Introduction to chemiluminescence immunoassay technology for mycotoxin

1、 Introduction to chemiluminescence immunoassay technology:

Immunology is an important fundamental and cutting-edge discipline. Immunological testing is a technique based on the principle of antigen antibody specific reaction, using various sensitive labeling and tracing techniques (such as radioactive isotopes, fluorescein, enzymes, lanthanide elements, luminescent substances, colloidal gold, etc.) to specifically analyze and detect targets. Chemiluminescence immunoassay (CLIA) technology started in the early 1980s and rapidly developed in the 1990s, becoming an emerging immunoassay technology after fluorescence immunoassay, radioimmunoassay, and enzyme-linked immunosorbent assay. CLIA has developed into a mature and advanced technology for detecting ultra trace active substances, with rapid growth in the past decade. It is the fastest developing and widely used immunoassay method, as well as the most advanced labeled immunoassay technology. Its sensitivity and accuracy are several orders of magnitude higher than enzyme immunoassay and fluorescence assay, and it can completely replace radioimmunoassay and completely eliminate enzyme-linked immunosorbent assay. CLIA mainly has the advantages of high sensitivity, strong specificity, good reagent stability and long validity period, wide detection linear range, simple operation, stable and fast method, multiple detection items, and high degree of automation. This technology has become the mainstream method of current immunoassay technology and is widely used in various fields of the detection industry.

2、 The principle of chemiluminescence immunoassay technology is to combine highly sensitive chemiluminescence detection technology with highly specific immune reactions, label antibodies or antigens with chemiluminescence related substances, react with the tested antigen or antibody, separate the free state chemiluminescence label, and add other related substances to the chemiluminescence system to produce chemiluminescence. It is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. The chemiluminescence immunoassay system consists of two parts, namely the immune response system and the chemiluminescence analysis system. Immunoassay principle: Immunoassay is a highly selective analytical method established by the specific binding of antigens and antibodies to form antigen antibody complexes. According to its detection methods, it can be divided into unlabeled immunoassay and labeled immunoassay. Non labeled immunoassay is the use of antigen antibody complexes formed by the binding of antigens and antibodies, which undergo changes in their physicochemical properties, resulting in visible precipitation, agglutination, and other phenomena. These phenomena are used to detect the analyte. Label immunoassay is a method of indirectly detecting a test substance by labeling a labeled substance on an antigen or antibody without affecting its biological activity, and then performing an immune reaction to form an antigen antibody complex. The labeled substance is then detected. The commonly used biomarkers currently include radioactive 125I, non radioactive alkaline phosphatase, horseradish peroxidase, lanthanide rare earth elements, etc. Principle of chemiluminescence: Chemiluminescence is the light radiation produced by a chemical substance in a specific chemical reaction. The formation of singlet molecules is excited in chemical reactions through the decomposition of high-energy intermediates. Once excited, the molecules become unstable and release excess energy to return to the ground state, with some of the energy being released in the form of luminescence.

The process can be roughly divided into the following steps:

(1 Immune response: The formation of antigen antibody complexes by specific antibodies or antigens that bind specifically to the target analyte. This specific binding is the basis of immune response and a key step in chemiluminescence immunoassay.     

(2 Separation and Purification: Using physical or chemical methods to separate the formed antigen antibody complex from complex biological samples, in order to achieve purification of the target analyte.     

(3 Chemiluminescence reaction: Under specific conditions, a chemiluminescence substrate is added to the purified antigen antibody complex. These substrates can undergo chemical reactions and generate light energy under the action of specific catalysts or oxidants. This light energy can be captured by specific detectors and converted into electrical signals, thereby achieving quantitative detection of the target analyte.

3、 The chemiluminescence immunoassay method for mycotoxin uses the principle of indirect competition to determine the content of mycotoxin. The principle is that mycotoxin in the test sample compete with biotinylated antigens bound to streptavidin magnetic particles to form binding complexes with bifunctional fusion proteins (AP labeled antibodies). After washing, the luminescent substrate is added, and the luminescent substrate is catalytically cleaved by enzymes in the complex, forming an unstable excited state intermediate. When the excited state intermediate returns to the ground state, photons are emitted. The number of photons generated is negatively correlated with the content of mycotoxin to be tested in the sample. Based on the built-in standard curve of the instrument, the content of mycotoxin in the sample is automatically calculated.

The use of magnetic particles expands the surface volume of the solid-phase carrier, thereby increasing the binding amount between proteins and enzymes, achieving signal amplification, and making it easier to separate the bound and unbound parts of antigens and antibodies, reducing the influence of interfering substances and improving the sensitivity and detection efficiency of CLIA.

4、 Characteristics of chemiluminescence immunoassay for mycotoxin

(1 Strong specificity: Chemiluminescence immunoassay utilizes specific immune reactions to identify and capture target analytes, with high specificity.     

(2 High sensitivity: High sensitivity is the key advantage of chemiluminescence immunoassay, with a sensitivity of 10-22mol/L, which can detect substances that cannot be detected by methods such as radioimmunoassay and enzyme-linked immunosorbent assay. The luminescence intensity of chemiluminescence immunoassay ranges from 4 to 6 orders of magnitude and shows a linear relationship with the concentration of the measured substance, which is significantly advantageous compared to the absorbance (OD value) range of 2.0 in colorimetric enzyme immunoassay.     

(3 Wide linear range: High sensitivity and specificity enable chemiluminescence immunoassay to achieve accurate measurements over a wide concentration range, suitable for sample analysis at different concentrations.     

(4 Good stability and reliability: The sample system directly emits light on its own without the need for any light source irradiation, and the light signal generated by CLIA has a long duration, eliminating the influence of various possible factors (light source stability, light scattering, light wave selector, etc.) on the analysis, making the analysis results sensitive, stable, and reliable.     

(5 Long term effectiveness of reagents: CLIA markers are stable and not easily affected by environmental factors, ensuring a long shelf life of the reagents.     

(6 High degree of automation: With the development of technology, chemiluminescence immunoassay has been able to achieve highly automated detection, reduce human error, and improve the efficiency and accuracy of analysis.