Journal: The Journal of Physical Chemistry C

Article Title: Frequency and Amplitude Optimizations for Magnetic Particle Spectroscopy Applications

doi: 10.1021/acs.jpcc.2c07534

Figure Lengend Snippet: Figure 2. (a) Photograph of the prepared single-core SHS30 (vials I and II) and multicore SuperMag50 (vials III and IV) samples. Complete sedimentation of the MNPs was observed from vials II and IV in which excess amounts of biotinylated antibodies were added. Schematic presentation of the cluster formation of single-core and multicore MNPs presented, respectively, in (b) and (c).

Article Snippet: Biotinylated antibody goat anti-mouse IFN-γ with an antibody concentration of 0.4 μg/mL (2.67 nM) was obtained from R&D systems, USA (ELISA kit product number DY485-05, part number 840123).

Techniques: Sedimentation

Journal: The Journal of Physical Chemistry C

Article Title: Frequency and Amplitude Optimizations for Magnetic Particle Spectroscopy Applications

doi: 10.1021/acs.jpcc.2c07534

Figure Lengend Snippet: Figure 9. (a) Schematic representation of a surface-conjugation-based MPS bioassay mechanism along with the harmonic signal generation for single- and multicore nanoparticles. (i) Capture antibodies are immobilized on a reaction surface. (ii) In addition to the biological test sample constituting a target analyte, the target analyte specifically binds to the capture antibodies. (iii) Biotinylated detection antibodies are immobilized on the target analyte completing the sandwich assay structure. Streptavidin-coated nanoparticles are then captured on the biotinylated detection antibodies; this process for single- and multicore MNPs has been shown in (iv) and (v), respectively, with corresponding resultant MPS harmonic spectra. (b) Harmonic amplitude per micro-mole of the bound state of MNPs for SHS30 and SuperMag50 has been depicted.

Article Snippet: Biotinylated antibody goat anti-mouse IFN-γ with an antibody concentration of 0.4 μg/mL (2.67 nM) was obtained from R&D systems, USA (ELISA kit product number DY485-05, part number 840123).

Techniques: Conjugation Assay, Bioassay

Journal:

Article Title: Herpes Simplex Virus Type 1 DNA Is Immunostimulatory In Vitro and In Vivo

doi: 10.1128/JVI.77.20.11158-11169.2003

Figure Lengend Snippet: In vitro cytokine response to CpG DNA. Splenocytes were cultured in the presence of ODNs or DNA for 48 h (which was determined to be the optimal time for cytokine production). Supernatants were analyzed for secreted IL-6 (A) and IFN-γ (B) mRNA transcripts or secreted protein (C). In panel B, the arrow marks the IFN-γ band and the asterisk marks the internal GAPDH control band.

Article Snippet: Cells were grown in cultures for 40 h to accumulate secreted IFN-γ, the cells were washed off the membrane, and bound IFN-γ was detected using a biotinylated mouse anti-IFN-γ monoclonal antibody (BAF 485; R&D Systems) using streptavidin-horseradish peroxidase and nickel-enhanced 3,3′-diaminobenzidine as the substrate.

Techniques: In Vitro, Cell Culture

Journal:

Article Title: Herpes Simplex Virus Type 1 DNA Is Immunostimulatory In Vitro and In Vivo

doi: 10.1128/JVI.77.20.11158-11169.2003

Figure Lengend Snippet: Macrophage responses to CpG ODNs. The RAW 264.7 macrophage cell line (A), primary C57BL/6 BM-derived macrophages (B), and peritoneal exudate macrophages (C) were activated overnight with recombinant IFN-γ (10 U/ml) prior to stimulation with the indicated ODNs at 1 μM or with 100 ng of LPS/ml as the positive control. RAW cells cultured with IFN-γ alone (10 U/ml) served as the negative control (Med). NO levels were determined using the Griess reaction, and cytokine levels were determined using conventional ELISAs (BioSource). Results are representative of four (A and B) or two (C) experiments..

Article Snippet: Cells were grown in cultures for 40 h to accumulate secreted IFN-γ, the cells were washed off the membrane, and bound IFN-γ was detected using a biotinylated mouse anti-IFN-γ monoclonal antibody (BAF 485; R&D Systems) using streptavidin-horseradish peroxidase and nickel-enhanced 3,3′-diaminobenzidine as the substrate.

Techniques: Derivative Assay, Recombinant, Positive Control, Cell Culture, Negative Control

Journal:

Article Title: Herpes Simplex Virus Type 1 DNA Is Immunostimulatory In Vitro and In Vivo

doi: 10.1128/JVI.77.20.11158-11169.2003

Figure Lengend Snippet: In vitro proliferative and cytokine responses to phosphodiester and phosphorothioate modified ODNs. PdE, phosphodiester ODN; 5/3-PT, ODN with terminal phosphorothioate linkages; cPTO, completely phosphorothioate ODN. (A) Cytokine production in spleen cell cultures treated with ODNs at 1.1 μM final concentration. Medium-treated samples contained 909 ± 22 pg of IFN-γ/ml and no IL-10. No IL-4 or IL-12 was detected in these samples (not shown). (B) Proliferative response of splenocyte cultures treated with various concentrations of ODN. ODNs were tested in triplicate in 48-h splenocyte cultures that were tritium labeled for the last 24 h. The stimulation index (SI) was calculated as test sample/medium control. nCpG, open circle; CpG, grey circle; gD, black square; ICP27, black circle. The SI for PHA (9.4) is shown as a solid horizontal line in each panel.

Article Snippet: Cells were grown in cultures for 40 h to accumulate secreted IFN-γ, the cells were washed off the membrane, and bound IFN-γ was detected using a biotinylated mouse anti-IFN-γ monoclonal antibody (BAF 485; R&D Systems) using streptavidin-horseradish peroxidase and nickel-enhanced 3,3′-diaminobenzidine as the substrate.

Techniques: In Vitro, Modification, Concentration Assay, Labeling

Journal:

Article Title: Herpes Simplex Virus Type 1 DNA Is Immunostimulatory In Vitro and In Vivo

doi: 10.1128/JVI.77.20.11158-11169.2003

Figure Lengend Snippet: Memory cytokine response after immunization with CpG DNA as the adjuvant. Mice were immunized as described for Fig. ​Fig.6.6. On days 10 to 12, splenocytes were cultured in the presence of OVA (200 μg/ml) or medium. Supernatants were collected after 72 h and analyzed by ELISA for IFN-γ and IL-4 levels (BioSource). The ratios of secreted IFN-γ and IL-4 are given between the paired IFN-γ (A and C) and IL-4 (B and D) panels. Asterisks indicate a ratio below 0.3. Results are representative of two experiments.

Article Snippet: Cells were grown in cultures for 40 h to accumulate secreted IFN-γ, the cells were washed off the membrane, and bound IFN-γ was detected using a biotinylated mouse anti-IFN-γ monoclonal antibody (BAF 485; R&D Systems) using streptavidin-horseradish peroxidase and nickel-enhanced 3,3′-diaminobenzidine as the substrate.

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

Journal: Infection and Immunity

Article Title: Mucosal Vaccination against Tuberculosis Using Inert Bioparticles

doi: 10.1128/IAI.00786-13

Figure Lengend Snippet: IFN-γ responses. IFN-γ was determined from splenocytes stimulated using an ELISA or ELISPOT assay. (a and b) ELISA detection of IFN-γ from splenocytes stimulated with rMPT64 (a) or with rAcr or rAg85B (b). (c) ELISPOT assay determination of the number of spot-forming units (SFU) in splenocytes stimulated with rMPT64 or rAcr-Ag85B. Spleens were taken from mice (n = 2) immunized using dosing regimen 1. The BCG group received one dose of BCG.

Article Snippet: Free binding sites were blocked with 10% FCS in PBS at RT for 1 h. Culture supernatants were tested at RT for 2 h, and IFN-γ was detected with a biotinylated anti-mouse IFN-γ–streptavidin–horseradish peroxidase conjugate (BD Biosciences) at RT for 1 h. Recombinant mouse IFN-γ (BD Biosciences) was used as a standard.

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

Journal: Infection and Immunity

Article Title: Mucosal Vaccination against Tuberculosis Using Inert Bioparticles

doi: 10.1128/IAI.00786-13

Figure Lengend Snippet: Gating strategy for polyfunctional T cell analysis. Representative FlowJo scatter plots of a naive mouse splenocyte sample are presented to show the gating strategy. Samples were first gated by forward scatter (FSC-A)/side scatter (SSC-A) to select lymphocytes and were then gated by FSC-H/FSC-A to gate for single cells. This population was then gated for CD3+ CD4+ or CD3+ CD8+ staining. These two populations were then analyzed for IFN-γ, IL-2, and TNF-α by staining.

Article Snippet: Free binding sites were blocked with 10% FCS in PBS at RT for 1 h. Culture supernatants were tested at RT for 2 h, and IFN-γ was detected with a biotinylated anti-mouse IFN-γ–streptavidin–horseradish peroxidase conjugate (BD Biosciences) at RT for 1 h. Recombinant mouse IFN-γ (BD Biosciences) was used as a standard.

Techniques: Staining

Journal: Cancer Immunity : a Journal of the Academy of Cancer Immunology

Article Title: Lung and splenic B cells facilitate diverse effects on in vitro measures of antitumor immune responses

doi:

Figure Lengend Snippet: Production of IFN-γ by lung lymphocytes in response to MADB106 tumor cells. Total lung lymphocytes were incubated in the presence of MADB106 tumor cells. At selected time intervals (2, 6, 24 and 96 h), the levels of IFN-γ cytokine were determined in culture supernatants. In addition, B cells were removed from total lung lymphocyte cultures. After 1 and 4 days of culture, supernatants were collected and the level of IFN-γ determined using ELISA techniques. Vertical bars and error bars represent the mean ± SE (n = 4). Asterisks indicate statistical differences (P less than or equal to 0.05) in the cytokine production by lung cells.

Article Snippet: 100 µl of biotinylated mouse anti-rat IFN-γ cytokine was added to the appropriate wells and incubated for 1 h. To detect cytokine levels, avidin-HRP and 3, 3', 5, 5'-tetramethylbenzidine substrate (BD PharMingen) were allowed to react on the plate for 30 minutes.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunity : a Journal of the Academy of Cancer Immunology

Article Title: Lung and splenic B cells facilitate diverse effects on in vitro measures of antitumor immune responses

doi:

Figure Lengend Snippet: Production of IFN-γ by spleen lymphocytes in response to MADB106 tumor cells. Total spleen lymphocytes were incubated in the presence of MADB106 tumor cells. At selected time intervals (2, 6, 24 and 96 h), the levels of IFN-γ cytokine were determined in culture supernatants. In addition, B cells were removed from total splenic lymphocyte cultures. After 1 and 4 days of culture, supernatants were collected and the level of IFN-γ determined using ELISA techniques. Vertical bars and error bars represent the mean ± SE (n = 4). N.D. indicates IFN-γ production was not detectable. An asterisk (*) indicates a statistical difference (P less than or equal to 0.05) in the cytokine production by spleen cells. Asterisks (**) indicate a statistical difference (P less than or equal to 0.05) in the cytokine production between total and B cell-depleted groups.

Article Snippet: 100 µl of biotinylated mouse anti-rat IFN-γ cytokine was added to the appropriate wells and incubated for 1 h. To detect cytokine levels, avidin-HRP and 3, 3', 5, 5'-tetramethylbenzidine substrate (BD PharMingen) were allowed to react on the plate for 30 minutes.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Human Gene Therapy

Article Title: Nonintegrating Lentiviral Vectors Can Effectively Deliver Ovalbumin Antigen for Induction of Antitumor Immunity

doi: 10.1089/hum.2009.012

Figure Lengend Snippet: Nonintegrating lentiviral vector expressing OVA antigen was comparable to its integrating counterpart in inducing antigen-specific CTLs and humoral responses in vivo. (A–G) B6 mice were immunized with either FKOVA/VSVG(IN+) or FKOVA/VSVG(IN–) via footpad injections. The amount of viral particles used for each immunization was quantified to be 900 ng of p24. Mice without immunization (No LV) were included as a control. Mice were analyzed 2 weeks postimmunization. (A) T cells were collected from spleens and examined for the presence of OVAp1-specific CD8+ T cells by tetramer staining. (B) Spleen cells from (A) were restimulated with OVAp1 for 6 hr and IFN-γ secretion was evaluated by intracellular staining. (C) IgG serum specific for OVA was assessed by ELISA. (D–G) On restimulation with OVAp1 or OVAp2, spleen cells from (A) were analyzed by ELISPOT to measure IFN-γ or IL-2 spot-forming cells.

Article Snippet: On the third day, biotinylated anti-mouse IFN-γ or IL-2 antibodies (BD Biosciences) were added to the plate, followed by the addition of streptavidin–alkaline phosphatase (Millipore) and BCIP/NBTplus substrate (Chemicon International, Temecula, CA) to develop spots.

Techniques: Plasmid Preparation, Expressing, In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Human Vaccines & Immunotherapeutics

Article Title: Intranasal immunization with a single dose of the fusion protein formulated with a combination adjuvant induces long-term protective immunity against respiratory syncytial virus

doi: 10.1080/21645515.2017.1349584

Figure Lengend Snippet: RSV ΔF-specific systemic humoral immune responses in mice. (A) Serum IgG1 and IgG2a titers before challenge (B/C), (B) Serum IgG1 and IgG2a titers after challenge (A/C) with RSV, (C) Serum VN antibody titers determined before (B/C) and after (A/C) RSV challenge, and (D) Numbers of IFN-γ and IL-5 secreting splenocytes determined in response to in vitro restimulation with ΔF protein. BALB/c mice were immunized once IN with ΔF formulated with TriAdj and challenged 3 weeks later with RSV. Control groups were immunized with PBS and challenged with RSV (Placebo) or mock-challenged (Placebo/mock). ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus neutralization titers are expressed as the highest dilution of serum that resulted in <50% of cells displaying cytopathic effects. Cytokine secreting cell numbers are expressed as the difference in the number of spots between ΔF-stimulated wells and medium-control wells. Bars indicate median values with interquartile ranges. **P < 0.01; ***P < 0.001.

Article Snippet: Briefly, splenocytes and LN cells were stimulated with ΔF protein (2 µg/ml) and spots were developed with biotinylated IFN-γ- or IL-5-specific goat anti-mouse IgG (BD Biosciences, 554410, 554397) or biotinylated goat anti-mouse IgA-specific antibody, followed by AP-conjugated streptavidin and BCIP/NBT (Sigma-Aldrich, B5655) as the substrate.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control, Neutralization

Journal: Human Vaccines & Immunotherapeutics

Article Title: Intranasal immunization with a single dose of the fusion protein formulated with a combination adjuvant induces long-term protective immunity against respiratory syncytial virus

doi: 10.1080/21645515.2017.1349584

Figure Lengend Snippet: Mucosal immune responses to RSV ΔF protein in mice after challenge with RSV. The IgA titers (A), virus titers (B), percentages of ΔF-specific CD8 + T cells (C), and ΔF-specific IFN-γ secreting CD8 + T cells (D) were measured in the lung after RSV challenge. Mice were immunized and challenged as described in the legend for Fig. 1 . ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus replication in the lungs is expressed as pfu per gram of lung tissue. The proportion of RSV F-specific CD8 + T cells was determined in the lung by KYKNAVTEL-MHC I pentamer staining. IFN-γ expression by CD8 + T cells was determined by intracellular cytokine staining. Bars represent median values with interquartile ranges. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Briefly, splenocytes and LN cells were stimulated with ΔF protein (2 µg/ml) and spots were developed with biotinylated IFN-γ- or IL-5-specific goat anti-mouse IgG (BD Biosciences, 554410, 554397) or biotinylated goat anti-mouse IgA-specific antibody, followed by AP-conjugated streptavidin and BCIP/NBT (Sigma-Aldrich, B5655) as the substrate.

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Staining, Expressing

Journal: Human Vaccines & Immunotherapeutics

Article Title: Intranasal immunization with a single dose of the fusion protein formulated with a combination adjuvant induces long-term protective immunity against respiratory syncytial virus

doi: 10.1080/21645515.2017.1349584

Figure Lengend Snippet: Systemic immune responses to RSV ΔF protein. (A) Serum IgG1 and IgG2a titers before challenge (B/C), (B) Serum IgG1 and IgG2a titers after RSV challenge (A/C), (C) Serum VN antibody titers after RSV challenge (A/C), and (D) Numbers of IFN-γ and IL-5 secreting splenocytes determined in response to in vitro restimulation with ΔF protein. BALB/c mice were immunized once IN with ΔF formulated with TriAdj and challenged with RSV on day 150. Control groups were immunized with PBS and challenged with RSV (Placebo) or mock-challenged (Placebo/mock). ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus neutralization titers are expressed as the highest dilution of serum that resulted in <50% of cells displaying cytopathic effects. Cytokine secreting cell numbers are expressed as the difference in the number of spots between ΔF-stimulated wells and medium-control wells. Bars indicate median values with interquartile ranges. ***P < 0.001.

Article Snippet: Briefly, splenocytes and LN cells were stimulated with ΔF protein (2 µg/ml) and spots were developed with biotinylated IFN-γ- or IL-5-specific goat anti-mouse IgG (BD Biosciences, 554410, 554397) or biotinylated goat anti-mouse IgA-specific antibody, followed by AP-conjugated streptavidin and BCIP/NBT (Sigma-Aldrich, B5655) as the substrate.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control, Neutralization