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Boster's Dibromo-tyrosine ELISA is a competitive assay that can be used for the quantification of 3,5-dibromo-tyrosine in urine, plasma, and other sample matrices. The ELISA utilizes an dibromo-tyrosine-coated plate and an biotin-conjugated antibody for detection which allows for an assay range of 0.078 - 5 μg/mL, with a sensitivity of 0.04 μg/mL. Additional kit highlights are quick incubation times, stable reagents, and an easy to use protocol. It is important to note that the dibromo-tyrosine antibody used in this assay recognizes both free dibromo-tyrosine and brominated residues within a protein. Since complex samples such as plasma, are comprised of mixtures of protein fragments and free 3,5-dibromo-tyrosine, concentrations of 3,5-dibromo-tyrosine reported by ELISA methodology may not coincide with literature values where the free residue is typically measured. This should be kept in mind when analyzing and interpreting experimental results.
| Product Name | Dibromo-tyrosine ELISA Kit |
|---|---|
| SKU/Catalog Number | EK7118 |
| Description | Colorimetric detection of Dibromo-tyrosine. 96wells/kit, with removable strips. |
| Cite This Product | Dibromo-tyrosine ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7118) |
| Validated Species | All Animals |
| Application | ELISA *Our Boster Guarantee covers the use of this product in the above tested applications. |
| Cross Reactivity | There is no detectable cross-reactivity. |
| Pack Size | 96wells/kit, with removable strips. |
| Sensitivity | 0.04 ?g/ml *Sensitivity, or Lower Limit of Detection (LLD), is the minimum level of target protein the ELISA assay can detect. We measure 20 blank wells and if the O.D. value is 2 standard deviations higher than the blanks' average O.D. the sample can be deemed positive. |
|---|---|
| Assay Range | 0.078 - 5 ?g/mL *This assay range is determined using common samples. For samples with low target protein concentrations, users can adjust the standard curve to extend the lower limit of assay range. |
| Sample Type | Plasma, Urine *The above listed samples are the ones valided with the assay. If you do not see your sample of interest listed, as long as there is enough level of target protein present in the sample, this Picokine? ELISA kit should detect it. **For protocol and tips regarding preparing your sample of interest, please check our ELISA sample preparation guide. |
| Storage | Store at 4°C and -20°C. (All reagents are stable as supplied at 4°C, except the Dibromo-tyrosine Standard, which should be stored at -20°C. ) |
| Description | Quantity |
| Dibromo-tyrosine: BSA Coated Plate | 1 Plate |
| Dibromo-tyrosine Standard | 1 vial/75 ?l |
| Dibromo-tyrosine: Biotin Conjugated Monoclonal Antibody | 1 vial/75?l |
| Streptavidin Poly HRP Concentrate | 1 vial/150 ?l |
| Streptavidin Poly HRP Diluent | 1 vial/13 ml |
| Sample and Standard Diluent (Red) | 1 vial/ 50mL |
| Dibromo-tyrosine Antibody Diluent (Blue) | 1 vial/ 13 ml |
| Wash Buffer Concentrate (10X) | 1 vial/ 50 ml |
| TMB Substrate | 1 vial/ 13 ml |
| Stop Solution | 1 vial/ 13 ml |
| Plate Cover | 2 |
1. A plate reader capable of measuring absorbance at 450 nm.
2. Adjustable pipettes and a repeat pipettor.
3. Deionized or distilled water
4. Materials used for Sample Preparation
Dibromo-tyrosine is produced by the oxidative bromination of tyrosine residues. This reaction occurs via eosinophil peroxidase (EPO), an enzyme released by activated eosinophils. Upon activation of eosinophils, a respiratory burst occurs releasing elevated levels of O2 and H202. In the oxidation of tyrosine, EPO utilizes H202 to catalyze the peroxidation of physiological levels of bromine found within plasma to generate the brominating reagent hypobromous acid (HOBr). Eosinophils play an immunomodulatory role through their recruitment to host sites of parasitic invasion. EPO levels also contribute to diseases such as asthma, cancers and allergic disorders where cellular activation is found to occur at pathological sites (6-10). Brominated products such as 3,5-dibromo-tyrosine serve as biological markers for in vivo eosinophil-mediated tissue damage which allows for understanding the overall roll oxidative stress has on pathways implicated in diseased states within organisms.
Click the images to enlarge.
Typical Standard Curve for the Dibromo-tyrosine ELISA Kit (Enzyme-Linked Immunosorbent Assay)–EK7118 Assay Type: Competitive ELISA. Detection Method: Colorimetric Assay. Assay Range: 0.078 – 5 ?g/ml.
Chemical Equation of the Bromination of Tyrosine.
Diagram of the Dibromo-tyrosine Competitive ELISA.
Recovery of dibromo-tyrosine from urine. Urine samples were spiked with dibromo-tyrosine, and diluted as described in the Sample Preparation section (page 10) and analyzed using the Dibromo-Tyrosine ELISA kit. The y-intercept corresponds to the amount of dibromo-tyrosine in unspiked urine.
Diagram of the Preparation of the Dibromo-tyrosine Standards.
Diagram of the Triplicate Sample Plate Format.
Graph of the Dibromo-tyrosine Spike Recovery in Human Plasma.
Graph of the sample comparison between diabetic smoker plasma and non-diabetic/non-smoker plasma.
Intra-Assay Precision: Three samples of known concentration were assayed thirty times on one plate; the intra-assay coefficient of variation of the Dibromo-tyrosine 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 Dibromo-tyrosine ELISA has been determined to be < 10%.
1. Prepare standard and samples in the Sample and Standard Diluent.
2. Add 50 ?L of prepared standards and samples in triplicate to appropriate wells.
3. Add 50 ?L of the diluted Dibromo-tyrosine-Biotin antibody to the appropriate wells.
4. Cover plate with Plate Cover and incubate at 37 °C for 1 hour.
5. Wash plate 4 times with 1X Wash Buffer.
6. Add 100 μL of Streptavidin-HRP Working Solution to each well.
7. Cover plate with Plate Sealer and incubate at room temperature for 30 minutes.
8. Wash plate 4 times with 1X Wash Buffer.
9. Add 100 ?L of TMB Substrate to each well.
10. Develop the plate in the dark at room temperature (20-25°C) for 30 minutes.
11. Stop reaction by adding 100 μL of Stop Solution to each well.
12. Measure absorbance on plate reader at 450 nm.
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