Tumor Necrosis Factor-alpha (TNF-alpha), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders. TNF-alpha, a 17 kDa cytokine, is mainly secreted by activated macrophages and monocytes in response to several inflammatory and immunological stimuli. To study the effect of environmental chemicals on TNF-alpha secretion, a homogeneous TNF-alpha assay was developed more ..
Target
| Sequence: tumor necrosis factor [Homo sapiens] Protein Family: TNF Gene:TNF Related Protein 3D Structures |
BioActive Compounds: 7
Description:
National Center for Advancing Translational Sciences [NCATS]
NIH Chemical Genomics Center [NCGC]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]
Assay Overview:
Tumor Necrosis Factor-alpha (TNF-alpha), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders. TNF-alpha, a 17 kDa cytokine, is mainly secreted by activated macrophages and monocytes in response to several inflammatory and immunological stimuli. To study the effect of environmental chemicals on TNF-alpha secretion, a homogeneous TNF-alpha assay was developed using Time Resolved fluorescence (HTRF) technology based on Time Resolved Fluorescent Resonance Energy Transfer (TR-FRET) (Leister et al., Current Chemical Genomics, 5:21-29, 2011). This assay allows measuring TNF-alpha secreted from the cells in a 1536-well plate format.
NIH Chemical Genomics Center [NCGC]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]
Assay Overview:
Tumor Necrosis Factor-alpha (TNF-alpha), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders. TNF-alpha, a 17 kDa cytokine, is mainly secreted by activated macrophages and monocytes in response to several inflammatory and immunological stimuli. To study the effect of environmental chemicals on TNF-alpha secretion, a homogeneous TNF-alpha assay was developed using Time Resolved fluorescence (HTRF) technology based on Time Resolved Fluorescent Resonance Energy Transfer (TR-FRET) (Leister et al., Current Chemical Genomics, 5:21-29, 2011). This assay allows measuring TNF-alpha secreted from the cells in a 1536-well plate format.
Protocol
Assay protocol summary:
THP-1 cells were dispensed at 3000 cells/5ul/well in 1536-well white solid plates. After addition of 23 nl of compound, positive controls and DMSO only, the assay plates were incubated at 37 C for 17 hrs, followed by addition of 5 uLof mixture of cryptate labeled and XL665 labeled anti-TNF-alpha antibodies. After the assay plates were incubated at room temperature for 3 hr, the plates were measured by an EnVision plate reader.
THP-1 cells were dispensed at 3000 cells/5ul/well in 1536-well white solid plates. After addition of 23 nl of compound, positive controls and DMSO only, the assay plates were incubated at 37 C for 17 hrs, followed by addition of 5 uLof mixture of cryptate labeled and XL665 labeled anti-TNF-alpha antibodies. After the assay plates were incubated at room temperature for 3 hr, the plates were measured by an EnVision plate reader.
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | Phenotype | Indicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive. | String | |||
| 2 | Potency* | Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed. | | Float | μM | |
| 3 | Efficacy | Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves. | | Float | % | |
| 4 | Analysis Comment | Annotation/notes on a particular compound's data or its analysis. | String | |||
| 5 | W665-Activity_Score | Activity score. | | Integer | ||
| 6 | W665-Curve_Description | A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control. | String | |||
| 7 | W665-Fit_LogAC50 | The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). | | Float | ||
| 8 | W665-Fit_HillSlope | The Hill slope from a fit of the data to the Hill equation. | | Float | ||
| 9 | W665-Fit_R2 | R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. | | Float | ||
| 10 | W665-Fit_InfiniteActivity | The asymptotic efficacy from a fit of the data to the Hill equation. | | Float | % | |
| 11 | W665-Fit_ZeroActivity | Efficacy at zero concentration of compound from a fit of the data to the Hill equation. | | Float | % | |
| 12 | W665-Fit_CurveClass | Numerical encoding of curve description for the fitted Hill equation. | | Float | ||
| 13 | W665-Excluded_Points | Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations. | String | |||
| 14 | W665-Max_Response | Maximum activity observed for compound (usually at highest concentration tested). | | Float | % | |
| 15 | W665-Activity at 0.0005899000 uM (0.0005899μM**) | % Activity at given concentration. | | Float | % | |
| 16 | W665-Activity at 0.00295 uM (0.002949μM**) | % Activity at given concentration. | | Float | % | |
| 17 | W665-Activity at 0.015 uM (0.01475μM**) | % Activity at given concentration. | | Float | % | |
| 18 | W665-Activity at 0.033 uM (0.03297μM**) | % Activity at given concentration. | | Float | % | |
| 19 | W665-Activity at 0.074 uM (0.07373μM**) | % Activity at given concentration. | | Float | % | |
| 20 | W665-Activity at 0.165 uM (0.1649μM**) | % Activity at given concentration. | | Float | % | |
| 21 | W665-Activity at 0.369 uM (0.3687μM**) | % Activity at given concentration. | | Float | % | |
| 22 | W665-Activity at 0.824 uM (0.8244μM**) | % Activity at given concentration. | | Float | % | |
| 23 | W665-Activity at 1.843 uM (1.843μM**) | % Activity at given concentration. | | Float | % | |
| 24 | W665-Activity at 4.122 uM (4.122μM**) | % Activity at given concentration. | | Float | % | |
| 25 | W665-Activity at 9.217 uM (9.217μM**) | % Activity at given concentration. | | Float | % | |
| 26 | W665-Activity at 20.61 uM (20.61μM**) | % Activity at given concentration. | | Float | % | |
| 27 | W665-Activity at 46.08 uM (46.08μM**) | % Activity at given concentration. | | Float | % | |
| 28 | W665-Activity at 92.17 uM (92.17μM**) | % Activity at given concentration. | | Float | % | |
| 29 | Ratio-Activity_Score | Activity score. | | Integer | ||
| 30 | Ratio-Curve_Description | A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control. | String | |||
| 31 | Ratio-Fit_LogAC50 | The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). | | Float | ||
| 32 | Ratio-Fit_HillSlope | The Hill slope from a fit of the data to the Hill equation. | | Float | ||
| 33 | Ratio-Fit_R2 | R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. | | Float | ||
| 34 | Ratio-Fit_InfiniteActivity | The asymptotic efficacy from a fit of the data to the Hill equation. | | Float | % | |
| 35 | Ratio-Fit_ZeroActivity | Efficacy at zero concentration of compound from a fit of the data to the Hill equation. | | Float | % | |
| 36 | Ratio-Fit_CurveClass | Numerical encoding of curve description for the fitted Hill equation. | | Float | ||
| 37 | Ratio-Excluded_Points | Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations. | String | |||
| 38 | Ratio-Max_Response | Maximum activity observed for compound (usually at highest concentration tested). | | Float | % | |
| 39 | Ratio-Activity at 0.0005899000 uM (0.0005899μM**) | % Activity at given concentration. | | Float | % | |
| 40 | Ratio-Activity at 0.00295 uM (0.002949μM**) | % Activity at given concentration. | | Float | % | |
| 41 | Ratio-Activity at 0.015 uM (0.01475μM**) | % Activity at given concentration. | | Float | % | |
| 42 | Ratio-Activity at 0.033 uM (0.03297μM**) | % Activity at given concentration. | | Float | % | |
| 43 | Ratio-Activity at 0.074 uM (0.07373μM**) | % Activity at given concentration. | | Float | % | |
| 44 | Ratio-Activity at 0.165 uM (0.1649μM**) | % Activity at given concentration. | | Float | % | |
| 45 | Ratio-Activity at 0.369 uM (0.3687μM**) | % Activity at given concentration. | | Float | % | |
| 46 | Ratio-Activity at 0.824 uM (0.8244μM**) | % Activity at given concentration. | | Float | % | |
| 47 | Ratio-Activity at 1.843 uM (1.843μM**) | % Activity at given concentration. | | Float | % | |
| 48 | Ratio-Activity at 4.122 uM (4.122μM**) | % Activity at given concentration. | | Float | % | |
| 49 | Ratio-Activity at 9.217 uM (9.217μM**) | % Activity at given concentration. | | Float | % | |
| 50 | Ratio-Activity at 20.61 uM (20.61μM**) | % Activity at given concentration. | | Float | % | |
| 51 | Ratio-Activity at 46.08 uM (46.08μM**) | % Activity at given concentration. | | Float | % | |
| 52 | Ratio-Activity at 92.17 uM (92.17μM**) | % Activity at given concentration. | | Float | % | |
| 53 | W615-Activity_Score | Activity score. | | Integer | ||
| 54 | W615-Curve_Description | A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control. | String | |||
| 55 | W615-Fit_LogAC50 | The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). | | Float | ||
| 56 | W615-Fit_HillSlope | The Hill slope from a fit of the data to the Hill equation. | | Float | ||
| 57 | W615-Fit_R2 | R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. | | Float | ||
| 58 | W615-Fit_InfiniteActivity | The asymptotic efficacy from a fit of the data to the Hill equation. | | Float | % | |
| 59 | W615-Fit_ZeroActivity | Efficacy at zero concentration of compound from a fit of the data to the Hill equation. | | Float | % | |
| 60 | W615-Fit_CurveClass | Numerical encoding of curve description for the fitted Hill equation. | | Float | ||
| 61 | W615-Excluded_Points | Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations. | String | |||
| 62 | W615-Max_Response | Maximum activity observed for compound (usually at highest concentration tested). | | Float | % | |
| 63 | W615-Activity at 0.0005899000 uM (0.0005899μM**) | % Activity at given concentration. | | Float | % | |
| 64 | W615-Activity at 0.00295 uM (0.002949μM**) | % Activity at given concentration. | | Float | % | |
| 65 | W615-Activity at 0.015 uM (0.01475μM**) | % Activity at given concentration. | | Float | % | |
| 66 | W615-Activity at 0.033 uM (0.03297μM**) | % Activity at given concentration. | | Float | % | |
| 67 | W615-Activity at 0.074 uM (0.07373μM**) | % Activity at given concentration. | | Float | % | |
| 68 | W615-Activity at 0.165 uM (0.1649μM**) | % Activity at given concentration. | | Float | % | |
| 69 | W615-Activity at 0.369 uM (0.3687μM**) | % Activity at given concentration. | | Float | % | |
| 70 | W615-Activity at 0.824 uM (0.8244μM**) | % Activity at given concentration. | | Float | % | |
| 71 | W615-Activity at 1.843 uM (1.843μM**) | % Activity at given concentration. | | Float | % | |
| 72 | W615-Activity at 4.122 uM (4.122μM**) | % Activity at given concentration. | | Float | % | |
| 73 | W615-Activity at 9.217 uM (9.217μM**) | % Activity at given concentration. | | Float | % | |
| 74 | W615-Activity at 20.61 uM (20.61μM**) | % Activity at given concentration. | | Float | % | |
| 75 | W615-Activity at 46.08 uM (46.08μM**) | % Activity at given concentration. | | Float | % | |
| 76 | W615-Activity at 92.17 uM (92.17μM**) | % Activity at given concentration. | | Float | % | |
| 77 | Compound QC | NCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling' | String |
* Activity Concentration. ** Test Concentration.
Data Table (Concise)
Classification
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