Direct chemiluminescence immunoassays

Chemiluminescent Immunoassay. Chemiluminescence is the emission of visible light resulting from a chemical reaction. The majority of such reactions are oxidations, using oxygen or peroxides, and among the first chemicals studied for chemiluminescence were luminol (5-amino-2,3-dihydro-l,4-phthalazinedione [521-31-3]) and its derivatives (see Luminescent materials, chemiluminescence). Luminol or isoluminol can be directly linked to antibodies and used in a system with peroxidase to detect specific antigens. One of the first appHcations of this approach was for the detection of biotin (31). 

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. 

An example for an anti-HBe test with a direct label is the electrochemiluminescence immunoassay ECLIA from Roche Diagnostics (automated on Elecsys immunoassay analyser), whereas the Im-mulite anti-HBe test from Diagnostics Products Corp. represents an enzyme-amplified chemiluminescence immunoassay with sustained signal (automated on the Immulite chemiluminescent system). 

De Boever J, Kohen F, Usanachitt C, Vandekerckhove D, Leyseele D, Vandewalle L. Direct chemiluminescence immunoassay for estradiol in serum. Clin Chem 1986 32 1895-900. 

De Boever, J., Kohen, F., Leyseele, D., and Vandekerckhove, D., Isoluminol-based direct chemiluminescence immunoassay for steroid hormones. In Luminescence Immunoassay and Molecular Applications (K. Van Dyke and R. Van Dyke, eds.), pp. 119-139. CRC Press, Boca Raton, FL, 1990. 

A variation of flow-injection analysis, sequential-injection analysis (SIA), has recently been introduced. Contrary to flow-injection analysis, where the components are injected into a continuous flowing stream, in SIA injections are done sequentially directly into the reactor/detector system. The instrumental setup usually consists of a syringe pump in combination with an electronically controlled multi-port valve. SIA systems have been shown to be more flexible regarding injection volumes and injection times. This technology has been demonstrated to be very efficient for carrying out immunoassays. Both fluorescence and chemiluminescence immunoassays have already been carried out with this technique. 

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. 

Antibody molecules have no inherent characteristic that facilitates their direct detection in immunoassays. A second important step in developing a successful immunoassay, therefore, involves the incorporation of a suitable marker . The marker serves to facilitate the rapid detection and quantification of antibody-antigen binding. Earlier immunoassay systems used radioactive labels as a marker (radioimmunoassay RIA) although immunoassay systems using enzymes (enzyme immunoassays EIA) subsequently have come to the fore. Yet additional immunoassay systems use alternative markers including fluorescent or chemiluminescent tags. 

Immunosensors have been designed which use both direct and indirect immunoassay technology to detect specific analytes within a minute or less in a variety of matrices (see Fig. 9). Indirect immunosensors may employ ELA, FLA, or CLIA principles whereby enzyme-, fluorophore- or chemiluminescent-labeled analyte competes with the target (nonlabeled) analyte for binding sites on the immobilized antibody. Unbound (free) labeled analyte is then quantitated using an electrochemical, optical, or electromechanical transducer and compared to the amount of target analyte in the sample. 

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

A number of nonisotopic immunoassays for estradiol have been developed and adapted for use on fuHy automated immunoassay systems. All are heterogeneous assays (separation step needed), but most are direct assays and do not require prehminary extraction. Most procedures offer the convenience of solid-phase separation methods. For routine clinical applications, the greatest experience is with enzyme immunoassays. Most commercial enzyme immunoassays use horseradish peroxidase or alkaline phosphatase to label estradiol antigens enzyme activity is determined using a variety of photometric,fluorescent,or chemiluminescent substrates. ... 

Chemiluminescence continues to be a very selective and sensitive detection tool in FIA immunoassays. The main component in the most commonly used heterogeneous systems is an immunoreactor column, consisting of PTFE tubing packed with immobilized antibodies or haptens. The immunoreac-tion and the chemiluminescence reaction takes place within the immunoreactor, and the emitted light is collected directly from the cell. 

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