In the detection experiment, the nature of the coating is crucial. The protein concentration can degrade over time, which affects whether the antibody you're developing can recognize it. Therefore, preserving the antigen is essential. When working with recombinant proteins, it's important to thaw them slowly in an ice bath to maintain their integrity.
The kit utilizes a double-antibody one-step sandwich ELISA method, suitable for measuring cytokine or receptor levels in cell culture supernatants, serum, plasma, and tissue fluids. It provides high sensitivity with minimal interference, detecting concentrations as low as nanograms per milligram.
Operation Skills:
1. After adding the enzyme reagent, gently blot the microplate surface with a paper towel to remove excess liquid.
2. Plan the number of samples to avoid long waiting times during washing, ensuring timely processing.
3. When pipetting, ensure the correct volume is used to minimize errors and improve accuracy.
4. Perform duplicate wells whenever possible to increase data reliability and reflect the precision of the kit.
5. Use a dosing device for sample diluent and verify its accuracy regularly.
6. To prevent evaporation, cover the plate with a lid or film and place it in a closed box with a damp cloth.
7. Store unused plates or reagents at 2–8°C. Do not reuse diluted horseradish peroxidase-labeled anti-human IgG solutions.
8. Incubate the plate immediately after sample addition, especially when handling multiple specimens. Follow the instructions strictly to avoid extended incubation times that may cause non-specific binding.
9. Sterilize all waste by autoclaving at 121°C for 30 minutes or treat with 5.0 g/L sodium hypochlorite for 30 minutes.
10. During washing, fill each well completely to avoid losing free enzymes from the wells.
11. Leave one well as a blank control without any reagent added, except for the substrate solution and 2N Hâ‚‚SOâ‚„. This helps zero the OD reading during measurement.
12. After manual washing, allow the washing buffer to sit in each well for 15–30 seconds. Avoid splashing between wells to prevent cross-contamination. Dry the plate on absorbent paper after removing the liquid.
13. If results are questionable, consider using alternative testing methods for confirmation.
14. In case deionized or distilled water is unavailable, use purified water like Wahaha, but never tap water.
15. Always replace pipette tips when moving between different bottles, even when handling standard solutions.
16. Avoid rapid aspiration to prevent air bubbles, which can affect the accuracy of the ELISA kit.
17. Use a pipette with the correct range and volume to reduce error and ensure precision.
Precautions:
1. Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use. If the concentrated wash solution crystallizes after refrigeration, warm it in a water bath until fully dissolved.
2. Immediately return unused strips to the ziplock bag and store in a cool, dry place.
3. Follow the instruction manual precisely for incubation time, liquid volume, and sequence of steps.
4. Shake all reagents thoroughly before use.
Kit Preparation:
Read the instructions carefully for each cytokine, paying attention to batch-specific details. Centrifuge the reagent bottle before use to recover as much cytokine as possible. Reconstitute lyophilized cytokines according to the provided instructions. A standard curve can typically be prepared by 8-fold serial dilution, ranging from 2000 pg/ml to 15 pg/ml. Sensitivity can be enhanced using standard ELISA procedures, amplification kits, third-party reagents, or modifications to the enzyme substrate system. For optimal performance, incubate standards and samples together. If peroxidase is used as the detection system, do not add sodium azide to the wash buffer or diluent, as it may inhibit the enzyme activity of the kit.
Plate Format:
The operation of the kit depends on the solid-phase carrier used. In domestic medical testing, microtiter plates are commonly used.
Washing Method:
Manual washing involves aspirating or removing liquid from the plate without touching the walls. Place the plate upside down on absorbent paper and inject at least 0.3 ml of recommended wash buffer into each well. Let it soak for 1–2 minutes, repeating the process as needed. For automated washing, use an automatic washer only after becoming familiar with the equipment.
ELISA Kit
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