Chemiluminescence refers to light emission produced during a chemical reaction; it is used extensively in automated immunoassays. This methodology has excellent sensitivity and dynamic range. It does not require sample radiation and nonselective excitation and source instability are eliminated. Most chemiluminescent reagents and conjugates are stable and relatively nontoxic.

In immunoassays, chemiluminescent labels can be attached to an antigen or antibody. Acridinium esters are highly specific activity labels that can be used to label antibodies and haptens. Chemiluminescent labels are used to detect proteins, viruses, oligonucleotides, and genomic nucleic acid sequences in an immunoassay. Two formats are used, competitive and sandwich immunoassays.

In a competitive immunoassay, a fixed amount of labeled antigen competes with unlabeled antigen from a patient specimen for a limited number of antibody-binding sites (Fig. 1). The amount of light emitted is inversely proportional to the amount of analyte (antigen) measured.

Fig1. Format for competitive immunoassays. (Adapted from Jandreski MA: Chemiluminescence technology in immunoassays, Lab Med 29:555, 1998.)

In a sandwich immunoassay, the sample antigen binds to an antibody fixed onto a solid phase; a second antibody, labeled with a chemiluminescent label, binds to the antigen-antibody complex on the solid phase (Fig. 2). In the sandwich assay, the emitted light is directly proportional to the analyte concentration. The detection device for analyses is a simple photo multiplier tube used to detect the emitted light.

Fig2. Format for sandwich immunoassays. (Adapted from Jandreski MA: Chemiluminescence technology in immunoassays, Lab Med 29(9):555, 1998.)

Chemiluminescent labels can be divided into five major groups: (1) luminol; (2) acridinium esters; (3) peroxyoxalates; (4) dioxetanes; and (5) tris(2,2′−bipyridyl)-ruthenium (II). Direct labels include luminol, acridinium ester, and electrogenerated luminescent chelate from ruthenium and tripropylamine (TPA) complex [Ru(bpy)3+]. These labels are attached directly to antigens, antibodies, or deoxyribonucleic acid (DNA) probes, depending on the assay format.

Oxidation of isoluminol by hydrogen peroxide (H2O2) in the presence of a catalyst (e.g., microperoxidase) produces a relatively long-lived emission at 425 nm. Oxidation of acridinium ester by alkaline H2O2 in the presence of detergent produces a rapid flash of light lasting from 1 to 5 seconds at 429 nm. Peak intensity can be used for the measurement. An alternate method is to use an integrator to measure the entire light output for greater sensitivity.

Enzymes are typically used for indirect labels. Indirect labels are attached to antibodies, antigens, and DNA probes, depending on the assay format. Enzyme labels often used in indirect procedures include the following:

 • Alkaline phosphatase (ALP).

 • Horseradish peroxidase (HRP)

• Beta-galactosidase (β-galactosidase)

An interesting label is native or recombinant apoaequorin (from the bioluminescent jellyfish, Aequorea). It is activated by reaction with coelenterazine. Light emission at 469 nm is triggered by reaction with calcium chloride.

Specific Clinical Applications

 One of many clinical applications of chemiluminescence is a third-generation serum IgE (sIgE) method, Immulite 2000 (Siemens Healthcare Diagnostics, Tarrytown, NY), a solid phase (bead), two-step chemiluminescent EIA. Allergens are covalently lined to a soluble polymer-ligand matrix, allowing immunochemical reactions to occur in liquid phases for random access automation.

Using the Ru(bpy) 3+ complex label, various assays have been developed in a flow cell using magnetic beads as the solid phase. Beads are captured at the electrode surface and unbound label is washed out of the cell by a wash buffer. Label bound to the bead undergoes an electrochemiluminescent reaction and the emitted light is measured by an adjacent photomultiplier tube.

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اختبارات ناجحة لمحرك "VK-800" الروسي وتطويره لطائرة بايكال الخفيفة

رئيس إنفيديا: الذكاء الاصطناعي الصيني "محفز للتقدم العالمي"

المزيد

مواضيع ذات صلة

Immunofluorescence

Enzyme Immunoassay

Capillary Electrophoresis

Antibody Structure and Function

B Cell Receptor for Antigen

Antigen Processing and Presentation

Methods of Enhancing Agglutination

Mechanisms of Agglutination

The Major Histocompatibility Complex

Cellular Basis of the Adaptive Immune Response

Mechanisms of Innate Immunity

Overview of Acute-Phase Proteins