Enzyme-linked immunosorbent assay for Antigen Detection.
Organism Species
Pan-species (General)
Format
48T, 96T, 96Tx5, 96Tx10, 96Tx100
Specificity
This assay has high sensitivity and excellent specificity for detection of High Sensitive Nitrotyrosine (NT) . No significant cross-reactivity or interference between High Sensitive Nitrotyrosine (NT) and analogues was observed.
Sensitivity
The minimum detectable dose of this kit is typically less than 0.52ng/mL
Detection Range
1.23-100ng/mL
Precision
Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level High Sensitive Nitrotyrosine (NT) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level High Sensitive Nitrotyrosine (NT) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV Inter-Assay: CV Inter-Assay: CV
Stability
The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.
Assay Principle
This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to High Sensitive Nitrotyrosine (NT) has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled High Sensitive Nitrotyrosine (NT) and unlabeled High Sensitive Nitrotyrosine (NT) (Standards or samples) with the pre-coated antibody specific to High Sensitive Nitrotyrosine (NT) . After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of High Sensitive Nitrotyrosine (NT) in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of High Sensitive Nitrotyrosine (NT) in the sample.
Assay Protocol
1. Prepare all reagents, samples and standards; 2. Add 50µ L standard or sample to each well. And then add 50µ L prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37° C; 3. Aspirate and wash 3 times; 4. Add 100µ L prepared Detection Reagent B. Incubate 30 minutes at 37° C; 5. Aspirate and wash 5 times; 6. Add 90µ L Substrate Solution. Incubate 10-20 minutes at 37° C; 7. Add 50µ L Stop Solution. Read at 450 nm immediately.
