Nitrotyrosine ELISA Kit

Catalog No. ABIN2964845

This Various Species Nitrotyrosine ELISA Kit is a Colorimetric ELISA Kit designed to quantify Various Species Nitrotyrosine.

Quick Overview for Nitrotyrosine ELISA Kit (ABIN2964845)

Target: Nitrotyrosine

Reactivity: Various Species

Detection Method: Colorimetric

Method Type: Competition ELISA

Detection Range: 62.5 nM - 8000 nM

Application: ELISA

Sample Type: Plasma, Serum, Cell Lysate, Urine

Product Details Nitrotyrosine ELISA Kit

Minimum Detection Limit: 62.5 nM

Purpose: Colorimetric detection of Nitrotyrosine

Analytical Method: Quantitative

Sensitivity: 50 nM

Characteristics: ELISA kit used to quantitate Nitrotyrosine in samples.

Components:

• 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

Material not included: - 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

Application Details

Assay Time: 1 h

Plate: Pre-coated

Protocol:

1. Prepare standard and samples in the Sample and Standard Diluent.
2. Add 50 μL of prepared standards and samples in triplicate to appropriate wells.
3. Add 50 μL of the diluted antibody preparation to the appropriate wells.
4. Cover plate with Plate Cover and incubate at room temperature (20-25 °C) for 1 hour.
5. Wash plate 4 times with 1X Wash Buffer.
6. Add 100 μL of TMB Substrate to each well.
7. Cover plate and develop the plate in the dark at room temperature for 30 minutes.
8. Add 100 μL of Stop Solution to each well.
9. Measure absorbance on a plate reader at 450 nm.
10. Plot the standard curve and calculate sample concentrations.

Assay Procedure:

1. Prepare standard and samples in the Sample and Standard Diluent.
2. Add 50 μL of prepared standards and samples in triplicate to appropriate wells.
3. Add 50 μL of the diluted antibody preparation to the appropriate wells.
4. Cover plate with Plate Cover and incubate at room temperature (20-25 °C) for 1 hour.
5. Wash plate 4 times with 1X Wash Buffer.
6. Add 100 μL of TMB Substrate to each well.
7. Cover plate and develop the plate in the dark at room temperature for 30 minutes.
8. Add 100 μL of Stop Solution to each well.
9. Measure absorbance on a plate reader at 450 nm.
10. Plot the standard curve and calculate sample concentrations.

Calculation of Results:

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.

Assay Precision: 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%.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Target Details for Nitrotyrosine

Target: Nitrotyrosine

Target Type: Chemical

Background: 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.