**Rat Calcitonin (CT) ELISA Kit Instruction Manual**
**Kit Specification:**
This ELISA kit is available in 48-well or 96-well configurations. The standard dilution is 1.5 ml × 1 bottle, while the enzyme standard reagent comes as 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well). This reagent is for research purposes only.
**Standard Curve Preparation:**
To determine the concentration of CT in the sample, prepare a standard curve by plotting the standard concentrations on the x-axis and the corresponding OD values on the y-axis. Alternatively, calculate the linear regression equation using the standard concentrations and their OD values. Then, input the sample’s OD value into the equation to determine the actual concentration, multiplied by the dilution factor.
**Kit Components:**
- Sealing film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 ml × 1 bottle (2700 ng/L)
- Enzyme Standard: 1×48 / 1×96
- Sample Diluent: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Developer A: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Chromogen B: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Stop Solution: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Concentrated Washing Solution: 20 ml × 20 times (48) / 20 ml × 30 times (96)
**Storage Conditions:**
All components should be stored at 2–8°C. The kit has a shelf life of 6 months from the date of receipt.
**Principle of Operation:**
The kit uses a double-antibody sandwich method to detect calcitonin (CT) levels in samples. Microplates are pre-coated with purified rat CT antibodies. After adding the sample and HRP-labeled CT antibody, a complex is formed. Following washing, TMB substrate is added, producing a color change that correlates with CT concentration. The absorbance is measured at 450 nm, and results are calculated using a standard curve.
**Purpose:**
This kit is designed for the quantitative determination of calcitonin (CT) in serum, plasma, urine, cell culture supernatants, and tissue homogenates.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix for 10–20 minutes, then centrifuge.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells via freeze-thaw cycles before centrifugation.
- **Tissue:** Homogenize in PBS, centrifuge, and collect supernatant.
**Important Notes:**
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN₃, as it may inhibit HRP activity.
- Always prepare a standard curve and perform duplicate measurements for accuracy.
- Ensure all reagents are at room temperature before use.
- Handle all waste materials as biohazardous.
- Do not mix reagents from different batches.
**Operation Steps:**
1. Prepare standard dilutions and load them into designated wells.
2. Add sample diluent and test samples to the microplate.
3. Seal and incubate at 37°C for 30 minutes.
4. Wash the plate five times with diluted washing solution.
5. Add enzyme-labeled reagent and incubate again.
6. Add chromogen A and B, incubate for 15 minutes at 37°C.
7. Stop the reaction with stop solution.
8. Measure OD values at 450 nm within 15 minutes.
**Performance Characteristics:**
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
**Service Commitment:**
We provide free technical support during working hours. Custom testing services are also available upon request.
**Note:**
Always read and follow the instructions carefully. Results must be based on instrument readings. If any discrepancies occur, contact our support team for assistance.
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