anti human igm igg  (Thermo Fisher)


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    Name:
    F ab 2 Goat anti Human IgG IgA IgM Secondary Antibody
    Description:
    F ab 2 Goat anti Human IgG IgA IgM Secondary Antibody for Western Blot IHC ELISA
    Catalog Number:
    a24494
    Price:
    None
    Applications:
    Build Your Own Immunoassay|Cell Analysis|Cellular Imaging|ELISA|IHC Staining & Detection|Immunohistochemistry (IHC)|Protein Assays and Analysis|Protein Biology|Ready-To-Use Immunoassay|Western Blotting
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher anti human igm igg
    IPI-3063 potently promotes mouse B cell antibody switching and inhibits plasmablast differentiation. Purified B cells were pretreated with inhibitors, then stimulated with αCD40 + IL-4 (B) or LPS + IL-4 (C) for 96 h to induce switching to <t>IgG1</t> or LPS (D) for 72 h to induce plasmablast differentiation. Class switching to IgG1 was measured by the 7AAD − CFSE lo B220 + IgG1 + cells [ (A) , upper panels]. Plasmablast percentages were calculated by % 7AAD − CFSE lo CD138 + B220 lo population [ (A) , lower]. Supernatant was harvested (E) for <t>IgM</t> ELISA. Concentrations used were 1 nM, 10 nM, 100 nM, 1 µM for IPI-443 and 1, 10, 30, 100 nM for IPI-3063. Low concentrations of IPI-3063 were 0.01 and 0.1 nM (D) . AS252424 concentrations were 100 nM, 300 nM, and 1 µM, and GDC-0941 was at 500 nM. Samples were collected by time (* P
    F ab 2 Goat anti Human IgG IgA IgM Secondary Antibody for Western Blot IHC ELISA
    https://www.bioz.com/result/anti human igm igg/product/Thermo Fisher
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    1) Product Images from "The Selective Phosphoinoside-3-Kinase p110δ Inhibitor IPI-3063 Potently Suppresses B Cell Survival, Proliferation, and Differentiation"

    Article Title: The Selective Phosphoinoside-3-Kinase p110δ Inhibitor IPI-3063 Potently Suppresses B Cell Survival, Proliferation, and Differentiation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00747

    IPI-3063 potently promotes mouse B cell antibody switching and inhibits plasmablast differentiation. Purified B cells were pretreated with inhibitors, then stimulated with αCD40 + IL-4 (B) or LPS + IL-4 (C) for 96 h to induce switching to IgG1 or LPS (D) for 72 h to induce plasmablast differentiation. Class switching to IgG1 was measured by the 7AAD − CFSE lo B220 + IgG1 + cells [ (A) , upper panels]. Plasmablast percentages were calculated by % 7AAD − CFSE lo CD138 + B220 lo population [ (A) , lower]. Supernatant was harvested (E) for IgM ELISA. Concentrations used were 1 nM, 10 nM, 100 nM, 1 µM for IPI-443 and 1, 10, 30, 100 nM for IPI-3063. Low concentrations of IPI-3063 were 0.01 and 0.1 nM (D) . AS252424 concentrations were 100 nM, 300 nM, and 1 µM, and GDC-0941 was at 500 nM. Samples were collected by time (* P
    Figure Legend Snippet: IPI-3063 potently promotes mouse B cell antibody switching and inhibits plasmablast differentiation. Purified B cells were pretreated with inhibitors, then stimulated with αCD40 + IL-4 (B) or LPS + IL-4 (C) for 96 h to induce switching to IgG1 or LPS (D) for 72 h to induce plasmablast differentiation. Class switching to IgG1 was measured by the 7AAD − CFSE lo B220 + IgG1 + cells [ (A) , upper panels]. Plasmablast percentages were calculated by % 7AAD − CFSE lo CD138 + B220 lo population [ (A) , lower]. Supernatant was harvested (E) for IgM ELISA. Concentrations used were 1 nM, 10 nM, 100 nM, 1 µM for IPI-443 and 1, 10, 30, 100 nM for IPI-3063. Low concentrations of IPI-3063 were 0.01 and 0.1 nM (D) . AS252424 concentrations were 100 nM, 300 nM, and 1 µM, and GDC-0941 was at 500 nM. Samples were collected by time (* P

    Techniques Used: Purification, Enzyme-linked Immunosorbent Assay

    IPI-3063 potently inhibits human B cell proliferation. (A–C) Purified human B cells were pretreated with inhibitors for 30 min, then stimulated with human CD40L + anti-human IgM/IgG + human IL-2 + human IL-21 for 120 h. Concentrations used were 1 nM, 10 nM, 100 nM, and 1 µM for IPI-443, and 0.1, 1, 10, and 100 nM for IPI-3063. AS252424 concentrations were 100 nM, 300 nM, and 1 µM, and GDC-0941 was at 500 nM. Total numbers and percent divided were determined by the number of CD19 + 7AAD − CFSE lo cells. Samples were collected by time (* P
    Figure Legend Snippet: IPI-3063 potently inhibits human B cell proliferation. (A–C) Purified human B cells were pretreated with inhibitors for 30 min, then stimulated with human CD40L + anti-human IgM/IgG + human IL-2 + human IL-21 for 120 h. Concentrations used were 1 nM, 10 nM, 100 nM, and 1 µM for IPI-443, and 0.1, 1, 10, and 100 nM for IPI-3063. AS252424 concentrations were 100 nM, 300 nM, and 1 µM, and GDC-0941 was at 500 nM. Total numbers and percent divided were determined by the number of CD19 + 7AAD − CFSE lo cells. Samples were collected by time (* P

    Techniques Used: Purification

    2) Product Images from "Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis"

    Article Title: Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis

    Journal: Autoimmunity

    doi: 10.3109/08916934.2012.750301

    Indirect fluorescence for binding of serum antibody to normal brain tissue. Serum from EAE affected WT mice show diffuse, strong white matter associated signal consistent with the presence of MOG specific IgG1. By contrast, corresponding signal is absent using serum from MOG induced KO mice, or healthy controls. There is no difference of signal intensity or distribution for IgM binding to brain, regardless of MOG induction or genotype. Representative images are shown.
    Figure Legend Snippet: Indirect fluorescence for binding of serum antibody to normal brain tissue. Serum from EAE affected WT mice show diffuse, strong white matter associated signal consistent with the presence of MOG specific IgG1. By contrast, corresponding signal is absent using serum from MOG induced KO mice, or healthy controls. There is no difference of signal intensity or distribution for IgM binding to brain, regardless of MOG induction or genotype. Representative images are shown.

    Techniques Used: Fluorescence, Binding Assay, Mouse Assay

    Generation and functional characterization of Aicda deficient mice. A) Gene targeting strategy used to delete the catalytic domain (exon-3) of Aicda locus. The structure of the AID domains is shown in top. The location of the homology arms in the targeting vector is indicated. The schematic of targeting construct used to delete the catalytic domain of AID (exon-3) is shown in the middle. The solid triangles represent Cre recombination sites and the Neomycin cassette was subsequently removed by expression of Cre-recombinase. Representative WT or KO PCR products using 3′ or 5′ arm analysis at the Aicda locus is shown in the right panel. The size of Aicda KO PCR product is indicated and corresponds to the modified locus. Notice that one primer is located in the Neomycin cassette and therefore only detects the targeted Aicda locus. B) FACS analysis of LPS/IL4 stimulated WT or AID KO B cells in vitro. Percentage of IgM or IgG1 are indicated in each quadrant. B220-FITC is used as a B-cell marker.
    Figure Legend Snippet: Generation and functional characterization of Aicda deficient mice. A) Gene targeting strategy used to delete the catalytic domain (exon-3) of Aicda locus. The structure of the AID domains is shown in top. The location of the homology arms in the targeting vector is indicated. The schematic of targeting construct used to delete the catalytic domain of AID (exon-3) is shown in the middle. The solid triangles represent Cre recombination sites and the Neomycin cassette was subsequently removed by expression of Cre-recombinase. Representative WT or KO PCR products using 3′ or 5′ arm analysis at the Aicda locus is shown in the right panel. The size of Aicda KO PCR product is indicated and corresponds to the modified locus. Notice that one primer is located in the Neomycin cassette and therefore only detects the targeted Aicda locus. B) FACS analysis of LPS/IL4 stimulated WT or AID KO B cells in vitro. Percentage of IgM or IgG1 are indicated in each quadrant. B220-FITC is used as a B-cell marker.

    Techniques Used: Functional Assay, Mouse Assay, Plasmid Preparation, Construct, Expressing, Polymerase Chain Reaction, Modification, FACS, In Vitro, Marker

    Characterization of anti-MOG antibodies using ELISA at day zero and 28. A) IgG anti-rhMOG, B) IgM anti-rhMOG, C) IgG anti-MOG peptide 35–55, D) IgM anti-MOG peptide 35–55. Mixed day 28 sera of WT or KO immunized mice were used as standard (arbitrarily defined as 100 U) for IgG or IgM antibodies, respectively. E) IgM binding to rhMOG coated at different concentrations. Sera from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Pooled mice sera IgM Abs normalized at 0.1 mg/ml in the assay. Anti-mouse IgM was used as detection to exclusively measure the binding of IgM antibodies in serum.
    Figure Legend Snippet: Characterization of anti-MOG antibodies using ELISA at day zero and 28. A) IgG anti-rhMOG, B) IgM anti-rhMOG, C) IgG anti-MOG peptide 35–55, D) IgM anti-MOG peptide 35–55. Mixed day 28 sera of WT or KO immunized mice were used as standard (arbitrarily defined as 100 U) for IgG or IgM antibodies, respectively. E) IgM binding to rhMOG coated at different concentrations. Sera from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Pooled mice sera IgM Abs normalized at 0.1 mg/ml in the assay. Anti-mouse IgM was used as detection to exclusively measure the binding of IgM antibodies in serum.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Binding Assay

    Surface plasmon resonance (SPR) analysis of serum antibody binding to MOG. A. SPR sensorgrams of serum samples binding to sensor immobilized MOG. Serum from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Start and stop injection times are indicated by arrows. Day 28 WT serum showed the highest binding response, and the majority of this binding signal could be competed with soluble MOG (2 uM) preincubated with the serum sample before injection. B. Detection of the major binding isotype in Day 28 WT serum. Subsequent injections of three anti-isotype antibodies were used to probe the MOG bound component. Start and stop injection times are indicated by arrows for each sample (Day 28 WT serum, anti-mIgM, anti-mIgG2a and anti-mIgG1). The data indicate that the majority of the binding signal is specific to IgG1. No background binding of anti-mIgG1 to MOG alone was detected (data not shown).
    Figure Legend Snippet: Surface plasmon resonance (SPR) analysis of serum antibody binding to MOG. A. SPR sensorgrams of serum samples binding to sensor immobilized MOG. Serum from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Start and stop injection times are indicated by arrows. Day 28 WT serum showed the highest binding response, and the majority of this binding signal could be competed with soluble MOG (2 uM) preincubated with the serum sample before injection. B. Detection of the major binding isotype in Day 28 WT serum. Subsequent injections of three anti-isotype antibodies were used to probe the MOG bound component. Start and stop injection times are indicated by arrows for each sample (Day 28 WT serum, anti-mIgM, anti-mIgG2a and anti-mIgG1). The data indicate that the majority of the binding signal is specific to IgG1. No background binding of anti-mIgG1 to MOG alone was detected (data not shown).

    Techniques Used: SPR Assay, Binding Assay, Mouse Assay, Injection

    3) Product Images from "DNA epitope vaccine containing complement component C3d enhances anti-amyloid-? antibody production and polarizes the immune response towards a Th2 phenotype"

    Article Title: DNA epitope vaccine containing complement component C3d enhances anti-amyloid-? antibody production and polarizes the immune response towards a Th2 phenotype

    Journal: Journal of neuroimmunology

    doi: 10.1016/j.jneuroim.2008.08.016

    3C3d molecular adjuvant significantly enhances anti-Aβ antibody response induced by DNA epitope vaccine and dramatically increased IgG1 (Th2)/IgG2a b (Th1) ratio. (A) Concentrations of anti-Aβ antibodies in the sera of mice immunized with
    Figure Legend Snippet: 3C3d molecular adjuvant significantly enhances anti-Aβ antibody response induced by DNA epitope vaccine and dramatically increased IgG1 (Th2)/IgG2a b (Th1) ratio. (A) Concentrations of anti-Aβ antibodies in the sera of mice immunized with

    Techniques Used: Mouse Assay

    4) Product Images from "Impairment of Granzyme B-Producing Regulatory B Cells Correlates with Exacerbated Rheumatoid Arthritis"

    Article Title: Impairment of Granzyme B-Producing Regulatory B Cells Correlates with Exacerbated Rheumatoid Arthritis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00768

    Correlation analysis of granzyme B (GrB)-producing regulatory B cell (Breg) with rheumatoid arthritis (RA) patient clinical manifestations. The frequencies of GrB-producing Breg were negatively correlated with RA patient ESR (A) , tender joint counts (B) , and DAS28-ESR (C) . The frequency difference of GrB-producing Breg was also analyzed between RA patients with high disease activity (DAS28 > 5.1) and non-high disease activity (DAS28 ≤ 5.1) (D) . The correlation of GrB-producing Breg with RA patient swollen joint counts (E) , CRP (F) , disease duration (G) , rheumatoid factor (RF) (H) , anti-CCP antibody (I) , serum IgA (J) , IgG (K) , and IgM (L) was also analyzed. * P
    Figure Legend Snippet: Correlation analysis of granzyme B (GrB)-producing regulatory B cell (Breg) with rheumatoid arthritis (RA) patient clinical manifestations. The frequencies of GrB-producing Breg were negatively correlated with RA patient ESR (A) , tender joint counts (B) , and DAS28-ESR (C) . The frequency difference of GrB-producing Breg was also analyzed between RA patients with high disease activity (DAS28 > 5.1) and non-high disease activity (DAS28 ≤ 5.1) (D) . The correlation of GrB-producing Breg with RA patient swollen joint counts (E) , CRP (F) , disease duration (G) , rheumatoid factor (RF) (H) , anti-CCP antibody (I) , serum IgA (J) , IgG (K) , and IgM (L) was also analyzed. * P

    Techniques Used: Electron Paramagnetic Resonance, Activity Assay

    5) Product Images from "Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans"

    Article Title: Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0180768

    [A] Correlation between anti-nonGal and anti-nonGal/nonNeu 5 Gc IgM and IgG antibody levels. When pRBCs were the target cells, there was no correlation between anti-nonGal IgM and anti-nonGal/nonNeu5Gc IgM (P > 0.05). There was significant positive correlation between anti-nonGal IgG and anti-nonGal/nonNeu5Gc IgG (P
    Figure Legend Snippet: [A] Correlation between anti-nonGal and anti-nonGal/nonNeu 5 Gc IgM and IgG antibody levels. When pRBCs were the target cells, there was no correlation between anti-nonGal IgM and anti-nonGal/nonNeu5Gc IgM (P > 0.05). There was significant positive correlation between anti-nonGal IgG and anti-nonGal/nonNeu5Gc IgG (P

    Techniques Used:

    [A] Human IgM/IgG binding to Gal and Neu 5 Gc (ELISA). The level of anti-Gal IgM/IgG antibodies were higher than those of anti-Neu5Gc antibodies (P
    Figure Legend Snippet: [A] Human IgM/IgG binding to Gal and Neu 5 Gc (ELISA). The level of anti-Gal IgM/IgG antibodies were higher than those of anti-Neu5Gc antibodies (P

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    Definitions of anti-pig IgM and IgG antibodies. Anti-pig antibodies (both IgM and IgG) such as anti-Gal, anti-Neu5Gc, anti-nonGal, and anti-nonGal/nonNeu5Gc were defined according to binding to various pig cells.
    Figure Legend Snippet: Definitions of anti-pig IgM and IgG antibodies. Anti-pig antibodies (both IgM and IgG) such as anti-Gal, anti-Neu5Gc, anti-nonGal, and anti-nonGal/nonNeu5Gc were defined according to binding to various pig cells.

    Techniques Used: Binding Assay

    [A] Human serum IgM/IgG binding to GTKO/CD46 and GTKO/CD46/Neu 5 GcKO pRBCs and pAECs (flow cytometry) . There was no significant difference in IgM or IgG binding to GTKO/CD46 pRBCs or pAECs (P > 0.05). IgM and IgG binding to GTKO/CD46/Neu5GcKO pRBCs were both significantly less than to the equivalent pAECs (P
    Figure Legend Snippet: [A] Human serum IgM/IgG binding to GTKO/CD46 and GTKO/CD46/Neu 5 GcKO pRBCs and pAECs (flow cytometry) . There was no significant difference in IgM or IgG binding to GTKO/CD46 pRBCs or pAECs (P > 0.05). IgM and IgG binding to GTKO/CD46/Neu5GcKO pRBCs were both significantly less than to the equivalent pAECs (P

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry

    6) Product Images from "Heparosan-coated liposomes for drug delivery"

    Article Title: Heparosan-coated liposomes for drug delivery

    Journal: Glycobiology

    doi: 10.1093/glycob/cwx070

    Immunological challenge in rats with HEP-DiPalm and ELISA assessment. A set of three rats ( #1, 2, 3 ) then tested for the induction of an IgM or IgG response by ELISA (representative set of assays with averaged triplicate wells with standard deviation is presented). Overall, the signal from sera of rats before and after injection with HEP-DiPalm micelles (prelipid, white ; postlipid, black ) tested in the control wells coated with bovine serum albumin ( BSA ) was equivalent to the wells coated with HEP-BSA ( HEP ) or HEP-DiPalm ( DiPalm ) indicating that HEP is not significantly immunogenic, therefore, should be useful in multidose or long-term therapeutics.
    Figure Legend Snippet: Immunological challenge in rats with HEP-DiPalm and ELISA assessment. A set of three rats ( #1, 2, 3 ) then tested for the induction of an IgM or IgG response by ELISA (representative set of assays with averaged triplicate wells with standard deviation is presented). Overall, the signal from sera of rats before and after injection with HEP-DiPalm micelles (prelipid, white ; postlipid, black ) tested in the control wells coated with bovine serum albumin ( BSA ) was equivalent to the wells coated with HEP-BSA ( HEP ) or HEP-DiPalm ( DiPalm ) indicating that HEP is not significantly immunogenic, therefore, should be useful in multidose or long-term therapeutics.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation, Injection

    Representation of HEP-liposome assembly. Models of HEP-coated liposome assemblies with 13.3-kDa ( top ) and 7-kDa ( bottom ) HEP-lipid, based on typical molecular configurations from equilibrated coarse-grained simulations. The liposome and sugars are represented graphically by equivalent spheres (roughly to scale), and part of the liposome has been cut-away for viewing purposes. Potential interaction proteins are depicted (human serum albumin, HSA ; immunoglobulins IgG or IgM ; middle ), drawn to exact scale using coordinates from the Protein Data Bank. Less surface area is available for direct protein binding to liposomes coated with 0.5 mol% 13.3-kDa HEP-lipid compared to 0.5 mol% 7-kDa HEP-lipid, suggesting increased liposomal protection from clearance by the immune system. The image was rendered using POV-Ray software.
    Figure Legend Snippet: Representation of HEP-liposome assembly. Models of HEP-coated liposome assemblies with 13.3-kDa ( top ) and 7-kDa ( bottom ) HEP-lipid, based on typical molecular configurations from equilibrated coarse-grained simulations. The liposome and sugars are represented graphically by equivalent spheres (roughly to scale), and part of the liposome has been cut-away for viewing purposes. Potential interaction proteins are depicted (human serum albumin, HSA ; immunoglobulins IgG or IgM ; middle ), drawn to exact scale using coordinates from the Protein Data Bank. Less surface area is available for direct protein binding to liposomes coated with 0.5 mol% 13.3-kDa HEP-lipid compared to 0.5 mol% 7-kDa HEP-lipid, suggesting increased liposomal protection from clearance by the immune system. The image was rendered using POV-Ray software.

    Techniques Used: Protein Binding, Software

    7) Product Images from "Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3"

    Article Title: Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-06682-2

    Serum survival of K. pneumoniae strains. Strains were incubated in different concentrations of human serum ( a ) or 75% serum complement-inactivated by heat (HI) or cobra venom factor (CVF) treatment ( b ) for 3 h at 37 °C. The recovered bacterial count was expressed as percentage of the initial inoculum. ATCC43816 (O1:K2) and PCM-27 (O2:K27) strains were used as comparators.Graphs show combined results of 2 and 3 experiments for the active and heat inactivated serum experiments, respectively. The same O3 isolates were stained with 10% human serum pool and IgG and IgM antibodies binding to live bacteria were detected with a labelled anti-human IgG or IgM by flow cytometry ( c ). The median fluorescent intensity is shown as arbitrary units. The difference between the staining intensity for O3 and O3b strains was not significant using the Mann Whitney test (p > 0.05; n.s.). O-serotype and capsule type of the strains are indicated in Table 2 .
    Figure Legend Snippet: Serum survival of K. pneumoniae strains. Strains were incubated in different concentrations of human serum ( a ) or 75% serum complement-inactivated by heat (HI) or cobra venom factor (CVF) treatment ( b ) for 3 h at 37 °C. The recovered bacterial count was expressed as percentage of the initial inoculum. ATCC43816 (O1:K2) and PCM-27 (O2:K27) strains were used as comparators.Graphs show combined results of 2 and 3 experiments for the active and heat inactivated serum experiments, respectively. The same O3 isolates were stained with 10% human serum pool and IgG and IgM antibodies binding to live bacteria were detected with a labelled anti-human IgG or IgM by flow cytometry ( c ). The median fluorescent intensity is shown as arbitrary units. The difference between the staining intensity for O3 and O3b strains was not significant using the Mann Whitney test (p > 0.05; n.s.). O-serotype and capsule type of the strains are indicated in Table 2 .

    Techniques Used: Incubation, Combined Bisulfite Restriction Analysis Assay, Staining, Binding Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    8) Product Images from "Immunogenicity of Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine"

    Article Title: Immunogenicity of Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine

    Journal: Pathogens

    doi: 10.3390/pathogens9070557

    The anti-FIS IgM and IgG subclasses (IgG1 and IgG2) ELISA titres of cattle are vaccinated at week 0 and 3 with either PrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), CrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), SLSV (n = 8) and Emulsigen-D ® /Alhydrogel ® adjuvants (NegCtl) (n = 4) are presented as box and whisker plots. The cattle sera samples were collected before the vaccinations at week 0 and 3 as well as at week 5 and 12. Sera dilution started at a concentration of 1:50 and values below the (
    Figure Legend Snippet: The anti-FIS IgM and IgG subclasses (IgG1 and IgG2) ELISA titres of cattle are vaccinated at week 0 and 3 with either PrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), CrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), SLSV (n = 8) and Emulsigen-D ® /Alhydrogel ® adjuvants (NegCtl) (n = 4) are presented as box and whisker plots. The cattle sera samples were collected before the vaccinations at week 0 and 3 as well as at week 5 and 12. Sera dilution started at a concentration of 1:50 and values below the (

    Techniques Used: Enzyme-linked Immunosorbent Assay, Whisker Assay, Concentration Assay

    The anti-rPA IgM and IgG subclasses (IgG1 and IgG2) ELISA titres of cattle are vaccinated at week 0 and 3 with either PrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), CrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), SLSV (n = 8) and Emulsigen-D ® /Alhydrogel ® adjuvants (NegCtl) (n = 4) are presented as box and whisker plots. The cattle sera samples were collected before the vaccinations at week 0 and 3 as well as at week 5 and 12. Sera dilution started at a concentration of 1:50 and values below the (
    Figure Legend Snippet: The anti-rPA IgM and IgG subclasses (IgG1 and IgG2) ELISA titres of cattle are vaccinated at week 0 and 3 with either PrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), CrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), SLSV (n = 8) and Emulsigen-D ® /Alhydrogel ® adjuvants (NegCtl) (n = 4) are presented as box and whisker plots. The cattle sera samples were collected before the vaccinations at week 0 and 3 as well as at week 5 and 12. Sera dilution started at a concentration of 1:50 and values below the (

    Techniques Used: Recombinase Polymerase Amplification, Enzyme-linked Immunosorbent Assay, Whisker Assay, Concentration Assay

    9) Product Images from "Requirement for NF-?B in osteoclast and B-cell development"

    Article Title: Requirement for NF-?B in osteoclast and B-cell development

    Journal: Genes & Development

    doi:

    Developmental arrest of B cells is due to defects that track with to the B-cell lineage. Lethally irradiated RAG-1-deficient mice were injected with BM cells isolated from 22-day-old double knockout mice (dKO) or p50(+/+), p52(+/−) or p50(+/−), p52(+/+) (WT; these mice are indistinguishable from wild type) littermate control donor mice, as indicated. Fifteen weeks later mice were challenged i.p. with TNP–KLH (100 μg) adsorbed to alum. Mice were analyzed 9-days after challenge. ( A ) Three-color FCM analysis of spleen and lymph nodes of adoptively transferred RAG-1-deficient mice. Two-color profiles or single-color histograms are displayed in black and white, and genotypes of donor animals are as indicated. The following antibodies (listed top to bottom and left to right ) were used. Spleen: IgM–biotin vs. B220–PE; IgD–FITC vs. B220–PE; IgD–FITC vs. IgM–biotin; CD23–PE vs. B220–biotin; I-A b –biotin vs. B220–PE, and CD4–biotin vs. CD8a–PE. Lymph nodes: CD4–biotin vs. CD8a–PE and IgM–biotin vs. B220–PE. Numbers in the quadrants reflect the percentage of total spleen and lymph node cells in that quadrant. FCM data are representative of three paired sets of adoptively transferred RAG-1 mice. ( B ) Stained cryosections from representative double knockout (dKO, right panels) and p50(+/−), p52(+/+) (WT, left panels) control littermate marrow-transferred RAG-1-deficient animals are shown. Splenic cryosections were stained with PNA (brown) and anti-B220 antibodies (red) ( top panels) or anti-CD-3 antibodies (blue; bottom panels), as indicated.
    Figure Legend Snippet: Developmental arrest of B cells is due to defects that track with to the B-cell lineage. Lethally irradiated RAG-1-deficient mice were injected with BM cells isolated from 22-day-old double knockout mice (dKO) or p50(+/+), p52(+/−) or p50(+/−), p52(+/+) (WT; these mice are indistinguishable from wild type) littermate control donor mice, as indicated. Fifteen weeks later mice were challenged i.p. with TNP–KLH (100 μg) adsorbed to alum. Mice were analyzed 9-days after challenge. ( A ) Three-color FCM analysis of spleen and lymph nodes of adoptively transferred RAG-1-deficient mice. Two-color profiles or single-color histograms are displayed in black and white, and genotypes of donor animals are as indicated. The following antibodies (listed top to bottom and left to right ) were used. Spleen: IgM–biotin vs. B220–PE; IgD–FITC vs. B220–PE; IgD–FITC vs. IgM–biotin; CD23–PE vs. B220–biotin; I-A b –biotin vs. B220–PE, and CD4–biotin vs. CD8a–PE. Lymph nodes: CD4–biotin vs. CD8a–PE and IgM–biotin vs. B220–PE. Numbers in the quadrants reflect the percentage of total spleen and lymph node cells in that quadrant. FCM data are representative of three paired sets of adoptively transferred RAG-1 mice. ( B ) Stained cryosections from representative double knockout (dKO, right panels) and p50(+/−), p52(+/+) (WT, left panels) control littermate marrow-transferred RAG-1-deficient animals are shown. Splenic cryosections were stained with PNA (brown) and anti-B220 antibodies (red) ( top panels) or anti-CD-3 antibodies (blue; bottom panels), as indicated.

    Techniques Used: Irradiation, Mouse Assay, Injection, Isolation, Double Knockout, Staining

    Defects of B and T lymphocytes in double knockout mice. Three-color FCM analysis of spleen and BM cell suspensions from 13- to 15-day-old double knockout mice (dKO, left panels) and p50(+/+), p52(+/−) littermate controls (WT, right panels). Two-color profiles or single-color histograms are displayed. The data are taken from analyses of three representative sets of mice, but the results were confirmed with two additional pairs. The following antibodies (listed top to bottom and left to right ) were used. Spleen: B220–biotin, IgD–FITC vs. IgM–biotin; IgD-FITC vs. B220–PE; IgM–biotin vs. B220–PE; κ-light chain (LC)–FITC vs. B220–PE; λ-LC–FITC vs. B220–PE; CD23–FITC vs. B220–biotin; I-A b -biotin vs. B220–FITC; B220–FITC vs. Mac-1–PE; Gr-1–FITC; Thy1.2–biotin; and CD4–biotin vs. CD8a–PE. Bone marrow: IgM–biotin vs. B220–PE; B220–FITC vs. class II (I-A b )–biotin; and Gr-1–FITC vs. Mac-1–PE. Numbers reflect the percentage of positively stained cells. The percentage of B220 + BM cells was modestly increased in mutant mice, whereas class II expression was more intense in wild-type BM, even though fully mature cells were nearly absent, as judged by lack of expression of IgD (not shown).
    Figure Legend Snippet: Defects of B and T lymphocytes in double knockout mice. Three-color FCM analysis of spleen and BM cell suspensions from 13- to 15-day-old double knockout mice (dKO, left panels) and p50(+/+), p52(+/−) littermate controls (WT, right panels). Two-color profiles or single-color histograms are displayed. The data are taken from analyses of three representative sets of mice, but the results were confirmed with two additional pairs. The following antibodies (listed top to bottom and left to right ) were used. Spleen: B220–biotin, IgD–FITC vs. IgM–biotin; IgD-FITC vs. B220–PE; IgM–biotin vs. B220–PE; κ-light chain (LC)–FITC vs. B220–PE; λ-LC–FITC vs. B220–PE; CD23–FITC vs. B220–biotin; I-A b -biotin vs. B220–FITC; B220–FITC vs. Mac-1–PE; Gr-1–FITC; Thy1.2–biotin; and CD4–biotin vs. CD8a–PE. Bone marrow: IgM–biotin vs. B220–PE; B220–FITC vs. class II (I-A b )–biotin; and Gr-1–FITC vs. Mac-1–PE. Numbers reflect the percentage of positively stained cells. The percentage of B220 + BM cells was modestly increased in mutant mice, whereas class II expression was more intense in wild-type BM, even though fully mature cells were nearly absent, as judged by lack of expression of IgD (not shown).

    Techniques Used: Double Knockout, Mouse Assay, Staining, Mutagenesis, Expressing

    10) Product Images from "Assessment of enhanced influenza vaccination finds that FluAd conveys an advantage in mice and older adults"

    Article Title: Assessment of enhanced influenza vaccination finds that FluAd conveys an advantage in mice and older adults

    Journal: Clinical & Translational Immunology

    doi: 10.1002/cti2.1107

    Lung elevated cytokine production and secretory‐specific IgA, along with high serum H3‐stem antibodies in A‐eIIV mice after challenge. Mice were challenged with 20LD 50 H3N2‐1968 virus 21 days or 6 months post‐vaccination, and inflammatory responses were sampled at day 7 post‐infection. Inflammatory cytokine and chemokine concentrations in individual lung homogenates ( n = 4) by CBA were normalised to fold change from PBS response (a) . H3N2‐1968 VNA endpoint titres from serum from short‐term challenge at day 7 post‐infection (b) . In vitro protection assay of H3N2‐1968‐infected Raji cells with mouse serums from 7 and 21 days post‐infection ( n = 3 or 4), responses were normalised to naïve mouse serum (c) . Day 7 post‐challenge, H3N2‐1968 HA‐specific secretory IgA in BALF for short‐ and long‐term memory recall measured by ELISA (d) , normalised to total protein concentration by BCA. H3N2‐1968 HA and H3‐stem‐specific IgG responses by ELISA for short‐ and long‐term memory recall at day 21 post‐challenge (e) . Data represent the mean, SEM and individual responses, n = 4 mice per group. *shows statistical significance of eIIV versus S‐IIV. * P
    Figure Legend Snippet: Lung elevated cytokine production and secretory‐specific IgA, along with high serum H3‐stem antibodies in A‐eIIV mice after challenge. Mice were challenged with 20LD 50 H3N2‐1968 virus 21 days or 6 months post‐vaccination, and inflammatory responses were sampled at day 7 post‐infection. Inflammatory cytokine and chemokine concentrations in individual lung homogenates ( n = 4) by CBA were normalised to fold change from PBS response (a) . H3N2‐1968 VNA endpoint titres from serum from short‐term challenge at day 7 post‐infection (b) . In vitro protection assay of H3N2‐1968‐infected Raji cells with mouse serums from 7 and 21 days post‐infection ( n = 3 or 4), responses were normalised to naïve mouse serum (c) . Day 7 post‐challenge, H3N2‐1968 HA‐specific secretory IgA in BALF for short‐ and long‐term memory recall measured by ELISA (d) , normalised to total protein concentration by BCA. H3N2‐1968 HA and H3‐stem‐specific IgG responses by ELISA for short‐ and long‐term memory recall at day 21 post‐challenge (e) . Data represent the mean, SEM and individual responses, n = 4 mice per group. *shows statistical significance of eIIV versus S‐IIV. * P

    Techniques Used: Mouse Assay, Infection, Crocin Bleaching Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Protein Concentration

    A‐eIIV induces high‐avidity H3‐2013 and H3‐stem IgG in humans and mice. A randomised clinical trial to assess eIIV immunogenicity compared with S‐IIV, sampled blood plasma at days 0 (baseline), 7, 30 and 365 after vaccination (a) . H3N2‐2013 HA titres (b) , high‐avidity H3N2‐2013 HA (c) and H3‐stem‐specific (d) IgG responses were measured by ELISA. The same vaccines were assessed in a mouse model of vaccination and infection (e) . Vaccine strains, H1N1‐2009 and H3N2‐2013 HA‐specific and H1N1‐2009 NA‐specific, day 7 and 21 post‐vaccination IgG titres by ELISA (f) and day 21 post‐vaccination by VNA (g) . Cross‐reactive, H3N2‐1968, H3‐stem HA‐specific and NP‐specific, day 7 and 21 post‐vaccination IgG responses by ELISA (h) . Proportion of low‐ and high‐avidity H3N2‐1968 HA and H3‐stem‐specific IgG (i) . Data represent the mean, SEM and individual responses, (b–d) n = 20 per group and (f–i) n = 4 or 5 per group. For b–d , *shows statistical significance by one‐way ANOVA with the Friedman test in each vaccine group day 0 versus days 7, 30 and 365. For (f–i) , *shows statistical significance by one‐way ANOVA (mixed model) for eIIV versus S‐IIV by Tukey's multiple comparison test. For (i) , # shows statistical significance for low‐avidity antibodies and *for high‐avidity antibodies for eIIV versus S‐IIV by one‐way ANOVA (mixed model) and Tukey's multiple comparison test. # or * P
    Figure Legend Snippet: A‐eIIV induces high‐avidity H3‐2013 and H3‐stem IgG in humans and mice. A randomised clinical trial to assess eIIV immunogenicity compared with S‐IIV, sampled blood plasma at days 0 (baseline), 7, 30 and 365 after vaccination (a) . H3N2‐2013 HA titres (b) , high‐avidity H3N2‐2013 HA (c) and H3‐stem‐specific (d) IgG responses were measured by ELISA. The same vaccines were assessed in a mouse model of vaccination and infection (e) . Vaccine strains, H1N1‐2009 and H3N2‐2013 HA‐specific and H1N1‐2009 NA‐specific, day 7 and 21 post‐vaccination IgG titres by ELISA (f) and day 21 post‐vaccination by VNA (g) . Cross‐reactive, H3N2‐1968, H3‐stem HA‐specific and NP‐specific, day 7 and 21 post‐vaccination IgG responses by ELISA (h) . Proportion of low‐ and high‐avidity H3N2‐1968 HA and H3‐stem‐specific IgG (i) . Data represent the mean, SEM and individual responses, (b–d) n = 20 per group and (f–i) n = 4 or 5 per group. For b–d , *shows statistical significance by one‐way ANOVA with the Friedman test in each vaccine group day 0 versus days 7, 30 and 365. For (f–i) , *shows statistical significance by one‐way ANOVA (mixed model) for eIIV versus S‐IIV by Tukey's multiple comparison test. For (i) , # shows statistical significance for low‐avidity antibodies and *for high‐avidity antibodies for eIIV versus S‐IIV by one‐way ANOVA (mixed model) and Tukey's multiple comparison test. # or * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Infection

    Elevated CD4 + Tfh and GC B cells by increased expression of SHM and CSR genes lead to early IgG class switch for A‐eIIV. Vaccine responses were assessed by FACS (a) for CD4 + Tfh cells (b) and GC B cells (c) at days 7 and 21 post‐vaccination in the iLN. Expression of AID, Bcl6, IRF4, Pax5 and c‐Myc in B cells from iLN (relative expression to GAPDH expression, expressed in 2 Δ C t ) at day 7 post‐vaccination (d) . Quantity (f) and proportion (g) of H3N2‐1968 HA‐specific IgM and IgG at days 7 and 21 post‐vaccination, measured by ELISA AUC endpoint titres (Supplementary figure 2 ). Data represent the mean, SEM and individual responses, n = 4 or 5 mice per group. *shows statistical significance by one‐way ANOVA (mixed model) for eIIV versus S‐IIV, * P
    Figure Legend Snippet: Elevated CD4 + Tfh and GC B cells by increased expression of SHM and CSR genes lead to early IgG class switch for A‐eIIV. Vaccine responses were assessed by FACS (a) for CD4 + Tfh cells (b) and GC B cells (c) at days 7 and 21 post‐vaccination in the iLN. Expression of AID, Bcl6, IRF4, Pax5 and c‐Myc in B cells from iLN (relative expression to GAPDH expression, expressed in 2 Δ C t ) at day 7 post‐vaccination (d) . Quantity (f) and proportion (g) of H3N2‐1968 HA‐specific IgM and IgG at days 7 and 21 post‐vaccination, measured by ELISA AUC endpoint titres (Supplementary figure 2 ). Data represent the mean, SEM and individual responses, n = 4 or 5 mice per group. *shows statistical significance by one‐way ANOVA (mixed model) for eIIV versus S‐IIV, * P

    Techniques Used: Expressing, FACS, Enzyme-linked Immunosorbent Assay, Mouse Assay

    Memory B‐cell responses and long‐term antibody responses are increased in A‐eIIV‐vaccinated mice. BALB/c mice were vaccinated twice i.m. with either PBS, S‐IIV, A‐eIIV, H‐eIIV or R‐eIIV vaccines 3 weeks apart. iLN was collected at days 7 and 21 post‐vaccination to quantify by FACS (a) , IgG + (b) and IgM + (c) B memory cells. Longitudinal serum samples were serially sampled from individual mice at 2, 4 and 6 months post‐vaccination for H3N2‐2013 (d) , H3N2‐1968 (e) and H3‐stem (f) IgG by ELISA for avidity. Data represent the mean, SEM and individual responses, n = 4–8 mice per group. For (b, c) , *shows statistical significance for eIIV versus S‐IIV by one‐way ANOVA (mixed model) and Tukey's multiple comparison test. For d–f , # shows statistical significance for low‐avidity antibodies and *for high‐avidity antibodies for eIIV versus S‐IIV by one‐way ANOVA (mixed model) and Tukey's multiple comparison test. # or * P
    Figure Legend Snippet: Memory B‐cell responses and long‐term antibody responses are increased in A‐eIIV‐vaccinated mice. BALB/c mice were vaccinated twice i.m. with either PBS, S‐IIV, A‐eIIV, H‐eIIV or R‐eIIV vaccines 3 weeks apart. iLN was collected at days 7 and 21 post‐vaccination to quantify by FACS (a) , IgG + (b) and IgM + (c) B memory cells. Longitudinal serum samples were serially sampled from individual mice at 2, 4 and 6 months post‐vaccination for H3N2‐2013 (d) , H3N2‐1968 (e) and H3‐stem (f) IgG by ELISA for avidity. Data represent the mean, SEM and individual responses, n = 4–8 mice per group. For (b, c) , *shows statistical significance for eIIV versus S‐IIV by one‐way ANOVA (mixed model) and Tukey's multiple comparison test. For d–f , # shows statistical significance for low‐avidity antibodies and *for high‐avidity antibodies for eIIV versus S‐IIV by one‐way ANOVA (mixed model) and Tukey's multiple comparison test. # or * P

    Techniques Used: Mouse Assay, FACS, Enzyme-linked Immunosorbent Assay

    H7N7 cross‐reactive A‐eIIV antibodies in mice and humans. H7N9‐HA‐specific IgG titres measured by ELISA in mice at 7 days post‐vaccination ( n = 5 mice per group) (a) . Correlation of H7N9‐HA and H3‐stem IgG titres in mice serum by the Pearson correlation, R 2 = 0.624, P
    Figure Legend Snippet: H7N7 cross‐reactive A‐eIIV antibodies in mice and humans. H7N9‐HA‐specific IgG titres measured by ELISA in mice at 7 days post‐vaccination ( n = 5 mice per group) (a) . Correlation of H7N9‐HA and H3‐stem IgG titres in mice serum by the Pearson correlation, R 2 = 0.624, P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Placental Malaria Induces Variant-Specific Antibodies of the Cytophilic Subtypes Immunoglobulin G1 (IgG1) and IgG3 That Correlate with Adhesion Inhibitory Activity "

    Article Title: Placental Malaria Induces Variant-Specific Antibodies of the Cytophilic Subtypes Immunoglobulin G1 (IgG1) and IgG3 That Correlate with Adhesion Inhibitory Activity

    Journal: Infection and Immunity

    doi: 10.1128/IAI.73.9.5903-5907.2005

    Associations between binding of IgM and IgG subtypes to VSA expressed by the P. falciparum line CS2 and the ability of serum or plasma to inhibit adhesion of CS2-infected erythrocytes to chondroitin sulfate A. Levels of antibodies in serum or plasma from
    Figure Legend Snippet: Associations between binding of IgM and IgG subtypes to VSA expressed by the P. falciparum line CS2 and the ability of serum or plasma to inhibit adhesion of CS2-infected erythrocytes to chondroitin sulfate A. Levels of antibodies in serum or plasma from

    Techniques Used: Binding Assay, Infection

    Levels of IgG subtypes and IgM binding to VSA expressed by the chondroitin sulfate A-adherent P. falciparum line CS2. Flow cytometry was used to measure antibodies to CS2-VSA in serum/plasma from PG and MG women with histologically confirmed placental
    Figure Legend Snippet: Levels of IgG subtypes and IgM binding to VSA expressed by the chondroitin sulfate A-adherent P. falciparum line CS2. Flow cytometry was used to measure antibodies to CS2-VSA in serum/plasma from PG and MG women with histologically confirmed placental

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry

    12) Product Images from "Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients"

    Article Title: Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0184738

    TAGLN2 mRNA is induced upon BCR stimulation and significantly expressed on activated B-cells in the germinal center. (A) Immunohistochemistry showing TAGLN2 localized in the germinal center in human secondary lymphoid tissues (left) and Bcl-6 expression, a marker of germinal center B-cells (right) (original magnification, x400). GC, germinal center. The figure is representative of 5 tonsils examined. (B) Double immunofluorescence analysis of TAGLN2 (green) and the B-cell marker CD20 (red), showing coexpression of TAGLN2 and CD20 in the germinal center. Nuclei were stained with DAPI (arrows, original magnification, x400). (C) TAGLN2 mRNA is induced upon BCR stimulation. TAGLN2 mRNA levels were measured in primary human B-cells from six different healthy donors at the indicated time points after IgM+IgG stimulation using qRT-PCR. At each time point, the TAGLN2 mRNA levels in anti-IgM+IgG stimulated cells (stim) are expressed fold change relative to the levels in non-stimulated (non-stim) cells. The mean TAGLN2 mRNA expression ratio was 0.93-fold at 1 h, 1.09-fold at 6 h, and 2.85-fold at 24 h after stimulation, suggesting that TAGLN2 mRNA was induced by 24-h B-cell activation (p
    Figure Legend Snippet: TAGLN2 mRNA is induced upon BCR stimulation and significantly expressed on activated B-cells in the germinal center. (A) Immunohistochemistry showing TAGLN2 localized in the germinal center in human secondary lymphoid tissues (left) and Bcl-6 expression, a marker of germinal center B-cells (right) (original magnification, x400). GC, germinal center. The figure is representative of 5 tonsils examined. (B) Double immunofluorescence analysis of TAGLN2 (green) and the B-cell marker CD20 (red), showing coexpression of TAGLN2 and CD20 in the germinal center. Nuclei were stained with DAPI (arrows, original magnification, x400). (C) TAGLN2 mRNA is induced upon BCR stimulation. TAGLN2 mRNA levels were measured in primary human B-cells from six different healthy donors at the indicated time points after IgM+IgG stimulation using qRT-PCR. At each time point, the TAGLN2 mRNA levels in anti-IgM+IgG stimulated cells (stim) are expressed fold change relative to the levels in non-stimulated (non-stim) cells. The mean TAGLN2 mRNA expression ratio was 0.93-fold at 1 h, 1.09-fold at 6 h, and 2.85-fold at 24 h after stimulation, suggesting that TAGLN2 mRNA was induced by 24-h B-cell activation (p

    Techniques Used: Immunohistochemistry, Expressing, Marker, Immunofluorescence, Staining, Quantitative RT-PCR, Activation Assay

    TAGLN2 colocalizes with F-actin and moves together to the periphery after stimulation. TAGLN2-GFP overlaps with LifeAct-RFP in Raji B cells at the outer actin ring. Raji cells co-transfected with TAGLN2-GFP and the actin probe LifeAct-RFP were stimulated with IgM+IgG, and the localization of each protein was then followed over the next 18 min using time lapse fluorescence microscopy. Top: TAGLN2-GFP (Green), middle: LifeAct-RFP (Magenta), bottom: merged image. The images marked as 0 were acquired before stimulation. Both the actin and TAGLN2 signals increased in intensity and thickness after IgM+IgG stimulation and were depleted from the central area of the cells and moved together to the periphery.
    Figure Legend Snippet: TAGLN2 colocalizes with F-actin and moves together to the periphery after stimulation. TAGLN2-GFP overlaps with LifeAct-RFP in Raji B cells at the outer actin ring. Raji cells co-transfected with TAGLN2-GFP and the actin probe LifeAct-RFP were stimulated with IgM+IgG, and the localization of each protein was then followed over the next 18 min using time lapse fluorescence microscopy. Top: TAGLN2-GFP (Green), middle: LifeAct-RFP (Magenta), bottom: merged image. The images marked as 0 were acquired before stimulation. Both the actin and TAGLN2 signals increased in intensity and thickness after IgM+IgG stimulation and were depleted from the central area of the cells and moved together to the periphery.

    Techniques Used: Transfection, Fluorescence, Microscopy

    TAGLN2 knockdown results in impaired phosphorylation of PLCγ, which is related to the actin-linked signaling pathway and inhibition of cell migration. (A) The total protein levels of PLCγ2, PI3K, ERK, and Akt and the levels of their phosphorylated forms after 10 min IgM+IgG stimulation of TAGLN2 -knockdown (KD) Raji cells transfected with TAGLN2 siRNA, or of control cells with scrambled siRNA (Scramble), were detected by immunoblotting. Actin was blotted as a loading control. Suppression of TAGLN2 protein expression and PLCγ2 phosphorylation was seen in the TAGLN2 -KD Raji cells compared with the scrambled siRNA transfected cells. (B) Normalized expression levels of each protein and each phosphorylated protein assessed using membrane densitometry. Three independent experiments were performed. *p
    Figure Legend Snippet: TAGLN2 knockdown results in impaired phosphorylation of PLCγ, which is related to the actin-linked signaling pathway and inhibition of cell migration. (A) The total protein levels of PLCγ2, PI3K, ERK, and Akt and the levels of their phosphorylated forms after 10 min IgM+IgG stimulation of TAGLN2 -knockdown (KD) Raji cells transfected with TAGLN2 siRNA, or of control cells with scrambled siRNA (Scramble), were detected by immunoblotting. Actin was blotted as a loading control. Suppression of TAGLN2 protein expression and PLCγ2 phosphorylation was seen in the TAGLN2 -KD Raji cells compared with the scrambled siRNA transfected cells. (B) Normalized expression levels of each protein and each phosphorylated protein assessed using membrane densitometry. Three independent experiments were performed. *p

    Techniques Used: Inhibition, Migration, Transfection, Expressing

    13) Product Images from "Differences in Immune Responses Induced by Oral and Rectal Immunizations with Salmonella typhi Ty21a: Evidence for Compartmentalization within the Common Mucosal Immune System in Humans"

    Article Title: Differences in Immune Responses Induced by Oral and Rectal Immunizations with Salmonella typhi Ty21a: Evidence for Compartmentalization within the Common Mucosal Immune System in Humans

    Journal: Infection and Immunity

    doi:

    Expression of the gut HR α4β7 on circulating specific ASC and total ISC in volunteers 7 days after oral (a; n = 4) or rectal (b; n = 9) vaccination with S. typhi Ty21a. The bars indicate arithmetic means (±SD) of percentages of cells expressing α4β7 among IgA (black bars), IgG (white bars), and IgM (hatched bars) ASC and ISC.
    Figure Legend Snippet: Expression of the gut HR α4β7 on circulating specific ASC and total ISC in volunteers 7 days after oral (a; n = 4) or rectal (b; n = 9) vaccination with S. typhi Ty21a. The bars indicate arithmetic means (±SD) of percentages of cells expressing α4β7 among IgA (black bars), IgG (white bars), and IgM (hatched bars) ASC and ISC.

    Techniques Used: Expressing

    Kinetics of the circulating specific ASC response after oral (a; n = 4) or rectal (b; n = 6) immunization of humans with live S. typhi Ty21a vaccine. Each volunteer received one dose (at least 10 9 live bacteria/dose) of vaccine on each of days 0, 2, 4, and 6. Each value represents the sum of IgA-, IgG-, and IgM ASC of one individual on a given day. PBMC, peripheral blood mononuclear cells.
    Figure Legend Snippet: Kinetics of the circulating specific ASC response after oral (a; n = 4) or rectal (b; n = 6) immunization of humans with live S. typhi Ty21a vaccine. Each volunteer received one dose (at least 10 9 live bacteria/dose) of vaccine on each of days 0, 2, 4, and 6. Each value represents the sum of IgA-, IgG-, and IgM ASC of one individual on a given day. PBMC, peripheral blood mononuclear cells.

    Techniques Used:

    S. typhi -specific antibodies in serum after oral ( n = 5) or rectal ( n = 11) immunization with S. typhi Ty21a. The bars indicate geometric mean concentrations (±SEM) of specific IgA, IgG, or IgM antibodies (ab) in serum before and after immunization. Significant increases from preimmune levels are indicated with asterisks.
    Figure Legend Snippet: S. typhi -specific antibodies in serum after oral ( n = 5) or rectal ( n = 11) immunization with S. typhi Ty21a. The bars indicate geometric mean concentrations (±SEM) of specific IgA, IgG, or IgM antibodies (ab) in serum before and after immunization. Significant increases from preimmune levels are indicated with asterisks.

    Techniques Used:

    14) Product Images from "Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis"

    Article Title: Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis

    Journal: Autoimmunity

    doi: 10.3109/08916934.2012.750301

    Indirect fluorescence for binding of serum antibody to normal brain tissue. Serum from EAE affected WT mice show diffuse, strong white matter associated signal consistent with the presence of MOG specific IgG1. By contrast, corresponding signal is absent using serum from MOG induced KO mice, or healthy controls. There is no difference of signal intensity or distribution for IgM binding to brain, regardless of MOG induction or genotype. Representative images are shown.
    Figure Legend Snippet: Indirect fluorescence for binding of serum antibody to normal brain tissue. Serum from EAE affected WT mice show diffuse, strong white matter associated signal consistent with the presence of MOG specific IgG1. By contrast, corresponding signal is absent using serum from MOG induced KO mice, or healthy controls. There is no difference of signal intensity or distribution for IgM binding to brain, regardless of MOG induction or genotype. Representative images are shown.

    Techniques Used: Fluorescence, Binding Assay, Mouse Assay

    Generation and functional characterization of Aicda deficient mice. A) Gene targeting strategy used to delete the catalytic domain (exon-3) of Aicda locus. The structure of the AID domains is shown in top. The location of the homology arms in the targeting vector is indicated. The schematic of targeting construct used to delete the catalytic domain of AID (exon-3) is shown in the middle. The solid triangles represent Cre recombination sites and the Neomycin cassette was subsequently removed by expression of Cre-recombinase. Representative WT or KO PCR products using 3′ or 5′ arm analysis at the Aicda locus is shown in the right panel. The size of Aicda KO PCR product is indicated and corresponds to the modified locus. Notice that one primer is located in the Neomycin cassette and therefore only detects the targeted Aicda locus. B) FACS analysis of LPS/IL4 stimulated WT or AID KO B cells in vitro. Percentage of IgM or IgG1 are indicated in each quadrant. B220-FITC is used as a B-cell marker.
    Figure Legend Snippet: Generation and functional characterization of Aicda deficient mice. A) Gene targeting strategy used to delete the catalytic domain (exon-3) of Aicda locus. The structure of the AID domains is shown in top. The location of the homology arms in the targeting vector is indicated. The schematic of targeting construct used to delete the catalytic domain of AID (exon-3) is shown in the middle. The solid triangles represent Cre recombination sites and the Neomycin cassette was subsequently removed by expression of Cre-recombinase. Representative WT or KO PCR products using 3′ or 5′ arm analysis at the Aicda locus is shown in the right panel. The size of Aicda KO PCR product is indicated and corresponds to the modified locus. Notice that one primer is located in the Neomycin cassette and therefore only detects the targeted Aicda locus. B) FACS analysis of LPS/IL4 stimulated WT or AID KO B cells in vitro. Percentage of IgM or IgG1 are indicated in each quadrant. B220-FITC is used as a B-cell marker.

    Techniques Used: Functional Assay, Mouse Assay, Plasmid Preparation, Construct, Expressing, Polymerase Chain Reaction, Modification, FACS, In Vitro, Marker

    Characterization of anti-MOG antibodies using ELISA at day zero and 28. A) IgG anti-rhMOG, B) IgM anti-rhMOG, C) IgG anti-MOG peptide 35–55, D) IgM anti-MOG peptide 35–55. Mixed day 28 sera of WT or KO immunized mice were used as standard (arbitrarily defined as 100 U) for IgG or IgM antibodies, respectively. E) IgM binding to rhMOG coated at different concentrations. Sera from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Pooled mice sera IgM Abs normalized at 0.1 mg/ml in the assay. Anti-mouse IgM was used as detection to exclusively measure the binding of IgM antibodies in serum.
    Figure Legend Snippet: Characterization of anti-MOG antibodies using ELISA at day zero and 28. A) IgG anti-rhMOG, B) IgM anti-rhMOG, C) IgG anti-MOG peptide 35–55, D) IgM anti-MOG peptide 35–55. Mixed day 28 sera of WT or KO immunized mice were used as standard (arbitrarily defined as 100 U) for IgG or IgM antibodies, respectively. E) IgM binding to rhMOG coated at different concentrations. Sera from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Pooled mice sera IgM Abs normalized at 0.1 mg/ml in the assay. Anti-mouse IgM was used as detection to exclusively measure the binding of IgM antibodies in serum.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Binding Assay

    Surface plasmon resonance (SPR) analysis of serum antibody binding to MOG. A. SPR sensorgrams of serum samples binding to sensor immobilized MOG. Serum from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Start and stop injection times are indicated by arrows. Day 28 WT serum showed the highest binding response, and the majority of this binding signal could be competed with soluble MOG (2 uM) preincubated with the serum sample before injection. B. Detection of the major binding isotype in Day 28 WT serum. Subsequent injections of three anti-isotype antibodies were used to probe the MOG bound component. Start and stop injection times are indicated by arrows for each sample (Day 28 WT serum, anti-mIgM, anti-mIgG2a and anti-mIgG1). The data indicate that the majority of the binding signal is specific to IgG1. No background binding of anti-mIgG1 to MOG alone was detected (data not shown).
    Figure Legend Snippet: Surface plasmon resonance (SPR) analysis of serum antibody binding to MOG. A. SPR sensorgrams of serum samples binding to sensor immobilized MOG. Serum from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Start and stop injection times are indicated by arrows. Day 28 WT serum showed the highest binding response, and the majority of this binding signal could be competed with soluble MOG (2 uM) preincubated with the serum sample before injection. B. Detection of the major binding isotype in Day 28 WT serum. Subsequent injections of three anti-isotype antibodies were used to probe the MOG bound component. Start and stop injection times are indicated by arrows for each sample (Day 28 WT serum, anti-mIgM, anti-mIgG2a and anti-mIgG1). The data indicate that the majority of the binding signal is specific to IgG1. No background binding of anti-mIgG1 to MOG alone was detected (data not shown).

    Techniques Used: SPR Assay, Binding Assay, Mouse Assay, Injection

    15) Product Images from "Alterations of Total Serum Immunoglobulin Concentrations in Pemphigus and Pemphigoid: Selected IgG2 Deficiency in Bullous Pemphigoid"

    Article Title: Alterations of Total Serum Immunoglobulin Concentrations in Pemphigus and Pemphigoid: Selected IgG2 Deficiency in Bullous Pemphigoid

    Journal: Frontiers in Medicine

    doi: 10.3389/fmed.2020.00472

    Serum immunoglobulin (Ig) concentrations in patients with pemphigus and pemphigoid disease. Serum concentration of IgM, IgA, IgG, and IgG1–4 were determined in healthy blood donors and patients with the indicated pemphigus and pemphigoid diseases by ELISA. The numbers below the boxes indicate the number of controls/patients included in each group. Data are shown as median (black line) and 25/75 percentiles (boxes); the whiskers extend no further than 1.5× IQR from a hinge (where IQR is the interquartile range) and outliers (points). ANOVA with Tukey's HSD procedure as post-hoc test was used for statistical analysis. *indicates p
    Figure Legend Snippet: Serum immunoglobulin (Ig) concentrations in patients with pemphigus and pemphigoid disease. Serum concentration of IgM, IgA, IgG, and IgG1–4 were determined in healthy blood donors and patients with the indicated pemphigus and pemphigoid diseases by ELISA. The numbers below the boxes indicate the number of controls/patients included in each group. Data are shown as median (black line) and 25/75 percentiles (boxes); the whiskers extend no further than 1.5× IQR from a hinge (where IQR is the interquartile range) and outliers (points). ANOVA with Tukey's HSD procedure as post-hoc test was used for statistical analysis. *indicates p

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Impact of age and sex on serum immunoglobulin (Ig) concentrations in healthy blood donors. Serum concentration of IgM, IgA, IgG, and IgG1–4 was determined in healthy blood donors by ELISA. (A) The median age of the entire cohort was 69 years. To determine the impact of age on serum Ig concentration, these were compared between healthy subjects aged
    Figure Legend Snippet: Impact of age and sex on serum immunoglobulin (Ig) concentrations in healthy blood donors. Serum concentration of IgM, IgA, IgG, and IgG1–4 was determined in healthy blood donors by ELISA. (A) The median age of the entire cohort was 69 years. To determine the impact of age on serum Ig concentration, these were compared between healthy subjects aged

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay

    16) Product Images from "Autoimmunity against Carbonic Anhydrase II Affects Retinal Cell Functions in Autoimmune Retinopathy"

    Article Title: Autoimmunity against Carbonic Anhydrase II Affects Retinal Cell Functions in Autoimmune Retinopathy

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2009.02.001

    Immunological analysis of patients’ autoantibodies: (A) Titration of autoantibodies by ELISA using human CAII on the plate. Serum from normal subject served as a control. (B) Distribution of anti-CAII autoantibodies subclasses by ELISA using human CAII on the plate. Secondary antibodies specific to different IgG isotypes, IgM and IgA were used. Note that all antibodies are dominated by IgG4 and IgG1 subclass.
    Figure Legend Snippet: Immunological analysis of patients’ autoantibodies: (A) Titration of autoantibodies by ELISA using human CAII on the plate. Serum from normal subject served as a control. (B) Distribution of anti-CAII autoantibodies subclasses by ELISA using human CAII on the plate. Secondary antibodies specific to different IgG isotypes, IgM and IgA were used. Note that all antibodies are dominated by IgG4 and IgG1 subclass.

    Techniques Used: Titration, Enzyme-linked Immunosorbent Assay

    17) Product Images from "Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance"

    Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038313

    HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.
    Figure Legend Snippet: HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.

    Techniques Used: Transfection, Immunofluorescence, Labeling, Expressing, Fluorescence, Western Blot, Recombinant, SDS-Gel, Molecular Weight

    HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.
    Figure Legend Snippet: HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Labeling, Molecular Weight, Immunofluorescence, Quantitation Assay, Immunocytochemistry

    HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.
    Figure Legend Snippet: HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

    Techniques Used: Transfection, Labeling, Fluorescence, Purification, SDS Page, Staining

    Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.
    Figure Legend Snippet: Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.

    Techniques Used: Knock-Out, Mouse Assay, Labeling

    18) Product Images from "HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis"

    Article Title: HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2015.18

    HAX1 interaction with the cytoplasmic domains of Ig subtypes. ( a ) The transmembrane (M1) and the cytoplasmic domains (M2) of human IgE were exchanged with the mouse IgM, IgG1, IgG2a, IgE, or IgA isotype, and the resulting chimeric plasmids were stably expressed in the A20 B lymphoma cell line. ( b ) The mean fluorescent intensities (MFI) reflecting the surface expression of the chimeric proteins are shown as bar graph, and ( c ) one representative FACS histogram is shown. ( d ) HAX1 was enriched from the stably transfected A20 cell lines using protein G Sepharose, and the binding of the chimeric human IgE/mouse tail receptors was determined using the anti-human IgE antibody. ( e ) The protein lysates used for co-IP were controlled for the presence of the HAX1 protein.
    Figure Legend Snippet: HAX1 interaction with the cytoplasmic domains of Ig subtypes. ( a ) The transmembrane (M1) and the cytoplasmic domains (M2) of human IgE were exchanged with the mouse IgM, IgG1, IgG2a, IgE, or IgA isotype, and the resulting chimeric plasmids were stably expressed in the A20 B lymphoma cell line. ( b ) The mean fluorescent intensities (MFI) reflecting the surface expression of the chimeric proteins are shown as bar graph, and ( c ) one representative FACS histogram is shown. ( d ) HAX1 was enriched from the stably transfected A20 cell lines using protein G Sepharose, and the binding of the chimeric human IgE/mouse tail receptors was determined using the anti-human IgE antibody. ( e ) The protein lysates used for co-IP were controlled for the presence of the HAX1 protein.

    Techniques Used: Stable Transfection, Expressing, FACS, Transfection, Binding Assay, Co-Immunoprecipitation Assay

    19) Product Images from "Antigen-Specific CD4+ T Cells Exhibit Distinct Kinetic and Phenotypic Patterns During Primary and Secondary Responses to Infection"

    Article Title: Antigen-Specific CD4+ T Cells Exhibit Distinct Kinetic and Phenotypic Patterns During Primary and Secondary Responses to Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.02125

    Ly6C + Tfh cells expand during the secondary response and require T-bet. (A) Representative flow cytometry plots demonstrating CXCR5 and Ly6C expression on 2W:I-A b -specific CD4 + T cells, excluding Foxp3 + Tregs, in the SLOs at 5 dpi following primary or secondary Lm infection. (B) Number of Ly6C + Tfh cells in SLO and blood. (C) Representative flow cytometry plots of CXCR5 and Ly6C expression on 2W:I-A b -specific cells, excluding Foxp3 + Tregs, in the SLOs at 0, 3, 5, 7, 14, 21, and 32 dpi following primary or secondary Lm infection. (D) Frequency of Tfh cells in the SLOs at 14 dpi including or excluding Ly6C + cells. (E) Representative flow cytometry plots demonstrating Ly6C expression on 2W + LLO:I-A b -specific CXCR5 + Tfh cells in the SLOs following secondary Lm infection of T-bet fl/fl or Lck-Cre;T-bet fl/fl mice at 5 dpi. (F) Frequency and number of Ly6C + Tfh cells as shown in panel (E) . (G) ELISA data assessing the presence of anti-LLO or anti-Lm IgG antibodies in the serum of T-bet fl/fl or Lck-Cre;T-bet fl/fl mice following secondary Lm infection at 10 dpi. (H) ELISA data assessing the presence of anti-Lm IgM, IgG2b or IgG2c antibodies as in panel (G) . Data are compiled from two to three independent experiments per time point with n = 1–4 mice per experiment (A–D) or from two independent experiments with n = 2–4 mice per group per experiment (E–H) . * P
    Figure Legend Snippet: Ly6C + Tfh cells expand during the secondary response and require T-bet. (A) Representative flow cytometry plots demonstrating CXCR5 and Ly6C expression on 2W:I-A b -specific CD4 + T cells, excluding Foxp3 + Tregs, in the SLOs at 5 dpi following primary or secondary Lm infection. (B) Number of Ly6C + Tfh cells in SLO and blood. (C) Representative flow cytometry plots of CXCR5 and Ly6C expression on 2W:I-A b -specific cells, excluding Foxp3 + Tregs, in the SLOs at 0, 3, 5, 7, 14, 21, and 32 dpi following primary or secondary Lm infection. (D) Frequency of Tfh cells in the SLOs at 14 dpi including or excluding Ly6C + cells. (E) Representative flow cytometry plots demonstrating Ly6C expression on 2W + LLO:I-A b -specific CXCR5 + Tfh cells in the SLOs following secondary Lm infection of T-bet fl/fl or Lck-Cre;T-bet fl/fl mice at 5 dpi. (F) Frequency and number of Ly6C + Tfh cells as shown in panel (E) . (G) ELISA data assessing the presence of anti-LLO or anti-Lm IgG antibodies in the serum of T-bet fl/fl or Lck-Cre;T-bet fl/fl mice following secondary Lm infection at 10 dpi. (H) ELISA data assessing the presence of anti-Lm IgM, IgG2b or IgG2c antibodies as in panel (G) . Data are compiled from two to three independent experiments per time point with n = 1–4 mice per experiment (A–D) or from two independent experiments with n = 2–4 mice per group per experiment (E–H) . * P

    Techniques Used: Flow Cytometry, Expressing, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    20) Product Images from "Natural IgM switches the function of LPS activated murine bone marrow dendritic cells (BMDC) to a “regulatory” DC that suppresses innate inflammation"

    Article Title: Natural IgM switches the function of LPS activated murine bone marrow dendritic cells (BMDC) to a “regulatory” DC that suppresses innate inflammation

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1500052

    BMDC that protect renal IRI in WT-B6 mice require decreased CD40 membrane expression. Increasing CD40 expression on IgM/LPS BMDC by either preventing CD40 downregulation with the agonistic anti-CD40 antibody (clone 1C10) ( A ) or by adding soluble CD40-Ig to 48 hour IgM/LPS activated cells ( B ), inhibits the IgM mediated protection after renal IRI (C) as well as inhibits the IgM mediated downregulation of phospho p65NF-κB ( D ). Anti-CD40 or soluble CD40-Ig (20µg) was present throughout the 48 hour IgM/LPS pretreatment of BMDC. Addition of isotype control IgG antibody (for clone 1C10) or human IgG (for CD40-Ig) to IgM/LPS pretreated WT-BMDC did not negate the protective effect of these BMDC (data not shown). Dead or Gr-1+ cells were gated out in all panels. P values were calculated using an unpaired Student’s t Test.
    Figure Legend Snippet: BMDC that protect renal IRI in WT-B6 mice require decreased CD40 membrane expression. Increasing CD40 expression on IgM/LPS BMDC by either preventing CD40 downregulation with the agonistic anti-CD40 antibody (clone 1C10) ( A ) or by adding soluble CD40-Ig to 48 hour IgM/LPS activated cells ( B ), inhibits the IgM mediated protection after renal IRI (C) as well as inhibits the IgM mediated downregulation of phospho p65NF-κB ( D ). Anti-CD40 or soluble CD40-Ig (20µg) was present throughout the 48 hour IgM/LPS pretreatment of BMDC. Addition of isotype control IgG antibody (for clone 1C10) or human IgG (for CD40-Ig) to IgM/LPS pretreated WT-BMDC did not negate the protective effect of these BMDC (data not shown). Dead or Gr-1+ cells were gated out in all panels. P values were calculated using an unpaired Student’s t Test.

    Techniques Used: Mouse Assay, Expressing

    21) Product Images from "Susceptibility of swine cells and domestic pigs to SARS-CoV-2"

    Article Title: Susceptibility of swine cells and domestic pigs to SARS-CoV-2

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1831405

    Serological response in pigs infected orally/intranasally/intratracheally with SARS-CoV-2 and sentinel contact pigs. Indirect ELISAs were performed against the SARS-CoV-2 antigens N (nucleocapsid protein [A, B]) and RBD (Spike protein receptor binding domain [C, D]) to detect antigen-specific IgG (A, C) or IgM (B, D) antibodies. Sera reactivity was determined for three principal infected pigs (#893 (red), #193 (dark red), #219 (orange)) and three sentinel pigs (#848 (blue), #172 (cyan), #894 dark blue)). The cutoff for a positive sample was determined by +3 standard deviations of 0 DPC samples (dotted line). Feline SARS-CoV-2-specific antibodies were used as positive controls from a separate study (left bar, [ 38 ]). Porcine positive control sera for IgG/IgM-specific antibodies were ASFV-infected pig sera detecting the ASFV-p54 antigen (right bar). Uninfected pigs were used as negative controls. O.D. – optical density.
    Figure Legend Snippet: Serological response in pigs infected orally/intranasally/intratracheally with SARS-CoV-2 and sentinel contact pigs. Indirect ELISAs were performed against the SARS-CoV-2 antigens N (nucleocapsid protein [A, B]) and RBD (Spike protein receptor binding domain [C, D]) to detect antigen-specific IgG (A, C) or IgM (B, D) antibodies. Sera reactivity was determined for three principal infected pigs (#893 (red), #193 (dark red), #219 (orange)) and three sentinel pigs (#848 (blue), #172 (cyan), #894 dark blue)). The cutoff for a positive sample was determined by +3 standard deviations of 0 DPC samples (dotted line). Feline SARS-CoV-2-specific antibodies were used as positive controls from a separate study (left bar, [ 38 ]). Porcine positive control sera for IgG/IgM-specific antibodies were ASFV-infected pig sera detecting the ASFV-p54 antigen (right bar). Uninfected pigs were used as negative controls. O.D. – optical density.

    Techniques Used: Infection, Binding Assay, Positive Control

    22) Product Images from "Humans Surviving Cholera Develop Antibodies against Vibrio cholerae O-Specific Polysaccharide That Inhibit Pathogen Motility"

    Article Title: Humans Surviving Cholera Develop Antibodies against Vibrio cholerae O-Specific Polysaccharide That Inhibit Pathogen Motility

    Journal: mBio

    doi: 10.1128/mBio.02847-20

    Convalescent-phase plasma of cholera patients recognizes V. cholerae OSP and inhibits V. cholerae motility in an OSP-dependent manner. (A) IgG, IgA, and IgM plasma responses targeting V. cholerae O1 Ogawa OSP at the acute (D2) and convalescent (D7) phase of cholera ( n = 10) as determined by enzyme-linked immunosorbent assay. (B) Motility of V. cholerae O1 Ogawa O395 assessed by high-speed video microscopy after a 5-min incubation with a subagglutinating dilution of plasma (1:256) of cholera patients ( n = 10) at a bacterial OD 600 of 0.1. Percentages of motile versus nonmotile bacteria after incubation with heat-inactivated plasma with and without adsorption with V. cholerae OSP are shown. Bars show the medians with interquartile ranges. Differences within groups were assessed using the Wilcoxon matched-pairs signed-rank sums test. D2, day 2; D7, day 7; OSP, O-specific polysaccharide.
    Figure Legend Snippet: Convalescent-phase plasma of cholera patients recognizes V. cholerae OSP and inhibits V. cholerae motility in an OSP-dependent manner. (A) IgG, IgA, and IgM plasma responses targeting V. cholerae O1 Ogawa OSP at the acute (D2) and convalescent (D7) phase of cholera ( n = 10) as determined by enzyme-linked immunosorbent assay. (B) Motility of V. cholerae O1 Ogawa O395 assessed by high-speed video microscopy after a 5-min incubation with a subagglutinating dilution of plasma (1:256) of cholera patients ( n = 10) at a bacterial OD 600 of 0.1. Percentages of motile versus nonmotile bacteria after incubation with heat-inactivated plasma with and without adsorption with V. cholerae OSP are shown. Bars show the medians with interquartile ranges. Differences within groups were assessed using the Wilcoxon matched-pairs signed-rank sums test. D2, day 2; D7, day 7; OSP, O-specific polysaccharide.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Microscopy, Incubation, Adsorption

    23) Product Images from "Impact of Influenza on Pneumococcal Vaccine Effectiveness during Streptococcus pneumoniae Infection in Aged Murine Lung"

    Article Title: Impact of Influenza on Pneumococcal Vaccine Effectiveness during Streptococcus pneumoniae Infection in Aged Murine Lung

    Journal: Vaccines

    doi: 10.3390/vaccines8020298

    Impact of influenza on pneumococcal vaccine effectiveness during S. pneumoniae infection in aged lung . ( A ) Aged adult (18–20 months) mice received a subcutaneous injection of PBS or Pneumovax (5 mg/mouse) on day 0. On day 14 post vaccination, mice were intranasally instilled with PBS or influenza (A/Puerto Rico/8/1934, H1N1) prior to secondary intranasal instillation with S. pneumoniae (1 × 10 3 CFU) on day 7 post influenza. ( B ) Total cell counts and ( C ) viral titer were assessed in BAL collected from PBS- and Pneumovax-treated mice on day of influenza. ( D ) IgM and ( E ) IgG PPS3 antibody levels were assessed in serum collected on day 7 and 14 post vaccination. ( F ) Total cell counts, ( G ) bacterial titers, and ( H ) protein concentration in BAL collected at 24 h post-secondary S. pneumoniae infection were quantified in aged adult lung. Student’s t -test: * p
    Figure Legend Snippet: Impact of influenza on pneumococcal vaccine effectiveness during S. pneumoniae infection in aged lung . ( A ) Aged adult (18–20 months) mice received a subcutaneous injection of PBS or Pneumovax (5 mg/mouse) on day 0. On day 14 post vaccination, mice were intranasally instilled with PBS or influenza (A/Puerto Rico/8/1934, H1N1) prior to secondary intranasal instillation with S. pneumoniae (1 × 10 3 CFU) on day 7 post influenza. ( B ) Total cell counts and ( C ) viral titer were assessed in BAL collected from PBS- and Pneumovax-treated mice on day of influenza. ( D ) IgM and ( E ) IgG PPS3 antibody levels were assessed in serum collected on day 7 and 14 post vaccination. ( F ) Total cell counts, ( G ) bacterial titers, and ( H ) protein concentration in BAL collected at 24 h post-secondary S. pneumoniae infection were quantified in aged adult lung. Student’s t -test: * p

    Techniques Used: Infection, Mouse Assay, Injection, Protein Concentration

    24) Product Images from "Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance"

    Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038313

    HL 2°Abs show a bias for immunohistochemistry labeling with mAbs of different IgG subclasses. Rat brain sections were labeled with the same concentrations of a single mAb, and a rabbit anti-Kv2.1 pAb, followed by detection with SCS (left column) or HL (right column) 2°Abs, (red), and anti-rabbit IgG (green), each at 1 µg/ml. Top row: anti-Kv4.2 IgG1; middle row: anti-BK channel IgG2a; and bottom row: anti-Kv1.2 IgG2b. Each row was imaged at the same exposure times. Scale bar = 50 µm for panels in top two rows, and 25 µm for panels in bottom row.
    Figure Legend Snippet: HL 2°Abs show a bias for immunohistochemistry labeling with mAbs of different IgG subclasses. Rat brain sections were labeled with the same concentrations of a single mAb, and a rabbit anti-Kv2.1 pAb, followed by detection with SCS (left column) or HL (right column) 2°Abs, (red), and anti-rabbit IgG (green), each at 1 µg/ml. Top row: anti-Kv4.2 IgG1; middle row: anti-BK channel IgG2a; and bottom row: anti-Kv1.2 IgG2b. Each row was imaged at the same exposure times. Scale bar = 50 µm for panels in top two rows, and 25 µm for panels in bottom row.

    Techniques Used: Immunohistochemistry, Labeling

    HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.
    Figure Legend Snippet: HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.

    Techniques Used: Transfection, Immunofluorescence, Labeling, Expressing, Fluorescence, Western Blot, Recombinant, SDS-Gel, Molecular Weight

    HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.
    Figure Legend Snippet: HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Labeling, Molecular Weight, Immunofluorescence, Quantitation Assay, Immunocytochemistry

    HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.
    Figure Legend Snippet: HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

    Techniques Used: Transfection, Labeling, Fluorescence, Purification, SDS Page, Staining

    Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.
    Figure Legend Snippet: Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.

    Techniques Used: Knock-Out, Mouse Assay, Labeling

    SCS 2°Abs yield robust and reliable simultaneous triple labeling with three mAbs on immunoblots and in rat brain sections. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid, probed with anti-PSD-95 (IgG2a, blue), anti-Kv1.2 (IgG2b, red) and anti-Kv2.1 (IgG1, green), and SCS 2°Abs. Multicolor panel is original immunoblot; single color panels are images of separated colors. Lane to left shows molecular weight standards in kDa. Note differential post-translational modification of target proteins in brain versus heterologous cells alters their relative electrophoretic mobility. B–E. Images show specific and non-overlapping labeling for (B) Kv4.2 (green), (C) QKI (red), (D) and BK channels (blue), and (E) merge of all three, in a rat brain section, showing the region containing the entire cerebellum. Inset in E shows boxed area of cerebellar cortex. Labels mark the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). Scale bar on Panel E = 500 µm.
    Figure Legend Snippet: SCS 2°Abs yield robust and reliable simultaneous triple labeling with three mAbs on immunoblots and in rat brain sections. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid, probed with anti-PSD-95 (IgG2a, blue), anti-Kv1.2 (IgG2b, red) and anti-Kv2.1 (IgG1, green), and SCS 2°Abs. Multicolor panel is original immunoblot; single color panels are images of separated colors. Lane to left shows molecular weight standards in kDa. Note differential post-translational modification of target proteins in brain versus heterologous cells alters their relative electrophoretic mobility. B–E. Images show specific and non-overlapping labeling for (B) Kv4.2 (green), (C) QKI (red), (D) and BK channels (blue), and (E) merge of all three, in a rat brain section, showing the region containing the entire cerebellum. Inset in E shows boxed area of cerebellar cortex. Labels mark the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). Scale bar on Panel E = 500 µm.

    Techniques Used: Labeling, Western Blot, Transfection, Expressing, Plasmid Preparation, Molecular Weight, Modification

    25) Product Images from "Dysregulation of Systemic and Mucosal Humoral Responses to Microbial and Food Antigens as a Factor Contributing to Microbial Translocation and Chronic Inflammation in HIV-1 Infection"

    Article Title: Dysregulation of Systemic and Mucosal Humoral Responses to Microbial and Food Antigens as a Factor Contributing to Microbial Translocation and Chronic Inflammation in HIV-1 Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006087

    Dysregulation of systemic and mucosal humoral responses to microbial antigens in HIV-1-infected individuals. Depletion of CD4 + T cells in the intestinal mucosa of HIV-1-infected individuals reduces the capacity of mucosal B cells to undergo class switch recombination resulting in an increased production of IgM. Accumulated microbiota-specific IgM may exacerbate inflammatory processes by the formation of inflammatory immune complexes [ 28 ]. Epithelial cell apoptosis and decreased availability of antigen-specific IgG and IgA in mucosal secretions result in enhanced translocation of microbial products and whole bacteria across the intestinal mucosal barrier into the systemic circulation.
    Figure Legend Snippet: Dysregulation of systemic and mucosal humoral responses to microbial antigens in HIV-1-infected individuals. Depletion of CD4 + T cells in the intestinal mucosa of HIV-1-infected individuals reduces the capacity of mucosal B cells to undergo class switch recombination resulting in an increased production of IgM. Accumulated microbiota-specific IgM may exacerbate inflammatory processes by the formation of inflammatory immune complexes [ 28 ]. Epithelial cell apoptosis and decreased availability of antigen-specific IgG and IgA in mucosal secretions result in enhanced translocation of microbial products and whole bacteria across the intestinal mucosal barrier into the systemic circulation.

    Techniques Used: Infection, Translocation Assay

    IgA/IgM and IgG/IgM ratios specific for antigens derived from intestinal microbiota are significantly decreased in intestinal secretions of HIV-1-infected individuals. IgA/IgM and IgG/IgM ratios of total (A) and antigen-specific immunoglobulin in plasma (B) and rectal washes (C). Error bars represent SEM, statistical significance relative to healthy donors was determined using Mann-Whitney U test (* p
    Figure Legend Snippet: IgA/IgM and IgG/IgM ratios specific for antigens derived from intestinal microbiota are significantly decreased in intestinal secretions of HIV-1-infected individuals. IgA/IgM and IgG/IgM ratios of total (A) and antigen-specific immunoglobulin in plasma (B) and rectal washes (C). Error bars represent SEM, statistical significance relative to healthy donors was determined using Mann-Whitney U test (* p

    Techniques Used: Derivative Assay, Infection, MANN-WHITNEY

    Correlation between the levels of plasma and intestinal IgG and IgA specific for microbial and food antigens, CD4 + T cell count, and plasma levels of sCD14. A) Total and mucosal antigen-specific IgA/IgM ratios in intestinal secretions of individuals not treated with ART (NC and VC) correlate with CD4 + T cell count / μl of blood. Correlations with LPS— E . coli , LPS— S . typhi , and yeast mannan antigens showed a similar trend but did not reach statistical significance. B, C) Plasma sCD14 and LPS levels are significantly increased in HIV-1-infected ART-treated and ART-naïve subjects. D) Rectal wash levels of total and mucosal-antigen specific IgA negatively correlate with plasma sCD14 in ART-naïve HIV-1-infected individuals. Correlations were performed using Spearman rank order test; solid lines represent linear regression analysis.
    Figure Legend Snippet: Correlation between the levels of plasma and intestinal IgG and IgA specific for microbial and food antigens, CD4 + T cell count, and plasma levels of sCD14. A) Total and mucosal antigen-specific IgA/IgM ratios in intestinal secretions of individuals not treated with ART (NC and VC) correlate with CD4 + T cell count / μl of blood. Correlations with LPS— E . coli , LPS— S . typhi , and yeast mannan antigens showed a similar trend but did not reach statistical significance. B, C) Plasma sCD14 and LPS levels are significantly increased in HIV-1-infected ART-treated and ART-naïve subjects. D) Rectal wash levels of total and mucosal-antigen specific IgA negatively correlate with plasma sCD14 in ART-naïve HIV-1-infected individuals. Correlations were performed using Spearman rank order test; solid lines represent linear regression analysis.

    Techniques Used: Cell Counting, Infection

    26) Product Images from "Bead Arrays for Antibody and Complement Profiling Reveal Joint Contribution of Antibody Isotypes to C3 Deposition"

    Article Title: Bead Arrays for Antibody and Complement Profiling Reveal Joint Contribution of Antibody Isotypes to C3 Deposition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096403

    Schematic representation of assay workflow. A ) Serum samples are diluted 1∶10, either in a Ca 2+ -Mg 2+ containing assay buffer for detection of complement activation, or in an EDTA containing assay buffer for antibody detection. B) The samples are pre-adsorbed against neutravidin-specific antibodies. C ) A mixture of beads coupled to various antigens is distributed into a 384-well plate and the pre-adsorbed samples are added to the bead array. D ) Complement activation driven C3 deposition and different antibody isotypes are detected in parallel with fluorescently labeled secondary antibodies dispensed into individual wells of each quadrant of the 384-well plate.
    Figure Legend Snippet: Schematic representation of assay workflow. A ) Serum samples are diluted 1∶10, either in a Ca 2+ -Mg 2+ containing assay buffer for detection of complement activation, or in an EDTA containing assay buffer for antibody detection. B) The samples are pre-adsorbed against neutravidin-specific antibodies. C ) A mixture of beads coupled to various antigens is distributed into a 384-well plate and the pre-adsorbed samples are added to the bead array. D ) Complement activation driven C3 deposition and different antibody isotypes are detected in parallel with fluorescently labeled secondary antibodies dispensed into individual wells of each quadrant of the 384-well plate.

    Techniques Used: Activation Assay, Labeling

    27) Product Images from "Synthesis and characterization of heparosan-granulocyte-colony stimulating factor conjugates: a natural sugar-based drug delivery system to treat neutropenia"

    Article Title: Synthesis and characterization of heparosan-granulocyte-colony stimulating factor conjugates: a natural sugar-based drug delivery system to treat neutropenia

    Journal: Glycobiology

    doi: 10.1093/glycob/cwx072

    for study design) for testing by ELISA (0.5 or 5 μL sera/well for IgM or IgG, respectively, in anti-HEP tests with 30 min colorimetric development; 1 μL sera/well for IgG in anti-G-CSF tests with 15 min development). Representative data from averaged triplicate wells from the preimmune ( white bars ) or the third, final ( black bars ) bleeds are shown. There were no anti-HEP-specific immunoglobulin IgM ( top ) or IgG ( bottom ) detected because the signals from wells coated with HEP-BSA (HEP) were not higher than the signal from the negative control BSA-coated wells (BSA). On the other hand, an anti-G-CSF response (G-CSF) was detected in all the three rats (due to the nonidentical sequence of the human protein drug). Overall, this data indicates that HEP itself is not immunogenic.
    Figure Legend Snippet: for study design) for testing by ELISA (0.5 or 5 μL sera/well for IgM or IgG, respectively, in anti-HEP tests with 30 min colorimetric development; 1 μL sera/well for IgG in anti-G-CSF tests with 15 min development). Representative data from averaged triplicate wells from the preimmune ( white bars ) or the third, final ( black bars ) bleeds are shown. There were no anti-HEP-specific immunoglobulin IgM ( top ) or IgG ( bottom ) detected because the signals from wells coated with HEP-BSA (HEP) were not higher than the signal from the negative control BSA-coated wells (BSA). On the other hand, an anti-G-CSF response (G-CSF) was detected in all the three rats (due to the nonidentical sequence of the human protein drug). Overall, this data indicates that HEP itself is not immunogenic.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Negative Control, Sequencing

    28) Product Images from "Loss of Fancc impairs antibody secreting cell differentiation in mice through deregulating Wnt signaling pathway"

    Article Title: Loss of Fancc impairs antibody secreting cell differentiation in mice through deregulating Wnt signaling pathway

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1501056

    Abnormality of B cells lineage in bone marrow of Fancc −/− Bone marrow (BM) was harvested from tibias and femurs of 6-8 weeks old WT or Fancc −/− mice. Mononuclear cells were separated by centrifugation on Ficoll and labelled with anti-B220-eFluor660 and anti-IgM-Fitc Abs. Before Facs acquisition, propidium iodide (PI) was added detect and exclude dead cells. (A) Representative FACS plot of the evaluation of Pre/Pro (B220 dim IgM − ), Immature (B200 dim IgM + ) and Mature (B220 + IgM + ) among Live B220 dim/+ mononuclear BM cells from Fancc −/− or WT littermate mice. (B) Quantification of BM B cells populations (n=6 mice/genotype) as described in (A). (C) Bone marrow cellularity from 2 tibias and femurs of WT and Fancc −/− Mice (n=6 mice/genotype). (D) Representative Facs histogram of total B cell lineage percentage in live mononuclear BM cells from Fancc −/− or WT mice. (E) Total B cell (B220 + ) number from 2 tibias and femurs. (n=6 mice/genotype). For all graphics, Bar represents mean value ± standard deviation (SD). Evaluation of differences between genotype has been conducted using Student’s T-test (*=p≤0.05)
    Figure Legend Snippet: Abnormality of B cells lineage in bone marrow of Fancc −/− Bone marrow (BM) was harvested from tibias and femurs of 6-8 weeks old WT or Fancc −/− mice. Mononuclear cells were separated by centrifugation on Ficoll and labelled with anti-B220-eFluor660 and anti-IgM-Fitc Abs. Before Facs acquisition, propidium iodide (PI) was added detect and exclude dead cells. (A) Representative FACS plot of the evaluation of Pre/Pro (B220 dim IgM − ), Immature (B200 dim IgM + ) and Mature (B220 + IgM + ) among Live B220 dim/+ mononuclear BM cells from Fancc −/− or WT littermate mice. (B) Quantification of BM B cells populations (n=6 mice/genotype) as described in (A). (C) Bone marrow cellularity from 2 tibias and femurs of WT and Fancc −/− Mice (n=6 mice/genotype). (D) Representative Facs histogram of total B cell lineage percentage in live mononuclear BM cells from Fancc −/− or WT mice. (E) Total B cell (B220 + ) number from 2 tibias and femurs. (n=6 mice/genotype). For all graphics, Bar represents mean value ± standard deviation (SD). Evaluation of differences between genotype has been conducted using Student’s T-test (*=p≤0.05)

    Techniques Used: Mouse Assay, Centrifugation, FACS, Standard Deviation

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    Article Snippet: .. Anti-human IgM + IgG for B-cell receptor (BCR) stimulation and APC-Cyanine7-anti-human CD19 antibody, FITC-anti-human CD4 antibody, PerCP-Cyanine5.5-anti-human CD4 antibody, FITC-anti-human CD14 antibody, PE-anti-human-IL-21 receptor (IL-21R) antibody, PE-Cyanine7-anti-human CD25 antibody, PE-anti-human GrB antibody, FITC-anti-human IFN-gamma antibody, and eFluor® 660-anti-human IL-17A antibody for immunostaining were purchased from eBioscience (San Diego, CA, USA). .. Alexa Fluor® 700-anti-human CD3 antibody, Brilliant Violet 570-anti-human CD56 antibody, APC-annexin V, and 7-AAD viability staining solution were purchased from Biolegend (San Diego, CA, USA).

    Staining:

    Article Title: Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3
    Article Snippet: .. Flow cytometry Bacteria at mid-log phase of growth were washed and reacted (2 × 106 CFU per reaction) with O3 specific mouse mAbs (40 µg/ml) or with 10% human serum pool in HBSS buffer with 0.5% BSA followed by staining with 4 μg/ml of Alexa Fluor® 488-conjugated goat anti-mouse IgG, anti-human IgG or anti-human IgM secondary antibodies (Thermo Scientific). .. Bacteria were counterstained with 5 μM SYTO-62 nucleic acid stain (Thermo Scientific) and measured in a CytoFlex Flow Cytometer (Beckman Coulter).

    Article Title: Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans
    Article Snippet: .. After further washing with staining buffer, FITC-conjugated goat-derived anti-human IgM (mμ chain-specific) or IgG (γ chain-specific) polyclonal antibody (concentration 1:100 for pRBCs and 1:50 for pAECs; Invitrogen) was added, and the cells were incubated for 30min at 4°C. .. After washing with staining buffer, 200μL fixation buffer was added, and the cells allowed to sit at 4°C for 30min before adding 100μL staining buffer.

    Binding Assay:

    Article Title: Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis
    Article Snippet: .. After washing with PBS, binding of IgG1 or IgM on brain tissue was detected using IgG1 and IgM specific secondary antibodies (Invitrogen, A21121 and A21042; 5 ug/ml) conjugated to Alexa Fluor 488. ..

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    Thermo Fisher igm
    Immunological challenge in rats with HEP-DiPalm and ELISA assessment. A set of three rats ( #1, 2, 3 ) then tested for the induction of an <t>IgM</t> or <t>IgG</t> response by ELISA (representative set of assays with averaged triplicate wells with standard deviation is presented). Overall, the signal from sera of rats before and after injection with HEP-DiPalm micelles (prelipid, white ; postlipid, black ) tested in the control wells coated with bovine serum albumin ( BSA ) was equivalent to the wells coated with HEP-BSA ( HEP ) or HEP-DiPalm ( DiPalm ) indicating that HEP is not significantly immunogenic, therefore, should be useful in multidose or long-term therapeutics.
    Igm, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunological challenge in rats with HEP-DiPalm and ELISA assessment. A set of three rats ( #1, 2, 3 ) then tested for the induction of an IgM or IgG response by ELISA (representative set of assays with averaged triplicate wells with standard deviation is presented). Overall, the signal from sera of rats before and after injection with HEP-DiPalm micelles (prelipid, white ; postlipid, black ) tested in the control wells coated with bovine serum albumin ( BSA ) was equivalent to the wells coated with HEP-BSA ( HEP ) or HEP-DiPalm ( DiPalm ) indicating that HEP is not significantly immunogenic, therefore, should be useful in multidose or long-term therapeutics.

    Journal: Glycobiology

    Article Title: Heparosan-coated liposomes for drug delivery

    doi: 10.1093/glycob/cwx070

    Figure Lengend Snippet: Immunological challenge in rats with HEP-DiPalm and ELISA assessment. A set of three rats ( #1, 2, 3 ) then tested for the induction of an IgM or IgG response by ELISA (representative set of assays with averaged triplicate wells with standard deviation is presented). Overall, the signal from sera of rats before and after injection with HEP-DiPalm micelles (prelipid, white ; postlipid, black ) tested in the control wells coated with bovine serum albumin ( BSA ) was equivalent to the wells coated with HEP-BSA ( HEP ) or HEP-DiPalm ( DiPalm ) indicating that HEP is not significantly immunogenic, therefore, should be useful in multidose or long-term therapeutics.

    Article Snippet: After thoroughly washing wells with PBST, horseradish peroxidase-conjugated goat polyclonal anti-rat IgG or IgM (Thermo Scientific) detection agents diluted in PBST with 1% BSA were added to the wells and the plates were incubated for 2 h. After final washing with PBST, a peroxidase substrate solution (TMB Substrate Kit, Thermo Scientific) for color development was added.

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Injection

    Representation of HEP-liposome assembly. Models of HEP-coated liposome assemblies with 13.3-kDa ( top ) and 7-kDa ( bottom ) HEP-lipid, based on typical molecular configurations from equilibrated coarse-grained simulations. The liposome and sugars are represented graphically by equivalent spheres (roughly to scale), and part of the liposome has been cut-away for viewing purposes. Potential interaction proteins are depicted (human serum albumin, HSA ; immunoglobulins IgG or IgM ; middle ), drawn to exact scale using coordinates from the Protein Data Bank. Less surface area is available for direct protein binding to liposomes coated with 0.5 mol% 13.3-kDa HEP-lipid compared to 0.5 mol% 7-kDa HEP-lipid, suggesting increased liposomal protection from clearance by the immune system. The image was rendered using POV-Ray software.

    Journal: Glycobiology

    Article Title: Heparosan-coated liposomes for drug delivery

    doi: 10.1093/glycob/cwx070

    Figure Lengend Snippet: Representation of HEP-liposome assembly. Models of HEP-coated liposome assemblies with 13.3-kDa ( top ) and 7-kDa ( bottom ) HEP-lipid, based on typical molecular configurations from equilibrated coarse-grained simulations. The liposome and sugars are represented graphically by equivalent spheres (roughly to scale), and part of the liposome has been cut-away for viewing purposes. Potential interaction proteins are depicted (human serum albumin, HSA ; immunoglobulins IgG or IgM ; middle ), drawn to exact scale using coordinates from the Protein Data Bank. Less surface area is available for direct protein binding to liposomes coated with 0.5 mol% 13.3-kDa HEP-lipid compared to 0.5 mol% 7-kDa HEP-lipid, suggesting increased liposomal protection from clearance by the immune system. The image was rendered using POV-Ray software.

    Article Snippet: After thoroughly washing wells with PBST, horseradish peroxidase-conjugated goat polyclonal anti-rat IgG or IgM (Thermo Scientific) detection agents diluted in PBST with 1% BSA were added to the wells and the plates were incubated for 2 h. After final washing with PBST, a peroxidase substrate solution (TMB Substrate Kit, Thermo Scientific) for color development was added.

    Techniques: Protein Binding, Software

    Serum survival of K. pneumoniae strains. Strains were incubated in different concentrations of human serum ( a ) or 75% serum complement-inactivated by heat (HI) or cobra venom factor (CVF) treatment ( b ) for 3 h at 37 °C. The recovered bacterial count was expressed as percentage of the initial inoculum. ATCC43816 (O1:K2) and PCM-27 (O2:K27) strains were used as comparators.Graphs show combined results of 2 and 3 experiments for the active and heat inactivated serum experiments, respectively. The same O3 isolates were stained with 10% human serum pool and IgG and IgM antibodies binding to live bacteria were detected with a labelled anti-human IgG or IgM by flow cytometry ( c ). The median fluorescent intensity is shown as arbitrary units. The difference between the staining intensity for O3 and O3b strains was not significant using the Mann Whitney test (p > 0.05; n.s.). O-serotype and capsule type of the strains are indicated in Table 2 .

    Journal: Scientific Reports

    Article Title: Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3

    doi: 10.1038/s41598-017-06682-2

    Figure Lengend Snippet: Serum survival of K. pneumoniae strains. Strains were incubated in different concentrations of human serum ( a ) or 75% serum complement-inactivated by heat (HI) or cobra venom factor (CVF) treatment ( b ) for 3 h at 37 °C. The recovered bacterial count was expressed as percentage of the initial inoculum. ATCC43816 (O1:K2) and PCM-27 (O2:K27) strains were used as comparators.Graphs show combined results of 2 and 3 experiments for the active and heat inactivated serum experiments, respectively. The same O3 isolates were stained with 10% human serum pool and IgG and IgM antibodies binding to live bacteria were detected with a labelled anti-human IgG or IgM by flow cytometry ( c ). The median fluorescent intensity is shown as arbitrary units. The difference between the staining intensity for O3 and O3b strains was not significant using the Mann Whitney test (p > 0.05; n.s.). O-serotype and capsule type of the strains are indicated in Table 2 .

    Article Snippet: Flow cytometry Bacteria at mid-log phase of growth were washed and reacted (2 × 106 CFU per reaction) with O3 specific mouse mAbs (40 µg/ml) or with 10% human serum pool in HBSS buffer with 0.5% BSA followed by staining with 4 μg/ml of Alexa Fluor® 488-conjugated goat anti-mouse IgG, anti-human IgG or anti-human IgM secondary antibodies (Thermo Scientific).

    Techniques: Incubation, Combined Bisulfite Restriction Analysis Assay, Staining, Binding Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    The anti-FIS IgM and IgG subclasses (IgG1 and IgG2) ELISA titres of cattle are vaccinated at week 0 and 3 with either PrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), CrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), SLSV (n = 8) and Emulsigen-D ® /Alhydrogel ® adjuvants (NegCtl) (n = 4) are presented as box and whisker plots. The cattle sera samples were collected before the vaccinations at week 0 and 3 as well as at week 5 and 12. Sera dilution started at a concentration of 1:50 and values below the (

    Journal: Pathogens

    Article Title: Immunogenicity of Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine

    doi: 10.3390/pathogens9070557

    Figure Lengend Snippet: The anti-FIS IgM and IgG subclasses (IgG1 and IgG2) ELISA titres of cattle are vaccinated at week 0 and 3 with either PrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), CrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), SLSV (n = 8) and Emulsigen-D ® /Alhydrogel ® adjuvants (NegCtl) (n = 4) are presented as box and whisker plots. The cattle sera samples were collected before the vaccinations at week 0 and 3 as well as at week 5 and 12. Sera dilution started at a concentration of 1:50 and values below the (

    Article Snippet: For IgG, the secondary antibody (goat anti-bovine IgG (ThermoFisher Scientific)) was diluted at the concentration of 1:10,000 in PTSMP, whereas for IgM, IgG1 and IgG2, sheep anti-bovine IgM (ThermoFisher Scientific), and sheep anti-bovine IgG1 and IgG2 (ThermoFisher Scientific) were all diluted at 1:4000 in PTSMP, respectively.

    Techniques: Enzyme-linked Immunosorbent Assay, Whisker Assay, Concentration Assay

    The anti-rPA IgM and IgG subclasses (IgG1 and IgG2) ELISA titres of cattle are vaccinated at week 0 and 3 with either PrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), CrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), SLSV (n = 8) and Emulsigen-D ® /Alhydrogel ® adjuvants (NegCtl) (n = 4) are presented as box and whisker plots. The cattle sera samples were collected before the vaccinations at week 0 and 3 as well as at week 5 and 12. Sera dilution started at a concentration of 1:50 and values below the (

    Journal: Pathogens

    Article Title: Immunogenicity of Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine

    doi: 10.3390/pathogens9070557

    Figure Lengend Snippet: The anti-rPA IgM and IgG subclasses (IgG1 and IgG2) ELISA titres of cattle are vaccinated at week 0 and 3 with either PrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), CrPA+FIS+Emulsigen-D ® /Alhydrogel ® adjuvants (n = 8), SLSV (n = 8) and Emulsigen-D ® /Alhydrogel ® adjuvants (NegCtl) (n = 4) are presented as box and whisker plots. The cattle sera samples were collected before the vaccinations at week 0 and 3 as well as at week 5 and 12. Sera dilution started at a concentration of 1:50 and values below the (

    Article Snippet: For IgG, the secondary antibody (goat anti-bovine IgG (ThermoFisher Scientific)) was diluted at the concentration of 1:10,000 in PTSMP, whereas for IgM, IgG1 and IgG2, sheep anti-bovine IgM (ThermoFisher Scientific), and sheep anti-bovine IgG1 and IgG2 (ThermoFisher Scientific) were all diluted at 1:4000 in PTSMP, respectively.

    Techniques: Recombinase Polymerase Amplification, Enzyme-linked Immunosorbent Assay, Whisker Assay, Concentration Assay

    Serum immunoglobulin (Ig) concentrations in patients with pemphigus and pemphigoid disease. Serum concentration of IgM, IgA, IgG, and IgG1–4 were determined in healthy blood donors and patients with the indicated pemphigus and pemphigoid diseases by ELISA. The numbers below the boxes indicate the number of controls/patients included in each group. Data are shown as median (black line) and 25/75 percentiles (boxes); the whiskers extend no further than 1.5× IQR from a hinge (where IQR is the interquartile range) and outliers (points). ANOVA with Tukey's HSD procedure as post-hoc test was used for statistical analysis. *indicates p

    Journal: Frontiers in Medicine

    Article Title: Alterations of Total Serum Immunoglobulin Concentrations in Pemphigus and Pemphigoid: Selected IgG2 Deficiency in Bullous Pemphigoid

    doi: 10.3389/fmed.2020.00472

    Figure Lengend Snippet: Serum immunoglobulin (Ig) concentrations in patients with pemphigus and pemphigoid disease. Serum concentration of IgM, IgA, IgG, and IgG1–4 were determined in healthy blood donors and patients with the indicated pemphigus and pemphigoid diseases by ELISA. The numbers below the boxes indicate the number of controls/patients included in each group. Data are shown as median (black line) and 25/75 percentiles (boxes); the whiskers extend no further than 1.5× IQR from a hinge (where IQR is the interquartile range) and outliers (points). ANOVA with Tukey's HSD procedure as post-hoc test was used for statistical analysis. *indicates p

    Article Snippet: Determination of Serum Ig Concentrations by ELISA Serum concentrations of IgM, IgA, IgG, IgG1, IgG2, IgG3, or IgG4 were measured in sera of donors using commercially available ELISA kits (Thermo Fisher Scientific, Vienna, Austria, Cat. Nos.

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Impact of age and sex on serum immunoglobulin (Ig) concentrations in healthy blood donors. Serum concentration of IgM, IgA, IgG, and IgG1–4 was determined in healthy blood donors by ELISA. (A) The median age of the entire cohort was 69 years. To determine the impact of age on serum Ig concentration, these were compared between healthy subjects aged

    Journal: Frontiers in Medicine

    Article Title: Alterations of Total Serum Immunoglobulin Concentrations in Pemphigus and Pemphigoid: Selected IgG2 Deficiency in Bullous Pemphigoid

    doi: 10.3389/fmed.2020.00472

    Figure Lengend Snippet: Impact of age and sex on serum immunoglobulin (Ig) concentrations in healthy blood donors. Serum concentration of IgM, IgA, IgG, and IgG1–4 was determined in healthy blood donors by ELISA. (A) The median age of the entire cohort was 69 years. To determine the impact of age on serum Ig concentration, these were compared between healthy subjects aged

    Article Snippet: Determination of Serum Ig Concentrations by ELISA Serum concentrations of IgM, IgA, IgG, IgG1, IgG2, IgG3, or IgG4 were measured in sera of donors using commercially available ELISA kits (Thermo Fisher Scientific, Vienna, Austria, Cat. Nos.

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay