mouse antihuman igg2 alexa fluor 647  (SouthernBiotech)

Journal: Neurology Neuroimmunology & Neuroinflammation

Article Title: Serological Markers of Clinical Improvement in MuSK Myasthenia Gravis

doi: 10.1212/nxi.0000000000200313

Figure Lengend Snippet: Figure 3 MuSK-IgG Subclasses and Clinical Outcomes

Article Snippet: The levels of each MuSK-IgG isotype were determined using the same protocol and the following secondary antibodies: mouse anti-human IgG1 Alexa Fluor 647 (1: 200, cat#: 9052-31, Southern Biotech, USA), mouse antihuman IgG2 Alexa Fluor 647 (1:200, cat#: 9070-31, Southern Biotech), mouse anti-human IgG3 Alexa Fluor 647 (1:200, cat#:9210-31, Southern Biotech), and mouse anti-human IgG4 PE (1:400, cat#: 9200-09, Southern Biotech).

Techniques:

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Serological Markers of Clinical Improvement in MuSK Myasthenia Gravis

doi: 10.1212/NXI.0000000000200313

Figure Lengend Snippet: Comparison of MuSK-IgG levels (antigen binding capacity, ABC) in serum samples collected from patients during an acute MG phase and at a second time point, when they either achieved a favorable (MGFA PIS: MM-or-better = YES) unfavorable (MGFA PIS: MM-or-better = NO) outcome. (A) A reduction of MuSK-IgG levels was found in patients who achieved a favorable clinical outcome ( p = 0.0295), with no significant changes in those who did not. (B) A decrease in MuSK-IgG CBA end-point titers was associated with the achievement of an MGFA PIS of MM-or-better ( p = 0.0156) while no significant changes were observed in patients who were still symptomatic after immunotherapy. (C) Mixed-effects modeling showed a significant positive correlation between MuSK-IgG levels (ABC) and clinical severity classified according to the MGFA clinical classification (r s = 0.3885, 95% CI 0.06383–0,6387, p = 0.0175; mixed-effects model estimate: 2.296e-06, std. error: 1.024e-06, t = 2.243, p = 0.032) (a simple linear regression line is plotted; dotted lines show 95% CI). (D) Mixed-effects modeling did not show a significant correlation between MuSK-IgG CBA end-point titers and clinical severity classified according to the MGFA clinical classification (r s = 0.2304, 95% CI-0.1110-0.5232, p = 0.1701; mixed-effects model estimate: 2.474e-04, std. error: 2.077e-04, t = 1.191, p = 0.242) (a simple linear regression line is plotted; dotted lines show 95% CI). MGFA = Myasthenia Gravis Foundation of America; MM = minimal manifestation; PIS = postintervention status.

Article Snippet: The levels of each MuSK-IgG isotype were determined using the same protocol and the following secondary antibodies: mouse anti-human IgG1 Alexa Fluor 647 (1:200, cat#: 9052-31, Southern Biotech, USA), mouse anti-human IgG2 Alexa Fluor 647 (1:200, cat#: 9070-31, Southern Biotech), mouse anti-human IgG3 Alexa Fluor 647 (1:200, cat#:9210-31, Southern Biotech), and mouse anti-human IgG4 PE (1:400, cat#: 9200-09, Southern Biotech).

Techniques: Comparison, Binding Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Serological Markers of Clinical Improvement in MuSK Myasthenia Gravis

doi: 10.1212/NXI.0000000000200313

Figure Lengend Snippet: Scatter plots showing MuSK-IgG subclass levels (ABC) measured by flow cytometry live CBA and disease severity assessed by MGFA classification at sampling. Mixed-effects modeling revealed no significant correlations between disease severity and the levels of MuSK-IgG1 (r s = 0.04202, 95% CI-0.295-0.3697, p = 0.8049; mixed-effects model estimate: 6.970e-05, std. error: 8.458e-05, t = 0.824, p = 0.416) (A), MuSK-IgG2 (r s = 0.2342, 95% CI −0.1070 to 0.5261, p = 0.1629; mixed-effects model estimate: 1.290e-05, std. error: 1.175e-05, t = 1.097, p = 0.280) (B), and MuSK-IgG3 levels (r s = −0.099, 95% CI −0.4181 to 0.2418, p = 0.5598; mixed-effects model estimate: −7.752e-06, std. error: 1.814e-05, t = −0.427, p = 0.671) (C) and a significant positive correlation with MuSK-IgG4 levels (r s = 0.3808, 95% CI 0.05487–0.6334, p = 0.0201; mixed-effects model estimate: 2.740e-06, std. error: 1.129e-06, t = 2.426, p = 0.018) (D). A simple linear regression line is plotted; dotted lines show 95% CI. MGFA = Myasthenia Gravis Foundation of America.

Article Snippet: The levels of each MuSK-IgG isotype were determined using the same protocol and the following secondary antibodies: mouse anti-human IgG1 Alexa Fluor 647 (1:200, cat#: 9052-31, Southern Biotech, USA), mouse anti-human IgG2 Alexa Fluor 647 (1:200, cat#: 9070-31, Southern Biotech), mouse anti-human IgG3 Alexa Fluor 647 (1:200, cat#:9210-31, Southern Biotech), and mouse anti-human IgG4 PE (1:400, cat#: 9200-09, Southern Biotech).

Techniques: Flow Cytometry, Sampling

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Serological Markers of Clinical Improvement in MuSK Myasthenia Gravis

doi: 10.1212/NXI.0000000000200313

Figure Lengend Snippet: (A) MuSK-IgG4 reduction was associated with achievement of a favorable clinical outcome ( p = 0.0245). (B) No significant changes in MuSK-IgG4 levels were observed in patients who were still symptomatic after immunotherapy ( p = 0.7422). (C) Pairwise comparison of MuSK-IgG2 levels show a significant reduction in patients who achieved an MGFA PIS of MM-or-better ( p = 0.0085). (D) No significant changes in MuSK-IgG2 levels were observed in patients with an unfavorable clinical outcome ( p = 0.5469). MGFA = Myasthenia Gravis Foundation of America; PIS = postintervention status.

Article Snippet: The levels of each MuSK-IgG isotype were determined using the same protocol and the following secondary antibodies: mouse anti-human IgG1 Alexa Fluor 647 (1:200, cat#: 9052-31, Southern Biotech, USA), mouse anti-human IgG2 Alexa Fluor 647 (1:200, cat#: 9070-31, Southern Biotech), mouse anti-human IgG3 Alexa Fluor 647 (1:200, cat#:9210-31, Southern Biotech), and mouse anti-human IgG4 PE (1:400, cat#: 9200-09, Southern Biotech).

Techniques: Comparison

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Serological Markers of Clinical Improvement in MuSK Myasthenia Gravis

doi: 10.1212/NXI.0000000000200313

Figure Lengend Snippet: (A) Affinity-concentration plot showing SAffCon assay results in longitudinal samples from the same patient, who had an MGFA PIS of MM-or-better at time of initial sampling, with antibody properties of K D = 62 nM and concentration (antibody-binding sites) = 1,900 nM. The second serum sample was collected during a phase of clinical exacerbation (classified as “acute phase”), with antibodies showing a 100-fold increase in affinity ( K D = 0.6 nM) and a concentration = 140 nM. The K D is plotted on the x-axis as –log10(M); thus, higher values correspond to lower K D (and hence tighter binding). Antibody concentration is plotted on the y-axis as –log10(M); thus, lower values correspond to higher antibody concentrations. (B) Total MuSK-IgG levels (ABC), CBA end-point titers, and proportion of specific IgG4 of the same patient and time points showed in panel A, demonstrating the same end-point titers, a mild decrease in MuSK-IgG levels, and predominance of IgG4 at both time points (90.8% and 81.3%, respectively). (C) Affinity-concentration plot showing SAffCon assay results in longitudinal serum specimens of another patient, who was sampled during the acute MG phase at the first time point, with antibody properties of K D = 2 nM and concentration = 160 nM. At the second time point, when a favorable clinical outcome was achieved (MGFA PIS: MM-or-better) after immunotherapy, the antibody properties were K D = 1.3 nM and concentration = 24 nM. K D and antibody concentrations are plotted as described above. (D) Total MuSK-IgG levels (ABC), CBA end-point titers, and proportion of specific IgG4 of the same patient and time points showed in panel C, demonstrating a reduction of both MuSK-IgG levels (ABC) and CBA end-point titers while the predominant MuSK-IgG subclass was IgG4 at both time points (99.6% and 99.1%, respectively). MGFA = Myasthenia Gravis Foundation of America; PIS = postintervention status.

Article Snippet: The levels of each MuSK-IgG isotype were determined using the same protocol and the following secondary antibodies: mouse anti-human IgG1 Alexa Fluor 647 (1:200, cat#: 9052-31, Southern Biotech, USA), mouse anti-human IgG2 Alexa Fluor 647 (1:200, cat#: 9070-31, Southern Biotech), mouse anti-human IgG3 Alexa Fluor 647 (1:200, cat#:9210-31, Southern Biotech), and mouse anti-human IgG4 PE (1:400, cat#: 9200-09, Southern Biotech).

Techniques: Concentration Assay, Sampling, Binding Assay

Journal: Brain, Behavior, & Immunity - Health

Article Title: Synapsin autoantibodies during pregnancy are associated with fetal abnormalities

doi: 10.1016/j.bbih.2023.100678

Figure Lengend Snippet: High prevalence of synapsin-I autoantibodies in pregnant women. (A) Representative examples of immunofluorescence stainings with human sera at 1:300 dilution (green) with (top row) or without binding (bottom row) to HEK293 cells overexpressing human synapsin-Ib. Protein expression is confirmed with a commercial synapsin-I/II antibody (red). Nuclei are stained with DAPI (blue). Scale bar = 20 μm. (B) Frequencies of synapsin-Ib autoantibodies in pregnant women and control cohorts, as determined by CBA. CBA scores: 0 = no binding, 1 = unspecific signal, 2 = intensive binding (positive); vertical dotted line represents cut-off for positivity. (C) Representative immunoblots of wild type (wt) and Syn1/2/3 triple knock out (TKO) mice cortex homogenates with a commercial synapsin-I/II antibody as positive control and sera from CBA-positive pregnant women (1:200 dilution). The strong band at ∼90 kDa corresponding to the molecular weight of synapsin-Ia/Ib was detected in wild type but not in TKO mouse tissue. Another band at ∼50 kDa likely represents the synapsin-IIb isoform or breakdown products of synapsin-I. Detection of GAPDH served as loading control. (D) Immunofluorescence staining on rat hippocampal neurons demonstrated strong IgG binding of human sera (green) in a characteristic synaptic pattern (insert shows high magnification, MAP2 [blue] staining for better visualization of neuronal processes). The punctate staining completely overlapped with a commercial synapsin antibody (red, insert for high magnification).

Article Snippet: IgG subclasses were detected using mouse anti-human-IgG1, -IgG2, -IgG3 and -IgG4 AF647-antibodies (SouthernBiotech #9052-31, #9070-31, #9210-31, #9200-31).

Techniques: Immunofluorescence, Binding Assay, Expressing, Staining, Western Blot, Knock-Out, Positive Control, Molecular Weight

Journal: Brain, Behavior, & Immunity - Health

Article Title: Synapsin autoantibodies during pregnancy are associated with fetal abnormalities

doi: 10.1016/j.bbih.2023.100678

Figure Lengend Snippet: Summary of experimental synapsin-Ib antibody findings.

Article Snippet: IgG subclasses were detected using mouse anti-human-IgG1, -IgG2, -IgG3 and -IgG4 AF647-antibodies (SouthernBiotech #9052-31, #9070-31, #9210-31, #9200-31).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Brain, Behavior, & Immunity - Health

Article Title: Synapsin autoantibodies during pregnancy are associated with fetal abnormalities

doi: 10.1016/j.bbih.2023.100678

Figure Lengend Snippet: Strong, titer-dependent association of synapsin-I autoantibodies with abnormalities of fetal development during pregnancy. (A-B) Several ultrasound parameters and status of having previous children with neuropsychiatric disorders were significantly associated with the presence of synapsin-I autoantibodies in serum of pregnant mothers (A), with odds ratios of 3–7 (B). Statistical analysis used Fisher's exact test when expected frequencies were <5 or Chi-square test with expected frequencies ≥5, * representing p < 0.05, **p < 0.01 and ***p < 0.001. (C) Synapsin autoantibodies reached the fetal circulation and were present with similar titers (±1 titer step, in one case 2 titer steps) in umbilical cord blood. (D) Synapsin autoantibodies were of IgG1 and/or IgG3 subclass in almost all pregnant women. (E) While the autoantibody titer alone did not discriminate between pathological and normal findings during fetal development (left), the combination of IgG1 subclass with high-level titers (≥1:30,000) was significantly associated with abnormal measurements (right).

Article Snippet: IgG subclasses were detected using mouse anti-human-IgG1, -IgG2, -IgG3 and -IgG4 AF647-antibodies (SouthernBiotech #9052-31, #9070-31, #9210-31, #9200-31).

Techniques:

Journal: Brain, Behavior, & Immunity - Health

Article Title: Synapsin autoantibodies during pregnancy are associated with fetal abnormalities

doi: 10.1016/j.bbih.2023.100678

Figure Lengend Snippet: Detection of synapsin-I autoantibodies by ELISA, immunoblotting and microarray-based epitope mapping. (A) ELISA-based screening of all human sera using recombinant synapsin-I correlated with autoantibody binding in the CBA. (B) Immunoblots of synapsin-Ib-transfected HEK293 cells confirm that some human sera (1:200 dilution) strongly detect the synapsin-Ib band (middle), while in others binding to non-conformational epitopes is lost (right). Commercial synapsin-I/II antibody served as positive control (left, GAPDH is exemplarily shown; UT = untransfected cells). (C) An overlapping peptide library containing 20-mer peptides with an off-set of three amino acids was designed to fully cover synapsin-I primary sequence. Microarrays were probed with autoantibody-positive and -negative serum. The representative example shows detection of IgG epitope 2 within the synapsin-I slides and a representative negative control serum. Heat map visualization: IgG binding intensities are displayed in grayscale (0 = no binding, 1 = maximal binding). The core shared motif is highlighted in red. (D) Synapsin-I domain architecture together with a cartoon representation of the alpha-fold model of synapsin-I visualized in Pymol. Mapped synapsin-I autoantibody epitopes (1–5, marked in red) are located in domain A-B (epitope 1, peptide sequence: 28 PQPPPPPPGAH 38 ), domain C (epitope 2, 124 TDWAKYFKGKK 134 ) and domain D (epitope 3: 472 PGPQRQGPPLQ 482 ; 4: 583 GGQQRQGPPQK 592 ; 5: 610 VPRTGPPTTQQPRP 623 ).

Article Snippet: IgG subclasses were detected using mouse anti-human-IgG1, -IgG2, -IgG3 and -IgG4 AF647-antibodies (SouthernBiotech #9052-31, #9070-31, #9210-31, #9200-31).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Microarray, Recombinant, Binding Assay, Transfection, Positive Control, Sequencing, Negative Control

Journal: Science immunology

Article Title: Association of HLA-dependent islet autoimmunity with systemic antibody responses to intestinal commensal bacteria in children

doi: 10.1126/sciimmunol.aau8125

Figure Lengend Snippet: (A) Schematic representation of the anti-commensal antibody assay. In a first step, bacteria targets of a specific strain pool are incubated with 4 serial dilutions of the serum sample allowing serum antibodies to bind bacteria surface antigens. Next fluorescently-labelled, secondary anti-isotype antibodies (anti-IgA, IgG1, IgG2 and total Ig) are added allowing the visualization of the bacteria through flow cytometry. (B) An example of the signal measured by flow cytometry for one bacteria pool and four serial dilutions of a serum sample. The first four panels show contour plots of the anti-isotype intensity vs forward scatter parameter. The last panel shows a histogram overlay of the anti-isotype signal intensities. (C) Schematic representation of the Response Index calculation. For every serum sample, bacteria strain pool and isotype the dilution curve of the mean fluorescence intensity (MFI) vs dilution factor are generated on a log-log scale (left panel, grey lines). The mean dilution curve is generated using the average MFI values for all serum samples (left panel, black line). For every sample the difference between its dilution AUC and the mean dilution AUC is calculated (ΔAUC, right panel). The resulting Response Index is calculated as 2ΔAUC. (D) Schematic representation of the NET and TrialNet cohort subjects analyzed in this study.

Article Snippet: Bacteria were then incubated (30 min at 4 °C) with 50 μL of fluorophore-conjugated anti-isotype antibodies (goat anti-human IgG (DyLight 405 conjugated, Jackson ImmunoResearch Laboratories, Inc.), mouse anti-human IgG1 (phycoertherin conjugated, SouthernBiotech), mouse anti-human IgG2 (AF647 conjugated, SouthernBiotech) or mouse anti-human IgA (FITC conjugated, Jackson ImmunoResearch Laboratories, Inc.) ( ).

Techniques: Bacteria, Incubation, Flow Cytometry, Fluorescence, Generated

Journal: Science immunology

Article Title: Association of HLA-dependent islet autoimmunity with systemic antibody responses to intestinal commensal bacteria in children

doi: 10.1126/sciimmunol.aau8125

Figure Lengend Snippet: (A) Box-and-whiskers plot of anti-MET-2 IgG2 response indices (Y axis), segregated by HLA haplotype (X axis). (B) Box-and-whiskers plots of anti-R.faecis IgG2 response indices (Y axis), segregated by HLA haplotype (X axis). (C) Interaction plot of anti-MET-2 IgG2 response indices. (D) Interaction plot of anti-R.faecis IgG2 response indices. (E) Linear regression of anti-MET-2 IgG2 response indices by age. (F) Linear regression of anti-R.faecis IgG2 response indices by age.

Article Snippet: Bacteria were then incubated (30 min at 4 °C) with 50 μL of fluorophore-conjugated anti-isotype antibodies (goat anti-human IgG (DyLight 405 conjugated, Jackson ImmunoResearch Laboratories, Inc.), mouse anti-human IgG1 (phycoertherin conjugated, SouthernBiotech), mouse anti-human IgG2 (AF647 conjugated, SouthernBiotech) or mouse anti-human IgA (FITC conjugated, Jackson ImmunoResearch Laboratories, Inc.) ( ).

Techniques:

Journal: Science immunology

Article Title: Association of HLA-dependent islet autoimmunity with systemic antibody responses to intestinal commensal bacteria in children

doi: 10.1126/sciimmunol.aau8125

Figure Lengend Snippet: Correlations between isotype-specific ACAb responses (columns, bottom) and islet auto-antibody positive status (rows, left) are shown in heatmap colors. ACAb responses and IABs were each clustered by similarity by hierarchical clustering (dendrograms top and right, respectively). ACAb isotypes: IgA •/ IgG1•/ IgG2 •.

Article Snippet: Bacteria were then incubated (30 min at 4 °C) with 50 μL of fluorophore-conjugated anti-isotype antibodies (goat anti-human IgG (DyLight 405 conjugated, Jackson ImmunoResearch Laboratories, Inc.), mouse anti-human IgG1 (phycoertherin conjugated, SouthernBiotech), mouse anti-human IgG2 (AF647 conjugated, SouthernBiotech) or mouse anti-human IgA (FITC conjugated, Jackson ImmunoResearch Laboratories, Inc.) ( ).

Techniques: