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. 

Synthesis of polycyclic pyridazinediones as chemiluminescent compounds 98JHC1219. 

Chemiluminescent compounds and their precursors in P. stipticus. Although P. stipticus is negative in the luciferin-luciferase reaction, crude extracts of this fungus are chemiluminescent, like the luciferin obtained from Ompbalia flavida by Kuwabara and Wassink (1966). The chemiluminescence is elicited by the addition of H2O2 and Fe2+ under a mild condition of pH 5-8, and the luminescence is strongly... 

Panal is stable in aqueous media (pH 1-5) at room temperature, except for a gradual hydration that can be reversed with an aqueous acid. Panal is activated into chemiluminescent compounds upon treatment with the salts of ammonia or primary amines. 

The activation product of an equal-amount mixture of PS-A and PS-B gave four major chemiluminescent compounds, designated PM-1, PM-2, PM-3 and PM-4 in an approximate ratio of 1 2 2 1. The activation product of PS-A gave only PM-1, and that of PS-B gave only PM-4. 

Laser devices are the most sophisticated image-acquisition tools. They are particularly useful for gels labeled with fluorescent dyes because the lasers can be matched to the excitation wavelengths of the fluorophores. Detection is generally with photomultiplier tubes. Some instruments incorporate storage phosphor screens for detection of radiolabeled and chemiluminescent compounds (not discussed in this chapter). Resolution depends on the scanning speed of the illumination module and can be as low as 10 pm. 

The oxides of nitrogen (NO and N02) can be determined with a method based on chemiluminescence, compound-specific light emission after excitation. The principle of this method is based on the following chemical reaction ... 

However, the use of enzyme labels in ILAs seems to be less practical for a number of reasons. First, they often work only in aqueous buffers as enzymes are more susceptible to inactivation than fluorescent or chemiluminescent compounds under harsh conditions such as organic solvents. Additionally, enzymes are invariably high molecular weight species that diffuse slowly into the MIP cavities and may have a greater tendency to increase the non-specific interactions of some other labels. Accordingly, few enzyme ILAs have been reported to date (Table 4) and, in... 

Figure 2-16. Bioluminescence and chemiluminescence. Compounds of biological origin like luciferin (left) or suitable chemical compounds (right) can be converted enzymatically to substances which undergo further decomposition with concomitant emission of light (hv).

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). 

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. ... 

Titrimetric luminescence methods record changes in the indicator emission of radiation during titration. This change is noted visually or by instruments normally used in luminescence analysis. Most luminescence indicators are complex organic compounds which are classified into fluorescent and chemiluminescent, compounds according to the type of emission of radiation. As in titrimetry with adsorption of colored indicators, luminescence titration makes use of acid-base, precipitation, redox, and complexation reactions. Unlike color reactions, luminescence indicators enable the determination of ions in turbid or colored media and permit the detection limit to be lowered by a factor of nearly one thousand. In comparison with direct luminescence determination, the luminescence titrimetric method is more precise. 

Various chemiluminescent compounds (e.g. luminol) have been studied in order to find suitable and specific probes for detection of ROS. Recently Cypridina luciferin analogues CLA and MCLA are thought to emit light only when reacting with superoxide anion and singlet oxygen. ... 

Of the artificial chemiluminescent compounds, luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) is the most popular, and has been applied not only in analytical chemistry but also in other fields. The luminol chemiluminescence is based on the light emission from an excited 3-aminophthalate ion generated by oxidation with hydrogen peroxide or atmospheric oxygen in the presence of bases and catalysts. This peculiar chemiluminescence property and its industrial value have attracted continuous interest and prompted many chemists to investigate its reaction in detail... 

The energy requirement is an obstacle to the physical characterization of chemiluminescent compounds because the availability of a reaction of high exothermicity renders most such reagents unstable. They are usually generated in situ by mixing stable precursors with appropriate reagents. The observation of blue chemiluminescence from a solution of oxalyl chloride and hydrogen peroxide was first reported in 1963,510 now well... 

The 1,2-dioxetanes are another important group of chemiluminescent compounds. These compounds are oxidatively cleaved thermally in a concerted fashion to yield two carbonyl moieties, one of which is excited. The mechanism of this cleavage has been described as a chemically initiated electron-exchange... 

Many recent applications of chemiluminescence enjoy the protection of patents and some of these applications have been commercialized, e.g., novel chemiluminescent compounds, detectors, and complete assay systems. It is not, however, the purpose of this article to focus on such commercialization and, in keeping with this precept, no patent citations will appear in the reference list. Because of space limitations, certain topics such as bioluminescence, electrochemiluminescence [recently reviewed by Greenway (G12)], and cellular chemiluminescence [see Stanley and Kricka (S41) for an update] will not be covered in this article. Even with the above omissions, it is difficult to provide a comprehensive review of all aspects of chemiluminescence, and one has to exercise a degree of selectivity. The focus will be on what is important in the field of clinical analysis, and an attempt will be made to cover the representative literature. 

Fig. 1. Growth in chemiluminescence publications since 1980. These data were obtained by a computer search for titles and key descriptors containing the word chemiluminescence or the name of any specific chemiluminescent compound.

The literature on organic chemistry abounds with examples of molecules that, in appropriate conditions, will exhibit chemiluminescence (Al, Ml7, M22). Nevertheless, the development of chemiluminescence as a diagnostic tool in immunology, clinical chemistry, and molecular biology has resulted almost entirely from the use of three or four compounds and their respective derivatives. There are several reasons for this dichotomy. First, many chemiluminescent compounds are inherently unstable and difficult to control. Conversely, the light-producing reactions of extremely stable compounds (such as some of the dioxetanes) may be so... 

The following account presents a more detailed look at those chemiluminescent compounds and their derivatives that are currently regarded as useful in clinical assays. An attempt will be made to evaluate the literature and to highlight the strengths and limitations of each class of compound. 

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