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The Principle and Application of Chemiluminescence Immunoassay (CLIA) Technology

Chemiluminescence immunoassay (CLIA) was born in 1977. According to the basic principle of radioimmunoassay, the chemiluminescence immunoassay was established by combining the highly sensitive chemiluminescence technique with the highly specific immune response. CLIA has the characteristics of high sensitivity, strong specificity, wide linear range, simple operation, and does not require very expensive equipment.

Chemiluminescence immunoassay is a combination of highly sensitive chemiluminescence assay technology and highly specific immune response, and is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. . It is the latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay.

CLIA has a wide range of applications, not only can detect antigens, haptens and antibodies of different molecular sizes, but also can be used for the detection of nucleic acid probes. Compared with radioimmunoassay (RIA), fluorescence immunoassay (IFA) and enzyme immunoassay (EIA), CLIA has the advantages of no radiation, long validity period of markers and full automation. CLIA provides a trace or ultra-trace nonisotopic immunoassay for veterinary, medical and food analytical testing and scientific research.

1. The basic principle of chemiluminescence immunoassay technology

Chemiluminescence immunoassay contains two systems, immunoassay and chemiluminescence assay. The immunoassay system uses chemiluminescent substances or enzymes as markers, which are directly labeled on the antigen or antibody, and the antigen-antibody immune complex is formed through the reaction between the antigen and the antibody. The chemiluminescence analysis system is a luminescent substrate added with an oxidant or an enzyme after the immune reaction. After the chemiluminescent substance is oxidized by the oxidant, an intermediate in an excited state is formed, which emits photons to release energy to return to a stable ground state. The luminescence intensity can be detected using a luminescence signal measuring instrument. According to the relationship between the chemiluminescence marker and the luminescence intensity, the content of the analyte can be calculated using the standard curve.

2. Types of Chemiluminescence Immunoassays

Chemiluminescence immunoassays can be divided into three categories according to the different markers, namely chemiluminescence immunoassays, chemiluminescence enzyme immunoassays and electrochemiluminescence immunoassays.

2.1 Chemiluminescence immunoassay

Chemiluminescent immunoassays are a class of immunoassays in which antibodies or antigens are directly labeled with chemiluminescent agents. At present, the common markers are mainly luminol and acridine ester chemiluminescent agents.

2.1.1 Luminol-labeled chemiluminescence immunoassay

The luminescence of luminol substances is oxidation reaction luminescence. In alkaline solution, luminol can be oxidized by many oxidants, among which H2o2 is the most commonly used. Due to the slow reaction rate of luminescence, some enzymes or inorganic catalysts need to be added. Enzymes are mainly horseradish peroxidase (HRP), inorganic types include O3, halogen and Fe3, Cu2, Co2 and their complexes.

In the early days, it was mainly used for the determination of inorganic and organic biological small molecules, and the sensitivity was reduced due to the decrease of luminescence intensity after labeling. It has been found that the addition of some phenols and their derivatives, amines and their derivatives and phenylboronic acid derivatives into the luminescent system can significantly enhance the luminescence of the system, the luminescence intensity can be increased by 1000 times, and the “background” luminescence Significantly lower, and the luminescence time is also extended. The use of these enhancers enables chemiluminescence immunoassays to be widely used in the fields of protein and nucleic acid analysis.

2.1.2 Chemiluminescence immunoassay labeled with acridinium esters: acridinium esters are used in CLIA due to their poor thermal stability, and more stable acridinium ester derivatives have been synthesized through research. Under the condition of H2o2 and OH, acridine ester compounds can emit light rapidly, and the quantum yield is very high. For example, the quantum yield of acridine aryl ester can reach 0.05. Acridine ester is used as a marker for immunoassay, and the luminescence system is simple. , fast, does not need to add catalyst, and has high marking efficiency and low background. These characteristics have aroused great interest of the majority of analytical and diagnostic workers.

2.2 Chemiluminescent enzyme immunoassay

Chemiluminescent enzyme immunoassay (cLEIA) is an enzyme immunoassay, except that the substrate of the enzymatic reaction is a luminescent agent. The enzyme then acts on the luminescent substrate, emits light under the action of the signal reagent, and performs luminescence measurement with a luminescence signal analyzer.

The commonly used labeling enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (ALP), which have their own luminescent substrates.

The most commonly used luminescent substrates for HRP are luminol and its derivatives. In CLEIA, peroxidase-labeled antibody is used, and after immunoreaction, luminol is used as a luminescent substrate, and luminol emits light under the action of peroxidase and starting luminescent reagents (NaOH and H2o2), and enzyme immunoreaction The concentration of the enzyme in the substance determines the intensity of the chemiluminescence. This traditional chemiluminescence system (HRP-H2O2-LUMINOL) flashes instantaneously within a few seconds, and has the disadvantages of low luminescence intensity and difficulty in measurement. Later, an enhanced luminescent agent was added to the luminescence system to enhance the luminescence signal and keep it stable for a longer period of time to facilitate repeated measurements, thereby improving analytical sensitivity and accuracy.

Alkaline phosphatase (ALP) has been widely used in enzyme-linked immunoassays and nucleic acid hybridization assays. Alkaline phosphatase and 1,2. The luminescent system composed of dioxetane is currently the most important and sensitive type of chemiluminescent system. The representative of such systems is the ALP-AMPPD luminescent system. The phosphate bond of AMPPD in solution is very stable, and the non-enzymatic hydrolysis is very slow. In 0.05 mol/L sodium carbonate buffer solution of pH12, the decomposition half-life can reach 74 years, and there is almost no luminescence background of the reagent itself. AMPPD is a direct luminescent substrate for phosphatase, which can be used to detect alkaline phosphatase or conjugates of antibodies, nucleic acid probes and other ligands. The ALP-AMPPD luminescence system has very high sensitivity, and the detection limit of the marker ALP reaches 10-21 mol, which is one of the most sensitive immunoassay methods. AMPPD was improved to obtain a new generation of products with better reaction kinetics and higher sensitivity: CSPD, CDP-Star. These systems have been widely used in the identification of various genes and pathogen DNA.

2.3 Electrochemiluminescence immunoassay

Electrochemiluminescence (ECL) refers to a chemiluminescence process caused by an electrochemical reaction. The reaction of ECL was carried out on the electrode surface, the luminescent substrate was ruthenium terpyridine [Ru(byp)2+3], and tripropylamine (TPA) was used to stimulate the photoreaction. At the anode surface, both species lose electrons at the same time. On the electrode plate, Ru(byp)2+3 is oxidized to Ru(byp)3+3, TPA is also oxidized to cation radical (TPA+#), TPA+# spontaneously releases a proton and becomes an unstable molecule (TPA * ), passing an electron to Ru(byp)3+3, forming an excited state Ru(byp)2+3. Ru(byp)3+3 emits a photon with a wavelength of 620 NM while decaying, and returns to the ground state Ru(byp)2+3. This process is repeated on the electrode surface, resulting in efficient and stable continuous luminescence, which is continuously enhanced.

The outstanding advantages of ECL are:

①The marker molecule is small, multi-labeling can be realized, and the marker is very stable;

②Long luminous time and high sensitivity;

③The optical signal has good linearity and wide dynamic range, more than 6 orders of magnitude;

④ Repeatable measurement, good reproducibility;

⑤ Multiplex detection and homogeneous immunoassay can be realized;

⑥Fast, it usually only takes 18 minutes to complete the analysis of a sample;

⑦Fully automated. Due to its superiority, electrochemiluminescence immunoassay is a promising immunoassay method, which has been paid more and more attention by people. It has been widely used in the immunodetection of antigens, haptens and antibodies.

3. Application

Chemiluminescence immunoassay (CLIA) and luminescence enzyme immunoassay (CLEIA) are two immunoassays commonly used in chemiluminescence immunoassays. The substrate is the basis of the luminescent agent. According to the detection automation of the instrument, the instrument can be divided into two types: automatic and semi-automatic. At present, most of the fully automatic products in the domestic clinical market are imported products; according to the separation technology, the instruments are divided into two types: magnetic bead separation and plastic orifice plates.

Chemiluminescence immunoassay technology has many applications in laboratory medicine, including tumor markers, cardiac markers, thyroid function, insulin and C-peptide and diabetes, infectious diseases, bone metabolism, cytokines, hormones, growth hormone systems , Anemia diagnosis and differential diagnosis, allergic reactions and monitoring of therapeutic drug concentration.