Enter the World of Chemiluminescence

What is Chemiluminescence

The term "chemiluminescence" was first coined by Eilhardt Weidemann (1888). Chemiluminescence, the phenomenon observed when a product that has been stimulated exogenously into an excited state relaxes to its ground state and emits photons, can be defined in simple terms: the emission of light in a chemical reaction.

Historical Background of Chemiluminescence

A breakthrough from traditional immunoassays was the pioneering work of Yalow and Berson, the American scientists who first used radioimmunoassay (RIA) for the determination of insulin in 1959. Since the 1970s, various non-radiolabeling methods have been gradually developed. In 1977, Tsuji et al. developed a non-radiolabeled immunoassay based on the basic principles of radioimmunoassay, using labeled luminol as a tracer signal. The chemiluminescence of enzymes was combined with the immune reaction to develop the chemiluminescence immunoassay (CLIA).

Advantages of Chemiluminescent Immunoassay Technology

The advantages of CLIA over RIA, EIA, and FIA are

(1) No radiation hazard;

(2) High sensitivity, with a linear range of detection of 6 orders of magnitude and a lower limit of 10-21 mol;

(3) Stable luminescent markers with long validity;

(4) Easy to achieve automated operation;

(5) Wide range of applications, can detect antigens, semi-antigens, and antibodies of different molecular sizes, and can also be used for the detection of nucleic acid probes.

Basic Principles of Chemiluminescence Immunoassay

Chemiluminescence analysis is a combination of chemiluminescence and immunoassay. It offers both the high sensitivity of chemiluminescence and the high selectivity of immunoassay.  CLIA involves the labeling of an antigen or antibody with a chemiluminescent reagent (luminol or catalyst, etc.), the labeling of the antigen/antibody and the substance to be measured after a series of immunological and physicochemical steps (centrifugation, washing, etc.), and the final determination of the substance to be measured in the form of luminescence intensity.
Take Getein's acridine ester direct luminescence methodology as an example: the antibodies are labeled separately with acridine ester and magnetic beads, and after the immunoconjugate reaction between the labeled antibody and the antigen in the substance to be tested, a bead-coated antibody-antigen-acridine ester labeled antibody complex is formed. All that is required is the addition of an oxidizing agent to create an alkaline environment with NaOH, and the acridine anchorite can then directly emit light. The photon energy produced per unit time is recorded and the amount of the substance to be measured can be calculated from the standard curve.

Main types of chemiluminescent immunoassays

Depending on the marker, CLIA can be divided into three main categories: direct chemiluminescence, enzymatic chemiluminescence and electrochemiluminescence immunoassays.

Direct chemiluminescence

Luminol and acridine chemiluminescent reagents are the most commonly used markers for luminescence.

Luminol, Isoluminol, and their derivatives were one of the first common luminescent substances used in CLIA and the luminescence of these substances is oxidative. Under alkaline conditions, it is oxidized by H2O2 under HBP catalysis to produce an excited intermediate, which emits a photon when the phase returns to the ground state.

In 1935 it was discovered that lucigenin had chemiluminescent properties, and later many acridines were found to have this property. These compounds can rapidly produce chemiluminescence with high quantum yields as long as they are in the presence of H2O2 and OH-. The chemistry of acridine esters as markers for immunoassays is simple, rapid, requires no catalyst, and has high labeling and luminescence efficiencies.

Getein's newest product, the MAGICL 6000, is a compact, fully automated chemiluminescence instrument that uses this marker to greatly improve end-use efficiency while maintaining accuracy and sensitivity and has been used in a wide range of applications in 13 areas, including tumor, thyroid, and cardiac markers. 

Chemiluminescence Enzyme Immunoassay (CLEIA)

CLEIA is an enzyme immunoassay from the point of view of labeling immunoassay, except that the substrate for the enzyme reaction is the luminol: the enzyme labels the biologically active substance for the immunoassay, the enzyme in the immunoassay complex then acts on the luminol substrate and the luminol signal is measured for quantitative analysis.

Currently, the commonly used markers are HRP and ALP, both with their own luminescent substrates.

Electrochemiluminescence immunoassay (ECL)

ECL is a process of chemiluminescence caused by an electrochemical reaction. When a certain voltage or current is applied to an electrode, an electrochemical reaction occurs at the electrode, and the reaction products enter the excited state by chemical reaction between the electrode reaction products, or between the electrode reaction products and a component of the solution, and the luminescence is produced when the excited state returns to the ground state.

CLIA has the advantages of high sensitivity and good specificity and is increasingly favored by clinical tests. In addition, CLIA has a wide range of analytical methods and is widely used for the immunoassay of antigens, antibodies, and semi-antigens, and its linear range is relatively wide to meet clinical needs. Getein now offers a comprehensive menu of over 100 marker assays across 13 fields. Full process automation and a dual consumable bin design allow for non-stop consumable changes, enabling better assay efficiency; with a measurement speed of 150 T/H, while ensuring accurate whole blood assays, it provides laboratories with a compact and innovative chemiluminescence solution.