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Pig albumin (ALB) enzyme-linked immunoassay kit instructions for use - Database & Sql Blog Articles
**Porcine Albumin (ALB) Enzyme-Linked Immunoassay Kit Instructions**
This kit is intended for research use only and not for human or animal diagnostic purposes. It utilizes a double-antibody sandwich ELISA method to quantitatively detect porcine albumin (ALB) in various biological samples.
The principle of the assay involves coating a microplate with a specific monoclonal antibody against ALB. After incubation, the sample containing ALB is added, allowing it to bind to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then introduced, forming a complex of antibody-antigen-enzyme-labeled antibody. Following thorough washing, the substrate TMB is added, which changes color under the action of HRP. The reaction is stopped by adding an acidic stop solution, causing the color to shift from blue to yellow. The intensity of the color is directly proportional to the concentration of ALB in the sample. Absorbance is measured at 450 nm using a microplate reader, and the concentration is determined by comparing the OD value to a standard curve.
**Kit Components**
- 130× Washing Solution: 20 mL × 1 bottle
- Stop Solution: 6 mL × 1 bottle
- Enzyme Standard Reagent: 6 mL × 1 bottle (1600 μg/mL)
- Enzyme-Labeled Coating Plate: 12 wells × 8 strips
- Sample Diluent: 6 mL × 1 bottle
- TMB Color Developer A: 6 mL × 1 bottle
- TMB Color Developer B: 6 mL × 1 bottle
- Standard Dilutions: 1.5 mL × 1 bottle
- Instruction Manual: 1 copy
- Sealing Film: 2 sheets
- Storage Bag: 1
**Sample Requirements**
- Samples should be processed as soon as possible after collection. If not used immediately, store at -20°C, avoiding repeated freeze-thaw cycles.
- Avoid using samples containing NaN₃, as it may inhibit HRP activity.
**Procedure**
1. **Standard Dilution**: Prepare a dilution series from the original standard (800 μg/mL) to 50 μg/mL using the provided diluent.
2. **Sample Loading**: Add 50 μL of standard, 40 μL of sample diluent, and 10 μL of sample to each well. Final dilution factor is 5×.
3. **Incubation**: Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing**: Wash the plate 5 times with diluted washing solution.
5. **Enzyme Addition**: Add 50 μL of enzyme-labeled reagent to all wells except blank.
6. **Second Incubation**: Repeat incubation at 37°C for 30 minutes.
7. **Color Development**: Add 50 μL of TMB A and B, incubate at 37°C for 10 minutes.
8. **Stop Reaction**: Add 50 μL of stop solution to each well.
9. **Reading**: Measure absorbance at 450 nm within 15 minutes of stopping the reaction.
**Calculation**
Plot the standard curve using OD values versus concentrations. Calculate the sample concentration by interpolating the OD value on the curve, adjusting for dilution factors.
**Precautions**
- Allow the kit to reach room temperature before use. Store unopened enzyme-labeled reagents in a sealed bag.
- Wash solution may crystallize; heat gently if needed.
- Use accurate pipettes and avoid cross-contamination.
- Always run a standard curve in duplicate. If sample OD exceeds that of the highest standard, perform a preliminary dilution.
- Discard sealing film after one use. Keep substrates away from light.
- Follow instructions strictly and confirm results with a microplate reader.
- Treat all waste as biohazardous material.
- Do not mix components from different batches.
**Storage and Shelf Life**
- Store the kit at 2–8°C.
- Shelf life: 6 months from the date of manufacture.