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**Rat E-cadherin (E-cad) ELISA Kit – Instructions for Use**
This kit is intended for research use only and is designed to quantify E-cadherin (E-cad) levels in rat serum, plasma, and other biological fluids. The method employed is a double-antibody sandwich ELISA, ensuring high specificity and sensitivity.
**Principle of the Assay**
The assay utilizes a microtiter plate pre-coated with a specific anti-E-cad antibody. After adding the sample, E-cad binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody then recognizes the captured E-cad, forming a complex. Following washing steps, TMB substrate is added, which changes color in the presence of HRP. The reaction is stopped, and the absorbance at 450 nm is measured. The intensity of the color is directly proportional to the E-cad concentration in the sample.
**Kit Components**
The kit includes:
- Microtiter plate (48 or 96 wells)
- Standard (180 ng/L)
- Standard diluent
- Enzyme-labeled reagent
- Sample diluent
- TMB substrate solution (A and B)
- Wash buffer (concentrated, 20x)
- Sealing film
- Storage instructions: 2–8°C
**Sample Preparation**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant, mix well, and centrifuge similarly.
- **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 homogenate**: Homogenize in PBS, centrifuge, and collect the supernatant.
**Storage and Handling**
Samples should be processed immediately after collection. If not tested right away, store at -20°C, avoiding repeated freezing and thawing. Avoid samples containing NaN3, as it inhibits HRP activity.
**Procedure Summary**
1. Prepare standard dilutions (120, 80, 40, 20, 10 ng/L).
2. Add sample diluent and test samples to designated wells.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add enzyme-labeled reagent and incubate again.
6. Add TMB substrate and develop color for 15 minutes.
7. Stop the reaction with stop solution and measure OD at 450 nm.
**Data Analysis**
Plot the standard curve using OD values versus E-cad concentrations. Calculate the sample concentration by interpolating from the curve, adjusting for any dilution factor. Ensure that each measurement is accompanied by a standard curve, preferably in duplicate.
**Notes and Warnings**
- Equilibrate the kit at room temperature before use.
- Do not reuse sealing films.
- Protect TMB from light.
- Follow the manual precisely.
- Treat all waste as biohazardous material.
- Do not mix reagents from different batches.
**Performance Characteristics**
- Linear range: 3–150 ng/L
- Correlation coefficient (R²): ≥0.92
- Intra-batch and inter-batch CV < 9% and 15%, respectively
**Storage and Shelf Life**
Store the kit at 2–8°C. The shelf life is 6 months from the date of manufacture.
This detailed guide ensures accurate and reliable detection of E-cadherin in various biological samples, supporting research in cell adhesion and related fields.