In the ELISA kit test, the nature of the coating is crucial. The protein concentration and its integrity directly affect whether the antibody you produce can recognize it. Therefore, preserving the antigen's structure is essential. When working with recombinant proteins, it's important to keep them on ice to prevent denaturation. Slow thawing in a water bath is recommended to maintain protein stability.
Blocking is an essential step that involves adding a large amount of unrelated protein to fill any available binding sites. This helps prevent non-specific binding during the ELISA process. However, sometimes the original coating may be blocked using a neutral buffer solution depending on the specific requirements of the test.
The ELISA kit uses a double-antibody one-step sandwich ELISA method, which is ideal for measuring cytokines or their receptors in samples such as cell culture supernatants, serum, plasma, and tissue fluids. It allows detection at levels as low as nanograms per milligram with minimal interference.
**Operational Considerations:**
1. Store the kit at 2–8°C and allow it to equilibrate at room temperature for 20 minutes before use. If the concentrated washing solution crystallizes after being taken out of the fridge, gently heat it in a water bath until fully dissolved.
2. Immediately return unused strips to the ziplock bag and store them in a cool, dry place.
3. Follow the incubation time, volume, and sequence strictly as outlined in the instructions.
4. Shake all liquid reagents well before use.
Prepare the required reagents according to the kit instructions. Use high-quality distilled or deionized water for all steps, including washing. Adjust the pH of the self-contained buffer using a pH meter. Allow reagents removed from the refrigerator to reach room temperature before use. Return any unused parts of the kit to the refrigerator promptly.
**Kit Preparation:**
Carefully read the instructions for each cytokine, paying attention to batch-specific details. Centrifuge the reagent vial before use to collect any residual cytokine. Reconstitute lyophilized cytokines as specified in the batch instructions. Typically, the standard curve covers a range from 2000 pg/ml to 15 pg/ml through eight-fold serial dilution. Sensitivity can be improved by using standard ELISA procedures, amplification kits, third-party reagents, or modifying the enzyme substrate system. To optimize sensitivity, it’s recommended to incubate both standards and samples. If horseradish peroxidase is used as the detection system, avoid adding sodium azide to the washing buffer or diluent, as it can inhibit the enzyme activity.
The operation of the ELISA kit varies depending on the solid-phase carrier used. In domestic medical testing, microtiter plates are commonly used.
**Washing Method:**
1. Wash the plate manually by aspirating the liquid without touching the walls. Place the plate upside down on absorbent paper and tap it several times. Use at least 0.3 ml of washing buffer per well, soak for 1–2 minutes, and repeat as needed.
2. For automated washing, use an automatic washer after becoming familiar with the procedure.
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