Immunoassay chemiluminescent labels

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 ... 

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. 

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) ... 

The analytical detection limits of competitive and noncompetitive immunoassays are determined principally by the affinity of the antibody and the detection limit of the label used, respectively. Calculations have indicated that a lower limit of detection of lOfmol/L (Le., 600,000 molecules of analyte in a typical sample volume of 100 jiL) is possible in a competitive assay using an antibody with an affinity of iO L/mol. Table 9-2 illustrates the detection limits for isotopic and nonisotopic labels. A radioactive label, such as l, has low specific activity (7.5 million labels necessary for detection of 1 disintegration/s) compared with enzyme labels and chemiluminescent and fluorescent labels. Enzyme labels provide an amplification (each enzyme label produces many detectable product molecules), and the detection limit for an enzyme can be improved by replacing the conventional photometric detection reaction by a chemiluminescent or bioluminescent reaction. The combination of amplification and an ultrasensitive detection reaction makes noncompetitive chemiluminescent EIAs among the most sensitive types of immunoassay. Fluorescent labels also have... 

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... 

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. 

As mentioned in Section 3.1, the derivatization of luminol results in a much reduced quantum yield. On the other hand, isoluminol compounds (derivatized at the 6-amino position) exhibit higher quantum yields than the parent compound. This fact dictates the way in which each compound is utilized as an analytical agent. Compounds such as ABEl (Fig. 14) can be covalently conjugated to a variety of substances, ranging in size and complexity from steroid hormones to immunoglobulins. These chemiluminescent labels can be subsequently utilized in immunoassays according to the general scheme outlined in Section 1.3.1. 

F., Chemiluminescent labels in immunoassay. In Bioluminescence and Chemiluminescence Basic Chemistry and Analytical Applications (M. A. DeLuca and F. McCapra, eds.), pp. 673-679. Academic Press, New York, 1981. 

The chemiluminescence intensity from the reaction of amines with tris(2,2 -bipyridyl)ruthenium(III) is generally in the order tertiary > secondary > primary, but no definitive mechanisms have been elucidated. Derivatives of tris(2,2 -bipyridyl)ruthenium(II) have been introduced as labels for immunoassay or DNA probes (e.g., (13)). The use of chemiluminescent labels is clearly advantageous due to their nonradio-active nature and they can be quantified at sub-pi-comolar levels via oxidation in the presence of tripropylamine. 

Chemiluminescence and bioluminescence immunoassay Chemiluminescent groups Luminometry Complicated labelling detection simple, but time dependent low detection limits possible, homogeneous methods (17), (18), (19), (20) (21), (22)... 

In the most common method for chemiluminescent immunoassay (GLIA), after the immunological reaction and any necessary separation steps, the labeled compounds or complexes react with an oxidizer, eg, hydrogen peroxide, and an enzyme, eg, peroxidase, or a chelating agent such as hemin or metal... 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Chemiluminescence reactions are currently exploited mainly either for analyte concentration measurements or for immunoanalysis and nucleic acid detection. In the latter case, a compound involved in the light emitting reaction is used as a label for immunoassays or for nucleic acid probes. In the former case, the analyte of interest directly participates in a chemiluminescence reaction or undergoes a chemical or an enzymatic transformation in such a way that one of the reaction products is a coreactant of a chemiluminescence reaction. In this respect, chemiluminescent systems that require H2O2 for the light emission are of particular interest in biochemical analysis. Hydrogen peroxide is in fact a product of several enzymatic reactions, which can be then coupled to a chemiluminescent detection. 

One of the spinoffs of this work has been the appreciation that bio- or chemiluminescence could be used as an aid in analytical chemistry. This has led to the successful applications of acridinum esters as labels in immunoassays [6]... 

Antibody molecules have no inherent characteristic that facilitates their direct detection in immunoassays. A second important step in developing a successful immunoassay, therefore, involves the incorporation of a suitable marker . The marker serves to facilitate the rapid detection and quantification of antibody-antigen binding. Earlier immunoassay systems used radioactive labels as a marker (radioimmunoassay RIA) although immunoassay systems using enzymes (enzyme immunoassays EIA) subsequently have come to the fore. Yet additional immunoassay systems use alternative markers including fluorescent or chemiluminescent tags. 

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