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Human β-lactoglobulin (β-Lg) elisa kit instruction manual
**Human Beta Lactoglobulin (β-Lg) ELISA Kit – Instructions for Use**
**Kit Specifications:**
- 48-well or 96-well configuration
- Standard Diluent: 1.5 ml × 1 bottle
- Enzyme Standard Reagent: 3 ml × 1 bottle (for 48-well) / 6 ml × 1 bottle (for 96-well)
- Storage Conditions: 2–8°C
**Calculation Method:**
To determine the concentration of β-Lg in the sample, plot the standard concentrations on the x-axis and the corresponding OD values on the y-axis. A standard curve is generated by plotting these points. The sample concentration can be determined either by reading off the curve or by calculating the linear regression equation using the standard concentrations and their OD values. Once the sample’s OD value is known, it is substituted into the equation to calculate the concentration, which is then multiplied by the dilution factor to obtain the actual sample concentration.
**Kit Composition:**
- Sealing Film: 2 pieces (for 48-well) / 2 pieces (for 96-well)
- Standard: 0.5 ml × 1 bottle (2700 ng/L)
- Enzyme Standard: 1×48 / 1×96
- Sample Diluent: 3 ml × 1 bottle (for 48-well) / 6 ml × 1 bottle (for 96-well)
- Developer A: 3 ml × 1 bottle / 6 ml × 1 bottle
- Chromogen B: 3 ml × 1 bottle / 6 ml × 1 bottle (store at -20°C)
- Stop Solution: 3 ml × 1 bottle / 6 ml × 1 bottle
- Concentrated Wash Solution: 20 ml × 20 times / 20 ml × 30 times (store at 2–8°C)
**Principle of the Assay:**
This ELISA kit utilizes the sandwich method to detect human β-lactoglobulin (β-Lg). The microplate is pre-coated with a specific antibody against β-Lg. After incubation with the sample, the β-Lg binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added, producing a blue color that turns yellow in the presence of acid. The intensity of the color is directly proportional to the amount of β-Lg in the sample. The absorbance is measured at 450 nm, and the concentration is calculated using a standard curve.
**Purpose:**
This kit is designed to quantitatively measure β-Lg levels in human serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Storage and Shelf Life:**
- Store the kit at 2–8°C
- Shelf life: 6 months from the date of manufacture
**Sample Preparation Guidelines:**
1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge similarly.
3. **Urine:** Collect in a sterile tube and centrifuge.
4. **Cell Culture Supernatant:** Centrifuge after collection; for intracellular components, lyse cells by freezing and thawing before centrifugation.
5. **Tissue:** Homogenize in PBS, centrifuge, and collect the supernatant.
6. **General Notes:** Process samples immediately after collection. If not tested right away, store at -20°C, avoiding repeated freeze-thaw cycles. Avoid samples containing NaN3, as it may inhibit HRP activity.
**Procedure Steps:**
1. **Standard Dilution and Loading:** Prepare a serial dilution of the standard and add to designated wells.
2. **Sample Loading:** Add 40 μl of sample diluent followed by 10 μl of the sample to each test well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute the concentrated wash solution and wash the plate 5 times.
5. **Enzyme Addition:** Add 50 μl of enzyme reagent to each well (except blank).
6. **Second Incubation:** Repeat the incubation step.
7. **Color Development:** Add 50 μl of TMB A and B, incubate for 15 minutes at 37°C.
8. **Stop Reaction:** Add 50 μl of stop solution to each well.
9. **Measurement:** Read OD values at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to reach room temperature before use.
- The washing solution may crystallize; warm gently if needed.
- Use separate pipettes for each step and ensure accuracy.
- Always run standards in duplicate and prepare a standard curve each time.
- Avoid cross-contamination by using a new sealing film for each test.
- Keep the substrate away from light.
- Follow the manual strictly and rely on the microplate reader for accurate results.
- Treat all waste materials as biohazardous.
- Do not mix reagents from different batches.
- In case of discrepancies, the English version of the manual takes precedence.
**Performance Characteristics:**
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²) ≥ 0.95
- Intra-batch CV < 9%, Inter-batch CV < 11%
**Service Commitment:**
- Free technical support during working hours
- Free sample testing service available upon request
- Delivery period: From payment to delivery
This kit is ideal for researchers conducting studies involving β-Lg detection in various biological matrices. Proper handling and following the instructions carefully will ensure reliable and reproducible results.