**Rat Calcitonin (CT) ELISA Kit Instruction Manual**
**Kit Specifications:**
This Rat Calcitonin (CT) ELISA Kit is available in 48-well or 96-well configurations. The kit includes:
- Standard Diluent: 1.5 mL × 1 vial
- Enzyme Standard Reagent: 3 mL × 1 vial (for 48-well) / 6 mL × 1 vial (for 96-well)
- Microplate: 1×48 or 1×96
- Sealing Film: 2 pieces for both 48 and 96-well configurations
- Standard: 0.5 mL × 1 vial, concentration: 2700 ng/L
- Sample Diluent: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Developer A: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Chromogen B: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Wash Solution: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Concentrated Wash Solution: 20 mL × 20 times (for 48-well) or 20 mL × 30 times (for 96-well)
**Storage Conditions:**
- All reagents should be stored at 2–8°C unless otherwise specified.
- The shelf life of the kit is 6 months from the date of receipt.
**Purpose:**
This ELISA kit is designed to quantitatively determine the level of calcitonin (CT) in serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids. It is intended for research use only.
**Principle of Operation:**
The kit uses a double-antibody sandwich ELISA method. The microplate is pre-coated with anti-rat CT antibodies. After adding the sample, CT binds to the immobilized antibody. Then, HRP-conjugated anti-CT antibody is added, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added, and the color develops under HRP catalysis. The reaction is stopped with a stop solution, and the absorbance is measured at 450 nm. The concentration of CT in the sample is determined by comparing the OD value to a standard curve.
**Sample Preparation:**
1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge similarly.
3. **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
4. **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells using repeated freeze-thaw cycles before centrifugation.
5. **Tissue Homogenate:** Weigh the tissue, add PBS, homogenize, and centrifuge.
6. **Storage:** Samples should be processed immediately. If not tested right away, store at -20°C. Avoid repeated freezing and thawing. Do not use samples containing NaN3.
**Operation Steps:**
1. **Standard Dilution:** Prepare serial dilutions of the standard (1800, 1200, 600, 300, 150 ng/L).
2. **Sample Loading:** Add 40 μL of sample diluent and 10 μL of sample to each well (final dilution 5×).
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Rinse 5 times with diluted wash buffer.
5. **Enzyme Addition:** Add 50 μL of enzyme-labeled reagent to each well (except blank wells).
6. **Second Incubation:** Repeat incubation at 37°C for 30 minutes.
7. **Color Development:** Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μL of stop solution to each well.
9. **Measurement:** Read OD at 450 nm within 15 minutes.
**Notes:**
- Equilibrate the kit at room temperature for 15–30 minutes before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Ensure accurate pipetting and avoid light exposure during color development.
- Always run standards in duplicate. If the sample OD exceeds the highest standard, dilute the sample before testing.
- Follow the manual strictly. Results must be based on the microplate reader readings.
- Discard all waste as biohazardous material.
- Do not mix reagents from different batches.
**Performance:**
- Linear regression correlation coefficient (R) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
**Service Commitment:**
We offer free technical support during working hours and can assist with sample testing to ensure optimal results.
**Delivery:** Shipped upon payment.
This manual provides detailed instructions to ensure accurate and reliable results when measuring rat calcitonin levels using the ELISA method.
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