote isotype switching to an immunosuppressive IgG4 antibody response through upregulation of IL-10 in triple negative breast cancers

doi: 10.1186/s12967-022-03319-5

Figure Lengend Snippet: Correlation of tumor expressed inflammatory cytokines and IgG4 + B cells in TNBC tissues. Immunohistochemical analysis of serial tissue sections taken from 4 patients (p1-p4). Tissues were independently stained for CD20, IgG4, IL-10 and IL-4 (red stain). Nuclei were counterstained with hematoxylin (blue). Images were taken with a 10× objective

Article Snippet: Primary antibodies were incubated overnight at 4 °C with a rabbit monoclonal antibody to IgG4 (Abcam, ab109493, 1:1000), a rabbit polyclonal antibody to IL-4 (Abcam, ab9622, 1:100), and mouse monoclonal antibodies to CD20 (Abcam, ab9475, 1:100) and IL-10 (Santa Cruz, sc-8438, 1:50).

Techniques: Immunohistochemical staining, Staining

Journal: Journal of Translational Medicine

Article Title: Tumor-B-cell interactions promote isotype switching to an immunosuppressive IgG4 antibody response through upregulation of IL-10 in triple negative breast cancers

doi: 10.1186/s12967-022-03319-5

Figure Lengend Snippet: IgG4 + cells correlate with medium–low B cell density. Quantification of CD20 positive cells in tissue sections by low, medium and high density. IgG + cells are represented in black, IgG4- cells are represented in white

Article Snippet: Primary antibodies were incubated overnight at 4 °C with a rabbit monoclonal antibody to IgG4 (Abcam, ab109493, 1:1000), a rabbit polyclonal antibody to IL-4 (Abcam, ab9622, 1:100), and mouse monoclonal antibodies to CD20 (Abcam, ab9475, 1:100) and IL-10 (Santa Cruz, sc-8438, 1:50).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Tumor-B-cell interactions promote isotype switching to an immunosuppressive IgG4 antibody response through upregulation of IL-10 in triple negative breast cancers

doi: 10.1186/s12967-022-03319-5

Figure Lengend Snippet: Correlation matrix

Article Snippet: Primary antibodies were incubated overnight at 4 °C with a rabbit monoclonal antibody to IgG4 (Abcam, ab109493, 1:1000), a rabbit polyclonal antibody to IL-4 (Abcam, ab9622, 1:100), and mouse monoclonal antibodies to CD20 (Abcam, ab9475, 1:100) and IL-10 (Santa Cruz, sc-8438, 1:50).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Tumor-B-cell interactions promote isotype switching to an immunosuppressive IgG4 antibody response through upregulation of IL-10 in triple negative breast cancers

doi: 10.1186/s12967-022-03319-5

Figure Lengend Snippet: Survival analysis of TNBC patients by expression of IgG4 + B cells, and inflammatory cytokines. Kaplan Meyer survival curves showing recurrence free and overall survival outcomes for patients with ( a ) high, intermediate or low densities of CD20, ( b ) IgG4 + or IgG4- B cells ( c ) high or low IL-10 expression ( d ) High or low IL-4 expression

Article Snippet: Primary antibodies were incubated overnight at 4 °C with a rabbit monoclonal antibody to IgG4 (Abcam, ab109493, 1:1000), a rabbit polyclonal antibody to IL-4 (Abcam, ab9622, 1:100), and mouse monoclonal antibodies to CD20 (Abcam, ab9475, 1:100) and IL-10 (Santa Cruz, sc-8438, 1:50).

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Tumor-B-cell interactions promote isotype switching to an immunosuppressive IgG4 antibody response through upregulation of IL-10 in triple negative breast cancers

doi: 10.1186/s12967-022-03319-5

Figure Lengend Snippet: Multivariate analysis

Article Snippet: Primary antibodies were incubated overnight at 4 °C with a rabbit monoclonal antibody to IgG4 (Abcam, ab109493, 1:1000), a rabbit polyclonal antibody to IL-4 (Abcam, ab9622, 1:100), and mouse monoclonal antibodies to CD20 (Abcam, ab9475, 1:100) and IL-10 (Santa Cruz, sc-8438, 1:50).

Techniques: