Mouse NOD-like Receptor-2 (NOD-2) ELISA Kit

The NOD-like receptor-2 (NOD-2) ELISA Kit is based on a double-antibody one-step sandwich ELISA technique. The microwells are pre-coated with mouse NOD-2-specific capture antibodies. After adding the samples, standards, and HRP-conjugated detection antibodies sequentially, the plate is incubated, washed thoroughly, and developed using TMB substrate. Under peroxidase catalysis, TMB turns blue and then yellow when an acid is added. The intensity of the color correlates directly with the concentration of NOD-2 in the sample. Absorbance is measured at 450 nm using a microplate reader to determine the sample concentration. Sample collection and handling procedures vary depending on the type of sample: 1. **Serum**: Collect blood in pyrogen-free tubes. Centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells. 2. **Plasma**: Use anticoagulants such as EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 minutes to collect the supernatant. 3. **Cell culture supernatant**: Centrifuge at 3000 rpm for 10 minutes to remove debris and polymers. 4. **Tissue homogenate**: Homogenize tissue in physiological saline, then centrifuge at 3000 rpm for 10 minutes to obtain the supernatant. 5. **Storage**: If not used immediately, aliquot the samples and store at -20°C. Avoid repeated freeze-thaw cycles. Thaw at room temperature before use. For optimal results, the following items are required: a microplate reader (set at 450 nm), precision pipettes (0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL), and a 37°C incubator. **Precautions during operation**: - Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use. - If the washing buffer crystallizes after removal from the fridge, warm it gently in a water bath before use. - Unused strips should be returned to the ziplock bag and stored at low temperature. - Do not dilute the samples unless specified. Add 10 µL directly. - Follow all instructions precisely, including incubation times, reagent volumes, and sequence. - Vortex all solutions before use to ensure uniform mixing. **Kit Components**: - Microporous plates (96-well or 48-well configuration) - Standards (200 ng/mL) - Standard dilutions - Sample diluent - Detection antibody-HRP - 20× Wash buffer - Substrate A and B - Stop solution - Seal film - Instructions and ziplock bags **Reagent Preparation**: - Dilute the 20× wash buffer with distilled water in a 1:20 ratio. **Washing Procedure**: - Manual: Fill each well with wash buffer, let stand for 1 minute, drain, and repeat 5 times. - Automated: Use 350 µL of wash buffer per well, soak for 1 minute, and wash 5 times. **Procedure**: 1. Remove the required number of wells from the foil pouch after 20 minutes at room temperature. 2. Set up standard, sample, and blank wells. 3. Add 50 µL of standard solutions and 10 µL of sample + 40 µL diluent. 4. Add 50 µL of HRP-labeled detection antibody to each well. 5. Incubate at 37°C for 60 minutes. 6. Wash 5 times. 7. Add 50 µL of TMB substrate A and B, incubate in the dark for 15 minutes. 8. Stop the reaction by adding 50 µL stop solution. 9. Measure OD at 450 nm within 15 minutes. **Result Interpretation**: Plot the standard curve in Excel with concentration on the x-axis and OD values on the y-axis. Use the linear regression equation to calculate sample concentrations. **Kit Performance**: - Accuracy: R² ≥ 0.9900 - Sensitivity: <1.0 ng/mL - Specificity: No cross-reactivity with other analogs - Repeatability: CV <15% between plates - Storage: 2–8°C, protected from light and moisture - Shelf life: 6 months - Detection range: 6.25 ng/mL – 200 ng/mL **Disclaimer**: This kit is for research purposes only. It must not be used in clinical trials or animal experiments. The user assumes all risks and responsibilities. Do not mix different batch numbers. Any deviation from the instructions will result in inaccurate results, and the company is not liable for any consequences.

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