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Human β-lactoglobulin (β-Lg) elisa kit instruction manual
**Human Beta Lactoglobulin (β-Lg) ELISA Kit – User Manual**
This ELISA kit is designed for the quantitative determination of human beta lactoglobulin (β-Lg) in various biological samples such as serum, plasma, urine, cell culture supernatants, and tissue homogenates. The kit uses a sandwich ELISA method, which ensures high specificity and sensitivity.
**Kit Specifications:**
- **Configuration:** 48-well or 96-well plate
- **Standard Dilution:** 1.5 mL × 1 vial
- **Enzyme Standard Reagent:** 3 mL × 1 vial (for 48-well), 6 mL × 1 vial (for 96-well)
- **Storage Conditions:** 2–8°C
- **Validity Period:** 6 months from the date of receipt
**Kit Components:**
- Sealing Film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 mL × 1 vial (2700 ng/L)
- Enzyme Standard: 1×48 / 1×96
- 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 (48-well), 20 mL × 30 times (96-well)
**Principle of Operation:**
The kit employs a double-antibody sandwich ELISA technique. A microtiter plate coated with anti-human β-Lg antibodies is used to capture β-Lg from the sample. After incubation, horseradish peroxidase (HRP)-labeled secondary antibodies are added, forming a complex. Following washing, TMB substrate is added, leading to a color change that is proportional to the amount of β-Lg present. The reaction is stopped with an acidic solution, and the absorbance is measured at 450 nm. A standard curve is then plotted using known concentrations, and the sample concentration is calculated accordingly.
**Sample Preparation:**
- **Serum/Plasma:** Centrifuge at 2000–3000 rpm for 20 minutes after clotting or anticoagulant addition.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge to remove debris. For intracellular components, lyse cells by repeated 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 tested immediately.
**Procedure:**
1. Prepare serial dilutions of the standard.
2. Add 40 μL of sample diluent and 10 μL of sample to each well.
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash solution.
5. Add 50 μL of enzyme-labeled reagent to each well.
6. Incubate again at 37°C for 30 minutes.
7. Add 50 μL of TMB A and B, incubate for 15 minutes at 37°C.
8. Stop the reaction with 50 μL stop solution.
9. Measure OD at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to equilibrate to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each test.
- Do not mix reagents from different batches.
- Ensure accurate pipetting and avoid light exposure during the color development step.
- All waste should be treated as biohazardous material.
**Performance:**
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²): ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
**Service Commitment:**
- Free technical support during working hours.
- Free sample testing service available upon request.
- Delivery time: From payment to delivery.
**Storage:**
Store all components at 2–8°C. Avoid freezing and thawing multiple times.
This kit provides a reliable and efficient method for detecting β-Lg levels in biological samples. Proper handling and adherence to the protocol are essential for accurate results.