il 17a neutralizing antibodies  (R&D Systems)

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: SDS-PAGE and Western blot analysis of hScFv-IL-17A proteins expressed in various E. coli strains. (a) SDS-PAGE of insoluble fractions; (b) SDS-PAGE of soluble fractions; (c) Western blot of insoluble fractions; (d) Western blot of soluble fractions. Lane 1: M (molecular weight marker); Lanes 2–4: hScFv-IL-17A-WT expressed in E. coli Origami B (DE3), SHuffle, and BL21 (DE3) with DisCoTune, respectively; Lanes 5–7: hScFv-IL-17A-C97S expressed in the same strains; Lane 8: purified hScFv M6-1B9 (positive control). Western blot detection was performed using an anti-His tag antibody. The arrow indicates the expected size of hScFv-IL-17A (∼28 kDa). Estimated amounts of hScFv-IL-17A are labeled. < LOD indicates signals below the limit of detection.

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: SDS Page, Western Blot, Molecular Weight, Marker, Purification, Positive Control, Labeling

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: The expression efficiency of soluble hScFv-IL-17A-WT (orange) and hScFv-IL-17A-C97S (green) in different expression systems: (WT/Ori and C97S/Ori) from Origami B (DE3); (WT/SHuffle and C97S/SHuffle) from SHuffle; and (WT/BL21Dc and C97S/BL21Dc) from BL21 (DE3) with DisCoTune. Data represent mean ± SD from three independent biological replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Expressing

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Indirect ELISA of hScFv-IL-17A binding to hIL-17A using an avidin–biotin system. Purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S were tested at 1 and 5 µg/mL, expressed from SHuffle (WT/SHuffle-1, WT/SHuffle-5; C97S/SHuffle-1, C97S/SHuffle-5; light/dark green) and BL21 (DE3) with DisCoTune (WT/BL21Dc-1, WT/BL21Dc-5; C97S/BL21Dc-1, C97S/BL21Dc-5; light/dark orange). An IL-17A mAb (dark grey) was used as a positive control. Data represent mean ± SD from three independent biological replicates. Statistical analysis was determined by unpaired two-tailed Student’s t -test. Significance levels are denoted as: ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Indirect ELISA, Binding Assay, Avidin-Biotin Assay, Purification, Positive Control, Two Tailed Test

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Binding assessment of hScFv-IL-17A-WT (a) and hScFv-IL-17A-C97S (b) from BL21 (DE3) with DisCoTune by competitive ELISA. Various concentrations (0.1, 1, and 2 µg/mL) of IL-17A mAb (neutralizing antibody) were tested in competition with hScFv-IL-17A. WT was used at 1 µg/mL, whereas C97S was used at 5 µg/mL to compensate for its reduced binding activity observed in indirect ELISA. An anti-IFN-γ mAb (clone B27) was used as an irrelevant antibody control. Data represent mean ± SD from three independent biological replicates.

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Binding Assay, Competitive ELISA, Activity Assay, Indirect ELISA, Control

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Structural alignment of hScFv-IL-17A-WT and hScFvIL-17A-C97S. The predicted structures were generated using ABodyBuilder2. The conformational structures of hScFv-IL-17A-WT and hScFv-IL-17A-C97S were compared using UCSF ChimeraX software. The red ribbon indicates hScFv-IL-17A-WT structure, and the gold ribbon indicates hScFv-IL-17A-C97S structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Generated, Software

Journal: PLOS ONE

Article Title: Effect of IL-17A on the immune response to pulmonary tuberculosis induced by high- and low-virulence strains of Mycobacterium bovis

doi: 10.1371/journal.pone.0307307

Figure Lengend Snippet: Primer sequences.

Article Snippet: To evaluate the immunological contribution of IL-17A to M . bovis infection, particularly in the development of necrosis and protection against infection, groups of BALB/c mice were infected with strain 04–303 and treated with 25 μg of IL-17A neutralizing antibodies (R&D Systems, Minneapolis, MN, USA; MAB421) or isotype control antibodies (R&D Systems; MAB006), administered directly to the lungs by endotracheal instillation every other day between 13 and 19 days post-infection.

Techniques:

Journal: PLOS ONE

Article Title: Effect of IL-17A on the immune response to pulmonary tuberculosis induced by high- and low-virulence strains of Mycobacterium bovis

doi: 10.1371/journal.pone.0307307

Figure Lengend Snippet: A) Quantitative expression of IL-17A gene transcription determined by RT-PCR in the infected lungs of mice with M . bovis attenuated strain 534 or highly virulent strain 04–303 (n = 3 per timepoint /group). B) IL-17A total intensity expression in lungs assessed by digital pathology during infection with the indicated M . bovis strains (n = 3 per timepoint /group). The immunohistochemically stained sections were digitized and analyzed using an Aperio Scanscope CS. An algorithm was developed to quantify the cytoplasmic protein expression related to stain intensity, and the average of different stained areas was analyzed and represented as total intensity expression (pixels/μm 2 ), which corresponds to the estimated concentration of the selected cytokine in the indicated lung compartment. C) Total intensity expression of IL-17A in granulomas and pneumonia induced by strain 04–303 (n = 3 per timepoint /group). One representative example is shown from two independent experiments. Data were analyzed by ANOVA followed by the Bonferroni multiple comparison test. Each graph represents the means ± SE of IL-17A cellular detection of three mice for each day of the time course. Asterisks represent statistical significance between strains (*p<0.05, **p<0.01, **p<0.001). D) Representative micrographs of IL-17A immunostaining in non-infected lungs showing positive labeling in the bronchial epithelium (arrow) and some lymphocytes, and, on Day 21 post-infection, showing numerous positive cells in granuloma induced by infection with strain 534 and in pneumonic areas induced by strain 04–303 near necrotic cells (200x and 400x magnification, respectively).

Article Snippet: To evaluate the immunological contribution of IL-17A to M . bovis infection, particularly in the development of necrosis and protection against infection, groups of BALB/c mice were infected with strain 04–303 and treated with 25 μg of IL-17A neutralizing antibodies (R&D Systems, Minneapolis, MN, USA; MAB421) or isotype control antibodies (R&D Systems; MAB006), administered directly to the lungs by endotracheal instillation every other day between 13 and 19 days post-infection.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Staining, Concentration Assay, Comparison, Immunostaining, Labeling

Journal: PLOS ONE

Article Title: Effect of IL-17A on the immune response to pulmonary tuberculosis induced by high- and low-virulence strains of Mycobacterium bovis

doi: 10.1371/journal.pone.0307307

Figure Lengend Snippet: A) Gating strategy of representative flow cytometry samples of lung lobes from three different BALB/c mice per group infected with M . bovis strains 534 or 04–303 (n = 3 per timepoint /group). First, the granulocyte zone was selected from the FSC-H vs FSC-A gate, and 100,000 live CD11b + cells were selected to obtain the percentage of Ly6G + IL-17A + neutrophils. Then, the lymphocyte zone was selected from the FSC-H vs FSC-A gate, and 100,000 CD3 + live cells were selected to obtain the percentage of CD8 + IFN-γ + lymphocytes. B) Frequency of neutrophils (CD11c + Ly6G + ). C) Frequency of IL-17A + neutrophils. D) Frequency of CD8 + T lymphocytes (CD3 + CD8 + IFN + ). One representative example is shown from two independent experiments. Data were analyzed with ANOVA followed by the Bonferroni multiple comparison test. Values are expressed as mean ± SE. Asterisks represent statistical significance between strains (*p<0.05).

Article Snippet: To evaluate the immunological contribution of IL-17A to M . bovis infection, particularly in the development of necrosis and protection against infection, groups of BALB/c mice were infected with strain 04–303 and treated with 25 μg of IL-17A neutralizing antibodies (R&D Systems, Minneapolis, MN, USA; MAB421) or isotype control antibodies (R&D Systems; MAB006), administered directly to the lungs by endotracheal instillation every other day between 13 and 19 days post-infection.

Techniques: Flow Cytometry, Infection, Comparison

Journal: PLOS ONE

Article Title: Effect of IL-17A on the immune response to pulmonary tuberculosis induced by high- and low-virulence strains of Mycobacterium bovis

doi: 10.1371/journal.pone.0307307

Figure Lengend Snippet: A) Gating strategy of representative flow cytometry samples of lung lobes from three different BALB/c mice per group infected with M . bovis strains 534 or 04–303 (n = 3 per timepoint /group). The lymphocyte zone was selected from the FSC-H vs FSC-A gate, and 100,000 alive cells were selected from the CD3 vs CD4 gate to obtain the percentage of CD4 + IL17A + , CD4 + IL4 + , or CD4 + IFN-γ + lymphocytes. B) Percentage of Th17 cells (CD3 + CD4 + IL-17A + ). C) Percentage of Th1 lymphocytes (CD3 + CD4 + IFN + ). D) Percentage of Th2 lymphocytes (CD3 + CD4 + IL-4 + ). One representative example is shown from two independent experiments. Data were analyzed with ANOVA followed by the Bonferroni multiple comparison test. Values are expressed as mean ± SE. Asterisks represent statistical significance between strains (*p<0.05, ****p<0.0001).

Article Snippet: To evaluate the immunological contribution of IL-17A to M . bovis infection, particularly in the development of necrosis and protection against infection, groups of BALB/c mice were infected with strain 04–303 and treated with 25 μg of IL-17A neutralizing antibodies (R&D Systems, Minneapolis, MN, USA; MAB421) or isotype control antibodies (R&D Systems; MAB006), administered directly to the lungs by endotracheal instillation every other day between 13 and 19 days post-infection.

Techniques: Flow Cytometry, Infection, Comparison

Journal: PLOS ONE

Article Title: Effect of IL-17A on the immune response to pulmonary tuberculosis induced by high- and low-virulence strains of Mycobacterium bovis

doi: 10.1371/journal.pone.0307307

Figure Lengend Snippet: BALB/c mice were infected with M . bovis strain 04–303 and treated with 25 μg of IL-17A neutralizing antibodies (R&D Systems; MAB421) or isotype control antibodies (R&D Systems; MAB006) administered directly to the lung by endotracheal instillation every third day from Days 13 to 19 post-infection. A) Survival curves: mice treated with blocking IL-17A antibodies show significantly higher mortality. B) Significantly higher bacillary loads are seen in mice after eight days of start anti-IL-17A antibody administration (n = 5 per timepoint /group). C) Automated morphometry measurement of necrosis confirms more tissue damage in treated than in control mice (n = 4 per timepoint /group). D) Representative micrographs of the lungs of treated and non-treated mice with neutralizing IL-17A antibodies: the mouse lungs after five days of treatment show more extensive tissue damage (white asterisks indicate necrotic areas). Bacillary loads and morphometry curves were measured in five animals per time point in two different experiments. One representative example is shown from two independent experiments. Data were analyzed by applying ANOVA followed by the Bonferroni multiple comparison test. Asterisks represent statistical significance between treatments; values are expressed as mean ± SE (*p<0.05, **p<0.005, ****p<0.0001).

Article Snippet: To evaluate the immunological contribution of IL-17A to M . bovis infection, particularly in the development of necrosis and protection against infection, groups of BALB/c mice were infected with strain 04–303 and treated with 25 μg of IL-17A neutralizing antibodies (R&D Systems, Minneapolis, MN, USA; MAB421) or isotype control antibodies (R&D Systems; MAB006), administered directly to the lungs by endotracheal instillation every other day between 13 and 19 days post-infection.

Techniques: Infection, Control, Blocking Assay, Comparison

Journal: PLOS ONE

Article Title: Effect of IL-17A on the immune response to pulmonary tuberculosis induced by high- and low-virulence strains of Mycobacterium bovis

doi: 10.1371/journal.pone.0307307

Figure Lengend Snippet: BALB/c mice were infected with low-virulence M . bovis strain 534 and treated with 25 μg of IL-17A neutralizing antibodies (R&D Systems; MAB421) or isotype control antibodies (R&D Systems; MAB006) administered directly to the lung by endotracheal instillation every third day from Days 6 to 12 post-infection. A) Survival rates of infected and treated mice. B) Lung bacillary loads of infected and treated mice (n = 5 per timepoint /group). C) Granuloma size of infected and treated lungs (n = 4 per timepoint /group). D) Representative micrographs of infected and treated mice with anti-IL-17A or IgG2a isotype on Day 7 post-infection, showing smaller granulomas and lower inflammatory infiltrate around the airways and alveolar-capillary interstitium in mice treated with IL-17A blocking antibodies (micrographs of hematoxylin/eosin staining, 200x magnification). One representative example is shown from two independent experiments Data were analyzed by applying ANOVA followed by the Bonferroni multiple comparison test.

Article Snippet: To evaluate the immunological contribution of IL-17A to M . bovis infection, particularly in the development of necrosis and protection against infection, groups of BALB/c mice were infected with strain 04–303 and treated with 25 μg of IL-17A neutralizing antibodies (R&D Systems, Minneapolis, MN, USA; MAB421) or isotype control antibodies (R&D Systems; MAB006), administered directly to the lungs by endotracheal instillation every other day between 13 and 19 days post-infection.

Techniques: Infection, Control, Blocking Assay, Staining, Comparison

Journal: PLOS ONE

Article Title: Effect of IL-17A on the immune response to pulmonary tuberculosis induced by high- and low-virulence strains of Mycobacterium bovis

doi: 10.1371/journal.pone.0307307

Figure Lengend Snippet: A) Quantitative gene expression of the indicated cytokines was determined by RT-PCR using total RNA isolated from the lungs of infected mice treated with IL-17A blocking antibodies and control animals that received IgG isotype. Blocking IL-17A induced significantly higher expression of IL-23, G-CSF, and the Th2 cytokine IL-13 than in control mice and non-significantly higher transcription of the pro-inflammatory cytokines IFN-γ and TNF-α and the Th2 cytokine IL-4 (n = 3 per timepoint /group). B) Frequency of Th1 lymphocytes (CD3 + CD4 + IFN + ), Th2 lymphocytes (CD3 + CD4 + IL-4 + ), Th17 lymphocytes (CD3 + CD4 + IL-17A + ), CD3 + CD4 + TNF + lymphocytes, CD8 + T lymphocytes (CD3 + CD8 + ), IFN + , TNF + , and IL-4 + , and neutrophils (CD11c+ Ly6G+), IL-17A+, and TNF+ (n = 3 per timepoint /group). One representative example is shown from two independent experiments. Data were analyzed with ANOVA followed by the Bonferroni multiple comparison test. Each graph represents the means ± SE of at least three mice for each of the time points. Asterisks represent statistical significance between groups (*p<0.05, **p<0.01).

Article Snippet: To evaluate the immunological contribution of IL-17A to M . bovis infection, particularly in the development of necrosis and protection against infection, groups of BALB/c mice were infected with strain 04–303 and treated with 25 μg of IL-17A neutralizing antibodies (R&D Systems, Minneapolis, MN, USA; MAB421) or isotype control antibodies (R&D Systems; MAB006), administered directly to the lungs by endotracheal instillation every other day between 13 and 19 days post-infection.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Infection, Blocking Assay, Control, Comparison