This reagent is intended for research use only and is designed to quantify bovine serum albumin (BSA) in bovine serum, plasma, and related liquid samples. The assay is based on the double-antibody sandwich immunoassay principle. A microplate is pre-coated with a purified BSA-specific antibody to form a solid-phase antibody. The sample containing BSA is then added, allowing it to bind to the immobilized antibody. Subsequently, HRP-conjugated BSA antibodies are introduced, forming an immune complex of antibody-antigen-enzyme-labeled antibody. After thorough washing, the substrate TMB is added, which changes color under the catalytic action of HRP. The reaction is stopped by adding an acidic solution, causing the blue color to turn yellow. The intensity of the yellow color is directly proportional to the concentration of BSA in the sample.
The absorbance at 450 nm is measured using a microplate reader, and the BSA concentration in the sample is determined by comparing the OD value to a standard curve. This method ensures high specificity and sensitivity for accurate quantification.
**Kit Components:**
- 48-well configuration: 1×48 enzyme-labeled plate, 1 standard (90 μg/L), 1 standard dilution (1.5 mL), 1 enzyme standard reagent (3 mL), 1 sample dilution (3 mL), 1 developer A (3 mL), 1 developer B (3 mL), 1 wash solution (20× diluted, 20 mL), 1 sealing film (2 pieces), 1 storage bag.
- 96-well configuration: 1×96 enzyme-labeled plate, 1 standard (90 μg/L), 1 standard dilution (1.5 mL), 1 enzyme standard reagent (6 mL), 1 sample dilution (6 mL), 1 developer A (6 mL), 1 developer B (6 mL), 1 wash solution (20× diluted, 20 mL), 2 sealing films, 1 storage bag.
**Storage Instructions:**
All components should be stored at 2–8°C. The kit has a shelf life of 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. Carefully collect the supernatant; if precipitate forms, re-centrifuge before testing.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully.
3. **Urine:** Collect in a sterile tube and centrifuge at 2000–3000 rpm for 20 minutes. Discard any precipitate and use the supernatant.
4. **Cell Culture Supernatant:** For secreted components, collect in a sterile tube and centrifuge. For intracellular components, lyse cells by repeated freeze-thaw cycles, then centrifuge and collect the supernatant.
5. **Tissue Specimens:** Homogenize tissue in PBS (pH 7.4), centrifuge, and collect the supernatant. Store at 2–8°C after thawing.
6. **General:** Samples should be processed as soon as possible. If not tested immediately, store at -20°C. Avoid repeated freezing and thawing. Do not use samples containing NaN3, as it inhibits HRP activity.
**Procedure Summary:**
1. Prepare standard dilutions and load them into the microplate.
2. Add sample diluent and test samples to designated wells.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate thoroughly with diluted wash buffer.
5. Add HRP-labeled antibody and incubate again.
6. Wash the plate once more.
7. Add TMB substrate and incubate for 15 minutes at 37°C.
8. Stop the reaction with stop solution.
9. Measure OD at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to reach room temperature before use.
- Ensure all reagents are properly mixed and handled.
- Use a pipette with accuracy and avoid cross-contamination.
- Always run a standard curve and consider sample dilution if necessary.
- Keep substrates away from light.
- Follow the manual instructions strictly for accurate results.
- All waste materials should be treated as biohazardous.
**Performance:**
- Correlation coefficient (R²) ≥ 0.990.
- Intra-batch and inter-batch variability < 9% and < 11%, respectively.
- Detection range: 3 μg/L – 70 μg/L.
This kit provides a reliable and efficient method for quantifying BSA in various biological matrices, making it suitable for research and diagnostic applications.
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