What should be noted when using luminol for chemiluminescence immunoassay?

Chemiluminescent immunoassay (CLIA) is a highly sensitive and specific immunoassay technique widely used in clinical diagnosis, drug screening, and scientific research. Luminol, a 3-aminobenzoylhydrazine compound, is one of the commonly used chemiluminescent agents in CLIA. When using luminol for chemiluminescence immunoassay, ensuring the accuracy and reliability of the experiment requires following a series of rigorous operating procedures and precautions.

1、 Properties and Storage of Luminol

Luminol, as a photosensitive substance, is extremely sensitive to light and moisture. Therefore, the correct storage method is crucial. Luminol should be stored in a cool, dry, and dark environment, preferably in a sealed container, to prevent its decomposition or degradation. Regularly check the purity and activity of luminol to ensure that it is in good condition to maintain the efficiency of the chemiluminescence reaction.

2、 Optimization of chemiluminescence reaction

The chemiluminescence reaction of luminol relies on the presence of an oxidant, typically using hydrogen peroxide (H ₂ O ₂) as the oxidant, and requires a catalytic enzyme such as horseradish peroxidase (HRP) to promote the luminescence reaction. In the experimental design phase, it is necessary to carefully regulate the pH value, temperature, and reactant concentration in the reaction system to achieve optimal luminescence efficiency. In addition, certain enhancers, such as bromophenol red, have been shown to significantly enhance the luminescence intensity of luminol, and can be added appropriately according to specific needs.

3、 Avoid non-specific binding

In chemiluminescence immunoassay, non-specific binding can lead to an increase in background signal, thereby affecting the accuracy of detection results. Using high-quality blocking reagents, optimizing the ratio of antibodies and antigens, and adopting strict washing steps can reduce non-specific binding, lower background noise, and improve the signal-to-noise ratio of detection.

4、 Safe operation and personal protection

Luminol and its solutions have a certain degree of strong acidity and irritability. When operating, protective goggles, lab coats, and gloves should be worn to avoid direct contact with the skin and eyes. Meanwhile, ensure good ventilation in the laboratory to reduce the accumulation of harmful gases.

5、 Calibration of instruments and testing conditions

Before conducting chemiluminescence detection, it is necessary to ensure that the detection instrument has been calibrated and that components such as the light source, detector, and filter are in optimal working condition. Adjust the sensitivity and wavelength selection of the detector to adapt to the luminescent properties of luminol, typically with the maximum emission wavelength around 425nm.

6、 Establish standard curve

In order to quantitatively analyze target molecules in a sample, a standard curve must be established using a known concentration standard. This process requires precise preparation of standard samples and high repeatability of experimental operations to ensure the linear range and accuracy of the standard curve.

7、 Repeatability of experiments and data processing

Repeatability is a key indicator for evaluating the quality of chemiluminescence immunoassay. Each experiment should include a control group and multiple parallel samples to verify the consistency of the results. When processing data, statistical methods should be used to analyze, eliminate outliers, calculate the mean and standard deviation, and ensure the reliability and scientificity of the results.

The application of luminol in chemiluminescence immunoassay not only requires experimenters to have solid professional knowledge, but also requires high experimental skills and meticulous experimental management. Only by fully considering and strictly adhering to the above precautions can we fully leverage the advantages of luminol in chemiluminescence immunoassay and achieve efficient and accurate detection results. In future scientific research and clinical practice, luminol will continue to be an indispensable tool in the field of chemiluminescence immunoassay, promoting the development of life sciences. As a manufacturer of luminescent reagents, Desheng has a complete range of luminol types, high sensitivity, and professional personnel to track and guide, helping to solve technical problems. If you have any relevant intentions, please click on the website to inquire about details!