peroxidase affinipure goat anti mouse igg  (Jackson Immuno)

Journal: Infection and Immunity

Article Title: The UspA2 Protein of Moraxella catarrhalis Is Directly Involved in the Expression of Serum Resistance

doi: 10.1128/IAI.73.4.2400-2410.2005

Figure Lengend Snippet: Killing of wild-type and mutant strains of M. catarrhalis by human sera. (A) Effect of various sera on selected strains. The wild-type strain O35E (1), the uspA2 mutant O35EΔ2 (2), and the wild-type strain MC317 (3) were incubated for 30 min at 37°C in VBS ++ containing 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), or 10% (vol/vol) factor B-depleted human serum (c) or in VBS containing 10 mM MgCl 2 , 10 mM EGTA, and 10% (vol/vol) NHS (d). Portions of these reaction mixtures were plated at time zero and after 30 min, and the percentage of survival is calculated relative to the original inoculum. The data presented here are the means of results from three independent experiments plus the standard errors. (B) Involvement of IgG in killing of the uspA2 mutant O35EΔ2. Wild-type strain O35E (1) and the uspA2 mutant O35EΔ2 (2) were incubated with the following sera: 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), 10% (vol/vol) IgG-depleted NHS (c), and 10% (vol/vol) IgG-depleted NHS mixed with heat-inactivated NHS as a source of IgG (d). The data presented here are the means of results from three independent experiments plus the standard errors.

Article Snippet: The secondary antibody used for Western blot analysis was goat anti-mouse immunoglobulin G (IgG) conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, Pa.).

Techniques: Mutagenesis, Incubation

Journal: Infection and Immunity

Article Title: The UspA2 Protein of Moraxella catarrhalis Is Directly Involved in the Expression of Serum Resistance

doi: 10.1128/IAI.73.4.2400-2410.2005

Figure Lengend Snippet: Comparison of UspA2 proteins from M. catarrhalis strains O35E and MC317. (A) Alignment of the deduced amino acid sequences. Identical amino acids are shaded in dark gray, while conserved amino acids are shaded with light gray. This figure was generated by using the ClustalW Alignment program in MacVector (version 6.5). (B) Relative amounts of UspA2 exposed on the surface of the following strains as measured by the indirect antibody accessibility assay: the M. catarrhalis uspA1 uspA2 mutant O35E.12 (1), the uspA1 mutant O35E.1 (2), and the uspA1 mutant MC317.1 (3). The counts per minute of radioiodinated goat anti-mouse IgG bound to MAb 17C7 on the bacterial cell surface (a) are plotted on the y axis. MAb 3F12, a murine IgG MAb specific for the major outer membrane protein of H. ducreyi ), was used as the negative control (b). These data represent the means of results from two independent experiments plus standard deviations.

Article Snippet: The secondary antibody used for Western blot analysis was goat anti-mouse immunoglobulin G (IgG) conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, Pa.).

Techniques: Generated, Mutagenesis, Negative Control

Journal: bioRxiv

Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

doi: 10.1101/2020.10.09.334136

Figure Lengend Snippet: Antibody and T-cell responses after GX-19 vaccination in macaques. Macaques (n=3) were immunized with 3 mg of GX-19 as described in the methods. Serum and PBMCs were collected before (wk 0), during (wk 4, and 5.5) and after (wk 8) vaccination and were assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) and neutralizing antibodies against SARS-CoV-2 live-virus (B) . Data represent mean SEM of individual macaques, and dashed line indicate the assay limits of detection. The number of SARS-CoV-2 S-specific IFN-γ secreting cells in PBMCs was determined by IFN-γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 PBMCS in triplicate wells (C) . The frequency of S-specific CD4 + or CD8 + T cells producing IFN-γ, TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (D) .

Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205), or IgG2a (Jackson ImmunoResearch Laboratories 115-035-206) or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P) for the NHP sera for 1 hour at 37°C.

Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Staining

Journal: bioRxiv

Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

doi: 10.1101/2020.10.09.334136

Figure Lengend Snippet: Immunization with GX-19 elicit Th1-biased T cell responses in mice. BALB/c mice (n=3-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 (empty control vector) as described in the methods (A-C) . Sera were collected 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG1 and IgG2a/b. Endpoint titers (A) , and endpoint tier ratios of IgG2a/b to IgG1 (B) were calculated. 2 weeks post-boost mouse splenocytes were isolated and re-stimulated with peptide pools spanning the SARS-CoV-2 S protein ex vivo . Indicated cytokines in the supernatants of culture were quantified using Th1/Th2 cytometric bead array kit (D) . T cell responses were measured by IFN-γ ELISPOT in splenocytes stimulated with peptide pools spanning the SARS-CoV-2 S protein (C) . Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (E) . Data representative of two independent experiments. P values determined by Mann-Whitney test.

Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205), or IgG2a (Jackson ImmunoResearch Laboratories 115-035-206) or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P) for the NHP sera for 1 hour at 37°C.

Techniques: Mouse Assay, Plasmid Preparation, Isolation, Ex Vivo, Enzyme-linked Immunospot, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

doi: 10.1101/2020.10.09.334136

Figure Lengend Snippet: Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) (A) . BALB/c mice (n=4-10/group) were immunized at week 0 and 2 with pGX27-S ΔTM , pGX27-S or pGX27 (empty control vector) as described in the methods. Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies (B) .

Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205), or IgG2a (Jackson ImmunoResearch Laboratories 115-035-206) or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P) for the NHP sera for 1 hour at 37°C.

Techniques: Expressing, Mouse Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

doi: 10.1101/2020.10.09.334136

Figure Lengend Snippet: GX-19 elicit robust binding and neutralizing antibody responses in mice. BALB/c mice (n=4-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 as described in the methods (A-C) . Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) , and for post-boost sera, neutralizing antibodies against SARS-CoV-2 live-virus (C) . BAL were collected 2 weeks post-boost and assayed for SARS-CoV-2 S-specific IgG antibodies by ELISA (B) . Data representative of two independent experiments. P values determined by Mann-Whitney test.

Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205), or IgG2a (Jackson ImmunoResearch Laboratories 115-035-206) or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P) for the NHP sera for 1 hour at 37°C.

Techniques: Binding Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: NPJ Vaccines

Article Title: Rabies virus-based COVID-19 vaccine CORAVAX™ induces high levels of neutralizing antibodies against SARS-CoV-2

doi: 10.1038/s41541-020-00248-6

Figure Lengend Snippet: Analysis of the immune response to CORAVAX in mice. Balb/C mice were immunized once with live CORAVAX or twice (days 0 and 21) with 10 μg chemically inactivated particles of CORAVAX with or without adjuvant. Serum was collected from each mouse at days 14, 21, 28, 42, and 56 for analysis by a SARS-CoV-2 S-specific ELISA. a Immunization schedule: live CORAVAX used once on day 0; the inactivated vaccines were dosed on days 0 and 28 (figure created with BioRender.com). b SARS CoV-2 S1 IgG responses represented as EC50 titers c SARS CoV-2 RBD IgG responses on day 56 sera d SARS CoV-2 S1 IgG2/1 isotype ratio. Two animals in the inactivated CORAVAX group did not make detectable IgG1 antibodies.

Article Snippet: Plates were washed three times the next day, followed by the addition of goat anti-mouse-IgG-Fc HRP antibody (Southern Biotech, Cat# 1033-05, 1:8000 in PBST) or goat anti-mouse-IgG1-Fc HRP antibody (Jackson ImmunoResearch, Cat# 115-035-205, 1:8000 in PBST) or goat anti-mouse-IgG2a-Fc HRP antibody (Jackson ImmunoResearch, Cat# 115-035-206, 1:8000 in PBST) for 2 h at RT.

Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: The PX-domain protein SNX17 interacts with members of the LDL receptor family and modulates endocytosis of the LDL receptor

doi: 10.1093/emboj/cdf435

Figure Lengend Snippet: Fig. 7. Binding of SNX17 and Dab1 to the intracellular domain of ApoER2. Microtiter plates (96-well) coated with a GST fusion protein containing the intracellular domain of ApoER2 were incubated ( A ) with the indicated amounts of purified Dab1–MBP expressed in E.coli or ( B ) with cell extracts derived from 293T cells expressing myc-tagged SNX17. Bound Dab1–MBP was visualized with the combination of an anti-MBP antibody and HRP-conjugated goat anti-rabbit IgG. Bound myc-SNX17 was visualized using the combination of an anti-myc antibody and HRP-conjugated goat anti-mouse IgG as described in Materials and methods. The color reaction was monitored by measuring absorption at 450 nm. ( C ) Competition of SNX17 binding by Dab1 was measured at two selected concentrations of myc-SNX17 (corresponding to 25 and 40% of cell lysate present in the incubation medium, respectively) in the absence and presence of 2 µg/ml Dab–MBP.

Article Snippet: The following antibodies were obtained commercially from the sources indicated: anti-HA tag (16B12; Babco), anti-Myc (9E10, used as hybridoma supernatant from the corresponding cells; ATCC), anti-EEA1 (Affinity Bioreagents, Inc.), Oregon Green 488-labeled goat anti-mouse IgG (Molecular Probes), Alexa 594-labeled goat anti-rabbit IgG (Molecular Probes), goat anti-rabbit biotinylated IgG (Sigma), anti-MBP (E8030S; New England Biolabs), peroxidase- conjugated goat anti-mouse IgG (Jackson Immuno Research Laboratories) and peroxidase-conjugated anti-protein A (Sigma).

Techniques: Binding Assay, Incubation, Purification, Derivative Assay, Expressing