Nitrotyrosine ELISA 试剂盒
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Quick Overview for Nitrotyrosine ELISA 试剂盒 (ABIN2964845)
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检测方法
实验类型
检测范围
应用范围
样品类型
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最低检测浓度
- 62.5 nM
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原理
- Colorimetric detection of Nitrotyrosine
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Analytical Method
- Quantitative
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灵敏度
- 50 nM
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产品特性
- ELISA kit used to quantitate Nitrotyrosine in samples.
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组件
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- Nitrosylated BSA Coated Plate
- Nitrotyrosine Standard
- Nitrotyrosine HRP Conjugated Monoclonal Antibody
- Sample and Standard Diluent (Red)
- Nitrotyrosine Antibody Diluent (Blue)
- Wash Buffer Concentrate (10X)
- TMB Substrate
- Stop Solution
- Plate Cover
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试剂未包括
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- A plate reader capable of measuring absorbance at 450 nm
- Adjustable pipettes and a repeat pipettor
- Deionized or distilled water
- Materials used for Sample Preparation
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Nitrotyrosine ELISA Kit
适用: 化学剂 Colorimetric Competition ELISA Plasma, Serum
Nitrotyrosine ELISA Kit适用: Various Species Colorimetric Competition ELISA 3.7 ng/mL - 300 ng/mL Plasma, Serum
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实验时间
- 1 h
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板类型
- Pre-coated
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实验流程
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- Prepare standard and samples in the Sample and Standard Diluent.
- Add 50 μL of prepared standards and samples in triplicate to appropriate wells.
- Add 50 μL of the diluted antibody preparation to the appropriate wells.
- Cover plate with Plate Cover and incubate at room temperature (20-25 °C) for 1 hour.
- Wash plate 4 times with 1X Wash Buffer.
- Add 100 μL of TMB Substrate to each well.
- Cover plate and develop the plate in the dark at room temperature for 30 minutes.
- Add 100 μL of Stop Solution to each well.
- Measure absorbance on a plate reader at 450 nm.
- Plot the standard curve and calculate sample concentrations.
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实验流程
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- Prepare standard and samples in the Sample and Standard Diluent.
- Add 50 μL of prepared standards and samples in triplicate to appropriate wells.
- Add 50 μL of the diluted antibody preparation to the appropriate wells.
- Cover plate with Plate Cover and incubate at room temperature (20-25 °C) for 1 hour.
- Wash plate 4 times with 1X Wash Buffer.
- Add 100 μL of TMB Substrate to each well.
- Cover plate and develop the plate in the dark at room temperature for 30 minutes.
- Add 100 μL of Stop Solution to each well.
- Measure absorbance on a plate reader at 450 nm.
- Plot the standard curve and calculate sample concentrations.
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结果分析
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This kit can be analyzed using any of the following methods:
A. Many plate readers come with data reduction software that plot data automatically.
B. The following procedure is recommended for preparation of the data prior to graphical analysis.
1. Calculate the average Net Optical Density (OD) bound for each standard and sample by subtracting the average Blank OD from the average OD bound.
2. Plot Net OD versus Concentration of Nitrotyrosine for the standards. Sample concentrations may be calculated off of Net OD values using the desired curve fitting.
3. Samples that read at concentrations outside of the standard curve range will need to be re-analyzed using a different dilution. Make sure to multiply sample concentrations calculated off the curve by the dilution factor used during sample preparation to get starting sample concentration. -
实验精密度
- Intra-Assay Precision: Three samples of known concentration were assayed thirty times on one plate. The intra-assay coefficient of variation of the Nitrotyrosine ELISA has been determined to be <10%. Inter-Assay Precision: Three samples of known concentration were assayed thirty times in three individual assays. The inter-assay coefficient of variation of the Nitrotyrosine ELISA has been determined to be <15%.
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限制
- 仅限研究用
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储存条件
- 4 °C
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- Nitrotyrosine
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物质类
- Chemical
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背景
- Nitrotyrosine has been identified as a marker of inflammation and NO production. Nitrotyrosine is formed in presence of the active metabolite NO. Various pathways including the formation of peroxinitrite lead to nitrotyrosine production. Since nitrotyrosine is a stable end product of peroxynitrite oxidation, assessment of its plasma concentration may be useful as a marker of NO-dependent damage in vivo. Since NOX is only an indicator for enhanced NO production, protein associated nitrotyrosine might be a more suitable marker for damage induced by reactive nitrogen intermediates derived from NO. Furthermore, most proteins have a longer half life in the circulation than NOX levels. The presence of nitrotyrosine has been detected in various inflammatory processes including atherosclerotic plaques, celiac disease, rheumatoid arthritis, chronic renal failure and septic shock. In normal plasma low, undetectable, levels of nitrotyrosine are present. Nitrosylation of the amino acid tyrosine occurs both for free tyrosine and for protein bound tyrosine.
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