Chemiluminescence acridinium esters

Although acridinium esters can be used to determine hydrogen peroxide, they are more commonly used as labels for immunoassay or DNA probes. As no catalyst is required, acridinium ester chemiluminescence has a lower chemical blank than the oxidation of luminol and therefore has superior detection limits. A strongly alkaline solution (pH 12-13) is required, but under these conditions acridinium esters undergo reversible conversion to the non-chemiluminescent pseudobase form (Scheme 1), which slowly decomposes. As a consequence, acidic hydrogen peroxide is added to reconvert the pseudobase to the acridinium ester before the chemiluminescence reaction is initiated with a sodium hydroxide solution. 

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. 

Figure 6 Chemiluminescent reaction of an acridinium ester and hydrogen peroxide in the presence of a base.

Acridinium esters have also been utilized for chemiluminescent detection of cDNA probes (Fig. 5) [9-11], The hydrolysis rate is much faster when the ester is conjugated to single-stranded DNA, rather than to double-stranded DNA. This means that the chemiluminescence from unhybridized acridinium ester-labeled probe is rapidly lost, whereas the chemiluminescence from the hybridized probe is minimally affected. This permits discrimination between hybridized and unhybridized acridinium ester-labeled DNA probes without separation steps. 

Therefore, chemiluminescent methods using an acridinium ester-labeled cDNA probe allow the discrimination of a mismatched DNA sequence in a homogeneous assay. 

Figure 5 Chemiluminescent reaction of acridinium ester and its hydrolysis.

CHEMILUMINESCENT DETECTION OF ACRIDINIUM ESTER-LABELED PROBE... 

The acridinium ester (AE) in an AE-labeled cDNA probe hybridized to target DNA is less likely to be hydrolyzed than in the unhybridized conformation (Fig. 10) [9-11]. Single-base mismatches in the duplex adjacent to the site of AE attachment disrupt this protection, resulting in rapid AE hydrolysis [11]. Hydrolysis by a weak base renders AE permanently nonchemiluminescent. After hydrolysis, it is possible to use the remaining chemiluminescence as a direct measure of the amount of hybrid present. This selective degradation process is a highly specific chemical hydrolysis reaction, which is sensitive to the local environment of the acridinium ester. The matched duplex can be detected and quantified readily, whereas the mismatched duplex produces a minimal signal. 

Figure 10 Mismatch detection by using a chemiluminescent AE-labeled cDNA probe. Procedure [9, 11] Acridinium ester-labeled probes specific for either wild-type or mutant sequence corresponding to a target DNA are hybridized with the sample DNA for 1.0 h at 60°C in a hybridization buffer (pH 5.2). Hybridized and nonhybridized probes are discriminated by the hydrolysis reaction for 12 min at 62.5°C in the presence of Na2B407 (pH 8.5) and Triton X-100. The chemiluminescence of each sample is then measured in a luminometer.

Chemiluminescence Luminol, acridinium esters, adamantyl dioxetane... 

In chemiluminescence immunoassay the antigen is tagged with a molecule such as luminol or an acridinium ester which emits light with a high quantum yield on oxidation. Alternatively, the antigen may be labelled with a bio-luminescent molecule such as luciferin, which emits light when oxidized by the enzyme luciferase. 

Figure 2 illustrates the reaction mechanisms of acridinium ester label probes and alkaline phosphatase probes using dioxetane chemiluminescent detection. Table 2 summarizes approaches for labeling DNA. 

Acridinium ester—labeled chemiluminescent probes have been utilized to detect the specific protein-coding transcripts and to distinguish between transcripts that code for the 190-kDa protein and the two closely related 210-kDa proteins. The assay is called the hybridization protection assay (D3). In this assay, RNA isolated from the patient s white blood cells is first amplified by PCR. The amplified product is incubated with the chemiluminescent probe. The unhybridized probe is removed by selective hydrolysis in sodium tetraborate buffer, containing surfactant Triton X-100 at pH 8.5, in an incubation step at 60°C for 6 min. After the sample is cooled to room temperature, the chemiluminescence of the hybridized probe is measured in a luminometer. The procedure is reported to detect one leukemic cell in a population of a million or more normal cells. It is also rapid, requiring less than 30 min. Its reliability has been attested to by correlation with results obtained on karyotypic and Southern blot analysis (D3). 

N4. Nelson, N. C., and Kacian, D. L., Chemiluminescent DNA probes A comparison of the acridinium ester and dioxetane detection systems and their use in clinical diagnostic assays. Clin. Chim. Acta 194, 73-90 (1990). 

Figure 3.29.A shows a flow-cell of 20 iL inner volume used to hold immobilized anti-mouse IgG bound to a rigid beaded support (activated Pierce trisacryl GF-2000). The cell was used to develop a two-site immunoassay for mouse IgG by consecutive injection of the sample, acridinium ester-labelled antibody and alkaline hydrogen peroxide to initiate the chemiluminescence, which started the reaction sequence shown in Fig. 3.29.B. Regenerating the sensor entailed subsequent injection of an acid solution, which resulted in a determination time of ca. 12 min (this varied as a fimction of the flow-rate used, which also determined the detection limit achieved, viz. 50 amol for an overall analysis time of 18 min) [218]. The sensor was used for at least one week with an inter-assay RSD of 5.9%. Attempts at automating the hydrodynamic system for use in routine analyses are currently under way.

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

Chemiluminescent Acridinium ester Phenanthridinium ester Ester hydrolysis Ester hydrolysis... 

Chemiluminescent Acridinium ester Isoluminol Ester hydrolysis Light emission... 

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