The human low-density lipoprotein (LDL) ELISA kit is based on a double-antibody one-step sandwich ELISA method. The process begins by adding the coated microwells, which are pre-coated with LDL capture antigen, followed by the addition of standards, samples, and HRP-labeled detection antibodies. After incubation and thorough washing, the substrate TMB is used to develop color. Under peroxidase catalysis, TMB turns blue and then changes to yellow when an acid is added. The intensity of the color is directly proportional to the concentration of LDL in the sample. The optical density (OD) at 450 nm is measured using a microplate reader to determine the sample concentration.
Sample collection and handling vary depending on the type of sample:
1. **Serum**: Use endotoxin-free tubes. After blood collection, centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells.
2. **Plasma**: Anticoagulants such as EDTA, citrate, or heparin should be used. Centrifuge at 3000 rpm for 30 minutes to collect the supernatant.
3. **Cell supernatant**: Centrifuge at 3000 rpm for 10 minutes to remove debris and polymers.
4. **Tissue homogenate**: Tissue is homogenized in physiological saline and centrifuged at 3000 rpm for 10 minutes to obtain the supernatant.
5. **Storage**: If not tested immediately, aliquot the samples and store at -20°C. Avoid repeated freeze-thaw cycles. Thaw at room temperature and ensure complete thawing before use.
For the experiment, you will need:
- A microplate reader set at 450 nm
- High-precision pipettes and tips (0.5–10 μL, 2–20 μL, 20–200 μL, 200–1000 μL)
- A 37°C incubator
**Precautions:**
- Store the kit at 2–8°C and allow it to equilibrate at room temperature for 20 minutes before use. If the washing buffer crystallizes after removal from the fridge, gently warm it in a water bath before use.
- Unused wells should be returned to the ziplock bag and stored at low temperature.
- No dilution is needed for pre-treated samples; add 10 μL directly.
- Follow the instructions strictly regarding timing, volume, and sequence.
- Shake all reagents well before use.
**Kit Components:**
- Microporous plates (96-well or 48-well configuration)
- Standards (300 ng/mL), standard dilutions, sample diluent, detection antibody-HRP, 20× wash buffer, substrates A and B, stop solution, seal film, and zipper bags
**Reagent Preparation:**
- Dilute 20× wash buffer with distilled water in a 1:20 ratio.
**Washing Procedure:**
- Manual: Wash 5 times by filling each well with washing solution, letting it sit for 1 minute, and discarding the liquid.
- Automatic: Use 350 μL of washing solution per well, soak for 1 minute, and repeat 5 times.
**Procedure Steps:**
1. Remove the required wells from the foil pouch after equilibration and store the rest in a sealed bag at 4°C.
2. Set up standard, sample, and blank wells. Add 50 μL of standard solutions at different concentrations.
3. Add 10 μL of sample and 40 μL of sample diluent to each sample well.
4. Add 50 μL of HRP-labeled detection antibody to standard and sample wells. Seal and incubate at 37°C for 60 minutes.
5. Wash the plate 5 times with washing solution.
6. Add 50 μL of substrates A and B, and incubate in the dark for 15 minutes.
7. Stop the reaction by adding 50 μL of stop solution, and measure OD at 450 nm within 15 minutes.
**Result Interpretation:**
Plot standard concentrations against their corresponding OD values in Excel to create a standard curve. Use the regression equation to calculate sample concentrations.
**Kit Performance:**
- Accuracy: R value ≥ 0.9900
- Sensitivity: <1.0 ng/mL
- Specificity: No cross-reactivity with other similar molecules
- Repeatability: CV <15%
- Storage: 2–8°C, away from light and moisture
- Shelf Life: 6 months
- Detection Range: 9.3 ng/mL – 300 ng/mL
**Disclaimer:**
This kit is for research use only. Not suitable for clinical trials or human experimentation. The user assumes full responsibility for any misuse. Do not mix different batch numbers. Failure to follow instructions may lead to inaccurate results.
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