Human Oxidized Low Density Lipoprotein (ox-LDL) elisa Kit Instructions
2025-09-15 05:23:28
**Human Oxidized Low Density Lipoprotein (ox-LDL) ELISA Kit Instructions – Guangrui Bio, China's Leading High-Quality ELISA Supplier**
This ELISA kit is intended for **research purposes only** and is designed to quantitatively measure **oxidized low-density lipoprotein (ox-LDL)** in human serum, plasma, urine, and other biological fluids. The method used is the **double-antigen sandwich immunoassay**, which ensures high specificity and sensitivity.
**Principle of Operation:**
The kit employs a microtiter plate pre-coated with **purified anti-ox-LDL antibodies**. After adding the sample, ox-LDL binds to the immobilized antibodies. A **HRP-conjugated secondary antibody** is then added, forming an antibody-antigen-enzyme complex. After washing, the substrate TMB is introduced, and the color develops under the action of HRP. The reaction is stopped with a stop solution, and the absorbance at 450 nm is measured. The intensity of the color is directly proportional to the ox-LDL concentration in the sample.
**Kit Components:**
- 48 or 96 wells (depending on the configuration)
- Coated microplate (1×48 or 1×96)
- Standard: 270 μg/L (0.5 mL × 1 vial)
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Reagent: 3 mL × 1 bottle (or 6 mL)
- Sample Diluent: 3 mL × 1 bottle (or 6 mL)
- TMB Substrate A & B: 3 mL × 1 bottle (or 6 mL)
- Stop Solution: 3 mL × 1 bottle (or 6 mL)
- Wash Buffer (concentrated): 20 mL × 20x or 30x (1 bottle)
- Sealing Film: 2 pieces (for 48 or 96 wells)
- Storage: All components should be stored at 2–8°C, except for the enzyme reagent, which must be kept at -20°C if not used immediately.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge similarly.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells by repeated freezing and thawing before centrifugation.
- **Tissue:** Homogenize in PBS, centrifuge, and collect the supernatant.
- **Storage:** Samples should be processed as soon as possible. If not tested immediately, store at -20°C. Avoid repeated freeze-thaw cycles.
**Procedure Summary:**
1. Prepare standard dilutions (from 180 to 15 μg/L).
2. Add samples and standards to the microplate.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add HRP-labeled antibody and incubate again.
6. Add TMB substrate and incubate for 15 minutes.
7. Stop the reaction with stop solution.
8. Measure OD at 450 nm using a microplate reader.
9. Calculate ox-LDL concentrations based on the standard curve.
**Notes:**
- Ensure all reagents are brought to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Always run a standard curve in duplicate for accurate results.
- Do not mix reagents from different batches.
- Keep the substrate away from light during incubation.
- Follow all safety protocols when handling biological samples.
**Performance Characteristics:**
- Linear range: 10–200 μg/L
- Correlation coefficient (R²): ≥ 0.990
- Intra-assay CV < 9%, Inter-assay CV < 11%
**Storage and Shelf Life:**
- Store the kit at 2–8°C.
- Shelf life: 6 months from the date of manufacture.
This ELISA kit provides a reliable and efficient method for measuring ox-LDL levels in clinical and research settings. It is ideal for studies related to oxidative stress, cardiovascular disease, and metabolic disorders.
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