Chemiluminescent tracers

CLIA is similar to EIA and ELISA techniques except that the final receptor enzyme assay is replaced with a chemiluminescent tracer followed by measurement of light released as a result of the chemical reaction. The principles of a chemiluminescence competitive binding assay are shown in Figure 8-8. 

Acridinium ester used as a chemiluminescent tracer. Labeled acridinium ester in the presence of alkaline hydrogen peroxide undergoes chemical changes producing light that is detected at 430 nm by a photomultiplier detector. 

P8. Pazzagli, M., Messeri, G., Caldini, A. L., Moneti, G., Martinazzo, G., and Serio, M., Preparation and evaluation of steroid chemiluminescent tracers. J. Steroid Biochem. 19, 407-412... 

Chemiluminescence tracer used for steroid hormone immunoassays. 

Concerns over safe handling of radioactive materials and issues around the cost and disposal of low level radioactive waste has stimulated the development of nonradiometric products and technologies with the aim of replacing radioactive tracers in research and medical diagnosis (25). However, for many of the appHcations described, radioactive tracer technology is expected to continue to be widely used because of its sensitivity and specificity when compared with colorimetric, fluorescent, or chemiluminescent detection methods. 

As mentioned earlier (see p. 122) the previously postulated dioxetane intermediate in firefly bioluminescence has been challenged as no 180 is in-corporated in the carbon dioxide released during oxidation of firefly luciferin with 18C>2. In view of the crucial significance of the 180. experiments De Luca and Dempsey 202> rigorously examined the reliability of their tracer method. They conclude from their experiments that all available evidence is in favour of a linear, not a cyclic peroxide intermediate — in contrast to Cypridina bioluminescence where at least part of the reaction proceeds via a cyclic peroxide (dioxetane) as concluded from the incorporation of 180 into the carbon dioxide evolved 202,203). However, the dioxetane intermediate is not absolutely excluded as there is the possibility of a non-chemiluminescent hydrolytic cleavage of the four-membered ring 204>. 

Copper 0.5-6 nmol/kg Tracers of air-sea interaction 0.1 nmol/kg 1/d Stripping voltamme chemiluminescent... 

Finally, we must point out that nowadays the widely extended RILAs are basically used for MIP characterization purposes. In a general way, the use of RILAs has been reduced drastically in the last few years. The literature shows that they have been replaced by other tracers with fluorescence, chemiluminescence or absorbance detection often combined with labeled enzymes. Simplicity in their use, easy access to these techniques, and the lack of radioactive compounds are the main reasons for such substitution. 

Surugiu et al. [67] have introduced an Enzyme Immuno-Like Assays (EzILA) for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The label was a 2,4-D conjugate with the tobacco peroxidase (TOP) enzyme, which allows for both colorimetric and chemiluminescent detection. In this case, the polymer imprinted with 2,4-D was synthesized in the form of microspheres. In contrast, despite its higher binding capacity for radiolabeled 2,4-D, a conventional MIP prepared by bulk polymerization showed only weak binding of the 2,4-D-TOP tracer. 

Adamczyk M, Chen YY, Fishpaugh JR, et al. Linker-mediated modulation of the chemiluminescent signal from N(10)-(3-SuIfopropyl)-N-sulfonylacridinium-9-carboxamide tracers. Bioconj Chem 2000 11 714-24. 

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. ... 

A direct chemiluminescence assay for urinary pregnanediol 3-glucuronide employs the isoluminol conjugate (31) as tracer, with monoclonal antibodies. Specificity is excellent, the assay being sensitive to 30 pg.143 A solid-phase chemiluminescence assay for plasma progesterone uses as tracer the isoluminol conjugate (32) derived from lla-hydroxyprogesterone.144... 

Competitive binding chemiluminescence assay. The chemiluminescent competitve binding assay is similar to a RIA. The acridinium ester-labeled substance and the endogenous substance compete with the specific antibody that is available in limited concentration. The concentration is determined by the quantity of tracer that binds to the antibody. The use of solid-phase antibody simplifies separation of free from bound tracer. 

We used an anti-TSH monoclonal antibody bound magnetizable microparticles as solid phase and alkaline phosphatase-labelled anti-TSH monoclonal antibody as a tracer. After 6 min incubation of solid phase, a tracer and sample or calibrator (30 fiL), microparticles were washed to remove unbound materials and were then incubated for 5 min with 50 /iL of chemiluminescence substrate, new 1,2-dioxetane derivative. The amount of enzyme-labeled monoclonal antibody that binds to the microparticles is directly proportional to the TSH concentration in the test sample. 

Electrocapillary methods, described in Sections 13.2 and 13.3, are very useful in the determination of relative surface excesses of specifically adsorbed species on mercury. As discussed in Section 13.4, such methods are less straightforward with solid electrodes. For electroactive species and products of electrode reactions, the faradaic response can frequently be used to determine the amount of adsorbed species (Section 14.3). Nonelectro-chemical methods can also be applied to both electroactive and electroinactive species. For example, the change in concentration of an adsorbable solution species after immersion of a large-area electrode and application of different potentials can be monitored by a sensitive analytical technique (e.g., spectrophotometry, fluorimetry, chemiluminescence) that can provide a direct measurement of the amount of substance that has left the bulk solution upon adsorption (7, 44). Radioactive tracers can be employed to determine the change in adsorbate concentration in solution (45). Radioactivity measurements can also be applied to electrodes removed from the solution, with suitable corrections applied for bulk solution still wetting the electrode (45). A general problem with such direct methods is the sensitivity and precision required for accurate determinations, since the bulk concentration changes caused by adsorption are usually rather small (see Problem 13.7). 

The Emneus group in Denmark has reported the sensing of atrazine in surface water using microfluidic enzyme immunoassay and chemiluminescence as the transduction technique [19]. Silicon microchips were functionalized with proteins A and G via a hydrophilic polymer layer. An affinity capture competitive immunoassay was performed using a mixture of enzyme tracer [horseradish peroxidase (HRP)], atrazine sample, and antiatra-zine antibody which was subsequently injected on the microchannel, followed by addition of the substrate mixture [luminol/p-iodophenol (PIP)/H202]. Chemiluminescent oxidation of luminol/PIP/H202 in the presence of HRP enzyme was monitored via PMT. 

McCullough et al heated NO/Ar mixtures to temperatures in the range 1750-2100 K in an alumina flow tube reactor and monitored the fractional decomposition of NO as a function of flow rate (residence time) in the reactor using a commercial chemiluminescent analyzer. Experiments at low temperatures were also performed but the data were excluded because of the influence of surface reactions. The high-temperature central section of the reactor was packed with small pieces of alumina to promote uniform flow, and pulsed tracer experiments were conducted to determine deviations from plug flow. A detailed kinetic and flow model was used, with some simplifications to reduce computing time, to calculate the fractional removal of NO versus flow rate. A... 

---