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goat polyclonal biotinylated antibody  (R&D Systems)


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    R&D Systems goat polyclonal biotinylated antibody
    Goat Polyclonal Biotinylated Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/biotinylated+tnf/Rat+TNF-alpha+Biotinylated+Antibody/pmc12861154-76-24-28
    Average 93 stars, based on 15 article reviews
    goat polyclonal biotinylated antibody - by Bioz Stars, 2026-10
    93/100 stars

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    Related Articles

    Cell Culture:

    Article Title: Inhibition of TBK1/IKKε mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing
    Article Snippet: .. 1 × 107 BLM or D10 cells (sgCtrl or sgTBK1/IKKε) were cultured for 24 h and subsequently incubated with 100 ng/mL biotinylated TNF (BT210–010, R&D Systems) or unlabeled TNF (300-01 A, PeproTech) for 10 min. .. Following this treatment, cells were harvested and lysed using a homemade IP lysis buffer (30 mM Tris-HCl pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100) containing a protease and phosphatase inhibitor cocktail (Roche).

    Article Title: RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells
    Article Snippet: .. 1×10 7 BLM cells (sgCtrl or sgRNF31) were cultured with the addition of HOIPIN-8 (20 μM) or vehicle for 24 h before treatment with 100 ng/mL biotinylated TNF (BT210–010, R&D Systems) or unlabeled TNF (Peprotech) for 10 min. .. Cells were then collected and lysed in IP lysis buffer (30 mM Tris-HCl pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100 and supplemented with protease inhibitor cocktail (04693159001, Roche)).

    Article Title: Inhibition of TBK1/IKKe mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing
    Article Snippet: .. 1×107 BLM or D10 cells (sgCtrl or sgTBK1/IKKe) were cultured for 24 hours and subsequently incubated with 100 ng/mL biotinylated TNF (BT210–010, R&D Systems) or unlabeled TNF (300-01A, PeproTech) for 10 minutes. .. Following this treatment, cells were harvested and lysed using a homemade IP lysis buffer (30 mM Tris-HCl pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100) containing a protease and phosphatase inhibitor cocktail (Roche).

    Article Title: Inhibition of TBK1/IKKε mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing.
    Article Snippet: .. 1 × 107 BLM or D10 cells (sgCtrl or sgTBK1/IKKε) were cultured for 24 h and subsequently incubated with 100 ng/mL biotinylated TNF (BT210–010, R&D Systems) or unlabeled TNF (300-01 A, PeproTech) for 10min. .. Following this treatment, cells were harvested and lysed using a homemade IP lysis buffer (30mM Tris-HCl pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100) containing a protease and phosphatase inhibitor cocktail (Roche).

    Incubation:

    Article Title: Inhibition of TBK1/IKKε mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing
    Article Snippet: .. 1 × 107 BLM or D10 cells (sgCtrl or sgTBK1/IKKε) were cultured for 24 h and subsequently incubated with 100 ng/mL biotinylated TNF (BT210–010, R&D Systems) or unlabeled TNF (300-01 A, PeproTech) for 10 min. .. Following this treatment, cells were harvested and lysed using a homemade IP lysis buffer (30 mM Tris-HCl pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100) containing a protease and phosphatase inhibitor cocktail (Roche).

    Article Title: Inhibition of TBK1/IKKe mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing
    Article Snippet: .. 1×107 BLM or D10 cells (sgCtrl or sgTBK1/IKKe) were cultured for 24 hours and subsequently incubated with 100 ng/mL biotinylated TNF (BT210–010, R&D Systems) or unlabeled TNF (300-01A, PeproTech) for 10 minutes. .. Following this treatment, cells were harvested and lysed using a homemade IP lysis buffer (30 mM Tris-HCl pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100) containing a protease and phosphatase inhibitor cocktail (Roche).

    Article Title: Inhibition of TBK1/IKKε mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing.
    Article Snippet: .. 1 × 107 BLM or D10 cells (sgCtrl or sgTBK1/IKKε) were cultured for 24 h and subsequently incubated with 100 ng/mL biotinylated TNF (BT210–010, R&D Systems) or unlabeled TNF (300-01 A, PeproTech) for 10min. .. Following this treatment, cells were harvested and lysed using a homemade IP lysis buffer (30mM Tris-HCl pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100) containing a protease and phosphatase inhibitor cocktail (Roche).

    other:

    Article Title: Zinc Oxide Nanoparticles Suppress LPS-Induced NF-κB Activation by Inducing A20, a Negative Regulator of NF-κB, in RAW 264.7 Macrophages.
    Article Snippet: Zinc contained in solar salt and bamboo salt plays a critical role in various immune responses.. Zinc oxide is a source of zinc, and recently it has been reported that zinc oxide nanoparticles (ZO-NP) more effectively decrease allergic inflammatory reactions than zinc oxide bulk material.. The aim of this work was to investigate the regulatory effect of ZO-NP on interferon (IFN)plus lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

    Recombinant:

    Article Title: Fritillaria ussuriensis extract inhibits the production of inflammatory cytokine and MAPKs in mast cells.
    Article Snippet: Fritillaria ussuriensis (FU, derived from the bulbs of various species of the genus Fritillaria, including Fritillaria thunbergii Miq.) is used in herbal medicine to treat conditions such as eczema, skin burns, and frostbite.. In this study, we investigated the mechanism of the anti-allergy effect of FU.. FU extract (80mg/kg), orally administered to Sprague-Dawley (SD) rats, significantly inhibited the passive cutaneous anaphylaxis (PCA) reaction.



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    (a) Schematic of laser-cut PDMS microwell array reversibly assembled on a polystyrene petri dish and workflow of enzymatically amplified silver metallization using Biotin-BSA and poly-HRP-SA. (b) Cell-phone image of silver metallized wells after assay. (c) Biotin-BSA dilution curve obtained from cell-phone image silver darkness quantification in ImageJ. (d) Assay stack for initial biotinyl tyramide signal amplification testing using <t>biotinylated</t> anti-IFN-γ as capture. (e) Dilution curve of silver darkness of increasing biotinylated anti-IFN-γ as target and 10 µg/mL, 1 µg/mL, and 0 µg/mL biotinyl tyramide. All assays were repeated three (n=3) times and fit lines are four-parameter logistic sigmoidal curves.
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    Fusion to TfR1b-Nb enables CNS delivery of TNFI-Nb1 via human TfR1-mediated transcytosis. a <t>TNFα-based</t> ELISA quantification of TNFI-Nb1 in serum and brain homogenates from Tfr1 h/w and Tfr1 w/w rats, 6 h after intravenous injection. TNFI-Nb1 was detectable in serum but absent in brain tissue, indicating lack of BBB permeability. b SDS-PAGE and SEC-HPLC analysis of the heterodimer composed of TNFI-Nb1 fused to TfR1b-Nb 04B05R3 (Family A), produced in CHO-S cells. C ) TNFα-based ELISA quantification of TNFI-Nb1 ~ 04B05R3 in serum, CSF, and brain homogenates of Tfr1 h/w and Tfr1 w/w rats, 48 h post-injection. The heterodimer was present in serum of both genotypes but detected in CSF and brain only in Tfr1 h/w rats, demonstrating human TfR1-dependent BBB transcytosis. The observed CSF/serum ratio (TNFα-based ELISA) of ~ 1 indicates efficient CNS delivery. Statistical analyses were conducted using unpaired two-tailed t-tests, with p-values reported in the figure panels. Data are presented as mean ± SEM
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    Fusion to TfR1b-Nb enables CNS delivery of TNFI-Nb1 via human TfR1-mediated transcytosis. <t>a</t> <t>TNFα-based</t> <t>ELISA</t> quantification of TNFI-Nb1 in serum and brain homogenates from Tfr1 h/w and Tfr1 w/w rats, 6 h after intravenous injection. TNFI-Nb1 was detectable in serum but absent in brain tissue, indicating lack of BBB permeability. b SDS-PAGE and SEC-HPLC analysis of the heterodimer composed of TNFI-Nb1 fused to TfR1b-Nb 04B05R3 (Family A), produced in CHO-S cells. C ) TNFα-based ELISA quantification of TNFI-Nb1 ~ 04B05R3 in serum, CSF, and brain homogenates of Tfr1 h/w and Tfr1 w/w rats, 48 h post-injection. The heterodimer was present in serum of both genotypes but detected in CSF and brain only in Tfr1 h/w rats, demonstrating human TfR1-dependent BBB transcytosis. The observed CSF/serum ratio (TNFα-based ELISA) of ~ 1 indicates efficient CNS delivery. Statistical analyses were conducted using unpaired two-tailed t-tests, with p-values reported in the figure panels. Data are presented as mean ± SEM
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    Image Search Results


    (a) Schematic of laser-cut PDMS microwell array reversibly assembled on a polystyrene petri dish and workflow of enzymatically amplified silver metallization using Biotin-BSA and poly-HRP-SA. (b) Cell-phone image of silver metallized wells after assay. (c) Biotin-BSA dilution curve obtained from cell-phone image silver darkness quantification in ImageJ. (d) Assay stack for initial biotinyl tyramide signal amplification testing using biotinylated anti-IFN-γ as capture. (e) Dilution curve of silver darkness of increasing biotinylated anti-IFN-γ as target and 10 µg/mL, 1 µg/mL, and 0 µg/mL biotinyl tyramide. All assays were repeated three (n=3) times and fit lines are four-parameter logistic sigmoidal curves.

    Journal: bioRxiv

    Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

    doi: 10.64898/2026.01.27.700981

    Figure Lengend Snippet: (a) Schematic of laser-cut PDMS microwell array reversibly assembled on a polystyrene petri dish and workflow of enzymatically amplified silver metallization using Biotin-BSA and poly-HRP-SA. (b) Cell-phone image of silver metallized wells after assay. (c) Biotin-BSA dilution curve obtained from cell-phone image silver darkness quantification in ImageJ. (d) Assay stack for initial biotinyl tyramide signal amplification testing using biotinylated anti-IFN-γ as capture. (e) Dilution curve of silver darkness of increasing biotinylated anti-IFN-γ as target and 10 µg/mL, 1 µg/mL, and 0 µg/mL biotinyl tyramide. All assays were repeated three (n=3) times and fit lines are four-parameter logistic sigmoidal curves.

    Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

    Techniques: Amplification

    (a) Schematic of immunoassay for cytokine detection. (b) Initial LOD optimization for the IFN-γ assay. (c-e) IFN-γ (n=2), IL-2 (n=5), TNF-α (n=2) dilution curves using recombinant cytokines (f-h) Correlation plots of ELISA absorbance values at 450 nm versus silver darkness values for IFN-γ (f), TNF-α (g), and IL-2 (h). All fit lines are four-parameter logistic sigmoidal curves.

    Journal: bioRxiv

    Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

    doi: 10.64898/2026.01.27.700981

    Figure Lengend Snippet: (a) Schematic of immunoassay for cytokine detection. (b) Initial LOD optimization for the IFN-γ assay. (c-e) IFN-γ (n=2), IL-2 (n=5), TNF-α (n=2) dilution curves using recombinant cytokines (f-h) Correlation plots of ELISA absorbance values at 450 nm versus silver darkness values for IFN-γ (f), TNF-α (g), and IL-2 (h). All fit lines are four-parameter logistic sigmoidal curves.

    Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

    (a) Schematic demonstrating the stimulation groups and controls. Dilution curves for IFN-γ (b-c), IL-2 (d-e), and TNF-α (f-g) in pooled LTB+ and LTB-clinical supernatant samples. Clinical samples consisted of NIL (negative control), Ag1, Ag2, and Mitogen (positive control) groups (n=2).

    Journal: bioRxiv

    Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

    doi: 10.64898/2026.01.27.700981

    Figure Lengend Snippet: (a) Schematic demonstrating the stimulation groups and controls. Dilution curves for IFN-γ (b-c), IL-2 (d-e), and TNF-α (f-g) in pooled LTB+ and LTB-clinical supernatant samples. Clinical samples consisted of NIL (negative control), Ag1, Ag2, and Mitogen (positive control) groups (n=2).

    Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

    Techniques: Negative Control, Positive Control

    Individual participant silver darkness values for IFN-γ (a), IL-2 (b), and TNF-α (c) detection from NIL, Ag1, Ag2, and Mitogen samples of IGRA+ and IGRA-patients using a sample dilution of 1:10. Average of n=2 repeats was plotted for each sample.

    Journal: bioRxiv

    Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

    doi: 10.64898/2026.01.27.700981

    Figure Lengend Snippet: Individual participant silver darkness values for IFN-γ (a), IL-2 (b), and TNF-α (c) detection from NIL, Ag1, Ag2, and Mitogen samples of IGRA+ and IGRA-patients using a sample dilution of 1:10. Average of n=2 repeats was plotted for each sample.

    Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

    Techniques:

    Fusion to TfR1b-Nb enables CNS delivery of TNFI-Nb1 via human TfR1-mediated transcytosis. a TNFα-based ELISA quantification of TNFI-Nb1 in serum and brain homogenates from Tfr1 h/w and Tfr1 w/w rats, 6 h after intravenous injection. TNFI-Nb1 was detectable in serum but absent in brain tissue, indicating lack of BBB permeability. b SDS-PAGE and SEC-HPLC analysis of the heterodimer composed of TNFI-Nb1 fused to TfR1b-Nb 04B05R3 (Family A), produced in CHO-S cells. C ) TNFα-based ELISA quantification of TNFI-Nb1 ~ 04B05R3 in serum, CSF, and brain homogenates of Tfr1 h/w and Tfr1 w/w rats, 48 h post-injection. The heterodimer was present in serum of both genotypes but detected in CSF and brain only in Tfr1 h/w rats, demonstrating human TfR1-dependent BBB transcytosis. The observed CSF/serum ratio (TNFα-based ELISA) of ~ 1 indicates efficient CNS delivery. Statistical analyses were conducted using unpaired two-tailed t-tests, with p-values reported in the figure panels. Data are presented as mean ± SEM

    Journal: Cell Communication and Signaling : CCS

    Article Title: The NewroBus platform: engineered humanized anti-TfR1 nanobodies for efficient brain delivery

    doi: 10.1186/s12964-025-02605-1

    Figure Lengend Snippet: Fusion to TfR1b-Nb enables CNS delivery of TNFI-Nb1 via human TfR1-mediated transcytosis. a TNFα-based ELISA quantification of TNFI-Nb1 in serum and brain homogenates from Tfr1 h/w and Tfr1 w/w rats, 6 h after intravenous injection. TNFI-Nb1 was detectable in serum but absent in brain tissue, indicating lack of BBB permeability. b SDS-PAGE and SEC-HPLC analysis of the heterodimer composed of TNFI-Nb1 fused to TfR1b-Nb 04B05R3 (Family A), produced in CHO-S cells. C ) TNFα-based ELISA quantification of TNFI-Nb1 ~ 04B05R3 in serum, CSF, and brain homogenates of Tfr1 h/w and Tfr1 w/w rats, 48 h post-injection. The heterodimer was present in serum of both genotypes but detected in CSF and brain only in Tfr1 h/w rats, demonstrating human TfR1-dependent BBB transcytosis. The observed CSF/serum ratio (TNFα-based ELISA) of ~ 1 indicates efficient CNS delivery. Statistical analyses were conducted using unpaired two-tailed t-tests, with p-values reported in the figure panels. Data are presented as mean ± SEM

    Article Snippet: The same procedure was used with the following modifications: plates were coated with 0.2 μg/mL biotinylated human TNFα (Acro Biosystems, TNA-H82E3-25ug) instead of TfR1.

    Techniques: Enzyme-linked Immunosorbent Assay, Injection, Permeability, SDS Page, Produced, Two Tailed Test

    TfR1 binding and TNFα inhibitory activity of optimized and humanized heterodimers. a Schematic representation of the 16 humanized and optimized TNFI–TfR1b heterodimers. b Flow cytometry analysis in HEK293 cells stably expressing human TfR1, comparing the binding of heterodimers to that of their parental humanized TfR1b-Nbs. c ) Dose–response apoptosis inhibition curves in WEHI-13VAR cells treated with human TNFα and twofold serial dilutions of TNFI–TfR1b heterodimers (100,000 to 12.21 pM). Apoptosis was measured using a fluorogenic caspase-3/7 activation assay. Caspase-3/7 activity in the TNFα-only samples was defined as 100%. The IC₅₀ values of the heterodimers were comparable to those of the parental humanized nanobodies TNFI-α (IC₅₀ = 48.84 pM, range: 42.11–56.56) and TNFI-β (IC₅₀ = 64.31 pM, range: 57.17–72.31).). Data are presented as mean ± SEM from triplicate measurements and were analyzed using the

    Journal: Cell Communication and Signaling : CCS

    Article Title: The NewroBus platform: engineered humanized anti-TfR1 nanobodies for efficient brain delivery

    doi: 10.1186/s12964-025-02605-1

    Figure Lengend Snippet: TfR1 binding and TNFα inhibitory activity of optimized and humanized heterodimers. a Schematic representation of the 16 humanized and optimized TNFI–TfR1b heterodimers. b Flow cytometry analysis in HEK293 cells stably expressing human TfR1, comparing the binding of heterodimers to that of their parental humanized TfR1b-Nbs. c ) Dose–response apoptosis inhibition curves in WEHI-13VAR cells treated with human TNFα and twofold serial dilutions of TNFI–TfR1b heterodimers (100,000 to 12.21 pM). Apoptosis was measured using a fluorogenic caspase-3/7 activation assay. Caspase-3/7 activity in the TNFα-only samples was defined as 100%. The IC₅₀ values of the heterodimers were comparable to those of the parental humanized nanobodies TNFI-α (IC₅₀ = 48.84 pM, range: 42.11–56.56) and TNFI-β (IC₅₀ = 64.31 pM, range: 57.17–72.31).). Data are presented as mean ± SEM from triplicate measurements and were analyzed using the "Inhibitor vs. Normalized Response" model in GraphPad Prism 10 software

    Article Snippet: The same procedure was used with the following modifications: plates were coated with 0.2 μg/mL biotinylated human TNFα (Acro Biosystems, TNA-H82E3-25ug) instead of TfR1.

    Techniques: Binding Assay, Activity Assay, Flow Cytometry, Stable Transfection, Expressing, Inhibition, Activation Assay, Software

    Fusion to TfR1b-Nb enables CNS delivery of TNFI-Nb1 via human TfR1-mediated transcytosis. a TNFα-based ELISA quantification of TNFI-Nb1 in serum and brain homogenates from Tfr1 h/w and Tfr1 w/w rats, 6 h after intravenous injection. TNFI-Nb1 was detectable in serum but absent in brain tissue, indicating lack of BBB permeability. b SDS-PAGE and SEC-HPLC analysis of the heterodimer composed of TNFI-Nb1 fused to TfR1b-Nb 04B05R3 (Family A), produced in CHO-S cells. C ) TNFα-based ELISA quantification of TNFI-Nb1 ~ 04B05R3 in serum, CSF, and brain homogenates of Tfr1 h/w and Tfr1 w/w rats, 48 h post-injection. The heterodimer was present in serum of both genotypes but detected in CSF and brain only in Tfr1 h/w rats, demonstrating human TfR1-dependent BBB transcytosis. The observed CSF/serum ratio (TNFα-based ELISA) of ~ 1 indicates efficient CNS delivery. Statistical analyses were conducted using unpaired two-tailed t-tests, with p-values reported in the figure panels. Data are presented as mean ± SEM

    Journal: Cell Communication and Signaling : CCS

    Article Title: The NewroBus platform: engineered humanized anti-TfR1 nanobodies for efficient brain delivery

    doi: 10.1186/s12964-025-02605-1

    Figure Lengend Snippet: Fusion to TfR1b-Nb enables CNS delivery of TNFI-Nb1 via human TfR1-mediated transcytosis. a TNFα-based ELISA quantification of TNFI-Nb1 in serum and brain homogenates from Tfr1 h/w and Tfr1 w/w rats, 6 h after intravenous injection. TNFI-Nb1 was detectable in serum but absent in brain tissue, indicating lack of BBB permeability. b SDS-PAGE and SEC-HPLC analysis of the heterodimer composed of TNFI-Nb1 fused to TfR1b-Nb 04B05R3 (Family A), produced in CHO-S cells. C ) TNFα-based ELISA quantification of TNFI-Nb1 ~ 04B05R3 in serum, CSF, and brain homogenates of Tfr1 h/w and Tfr1 w/w rats, 48 h post-injection. The heterodimer was present in serum of both genotypes but detected in CSF and brain only in Tfr1 h/w rats, demonstrating human TfR1-dependent BBB transcytosis. The observed CSF/serum ratio (TNFα-based ELISA) of ~ 1 indicates efficient CNS delivery. Statistical analyses were conducted using unpaired two-tailed t-tests, with p-values reported in the figure panels. Data are presented as mean ± SEM

    Article Snippet: Biotinylated TNFα , ELISA capture antigen , Acro Biosystems , TNA-H82E3-25ug.

    Techniques: Enzyme-linked Immunosorbent Assay, Injection, Permeability, SDS Page, Produced, Two Tailed Test