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  • 96
    Thermo Fisher igg3
    Igg3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore igg3
    Results for immunoglubin G subtypes evaluation <t>IgG</t> subtypes in mice serum samples of different experimental groups were assayed against rEG95 by ELISA. Data from each group was compared by other groups with LSD test via one-way analysis of variance (one-way ANOVA). P -values less than 0.05 were recognized as significant. Freund’s : sera from mice immunized with rEG95 formulated with FA. Alum : sera from mice immunized with rEG95 formulated with alum. Trx : sera from mice immunized with Trx pET32 *: significant difference with non-immunized (negative control) group ( P
    Igg3, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson igg3
    GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total IgG1, IgG2a, IgM and <t>IgG3</t> on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p
    Igg3, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 866 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems mouse igg1 isotype control
    GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total IgG1, IgG2a, IgM and <t>IgG3</t> on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p
    Mouse Igg1 Isotype Control, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 990 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    SouthernBiotech igg3
    Antigen-specific <t>IgG</t> subclasses during follow-up and relapse. The PLA 2 R1- (A) and THSD7A-specific (B) IgG subclasses did not significantly change during the follow-up period. At the time of relapse of PLA 2 R1-ab, antigen-specific IgG subclasses were similar to the distribution observed at baseline (C) .
    Igg3, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 95/100, based on 2418 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad igg3
    (A) Analysis of the <t>IgG</t> subclass of anti-TRIM9 Abs in patient CSF. HEK cells expressing TRIM9 were incubated with patient CSF and revealed with subclass-specific anti-human IgG antibodies. Positive signal was observed for all IgG subclasses; the strongest signal was found for <t>IgG1.</t> (B) Epitope mapping of anti-TRIM9 Abs. HEK cells were transfected with plasmids coding for different domains of TRIM9 and then lysed and immuno-blotted with CSF from patient 1 that recognized all TRIM9 domains except the B30.2 domain. The Abs are polyclonal.
    Igg3, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    igg3  (Abcam)
    99
    Abcam igg3
    Expression and purification of the recombinant gH/gL/gQ1/gQ2 complex. (A) The constructions of gH, gL, gQ1, and gQ2 are shown. gH is modified to have an N-terminal IL-2 signal sequence and a C-terminal human <t>IgG1</t> Fc (hFc) and His 6 sequence replacing the intrinsic gH signal sequence and transmembrane-cytoplasmic tail domains, respectively. ( B) Cell binding assay was performed using hCD134 expressing JJhan cells or JJhan cells (negative control). After incubation with the tetramer-hFc containing medium, the interaction between cell expressing hCD134 and tetramer-hFc was detected using Alexa 488 conjugated anti-human <t>IgG</t> targeting the hFc by flow cytometry. The hFc protein (without tetramer) was used as the non-binding negative control. ( C) Size exclusion column chromatography of the purified tetramer. ( D) Western blotting analysis of the tetramer purified by the size exclusion column chromatography. gH, gQ1, gL, and gQ2 were detected by the respective antibodies. ( E) Purified gH/gL/gQ1/gQ2 complex detected by Coomassie brilliant blue staining.
    Igg3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher control mouse igg
    Expression and purification of the recombinant gH/gL/gQ1/gQ2 complex. (A) The constructions of gH, gL, gQ1, and gQ2 are shown. gH is modified to have an N-terminal IL-2 signal sequence and a C-terminal human <t>IgG1</t> Fc (hFc) and His 6 sequence replacing the intrinsic gH signal sequence and transmembrane-cytoplasmic tail domains, respectively. ( B) Cell binding assay was performed using hCD134 expressing JJhan cells or JJhan cells (negative control). After incubation with the tetramer-hFc containing medium, the interaction between cell expressing hCD134 and tetramer-hFc was detected using Alexa 488 conjugated anti-human <t>IgG</t> targeting the hFc by flow cytometry. The hFc protein (without tetramer) was used as the non-binding negative control. ( C) Size exclusion column chromatography of the purified tetramer. ( D) Western blotting analysis of the tetramer purified by the size exclusion column chromatography. gH, gQ1, gL, and gQ2 were detected by the respective antibodies. ( E) Purified gH/gL/gQ1/gQ2 complex detected by Coomassie brilliant blue staining.
    Control Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Pharmingen igg3
    A metalloprotease inhibitor, GI129471, blocks the production of IgE and <t>IgG1</t> enhanced by RMCP-I. B cells enriched from BALB/c spleen cells were cultured with (open symbols) or without (closed symbols) 1 µg/ml RMCP-I in the presence of LPS plus IL-4 for 7 days. Indicated concentration of GI129471 was added to some culture. Concentrations of IgE (a) and IgG1 (b) in the supernatant were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing GI129471 were compared with values from the culture without the inhibitor by Dunnett's method (*, P
    Igg3, supplied by Pharmingen, used in various techniques. Bioz Stars score: 93/100, based on 216 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems mouse igg3
    A metalloprotease inhibitor, GI129471, blocks the production of IgE and <t>IgG1</t> enhanced by RMCP-I. B cells enriched from BALB/c spleen cells were cultured with (open symbols) or without (closed symbols) 1 µg/ml RMCP-I in the presence of LPS plus IL-4 for 7 days. Indicated concentration of GI129471 was added to some culture. Concentrations of IgE (a) and IgG1 (b) in the supernatant were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing GI129471 were compared with values from the culture without the inhibitor by Dunnett's method (*, P
    Mouse Igg3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Bio-Rad goat anti mouse igg conjugated
    A metalloprotease inhibitor, GI129471, blocks the production of IgE and <t>IgG1</t> enhanced by RMCP-I. B cells enriched from BALB/c spleen cells were cultured with (open symbols) or without (closed symbols) 1 µg/ml RMCP-I in the presence of LPS plus IL-4 for 7 days. Indicated concentration of GI129471 was added to some culture. Concentrations of IgE (a) and IgG1 (b) in the supernatant were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing GI129471 were compared with values from the culture without the inhibitor by Dunnett's method (*, P
    Goat Anti Mouse Igg Conjugated, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher igg3 antibodies
    A metalloprotease inhibitor, GI129471, blocks the production of IgE and <t>IgG1</t> enhanced by RMCP-I. B cells enriched from BALB/c spleen cells were cultured with (open symbols) or without (closed symbols) 1 µg/ml RMCP-I in the presence of LPS plus IL-4 for 7 days. Indicated concentration of GI129471 was added to some culture. Concentrations of IgE (a) and IgG1 (b) in the supernatant were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing GI129471 were compared with values from the culture without the inhibitor by Dunnett's method (*, P
    Igg3 Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Athens Research igg3
    Figure 1. Schematic depiction of multiplexed assessment of <t>IgG</t> interactions with FcR and lectins. Up to 500 bead sets, each coupled with an FcR or lectin of interest, can be combined into one well containing an antibody sample of interest. Bound
    Igg3, supplied by Athens Research, used in various techniques. Bioz Stars score: 92/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Immunotec igg3
    Total <t>IgG</t> and IgG subclass levels in sera of individual patient of sparganosis.
    Igg3, supplied by Immunotec, used in various techniques. Bioz Stars score: 92/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems igg3
    Histopathology and immunofluorescence analysis of kidneys. ( A ) Hematoxylin and eosin (H E), periodic acid-Schiff (PAS) reagent, and Masson’s trichrome staining of kidneys obtained from mice at the end of the experiment (original magnification: ×400). Inflammatory cell infiltration and mesangial proliferation were scored on a graded scale from 0 (no) to 4 (severe). ( B ) <t>IgG</t> and C3 deposition in kidneys (original magnification: ×200). Fluorescence staining intensities of IgG and C3 deposits were graded as 0 (none), 1+ (mild), 2+ (moderate), 3+ (moderate to strong), or 4+ (strong). Intergroup analysis was performed using ANOVA followed by Tukey’s multiple comparison post-hoc tests. * p
    Igg3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Alpha Diagnostics igg3
    Depletion and ablation of IL-4 shifts post-vaccination <t>IgG</t> subclass distribution. ( A – E ) Serum anti-oxycodone IgG antibodies were analyzed for IgG 1 , IgG 2a , and IgG 3 subclass in mice immunized with OXY-KLH ± αIL-4 mAb from experiments shown in Figs 1A–H and 2A–C . ( A ) oxycodone-specific IgG titers in mice immunized with OXY-KLH s.c., n = 49; OXY-KLH s.c. + IL-4 mAb i.p., n = 38; OXY-KLH i.m., n = 8; OXY-KLH i.m. + IL-4 mAb i.m., n = 8). IgG subclass analysis was performed on a randomly selected sample from each immunization group (n = 5–6 mice/group): ( B ) IgG 1 , ( C ) IgG 2a , ( D ) IgG 3 , ( E ) IgG 1 /((IgG 2a + IgG 3 )/2). ( F – H ) In a separate study, IL-4 −/− and wild-type mice controls were immunized with KLH or OXY-KLH in alum (s.c., n = 5 each group). ( F ) oxycodone-specific IgG titers by subclass, ( G ) IgG 1 /((IgG 2a + IgG 3 )/2), and ( H ) effect of immunization on oxycodone distribution to serum and brain. ( I ) In a follow-up independent experiment, mice were immunized with either KLH (s.c., n = 4) or OXY-KLH (s.c., n = 10). The OXY-KLH group received either saline or liposome-encapsulated clodronate to deplete macrophages (n = 5), and 24 hrs later challenged with 5.0 mg/kg oxycodone. Data are from one experiment. ( I ) vaccine efficacy in blocking oxycodone distribution to the brain (left) and the subset (%) of immunized mice showing efficacy (right). Data are mean ± SEM. ( A – G ) unpaired two-tailed t-test. ( H , I ) One-way ANOVA paired with Tukey’s test, and chi-square test (χ 2 = 8.00, df = 1, p = 0.0047). *p
    Igg3, supplied by Alpha Diagnostics, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Bio-Rad mouse igg
    Depletion and ablation of IL-4 shifts post-vaccination <t>IgG</t> subclass distribution. ( A – E ) Serum anti-oxycodone IgG antibodies were analyzed for IgG 1 , IgG 2a , and IgG 3 subclass in mice immunized with OXY-KLH ± αIL-4 mAb from experiments shown in Figs 1A–H and 2A–C . ( A ) oxycodone-specific IgG titers in mice immunized with OXY-KLH s.c., n = 49; OXY-KLH s.c. + IL-4 mAb i.p., n = 38; OXY-KLH i.m., n = 8; OXY-KLH i.m. + IL-4 mAb i.m., n = 8). IgG subclass analysis was performed on a randomly selected sample from each immunization group (n = 5–6 mice/group): ( B ) IgG 1 , ( C ) IgG 2a , ( D ) IgG 3 , ( E ) IgG 1 /((IgG 2a + IgG 3 )/2). ( F – H ) In a separate study, IL-4 −/− and wild-type mice controls were immunized with KLH or OXY-KLH in alum (s.c., n = 5 each group). ( F ) oxycodone-specific IgG titers by subclass, ( G ) IgG 1 /((IgG 2a + IgG 3 )/2), and ( H ) effect of immunization on oxycodone distribution to serum and brain. ( I ) In a follow-up independent experiment, mice were immunized with either KLH (s.c., n = 4) or OXY-KLH (s.c., n = 10). The OXY-KLH group received either saline or liposome-encapsulated clodronate to deplete macrophages (n = 5), and 24 hrs later challenged with 5.0 mg/kg oxycodone. Data are from one experiment. ( I ) vaccine efficacy in blocking oxycodone distribution to the brain (left) and the subset (%) of immunized mice showing efficacy (right). Data are mean ± SEM. ( A – G ) unpaired two-tailed t-test. ( H , I ) One-way ANOVA paired with Tukey’s test, and chi-square test (χ 2 = 8.00, df = 1, p = 0.0047). *p
    Mouse Igg, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies igg3
    Expression of fibroblast activation marker Podoplanin (PDPN) and nociceptive neuromodulator NMDAR-1 in diseased supraspinatus tendon tissues. a – b Representative immunofluorescence images showing staining of cell nuclei (POPO-1, cyan) and PDPN (green) with a expression of NMDAR-1 (red) and PGIS (violet) and b expression of TLR4 (red) and IL-1R (violet). c Representative confocal immunofluorescence images showing merged image of diseased tendon sections stained with isotype control antibodies for mouse <t>IgG1,</t> <t>IgG2a,</t> and <t>IgG2b</t> and rabbit <t>IgG</t> fractions. Cyan represents POPO-1 nuclear counterstain. Scale bar, 20 μm
    Igg3, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher fitc conjugated anti mouse igg
    Expression of fibroblast activation marker Podoplanin (PDPN) and nociceptive neuromodulator NMDAR-1 in diseased supraspinatus tendon tissues. a – b Representative immunofluorescence images showing staining of cell nuclei (POPO-1, cyan) and PDPN (green) with a expression of NMDAR-1 (red) and PGIS (violet) and b expression of TLR4 (red) and IL-1R (violet). c Representative confocal immunofluorescence images showing merged image of diseased tendon sections stained with isotype control antibodies for mouse <t>IgG1,</t> <t>IgG2a,</t> and <t>IgG2b</t> and rabbit <t>IgG</t> fractions. Cyan represents POPO-1 nuclear counterstain. Scale bar, 20 μm
    Fitc Conjugated Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 298 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology igg3
    Duration of serum FMDV-specific <t>IgG</t> responses. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological
    Igg3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Results for immunoglubin G subtypes evaluation IgG subtypes in mice serum samples of different experimental groups were assayed against rEG95 by ELISA. Data from each group was compared by other groups with LSD test via one-way analysis of variance (one-way ANOVA). P -values less than 0.05 were recognized as significant. Freund’s : sera from mice immunized with rEG95 formulated with FA. Alum : sera from mice immunized with rEG95 formulated with alum. Trx : sera from mice immunized with Trx pET32 *: significant difference with non-immunized (negative control) group ( P

    Journal: Iranian Journal of Parasitology

    Article Title: Evaluation of Immunogenicity of Novel Isoform of EG95 (EG95-5G1) From Echinococcus granulosus in BALB/C Mice

    doi:

    Figure Lengend Snippet: Results for immunoglubin G subtypes evaluation IgG subtypes in mice serum samples of different experimental groups were assayed against rEG95 by ELISA. Data from each group was compared by other groups with LSD test via one-way analysis of variance (one-way ANOVA). P -values less than 0.05 were recognized as significant. Freund’s : sera from mice immunized with rEG95 formulated with FA. Alum : sera from mice immunized with rEG95 formulated with alum. Trx : sera from mice immunized with Trx pET32 *: significant difference with non-immunized (negative control) group ( P

    Article Snippet: Following overnight incubation at 4°C, the plates were washed and incubated for 1 hr at 37°C with goat anti-mouse IgG1, IgG2a, IgG2b and IgG3 (ISO-2, Sigma) at 1/2000 dilution in 1% BSA-PBST (50μl per well).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total IgG1, IgG2a, IgM and IgG3 on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total IgG1, IgG2a, IgM and IgG3 on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection

    The development of kidney disease in GaMD-immunized γ1 - mice is independent of IFN-γ, IgG2a, C3 and FcRγ BALB/c WT and γ1 - mice (5/gp) were immunized with GaMD on d0 and injected with 1 mg of either anti-IFN-γ or control mAb on days 0 and 5. a , Total levels of all Ig isotypes were determined in 24 hr culture supernatants of spleen cells harvested on days shown. b , GaMD-immunized γ1 - , γ1 - /FcRγ - , γ1 - /C3 - , C3 - / FcRγ - and γ1 - /C3 - /FcγR - mice (5/gp) had their urine tested for LE and blood on days shown. c , d , BALB/c WT and γ1 - mice (5/gp) were immunized with GaMD on d0 and injected with 1 mg of either anti-IFN-γ or control mAb on days 0 and 5. c , Urine obtained on days indicated was assayed for protein, LE and blood. d , BUN levels were determined prior to and 10 days after GaMD immunization. * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: The development of kidney disease in GaMD-immunized γ1 - mice is independent of IFN-γ, IgG2a, C3 and FcRγ BALB/c WT and γ1 - mice (5/gp) were immunized with GaMD on d0 and injected with 1 mg of either anti-IFN-γ or control mAb on days 0 and 5. a , Total levels of all Ig isotypes were determined in 24 hr culture supernatants of spleen cells harvested on days shown. b , GaMD-immunized γ1 - , γ1 - /FcRγ - , γ1 - /C3 - , C3 - / FcRγ - and γ1 - /C3 - /FcγR - mice (5/gp) had their urine tested for LE and blood on days shown. c , d , BALB/c WT and γ1 - mice (5/gp) were immunized with GaMD on d0 and injected with 1 mg of either anti-IFN-γ or control mAb on days 0 and 5. c , Urine obtained on days indicated was assayed for protein, LE and blood. d , BUN levels were determined prior to and 10 days after GaMD immunization. * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection

    IgG1 inhibits IgG3-induced cryoglobulin kidney disease independent of complement and FcγRIIB and better than IgG2a and IgG2b a , WT mice (4/gp) were injected i.v. with 4 mg of mouse IgG1, IgG2a, IgG2b, or IgG3 anti-TNP mAb and s.c. with 100 μl of TNP-goat serum on days 0 and 1. Urine LE and blood measured prior to injections and on d1 and d2. b , Urine LE and blood for BALB/c WT and C3 - mice (4/gp) injected i.v. with 4 mg of IgG3 anti-TNP mAb and s.c. with 400 μl of TNP-goat serum on d0 and 1. c , WT and FcγRIIB-deficient (FcγRIIB - ) mice (4/gp) were injected s.c. with 100 μl of TNP-goat serum and i.v. with 4 mg of IgG3 anti-TNP ± 5 mg of IgG1 anti-TNP on d0 and 1. Urinalysis on d0, 1 and 2. d , BALB/c mice were injected i.v. with 4 mg of IgG3 anti-TNP and s.c. with 1.4 mg of TNP-BSA on days 0 and 1. Some mice were also injected with 0.625, 1.25, 2.5, or 5 mg of switch variants of IgG1, IgG2a or IgG2b anti-TNP mAbs on d0 and 1. Urine protein was determined on d0 (not shown), d1 (upper panel) and d2 (lower panel). Results are pooled from a total of 7 experiments. Group size: IgG3 alone: 19 mice; 0.625 mg of IgG1, IgG2a, or IgG2b: 4 mice; 1.25 mg of IgG1, IgG2a or IgG2b: 8 mice; 2.5 mg of IgG1, IgG2a or IgG2b: 6 mice; 5 mg of IgG1, IgG2a or IgG2b: 8 or 9 mice. The significance of differences between treatment groups was determined as described in the legend to Fig. 4f . # p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: IgG1 inhibits IgG3-induced cryoglobulin kidney disease independent of complement and FcγRIIB and better than IgG2a and IgG2b a , WT mice (4/gp) were injected i.v. with 4 mg of mouse IgG1, IgG2a, IgG2b, or IgG3 anti-TNP mAb and s.c. with 100 μl of TNP-goat serum on days 0 and 1. Urine LE and blood measured prior to injections and on d1 and d2. b , Urine LE and blood for BALB/c WT and C3 - mice (4/gp) injected i.v. with 4 mg of IgG3 anti-TNP mAb and s.c. with 400 μl of TNP-goat serum on d0 and 1. c , WT and FcγRIIB-deficient (FcγRIIB - ) mice (4/gp) were injected s.c. with 100 μl of TNP-goat serum and i.v. with 4 mg of IgG3 anti-TNP ± 5 mg of IgG1 anti-TNP on d0 and 1. Urinalysis on d0, 1 and 2. d , BALB/c mice were injected i.v. with 4 mg of IgG3 anti-TNP and s.c. with 1.4 mg of TNP-BSA on days 0 and 1. Some mice were also injected with 0.625, 1.25, 2.5, or 5 mg of switch variants of IgG1, IgG2a or IgG2b anti-TNP mAbs on d0 and 1. Urine protein was determined on d0 (not shown), d1 (upper panel) and d2 (lower panel). Results are pooled from a total of 7 experiments. Group size: IgG3 alone: 19 mice; 0.625 mg of IgG1, IgG2a, or IgG2b: 4 mice; 1.25 mg of IgG1, IgG2a or IgG2b: 8 mice; 2.5 mg of IgG1, IgG2a or IgG2b: 6 mice; 5 mg of IgG1, IgG2a or IgG2b: 8 or 9 mice. The significance of differences between treatment groups was determined as described in the legend to Fig. 4f . # p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection

    Concurrent injection of WT mice with IgG3 anti-TNP mAb and TNP-goat serum induces glomerulopathy a , WT mice (4/gp) were injected i.v.with 4 mg of mouse IgG1, IgG2a, IgG2b, or IgG3 anti-TNP mAb and s.c. with 100 μl of TNP-goat serum on days 0 and 1. Urine protein measured prior to injections and on d1 and d2. b , c , WT mice (4/gp) were injected with mouse IgG3 anti-TNP mAb +/- TNP-goat serum as in “a.” Day 2 mouse sera were analyzed for BUN (b). Day 2 kidneys were stained with PAS (c, panel 1: glomerulus from mouse that received only IgG3; panels 2: glomerulus from mouse that received IgG3 + TNP-goat serum). Representative of 3 mice/group. d , Urine protein for BALB/c WT and C3 - mice (4/gp) injected i.v. with 4 mg of IgG3 anti-TNP mAb and s.c. with 400 μl of TNP-goat serum on d0 and 1. * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Concurrent injection of WT mice with IgG3 anti-TNP mAb and TNP-goat serum induces glomerulopathy a , WT mice (4/gp) were injected i.v.with 4 mg of mouse IgG1, IgG2a, IgG2b, or IgG3 anti-TNP mAb and s.c. with 100 μl of TNP-goat serum on days 0 and 1. Urine protein measured prior to injections and on d1 and d2. b , c , WT mice (4/gp) were injected with mouse IgG3 anti-TNP mAb +/- TNP-goat serum as in “a.” Day 2 mouse sera were analyzed for BUN (b). Day 2 kidneys were stained with PAS (c, panel 1: glomerulus from mouse that received only IgG3; panels 2: glomerulus from mouse that received IgG3 + TNP-goat serum). Representative of 3 mice/group. d , Urine protein for BALB/c WT and C3 - mice (4/gp) injected i.v. with 4 mg of IgG3 anti-TNP mAb and s.c. with 400 μl of TNP-goat serum on d0 and 1. * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Injection, Mouse Assay, Staining

    Glomerulopathy in GaMD-immunized γ1 - mice is complement- and FcRγ -independent and associated with IgG3 cryoglobulinemia a . Serum anti-goat IgG titers in WT and γ1 - mice (4/gp) 8d after GaMD immunization. b , c . Urine protein (b) and BUN (c) of GaMD-immunized γ1 - , γ1 - /FcRγ - , γ1 - /C3 - , C3 - / FcRγ - and γ1 - /C3 - /FcγR - mice (5/gp). d , e . Serum cryoprecipitate protein and Ig isotype concentrations 6-7 d after GaMD immunization of WT and γ1 - mice (7 or 8/gp). Only cryoprecipitates from γ1 - mice contained detectable Ig. f. IgG3 (brown color) in glomerular capillaries (arrows) of γ1 - mice 8d after GaMD (panels 1, low magnification; panel 2, high magnification). * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Glomerulopathy in GaMD-immunized γ1 - mice is complement- and FcRγ -independent and associated with IgG3 cryoglobulinemia a . Serum anti-goat IgG titers in WT and γ1 - mice (4/gp) 8d after GaMD immunization. b , c . Urine protein (b) and BUN (c) of GaMD-immunized γ1 - , γ1 - /FcRγ - , γ1 - /C3 - , C3 - / FcRγ - and γ1 - /C3 - /FcγR - mice (5/gp). d , e . Serum cryoprecipitate protein and Ig isotype concentrations 6-7 d after GaMD immunization of WT and γ1 - mice (7 or 8/gp). Only cryoprecipitates from γ1 - mice contained detectable Ig. f. IgG3 (brown color) in glomerular capillaries (arrows) of γ1 - mice 8d after GaMD (panels 1, low magnification; panel 2, high magnification). * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay

    Delayed antigen elimination does not account for renal disease in GaMD-immunized γ1 - mice a . BALB/c WT and γ1 - mice (10/gp) were immunized s.c. withGaMD. Sera obtained 5, 6, 7 and 9 days later were evaluated by gel double diffusion for the presence of goat IgG. b-e. BALB/c WT mice (4 or 5/gp) were injected s.c. with a total of 0.2 ml of different mixtures of GaMD and goat anti-KLH antisera. b, c , Mouse sera collected 9d later were assayed for BUN ( b ) and IgG1 anti-goat IgG Ab ( c ). d , Sera obtained 6-13d post-immunization were evaluated by gel double diffusion for the presence of goat IgG. e , Urine samples collected 4-12 days post-immunization were analyzed for protein. * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Delayed antigen elimination does not account for renal disease in GaMD-immunized γ1 - mice a . BALB/c WT and γ1 - mice (10/gp) were immunized s.c. withGaMD. Sera obtained 5, 6, 7 and 9 days later were evaluated by gel double diffusion for the presence of goat IgG. b-e. BALB/c WT mice (4 or 5/gp) were injected s.c. with a total of 0.2 ml of different mixtures of GaMD and goat anti-KLH antisera. b, c , Mouse sera collected 9d later were assayed for BUN ( b ) and IgG1 anti-goat IgG Ab ( c ). d , Sera obtained 6-13d post-immunization were evaluated by gel double diffusion for the presence of goat IgG. e , Urine samples collected 4-12 days post-immunization were analyzed for protein. * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Diffusion-based Assay, Injection

    Ag-specific IgG1 prevents IgG3-mediated glomerulopathy BALB/c γ1 - mice (5/gp) were injected with GaMD on day 0 and/or GaMD-immune or rabbit anti-mouse IgD (RaMD) immune WT serum daily on d4-7. a , b , Urine protein ( a ) and d12 serum albumin and BUN levels ( b ). * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Ag-specific IgG1 prevents IgG3-mediated glomerulopathy BALB/c γ1 - mice (5/gp) were injected with GaMD on day 0 and/or GaMD-immune or rabbit anti-mouse IgD (RaMD) immune WT serum daily on d4-7. a , b , Urine protein ( a ) and d12 serum albumin and BUN levels ( b ). * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection

    GaMD immunization of γ1 - mice induces renal dysfunction and glomerular deposition of PAS + material that includes IgG and complement a , WT and γ1 - mice (4/gp) were immunized with GaMD. Urine LE and blood were obtained. b , Representative photomicrographs of glomeruli stained for C3 (top panels) or total mouse IgG (bottom panels) from WT (right panels) and γ1 -/- mice (left panels) 12 d post-GaMD immunization 3 mice/group. c , Deposition of amorphous PAS + material in glomeruli of γ1 - , but not WT begins ∼7 days post GaMD-immunization and leads to glomerular destruction by day 9. Note the scarcity of inflammatory cells in glomeruli. Representative data of 6 mice/group.

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: GaMD immunization of γ1 - mice induces renal dysfunction and glomerular deposition of PAS + material that includes IgG and complement a , WT and γ1 - mice (4/gp) were immunized with GaMD. Urine LE and blood were obtained. b , Representative photomicrographs of glomeruli stained for C3 (top panels) or total mouse IgG (bottom panels) from WT (right panels) and γ1 -/- mice (left panels) 12 d post-GaMD immunization 3 mice/group. c , Deposition of amorphous PAS + material in glomeruli of γ1 - , but not WT begins ∼7 days post GaMD-immunization and leads to glomerular destruction by day 9. Note the scarcity of inflammatory cells in glomeruli. Representative data of 6 mice/group.

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Staining

    Antigen-specific IgG1 can prevent IgG3 immune complex glomerular deposition BALB/c WT mice were injected i.v.with mouse IgG1 and/or IgG3 anti-TNP mAb with or without s.c.injection of TNP-BSA on days 0 and 1. a. Kidneys were stained with PAS on day 2. Representative micrographs from 3 mice/group are shown. b. Kidney serial sections were stained with PAS or for IgG3 or IgG1 (brown pigment). Representative micrographs from 4 mice/group are shown.

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Antigen-specific IgG1 can prevent IgG3 immune complex glomerular deposition BALB/c WT mice were injected i.v.with mouse IgG1 and/or IgG3 anti-TNP mAb with or without s.c.injection of TNP-BSA on days 0 and 1. a. Kidneys were stained with PAS on day 2. Representative micrographs from 3 mice/group are shown. b. Kidney serial sections were stained with PAS or for IgG3 or IgG1 (brown pigment). Representative micrographs from 4 mice/group are shown.

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection, Staining

    IgG3 IC persist and accumulate in the glomeruli of GaMD-immunized γ1 - BALB/c WT and γ1 - mice were left untreated or were immunized with GaMD. Kidney sections were stained for mouse IgG1, IgG2a, IgG2b, IgG3 and IgM 8 and 12d later. Representative photomicrographs from 3 GaMD-immunized mice are shown. Insets show magnified views. No staining was observed with sections from unimmunized mice (not shown).

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: IgG3 IC persist and accumulate in the glomeruli of GaMD-immunized γ1 - BALB/c WT and γ1 - mice were left untreated or were immunized with GaMD. Kidney sections were stained for mouse IgG1, IgG2a, IgG2b, IgG3 and IgM 8 and 12d later. Representative photomicrographs from 3 GaMD-immunized mice are shown. Insets show magnified views. No staining was observed with sections from unimmunized mice (not shown).

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Staining

    GaMD-immunized γ1 +/- mice generate large IgG3 responses but develop mild renal disease BALB/c mice homozygous (γ1 +/+ ), heterozygous (γ1 +/- ) and null (γ1 -/- ) for a functional γ1 allele (6/gp) were injected s.c. with GaMD. a , Sera were titered for goat IgG-specific IgG1, IgG2a and IgG3 0, 8 and 12 days later. Day 0 titers were zero for all Ig isotypes (data not shown). b , Urine samples from the same mice were assayed for protein and leukocyte esterase. ND = none detected.

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: GaMD-immunized γ1 +/- mice generate large IgG3 responses but develop mild renal disease BALB/c mice homozygous (γ1 +/+ ), heterozygous (γ1 +/- ) and null (γ1 -/- ) for a functional γ1 allele (6/gp) were injected s.c. with GaMD. a , Sera were titered for goat IgG-specific IgG1, IgG2a and IgG3 0, 8 and 12 days later. Day 0 titers were zero for all Ig isotypes (data not shown). b , Urine samples from the same mice were assayed for protein and leukocyte esterase. ND = none detected.

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Functional Assay, Injection

    Disease phenotype and severity of experimental epidermolysis bullosa acquisita induced by the passive transfer of collagen VII-specific IgG. ( A ) BALB/c mice received injections of IgG purified from rabbits immunized with different glutathione-S-transferase (GST)-fusion proteins containing fragments of murine collagen VII (mCVII-1, n = 10; mCVII-2, n = 10; mCVII-3, n = 10; mCVII-4, n = 10; mCVII-5, n = 6; mCVII-z, n = 5) or GST alone ( n = 8), or from pre-immune animals ( n = 8). Mice injected with rabbit IgG against mCVII-1, mCVII-2, mCVII-3, mCVII-4 and mCVII-5, but not against mCVII-z and GST or injected with pre-immune IgG, developed skin blisters and erosions covered by crusts. ( B ) The disease severity, based on a scoring system (4 being the highest score) and depicted as means of individual clinical scores ± SEM is shown before the first injection and every other following day for 12 days. Significantly more extensive disease was induced by injecting IgG against fragments mCVII-2, mCVII-3, mCVII-4 and mCVII-1, P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Blister-inducing antibodies target multiple epitopes on collagen VII in mice

    doi: 10.1111/jcmm.12338

    Figure Lengend Snippet: Disease phenotype and severity of experimental epidermolysis bullosa acquisita induced by the passive transfer of collagen VII-specific IgG. ( A ) BALB/c mice received injections of IgG purified from rabbits immunized with different glutathione-S-transferase (GST)-fusion proteins containing fragments of murine collagen VII (mCVII-1, n = 10; mCVII-2, n = 10; mCVII-3, n = 10; mCVII-4, n = 10; mCVII-5, n = 6; mCVII-z, n = 5) or GST alone ( n = 8), or from pre-immune animals ( n = 8). Mice injected with rabbit IgG against mCVII-1, mCVII-2, mCVII-3, mCVII-4 and mCVII-5, but not against mCVII-z and GST or injected with pre-immune IgG, developed skin blisters and erosions covered by crusts. ( B ) The disease severity, based on a scoring system (4 being the highest score) and depicted as means of individual clinical scores ± SEM is shown before the first injection and every other following day for 12 days. Significantly more extensive disease was induced by injecting IgG against fragments mCVII-2, mCVII-3, mCVII-4 and mCVII-1, P

    Article Snippet: For IgG subclass identification, the incubation with the 100 times diluted mouse sera was followed by incubation with 250 times diluted biotinylated monoclonal rat antimouse IgG1, IgG2a, IgG2b and IgG3 Abs (all from BD Pharmingen, Heidelberg, Germany), and HRP-conjugated streptavidine (Dianova, Hamburg, Germany).

    Techniques: Mouse Assay, Purification, Injection

    Complement activation triggered by collagen-specific antibodies ex vivo and in vivo . ( A ) Linear depositions of rabbit IgG at the epidermal basement membrane in frozen sections of perilesional mouse skin biopsies were visualized by direct IF microscopy and the staining intensity measured using ImageJ was expressed as mean fluorescence density arbitrary units ± SEM. ( B ) Linear depositions of murine C3 at the epidermal basement membrane in frozen sections of perilesional mouse skin biopsies were visualized by direct IF microscopy and the staining intensity was semi-quantitatively defined by scoring. Fluorescence intensities were expressed as mean score values ± SEM. Fluorescence intensities were expressed as mean score values ± SEM. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Blister-inducing antibodies target multiple epitopes on collagen VII in mice

    doi: 10.1111/jcmm.12338

    Figure Lengend Snippet: Complement activation triggered by collagen-specific antibodies ex vivo and in vivo . ( A ) Linear depositions of rabbit IgG at the epidermal basement membrane in frozen sections of perilesional mouse skin biopsies were visualized by direct IF microscopy and the staining intensity measured using ImageJ was expressed as mean fluorescence density arbitrary units ± SEM. ( B ) Linear depositions of murine C3 at the epidermal basement membrane in frozen sections of perilesional mouse skin biopsies were visualized by direct IF microscopy and the staining intensity was semi-quantitatively defined by scoring. Fluorescence intensities were expressed as mean score values ± SEM. Fluorescence intensities were expressed as mean score values ± SEM. * P

    Article Snippet: For IgG subclass identification, the incubation with the 100 times diluted mouse sera was followed by incubation with 250 times diluted biotinylated monoclonal rat antimouse IgG1, IgG2a, IgG2b and IgG3 Abs (all from BD Pharmingen, Heidelberg, Germany), and HRP-conjugated streptavidine (Dianova, Hamburg, Germany).

    Techniques: Activation Assay, Ex Vivo, In Vivo, Microscopy, Staining, Fluorescence

    Disease phenotype and severity of experimental EBA induced by immunization with recombinant protein corresponding to different fragments of murine collagen VII. ( A ) SJL-1 mice ( n = 10/fragment) immunized with an equimolar mix or individual proteins of recombinant collagen VII developed at different extent lesions on the skin including alopecia, blisters, erosions and crusts. Control mice immunized with GST ( n = 10) had a normal appearance. ( B ) The disease severity, based on a scoring system (4 being the highest score) and depicted as means of individual clinical scores ± SEM is shown before the first injection and every following week for 12 weeks. Mice immunized with GST-mCVII-3 and GST-mCVII-1 showed the highest clinical scores, whereas less or no disease activity is seen in the other experimental groups. ( C ) Levels of collagen VII-specific mouse IgG in serum samples of mice immunized with recombinant forms of collagen VII ( n = 10/fragment) or GST ( n = 10) were measured by ELISA. Data are shown as mean OD reading ± SEM.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Blister-inducing antibodies target multiple epitopes on collagen VII in mice

    doi: 10.1111/jcmm.12338

    Figure Lengend Snippet: Disease phenotype and severity of experimental EBA induced by immunization with recombinant protein corresponding to different fragments of murine collagen VII. ( A ) SJL-1 mice ( n = 10/fragment) immunized with an equimolar mix or individual proteins of recombinant collagen VII developed at different extent lesions on the skin including alopecia, blisters, erosions and crusts. Control mice immunized with GST ( n = 10) had a normal appearance. ( B ) The disease severity, based on a scoring system (4 being the highest score) and depicted as means of individual clinical scores ± SEM is shown before the first injection and every following week for 12 weeks. Mice immunized with GST-mCVII-3 and GST-mCVII-1 showed the highest clinical scores, whereas less or no disease activity is seen in the other experimental groups. ( C ) Levels of collagen VII-specific mouse IgG in serum samples of mice immunized with recombinant forms of collagen VII ( n = 10/fragment) or GST ( n = 10) were measured by ELISA. Data are shown as mean OD reading ± SEM.

    Article Snippet: For IgG subclass identification, the incubation with the 100 times diluted mouse sera was followed by incubation with 250 times diluted biotinylated monoclonal rat antimouse IgG1, IgG2a, IgG2b and IgG3 Abs (all from BD Pharmingen, Heidelberg, Germany), and HRP-conjugated streptavidine (Dianova, Hamburg, Germany).

    Techniques: Recombinant, Mouse Assay, Injection, Activity Assay, Enzyme-linked Immunosorbent Assay

    Antigen-specific IgG subclasses during follow-up and relapse. The PLA 2 R1- (A) and THSD7A-specific (B) IgG subclasses did not significantly change during the follow-up period. At the time of relapse of PLA 2 R1-ab, antigen-specific IgG subclasses were similar to the distribution observed at baseline (C) .

    Journal: Frontiers in Immunology

    Article Title: Antigen-Specific IgG Subclasses in Primary and Malignancy-Associated Membranous Nephropathy

    doi: 10.3389/fimmu.2018.03035

    Figure Lengend Snippet: Antigen-specific IgG subclasses during follow-up and relapse. The PLA 2 R1- (A) and THSD7A-specific (B) IgG subclasses did not significantly change during the follow-up period. At the time of relapse of PLA 2 R1-ab, antigen-specific IgG subclasses were similar to the distribution observed at baseline (C) .

    Article Snippet: For detection of subclass-specific IgG in Western blot analyses, horseradish peroxidase-conjugated monoclonal anti-human IgG1 (9054-05), IgG2 (9060-05), IgG3 (9210-05), IgG4 (9200-05), and IgG (9040-05) antibodies from mouse were used (IgG1, IgG2, and IgG3 at a 1:5,000 dilution; IgG4 at a 1:10,000 dilution; IgG at a 1:20,000 dilution, all SouthernBiotech, Birmingham, USA), diluted in 5% dry milk in PBS-T.

    Techniques: Proximity Ligation Assay

    Representative Western blots of patients with primary MN and malignancy-associated MN. Representative Western blot analyses of patients with all 4 subclasses of antigen-specific IgG binding. (A) Patient 1 with PLA 2 R1-associated MN and malignancy. (B) Patient 2 with PLA 2 R1-associated MN and no malignancy. (C) Patient 3 with THSD7A-associated MN and malignancy. (D) Patient 4 with THSD7A-associated MN and no malignancy.

    Journal: Frontiers in Immunology

    Article Title: Antigen-Specific IgG Subclasses in Primary and Malignancy-Associated Membranous Nephropathy

    doi: 10.3389/fimmu.2018.03035

    Figure Lengend Snippet: Representative Western blots of patients with primary MN and malignancy-associated MN. Representative Western blot analyses of patients with all 4 subclasses of antigen-specific IgG binding. (A) Patient 1 with PLA 2 R1-associated MN and malignancy. (B) Patient 2 with PLA 2 R1-associated MN and no malignancy. (C) Patient 3 with THSD7A-associated MN and malignancy. (D) Patient 4 with THSD7A-associated MN and no malignancy.

    Article Snippet: For detection of subclass-specific IgG in Western blot analyses, horseradish peroxidase-conjugated monoclonal anti-human IgG1 (9054-05), IgG2 (9060-05), IgG3 (9210-05), IgG4 (9200-05), and IgG (9040-05) antibodies from mouse were used (IgG1, IgG2, and IgG3 at a 1:5,000 dilution; IgG4 at a 1:10,000 dilution; IgG at a 1:20,000 dilution, all SouthernBiotech, Birmingham, USA), diluted in 5% dry milk in PBS-T.

    Techniques: Western Blot, Binding Assay, Proximity Ligation Assay

    Binding of antigen-specific IgG subclasses to human glomerular and lung tissue. Representative Western blot analyses of the antigen-specific IgG subclasses binding to lung tissue extracts (LTE) and human glomerular extracts (HGE). (A) Serum from patient 1 was positive for all THSD7A-specific IgG subclasses. (B) Serum from patient 2 showed detectability for THSD7A-specific IgG3 and IgG4. (C) Serum from patient 3 was positive for PLA 2 R1-specific IgG3 and IgG4.

    Journal: Frontiers in Immunology

    Article Title: Antigen-Specific IgG Subclasses in Primary and Malignancy-Associated Membranous Nephropathy

    doi: 10.3389/fimmu.2018.03035

    Figure Lengend Snippet: Binding of antigen-specific IgG subclasses to human glomerular and lung tissue. Representative Western blot analyses of the antigen-specific IgG subclasses binding to lung tissue extracts (LTE) and human glomerular extracts (HGE). (A) Serum from patient 1 was positive for all THSD7A-specific IgG subclasses. (B) Serum from patient 2 showed detectability for THSD7A-specific IgG3 and IgG4. (C) Serum from patient 3 was positive for PLA 2 R1-specific IgG3 and IgG4.

    Article Snippet: For detection of subclass-specific IgG in Western blot analyses, horseradish peroxidase-conjugated monoclonal anti-human IgG1 (9054-05), IgG2 (9060-05), IgG3 (9210-05), IgG4 (9200-05), and IgG (9040-05) antibodies from mouse were used (IgG1, IgG2, and IgG3 at a 1:5,000 dilution; IgG4 at a 1:10,000 dilution; IgG at a 1:20,000 dilution, all SouthernBiotech, Birmingham, USA), diluted in 5% dry milk in PBS-T.

    Techniques: Binding Assay, Western Blot, Proximity Ligation Assay

    THSD7A and PLA 2 R1 are differentially glycosylated in human lung and kidney. (A) The THSD7A-ab positive serum shows positive binding to the antigen of human lung tissue (LTE) and glomerular extract (HGE), however, the antigen migrates differentially in both tissues. The renal antigen showed a higher molecular weight compared to the lung antigen. (B) Upon addition of deglycosylating enzymes THSD7A shows the same migration pattern in both tissues, suggesting a polypeptide backbone with different glycosylation in the lung and the kidney. (C,D) Similar observations were made for PLA 2 R1 in lung and kidney tissue. 140 kDa band in LTE represents residual IgG, which was also detected in negative controls (healthy serum, n = 5, data not shown).

    Journal: Frontiers in Immunology

    Article Title: Antigen-Specific IgG Subclasses in Primary and Malignancy-Associated Membranous Nephropathy

    doi: 10.3389/fimmu.2018.03035

    Figure Lengend Snippet: THSD7A and PLA 2 R1 are differentially glycosylated in human lung and kidney. (A) The THSD7A-ab positive serum shows positive binding to the antigen of human lung tissue (LTE) and glomerular extract (HGE), however, the antigen migrates differentially in both tissues. The renal antigen showed a higher molecular weight compared to the lung antigen. (B) Upon addition of deglycosylating enzymes THSD7A shows the same migration pattern in both tissues, suggesting a polypeptide backbone with different glycosylation in the lung and the kidney. (C,D) Similar observations were made for PLA 2 R1 in lung and kidney tissue. 140 kDa band in LTE represents residual IgG, which was also detected in negative controls (healthy serum, n = 5, data not shown).

    Article Snippet: For detection of subclass-specific IgG in Western blot analyses, horseradish peroxidase-conjugated monoclonal anti-human IgG1 (9054-05), IgG2 (9060-05), IgG3 (9210-05), IgG4 (9200-05), and IgG (9040-05) antibodies from mouse were used (IgG1, IgG2, and IgG3 at a 1:5,000 dilution; IgG4 at a 1:10,000 dilution; IgG at a 1:20,000 dilution, all SouthernBiotech, Birmingham, USA), diluted in 5% dry milk in PBS-T.

    Techniques: Proximity Ligation Assay, Binding Assay, Molecular Weight, Migration

    Distribution of PLA 2 R1- and THSD7A-specific IgG subclasses at baseline. (A) Detection of antigen-specific IgG subclasses in the complete study cohort by Western Blot using recombinant proteins. All patients were found to be positive for antigen-specific IgG4-antibodies. Antigen-specific IgG3 antibodies were more common than antigen-specific IgG1, while IgG2 was detected less often. The distribution of (B) PLA 2 R1- and (C) THSD7A-specific IgG subclasses was not different in patients with or without malignancy. Patients with (D) PLA 2 R1- or (E) THSD7A-associated MN were grouped depending on the clinical outcome: negativity of autoantibodies during follow-up, remission of proteinuria, doubling of serum creatinine. IgG1-3 depicts the percentage of patients in whom at least one antigen-specific IgG subclass (IgG1, IgG2, or IgG3) was detected in addition to IgG4.

    Journal: Frontiers in Immunology

    Article Title: Antigen-Specific IgG Subclasses in Primary and Malignancy-Associated Membranous Nephropathy

    doi: 10.3389/fimmu.2018.03035

    Figure Lengend Snippet: Distribution of PLA 2 R1- and THSD7A-specific IgG subclasses at baseline. (A) Detection of antigen-specific IgG subclasses in the complete study cohort by Western Blot using recombinant proteins. All patients were found to be positive for antigen-specific IgG4-antibodies. Antigen-specific IgG3 antibodies were more common than antigen-specific IgG1, while IgG2 was detected less often. The distribution of (B) PLA 2 R1- and (C) THSD7A-specific IgG subclasses was not different in patients with or without malignancy. Patients with (D) PLA 2 R1- or (E) THSD7A-associated MN were grouped depending on the clinical outcome: negativity of autoantibodies during follow-up, remission of proteinuria, doubling of serum creatinine. IgG1-3 depicts the percentage of patients in whom at least one antigen-specific IgG subclass (IgG1, IgG2, or IgG3) was detected in addition to IgG4.

    Article Snippet: For detection of subclass-specific IgG in Western blot analyses, horseradish peroxidase-conjugated monoclonal anti-human IgG1 (9054-05), IgG2 (9060-05), IgG3 (9210-05), IgG4 (9200-05), and IgG (9040-05) antibodies from mouse were used (IgG1, IgG2, and IgG3 at a 1:5,000 dilution; IgG4 at a 1:10,000 dilution; IgG at a 1:20,000 dilution, all SouthernBiotech, Birmingham, USA), diluted in 5% dry milk in PBS-T.

    Techniques: Proximity Ligation Assay, Western Blot, Recombinant

    (A) Analysis of the IgG subclass of anti-TRIM9 Abs in patient CSF. HEK cells expressing TRIM9 were incubated with patient CSF and revealed with subclass-specific anti-human IgG antibodies. Positive signal was observed for all IgG subclasses; the strongest signal was found for IgG1. (B) Epitope mapping of anti-TRIM9 Abs. HEK cells were transfected with plasmids coding for different domains of TRIM9 and then lysed and immuno-blotted with CSF from patient 1 that recognized all TRIM9 domains except the B30.2 domain. The Abs are polyclonal.

    Journal: Cerebellum (London, England)

    Article Title: TRIM9 and TRIM67 are new targets in paraneoplastic cerebellar degeneration

    doi: 10.1007/s12311-018-0987-5

    Figure Lengend Snippet: (A) Analysis of the IgG subclass of anti-TRIM9 Abs in patient CSF. HEK cells expressing TRIM9 were incubated with patient CSF and revealed with subclass-specific anti-human IgG antibodies. Positive signal was observed for all IgG subclasses; the strongest signal was found for IgG1. (B) Epitope mapping of anti-TRIM9 Abs. HEK cells were transfected with plasmids coding for different domains of TRIM9 and then lysed and immuno-blotted with CSF from patient 1 that recognized all TRIM9 domains except the B30.2 domain. The Abs are polyclonal.

    Article Snippet: Patients’ antibody IgG subtypes contained in serum or CSF were identified using full-length TRIM9 -transfected HEK 293 cells and secondary anti-human antibodies specific for IgG1 (MCA4774, Bio-Rad, Hercules, CA), IgG2 (555873, BD Pharmingen, Le Pont de Claix, France), IgG3 (5247-9850, ABD serotec, Kidlington, OX) and IgG4 (555881, BD pharmingen).

    Techniques: Expressing, Incubation, Transfection

    Expression and purification of the recombinant gH/gL/gQ1/gQ2 complex. (A) The constructions of gH, gL, gQ1, and gQ2 are shown. gH is modified to have an N-terminal IL-2 signal sequence and a C-terminal human IgG1 Fc (hFc) and His 6 sequence replacing the intrinsic gH signal sequence and transmembrane-cytoplasmic tail domains, respectively. ( B) Cell binding assay was performed using hCD134 expressing JJhan cells or JJhan cells (negative control). After incubation with the tetramer-hFc containing medium, the interaction between cell expressing hCD134 and tetramer-hFc was detected using Alexa 488 conjugated anti-human IgG targeting the hFc by flow cytometry. The hFc protein (without tetramer) was used as the non-binding negative control. ( C) Size exclusion column chromatography of the purified tetramer. ( D) Western blotting analysis of the tetramer purified by the size exclusion column chromatography. gH, gQ1, gL, and gQ2 were detected by the respective antibodies. ( E) Purified gH/gL/gQ1/gQ2 complex detected by Coomassie brilliant blue staining.

    Journal: PLoS Pathogens

    Article Title: Tetrameric glycoprotein complex gH/gL/gQ1/gQ2 is a promising vaccine candidate for human herpesvirus 6B

    doi: 10.1371/journal.ppat.1008609

    Figure Lengend Snippet: Expression and purification of the recombinant gH/gL/gQ1/gQ2 complex. (A) The constructions of gH, gL, gQ1, and gQ2 are shown. gH is modified to have an N-terminal IL-2 signal sequence and a C-terminal human IgG1 Fc (hFc) and His 6 sequence replacing the intrinsic gH signal sequence and transmembrane-cytoplasmic tail domains, respectively. ( B) Cell binding assay was performed using hCD134 expressing JJhan cells or JJhan cells (negative control). After incubation with the tetramer-hFc containing medium, the interaction between cell expressing hCD134 and tetramer-hFc was detected using Alexa 488 conjugated anti-human IgG targeting the hFc by flow cytometry. The hFc protein (without tetramer) was used as the non-binding negative control. ( C) Size exclusion column chromatography of the purified tetramer. ( D) Western blotting analysis of the tetramer purified by the size exclusion column chromatography. gH, gQ1, gL, and gQ2 were detected by the respective antibodies. ( E) Purified gH/gL/gQ1/gQ2 complex detected by Coomassie brilliant blue staining.

    Article Snippet: For the measurement of IgG1, IgG2a and IgG3, the first antibodies were diluted to 1:2560 and the secondary antibodies were added to HRP-conjugated anti-mouse IgG1, IgG2a or IgG3 (Abcam, Cambridge, UK).

    Techniques: Expressing, Purification, Recombinant, Modification, Sequencing, Cell Binding Assay, Negative Control, Incubation, Flow Cytometry, Binding Assay, Column Chromatography, Western Blot, Staining

    Humoral immunity induced by immunization with the tetramer and the combination of Alum and CpG adjuvant. (A) Immunization schedule indicated as the weeks of immunization of four-week-old female BALB/c mice (n = 5). ( B) The amounts of the anti-tetramer antibodies in the sera collected from mice immunized with the indicated combination of adjuvants at the eight weeks after the third immunization were evaluated for the further ELISA assay using purified tetramer. The titers were calculated as the maximum dilution at which the value of OD405 was higher than that of the mean+2SD of the group immunized with PBS. ( C) The IgG isotypes of the anti-tetramer antibodies were analyzed by the ELISA assay at the dilution of 2560. IgG1, 1gG2a and IgG3 were detected separately from the same sera. The values of serum estimated IgG concentration were obtained from standard curves for the isotype control antibodies of mouse IgG1, IgG2a and IgG3, and presented as the mean±SD. (D) The neutralizing activities of the sera from the immunized mice indicated were measured in MT4 cells. Titers are presented as the maximum dilution at which the HHV-6B IE1 protein could not be detected. Data are the mean±SD of each group. *p

    Journal: PLoS Pathogens

    Article Title: Tetrameric glycoprotein complex gH/gL/gQ1/gQ2 is a promising vaccine candidate for human herpesvirus 6B

    doi: 10.1371/journal.ppat.1008609

    Figure Lengend Snippet: Humoral immunity induced by immunization with the tetramer and the combination of Alum and CpG adjuvant. (A) Immunization schedule indicated as the weeks of immunization of four-week-old female BALB/c mice (n = 5). ( B) The amounts of the anti-tetramer antibodies in the sera collected from mice immunized with the indicated combination of adjuvants at the eight weeks after the third immunization were evaluated for the further ELISA assay using purified tetramer. The titers were calculated as the maximum dilution at which the value of OD405 was higher than that of the mean+2SD of the group immunized with PBS. ( C) The IgG isotypes of the anti-tetramer antibodies were analyzed by the ELISA assay at the dilution of 2560. IgG1, 1gG2a and IgG3 were detected separately from the same sera. The values of serum estimated IgG concentration were obtained from standard curves for the isotype control antibodies of mouse IgG1, IgG2a and IgG3, and presented as the mean±SD. (D) The neutralizing activities of the sera from the immunized mice indicated were measured in MT4 cells. Titers are presented as the maximum dilution at which the HHV-6B IE1 protein could not be detected. Data are the mean±SD of each group. *p

    Article Snippet: For the measurement of IgG1, IgG2a and IgG3, the first antibodies were diluted to 1:2560 and the secondary antibodies were added to HRP-conjugated anti-mouse IgG1, IgG2a or IgG3 (Abcam, Cambridge, UK).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Purification, Concentration Assay

    A metalloprotease inhibitor, GI129471, blocks the production of IgE and IgG1 enhanced by RMCP-I. B cells enriched from BALB/c spleen cells were cultured with (open symbols) or without (closed symbols) 1 µg/ml RMCP-I in the presence of LPS plus IL-4 for 7 days. Indicated concentration of GI129471 was added to some culture. Concentrations of IgE (a) and IgG1 (b) in the supernatant were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing GI129471 were compared with values from the culture without the inhibitor by Dunnett's method (*, P

    Journal: Immunology

    Article Title: Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4

    doi: 10.1046/j.1365-2567.2001.01320.x

    Figure Lengend Snippet: A metalloprotease inhibitor, GI129471, blocks the production of IgE and IgG1 enhanced by RMCP-I. B cells enriched from BALB/c spleen cells were cultured with (open symbols) or without (closed symbols) 1 µg/ml RMCP-I in the presence of LPS plus IL-4 for 7 days. Indicated concentration of GI129471 was added to some culture. Concentrations of IgE (a) and IgG1 (b) in the supernatant were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing GI129471 were compared with values from the culture without the inhibitor by Dunnett's method (*, P

    Article Snippet: Mouse myeloma IgG1 (clone MOPC-21; PharMingen) and IgG3 (clone J606; PharMingen) were employed as standards.

    Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Bovine chymotrypsin enhances the production of IgE and IgG1 by murine spleen cells stimulated with LPS plus IL-4. Spleen cells from BALB/c mice were cultured with or without indicated concentration of bovine chymotrypsin (open squares) or DFP-treated chymotrypsin (closed squares) in the presence of LPS plus IL-4 for 7 days. IgE (a) and IgG1 (b) in the supernatant were quantitated by ELISA. Each symbol and bar represent mean and standard error of mean (SEM; n = 6). Values obtained from the culture containing chymotrypsin were compared with values from the culture without chymotrypsin by Dunnett's method (*, P

    Journal: Immunology

    Article Title: Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4

    doi: 10.1046/j.1365-2567.2001.01320.x

    Figure Lengend Snippet: Bovine chymotrypsin enhances the production of IgE and IgG1 by murine spleen cells stimulated with LPS plus IL-4. Spleen cells from BALB/c mice were cultured with or without indicated concentration of bovine chymotrypsin (open squares) or DFP-treated chymotrypsin (closed squares) in the presence of LPS plus IL-4 for 7 days. IgE (a) and IgG1 (b) in the supernatant were quantitated by ELISA. Each symbol and bar represent mean and standard error of mean (SEM; n = 6). Values obtained from the culture containing chymotrypsin were compared with values from the culture without chymotrypsin by Dunnett's method (*, P

    Article Snippet: Mouse myeloma IgG1 (clone MOPC-21; PharMingen) and IgG3 (clone J606; PharMingen) were employed as standards.

    Techniques: Mouse Assay, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    RMCP-I enhances the production of IgE and IgG1 by enriched B cells. B cells enriched from BALB/c spleen cells were cultured with or without indicated concentration of either RMCP-I (open symbols) or PMSF-treated RMCP-I (closed symbols) in the presence of LPS plus IL-4 for 7 days. Concentrations of IgE (a) and IgG1 (b) in the supernatant were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing chymase were compared with values from the culture without chymase by Dunnett's method (*, P

    Journal: Immunology

    Article Title: Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4

    doi: 10.1046/j.1365-2567.2001.01320.x

    Figure Lengend Snippet: RMCP-I enhances the production of IgE and IgG1 by enriched B cells. B cells enriched from BALB/c spleen cells were cultured with or without indicated concentration of either RMCP-I (open symbols) or PMSF-treated RMCP-I (closed symbols) in the presence of LPS plus IL-4 for 7 days. Concentrations of IgE (a) and IgG1 (b) in the supernatant were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing chymase were compared with values from the culture without chymase by Dunnett's method (*, P

    Article Snippet: Mouse myeloma IgG1 (clone MOPC-21; PharMingen) and IgG3 (clone J606; PharMingen) were employed as standards.

    Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Enzymatic activity of RMCP-I is required for enhancing IgE and IgG1. BALB/c spleen cells were cultured with (grey column) or without (open column) 1 µg/ml RMCP-I in the presence of LPS plus IL-4 for 7 days. Indicated concentration of either chymostatin (closed symbols) or Y-40613 (open symbols) was added to some culture with RMCP-I. Concentrations of IgE (a) and IgG1 (b) were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). The data obtained in the presence of the inhibitors were compared to the data obtained in the absence of the inhibitors by Williams' Multiple Comparison Test (*, P

    Journal: Immunology

    Article Title: Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4

    doi: 10.1046/j.1365-2567.2001.01320.x

    Figure Lengend Snippet: Enzymatic activity of RMCP-I is required for enhancing IgE and IgG1. BALB/c spleen cells were cultured with (grey column) or without (open column) 1 µg/ml RMCP-I in the presence of LPS plus IL-4 for 7 days. Indicated concentration of either chymostatin (closed symbols) or Y-40613 (open symbols) was added to some culture with RMCP-I. Concentrations of IgE (a) and IgG1 (b) were determined by ELISA. Each symbol and bar represents mean and SEM ( n = 6). The data obtained in the presence of the inhibitors were compared to the data obtained in the absence of the inhibitors by Williams' Multiple Comparison Test (*, P

    Article Snippet: Mouse myeloma IgG1 (clone MOPC-21; PharMingen) and IgG3 (clone J606; PharMingen) were employed as standards.

    Techniques: Activity Assay, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    RMCP-I enhances the production of IgE and IgG1, but not IgG3. Spleen cells from BALB/c mice were cultured with or without indicated concentration of RMCP-I in the presence of either LPS plus IL-4 (a and b) or LPS alone (c) for 7 days. IgE (a), IgG1 (b) and IgG3 (c) in the supernatant were quantitated by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing chymase were compared with values from the culture without chymase by Dunnett's method (*, P

    Journal: Immunology

    Article Title: Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4

    doi: 10.1046/j.1365-2567.2001.01320.x

    Figure Lengend Snippet: RMCP-I enhances the production of IgE and IgG1, but not IgG3. Spleen cells from BALB/c mice were cultured with or without indicated concentration of RMCP-I in the presence of either LPS plus IL-4 (a and b) or LPS alone (c) for 7 days. IgE (a), IgG1 (b) and IgG3 (c) in the supernatant were quantitated by ELISA. Each symbol and bar represents mean and SEM ( n = 6). Values obtained from the culture containing chymase were compared with values from the culture without chymase by Dunnett's method (*, P

    Article Snippet: Mouse myeloma IgG1 (clone MOPC-21; PharMingen) and IgG3 (clone J606; PharMingen) were employed as standards.

    Techniques: Mouse Assay, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Figure 1. Schematic depiction of multiplexed assessment of IgG interactions with FcR and lectins. Up to 500 bead sets, each coupled with an FcR or lectin of interest, can be combined into one well containing an antibody sample of interest. Bound

    Journal: mAbs

    Article Title: Highly parallel characterization of IgG Fc binding interactions

    doi: 10.4161/mabs.28808

    Figure Lengend Snippet: Figure 1. Schematic depiction of multiplexed assessment of IgG interactions with FcR and lectins. Up to 500 bead sets, each coupled with an FcR or lectin of interest, can be combined into one well containing an antibody sample of interest. Bound

    Article Snippet: IgG1, IgG2, IgG4 (Athens Research) and IgG3 (Sigma Aldrich) from myeloma plasma and serum IgG1, IgG2 and IgG3 from healthy donors (Athens Research) were also purified using a Sephacryl S-200 column to remove aggregates.

    Techniques:

    Figure 4. Probing glycan dependence of polyclonal antibody samples. A,B : FcγR ( A ) or lectin ( B ) conjugated beads were incubated with 267 nM of native IgG pooled from HIV positive donors (HIVIG) or HIVIG treated with PNGaseF to remove N-linked

    Journal: mAbs

    Article Title: Highly parallel characterization of IgG Fc binding interactions

    doi: 10.4161/mabs.28808

    Figure Lengend Snippet: Figure 4. Probing glycan dependence of polyclonal antibody samples. A,B : FcγR ( A ) or lectin ( B ) conjugated beads were incubated with 267 nM of native IgG pooled from HIV positive donors (HIVIG) or HIVIG treated with PNGaseF to remove N-linked

    Article Snippet: IgG1, IgG2, IgG4 (Athens Research) and IgG3 (Sigma Aldrich) from myeloma plasma and serum IgG1, IgG2 and IgG3 from healthy donors (Athens Research) were also purified using a Sephacryl S-200 column to remove aggregates.

    Techniques: Incubation

    Figure 2. Multiplexed assessment of IgG subclass specificity of FcγR and complement. Extracellular domain allotypes of human FcγR and C1q were simultaneously screened for the ability to bind to fractionated serum IgG1, IgG2, IgG3,

    Journal: mAbs

    Article Title: Highly parallel characterization of IgG Fc binding interactions

    doi: 10.4161/mabs.28808

    Figure Lengend Snippet: Figure 2. Multiplexed assessment of IgG subclass specificity of FcγR and complement. Extracellular domain allotypes of human FcγR and C1q were simultaneously screened for the ability to bind to fractionated serum IgG1, IgG2, IgG3,

    Article Snippet: IgG1, IgG2, IgG4 (Athens Research) and IgG3 (Sigma Aldrich) from myeloma plasma and serum IgG1, IgG2 and IgG3 from healthy donors (Athens Research) were also purified using a Sephacryl S-200 column to remove aggregates.

    Techniques:

    Figure 8. Avidity differentially modulates FcγR:antibody ligation among IgG subclasses and FcγR. Myeloma derived IgG1, IgG2, and IgG4 and pooled serum derived IgG3 were separated into monomeric and multimeric fractions by SEC.

    Journal: mAbs

    Article Title: Highly parallel characterization of IgG Fc binding interactions

    doi: 10.4161/mabs.28808

    Figure Lengend Snippet: Figure 8. Avidity differentially modulates FcγR:antibody ligation among IgG subclasses and FcγR. Myeloma derived IgG1, IgG2, and IgG4 and pooled serum derived IgG3 were separated into monomeric and multimeric fractions by SEC.

    Article Snippet: IgG1, IgG2, IgG4 (Athens Research) and IgG3 (Sigma Aldrich) from myeloma plasma and serum IgG1, IgG2 and IgG3 from healthy donors (Athens Research) were also purified using a Sephacryl S-200 column to remove aggregates.

    Techniques: Ligation, Derivative Assay, Size-exclusion Chromatography

    Total IgG and IgG subclass levels in sera of individual patient of sparganosis.

    Journal: The Korean Journal of Parasitology

    Article Title: IgG antibody responses in early experimental sparganosis and IgG subclass responses in human sparganosis

    doi: 10.3347/kjp.2000.38.3.145

    Figure Lengend Snippet: Total IgG and IgG subclass levels in sera of individual patient of sparganosis.

    Article Snippet: Mouse anti-human IgG subclasses were reacted for 2 hr in the following dilutions; 1:500 for anti-human IgG1 (JL512) and 1:1000 for anti-human IgG2 (GoM1), IgG3 (ZG4), and IgG4 (RJ4) (Immunotech, Marseille, France).

    Techniques:

    Representative examples of specific IgG subclass immunoblot in human sparganosis. Main reactions are shown at 36-26 kDa and 100-70 kDa bands against both whole IgG and IgG4 antibodies. Reactions in three different patients were shown. G, 1, 2, 3, and 4 at the top of each lane indicate total IgG, IgG1, IgG2, IgG3 and IgG4, respectively.

    Journal: The Korean Journal of Parasitology

    Article Title: IgG antibody responses in early experimental sparganosis and IgG subclass responses in human sparganosis

    doi: 10.3347/kjp.2000.38.3.145

    Figure Lengend Snippet: Representative examples of specific IgG subclass immunoblot in human sparganosis. Main reactions are shown at 36-26 kDa and 100-70 kDa bands against both whole IgG and IgG4 antibodies. Reactions in three different patients were shown. G, 1, 2, 3, and 4 at the top of each lane indicate total IgG, IgG1, IgG2, IgG3 and IgG4, respectively.

    Article Snippet: Mouse anti-human IgG subclasses were reacted for 2 hr in the following dilutions; 1:500 for anti-human IgG1 (JL512) and 1:1000 for anti-human IgG2 (GoM1), IgG3 (ZG4), and IgG4 (RJ4) (Immunotech, Marseille, France).

    Techniques:

    Histopathology and immunofluorescence analysis of kidneys. ( A ) Hematoxylin and eosin (H E), periodic acid-Schiff (PAS) reagent, and Masson’s trichrome staining of kidneys obtained from mice at the end of the experiment (original magnification: ×400). Inflammatory cell infiltration and mesangial proliferation were scored on a graded scale from 0 (no) to 4 (severe). ( B ) IgG and C3 deposition in kidneys (original magnification: ×200). Fluorescence staining intensities of IgG and C3 deposits were graded as 0 (none), 1+ (mild), 2+ (moderate), 3+ (moderate to strong), or 4+ (strong). Intergroup analysis was performed using ANOVA followed by Tukey’s multiple comparison post-hoc tests. * p

    Journal: Scientific Reports

    Article Title: CKD-506, a novel HDAC6-selective inhibitor, improves renal outcomes and survival in a mouse model of systemic lupus erythematosus

    doi: 10.1038/s41598-018-35602-1

    Figure Lengend Snippet: Histopathology and immunofluorescence analysis of kidneys. ( A ) Hematoxylin and eosin (H E), periodic acid-Schiff (PAS) reagent, and Masson’s trichrome staining of kidneys obtained from mice at the end of the experiment (original magnification: ×400). Inflammatory cell infiltration and mesangial proliferation were scored on a graded scale from 0 (no) to 4 (severe). ( B ) IgG and C3 deposition in kidneys (original magnification: ×200). Fluorescence staining intensities of IgG and C3 deposits were graded as 0 (none), 1+ (mild), 2+ (moderate), 3+ (moderate to strong), or 4+ (strong). Intergroup analysis was performed using ANOVA followed by Tukey’s multiple comparison post-hoc tests. * p

    Article Snippet: Serum immunoglobulin concentrations were determined with mouse IgG1, IgG2A, IgG2B, IgG3, and IgM Isotyping kits and a mouse Mag Isotyping assay base kit (R & D Systems, Minneapolis, MN).

    Techniques: Histopathology, Immunofluorescence, Staining, Mouse Assay, Fluorescence

    Depletion and ablation of IL-4 shifts post-vaccination IgG subclass distribution. ( A – E ) Serum anti-oxycodone IgG antibodies were analyzed for IgG 1 , IgG 2a , and IgG 3 subclass in mice immunized with OXY-KLH ± αIL-4 mAb from experiments shown in Figs 1A–H and 2A–C . ( A ) oxycodone-specific IgG titers in mice immunized with OXY-KLH s.c., n = 49; OXY-KLH s.c. + IL-4 mAb i.p., n = 38; OXY-KLH i.m., n = 8; OXY-KLH i.m. + IL-4 mAb i.m., n = 8). IgG subclass analysis was performed on a randomly selected sample from each immunization group (n = 5–6 mice/group): ( B ) IgG 1 , ( C ) IgG 2a , ( D ) IgG 3 , ( E ) IgG 1 /((IgG 2a + IgG 3 )/2). ( F – H ) In a separate study, IL-4 −/− and wild-type mice controls were immunized with KLH or OXY-KLH in alum (s.c., n = 5 each group). ( F ) oxycodone-specific IgG titers by subclass, ( G ) IgG 1 /((IgG 2a + IgG 3 )/2), and ( H ) effect of immunization on oxycodone distribution to serum and brain. ( I ) In a follow-up independent experiment, mice were immunized with either KLH (s.c., n = 4) or OXY-KLH (s.c., n = 10). The OXY-KLH group received either saline or liposome-encapsulated clodronate to deplete macrophages (n = 5), and 24 hrs later challenged with 5.0 mg/kg oxycodone. Data are from one experiment. ( I ) vaccine efficacy in blocking oxycodone distribution to the brain (left) and the subset (%) of immunized mice showing efficacy (right). Data are mean ± SEM. ( A – G ) unpaired two-tailed t-test. ( H , I ) One-way ANOVA paired with Tukey’s test, and chi-square test (χ 2 = 8.00, df = 1, p = 0.0047). *p

    Journal: Scientific Reports

    Article Title: Blocking interleukin-4 enhances efficacy of vaccines for treatment of opioid abuse and prevention of opioid overdose

    doi: 10.1038/s41598-018-23777-6

    Figure Lengend Snippet: Depletion and ablation of IL-4 shifts post-vaccination IgG subclass distribution. ( A – E ) Serum anti-oxycodone IgG antibodies were analyzed for IgG 1 , IgG 2a , and IgG 3 subclass in mice immunized with OXY-KLH ± αIL-4 mAb from experiments shown in Figs 1A–H and 2A–C . ( A ) oxycodone-specific IgG titers in mice immunized with OXY-KLH s.c., n = 49; OXY-KLH s.c. + IL-4 mAb i.p., n = 38; OXY-KLH i.m., n = 8; OXY-KLH i.m. + IL-4 mAb i.m., n = 8). IgG subclass analysis was performed on a randomly selected sample from each immunization group (n = 5–6 mice/group): ( B ) IgG 1 , ( C ) IgG 2a , ( D ) IgG 3 , ( E ) IgG 1 /((IgG 2a + IgG 3 )/2). ( F – H ) In a separate study, IL-4 −/− and wild-type mice controls were immunized with KLH or OXY-KLH in alum (s.c., n = 5 each group). ( F ) oxycodone-specific IgG titers by subclass, ( G ) IgG 1 /((IgG 2a + IgG 3 )/2), and ( H ) effect of immunization on oxycodone distribution to serum and brain. ( I ) In a follow-up independent experiment, mice were immunized with either KLH (s.c., n = 4) or OXY-KLH (s.c., n = 10). The OXY-KLH group received either saline or liposome-encapsulated clodronate to deplete macrophages (n = 5), and 24 hrs later challenged with 5.0 mg/kg oxycodone. Data are from one experiment. ( I ) vaccine efficacy in blocking oxycodone distribution to the brain (left) and the subset (%) of immunized mice showing efficacy (right). Data are mean ± SEM. ( A – G ) unpaired two-tailed t-test. ( H , I ) One-way ANOVA paired with Tukey’s test, and chi-square test (χ 2 = 8.00, df = 1, p = 0.0047). *p

    Article Snippet: Primary antibodies were incubated with goat-anti-mouse IgG, IgG1 , IgG2a , or IgG3 conjugated to horseradish peroxidase to measure titers of oxycodone- or TT-specific IgG and individual IgG subclasses (Alpha Diagnostic International, Inc., San Antonio, TX).

    Techniques: Mouse Assay, Blocking Assay, Two Tailed Test

    Modulation of IL-4 signaling enhances anti-oxycodone antibody, vaccine efficacy against oxycodone, and the subset of immunized subjects that showed vaccine efficacy. Three independent cohorts of male BALB/c mice were immunized s.c. with OXY-KLH and KLH adsorbed on alum adjuvant and challenged a week after the 3 rd immunization with increasing doses of oxycodone (2.25–10 mg/kg s.c.). Interleukins were administered at days 0, 14 and 28 in combination with each immunization. The mAb was administered 2 days prior and 1 day after the 1 st immunization. Experimental groups: ( A – C ) KLH (n = 24), OXY-KLH (n = 25), and OXY-KLH plus: rIL-4 (30,000 IU, s.c.), rIL-4 (60,000 IU, s.c.), IL-4 plus IL-21 (60,000 IU total, s.c.), αIL-2 alpha receptor mAb (αCD25, 1.0 mg, i.p., n = 10), αCD25 mAb (1.0 mg, i.p.) plus IL-4 (30,000 IU, s.c., n = 10), αIL-4 mAb (1.0 mg, i.p., n = 10), or IL-4:αIL-4 mAb (30,000 IU and 0.5 mg of mAb mixed prior to injection, s.c., n = 5), and challenged with 2.25 mg/kg oxycodone. ( D–F ) KLH, OXY-KLH, and OXY-KLH plus αCD25 mAb (1.0 mg, i.p.), or αIL-4 mAb (1.0 mg, i.p.) and challenged with 5 mg/kg oxycodone (n = 10/group). ( G – I ) KLH, OXY-KLH, and OXY-KLH plus αIL-4 mAb (1.0 mg, i.p.) and challenged with 10 mg/kg oxycodone (n = 10/group). Shown oxycodone-specific IgG titers, oxycodone distribution to the brain, and fraction (%) of immunized subjects that showed more than 50% reduction in oxycodone distribution to the brain compared to the mean brain concentration in the KLH control group. Data are mean ± SEM. Data shown are from at least two independent experiments. Titers and oxycodone concentrations analyzed by one-way ANOVA and Tukey’s multiple comparisons test or unpaired two-tailed t-test. Chi-square test: ( C ) (χ 2 = 282.8, df = 8), F) (χ 2 = 120.0, df = 2), and I) (χ 2 = 12.5, df = 1). Percentages (%) above bars indicate reduction compared to KLH. *p

    Journal: Scientific Reports

    Article Title: Blocking interleukin-4 enhances efficacy of vaccines for treatment of opioid abuse and prevention of opioid overdose

    doi: 10.1038/s41598-018-23777-6

    Figure Lengend Snippet: Modulation of IL-4 signaling enhances anti-oxycodone antibody, vaccine efficacy against oxycodone, and the subset of immunized subjects that showed vaccine efficacy. Three independent cohorts of male BALB/c mice were immunized s.c. with OXY-KLH and KLH adsorbed on alum adjuvant and challenged a week after the 3 rd immunization with increasing doses of oxycodone (2.25–10 mg/kg s.c.). Interleukins were administered at days 0, 14 and 28 in combination with each immunization. The mAb was administered 2 days prior and 1 day after the 1 st immunization. Experimental groups: ( A – C ) KLH (n = 24), OXY-KLH (n = 25), and OXY-KLH plus: rIL-4 (30,000 IU, s.c.), rIL-4 (60,000 IU, s.c.), IL-4 plus IL-21 (60,000 IU total, s.c.), αIL-2 alpha receptor mAb (αCD25, 1.0 mg, i.p., n = 10), αCD25 mAb (1.0 mg, i.p.) plus IL-4 (30,000 IU, s.c., n = 10), αIL-4 mAb (1.0 mg, i.p., n = 10), or IL-4:αIL-4 mAb (30,000 IU and 0.5 mg of mAb mixed prior to injection, s.c., n = 5), and challenged with 2.25 mg/kg oxycodone. ( D–F ) KLH, OXY-KLH, and OXY-KLH plus αCD25 mAb (1.0 mg, i.p.), or αIL-4 mAb (1.0 mg, i.p.) and challenged with 5 mg/kg oxycodone (n = 10/group). ( G – I ) KLH, OXY-KLH, and OXY-KLH plus αIL-4 mAb (1.0 mg, i.p.) and challenged with 10 mg/kg oxycodone (n = 10/group). Shown oxycodone-specific IgG titers, oxycodone distribution to the brain, and fraction (%) of immunized subjects that showed more than 50% reduction in oxycodone distribution to the brain compared to the mean brain concentration in the KLH control group. Data are mean ± SEM. Data shown are from at least two independent experiments. Titers and oxycodone concentrations analyzed by one-way ANOVA and Tukey’s multiple comparisons test or unpaired two-tailed t-test. Chi-square test: ( C ) (χ 2 = 282.8, df = 8), F) (χ 2 = 120.0, df = 2), and I) (χ 2 = 12.5, df = 1). Percentages (%) above bars indicate reduction compared to KLH. *p

    Article Snippet: Primary antibodies were incubated with goat-anti-mouse IgG, IgG1 , IgG2a , or IgG3 conjugated to horseradish peroxidase to measure titers of oxycodone- or TT-specific IgG and individual IgG subclasses (Alpha Diagnostic International, Inc., San Antonio, TX).

    Techniques: Mouse Assay, Injection, Concentration Assay, Two Tailed Test

    IL-4 depletion does not impair post-immunization hapten-specific B cell early expansion, increases peptide-specific T cell differentiation, and enhances efficacy of a tetanus-diphtheria-pertussis vaccine. ( A , B ) BALB/c mice immunized once with KLH (s.c., n = 4), OXY-KLH (s.c., n = 20), and OXY-KLH (s.c.) plus αIL-4 mAb (i.p, n = 20). At 7 days after immunization, analysis of OXY-specific B cells in pooled lymph nodes and spleen by flow cytometry: ( A ) Antibody secreting cells, and ( B ) GC B cells. Data shown are from three experiments. ( C – F ) C57Bl/6 naïve mice (n = 5) and mice immunized once with PE-SA 2W (n = 8) and PE-SA 2W plus αIL-4 mAb (n = 8). At 7 days after immunization, analysis of 2W-specific CD4 + T cells in pooled lymph nodes and spleen by flow cytometry: ( C ) non-Tfh cells, ( D ) Tfh cells, ( E ) GC-Tfh cells, ( F ) (Tfh + GC- Tfh)/non-Tfh. Data shown are from two experiments. ( G – I ) Mice immunized with TDaP and TDaP plus αIL-4 mAb (n = 10 each group). ( G ) TT-specific IgG titer over time, ( H ) TT-specific IgG 1 and IgG 2a titers on day 28 after the first immunization, and ( I ) subset (%) of subjects showing anti-diphtheria protective antibodies. Data shown are from one experiment. Data are mean ± SEM. ( A – E ) One-way ANOVA paired with Tukey’s multiple comparisons test. ( F and H ) unpaired two tailed t-test. ( G ) Two-way ANOVA paired with Tukey’s multiple comparisons test. ( I ) Chi-square (χ 2 = 52.43, df = 1). *p

    Journal: Scientific Reports

    Article Title: Blocking interleukin-4 enhances efficacy of vaccines for treatment of opioid abuse and prevention of opioid overdose

    doi: 10.1038/s41598-018-23777-6

    Figure Lengend Snippet: IL-4 depletion does not impair post-immunization hapten-specific B cell early expansion, increases peptide-specific T cell differentiation, and enhances efficacy of a tetanus-diphtheria-pertussis vaccine. ( A , B ) BALB/c mice immunized once with KLH (s.c., n = 4), OXY-KLH (s.c., n = 20), and OXY-KLH (s.c.) plus αIL-4 mAb (i.p, n = 20). At 7 days after immunization, analysis of OXY-specific B cells in pooled lymph nodes and spleen by flow cytometry: ( A ) Antibody secreting cells, and ( B ) GC B cells. Data shown are from three experiments. ( C – F ) C57Bl/6 naïve mice (n = 5) and mice immunized once with PE-SA 2W (n = 8) and PE-SA 2W plus αIL-4 mAb (n = 8). At 7 days after immunization, analysis of 2W-specific CD4 + T cells in pooled lymph nodes and spleen by flow cytometry: ( C ) non-Tfh cells, ( D ) Tfh cells, ( E ) GC-Tfh cells, ( F ) (Tfh + GC- Tfh)/non-Tfh. Data shown are from two experiments. ( G – I ) Mice immunized with TDaP and TDaP plus αIL-4 mAb (n = 10 each group). ( G ) TT-specific IgG titer over time, ( H ) TT-specific IgG 1 and IgG 2a titers on day 28 after the first immunization, and ( I ) subset (%) of subjects showing anti-diphtheria protective antibodies. Data shown are from one experiment. Data are mean ± SEM. ( A – E ) One-way ANOVA paired with Tukey’s multiple comparisons test. ( F and H ) unpaired two tailed t-test. ( G ) Two-way ANOVA paired with Tukey’s multiple comparisons test. ( I ) Chi-square (χ 2 = 52.43, df = 1). *p

    Article Snippet: Primary antibodies were incubated with goat-anti-mouse IgG, IgG1 , IgG2a , or IgG3 conjugated to horseradish peroxidase to measure titers of oxycodone- or TT-specific IgG and individual IgG subclasses (Alpha Diagnostic International, Inc., San Antonio, TX).

    Techniques: Cell Differentiation, Mouse Assay, Flow Cytometry, Cytometry, Two Tailed Test

    The lead formulation of OXY-KLH plus αIL-4 mAb is effective across clinically-relevant immunization regimens and attenuated oxycodone-induced physiological and behavioral effects. BALB/c mice were immunized with OXY-KLH or KLH on days 0, 14 and 28, and tested for vaccine efficacy a week after the third immunization. Mice received KLH or OXY-KLH s.c. or i.m., whereas αIL-4 mAb was administered i.p., i.v., or i.m. In a first cohort, mice were challenged with 5 mg/kg oxycodone: ( A ) oxycodone-specific IgG titers, ( B ) oxycodone brain concentrations, and ( C ) subjects that showed more than 50% reduction in oxycodone distribution to the brain compared to the KLH group. Data are from two independent experiments and expressed as mean ± SEM. Percentages (%) above bars indicate reduction compared to KLH. One-way ANOVA paired with Tukey’s multiple comparisons test or unpairedtwo-tailed t-test. Chi-square test (χ 2 = 62.66, df = 6). *p

    Journal: Scientific Reports

    Article Title: Blocking interleukin-4 enhances efficacy of vaccines for treatment of opioid abuse and prevention of opioid overdose

    doi: 10.1038/s41598-018-23777-6

    Figure Lengend Snippet: The lead formulation of OXY-KLH plus αIL-4 mAb is effective across clinically-relevant immunization regimens and attenuated oxycodone-induced physiological and behavioral effects. BALB/c mice were immunized with OXY-KLH or KLH on days 0, 14 and 28, and tested for vaccine efficacy a week after the third immunization. Mice received KLH or OXY-KLH s.c. or i.m., whereas αIL-4 mAb was administered i.p., i.v., or i.m. In a first cohort, mice were challenged with 5 mg/kg oxycodone: ( A ) oxycodone-specific IgG titers, ( B ) oxycodone brain concentrations, and ( C ) subjects that showed more than 50% reduction in oxycodone distribution to the brain compared to the KLH group. Data are from two independent experiments and expressed as mean ± SEM. Percentages (%) above bars indicate reduction compared to KLH. One-way ANOVA paired with Tukey’s multiple comparisons test or unpairedtwo-tailed t-test. Chi-square test (χ 2 = 62.66, df = 6). *p

    Article Snippet: Primary antibodies were incubated with goat-anti-mouse IgG, IgG1 , IgG2a , or IgG3 conjugated to horseradish peroxidase to measure titers of oxycodone- or TT-specific IgG and individual IgG subclasses (Alpha Diagnostic International, Inc., San Antonio, TX).

    Techniques: Mouse Assay

    Expression of fibroblast activation marker Podoplanin (PDPN) and nociceptive neuromodulator NMDAR-1 in diseased supraspinatus tendon tissues. a – b Representative immunofluorescence images showing staining of cell nuclei (POPO-1, cyan) and PDPN (green) with a expression of NMDAR-1 (red) and PGIS (violet) and b expression of TLR4 (red) and IL-1R (violet). c Representative confocal immunofluorescence images showing merged image of diseased tendon sections stained with isotype control antibodies for mouse IgG1, IgG2a, and IgG2b and rabbit IgG fractions. Cyan represents POPO-1 nuclear counterstain. Scale bar, 20 μm

    Journal: Arthritis Research & Therapy

    Article Title: Divergent roles of prostacyclin and PGE2 in human tendinopathy

    doi: 10.1186/s13075-019-1855-5

    Figure Lengend Snippet: Expression of fibroblast activation marker Podoplanin (PDPN) and nociceptive neuromodulator NMDAR-1 in diseased supraspinatus tendon tissues. a – b Representative immunofluorescence images showing staining of cell nuclei (POPO-1, cyan) and PDPN (green) with a expression of NMDAR-1 (red) and PGIS (violet) and b expression of TLR4 (red) and IL-1R (violet). c Representative confocal immunofluorescence images showing merged image of diseased tendon sections stained with isotype control antibodies for mouse IgG1, IgG2a, and IgG2b and rabbit IgG fractions. Cyan represents POPO-1 nuclear counterstain. Scale bar, 20 μm

    Article Snippet: Isotype control antibodies were a cocktail of mouse immunoglobulin G (IgG1 ), IgG2a , IgG2b , IgG3 , and IgM (Dako) and rabbit immunoglobulin fraction of serum from non-immunized rabbits, solid-phase absorbed (Dako).

    Techniques: Expressing, Activation Assay, Marker, Immunofluorescence, Staining

    Duration of serum FMDV-specific IgG responses. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Duration of serum FMDV-specific IgG responses. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay, Injection

    Numbers of FMDV-specific IgG-secreting cells in bone marrow. Mice ( n = 8/group) were s.c. immunized with FMDV antigen plus physiological saline, RO, or RO containing GS-R (4 μg) on days 1 and 21. Bone marrow cells were prepared 8 weeks after boosting,

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Numbers of FMDV-specific IgG-secreting cells in bone marrow. Mice ( n = 8/group) were s.c. immunized with FMDV antigen plus physiological saline, RO, or RO containing GS-R (4 μg) on days 1 and 21. Bone marrow cells were prepared 8 weeks after boosting,

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay

    Serum FMDV-specific IgG titers. Mice ( n = 8/group) were s.c. immunized with FMDV antigen plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Sera were collected 2 weeks after boosting, and serum FMDV-specific

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Serum FMDV-specific IgG titers. Mice ( n = 8/group) were s.c. immunized with FMDV antigen plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Sera were collected 2 weeks after boosting, and serum FMDV-specific

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay

    Serum FMDV-specific IgG levels. Mice ( n = 8/group) were s.c. injected with FMDV antigen plus physiological saline, RO, or RO containing GSLS (2, 4, or 6 μg) or GS-R (2, 4, or 6 μg) on days 1 and 21. Sera were collected 3 and 7 days after

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Serum FMDV-specific IgG levels. Mice ( n = 8/group) were s.c. injected with FMDV antigen plus physiological saline, RO, or RO containing GSLS (2, 4, or 6 μg) or GS-R (2, 4, or 6 μg) on days 1 and 21. Sera were collected 3 and 7 days after

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay, Injection

    Serum FMDV-specific IgG isotype levels. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological saline

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Serum FMDV-specific IgG isotype levels. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological saline

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay, Injection