Chemiluminescent Assay


Luminescent immunoassays are variations of the standard ELISA, just like fluorescent immunoassays. An enzyme converts a substrate to a reaction product that emits photons of light instead of developing a visible color. Luminescence is described as the emission of light from a substance as it returns from an electronically excited state to ground state. The various forms of luminescence (bioluminescence, chemiluminescence, photoluminescence) differ in the way the excited state is reached. For example, photoluminescence is simply fluorescence; the excitation is initiated by light at a particular wavelength. Bioluminescence is characterized by the use of a bioluminescent compound, such as luciferin and firefly luciferase. Chemiluminescence is light produced by a chemical reaction. The chemiluminescent substance is excited by the oxidation and catalysis forming intermediates. When the excited intermediates return back to their stable ground state, a photon is released, which is detected by the luminescent signal instrument.

Both bioluminescence and chemiluminescence are widely used for immunoassays and will be discussed as “luminescence”. Luminescent assays, in particular enhanced luminescent assays, are very sensitive and have a wide dynamic range. It is believed that luminescence is the most sensitive detection method currently in use due to the ability of signal multiplication and amplification. Luminescent reactions are measured in relative light units (RLU) that are typically proportionate to the amount of analyte present in a sample.

Principle of luminescent immunoassays

The widely used enzymes for luminescent immunoassays are also AP and HRP. HRP can be used with either bio- or chemiluminescent systems and is easily enhanced to allow prolonged detection of intense light (glow luminescence) which makes it compatible with all size microplate assay formats.

The suitable substrates for peroxidase include (i) luminal (3-Aminophthalhydrazide), (ii) polyphenols and acridine esters, and (iii) luciferin. The reaction of peroxidase with luciferin is considered bioluminescence. In this reaction, peroxidase replaces the in vivo enzyme, luciferase. The other substrates are chemiluminescent compounds. Polyphenols are actually a class of substrates that include pyrogallol, purpurogallin, gallic acid, and umbelliferone. All polyphenols are known for their excellent signal to noise ratio and extremely rapid light decay. Polyphenol and acridine ester substrates can only be used in conjunction with luminescent detectors equipped to handle “flash” reactions. The most popular substrate used for immunoassays is luminal or its derivatives such as isoluminol (4-Aminophthalhydrazide). It is the most suited for clinical diagnostic tests due to its properties when used in an enhanced luminescence system. Commercially available luminol is provided with an enhancer (phenols, naphthols, aromatic amines, or benzothiazoles) that acts as an enzyme protector and allows the reaction to proceed for many minutes without substantial decay in light output. Luminol oxidation reaction is carried out in an alkaline buffer, peroxidase enzymes and reactive oxygen species [peroxide anion (O2-), singlet oxygen (1O2), hydroxyalkyl radical (OH •), peroxide hydrogen (H2O2)], to generate excited state intermediates. When they return to the ground state, a wavelength of 425 nm is emitted. Typically, light emission stabilizes in less than 2 minutes, and sustained emission lasts for approximately 20 minutes or more.

Enhanced luminescence is characterized by the following desirable features: intense light emission, prolonged light emission, low background, no preincubation step, and substrate that can be added several minutes prior to detection. As long as commercial preparations of luminol are used, control of the reaction pH is not a concern. However, if the substrate is a “home-brew” preparation, pH must be stabilized at about 8.5 to allow both peroxidase activity (optimal at pH 5.5) and light emission (optimal at pH 12.0) to occur. If the pH varies much above or below 8.5, either the enzymatic activity or the luminescent detection will be negatively affected. As mentioned, luminolbased chemiluminescence is well suited for microplate-based immunoassays; in addition, this system is also recommended for DNA probe assays. The semi-automatic analysis system Kodak Am erliteTM is specially designed for this system.

AP and galactosidase each have one preferred substrate. AMPPD (3-(2'-spiroadamantane)-4- methyl-4-(3'-phosphoryloxyphenyl-1, 2- dioxetane, disodium salt) is the substrate most commonly used with alkaline phosphatase. A similar substrate, AMPGD (3-(2'-spiroadamantane)-4-methoxy-4- (3'-ß-D-galactopyranosyloxyphenyl-1,2-dioxetane), is routinely used with ß-galactosidase. Both substrates are compatible with commercially available enhancers.

Properties: ultra-high sensitivity (10, 000 times higher than and the light absorption method and 1000 times higher than the fluorescence detection method); wider dynamic range; show a linear relationship between luminous intensity (6-8 orders of magnitude) and the concentration of measured substance; lower background; no background expression of luciferase in mammals