**Squid Vitellogenin (VTG) ELISA Kit Instruction Manual**
**Kit Specification:**
This ELISA kit is available in either 48-well or 96-well configurations. The standard dilution provided is 1.5 ml × 1 bottle. The enzyme standard reagent comes in 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well). This reagent is intended for research use only.
**Standard Curve Preparation:**
The standard curve should be plotted with the concentration of the standard on the x-axis and the OD value on the y-axis. Alternatively, a linear regression equation can be calculated from the standard concentrations and their corresponding OD values. The OD value of the sample is then used to determine its concentration by substituting into the equation, followed by multiplying by the dilution factor to obtain the actual sample concentration.
**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-well) / 6 ml × 1 bottle (96-well)
- Developer A: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well)
- Chromogen B: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well)
- Stop Solution: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well)
- Concentrated Wash Solution: 20 ml × 20 times (48-well) / 20 ml × 30 times (96-well)
**Storage Conditions:**
All components should be stored at 2–8°C. The shelf life of the kit is 6 months.
**Principle of the Assay:**
This kit utilizes a double-antibody sandwich ELISA method to quantify vitellogenin (VTG) in the sample. The microtiter plate is pre-coated with a specific anti-VTG antibody. After adding the sample and HRP-labeled anti-VTG antibody, a complex forms. After washing, TMB substrate is added, producing a color change that is proportional to the VTG concentration. The reaction is stopped, and absorbance is measured at 450 nm using a microplate reader. The concentration is determined from the standard curve.
**Purpose:**
This kit is designed to measure the levels of vitellogenin (VTG) in salmon 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 well and centrifuge as above.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via freeze-thaw cycles before centrifugation.
- **Tissue Homogenate:** Weigh the tissue, add PBS, homogenize, and centrifuge. Store the supernatant at 2–8°C or -20°C if not used immediately.
**Notes and Recommendations:**
- Ensure all reagents are brought to room temperature before use.
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN₃, as it inhibits HRP activity.
- Always prepare a standard curve and perform duplicate measurements.
- Keep the substrate away from light.
- Strictly follow the protocol. Results must be based on microplate reader readings.
- All waste materials should be handled as biohazardous.
- Do not mix reagents from different batches.
**Performance Characteristics:**
- The correlation coefficient (R²) for the standard curve should be ≥ 0.95.
- Intra-assay and inter-assay variation should be < 9% and < 11%, respectively.
- Detection range: 0.2 IU/L – 6 IU/L.
**Service Commitment:**
We offer free technical support during working hours and sample testing services upon request to ensure optimal experimental results. Our delivery time starts from payment confirmation.
**Operation Steps:**
1. Prepare standard dilutions and load them into the wells.
2. Add sample diluent and test samples.
3. Incubate the plate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash solution.
5. Add enzyme conjugate and incubate again.
6. Add TMB substrate and develop the color.
7. Stop the reaction and measure the absorbance at 450 nm within 15 minutes.
This manual provides a comprehensive guide to the proper use of the Squid Vitellogenin (VTG) ELISA Kit. Following these instructions will help ensure accurate and reliable results in your research.
Paper Eyelash Box,Holographic Lash Boxes,Square Lash Boxes,Paper Lash Boxes
Zhengzhou Cuka Electronic Commerce Co., Ltd. , https://www.cukalashes.com