Chemiluminescent labeling

Chemiluminescent labels, in which the luminescence is generated by a chemical oxidation step, and bioluminescent labels, where the energy for light emission is produced by an enzyme-substrate reaction, are additional labeling types (39,42). Luminol [521 -31 -3] CgHyN202, and acridine [260-94-6] C H N, derivatives are often used as chemiluminescent labels. 

The unsubstituted phthalic acid hydrazide and several nonaromatic cyclic hydrazides such as maleic acid hydrazide or succinic acid hydrazide are either nonchemiluminescent or show extremely weak CL. However, the 6-amino isomer of luminol, which is called isoluminol, is chemiluminescent to about the same extent as is luminol. Isoluminol has been used in many chemiluminescent studies, and because the amino group is less sterically hindered than that of luminol, it is probably derivatized for chemiluminescent labeling far more often than is luminol (Fig. 3). 

Ru(bpy)32+ itself can be determined with great sensitivity in an excess of an amine to subpicomolar levels [39], This has led to the development of electro-chemiluminescent labels based on Ru(bpy)32+ derivatives that have found successful applications in ECL immunoassay and DNA probe analysis. These are discussed in Sec. 9. 

Acridinium derivatives, specifically the 9-carboxyacridinium phenyl esters, are one of the most efficient chemiluminescent labels used in immunoassays since 1980 ... 

Chemiluminescent labeling systems have been developed, based on the incorporation of fluorescein-11-dUTP into a DNA probe. An anti-fluorescein antibody covalently bound to the enzyme HRP is then bound to the incorporated fluorescein label. HRP catalyses the breakdown of luminol and the chemiluminescent signal is detected by autoradiography with X-ray film or by fluorescence scanning instrumentation. Chemiluminescence is more sensitive than enzyme-based color detection systems. Furthermore the labeled gene probes are stable and give results quickly (160). 

Immunosensors have been designed which use both direct and indirect immunoassay technology to detect specific analytes within a minute or less in a variety of matrices (see Fig. 9). Indirect immunosensors may employ ELA, FLA, or CLIA principles whereby enzyme-, fluorophore- or chemiluminescent-labeled analyte competes with the target (nonlabeled) analyte for binding sites on the immobilized antibody. Unbound (free) labeled analyte is then quantitated using an electrochemical, optical, or electromechanical transducer and compared to the amount of target analyte in the sample. 

Methods based on chemiluminescent labels are other non-isotopic IAs and ILAs that continue to be the focus of active research. Most common approaches under this category are direct measurements of the chemiluminescence accomplished by the action of the imprinted polymer (e.g., catalytic effect) or by indirect methods, such as competitive CILAs. 

Chemiluminescence immunoassay These assays rely on the use of chemiluminescent labels, which may be labelled antigens or antibodies. For example, the chemiluminescent label isoluminol is oxidized in the presence of hydrogen peroxide and a catalyst, producing long-lived light emission that is measured, thus allowing determination of the unknown antigen or antibody concentration in a sample. Another useful variant is electrochemiluminescence immunoassay that is commonly used in biomolecular detection, and in particular the measurement of native and recombinant peptides and proteins. 

Chemiluminescent labels may be employed in sandwich or competitive antigen assays. In sandwich assays, a solid support holds a primary antibody, and incubation with ligand yields a species that is detectable following a second incubation step with a labeled second antibody. Luminol has been tested as an immunoassay label it may be coupled to proteins through its primary amino group. Luminol reacts with hydrogen peroxide and hydroxide in a microperoxidase-catalyzed reaction, which yields light at 430 nm (Eq. 6.8) ... 

Chemiluminescent labels may also be used in labeled-antigen (competitive) assays. The antigen (analyte) competes with the labeled analyte for immobilized antibody, and, following a rinse step, reagents are added to generate chemiluminescence from the labels. 

Immunometric assays for TSH are available commercially as manual kit procedures or for use on automated systems. For practical reasons, nonisotopic methods dominate the market and have replaced radioactive tracer methods in most routine laboratories. The majority of immunometric TSH assays label the detection antibody with chemiluminescent labelled molecules other labels include peroxidase or alkaline phosphatase and sensitive photo-metric and fluorescenri molecules. Other assays are based on the use of fluorescent labels using europium chelates chemiluminescent compounds such as acri-dinium esters or ruthenium or bioluminescent molecules such as recombinant aequorin. ... 

Christofides ND, Sheehan CP. Enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine Design, development, and technical evaluation. Clin Chem 1995 41 12-23. 

Chemiluminescence-labelling has been applied to the immunoassay of oestriol 16a-glucuronide by synthesis of the A-aminobutyl-A-ethylisoluminol derivative (30), which emits light when oxidized with H202-microperoxidase. Binding of the conjugate (30) to an antibody for the steroid enhances light emission. Competitive... 

Chemiluminescence offers a useful alternative to both fluorescence and radioactivity, as it does not require an excitation source, there is less light scattering, and the problem of source instability is absent. The radiation hazard is also absent from chemiluminescent molecules. Most chemiluminescent reagents and their conjugates are stable and can be applied to both homogeneous and heterogenous assays. The chemiluminescent label may be attached to an enzyme or may serve either as a substrate or as a cofactor for the enzyme. 

Only one acridinium phenyl carboxylate has found significant use as a chemiluminescent label. 4-(2-succinimidyloxycarbonylethyl)-phenyl-10-methyl-acridinium-9-carboxylate fluorosulphonate has been employed for the determination of albumin and thyroxine and a-fetoprotein. 

In contrast, the oxalate esters have hardly found use as chemiluminescent labels in immunoassays, despite their greater chemiluminescence efficiency. This is probably a consequence of the need to have a fluorescer in the medium with the oxalate ester to generate a measurable light signal. 

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