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BH4(Tetrahydrobiopterin) ELISA Kit
SKU: E-EL-0110
BH4(Tetrahydrobiopterin) ELISA Kit
| Detection Range | 31.25-2000 pg/mL |
| Sensitivity | 18.75 pg/mL |
Product Details
Properties
| Assay type | Competitive-ELISA |
| Format | 96T/48T |
| Assay time | 2.0h |
| Detection range | 31.25-2000 pg/mL |
| Sensitivity | 18.75 pg/mL |
| Sample type &Sample volume | serum, plasma and other biological fluids; 50μL |
| Specificity | This kit recognizes BH4 in samples. No significant cross-reactivity or interference between BH4 and analogues was observed. |
| Reproducibility | Both intra-CV and inter-CV are < 10%. |
| Application | This ELISA kit applies to the in vitro quantitative determination of BH4 concentrations in serum, plasma and other biological fluids. |
Test Principle
This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with BH4. During the reaction, BH4 in the sample or standard competes with a fixed amount of BH4 on the solid phase supporter for sites on the Biotinylated Detection Ab specific to BH4. Excess conjugate and unbound sample or standard are washed away, and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns from blue to yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of BH4 in tested samples can be calculated by comparing the OD of the samples to the standard curve.
Kit Components and Storage
An unopened kit can be stored at 2-8℃ for 1 month. If the kit is not supposed to be used within 1 month, store the components separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro ELISA Plate(Dismountable) | 96T: 8 wells ×12 strips | -20℃, 6 months |
| 48T: 8 wells ×6 strips | ||
| Reference Standard | 96T: 2 vials | |
| 48T: 1 vial | ||
| Concentrated Biotinylated Detection Ab (100×) | 96T: 1 vial, 120 μL | |
| 48T: 1 vial, 60 μL | ||
| Concentrated HRP Conjugate (100×) | 96T: 1 vial, 120 μL | -20℃(Protect from light), 6 months |
| 48T: 1 vial, 60 μL | ||
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 2-8°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 2-8℃ (Protect from light) |
| Stop Solution | 1 vial, 10 mL | 2-8℃ |
| Plate Sealer | 5 pieces | |
| Manual | 1 copy | |
| Certificate of Analysis | 1 copy |
Technical Data:
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level BH4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level BH4 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 105.95 | 238.37 | 995.86 | 112.62 | 233.33 | 1067.77 |
| Standard deviation | 6.79 | 13.40 | 50.39 | 6.27 | 13.91 | 50.83 |
| CV (%) | 6.41 | 5.62 | 5.06 | 5.57 | 5.96 | 4.76 |
Recovery
The recovery of BH4 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
|---|---|---|
| Serum(n=8) | 89-104 | 96 |
| EDTA plasma(n=8) | 88-103 | 96 |
| Cell culture media(n=8) | 93-108 | 100 |
Target Information
| Synonyms | THB, sapropterin |
Assay Procedures
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1. Add 50μL standard or sample to the wells, immediately add 50μL Biotinylated Detection Ab working solution to each well. Incubate for 45 min at 37°C |
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2. Aspirate and wash the plate for 3 times |
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3. Add 100μL HRP conjugate working solution. Incubate for 30 min at 37°C. Aspirate and wash the plate for 5 times |
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4. Add 90μL Substrate Reagent. Incubate for 15 min at 37°C |
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5. Add 50μL Stop Solution |
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6. Read the plate at 450nm immediately. Calculation of the results |