• Various species
• High sensitivity
• Rapid detection within 2.5 hours
• Good characteristics in terms of linearity, stability, precision, and spiking & recovery

Among the molecular signatures of nitro-oxidative damage, 3-nitrotyrosine (3NT) has emerged as a biologically relevant marker of protein nitration. Its exceptional chemical stability and analytical accessibility make it particularly suitable for quantitative detection. Accumulation of 3NT reflects oxidative damage and modulates protein function across diverse pathological conditions characterized by inflammation and redox imbalance, establishing it as a promising biomarker. Furthermore, 3NT serves as an indicator of inflammatory activity, cellular injury, and nitric oxide metabolism.

The RayBio^® 3NT Enzyme Immunoassay (EIA) Kit is a competitive immunoassay that quantifies 3NT in serum or plasma from human, mouse, rat, equine, and bovine samples, as well as urine samples within 2.5 hours. Absorbance is measured at 450 nm, and concentrations are interpolated from a ready-to-use 3NT standard curve. The assay demonstrates a sensitivity of 1.9716 pg/ml and has been validated for linearity, stability, precision, and spike-recovery performance. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples.

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1-liter graduated cylinders
• Distilled or deionized water
• Tubes to prepare standard or sample dilutions
• Orbital shaker
• GraphPad Prism or SigmaPlot software

1. Prepare all reagents, samples and standards as instructed.
2. Add 50 µL standards or samples to appropriate well. Incubate for 10 minutes at room temperature.
3. Add 50 µL anti-3NT to appropriate well. Incubate 1 hour at room temperature. And wash 5 times.
4. Add 100 µL prepared HRP-Secondary antibody solution. Incubate 1 hour at room temperature. And wash 5 times.
5. Add 100 µL TMB One-Step Substrate Reagent to each well. Incubate 10-25 minutes at room temperature.
6. Add 50 µL Stop Solution to each well. Read at 450 nm immediately.