Types and Future Directions of Chemiluminescence Immunoassay Methods

In recent years, chemiluminescence immunoassay has been increasingly favored by people because of its high sensitivity, strong specificity, wide application range, simple equipment and wide linear range. It is widely used in life sciences, clinical medicine, environment, food, drugs and other fields. chemiluminescence immunoassay is gradually developing in the direction of rapid, high-throughput, and highly sensitive detection because actual detection often requires the measurement of large, complex, low-abundance samples.

Category of Chemiluminescence Immunoassay

According to the different markers selected in the chemiluminescence immunoassay system, the chemiluminescence immunoassay method is divided into three categories: chemiluminescence immunoassay, chemiluminescent enzyme immunoassay and electrochemiluminescence immunoassay.

Chemiluminescence immunoassays are a type of immunoassay that directly labels antibodies or antigens with chemiluminescent labeling substances. As chemiluminescent labels, the most commonly used materials are luminol and acridinium esters.

(1) The chemiluminescence of luminol-like substance is an oxidative reaction. In an alkaline environment, luminol can be oxidized by many substances, of which hydrogen peroxide (H₂O₂) is most commonly used. In order to increase the rate of chemiluminescence reaction, certain enzymes or inorganic catalysts are added to this reaction system. The enzymes for catalysis mainly select horseradish peroxidase (HRP), and the inorganic catalyst mainly includes O₃, halogen element, and Fe³⁺, Cu²⁺, Co²⁺ and their complexes.

(2) Chemiluminescence of acridine esters: in an alkaline environment, acridine esters are oxidized by H₂O₂ to rapidly generate photons, which has a good quantum yield. The acridine ester substance is used as a chemiluminescent label for chemiluminescence immunoassay, and has the advantages of simple luminescence system, fast reaction rate, no catalyst, high labeling efficiency, and low luminescent background.

Chemiluminescent enzyme immunoassays are a type of chemiluminescent immunoassay for labeling antigens or antibodies with enzymes. After the enzyme-labeled antigen or antibody is subjected to a specific immunological reaction, the enzyme-catalyzed chemiluminescent substrate on the immunoreactive complex emits light under the action of a signal reagent. The most widely used labeling enzymes are HRP and alkaline phosphatase (ALP), both of which have their own luminescent substrates. The most commonly used luminescent substrate for HRP is luminol and its derivatives. In the chemiluminescent enzyme immunoassay, HRP-labeled antibody or antigen is used. After the immune reaction, luminol is used as a luminescent substrate. In the alkaline environment and HRP, luminol is oxidized by H₂O₂, and the concentration of HRP determine the intensity of chemiluminescence. This conventional chemiluminescence system (HRP-H₂O₂-Luminol) is a flash-type luminescence that lasts for a few seconds, and has the disadvantages of low luminous intensity and difficulty in measurement. The addition of a chemiluminescence enhancer to the chemiluminescence reaction system can greatly enhance the luminescence signal and maintain the luminescence stability for a long period of time, thereby improving the sensitivity and accuracy of the chemiluminescence reaction system. The most commonly used luminescent substrate for ALP is 1,2-dioxane. The most classic of these chemiluminescence systems is the 3-(2-helixadamantane)-4-methoxy-4-(3-phosphonooxy)-phenyl-1,2-dioxane Ethane disodium salt (AMPPD)-ALP luminescence system proposed by Bronstein et al. This chemiluminescent reaction system is characterized by a fast reaction rate and a short time to provide a test result.

Electrochemiluminescence immunoassay is a type of chemiluminescence immunoassay that combines electrochemiluminescence with immunoassay. Electrochemiluminescence is a specific chemiluminescence reaction initiated by electrochemistry on the surface of an electrode. The electrochemiluminescence reaction is easy to precisely control. The most commonly used luminescent substrate for electrochemiluminescence immunoassays is the terpyridine pyridine [Ru(byp)₃²⁺], TPA is used to stimulate the luminescence reaction of this system. On the surface of the anode, Ru(byp)₃²⁺ and TPA lose electrons at the same time, Ru(byp)₃²⁺ is oxidized to Ru(byp)₃³⁺, TPA is oxidized to cationic radical (TPA⁺*), and TPA⁺* will spontaneously release a proton becomes an unstable molecule (TPA*), and an electron is transferred to Ru(byp)₃³⁺ to form an excited state of Ru(byp)₃²⁺*, and Ru(byp)₃²⁺* emits while attenuating a photon with a wavelength of 620nm returns to the ground state Ru(byp)₃²⁺. This reaction process is repeated on the surface of the electrode to produce efficient and stable continuous luminescence, and the luminescence intensity is continuously enhanced.

Development direction of chemiluminescence immunoassay

On the whole, the chemiluminescence immunoassay method has higher sensitivity than the traditional enzyme-linked immunoassay method, but the intra-assay coefficient of variation of the method is higher than that of the enzyme-linked immunosorbent assay. The main reason is that the existing chemiluminescent label is not very stable after the luminescence reaches the peak attenuation. Therefore, the chemiluminescence immunoassay method still needs to be studied in the following aspects. 1) continue to screen new chemiluminescent labels; 2) carry out research on chemiluminescence sensitization system in the existing chemiluminescence reaction system; 3) strengthen the application research of electrochemiluminescence immunoassay in the field of food safety detection. It can be expected that as scientists pay more attention to solving the problem of luminescence stability of chemiluminescent labels, chemiluminescence immunoassay methods will be more widely used in other fields.

Edited by Suzhou Yacoo Science Co., Ltd.