**Rat Growth Hormone Releasing Polypeptide (GHRP) ELISA Kit Instruction Manual**
**Kit Specification:**
This kit is available in 48-well or 96-well configurations. It includes the following components:
- Standard Diluent: 1.5 ml × 1 bottle
- Enzyme Standard Reagent: 3 ml × 1 bottle (for 48-well) / 6 ml × 1 bottle (for 96-well)
- Sealing Film: 2 pieces (for 48-well) / 2 pieces (for 96-well)
- Standard: 0.5 ml × 1 bottle, concentration of 2700 ng/L
- Sample Diluent: 3 ml × 1 bottle (for 48-well) / 6 ml × 1 bottle (for 96-well)
- Developer A: 3 ml × 1 bottle (for 48-well) / 6 ml × 1 bottle (for 96-well)
- Chromogen B: 3 ml × 1 bottle (for 48-well) / 6 ml × 1 bottle (for 96-well)
- Stop Solution: 3 ml × 1 bottle (for 48-well) / 6 ml × 1 bottle (for 96-well)
- Concentrated Washing Solution: 20 ml × 20 times (for 48-well) / 20 ml × 30 times (for 96-well)
**Storage Conditions and Expiry:**
- Storage: 2–8°C
- Shelf Life: 6 months from the date of manufacture
**Purpose:**
This ELISA kit is designed for the quantitative determination of Rat Growth Hormone Releasing Polypeptide (GHRP) in serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Principle of Operation:**
The kit utilizes a sandwich ELISA method. The microtiter plate is pre-coated with a specific antibody against GHRP. After incubation with the sample, the target antigen binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an immune complex. Following washing, TMB substrate is added, which turns blue under HRP catalysis and changes to yellow upon acid termination. The color intensity is directly proportional to the GHRP concentration in the sample. The optical density (OD) at 450 nm is measured, and the concentration is determined by comparing it to a standard curve.
**Sample Preparation Guidelines:**
- **Serum:** Collect blood at room temperature, allow clotting for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well, centrifuge, and collect the supernatant.
- **Urine:** Collect using a sterile container, centrifuge, and retain the supernatant.
- **Cell Culture Supernatant:** Centrifuge and collect the supernatant. For intracellular components, lyse cells via repeated freeze-thaw cycles.
- **Tissue Homogenate:** Weigh the tissue, add PBS, homogenize, centrifuge, and collect the supernatant.
**Precautions and Notes:**
- Ensure all reagents are equilibrated to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each test.
- Do not mix reagents from different batches.
- Handle the substrate away from light.
- Always prepare a standard curve and run samples in duplicate.
- If the sample OD exceeds that of the highest standard, dilute the sample before testing.
**Performance Characteristics:**
- Intra-assay and inter-assay coefficients of variation < 9% and < 11%, respectively.
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²) ≥ 0.95
**Service Commitment:**
- Free technical support during working hours.
- Sample testing services available upon request.
- Delivery within the agreed period after payment.
**Usage Instructions:**
Follow the step-by-step procedure provided in the manual. Ensure accurate pipetting, proper incubation times, and thorough washing steps. Read the absorbance at 450 nm within 15 minutes of adding the stop solution.
**Note:** This kit is intended for research purposes only and should not be used for diagnostic or clinical applications. All waste materials should be handled as biohazardous waste.
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