Chemiluminescence immunoassay chemiluminescent compounds

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. 

More recently, noncompetitive methods have been developed for measuring human OC. These immunoassays use monoclonal antibodies and affinity-purified polyclonal antisera against synthetic peptides of human OC or intact human or bovine OC. Many antibody pairs have been reported including capture [and signal] antibodies, respectively, against amino acid sequences 12-33 [and 34-49], 43-49 [and 5-13], 25-37 [and 5-13], 20-43 [and 7-19], 1-19 [and 20-49], and 1-12 [and 15-30 or 38-49], These noncompetitive methods have used signal antibodies labeled with radioactivity (IRMA), enzymes (EIA/ELISA), or chemiluminescent compounds (ICMA). 

Some analytical methods, for example, for detection of trace metals, have been devised based on this reaction. Luminol has also been suggested as a labeling compound for the ECL immunoassay. In addition to luminol there are a number of analogous chemiluminescent compounds that require hydrogen peroxide in their luminescent reactions. Among these compounds the acridine derivatives lucigenin and acridinium esters have been used in ECL methods. 

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... 

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. 

A. E. Botchkareva, F. Fini, S. Eremin, J. V. Mercader, A. Montoya, S. Girotti, Development of a heterogeneous chemiluminescent flow immunoassay for DDT and related compounds, ka/. Chim. Acta, 43-52 (2002). 

Signal antibodies have most often been radiolabeled (for immunoradiometric assays [IRMA]) with ( 25j) 7,62,i%,344,4i4 labeled with a chemiluminescent (for immunochemilummo-metric assays [ICMA]) compound, such as acridinium ester, or an enzyme (enzyme-linked immunosorbent assay [ELISA] or enzyme immunoassay [EIA]), such as ALP, converting a substrate (1,2-dioxetane phosphate) to a chemiluminescent product. 

Chemiluminescence methods are known for their high sensitivities. Typical detection limits range from parts per million to parts per billion or lower. Applications include the determination of gases, such as oxides of nitrogen, ozone, and sulfur compounds, determination of inorganic species such as hydrogen peroxide and some metal ions, immunoassay techniques, DNA probe assays, and polymerase chain reacrion methods.- ... 

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. 

Enzymes. Enzymes such as horseradish peroxidase and alkaline phosphatase are the most commonly used labels. They give name to the most common immunoassay, enzyme-hnked immunosorhent assay (ELISA). The enzyme catalyzes a substrate, producing a measurable product. Two t) pes of substrates are used for ELISA those that produce a colored product (colorimetric assay) and thoes that form a self-glowing compound (chemiluminescence assay). 

Selectivity of chemiluminescence reactions can be a concern. Some reactions are essentially compound-specific. An example is tetrakis(dime-thylamino)ethylene, which undergoes chemiluminescence reaction only with Oz- Although such specificity provides freedom from measurement interference, that chemiluminescence reaction then lacks universal application. In other cases, several species could yield emission with a given reagent. These situations then require coupling of the chemiluminescence detection with some sort of highly selective physical or chemical step (such as chromatography, immunoassay, enzyme reactions) to achieve an interference-free measurement. An... 

---