Human Low Density Lipoprotein (LDL) ELISA Test Kit

The human low-density lipoprotein (LDL) ELISA kit is based on a double-antibody one-step sandwich ELISA technique. The microplate wells are pre-coated with LDL capture antibodies. After adding the samples, standards, and HRP-labeled detection antibodies sequentially, the plate is incubated, washed thoroughly, and developed using TMB substrate. Under the action of peroxidase, TMB turns blue and then yellow when an acid is added. The intensity of the color is directly proportional to the concentration of LDL in the sample. The absorbance at 450 nm is measured using a microplate reader to determine the sample concentration. **Sample Collection and Handling:** 1. **Serum:** Collect blood in pyrogen-free tubes, centrifuge at 3000 rpm for 10 minutes, and carefully separate serum from red blood cells. 2. **Plasma:** Use EDTA, citrate, or heparin as anticoagulants, then centrifuge at 3000 rpm for 30 minutes to collect plasma. 3. **Cell Supernatant:** Centrifuge at 3000 rpm for 10 minutes to remove debris. 4. **Tissue Homogenate:** Mix tissue with physiological saline, homogenize, and centrifuge at 3000 rpm for 10 minutes. 5. **Storage:** Store samples at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles. Thaw at room temperature before use. **Required Equipment:** - Microplate reader (450 nm) - Precision pipettes (0.5–10 μL, 2–20 μL, 20–200 μL, 200–1000 μL) - Incubator at 37°C **Precautions:** - Store the kit at 2–8°C and allow it to equilibrate at room temperature for 20 minutes before use. - Reconstituted washing buffer may crystallize; warm it gently before use. - Unused wells should be resealed and stored in a ziplock bag. - Do not dilute samples unless specified. - Follow the protocol strictly regarding timing, volume, and sequence. - Shake all reagents well before use. **Kit Components (96-well):** - Microwells: 12×8 - Standards (300 ng/mL): 0.5 mL - Standard Dilutions: 6 mL - Sample Diluent: 6 mL - Detection Antibody-HRP: 6 mL - 20× Wash Buffer: 20 mL - Substrate A & B: 6 mL each - Stop Solution: 6 mL - Seal Film: 2 sheets - Ziplock Bag: 1 piece **Reagent Preparation:** Dilute 20× wash buffer by mixing 1 part with 19 parts distilled water. **Washing Procedure:** Manual: Wash 5 times, 1 minute per cycle. Automatic: Add 350 μL per well, soak for 1 minute, and repeat 5 times. **Procedure Steps:** 1. Remove the required number of wells from the foil pouch and reseal the rest. 2. Set up standard, sample, and blank wells. 3. Add 50 μL of standards and 10 μL of sample plus 40 μL diluent. 4. Add 50 μL of HRP-labeled antibody, seal, and incubate at 37°C for 60 minutes. 5. Wash 5 times. 6. Add 50 μL of TMB A and B, incubate in the dark for 15 minutes. 7. Add 50 μL stop solution and measure OD at 450 nm within 15 minutes. **Result Interpretation:** Plot standard concentrations vs. OD values in Excel, generate a standard curve, and calculate sample concentrations. **Kit Performance:** - Accuracy: R ≥ 0.9900 - Sensitivity: <1.0 ng/mL - Specificity: No cross-reactivity - Reproducibility: CV <15% - Storage: 2–8°C, protected from light and moisture - Shelf Life: 6 months - Detection Range: 9.3–300 ng/mL **Disclaimer:** This kit is for research purposes only. Not intended for clinical use. The user assumes all risks. Follow instructions strictly. Do not mix different batch numbers. The company is not responsible for misuse.

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