Fiber Optic Cabinet,Fiber Cabinet,Fiber Distribution Cabinet,Outdoor Fiber Cabinet Cixi Dani Plastic Products Co.,Ltd , https://www.danifiberoptic.com
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 diagnostic or therapeutic purposes. The method used in this assay is the double-antibody sandwich ELISA, which allows for the accurate quantification of porcine albumin (ALB) in various biological samples.
The principle of the assay involves coating a microtiter plate with a specific monoclonal antibody against porcine ALB. After incubation, the sample containing ALB is added to the wells, 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 washing steps to remove unbound components, the substrate TMB is added. TMB turns blue under HRP catalysis and changes to yellow when an acid stop solution is applied. The intensity of the color is directly proportional to the concentration of ALB in the sample. The absorbance is measured at 450 nm using a microplate reader, and the concentration of ALB in the sample is determined by comparing the OD values 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 × 8 wells
- Standard Dilutions – 1.5 mL × 1 bottle
- Sample Diluent – 6 mL × 1 bottle
- TMB Substrate A & B – 6 mL × 1 bottle each
- Instruction Manual – 1 copy
- Sealing Film – 2 sheets
- Zip Lock Bag – 1
**Sample Requirements:**
- Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles.
- Do not use samples containing NaN₃, as it may inhibit HRP activity.
**Assay Procedure:**
1. **Standard Dilution:** Prepare a series of standards by diluting the original stock according to the provided chart.
2. **Sample Loading:** Add 50 μL of standard, 40 μL of sample diluent, and 10 μL of sample to the appropriate wells (final dilution factor of 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 controls.
6. **Incubation:** Repeat the incubation step at 37°C for 30 minutes.
7. **Washing:** Perform the washing step again.
8. **Color Development:** Add 50 μL of TMB A and B to each well, mix gently, and incubate at 37°C for 10 minutes.
9. **Stop Reaction:** Add 50 μL of stop solution to each well. The color will change from blue to yellow.
10. **Measurement:** Read the absorbance at 450 nm within 15 minutes of adding the stop solution.
**Data Analysis:**
Plot the OD values against the standard concentrations to create a standard curve. Use linear regression to calculate the unknown sample concentration. Multiply the result by the dilution factor to obtain the actual concentration.
**Precautions:**
- Allow the kit to reach room temperature before use (15–30 minutes). Store unopened enzyme reagents in a sealed bag.
- The washing solution may crystallize; if so, warm it in a water bath before use.
- Use separate pipettes for each step and ensure accuracy. For large sample numbers, use a multichannel pipette.
- Always run a standard curve in duplicate. If the sample OD exceeds the highest standard, dilute the sample and adjust the calculation accordingly.
- Use the sealing film only once to prevent cross-contamination.
- Keep the TMB substrate away from light.
- Follow the manual instructions carefully and verify results with a microplate reader.
- Treat all waste materials as biohazardous.
**Storage Conditions:**
- Store the kit at 2–8°C.
- Shelf life: 6 months from the date of manufacture.