In the ELISA kit test, the nature of the coating is crucial. The protein concentration and its integrity are directly related to whether the antibody you produce can effectively recognize it. Therefore, preserving the antigen's structure is essential for accurate results. When working with recombinant proteins, it’s important to keep them on ice to prevent degradation. Slow melting in a water bath is often necessary to maintain their stability.
Blocking is an essential step that involves adding excess non-specific proteins to fill any available binding sites on the plate. This prevents unwanted substances from interfering during the ELISA process. However, sometimes, depending on the test requirements, the original coating may be preserved using a neutral buffer solution to ensure specificity.
The ELISA kit employs a double-antibody one-step sandwich ELISA method, making it ideal for measuring cytokines or receptors in various biological samples such as cell culture supernatants, serum, plasma, and tissue fluids. It allows for the detection of low levels of analytes—often in the range of nanograms per milligram—with minimal interference.
**Operational Notes:**
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, it's normal. Warm it gently in a water bath until fully dissolved before use.
2. Unused microtiter plates should be immediately returned to the ziplock bag and stored under low-temperature, dry conditions to maintain their quality.
3. Follow the incubation time, volume, and sequence strictly as outlined in the instructions. Any deviation can affect the accuracy of the results.
4. Shake all liquid reagents thoroughly before use. Prepare the required reagents according to the kit instructions. Use high-quality distilled or deionized water for all steps, including washing. Ensure that self-contained buffers are properly calibrated using a pH meter.
5. Allow reagents removed from the refrigerator to reach room temperature before use. Return unused parts of the kit to the refrigerator promptly to avoid contamination or degradation.
**Kit Preparation:**
For each cytokine, carefully read the instructions, paying attention to batch-specific details. Centrifuge the reagent bottle before use to collect any residual cytokine. Reconstitute lyophilized cytokines according to the specific instructions provided with each batch. Typically, the standard curve covers a linear range from 2000 pg/ml down to 15 pg/ml through eight serial dilutions.
To enhance sensitivity, consider using standard ELISA procedures, amplification kits, third-party reagents, or modifying the enzyme substrate system. For optimal performance, it's recommended to incubate both standards and samples. If peroxidase is used as the detection system, avoid adding sodium azide to the washing buffer or diluent, as it can inhibit the enzyme activity of the ELISA kit.
The operation of the ELISA kit may vary based on the type of solid-phase carrier used. In domestic medical testing, microtiter plates are commonly used.
**Washing Procedure:**
1. Wash the plate manually by aspirating the liquid (without touching the well walls) or by gently removing it. Place the plate upside down on absorbent paper and blot several times. Use at least 0.3 ml of washing buffer per well, let it soak for 1–2 minutes, and repeat the process as needed.
2. If an automated washer is available, use it only after becoming proficient with it to ensure consistent and accurate results.
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