Structured Review

Becton Dickinson igg enzyme linked immunosorbent assays
Impaired induction of T follicular helper (TFH) cells in 6F/6F mice. 6Y/6Y and 6F/6F mice were injected with sheep red blood cells (SRBCS) and 10 days later spleens were harvested for analysis. Percentage and numbers of ( a ) TFH (CD4 + CXCR5 + PD-1 + ) cells and ( b ) germinal centre B (GC B) cells (B220 + Fas + GL-7 + ) detected in 6Y/6Y and 6F/6F mice. N =9 6Y/6Y and 9 6F/6F mice. ( c ) Immunofluorescence staining of spleens from 6Y/6Y and 6F/6F mice 1 week after immunization with SRBCS. One representative of three experiments. Scale bar, 200 μm. ( d ) Mean fluorescence intensity of anti-Bcl6 mAb staining of conventional CD4-SP T-cells (CD4 + CXCR5 − PD-1 − ) and TFH cells (CD4 + CXCR5 + PD-1 + ). ( e – g ) 6Y/6Y and 6F/6F mice (six each) were immunized with NP-KLH or NP-CGG. One week after immunization, serum was obtained by tail bleeding and analysed for NP-reactive <t>IgG</t> by enzyme-linked <t>immunosorbent</t> assay (ELISA). ( e ) Anti-NP IgG1. ( f ) Anti-NP IgG2a. ( g ) Ratio of high affinity/low affinity anti-NP IgG1 determined by ELISA with plates coated with NP(4) or NP(32). For bar graphs, data were analysed by unpaired t -test (two tailed) and are represented as mean±s.d. * P
Igg Enzyme Linked Immunosorbent Assays, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "TCR ITAM multiplicity is required for the generation of follicular helper T-cells"

Article Title: TCR ITAM multiplicity is required for the generation of follicular helper T-cells

Journal: Nature Communications

doi: 10.1038/ncomms7982

Impaired induction of T follicular helper (TFH) cells in 6F/6F mice. 6Y/6Y and 6F/6F mice were injected with sheep red blood cells (SRBCS) and 10 days later spleens were harvested for analysis. Percentage and numbers of ( a ) TFH (CD4 + CXCR5 + PD-1 + ) cells and ( b ) germinal centre B (GC B) cells (B220 + Fas + GL-7 + ) detected in 6Y/6Y and 6F/6F mice. N =9 6Y/6Y and 9 6F/6F mice. ( c ) Immunofluorescence staining of spleens from 6Y/6Y and 6F/6F mice 1 week after immunization with SRBCS. One representative of three experiments. Scale bar, 200 μm. ( d ) Mean fluorescence intensity of anti-Bcl6 mAb staining of conventional CD4-SP T-cells (CD4 + CXCR5 − PD-1 − ) and TFH cells (CD4 + CXCR5 + PD-1 + ). ( e – g ) 6Y/6Y and 6F/6F mice (six each) were immunized with NP-KLH or NP-CGG. One week after immunization, serum was obtained by tail bleeding and analysed for NP-reactive IgG by enzyme-linked immunosorbent assay (ELISA). ( e ) Anti-NP IgG1. ( f ) Anti-NP IgG2a. ( g ) Ratio of high affinity/low affinity anti-NP IgG1 determined by ELISA with plates coated with NP(4) or NP(32). For bar graphs, data were analysed by unpaired t -test (two tailed) and are represented as mean±s.d. * P
Figure Legend Snippet: Impaired induction of T follicular helper (TFH) cells in 6F/6F mice. 6Y/6Y and 6F/6F mice were injected with sheep red blood cells (SRBCS) and 10 days later spleens were harvested for analysis. Percentage and numbers of ( a ) TFH (CD4 + CXCR5 + PD-1 + ) cells and ( b ) germinal centre B (GC B) cells (B220 + Fas + GL-7 + ) detected in 6Y/6Y and 6F/6F mice. N =9 6Y/6Y and 9 6F/6F mice. ( c ) Immunofluorescence staining of spleens from 6Y/6Y and 6F/6F mice 1 week after immunization with SRBCS. One representative of three experiments. Scale bar, 200 μm. ( d ) Mean fluorescence intensity of anti-Bcl6 mAb staining of conventional CD4-SP T-cells (CD4 + CXCR5 − PD-1 − ) and TFH cells (CD4 + CXCR5 + PD-1 + ). ( e – g ) 6Y/6Y and 6F/6F mice (six each) were immunized with NP-KLH or NP-CGG. One week after immunization, serum was obtained by tail bleeding and analysed for NP-reactive IgG by enzyme-linked immunosorbent assay (ELISA). ( e ) Anti-NP IgG1. ( f ) Anti-NP IgG2a. ( g ) Ratio of high affinity/low affinity anti-NP IgG1 determined by ELISA with plates coated with NP(4) or NP(32). For bar graphs, data were analysed by unpaired t -test (two tailed) and are represented as mean±s.d. * P

Techniques Used: Mouse Assay, Injection, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Two Tailed Test

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Article Title: Therapeutic Administration of Broadly Neutralizing FI6 Antibody Reveals Lack of Interaction Between Human IgG1 and Pig Fc Receptors
Article Snippet: .. The IgG ELISA was performed in 96-well ELISA plates (BD Biosciences) coated with 1 × 106 PFU/ml of A/swine/England/1353/09 over night at 4°C. ..

Article Title: Attenuated Toxoplasma gondii therapy of disseminated pancreatic cancer generates long-lasting immunity to pancreatic cancer
Article Snippet: .. For IgG ELISA, purified mouse IgG was diluted in coating buffer (BD Bioscience) at 500 ng/mL. .. Following overnight incubation, cells were blocked with animal serum in a buffered solution with ProClin®-150 (assay diluent) (BD Bioscience), followed by three washes with 1X wash buffer (concentrated detergent solution with ProClin®-150 as a preservative).

Purification:

Article Title: Attenuated Toxoplasma gondii therapy of disseminated pancreatic cancer generates long-lasting immunity to pancreatic cancer
Article Snippet: .. For IgG ELISA, purified mouse IgG was diluted in coating buffer (BD Bioscience) at 500 ng/mL. .. Following overnight incubation, cells were blocked with animal serum in a buffered solution with ProClin®-150 (assay diluent) (BD Bioscience), followed by three washes with 1X wash buffer (concentrated detergent solution with ProClin®-150 as a preservative).

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    Becton Dickinson ev71 specific igg1
    Comparison of the effects of the intranasal (IN) and intramuscular (IM) immunisation routes on <t>EV71-specific</t> antibody titres and cytokine production. Mice were IN or IM immunised with PBS or formalin-inactivated EV71 (5 μg/dose) alone or adjuvanted with CpG (20 μg/dose) three times at 3-week intervals. ELISA was used to measure EV71-specific IgA in the nasal wash ( a ), BALF ( b ), faeces ( c ), and serum ( d ) and EV71-specific <t>IgG</t> in the serum ( e ) of mice at 2 weeks after the third immunisation. Splenocytes from immunised mice were harvested and cultured for 2 days in medium containing 10 μg/mL heat-inactivated EV71, after which culture supernatants were analysed for IFN-γ ( f ), IL-4 ( g ), and IL-17 ( h ) using ELISA. One representative study of two with similar results is shown. Values shown are means ± SEMs of five mice in each experimental group; * p
    Ev71 Specific Igg1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson igg isotypes
    Durability of anti-gp120 <t>IgG</t> and serum and mucosal IgA responses following immunization of mice via parenteral, mucosal and combination routes. Anti-gp120 IgG (a, left panel) and IgA (a, right panel) responses, assayed by ELISA, were determined for serum samples of DNA prime–protein boosted mice at weeks 13, 15, 18, 22 and 34 of the study. IgA was also measured at weeks 13 and 34 of the study for saliva (b, 1 : 5 dilution) and vaginal-wash samples (c, 1 : 2 dilution). ELISA titres and A 450 are reported.
    Igg Isotypes, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson igg3
    Defective humoral responses in Itsn2 -/- animals ( A ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and spleens of immunised animals analysed by flow cytometry 13d after immunisation. Tfh cells (CD4 + CXCR5 + PD-1 + ). Panels on the right show the show percentage of cells in the indicated gates (each dot represents a different animal). ( B ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and 13 days post-immunisation, NP-specific antibody secreting cells were detected in the spleen by ELISPOT. ( F ) WT and Itsn2 -/- littermates were immunised with NP-LPS and titres of NP 23 -specific IgM and <t>IgG3</t> were measured by ELISA. Data are representative of at least 2 independent experiments with more than three animals in each group. Student’s t-test, ns p > 0.05, *p
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    Becton Dickinson rat igg2a isotype pe
    GM-CSF controls IgM production. (A) In vitro culture of CD19 + serosal B cells. Gating strategy and phenotyping of IRA B cells 2 d after culture in medium or with 10 µg/ml LPS. A representative contour plot of n > 5 is shown. (B) CD131 (Csf2rb) expression on selected cells from WT mice ( n = 4). (C) In vitro culture of serosal B1a cells sorted from WT, Csf2 −/− , and Csfrb −/− mice. Data show intracellular IgM and GM-CSF in cells cultured in medium alone or after LPS (10 µg/ml) stimulation after 1 d of culture ( n = 3–5). The gate for GM-CSF was set using an isotype control <t>(IgG2a)</t> and the gate for intracellular IgM represents the upper 99% limit of intracellular IgM staining at baseline. (D) In vitro culture of serosal B1a cells sorted from WT, Csf2 −/− , and Csfrb −/− mice, and B2 cells sorted from WT mice. Percentage of IgM(ic) high cells cultured for 1 d in medium, after stimulation with 10 µg/ml LPS alone, or 10 µg/ml LPS + rGM-CSF, or with a Stat5 inhibitor ( n = 3–8, mean ± SD). ns, not significant. (E) IgM ELISA of culture supernatants from the same groups as in D ( n = 3–8, mean ± SD). ns, not significant. (F) IgM ELISPOT of cultured B1a cells. A representative ELISPOT of n = 3 is shown. (G) Quantitative RT-PCR analysis of Csf2 and Prdm1 expression in cultured serosal B1a cells from WT, Csf2 −/− , and Csf2rb −/− mice with and without LPS (10 µg/ml) stimulation for 1 d ( n = 3). Csf2 expression levels after LPS stimulation is shown relative to WT LPS as mean ± SD. Prdm1 expression after LPS stimulation is shown relative to WT medium as mean ± SD (nd, not detected). *, P
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    Comparison of the effects of the intranasal (IN) and intramuscular (IM) immunisation routes on EV71-specific antibody titres and cytokine production. Mice were IN or IM immunised with PBS or formalin-inactivated EV71 (5 μg/dose) alone or adjuvanted with CpG (20 μg/dose) three times at 3-week intervals. ELISA was used to measure EV71-specific IgA in the nasal wash ( a ), BALF ( b ), faeces ( c ), and serum ( d ) and EV71-specific IgG in the serum ( e ) of mice at 2 weeks after the third immunisation. Splenocytes from immunised mice were harvested and cultured for 2 days in medium containing 10 μg/mL heat-inactivated EV71, after which culture supernatants were analysed for IFN-γ ( f ), IL-4 ( g ), and IL-17 ( h ) using ELISA. One representative study of two with similar results is shown. Values shown are means ± SEMs of five mice in each experimental group; * p

    Journal: Scientific Reports

    Article Title: A CpG-adjuvanted intranasal enterovirus 71 vaccine elicits mucosal and systemic immune responses and protects human SCARB2-transgenic mice against lethal challenge

    doi: 10.1038/s41598-018-28281-5

    Figure Lengend Snippet: Comparison of the effects of the intranasal (IN) and intramuscular (IM) immunisation routes on EV71-specific antibody titres and cytokine production. Mice were IN or IM immunised with PBS or formalin-inactivated EV71 (5 μg/dose) alone or adjuvanted with CpG (20 μg/dose) three times at 3-week intervals. ELISA was used to measure EV71-specific IgA in the nasal wash ( a ), BALF ( b ), faeces ( c ), and serum ( d ) and EV71-specific IgG in the serum ( e ) of mice at 2 weeks after the third immunisation. Splenocytes from immunised mice were harvested and cultured for 2 days in medium containing 10 μg/mL heat-inactivated EV71, after which culture supernatants were analysed for IFN-γ ( f ), IL-4 ( g ), and IL-17 ( h ) using ELISA. One representative study of two with similar results is shown. Values shown are means ± SEMs of five mice in each experimental group; * p

    Article Snippet: For measuring EV71-specific IgG1 or IgG2a, 1 μg/mL biotinylated anti-mouse IgG1 (1:5000; A85-1; Becton, Dickinson and Company, Franklin Lakes, NY, USA) or anti-mouse IgG2a (1:1000; R19-15; Becton, Dickinson and Company) was used; this was followed by incubation for 1 h with HRP-conjugated streptavidin (R & D Systems, Minneapolis, MN, USA).

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

    EV71-specific antibody responses, neurological symptoms, pathological changes, and lethality in mice infected with EV71 of the C2 genotype. hSCARB2-Tg mice were intranasally immunised twice with PBS, EV71 (1 μg), or EV71 (1 μg) plus CpG (4 μg) on postnatal days 1 and 2 and were then s.c. challenged with 3 × 10 6 pfu of the 5746 EV71 strain. ( a ) Serum samples were collected from each mouse on postnatal day 8 before the mouse was s.c. challenged with 3 × 10 6 pfu of the 5746 EV71 strain, and the collected samples were assayed to determine the titres of EV71-specific IgG. ( b ) CNS disorder-like limb paralysis was scored according to the criteria described in the Methods section. Representative images showing symptoms of ( c ) the PBS-immunised group with limb paralysis and ( d ) protected mice in the EV71 or EV71 + CpG immunised group at day 7 postinfection with EV71 virus. ( e ) Histological examination of EV71-infected mice. The animals were sacrificed in the moribund state, and paraffin sections of the organs, including the muscle, intestine, brainstem, and spinal cord, were stained with H E. Normal tissues were collected from mice without EV71 challenge as a mock control. ( f ) Daily survival rate, monitored after 8-day-old hSCARB2-Tg mice were s.c. infected with 3 × 10 6 pfu of the 5746 EV71 strain. Values shown are means ± SEMs of 8–10 mice in each experimental group; * p

    Journal: Scientific Reports

    Article Title: A CpG-adjuvanted intranasal enterovirus 71 vaccine elicits mucosal and systemic immune responses and protects human SCARB2-transgenic mice against lethal challenge

    doi: 10.1038/s41598-018-28281-5

    Figure Lengend Snippet: EV71-specific antibody responses, neurological symptoms, pathological changes, and lethality in mice infected with EV71 of the C2 genotype. hSCARB2-Tg mice were intranasally immunised twice with PBS, EV71 (1 μg), or EV71 (1 μg) plus CpG (4 μg) on postnatal days 1 and 2 and were then s.c. challenged with 3 × 10 6 pfu of the 5746 EV71 strain. ( a ) Serum samples were collected from each mouse on postnatal day 8 before the mouse was s.c. challenged with 3 × 10 6 pfu of the 5746 EV71 strain, and the collected samples were assayed to determine the titres of EV71-specific IgG. ( b ) CNS disorder-like limb paralysis was scored according to the criteria described in the Methods section. Representative images showing symptoms of ( c ) the PBS-immunised group with limb paralysis and ( d ) protected mice in the EV71 or EV71 + CpG immunised group at day 7 postinfection with EV71 virus. ( e ) Histological examination of EV71-infected mice. The animals were sacrificed in the moribund state, and paraffin sections of the organs, including the muscle, intestine, brainstem, and spinal cord, were stained with H E. Normal tissues were collected from mice without EV71 challenge as a mock control. ( f ) Daily survival rate, monitored after 8-day-old hSCARB2-Tg mice were s.c. infected with 3 × 10 6 pfu of the 5746 EV71 strain. Values shown are means ± SEMs of 8–10 mice in each experimental group; * p

    Article Snippet: For measuring EV71-specific IgG1 or IgG2a, 1 μg/mL biotinylated anti-mouse IgG1 (1:5000; A85-1; Becton, Dickinson and Company, Franklin Lakes, NY, USA) or anti-mouse IgG2a (1:1000; R19-15; Becton, Dickinson and Company) was used; this was followed by incubation for 1 h with HRP-conjugated streptavidin (R & D Systems, Minneapolis, MN, USA).

    Techniques: Mouse Assay, Infection, Staining

    EV71-specific IgG1 and IgG2a responses in the serum. Effects of CpG as an adjuvant on EV71-specific IgG1 ( a ), EV71-specific IgG2a ( b ), and EV71-specific IgG2a-to-IgG1 ratio ( c ) in the serum of BALB/c mice at 2 weeks after the third immunisation with PBS or with formalin-inactivated EV71 alone or with CpG. Values shown are means ± SEMs of six mice in each experimental group; * p

    Journal: Scientific Reports

    Article Title: A CpG-adjuvanted intranasal enterovirus 71 vaccine elicits mucosal and systemic immune responses and protects human SCARB2-transgenic mice against lethal challenge

    doi: 10.1038/s41598-018-28281-5

    Figure Lengend Snippet: EV71-specific IgG1 and IgG2a responses in the serum. Effects of CpG as an adjuvant on EV71-specific IgG1 ( a ), EV71-specific IgG2a ( b ), and EV71-specific IgG2a-to-IgG1 ratio ( c ) in the serum of BALB/c mice at 2 weeks after the third immunisation with PBS or with formalin-inactivated EV71 alone or with CpG. Values shown are means ± SEMs of six mice in each experimental group; * p

    Article Snippet: For measuring EV71-specific IgG1 or IgG2a, 1 μg/mL biotinylated anti-mouse IgG1 (1:5000; A85-1; Becton, Dickinson and Company, Franklin Lakes, NY, USA) or anti-mouse IgG2a (1:1000; R19-15; Becton, Dickinson and Company) was used; this was followed by incubation for 1 h with HRP-conjugated streptavidin (R & D Systems, Minneapolis, MN, USA).

    Techniques: Mouse Assay

    EV71-specific IgG antibody-secreting cells (ASCs) and IgA ASCs in mouse spleens. Mice were intranasally immunised with PBS or formalin-inactivated EV71 without/with CpG three times at 3-week intervals. Spleens were isolated at 2 weeks after the third immunisation, and the collected samples were used in EV71-specific ELISPOT assays to determine the numbers of IgG ASCs ( a , b ) and IgA ASCs ( c , d ). Values shown are means ± SEMs of six mice in each experimental group; * p

    Journal: Scientific Reports

    Article Title: A CpG-adjuvanted intranasal enterovirus 71 vaccine elicits mucosal and systemic immune responses and protects human SCARB2-transgenic mice against lethal challenge

    doi: 10.1038/s41598-018-28281-5

    Figure Lengend Snippet: EV71-specific IgG antibody-secreting cells (ASCs) and IgA ASCs in mouse spleens. Mice were intranasally immunised with PBS or formalin-inactivated EV71 without/with CpG three times at 3-week intervals. Spleens were isolated at 2 weeks after the third immunisation, and the collected samples were used in EV71-specific ELISPOT assays to determine the numbers of IgG ASCs ( a , b ) and IgA ASCs ( c , d ). Values shown are means ± SEMs of six mice in each experimental group; * p

    Article Snippet: For measuring EV71-specific IgG1 or IgG2a, 1 μg/mL biotinylated anti-mouse IgG1 (1:5000; A85-1; Becton, Dickinson and Company, Franklin Lakes, NY, USA) or anti-mouse IgG2a (1:1000; R19-15; Becton, Dickinson and Company) was used; this was followed by incubation for 1 h with HRP-conjugated streptavidin (R & D Systems, Minneapolis, MN, USA).

    Techniques: Mouse Assay, Isolation, Enzyme-linked Immunospot

    EV71-specific antibody responses measured in serum from BALB/c mice immunised with CpG as an adjuvant for a formalin-inactivated EV71 vaccine. Mice were intranasally immunised with PBS, formalin-inactivated EV71 (5 μg/dose), or formalin-inactivated EV71 (5 μg/dose) plus CpG (20 μg/dose) three times at 3-week intervals. ELISA was used to measure EV71-specific IgG ( a ) and EV71-specific IgA ( b ) in the serum of mice at 2 weeks after the third immunisation. Values shown are means ± SEMs of six mice in each experimental group; * p

    Journal: Scientific Reports

    Article Title: A CpG-adjuvanted intranasal enterovirus 71 vaccine elicits mucosal and systemic immune responses and protects human SCARB2-transgenic mice against lethal challenge

    doi: 10.1038/s41598-018-28281-5

    Figure Lengend Snippet: EV71-specific antibody responses measured in serum from BALB/c mice immunised with CpG as an adjuvant for a formalin-inactivated EV71 vaccine. Mice were intranasally immunised with PBS, formalin-inactivated EV71 (5 μg/dose), or formalin-inactivated EV71 (5 μg/dose) plus CpG (20 μg/dose) three times at 3-week intervals. ELISA was used to measure EV71-specific IgG ( a ) and EV71-specific IgA ( b ) in the serum of mice at 2 weeks after the third immunisation. Values shown are means ± SEMs of six mice in each experimental group; * p

    Article Snippet: For measuring EV71-specific IgG1 or IgG2a, 1 μg/mL biotinylated anti-mouse IgG1 (1:5000; A85-1; Becton, Dickinson and Company, Franklin Lakes, NY, USA) or anti-mouse IgG2a (1:1000; R19-15; Becton, Dickinson and Company) was used; this was followed by incubation for 1 h with HRP-conjugated streptavidin (R & D Systems, Minneapolis, MN, USA).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Durability of anti-gp120 IgG and serum and mucosal IgA responses following immunization of mice via parenteral, mucosal and combination routes. Anti-gp120 IgG (a, left panel) and IgA (a, right panel) responses, assayed by ELISA, were determined for serum samples of DNA prime–protein boosted mice at weeks 13, 15, 18, 22 and 34 of the study. IgA was also measured at weeks 13 and 34 of the study for saliva (b, 1 : 5 dilution) and vaginal-wash samples (c, 1 : 2 dilution). ELISA titres and A 450 are reported.

    Journal: The Journal of General Virology

    Article Title: Induction of mucosal and systemic antibody and T-cell responses following prime-boost immunization with novel adjuvanted human immunodeficiency virus-1-vaccine formulations

    doi: 10.1099/vir.0.023242-0

    Figure Lengend Snippet: Durability of anti-gp120 IgG and serum and mucosal IgA responses following immunization of mice via parenteral, mucosal and combination routes. Anti-gp120 IgG (a, left panel) and IgA (a, right panel) responses, assayed by ELISA, were determined for serum samples of DNA prime–protein boosted mice at weeks 13, 15, 18, 22 and 34 of the study. IgA was also measured at weeks 13 and 34 of the study for saliva (b, 1 : 5 dilution) and vaginal-wash samples (c, 1 : 2 dilution). ELISA titres and A 450 are reported.

    Article Snippet: Specific IgG isotypes were detected using rat anti-mouse IgG1–HRP (BD Pharmigen), rat anti-mouse IgG2a–HRP (BD Pharmigen), goat anti-mouse IgG2b–HRP (AbD Serotec) and goat anti-mouse IgG3–HRP (AbD Serotec).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Defective humoral responses in Itsn2 -/- animals ( A ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and spleens of immunised animals analysed by flow cytometry 13d after immunisation. Tfh cells (CD4 + CXCR5 + PD-1 + ). Panels on the right show the show percentage of cells in the indicated gates (each dot represents a different animal). ( B ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and 13 days post-immunisation, NP-specific antibody secreting cells were detected in the spleen by ELISPOT. ( F ) WT and Itsn2 -/- littermates were immunised with NP-LPS and titres of NP 23 -specific IgM and IgG3 were measured by ELISA. Data are representative of at least 2 independent experiments with more than three animals in each group. Student’s t-test, ns p > 0.05, *p

    Journal: eLife

    Article Title: Tuning of in vivo cognate B-T cell interactions by Intersectin 2 is required for effective anti-viral B cell immunity

    doi: 10.7554/eLife.26556

    Figure Lengend Snippet: Defective humoral responses in Itsn2 -/- animals ( A ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and spleens of immunised animals analysed by flow cytometry 13d after immunisation. Tfh cells (CD4 + CXCR5 + PD-1 + ). Panels on the right show the show percentage of cells in the indicated gates (each dot represents a different animal). ( B ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and 13 days post-immunisation, NP-specific antibody secreting cells were detected in the spleen by ELISPOT. ( F ) WT and Itsn2 -/- littermates were immunised with NP-LPS and titres of NP 23 -specific IgM and IgG3 were measured by ELISA. Data are representative of at least 2 independent experiments with more than three animals in each group. Student’s t-test, ns p > 0.05, *p

    Article Snippet: NP-specific antibody titres were detected by ELISA, using NP20-BSA or NP4-BSA for capture, and biotinylated anti–mouse IgM (Southern Biotech, Birmingham, Alabama), IgG (Southern Biotech), IgG2b (Southern Biotech), IgG2c (Southern Biotech), and IgG3 (BD, Franklin Lakes, New Jersey) for detection.

    Techniques: Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

    GM-CSF controls IgM production. (A) In vitro culture of CD19 + serosal B cells. Gating strategy and phenotyping of IRA B cells 2 d after culture in medium or with 10 µg/ml LPS. A representative contour plot of n > 5 is shown. (B) CD131 (Csf2rb) expression on selected cells from WT mice ( n = 4). (C) In vitro culture of serosal B1a cells sorted from WT, Csf2 −/− , and Csfrb −/− mice. Data show intracellular IgM and GM-CSF in cells cultured in medium alone or after LPS (10 µg/ml) stimulation after 1 d of culture ( n = 3–5). The gate for GM-CSF was set using an isotype control (IgG2a) and the gate for intracellular IgM represents the upper 99% limit of intracellular IgM staining at baseline. (D) In vitro culture of serosal B1a cells sorted from WT, Csf2 −/− , and Csfrb −/− mice, and B2 cells sorted from WT mice. Percentage of IgM(ic) high cells cultured for 1 d in medium, after stimulation with 10 µg/ml LPS alone, or 10 µg/ml LPS + rGM-CSF, or with a Stat5 inhibitor ( n = 3–8, mean ± SD). ns, not significant. (E) IgM ELISA of culture supernatants from the same groups as in D ( n = 3–8, mean ± SD). ns, not significant. (F) IgM ELISPOT of cultured B1a cells. A representative ELISPOT of n = 3 is shown. (G) Quantitative RT-PCR analysis of Csf2 and Prdm1 expression in cultured serosal B1a cells from WT, Csf2 −/− , and Csf2rb −/− mice with and without LPS (10 µg/ml) stimulation for 1 d ( n = 3). Csf2 expression levels after LPS stimulation is shown relative to WT LPS as mean ± SD. Prdm1 expression after LPS stimulation is shown relative to WT medium as mean ± SD (nd, not detected). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Pleural innate response activator B cells protect against pneumonia via a GM-CSF-IgM axis

    doi: 10.1084/jem.20131471

    Figure Lengend Snippet: GM-CSF controls IgM production. (A) In vitro culture of CD19 + serosal B cells. Gating strategy and phenotyping of IRA B cells 2 d after culture in medium or with 10 µg/ml LPS. A representative contour plot of n > 5 is shown. (B) CD131 (Csf2rb) expression on selected cells from WT mice ( n = 4). (C) In vitro culture of serosal B1a cells sorted from WT, Csf2 −/− , and Csfrb −/− mice. Data show intracellular IgM and GM-CSF in cells cultured in medium alone or after LPS (10 µg/ml) stimulation after 1 d of culture ( n = 3–5). The gate for GM-CSF was set using an isotype control (IgG2a) and the gate for intracellular IgM represents the upper 99% limit of intracellular IgM staining at baseline. (D) In vitro culture of serosal B1a cells sorted from WT, Csf2 −/− , and Csfrb −/− mice, and B2 cells sorted from WT mice. Percentage of IgM(ic) high cells cultured for 1 d in medium, after stimulation with 10 µg/ml LPS alone, or 10 µg/ml LPS + rGM-CSF, or with a Stat5 inhibitor ( n = 3–8, mean ± SD). ns, not significant. (E) IgM ELISA of culture supernatants from the same groups as in D ( n = 3–8, mean ± SD). ns, not significant. (F) IgM ELISPOT of cultured B1a cells. A representative ELISPOT of n = 3 is shown. (G) Quantitative RT-PCR analysis of Csf2 and Prdm1 expression in cultured serosal B1a cells from WT, Csf2 −/− , and Csf2rb −/− mice with and without LPS (10 µg/ml) stimulation for 1 d ( n = 3). Csf2 expression levels after LPS stimulation is shown relative to WT LPS as mean ± SD. Prdm1 expression after LPS stimulation is shown relative to WT medium as mean ± SD (nd, not detected). *, P

    Article Snippet: Human: anti–CD19-APC-A700, J3-119 (Beckman Coulter); anti–GM-CSF-PE, BVD2-21C11 (BD); anti–CD20-PE-Cy7, L27 (BD); anti–IgM-PacBlue, G20-127 (BD); anti–GM-CSF (blocking), 3209 (R & D Systems); anti–GM-CSFRα (blocking; CD116), K2B7.17A (Millipore); Aqua Live/Dead Stain (Life Technologies), rat IgG2a-isotype-PE (BD).

    Techniques: In Vitro, Expressing, Mouse Assay, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Quantitative RT-PCR