BH4 (Tetrahydrobiopterin) ELISA Kit (AEES00884)
- SKU:
- AEES00884
- Size:
- 96 Assays
- Reactivity:
- Universal
- Assay Type:
- Competitive-ELISA
- Sensitivity:
- 18.75 pg/mL
- Range:
- 31.25-2000 pg/mL
- Recovery:
- 80%-120%
Description
| Product Name: | BH4 (Tetrahydrobiopterin) ELISA Kit |
| SKU: | AEES00884 |
| Size: | 96 Assays |
| Detection Method: | Colorimetric method, ELISA, Competitive |
| Assay Type: | Competitive-ELISA |
| Sensitivity: | 18.75 pg/mL |
| Range: | 31.25-2000 pg/mL |
| Reactivity: | Universal |
| Sample Volume: | 50 μL |
| Total Assay Time: | 2.0h |
| Recovery: | 80%-120% |
| Kit Components: |
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This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with Universal BH4. During the reaction, Universal BH4 in the sample or standard competes with a fixed amount of Universal BH4 on the solid phase supporter for sites on the Biotinylated Detection Ab specific to Universal 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 Universal BH4 in tested samples can be calculated by comparing the OD of the samples to the standard curve.
| Standard: |
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| Linearity: |
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| Recovery: |
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| Precision: |
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*Note:The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Protocol |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Note: add all solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensure solutions do not foam when adding to the wells. |
| 2. | Add 50µL of Standard, Blank or Sample to their respective wells. The blank well is added with Sample / Standard dilution buffer. |
| 3. | Immediately add 50 µL of Biotin-detection antibody working solution to each well. |
| 4. | Cover with a plate seal and gently tap the plate to ensure thorough mixing. Incubate for 45 minutes at 37°C. Note: solutions should be added to the bottom of the micro-ELISA plate well, avoid touching the inside wall and causing foaming as much as possible. |
| 5. | Aspirate or decant the solution from the plate and add 350µL of wash buffer to each well and incubate for 1-2 minutes at room temperature. Aspirate the solution from each well and clap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplate washer can be used in this step and other wash steps. |
| 6. | Add 100µL of HRP Conjugate working solution to each well and cover with a plate seal. Incubate for 30 minutes at 37°C. |
| 7. | Repeat the aspiration/wash process 5 times according to step 5. |
| 8. | Add 90µL of the Substrate reagent to each well and cover with a new plate seal. Incubate for approximately 15 minutes at 37°C and protect from light. The reaction time can be shortened or extended according to the colour change, but not by more than 30 minutes. When apparent gradient appears in standard wells, terminate the reaction. |
| 9. | Stop: Add 50µL of Stop Solution to each well (wells will develop a yellow colour immediately). Note: Adding the stop solution should be done in the same order as the substrate solution. |
| 10. | Determine the optical density (OD value) of each well immediately with a microplate reader set at 450 nm. In advance, preheat the instrument and set the testing parameters. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 15 min at 1000×g at 2~8°C. Collect the supernatant to carry out the assay. Blood collection tubes should be disposable and endotoxin-free. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 min at 1000×g at 2~8°C within 30 min of collection. Collect the supernatant to carry out the assay. Hemolysed samples are not suitable for ELISA assay. |
| Cell lysates | For adherent cells, gently wash the cells with pre-cooled PBS and dissociate the cells using trypsin. Collect the cell suspension in a tube and centrifuge for 5 min at 1000×g. Discard the medium and wash the cells 3 times with precooled PBS. For each 1×106 cells, add 150-250µL of pre-cooled PBS to keep the cells suspended. Optimal cell concentration is 1 million/ml. To release cellular components, dilute the cell pellet in PBS and use 3-4 freeze-thaw cycles in liquid Nitrogen (commercial lysis buffers can be used according to manufacturer’s instructions). Centrifuge at 4°C for 20 mins at 2000-3000 rpm to pellet debris and remove clear supernatant to clean microcentrifuge tube for ELISA or storage. |
| Tissue homogenates | It is recommended to get detailed references from the literature before analyzing different tissue types. For general information, hemolysed blood may affect the results, so the tissues should be minced into small pieces and rinsed in ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then homogenized in PBS (tissue weight (g): PBS (mL) volume=1:9) with a glass homogenizer on ice. To further break down the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5 min at 5000×g to get the supernatant. |
| Cell culture supernatant or other biological fluids | Centrifuge samples for 20 min at 1000×g at 2~8°C. Collect the supernatant to carry out the assay. |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Universal BH4 in samples.No significant cross-reactivity or interference between Universal BH4 and analogues was observed |
| Storage: | 2-8℃/-20℃ |
| Expiration Date: | 12 months |
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